Combined Protocols

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Contents
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1.
1.1.
1.2.
1.3.
Introduction
Technology for routine three dimensional (3D) cell culture
Choosing the right AlvetexScaffold format
Handling AlvetexScaffold
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2. 3D Cell Culture Set Up

2.1. Use of AlvetexScaffold in 24-well plate format (AVP006)
2.2. Use of AlvetexScaffold in 12-well plate format (AVP002)
2.3. Use of AlvetexScaffold in well insert formats (AVP004-3 and AVP005-3)
2.4. Use of well Insert holder in deep Petri dish (AVP015)
2.5. Examples of 3D cell cultures on AlvetexScaffold presented in different formats
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3. Use of Coating Reagents with AlvetexScaffold

3.1. Poly-D or L-lysine coating of AlvetexScaffold
3.2. Collagen I coating of AlvetexScaffold
3.3. Matrigel coating of AlvetexScaffold
3.4. Fibronectin Coating of AlvetexScaffold
3.5. Collagen I Thin Gel Coating on AlvetexScaffold
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4. Cell Visualisation & Monitoring Cell Attachment/Growth

4.1. MTT cell viability assay of cultures grown on AlvetexScaffold in 3D
4.2. Simple Visualisation of Cells on AlvetexScaffold Using Light Microscopy
4.3. Live Cell Imaging of 3D Cultures Grown on AlvetexScaffold Using
Confocal Microscopy
4.4. Immunofluorescence confocal microscopy of 3D cultures grown on AlvetexScaffold
4.5. AlamarBlue Cell Viability Assay of upcyte hepatocyte Cultures Grown on
AlvetexScaffold in 3D
4.6. Determination of Cell Number and Growth Characteristics within AlvetexScaffold
using the PicoGreen Assay.
4.7. MTS Cell Viability Assay of upcyte hepatocyte Cultures Grown on
AlvetexScaffold in 3D
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10
Note: Please See Section 10: A Review of Imaging Techniques Compatible with Three
Dimensional Culture of Cells Grown in AlvetexScaffold)
HPM/18/09/2012
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5. Histology of Cell Cultures Grown on AlvetexScaffold

5.1. Choosing the Right Fixative to Preserve 3D Cell Cultures
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5.2. Processing AlvetexScaffold 3D Cultures for PFA Fixation and Paraffin Wax
Embedding for Immunofluorescence
5.3. Processing AlvetexScaffold 3D Cultures for Bouins Fixation and Paraffin Wax
Embedding for Haematoxylin and Eosin Staining
5.4. Processing AlvetexScaffold 3D Cultures for Resin Embedding and Toluidine Blue
Staining for Light Microscopy
5.5. Processing AlvetexScaffold 3D Cultures for Scanning Electron Microscopy (SEM)
5.6. Processing AlvetexScaffold 3D Cultures for Cryosectioning
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6. Methods for Molecular Analysis of Cells Cultured in AlvetexScaffold

6.1. In Situ Total Protein Extraction from Cells Cultured in AlvetexScaffold
6.2. In Situ Extraction of RNA from Cells Cultured in AlvetexScaffold
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8. Specialised Applications using AlvetexScaffold

8.1. Transfection of cells in AlvetexScaffold 3D Cell Culture
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9. Example Protocols for the Growth of Specific Cell Types on AlvetexScaffold

9.1. Culture of the 3T3 cell line on AlvetexScaffold in 12-well plate (AVP002) and
6-well insert format (AVP004-03)
9.2. Culture of the TERA2.cl.SP12 cell line on AlvetexScaffold in 12-well plate
format (AVP002) and 6-well insert format (AVP004-03)
9.3. Epidermal Equivalent HaCaT Cells
9.4. Culture of the HepG2 cell line on AlvetexScaffold 12-well plate format
(AVP002) & 6-well insert format (AVP004-03)
9.5. Culture of the CHO-K1 Cell Line
9.6. Culture of MCF-7 Cells
9.7. Culture of Primary Rat MSCs on AlvetexScaffold in Well Insert Formats
9.8. Culture of the MG63 Cell Line on AlvetexScaffold in Well Insert Formats
9.9. Culture of SW620 Cell Line
9.10. Culture of SW480 Cell Line
9.11. Co-culture of SW620 or SW480 with 3T3 Fibroblasts
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Retrieval of Cells from AlvetexScaffold
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10. White Papers

10.1. A Review of Imaging Techniques Compatible with Three Dimensional Culture
of Cells Grown in AlvetexScaffold
11. Publications
Data on the use of AlvetexScaffold have been published extensively in peer
reviewed publications exemplifying its use with a large number of celltypes
(including hepatocytes, fibroblasts, stem cells, tumour cells). These publications
also cover the use of AlvetexScaffold in a range of applications, particularly
those relevant to drug development (e.g. disease modelling and toxicity screening).
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1.
Schutte et al (2011). Rat primary hepatocytes show enhanced performance and

sensitivity to acetaminophen during three-dimensional culture on apolystyrene
scaffold designed for routine use. Assay Drug Dev. Technol. Epub ahead of print.
2.
Neofytou et al (2011). Adipose tissue-derived stem cells display a proangiogenic

phenotype on 3D scaffolds. J. Biomedical Materials Research. Epub.
3.
Rajan et al (2011). Dysregulated TRK signalling is a therapeutic target in CYLD

defective tumours. Oncogene. Epub ahead of print.
4.
Fox et al (2010). Validation of reference gene stability for APAP hepatotoxicity studies
in different in vitro systems and identification of novel potential toxicity biomarkers.
In Vitro Toxicology, 24 (7), 1962-1970.
5.
Maltman and Przyborski (2010). Developments in three dimensional cell culture

technology aimed at improving the accuracy of in vitro analyses.Biochem. Soc. Trans.,
38 (4), 1072-1075.
6.
Bokhari et al (2007). Novel cell culture device enabling three-dimensional cell growth
and improved cell function. Biochem. Biophys. Res. Comm.,354, 1095-1100.
7.
Bokhari et al (2007). Culture of HepG2 liver cells on three-dimensional polystyrene

scaffolds enhances cell structure and function during toxicological challenge.
J. Anatomy, 211 (4), 567-76.
8.
Bokhari et al (2007). Emulsion templated porous polymers as scaffolds for

three-dimensional cell culture: effect of synthesis parameters on scaffold formation
and homogeneity. J. Materials Chem. 17, 4088-4094.
9.
Carnachan et al (2006). Tailoring the morphology of emulsion-templated porous

polymers. Soft Matter, 2, 608-616.
10. Barbetta et al (2005). Porous polymers by emulsion templating. Macromolecular

Symposia, 226, 203-211.
11. Hayman et al (2005). Growth of human stem cell-derived neurons on solid
three-dimensional polymers. J. Biochem. Biophys. Methods, 62, 231-240.
12. Hayman et al (2004). Enhanced neurite outgrowth by human neurons grown on
solid three-dimensional scaffolds. Biochem. Biophys. Res.Comm., 314, 483-488.
General Information
Product Descriptions:
Description
Catalogue Number
Contains:
AlvetexScaffold
12 Well Plate Starter Pack
AVP002-2
2 X 12 Well Plates
(AVP002)
AlvetexScaffold
Well Insert Holder
Starter Pack
AVP015-2
2 x Well Insert Holder

in Deep Petri Dish
(AVP015)
AlvetexScaffold
6 Well Insert Starter Pack
AVP004-12
12 X 6 Well Inserts
(AVP004)
AlvetexScaffold
12 Well Insert Starter Pack
AVP005-12
12 X 12 Well Inserts
(AVP005)
Intended Use
AlvetexScaffold is intended for in vitro research only.
CAUTION: Not intended for human or animal diagnostic or therapeutic uses.
Protocols and Instructions
To view or download the AlvetexScaffold protocol that best suits your chosen cell type
and experimental design simply visit: www.reinnervate.com/alvetex/protocols and
choose from the following categories:
Preparation and use of AlvetexScaffold Products
Example Protocols for the Growth of Specific Cell Types
Protocols for Analytical Techniques
Protocols for Specialised Applications
Storage & Shelf Life
Store at room temperature. The product is sterile and is expected to be stable indefinitely
whilst the blister pack and peel away lid are intact.
Precautions
All handling of AlvetexScaffold products should be performed wearing gloves according
to standard aseptic methods required for cell culture in a Class I/II cabinet. All products
have been terminally sterilised by gamma irradiation and remain sterile until opened.
Contact Information for placing orders
For all other enquiries: NETPark Incubator, Thomas Wright Way, Sedgefield, Co Durham,
TS21 3FD, United Kingdom t: +44 (0)1740 625266 f: +44 (0)1740 625251
e.mail: info@reinnervate.com
Contact Information for technical assistance
Technical Services: techsupport@reinnervate.com
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Protocol Booklet
Introduction
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Technology for Routine Three

Dimensional (3D) Cell Culture
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What is 3D cell culture?

3D cell culture is about creating suitable surroundings for optimal cell growth, differentiation
and function by:
Allowing individual cells to maintain their normal 3D shape and structure with minimal
exogenous support and interference,
Encouraging cells to form complex interactions with adjacent cells and receive and
transmit signals,
Enabling a more natural environment to foster the creation of native architecture found
in tissue structures,
Reducing stress and artificial responses as a result of cell adaptation to flat, 2D growth
surfaces.
What is AlvetexScaffold?
AlvetexScaffold is a highly porous, cross-linked polystyrene scaffold, which has been
sectioned into 200 m thick membranes (below left). The resulting material is inert and does
not degrade during normal use. It has been adapted to fit a variety of conventional cell culture
plastic-ware formats. AlvetexScaffold provides a suitable 3D structure in which cells can
proliferate, migrate, differentiate and function in an appropriate niche environment. Cells
maintain a 3D shape and form close interactions with adjacent cells (below right,
TERA2.cl.SP12 cells maintained for 12 days).
The product has been terminally sterilised by gamma irradiation and remains sterile until
opened. AlvetexScaffold requires an ethanol (EtOH) wash prior to use to render it
hydrophilic. The material is compatible with a broad range of standard molecular, cellular and
histological techniques.
In deciding which AlvetexScaffold format to use, the following factors should be considered
in combination:
Cell type and duration of culture,
The desired depth of cell penetration in the 3D cell culture,
The type of assay to be performed or application to be used for.
The rate of cell growth in AlvetexScaffold is affected by cell metabolism, proliferation rate,
motility and cell size. It is therefore important to choose the AlvetexScaffold format that will
achieve optimal growth for the chosen cell type.
For example, in Figure 1. two different cell types were grown for the same time period using
AlvetexScaffold 12-well plates (AVP002), resulting in two cultures with different characteristics:
Figure 1. 3T3 (A.) and HepG2 (B.) cultures grown on AlvetexScaffold in 12-well plate format
for 7 days.
3T3 cells are small, highly proliferative and invasive, and therefore readily penetrated through
the entire scaffold. In contrast, HepG2 cells are slower growing cells that have a high tendency
for cell-cell attachment and consequently penetrated only the top 30% of the AlvetexScaffold
membrane. In both cases 3D cell culture was achieved. Micrographs taken at 20x magnification.
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AlvetexScaffold is available in a variety of formats, each specifically designed for different

3D cell culture applications. In the 12-well plate (AVP002) AlvetexScaffold is located at the
bottom of each well, whereas in well-inserts (AVP004-3 and AVP005-3) it is suspended. The
well inserts are easy to use and are highly versatile. They fit a range of culture plates from
different manufacturers as well as Reinnervates custom-made Well insert holder in a deep
Petri dish (AVP015).
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AlvetexScaffold formats have been specifically designed to enable optimal 3D cell culture in
both short and long term experiments. The 12-well plate format is ideal for short term cell
cultures where the medium is replaced every 1 to 2 days. The AlvetexScaffold membrane sits
at the bottom of each well and the cells are exposed to the medium only from above. This is
desirable for studies where easy access is required to the cells as they predominantly reside in
the top portion of the scaffold (e.g. for transfection studies). Alternatively a shorter experiment
in well inserts is also suitable, as the cells will not have had time to penetrate the scaffold.
The well inserts are capable of supporting cell growth for up to 3 weeks for assays and
applications where higher cell numbers are desirable (Table 1.). The design of the well insert
allows for greater cell penetration into the scaffold and higher cell yields because
AlvetexScaffold is suspended in the medium such that cells receive nutrients from above
and below. Therefore, they sustain optimal growth for longer and achieve greater
differentiation creating a cultured tissue that more closely resembles the growth of cells in
the body. Well inserts can be used to create co-cultures either in plates or in the well insert
holder in the Petri dish.
Application / AlvetexScaffold format
AVP002
(12-well plate)
AVP004-3
(6-well insert)
AVP005-3
(12-well insert)
Histology (10 m sections)

Immunohistochemistry
Confocal microscopy
Light microscopy (e.g. phase contrast)*
Viability assay
Toxicity assay
Proliferation assay
Metabolic activity assay
Gene expression / Microarray
Protein expression (e.g. Western Blotting)
Air-liquid interface differentiation
Cell signalling assay
Permeability assay
Transfection
Co-culture
Invasion assay
Migration assay
+++
+++
+++
N/A
++
++
++
++
++
++
N/A
++
N/A
+++
+
+
+
+++
+++
+++
N/A
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+++
+++
+++
+++
+++
+++
N/A
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+++
+++
+++
Table 1. Suggested guidelines for the use of AlvetexScaffold formats for cell applications and assays. + + + most suitable,
+ + suitable, + least suitable, N/A= not applicable.
Ranking is based on AlvetexScaffold disc format suitability, the likely cell yields and therefore signal generation, and whether
exogenously added chemicals/cells can be contained to only one side of the membrane.
*The growth of cells cannot be followed by traditional light microscopy as in 2D, but as with ex-vivo tissues,
3D structures have to be evaluated using histology or confocal microscopy. Alternatively cell
proliferation can be monitored using a viability assay such as the MTT.
Figure 2. HaCaT cells grown on AlvetexScaffold in 12 well insert (AVP005-3) in a 6-well plate
(A) and (B) in well insert holder in deep Petri dish (AVP015). Note significantly more proliferation
and cell invasion in the cultures grown in the well insert holder system, where more nutrients
were available. Micrographs taken at 20x magnification.
Thus, AlvetexScaffold products provide cell biologists with a broad range of choice and great
flexibility when designing their cell culture experiments.
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The well insert holder in a deep Petri dish (AVP015) is designed to hold well inserts in a large
volume Petri dish reducing the need for frequent media changes. This format should be used
for prolonged cell growth of highly proliferative and demanding cell types (Figure 2.). The
well inserts can be positioned at three different levels in the insert holder to allow for cultures
to be raised to the air liquid interface (for air liquid interface differentiation) and subsequent
permeability / barrier testing (Table 1.). 3D co-cultures can also be set-up in one or two of the
well inserts within the same Petri dish.
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All procedures concerning the handling of AlvetexScaffold should be performed wearing

gloves according to standard aseptic methods required for cell culture in a Class I/II cabinet.
When dry, AlvetexScaffold is reasonably fragile with a wafer-like consistency; however
once rehydrated the discs become much more robust. Therefore handle the material
carefully when performing any manipulation including media changes, transferring the
discs for analysis, fixing and embedding for histology, etc. When using forceps, exercise
care as manipulating the scaffold can damage its structure. Try to handle the
AlvetexScaffold discs around the edges only.
When dispensing liquids (e.g. 70% EtOH, PBS and medium) over AlvetexScaffold, place
the end of the pipette tip towards the wall of the culture vessel avoiding touching the
scaffold. If using the 12-well plate format, retain cylindrical clip in place. Well inserts
should be fed from the outside: place the end of the pipette tip towards the wall of the
culture vessel (either by going through the window of the well insert or beside it). Let
the liquid rise gently to touch the base of the well insert and if required dispense the rest
of the solution into the well insert to prevent it from floating. Seed cells on the middle
of the disc without touching the membrane itself.
In the 12-well plate format, the AlvetexScaffold is held in place by a polystyrene clip that can
easily be removed to release the disc for analysis of the cultured cells (Figure 1.)
Figure 1. AlvetexScaffold discs can easily be removed from the 12-well plate using flat ended
forceps (Fisher Scientific, MNK-155-M).
Figure 2. Removal of AlvetexScaffold disc from well-insert. Unclip the base of the well
insert all the way around (A), and pull down to release the disc (B). Alternatively the disc can
be cut out by holding the well insert upright (C) or upside down (D).
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In well insert formats, the AlvetexScaffold membrane can be removed using forceps (Figure
2. A & B) or can be cut out using a scalpel with a size 11 blade (C & D).
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3D Cell Culture Set Up
Use of AlvetexScaffold in 24-Well Plate Format (AVP006)

AlvetexScaffold 24-well plate format:
Figure 1. Presentation of AlvetexScaffold in 24-well plate format.

Preparing AlvetexScaffold (in 24-Well Plates) for First Use and Cell Seeding:
Open the 24-well plate carefully to ensure that the clips holding the
membranes are not displaced.
Add approximately 1 ml of 70 % EtOH to each well to pre-treat the

AlvetexScaffold in preparation for incubation in aqueous solutions (e.g. PBS, culture
medium).
Carefully aspirate the EtOH solution and immediately wash the membrane in
~1-1.5 ml of appropriate medium for ~1 min.
Carefully aspirate medium wash and replace with final wash medium (use
same type as for cell seeding). The AlvetexScaffold is now ready: aspirate medium
just before application of cells. If preparation of cell suspension is delayed, incubate
plate with medium at 37 C, 5 % CO2 until further use.
Similarly to 2D culture, if using serum-free medium, consider the use of

coating agents to enhance cell attachment. Prior to cell seeding, AlvetexScaffold
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The 24-well plate format is a simple presentation of AlvetexScaffold

technology: it comprises a single loose membrane and clip per well (Figure 1.). The
clip holds AlvetexScaffold in position during transit and use. The clip is made from
polystyrene, it is sterile and inert, and can easily be removed to release the
membrane. The 24-well plate format is primarily suitable for short term culture
experiments where the medium is replaced every 1-2 days.
can be pre-coated with standard cell culture reagents such as collagen, fibronectin,
laminin, poly-D/L-lysine, poly-L-ornithine and Matrigel to encourage cell adhesion,
differentiation and optimise function. Perform this step after the EtOH treatment and
appropriate buffer wash steps instead of medium. For specific coating protocols
please refer to www.reinnervate.com/workflow.
03
3D Cell Culture Using the AlvetexScaffold 24-Well Format:
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Protocol Booklet
3D cell culture is different to conventional 2D cell culture and as such requires

optimisation according to cell type:
For most applications initial cell seeding densities of 0.25-1.0x106 cells in 5075 l per disc are recommended. Seeding in a low volume enables cells to attach
predominantly to the AlvetexScaffold and avoids cell loss on other surfaces.
When inoculating, aspirate washing medium thoroughly from the plate and
carefully dispense cells on the middle of the membranes. Replace lid and incubate in
a humidified incubator at 37 C with 5 % CO2 for 30 minutes to 3 hours to facilitate
cell attachment.
After this time gently flood the wells with medium by dispensing up to 1.5 ml of
medium per well.
With 3D cell culture there will be many more cells growing per unit volume of
medium. Therefore, users must refresh media more frequently; ideally once a day,
however this will also depend on the population doubling rate and nutrient demands
of the cell type cultured.
Use of AlvetexScaffold in 12-Well

Plate Format (AVP002)
Figure 1. Presentation of AlvetexScaffold in 12-well plate format.

Preparing AlvetexScaffold (in 12-well plates) for first use and cell seeding
Open the 12-well plate carefully to ensure that the clips holding the AlvetexScaffold
discs are not displaced.
Add approximately 2 ml of 70% EtOH to each well to pre-treat the AlvetexScaffold disc
in preparation for incubation in aqueous solutions (e.g. PBS, culture medium).
Carefully aspirate the EtOH solution and immediately wash the AlvetexScaffold disc in
~2-3 ml of appropriate medium for ~1 min.
Carefully aspirate medium wash and replace with final wash medium (use same type of
medium as for cell seeding). The AlvetexScaffold disc is now ready for cell seeding:
aspirate medium just before application of cells. If preparation of cell suspension is
delayed, incubate plate with medium at 37 oC with 5% CO2 until further use.
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AlvetexScaffold 12-well plate format

The 12-well plate format is a simple presentation of AlvetexScaffold technology: it comprises a
single loose disc and clip per well (Figure 1.). The clip holds AlvetexScaffold in position during
transit and use. The clip is made from polystyrene, it is sterile and inert, and can easily be removed
to release the AlvetexScaffold disc. The 12-well plate format is primarily suitable for short term
culture experiments where the medium is replaced every 1-2 days.
Use of AlvetexScaffold in 12-Well

Plate Format (AVP002)
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Similarly to 2D culture, if using serum-free medium, consider the use of coating agents
to enhance cell attachment.
Prior to cell seeding, AlvetexScaffold can also be pre-coated with standard cell culture
reagents such as collagen, fibronectin, laminin, poly-D/L-lysine, poly-L-ornithine and
MatrigelTM to encourage cell adhesion, differentiation and optimise function. Perform
this step after the EtOH treatment and appropriate buffer wash steps instead of medium.
Optimisation of seeding and 3D cell culture using the AlvetexScaffold 12-well format
3D cell culture is different to conventional 2D cell culture and as such requires optimisation
according to cell type:
For most applications initial cell seeding densities of 0.5-2.0x106 cells in 100-150 l per
disc are recommended. Seeding in a low volume enables cells to attach predominantly
to the AlvetexScaffold disc and avoids cell loss on other surfaces.
When inoculating, aspirate washing medium thoroughly from the plate and carefully
dispense cells on the middle of the discs. Replace lid and incubate in a humidified
incubator at 37 oC with 5% CO2 for 30 minutes to 3 hours to facilitate cell attachment.
After this time gently flood the wells with medium by dispensing up to 4 ml of medium
per well.
With 3D cell culture there will be many more cells growing per unit volume of medium.
Therefore, users must refresh media more frequently; ideally once a day, however this
will also depend on the population doubling rate and nutrient demands of the cell type
cultured.
Use of AlvetexScaffold in well insert formats
Figure 1. Presentation of AlvetexScaffold in 6- and 12-well formats.

Preparing AlvetexScaffold (6-well and 12-well inserts) for first use and cell seeding
Open the required number of blister packs carefully and pick up the well insert(s) using
forceps.
Immersion in 70% EtOH will instantly pre-treat AlvetexScaffold in preparation for
incubation in aqueous solutions (e.g. PBS, culture medium). This can be done by dipping
the well insert into a beaker containing 70% EtOH before placing it into the chosen
holder vessel. Gently shake or tap the well insert to remove excess ethanol.
Alternatively EtOH treatment can be performed in situ, once the well insert is positioned
in the plate. Add sufficient 70% EtOH to the well so that the level of the liquid rises
above the membrane (for 6-well plates add approximately 5 ml/well, for 12-well plates
add approximately 2 ml/well).
Carefully aspirate to waste, leaving no excess liquid and immediately wash
AlvetexScaffold in an appropriate medium (for 6-well plates use 7 ml/well, for 12-well
plates use 2.5 ml/well) for ~1 min.
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AlvetexScaffold 6-well insert (AVP004-3) and 12-well insert (AVP005-3)

The presentation of AlvetexScaffold in well insert formats is versatile (Figure 1), enabling
long term 3D culture as cells can receive nutrients from media above and below the
membrane, sustaining optimal 3D cell growth.
Currently there are two well insert sizes available: AVP004-3 (22 mm diameter, A) and AVP0053 (15 mm diameter, B). Both are supplied in blister packs with three individually sealed inserts
containing AlvetexScaffold. The 6- and 12-well inserts are designed to fit into most 6-well
plates or Reinnervates custom-made Well Insert Holder in Deep Petri Dish (AVP015).
Snapping the extended wings of AVP005-3 will also enable it to fit into a 12-well plate. Note that
plates and well insert holders are not supplied with the product and have to be sourced separately.
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Aspirate and replace with final wash medium (use same type of medium as for cell
seeding). The scaffold is now ready for cell seeding: aspirate medium just before
application of cells. If preparation of cell suspension is delayed, incubate plate with
medium at 37 C with 5% CO2 until further use.
Similarly to 2D culture, if using serum-free medium, consider the use of coating agents to
enhance cell attachment. Prior to cell seeding, AlvetexScaffold can also be pre-coated with
standard cell culture reagents such as collagen, fibronectin, laminin, poly-D/L-lysine, poly-Lornithine and MatrigelTM to encourage cell adhesion, differentiation and optimise function.
Perform this step after the EtOH treatment followed by an appropriate buffer wash step instead
of medium.
Optimisation of seeding and 3D cell culture using the AlvetexScaffold 6-well and
12-well insert formats
3D cell culture is different to conventional 2D and as such requires optimisation according to
cell type, assay being performed and insert configuration used (Figure 2):
i. Media from
below only:
for cells grown in 3D
at air-liquid interface
ii. Media from

above and below:
for routine 3D growth of
cells with lower-average
metabolic activity/
proliferation rate OR for
experiments where cells
are incubated with test
substrate in top chamber
only for permeability
investigations.
iii. Media
interconnected:
for routine 3D growth
of cells with high
metabolic activity/
proliferation rate
Figure 2. Media filling levels and well insert configurations.
12-well insert in 6-well plate:

For most applications initial cell seeding densities of 0.25-1.0x106 cells in 50-75 l per disc
are suitable. Seeding in a low volume enables cells to attach predominantly to the disc
and avoids cell loss on other surfaces.
When inoculating, aspirate washing media thoroughly from the plate and carefully
dispense cells on the middle of the discs without touching the membrane. Replace lid
and incubate in a humidified incubator at 37 C with 5% CO2 for 30 to 90 minutes to
facilitate cell attachment.
After this time gently flood the wells with media by dispensing 3.0-10.5 ml
of medium per well. Fill up the wells carefully beside the insert, so the medium comes
up from the bottom to gently contact the cellularised AlvetexScaffold disc and gradually
floods the insert itself. The volume of medium required will depend on user requirements
and recommendations are outlined in Table 1 below.
For most applications initial cell seeding densities of 0.25-1.0x106 cells in 50-75 l per disc
are suitable. Seeding in a low volume enables cells to attach predominantly to the disc
and avoids cell loss on other surfaces.
When inoculating, aspirate washing media thoroughly from the plate and carefully
After this time gently flood the wells with media by dispensing 1.4-4.0 ml of medium per
well: Fill up the wells carefully beside the insert, so the medium comes up from the bottom
to gently contact the cellularised AlvetexScaffold disc and gradually floods the insert itself.
The volume of medium required will depend on user requirements and recommendations
are outlined in Table 1.
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For most applications initial cell seeding densities of 0.5-2.0x106 cells in 100-150 l per
disc are suitable. Seeding in a low volume enables cells to attach predominantly to the
disc and avoids cell loss on other surfaces.
When inoculating, aspirate washing medium thoroughly from the plate and carefully
After this time gently flood the wells with medium by dispensing 3.0-10.5 ml of medium
per well: Fill up the wells carefully beside the insert, so the medium comes up from the
bottom to gently contact the cellularised AlvetexScaffold disc and gradually floods the
insert itself. The volume of medium required will depend on user requirements and
recommendations are outlined in Table 1 below.
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Well insert and holder type
Feeding volumes
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Protocol Booklet
Below only(i)
Above and
Above and below
below separately(ii) interconnected(iii)
6-well insert in a 6-well plate
3.5 0.5 ml/well
7 1 ml/well
10 0.5 ml/well
12-well insert in a 6 well plate
3.5 0.5 ml/well
7 1 ml/well
10 0.5 ml/well
12-well insert in a 12-well plate
1.6 0.2 ml/well
2.4 0.2 ml/well
3.8 0.2 ml/well
Table 1. Feeding volumes for the different well insert configurations (i-iii, see also Figure 2.)
In 3D cell culture there will be more cells per unit volume of medium. Therefore, users must
refresh medium more frequently typically every 21 days, however this will also depend on the
population doubling rate, nutrient demands of the cell type cultured and the volume of medium
used as described above.
If any signs of cell attachment and growth are evident on the bottom of the plate, transfer the
well inserts into a new plate, re-feed and then incubate as usual.
Use of Well Insert Holder in

Deep Petri Dish (AVP015)
Figure 1. AVP005-3 (left) and AVP004-3 (right) in well insert holder system in deep Petri dishes.
AlvetexScaffold well insert formats and their use in the well insert holder
The deep Petri dish enables users to grow their 3D cultures in larger volumes of media
compared to an ordinary multiwell plate. Up to 95 ml of media can be used in the deep Petri
dish and is therefore capable of sustaining long term 3D culture experiments (3-4 weeks) and
reducing the frequency of medium exchanges. If required, a magnetic stirrer bar can be
placed in the bottom of the dish to circulate media and facilitate exchange.
The well insert can be positioned at three different levels in the insert holder: high, medium
and low (Figure 2).
High
Medium
Low
Figure 2. Well insert settings within the well insert holder (AVP015).
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A single well insert holder capable of housing up to three well inserts (6- or 12-well inserts,
Figure 1.) in a deep Petri dish is supplied in a pack. The Petri dish itself is not tissue culture
treated. The whole product has been terminally sterilised by gamma irradiation and remains
sterile until opened.

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This feature allows cultures to be raised to the air liquid interface by moving the insert to a
different level within the same holder.
Positioning the well inserts at different levels may be used to conserve expensive media or
allow for increasing media volumes for demanding cell types over the course of a long term
experiment.
Additionally, cultures can be fed from below the well insert only (i.), media from above and
below separately (ii.) and media interconnected (iii.) through the windows at the side of the
well inserts (Figure 3):
i.
ii.
iii.
Figure 3. Media filling levels and well insert configurations.

Recommended volumes for AVP004-3 (6-well insert):
Feeding Volume
Well insert setting within holder
Below only (i.)
Above and below

separately (ii.)
Above and below

interconnected (iii.)
Low
20ml 1ml
40ml 3ml
70ml 5ml
Medium
34ml 2ml
50ml 3ml
80ml 3ml
High
48ml 2ml
70ml 5ml
92ml 3ml

12
Recommended volumes for AVP005-3 (12-well insert):
Well insert setting within holder
Below only (i.)
Above and below

separately (ii.)
Above and below

interconnected (iii.)
Low
20ml 1ml
40ml 3ml
72ml 5ml
Medium
34ml 2ml
52ml 3ml
82ml 3ml
High
48ml 2ml
70ml 5ml
92ml 3ml
The well insert holder system also allows for the 3D co-culture of more than one cell type
by seeding different cells in one or two of the well inserts within the same Petri dish. 3D cocultures can also be set up within the same well insert. Alternatively, a support cell line can
be cultured at the base of the Petri dish in 2D and another in 3D in the well inserts.
As the Petri dishes are untreated, coat with poly-L-lysine, or similar, to facilitate cell
attachment. Ensure that suitable media is chosen that will simultaneously support the growth
of both cell types cultured. The well insert holder will fit into deep Petri dishes with
approximate dimensions of > 86 mm (internal diameter) x > 25 mm (height).
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Feeding Volume
Examples of 3D cell cultures on AlvetexScaffold

presented in different formats
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Comparison of 3D cell growth patterns on AlvetexScaffold 12-well plate format (AVP002)

AlvetexScaffold in the 12-well plate format (AVP002) was treated with EtOH and washed with
complete medium prior to cell seeding. [Complete medium consisted of: DMEM, 10% FBS, 2
mM L-glutamine and 100 U/ml Penicillin & Streptomycin]. HaCaT cells (a human keratinocyte cell
line) were plated at a density of 0.5x106 cells in 150 l per well while HepG2 cells (a human liver
cell line) were seeded at 2x106 cells in 150 l per well. Plates were incubated for three hours
before flooding with further media and maintained for 7 days. After preserving in Bouins fixative
the discs were paraffin embedded, sectioned (10 m) and counterstained with Haematoxylin
and Eosin. HaCaT cultures demonstrated significant cell invasion into the matrix, while HepG2
cells remained resident in the top 25% of the matrix.
HaCaT cells
HepG2 cells
Figure 1. Comparison of 3D cell growth patterns on AlvetexScaffold12-well plate format

(AVP002). Micrographs taken at 20x magnification.

12 well plate (AVP002)
6-well insert (AVP004-3) in a 6-well plate
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Comparison of 3D cell growth patterns of HaCaT cells on AlvetexScaffold presented

in various formats
HaCaT cells (a human keratinocyte cell line) were seeded (0.5x106 cells in 150 l per well) on
EtOH-treated and complete medium washed AlvetexScaffold scaffolds in the following
formats: 12-well plate (AVP002), 6-well inserts (AVP004-3) in 6-well plate and 12-well inserts
(AVP005-3) in well insert holder in deep Petri dish (AVP015). Cultures were maintained for 7
days. [Complete medium consisted of: DMEM, 10% FBS, 2 mM L-glutamine and 100 U/ml
Penicillin & Streptomycin]. After preserving in Bouins fixative the discs were paraffin embedded,
sectioned (10 m) and counterstained with Haematoxylin and Eosin.

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12-well inserts (AVP005-3) in well insert holder in deep Petri dish (AVP015)
Figure 2. Comparison of 3D cell growth patterns of HaCaT cells grown on various

AlvetexScaffold formats. Micrographs taken at 20x magnification.
Note significantly more proliferation and cell invasion into the AlvetexScaffold scaffold in
cultures grown in well-inserts, (media interconnected feeding regime) due to nutrient availability
from above and below the scaffold. Thus, as media volume and availability increased so did cell
proliferation and scaffold penetration. In the case of well inserts contained in a well insert holder
in a deep Petri dish, this resulted in the formation of a slab of tissue-like material.
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Use of Coating Reagents

with AlvetexScaffold
Poly-D or L-lysine Coating of AlvetexScaffold
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Introduction
The following protocol outlines how to coat AlvetexScaffold membranes with poly-D or Llysine in order to facilitate cell attachment and migration within the scaffold.
Poly-L-lysine is a synthetic amino acid that has been widely used as a coating agent in tissue
culture to enhance cell attachment to untreated plastic and glass. When Poly-L-lysine is applied,
it renders the material positively charged and increases the electrostatic attraction between the
surface and the negatively charged cells thus improving adhesion. The use of Poly-D-lysine is
favoured with cells displaying high proteolytic activity as it is less prone to breakdown than the
naturally-occurring L-enantiomer. Otherwise, Poly-D-Lysine and Poly-L-Lysine are equivalent for
these purposes. Refer to manufacturers instructions when choosing poly-lysine formats and
dilutions required for the desired cell type.
Note also that the available molecular weight (MW) ranges of poly-lysine vary. The lower the
MW, the less viscous the solution, while the higher the MW, the more binding sites per
molecule. In this protocol 3T3 fibroblasts were grown on poly-L-lysine (Sigma-Aldrich, P4707)
coated AlvetexScaffold in 12-well plate (AVP002) format for 7 days. The MW of poly-L-lysine
ranged from 70-150 kDa, which remained easy to handle while providing sufficient binding
sites for cell attachment.
Method:
1. Prepare AlvetexScaffold for coating by first treating with 70% ethanol followed by two
PBS washes as described in the relevant product information leaflet. Leave
AlvetexScaffold in the second PBS wash until ready to apply the poly-L-lysine solution.
2. Aspirate the second PBS wash and add 500 l of poly-D or L-lysine per well. Replace plate
lids and leave to stand for 1 hour at room temperature.
3. Tilt the 12-well plate and gently aspirate any excess fluid from the edge of the wells. If
using well insert AlvetexScaffold formats, remove excess fluid from AlvetexScaffold by
gently tapping the plate or Petri dish on the worktop. Check that no residual fluid is hanging
from the base of the well inserts. Aspirate to remove any residual coating agent from the
bottom of the wells.
4. Prepare cells for seeding in the appropriate culture media and seed directly on the wet polyL-lysine coated AlvetexScaffold membrane in the volumes relevant for the
AlvetexScaffold product format. Allow the cells to settle for 30-90 minutes in an incubator
(5 % CO2, 37 C) before flooding with media.
Poly-D or L-lysine Coating of AlvetexScaffold
03
Example: Growth of 3T3 Cells in poly-L-lysine Coated AlvetexScaffold
Results:
Pre-coating of AlvetexScaffold discs with poly-L-lysine resulted in enhanced infiltration of
3T3 cells into the scaffold compared with control cultures in uncoated AlvetexScaffold. Cells
were seen to reside deep within the scaffold and in greater abundance after 7 days of growth
in treated discs. These findings indicate that pre-treatment of AlvetexScaffold with well
established tissue culture coating agents is able to enhance the attachment and growth of
appropriate cell types into the 3D structure.
Uncoated AlvetexScaffold control
100 m
50 m
Poly L lysine-coated AlvetexScaffold

100 m
50 m
Figure 1. Brightfield micrographs showing the structure of 3T3 cells cultured on non-coated
and poly-L-lysine coated AlvetexScaffold discs in 12 well plate format
(AVP002) after 7 days culture
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Protocol Booklet
Cell Culture details:

3T3 cells were routinely maintained in T-75 flasks. Complete media consisted of: DMEM media
supplemented with 10 % v/v FBS, 2 mM L-glutamine and 100 U/ml Penicillin/Streptomycin.
AlvetexScaffold 12-well plates (AVP002) were coated in poly-L-lysine (Sigma-Aldrich, P4707, MW:
70-150 kDa, 0.01% solution) as described above.
Cells were seeded at a density of 0.5 x 106 cells in 125 l media suspension per disc and were left
to settle for 60 minutes in an incubator (5 % CO2, 37 C). Media was carefully added to each sample
(9 ml per well). Cultures were maintained for 7 days, with media changes every second day.
Collagen I Coating of AlvetexScaffold
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Introduction
The following protocol outlines how to coat AlvetexScaffold membranes with a thin layer of
collagen I in order to facilitate and enhance cell attachment and migration within the scaffold.
Example data shown herein was obtained using this protocol to grow HepG2 hepatocytes on
collagen I coated AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in 6-well plate format.
Method:
AlvetexScaffold in the second PBS wash until ready to apply the collagen solution.
2. Dilute rat tail collagen I (BD Biosciences, 354236) to a concentration of 0.8 mg/ml using
cell culture grade water. Handle the reagents on ice, using pre-chilled pipette tips to
perform the dilution and subsequent application onto AlvetexScaffold.
3. Aspirate the second PBS wash from AlvetexScaffold disc and carefully pipette 500 l of
the diluted collagen solution onto each disc. Replace plate lids and leave to stand for 1
hour at room temperature.
4. Remove excess fluid from AlvetexScaffold in well insert format by gently tapping the
plate or Petri dish on the worktop. Check that no residual fluid is hanging from the base of
the well inserts. Aspirate to remove any residual coating agent from the bottom of the
wells. If using AlvetexScaffold in 12-well plate format, tilt the plate and gently aspirate any
excess fluid from the edge of the wells.
5. Prepare cells for seeding in the appropriate culture media and seed directly on the wet
collagen coated AlvetexScaffold membrane in the volumes relevant for AlvetexScaffold
product format. Allow the cells to settle for 30-90 minutes in an incubator (5 % CO2, 37
C) before flooding with media.
Collagen I Coating of AlvetexScaffold
05
Example: Growth of HepG2 Hepatocyte Cell Line in Collagen I Coated AlvetexScaffold
Results:
Pre-coating of AlvetexScaffold discs with collagen I resulted in enhanced infiltration of HepG2
cells into the scaffold compared with control cultures in untreated AlvetexScaffold. Cells were
seen to reside deep within the scaffold after 7 days of growth in treated discs, while cells
grown in untreated AlvetexScaffold occupied only the upper half of the scaffold. These findings
indicate that pre-treatment of AlvetexScaffold with extracellular matrix products is able to
enhance the attachment and growth of appropriate cell types into the 3D structure.
3 Day
200 m
7 Day Culture
50 m
200 m
50 m
Collagen I-coated AlvetexScaffold

3 Day
200 m
7 Day Culture
50 m
200 m
50 m
Figure 1. Brightfield micrographs at low (10x) and high (40x) magnification showing HepG2
cells cultured for up to 7 days on 22 mm diameter AlvetexScaffold discs presented in 6-well
insert (AVP004-3) in 6-well plate format. Cells were fixed, sectioned and counterstained with
haematoxylin and eosin.
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Protocol Booklet

HepG2 cells (ATCC, HB-8065) were routinely maintained in T-75 flasks. HepG2 complete media
consisted of: MEM media (Gibco, 21090) supplemented with 10 % v/v FBS, 2 mM L-glutamine
and 100 U/ml Penicillin/Streptomycin. AlvetexScaffold 6-well inserts (AVP004-3) in 6-well
plates, were coated in collagen I as described above.
Cells were seeded at a density of 1 x 106 cells in 150 l media suspension per disc and were
left to settle for 90 minutes in an incubator (5 % CO2, 37 C). Media was carefully added to each
sample (9 ml per well). Cultures were maintained for 7 days, with media changes on days 2,
3, and 6.
MatrigelTM Coating of AlvetexScaffold
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Introduction
The following protocol outlines how to coat AlvetexScaffold membranes with MatrigelTM to
facilitate and enhance attachment and differentiation of both normal and transformed anchorage
dependent cells. Example data shown herein was obtained using this protocol to grow HepG2
hepatocytes in AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in 6-well plate format.
Method:
AlvetexScaffold in the second PBS wash until ready to apply the MatrigelTM solution.
2. Dilute Matrigel (BD Biosciences, 356234; pre-thawed overnight on ice) to a concentration
of 0.8 mg/ml (1 in 10 dilution) using appropriate cell culture media (e.g. MEM for the
example below). Handle the reagents on ice, using pre-chilled pipette tips to perform the
dilution and subsequent application onto AlvetexScaffold.
3. Aspirate the second PBS wash from AlvetexScaffold disc and carefully pipette 350 l of
the diluted Matrigel solution onto each disc. Replace plate lids and leave to stand for 12 hours at room temperature.
4. Remove excess fluid from AlvetexScaffold in well insert format by gently tapping the
plate or Petri dish on the worktop. Check that no residual fluid is hanging from the base of
the well inserts. Aspirate to remove any residual coating agent from the bottom of the
wells. If using AlvetexScaffold in 12-well plate format, tilt the plate and gently aspirate any
excess fluid from the edge of the wells.
5. Prepare cells for seeding in the appropriate culture media and seed directly on the wet
Matrigel coated AlvetexScaffold membrane in the volumes relevant to the
AlvetexScaffold product format. Allow the cells to settle for 30-90 minutes in an incubator
(5 % CO2, 37 C) before flooding with media.
MatrigelTM Coating of AlvetexScaffold
Example: Growth of HepG2 Hepatocyte Cell Line in MatrigelTM Coated AlvetexScaffold
Results:
Pre-coating of AlvetexScaffold discs with Matrigel resulted in enhanced infiltration of cells
into the scaffold compared with control cultures in untreated AlvetexScaffold. Cells were seen
to occupy the entire depth of the scaffold after 7 days of growth in Matrigel-coated discs,
while cells grown in untreated AlvetexScaffold occupied only the upper half of the scaffold.
These findings indicate that pre-treatment of AlvetexScaffold with extracellular matrix products
is able to enhance the growth of appropriate cell types into the 3D structure.
3 Day
200 m
7 Day Culture
50 m
200 m
50 m
Matrigel-coated AlvetexScaffold
3 Day
200 m
7 Day Culture
50 m
200 m
50 m
Figure 1. Brightfield micrographs at low (10x) and high (40x) magnification showing HepG2
cells cultured for up to 7 days on 22 mm diameter AlvetexScaffold discs presented in 6-well
insert (AVP004-3) in well insert holders in 6-well plate format. Cells were fixed, sectioned and
counterstained with haematoxylin and eosin.
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Protocol Booklet

and 100 U/ml Penicillin/Streptomycin. AlvetexScaffold 6-well inserts (AVP004-3) in 6-well
plates, were coated with MatrigelTM as described above.
Cells were seeded at a density of 1 x 106 cells in 150 l media suspension per disc and were
left to settle for 60 minutes in an incubator (5 % CO2, 37 C).
Media was carefully added to each sample well (9 ml per well). Cultures were maintained for
7 days, with media changed on days 3 and 5.
07
Fibronectin Coating of AlvetexScaffold
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Introduction
The following protocol outlines how to coat AlvetexScaffold membranes with fibronectin
in order to facilitate and enhance cell attachment and migration within the scaffold.
Example data shown herein was obtained using this protocol to grow HepG2 hepatocytes
on fibronectin coated AlvetexScaffold for 7 days in 6-well inserts (AVP004) in a 6-well
plate format.
Method
1.
Prepare AlvetexScaffold for coating by first treating with 70 % ethanol followed by

two PBS washes as described in the relevant product information leaflet. Leave
AlvetexScaffold in the second PBS wash until ready to apply the fibronectin solution.
2.
Reconstitute fibronectin (BD Biosciences, 356008) to a concentration of 0.5 mg/ml

using PBS.
3.
Aspirate the second PBS wash from AlvetexScaffold disc and carefully pipette 300 l
of the diluted fibronectin solution onto each disc. Replace plate lids and leave to stand
for 1 hour at room temperature.
4.
Remove excess fluid from AlvetexScaffold in well insert format by gently tapping the
plate or Petri dish on the worktop. Check that no residual fluid is hanging from the
base of the well inserts. Aspirate to remove any residual coating agent from the
bottom of the wells. If using AlvetexScaffold in 12-well plate format, tilt the plate and
gently aspirate any excess fluid from the edge of the wells.
5.
Prepare cells for seeding in the appropriate culture media and seed directly on the wet
fibronectin coated AlvetexScaffold membrane. Seed cells in volumes relevant for the
specific AlvetexScaffold format being used (see product information booklet for
volume details). Allow the cells to settle for 30-90 minutes in an incubator (5 % CO2,
37 C) before flooding with media.
Example Growth of HepG2 Hepatocyte Cell Line in Fibronectin Coated AlvetexScaffold

HepG2 cells (ATCC, HB-8065) were routinely maintained in T-75 flasks. HepG2 complete
media consisted of: MEM media (Gibco, 21090) supplemented with 10 % v/v FBS, 2 mM
L-glutamine and 100 U/ml Penicillin/Streptomycin. AlvetexScaffold 6-well inserts
(AVP004) in 6-well plates, were coated in fibronectin as described above.
Cells were seeded at a density of 1 x 106 cells in 150 l media suspension per disc and
were left to settle for 60 minutes in an incubator (5 % CO2, 37 C).
Media was carefully added to each sample (10 ml per well). Cultures
were maintained for 7 days with media changes on days 2 and 5.
Fibronectin Coating of AlvetexScaffold

7 Day
Fibronectin coated AlvetexScaffold

7 Day
Figure 1. Brightfield micrographs at low (10x) and high (40x) magnification showing HepG2 cells cultured
for up to 7 days on 22 mm diameter AlvetexScaffold discs presented in 6-well insert (AVP004) in 6-well
plate format. Cells were fixed, sectioned and counterstained with Haematoxylin and Eosin.
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Results
Pre-coating of AlvetexScaffold discs with fibronectin resulted in enhanced infiltration of
HepG2 cells into the scaffold compared with control cultures in untreated AlvetexScaffold.
Cells were seen to have reached deeper within the scaffold after 7 days of growth in treated
discs, while cells grown in untreated AlvetexScaffold occupied only the upper part of the
scaffold. These findings indicate that pre-treatment of AlvetexScaffold with extracellular
matrix products is able to enhance the attachment and growth of appropriate cell types
into the 3D structure.
Collagen I Thin Gel Coating on AlvetexScaffold
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Introduction:
Extra-cellular matrix (ECM) coating of in vitro culture surfaces is commonly used to enhance
cell-substrate adhesion, encourage cell-matrix signalling and to protect shear-sensitive cells
in perfusion devices. The following protocol outlines how to coat the top surface of
AlvetexScaffold membranes with a thin gel layer of collagen I. This method can be used to
promote adhesion and culture of a monolayer of epithelial cells at the surface of
AlvetexScaffold, either as a monoculture for diffusion assays, or as co-culture for tissue
engineering (e.g. a surface epithelial compartment layered on top of a fibroblastic
compartment grown inside AlvetexScaffold). The data shown exemplify the application of
this protocol to grow colorectal adenocarcinoma CaCo-2 cells on collagen I coated
AlvetexScaffold for 4 days in 6-well inserts (AVP004).
Method:
1.
Prepare AlvetexScaffold for coating by first treating with 70 % ethanol followed

by two PBS washes as described in the Quick Start Protocol available at
www.reinnervate.com/alvetex/workflow. Leave AlvetexScaffold in the second
PBS wash until ready to apply the collagen solution.
2.
Note: Ensure all reagents are maintained on ice and use pre-chilled pipette tips
to perform the dilution and subsequent application onto AlvetexScaffold.
Dilute rat tail collagen I (BD Biosciences, 354236) to a concentration of 2 mg/ml using
cell culture grade water and adjust the pH with NaOH according to manufacturers
instructions.
Note: Manufacturers instructions for BD Biosciences rat tail collagen (cat # 354236) can be
found at: www.bdbiosciences.com/external_files/dl/doc/manuals/live/web_enabled/354236
_pug.pdf
3.
Aspirate the second PBS wash from AlvetexScaffold disc. Do not allow the scaffold
to dry out. Immediately and carefully pipette an appropriate volume of the diluted
collagen solution onto each disc. Depending on the desired thickness of the collagen
layer, use 100-400 L of collagen solution per disc for the 12-well plate format
(AVP002) and the 6-well insert format (AVP004) or 75-200 L of collagen solution for
the 24-well plate (AVP006) and 12-well insert format (AVP005).
4.
Replace plate lids and leave to stand for 3 hours at 37 C.
5.
Remove excess fluid from AlvetexScaffold in well insert format by gently tapping the
plate or Petri dish on the worktop. Check that no residual fluid is hanging from the
HPM/18/07/2012
6.
Keep collagen-layered AlvetexScaffold hydrated by flooding the membrane with

medium until cells are ready for seeding.
7.
Prepare cells for seeding in the appropriate culture media and seed directly on the
wet collagen-layered AlvetexScaffold membrane, in the volumes relevant for the
AlvetexScaffold product format used. See the Quick Start protocol available from
www.reinnervate.com/alvetex/workflow for details.
Example Data: Collagen I Coating of AlvetexScaffold.

Pre-coating of AlvetexScaffold discs with a 2 mg/ml collagen I solution resulted in a layer
of even thickness (Figure 1) and covering the whole surface of the scaffold (Figure 2). The
integrity of the collagen I layer was demonstrated by the absence of Crystal Violet dye
leakage when the collagen-coated AlvetexScaffold discs were placed in a Side-Bi-Side
PermeGear diffusion chamber (Figure 3).
Figure 1. Thickness of Collagen I Coated

AlvetexScaffold. A,B: Brightfield
micrographs showing low (A) and high (B)
magnification images of PhastGel Blue R
staining of sectioned 6-well insert (AVP004)
coated with 100 l of 2 mg/ml collagen I. C:
Scanning electron microscopy showing sideview of collagen I 6-well insert (AVP004)
coated with 100 l of 2 mg/ml collagen I.
Note the constant thickness and smooth
surface of the collagen coating across
AlvetexScaffold. Scale bars: (A) 50 m;
(B) 8 m; (C) 100 m.
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base of the well inserts. Aspirate to remove any residual gelling agent from the
bottom of the wells. If using AlvetexScaffold in 24- or 12-well plate format, tilt the
plate and gently aspirate any excess fluid from the edge of the wells.
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Figure 2. Uniform Coverage of Collagen I Coated AlvetexScaffold.

Brightfield micrographs showing PhastGel Blue R staining of whole 12-well inserts
(AVP005) coated with 100 l of 2 mg/ml collagen I (A) compared to uncoated controls
(B). Note the complete coverage of the collagen-coated AlvetexScaffold disc.
Figure 3. Integrity of Collagen I Coated AlvetexScaffold. Crystal Violet Dye diffusion

test in Side-Bi-Side PermeGear chamber using whole 12-well inserts (AVP005) either
uncoated (A) or coated with 200 l of 2 mg/ml collagen I (B) to separate donor (left)
from recipient (right) chambers. Diffusion was assessed with mechanical agitation of
both chambers. Timepoint: (A) 30 seconds after dye introduction, (B) 2 minutes after
dye introduction. Note that the dye is excluded from the recipient chamber in the
presence of collagen-coated AlvetexScaffold, whereas it has freely entered the
recipient chamber in the absence of collagen I coating.
Example Data: Growth of CaCo-2 Colorectal Adenocarcinoma Cell Line on Collagen I

Thin Gel on AlvetexScaffold.
CaCo-2 cells (ATCC, HTB-37TM) were routinely maintained in T-75 flasks. CaCo-2 complete
medium consisted of: high-glucose DMEM media (Lonza, BE12-614F) supplemented with 20
% v/v FBS, 0.1 mM non-essential amino acids, 10 mM/L HEPES (pH 7.4), 2 mM L-glutamine
and 100 U/ml Penicillin/Streptomycin. AlvetexScaffold 6-well inserts (AVP004) in 6-well
plates, were layered with 200 l of 2 mg/ml collagen I as described above.
5 ml medium was added to the bottom of each well, i.e on the outside of the
AlvetexScaffold insert, taking care not to trap air bubbles underneath the insert. Cells were
seeded on top of each disc at a density of 0.5 x 106 cells in 500 l medium suspension and
were left to attach overnight in an incubator (5 % CO2, 37 C). The following day, medium
was carefully added to each sample up to 10 ml per well. Culture medium was changed
every two days. After 4 days and 10 days culture, samples were fixed, embedded, sectioned
and stained for hematoxylin & eosin or toluidine blue, according to established protocols
(see www.reinnervate.com/alvetex/workflow).
Results:
Culture of CaCo-2 cells on collagen I coated AlvetexScaffold resulted in an even cell
monolayer of epithelial morphology. Apical structures characteristic of differentiated intestinal
epithelium (microvilli), were evident after 10 days culture (Figure 4).
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Figure 4. Epithelial Morphology of Caco-2 Cells Grown on Collagen I Coated

AlvetexScaffold. A: Brightfield micrographs of H&E-stained paraffin-embedded sections
showing CaCo-2 cells cultured for 4 days. Note that cells form a densely-packed monolayer.
B: Brightfield micrographs of Toluidine Blue stained resin-embedded sections showing
CaCo-2 cells cultured for 10 days. Note the presence of microvilli on the apical surface
forming the brush border. Scale bars: (A) 25 m; (B) 8 m.-
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01
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Protocol Booklet
Cell Visualisation & Monitoring

Cell Attachment/Growth
Within AlvetexScaffold
MTT cell viability assay of cultures grown

on AlvetexScaffold in 3D
Method:
1.
Perform a PBS wash prior to running this assay if cells in AlvetexScaffold were cultured
in phenol-red containing medium. If using the AlvetexScaffold 12-well plate format,
gently remove the discs using flat-ended forceps, gently dip in PBS and place into a new
12-well plate. If using well inserts, dip in PBS and then unclip the base of the insert to
release the disc.
2.
Place each disc into a fresh 12-well plate and set up one well without AlvetexScaffold
as the negative control.
3.
Prepare 1 mg/ml MTT reagent (Sigma, M5655) solution in Phenol Red-free DMEM
(Lonza, BE12-917F) and filter sterilise.
4.
Add 1ml of MTT reagent to each well and cover with aluminium foil as it is light sensitive.
5.
Incubate at 37 C and 5% CO2 for 1 hour.
6.
Prepare an acidified isopropanol solution (acidified isopropanol = 1 l concentrated

hydrochloric acid per 1 ml isopropanol)
7.
Aspirate off the MTT solution from each disc and replace by pipetting 1 ml of acidified
isopropanol onto each disc plus the negative control.
8.
Cover plate in aluminium foil and place on rotating platform for 10 minutes at 100 rpm
to ensure blue formazan product is fully solubilised.
9.
Pipette 20 l of sample containing the formazan dye into a 96-well plate and add 180 l
of isopropanol to each well (This represents a 1 in 10 dilution of the formed product.
Different dilutions may be required depending on the viable cell numbers expected.)
10. Read absorbances at 570 nm.
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Introduction
This chromogenic assay involves the conversion of a yellow, water soluble compound, MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) to a purple, insoluble
formazan. This reaction only takes place when mitochondrial reductase enzymes are active,
and therefore the conversion can be directly related to the number of viable (living) cells. The
MTT reagent alone results in very low background absorbance values in the absence of cells
on AlvetexScaffold. It is recommended that for each cell type the linear relationship between
cell number and signal produced should be first established in order to investigate the limits
of the assay.
This is an example protocol, which may require further optimisation depending on the rate
of cell growth of a particular cell type on AlvetexScaffold.
MTT cell viability assay of cultures grown

on AlvetexScaffold in 3D
Example data set:

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Figure 1. Biochemical analysis

of cell viability using a standard
MTT assay. Data from 3 sample
replicates of HaCaT cells are
show (n=3, mean SD). Cells
were cultured for 3 days on 22
mm AlvetexScaffold discs
presented in the 12-well plate
format. Note good reproducibility
between samples.
Figure 2. Biochemical analysis of

cell viability using a standard
replicates of HaCaT cells are
shown (n=3, mean SD). Cells
were cultured for 3 days on
22 mm AlvetexScaffold discs
presented in 6-well inserts in
6-well plates. Note good
reproducibility between samples.
Figure 3. HaCaT cells cultured

for 1 day on AlvetexScaffold
22 mm discs in 12-well plate
format (AVP002; n=3, mean
SE). Good linearity was
observed (R2>0.9) between cell
number and signal produced
when cells were seeded at 0.5,
0.75, 1 and 1.25 x 106 viable
cells per well as determined by
the Trypan Blue exclusion assay.
Simple Visualisation of Cells on

Alvetex Scaffold Using Light Microscopy
Below are example protocols for visualisation of 3D cultures in AlvetexScaffold using

Methylene Blue (a destructive stain) and Neutral Red (a non-toxic stain). It is recommended
to use a fresh AlvetexScaffold disc for each time check, as the concentrations and/or nature
of the dye used in the example protocol below can be either destructive of perturb the
experiment. Imaging of the specimens will also need to be optimised according to the
specifications of the microscope used.
Cell Staining Protocol

1. At the desired time point, remove medium from the culture to be stained
and wash once with PBS.
2. Transfer the AlvetexScaffold disc/well inserts to be stained to a fresh plate and
return the remaining cultures to the 37 C, 5 % CO2 cell culture incubator.
3. Perform staining by the addition of Neutral Red solution (Sigma, N6264),
or Methylene Blue solution (Sigma, 03978). Dilute latter 1:1 with PBS
before use.
4. Add carefully 500 L of staining solution for cultures grown in 12-well plate
formats (AVP002), whereas AlvetexScaffold in 6 and 12-well insert formats
(AVP004 and AVP005), will require 150 and 100 L respectively.
5. Leave the cultures in staining solution for 5 minutes at room temperature.
6. Wash the cultures carefully and gently with PBS. For cultures grown in
12-well plate formats, perform 2 washes in PBS (4 ml/well), with gentle
agitation (e.g. 100 rpm) for 5 minutes for each wash on a platform shaker.
7. For cultures grown in multiwell insert formats, place the well insert into
a Petri dish with copious amounts of PBS and perform a single wash with
gentle agitation (e.g. 100 strokes/min on reciprocating shaker) for 5 minutes.
Note that Methylene Blue-stained cultures may require an additional

washing step if the second wash is still very blue in appearance.
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Introduction:
Cell culture in AlvetexScaffold allows the formation of multilayered, high-density cell
populations which approximate the complexity and structure of in vivo tissues. When viewing
an unstained, unsectioned AlvetexScaffold 3D culture under a standard brightfield
microscope, the combined density and thickness of the scaffold and the 3D culture within it
prevent the clear visualisation of individual cells. However, common visible dyes can be
successfully used to confirm cell attachment or to check confluency.

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8. After removing the last PBS wash, add 150 L of PBS to each culture and
proceed with visualisation.
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Protocol Booklet
9. Depending on intended use, i.e. macroscopic whole disc confluency or

microscopic individual cell morphology, visualisation can be performed in
different ways:
Cultures in their original holders can be viewed directly by eye or photographed
(See 3.0. Macroscopic visualisation of cells on AlvetexScaffold).
Cultures in their original holders can be checked under brightfield illumination
on a conventional microscope compatible with tissue-culture plastic.
(See 4.0. Microscopic visualisation of cells on AlvetexScaffold).
Cultures can be separated from their holders, placed on a microscope glass
slide with a drop of PBS and checked under brightfield illumination on a
conventional microscope compatible with glass coverslips.
(See 4.0. Microscopic visualisation of cells on AlvetexScaffold).
10.As the method described above is intended for quick and convenient
cell visualisation, the cells are not fixed. They must therefore be kept
wet with a small amount of PBS and be examined immediately
after staining.
Example results
1.0 Preparation of CHO-K1 Cultures on AlvetexScaffold
CHO-K1 cells (ATCC, CCL-61) were seeded on AlvetexScaffold 12-well plate format
(AVP002) at a density of 0 cells, 0.5 million cells and 2 million cells per scaffold, in a
seeding volume of 150 L with a settling time of 30 minutes before adding 4 ml Kaighns
modified nutrient mixture consisting of F-12K (Gibco, 21127022) supplemented with 10 %
(v/v) foetal calf serum, 100 g/ml penicillin and 10 g/ml streptomycin. (See also Example
protocols for the culture of the CHO-K1 cell line on AlvetexScaffold on
www.reinnervate.com/alvetex/protocols). Cells were visualised using the Neutral Red
staining technique after 1 day.
CHO-K1 cells were also seeded on AlvetexScaffold 6-well plate format (AVP004) at a density
of 0.5 million cells per scaffold as above and were maintained by complete media exchange
every two days for 6 days. Cells were visualised using the Neutral Red staining technique
after 2, 4 and 6 days.

2.0 Preparation of HepG2 Cultures on AlvetexScaffold
3.0 Macroscopic Visualisation of Cells

Immediately after staining, the gross appearance of the cultures were photographed (Figure
1.) Both Neutral Red and Methylene Blue are equally suitable for quick visualisation of cultures
on AlvetexScaffold discs.
Figure 1. Macroscopic appearance of cultures on AlvetexScaffold. A. Neutral Red staining of CHO-K1 cells
after 24 hours. B. Methylene Blue staining of HepG2 cells after 24 hours. Note the increase in staining
intensity with higher cell numbers.
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Protocol Booklet
HepG2 cells (ATCC, HB-8065) were seeded on AlvetexScaffold 12-well plate format
(AVP002) at a density of 0 cells, 0.5 million cells and 2 million cells per scaffold, in a
seeding volume of 150 L and with a settling time of 30 minutes before adding 4 ml
of EMEM cell culture medium (Gibco, 21090055) supplemented with 10 % (v/v) foetal
calf serum, 2 mM L-glutamine, 100 g/ml Penicillin and 10 g/ml Streptomycin. (See also
Example protocols for the culture of the HepG2 cell line on AlvetexScaffold on
www.reinnervate.com/alvetex/protocols). Cells were visualised using the Methylene Blue
staining technique after 1 day.
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4.0 Microscopic Visualisation of Cells
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Protocol Booklet
Reinnervate recommends the use of Neutral Red staining for the visualisation of individual
cells within the scaffold over Methylene Blue. Immediately after staining with Neutral Red,
the microscopic appearance of the cultures was observed using a Leica ICC50HD
microscope using LAS EZ software.
Figure 2. Microscopic appearance of CHO-K1 cells on AlvetexScaffold after 24 hours as visualised by

Neutral Red staining. Scalebar 200 m. Note the increase in staining intensity with higher cell numbers.
Neutral Red solution can be used as a very quick and simple staining technique to follow culture
growth and cell survival within AlvetexScaffold over a time course. Note as cell density
increased with time, the staining intensity also increased both macroscopically and
microscopically (Figures 3 and 4). Although Neutral Red can be compatible with live cell
experiments as it is non-toxic at low concentrations, it is recommended that the user should
check the effect of the dye on culture growth and cell survival first. Reinnervate recommends
setting up extra scaffolds for dye analysis alongside with the experimental setup.

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Figure 3. Neutral red staining of CHO-K1 cells cultured in AlvetexScaffold 6-well inserts (AVP004) in 6 wellplate format for up to 6 days. Cells were seeded at an initial seeding density of 0.5 x106 cells/well. Note the
intensity of the staining and the cell surface coverage increase with greater culture period. Scalebar 200 m.

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Figure 4. Close up view of Fig 3. Day 2 cells viewed by 20x objective. Note that this staining method clearly
enables the visualisation of individual cell nuclear morphologies. Scale bar 200 microns.
Live Cell Imaging of 3D Cultures Grown on

AlvetexScaffold Using Confocal Microscopy
Below are example results for live cell labelling of 3D cultures in AlvetexScaffold. It is
recommended that for each staining agent used, the original manufacturers instructions be
followed. Imaging of the specimens will also need to be optimised according to the
specifications of the microscope used.
General Recommendations for Live Cell Imaging

Successful live cell imaging requires the careful optimisation of experimental conditions and
microscope set-ups. In addition to following manufacturers instructions, below are a few
examples of simple parameters that can greatly affect the quality of the results obtained.
These are even more important for long exposures, i.e. experiments ranging from overnight
to several days.
Stage inserts designed to maintain the cell culture plate at a temperature of 37C require
overnight equilibration to allow for the expansion of the metal frame. Insufficient equilibration
times will not result in full expansion of the frame and lead to a continuous loss of focus
until equilibrium is reached, i.e. up to several hours.
CO2 supply designed to maintain a suitable pH within the cultures requires a few hours
runtime prior to introduction of the cultures to the containment cage. In cases when the CO2
supply is absent or unstable, HEPES-buffered cell culture media can be used.
Repeated and/or long-term exposure of dyes and live cells to fluorescent light can lead to
photobleaching and light toxicity, respectively. These problems can be reduced by optimising
laser settings and making sure that shutters are closed in between image acquisitions.
Figure 1: CHO-K1 cultures grown

in 6-well inserts were transferred
to a 35mm plastic cell culture
dish (Nunc, TKT100030M) and
placed on a heated stage insert
fitted to a ZEISS LSM 510
confocal microscope with live
cell imaging capability. The
culture was maintained in
HEPES-buffered F-10 medium
during imaging.
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Introduction
Live cell imaging allows real time monitoring of cell shape and form during cell differentiation,
proliferation and migration. AlvetexScaffold offers a unique ability to study cell growth in 3D
by providing an environment through which individual cells can navigate for extended periods
of time and where confluent cell populations routinely multilayer.

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Recommendations Specific to Cultures Grown in AlvetexScaffold

It is widely appreciated that visualisation of cells inside intact tissues is limited using
conventional microscopy methods. Similarly, when viewing an unstained, unsectioned
AlvetexScaffold 3D culture under a standard light microscope, the combined density and
thickness of the scaffold and the 3D culture within it prevent the clear visualisation of
individual cells. Therefore, cells grown in AlvetexScaffold must be stained prior to imaging
and, whenever possible, best results will be obtained by using fluorescent probes combined
with confocal microscopy.
AlvetexScaffold discs are supplied at a thickness of 200m, which might exceed the Z-stack
capacity of some models of confocal microscopes. The exact depth of sample that can be
successfully imaged will vary according to the density of the cell culture and the fluorophore
used, so that depths of between 50 and 100m can be achieved from a cell-seeded
AlvetexScaffold disc. As high-density cell cultures grown in AlvetexScaffold approximate
the complexity and structure of in vivo tissues, fluorophores specifically developed for in vivo
deep imaging can be used to improve performance if needed.
The material used to manufacture AlvetexScaffold will produce minimal autofluorescence
and will not result in background signal when using confocal microscopy. However, any
hydrophobic dye (e.g. DiI, calcein) will bind strongly to AlvetexScaffold and must therefore
be added to the cells prior to seeding into AlvetexScaffold.
Correct focus will only be achieved within the working range of the objective. Therefore if
using AlvetexScaffold in an insert format, it might be necessary to lift the insert from its
holder and to place it at the bottom of a Petri dish filled with the appropriate cell culture
medium. Then image the 3D culture in the Petri dish and return the AlvetexScaffold disc to
its original holder afterwards if applicable.
Materials and Methods

General Materials
Cells: e.g. CHO-K1 cells (ATCC, CCL-61).
Imaging media: e.g. for CHO-K1: Hams F-10 nutrient mixture, HEPES-buffered (Gibco,
Catalogue No: 22390025), supplemented with 10% (v/v) foetal calf serum, 100g/ml
penicillin and 10g/ml streptomycin. Note: Gibcos F-10 already contains L-glutamine.
AlvetexScaffold in 6- or 12-well inserts (AVP004/AVP005).

Materials Required for Fluorescent Labelling
Transfection reagent: e.g. linear, 25 kDa polyethylenimine (PEI) (Park Scientific Ltd.,
Catalogue No: 23966-2), diluted to a stock concentration of 0.9mg/ml in
dimethylsulfoxide (DMSO) (Sigma-Aldrich, Catalogue No: D-8779), followed by
filtration through a 0.2m mesh, aliquotted and stored at -20C.
Vector DNA: e.g. gWIZ GFP mammalian expression vector (Amsbio, Catalogue No:
P040400), provided at working concentration (1g/l) and stored at -20oC.
DiI Labelling:
CellTrackerTM CM-DiI (Invitrogen, Catalogue No: C-7000), diluted to a stock concentration
of 2mg/ml in dimethylformamide (DMF) (Sigma-Aldrich, Catalogue No: D-4551) and stored
at -20C.
Hoechst Counterstaining:
Hoechst 33342 (Invitrogen, Catalogue No: H-3570), as supplied at the stock concentration of
10mg/ml and stored at 4oC.
Equipment and Materials Required for Imaging:
Confocal fluorescent microscope with live cell imaging capacity, i.e. time-lapse acquisition,
temperature-controlled stage and CO2 supply. HEPES-buffered cell culture media can be
used in cases where a CO2 supply is unavailable. In the example results below, a Zeiss LSM
510 was used.
Methods
1.0. DiI Labelling
Note: Hydrophobic dyes (eg. DiI, calcein) can be used to label cells grown on
AlvetexScaffold , but the dye must be added prior to cell seeding and the cells thoroughly
washed after staining to minimise binding of residual dye to AlvetexScaffold. As some
hydrophobic dyes will partition between dividing cells, the time in culture before imaging
must also be kept in mind to prevent excessive dye dilution within a growing cell population.
1.1. Expand populations of cells in culture medium as monolayer cultures in
conventional 2D plastic-ware according to standard procedures.
1.2. See further information at www.reinnervate.com/alvetex/protocols on how
to choose, handle and prepare the AlvetexScaffold for cell culture.
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GFP Labelling:
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1.3. Prepare a single-cell suspension of cells in media using trypsin-EDTA to

remove cells from 2D plastic-ware. Count cells and prepare seeding
suspensions in medium to allow for a seeding density of 1x106 cells in 3.5ml,
per well.
1.4. Add CellTrackerTM CM-DiI at a dilution of 1/1000 of the stock solution. Mix
and keep at 37C for 5 minutes. Protect from light.
1.5. Spin down for 5 minutes at 1000rpm. Remove supernatant and resuspend
in 3.5ml of appropriate cell culture medium. Spin down again for 5 minutes
at 1000rpm. Remove supernatant and resuspend to allow for a seeding
density compatible with the AlvetexScaffold format used.
1.6. Seed cells on AlvetexScaffold according to the format used and leave the cultures
in a 37C, 5% CO2 cell culture incubator until the desired imaging
timepoint. Protect from light.
2.0. GFP Labelling
Note: Although this example protocol describes the transfection of live cells in suspension
before seeding into AlvetexScaffold, adherent 3D AlvetexScaffold cultures can also be
transfected and imaged.
2.1. Expand populations of cells in culture medium as monolayer cultures in
conventional 2D plastic-ware according to standard procedures.
2.2. See further information at www.reinnervate.com/alvetex/protocols on how to
choose, handle and prepare the AlvetexScaffold for cell culture.
2.3. In a sterile 1.5ml tube, mix 2l of vector DNA with 11.11l of PEI solution, i.e.
equivalent to a DNA:PEI ratio of 1:5 (w/w), per well. Leave for 15 minutes at room
temperature.
2.4. Meanwhile, prepare a single-cell suspension of cells in media using Trypsin-EDTA
to remove cells from 2D plastic-ware. Count cells and prepare seeding suspensions
in medium to allow for a seeding density of 1x106 cells in 0.5ml, per well.
2.5. Add 0.5ml of cell suspension to the DNA:PEI solution and mix gently. This is
equivalent to 2g DNA per million cells.
2.6. Seed cells on AlvetexScaffold according to the format used and leave the cultures
in a 37C, 5% CO2 cell culture incubator. Replace the medium with appropriate
fresh cell culture medium the following day and return the cultures to the 37C,
5% CO2 cell culture incubator until the desired imaging timepoint. Protect
from light.

3.0. Hoechst 33342 Counterstaining
Example Results
CHO-K1 cells (ATCC, CCL-61) were maintained in F-12K medium (Kaighns modification,
Gibco, 21127022) supplemented with 10% (v/v) foetal calf serum, 100g/ml penicillin and
10g/ml streptomycin (for full methods refer to Example protocol for the culture of CHO-K1
cells on AlvetexScaffold located at www.reinnervate.com/alvetex/protocols). For imaging,
cultures were switched to Hams F-10 nutrient mixture buffered with HEPES (Gibco,
22390025), supplemented with 10% (v/v) foetal calf serum, 100g/ml penicillin and 10g/ml
streptomycin.
Below are captured images of live cells over a time-course: labelled with CellTracker
(Figure 2.) and cells transfected with GFP (Figure 3.).
Figure 2: CHO-K1 cells were stained with CellTrackerTM CM-DiI (1/1000 dilution) immediately
prior to seeding onto AlvetexScaffold 6-well inserts. After five days, cultures were imaged
on a ZEISS LSM 510 confocal microscope. Cells were stained with Hoechst 33342 the
evening prior to imaging. Note the dividing cell (arrows).
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Protocol Booklet
For all the example labelling protocols described above (sections 1.0 and 2.0), cells can be
counterstained with Hoechst 33342 to check for labelling efficiency. Shortly prior to imaging,
add Hoechst 33342 at a dilution of 1/1000 of the supplied stock solution. Leave for 30
minutes at room temperature, then replace the medium with fresh cell culture medium and
proceed with imaging. Protect from light until imaging.
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Figure 3: CHO-K1 cells were grown on AlvetexScaffold 6-well inserts. After five days,
cultures were transfected with G-Wiz GFP construct and left to recover for a further 24 hours
before imaging on a ZEISS LSM 510 confocal microscope. Pictures shown are flattened
Z-stacks (6 sections, total height 18m). Note the development of a cytoplasmic extension
during the 3:30 hour recording period (arrows).
Immunofluorescence Confocal Microscopy

of 3D Cultures Grown on AlvetexScaffold
2.0. Method
2.1. If using the AlvetexScaffold 12-well plate format (AVP002, 22 mm disc), gently remove the
clip and lift the 3D culture using flat-ended metal forceps. If using well inserts (AVP004-3, 22
mm disc or AVP005-3, 15 mm disc) unclip the base of the insert to release the 3D culture.
2.2. Place each 3D culture into a fresh well
Note: To improve the efficiency of the subsequent washing, fixing and blocking steps of this
protocol, each 3D culture should be homogenously exposed to a suitably large volume of
solution. Therefore it is recommended that at this stage 3D cultures grown on 22 mm discs
be placed into 6-well plates and 3D cultures grown on 15 mm discs be placed into 12-well
plates. The plates maybe gently rocked to facilitate diffusion.
2.3. Wash the 3D culture in PBS, carefully aspirate and repeat the PBS wash twice
Note: Thorough washing of 3D cultures requires an excess volume of PBS. Therefore it is
recommended to use at least 5 ml of solution for 3D cultures placed into 6-well plates and at
least 3 ml of solution for 3D cultures placed into 12-well plates for each washing step.
2.4. Fix the 3D culture using either a fresh 4% paraformaldehyde solution (in PBS) for 10 min
at room temperature or an ice-cold methanol/acetone (1:1 v/v) solution for 20 min in the fridge.
Note: The choice of the fixative solution should be in accordance with the manufacturers
instructions for the antibodies used. As above, use excess volume of fixing solution. Use a
glass vessel if fixing with methanol/acetone mixture.
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1.0. Introduction
Immunofluorescence uses the recognition of cellular targets by fluorescent dyes or antigenspecific antibodies coupled to fluorophores. Depending on the antibody or dye used, proteins,
lipids and DNA can be visualised within individual cells and tissues. For 2D cell cultures, 3D cell
culture sections or tissue sections, conventional microscopy is generally sufficient to visualise
samples processed by immunofluorescence. For thicker specimens or when sectioning is not
desired, confocal microscopy is to be preferred. Confocal microscopy relies on the combination
of point illumination and a pinhole to eliminate most of the out-of-focus light signal and allows
for reconstruction of 3D volumes, making it ideal to image cultures grown in full-thickness
AlvetexScaffold.
Below is an example protocol. It is recommended that for each antibody or dye used, the
original manufacturers instructions for fixing, blocking and incubating methods be followed.
Imaging of the specimens will also need to be optimised according to the specifications of the
confocal microscope used.

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2.5. If using paraformaldehyde, an additional permeabilisation step is required. At the end of

the fixation period, wash the 3D culture three times in a wash solution containing 0.02% (w/v)
bovine serum albumin (BSA) in PBS, and then permeabilise cells with 1% (w/v) Triton-X100 in
PBS for 15 min at room temperature. As above, use excess volume of both permeabilisation
and washing solutions.
Note: If using methanol/acetone fixation, no permeabilisation step is required.
2.6. Wash the 3D culture three times in the PBS/BSA wash solution. As above, use excess
volume of washing solution.
2.7. Block the 3D culture using a freshly prepared blocking solution consisting of PBS
supplemented with 5% (v/v) normal goat serum (NGS), 1% (w/v) BSA and 0.2% (w/v) TritonX100 for 15 min at room temperature. This blocking step will decrease non-specific binding of
the antibodies used in the later steps of this protocol. As above, use excess volume of blocking
solution.
2.8. While the 3D culture is in blocking solution, dilute the primary antibodies or dyes in the
PBS/BSA wash solution at the working concentrations recommended by their manufacturers.
Note: The volume of antibody solution required for each 3D culture will vary with the format
of AlvetexScaffold used. If using 3D cultures grown on 22 mm discs, a minimum of 150 L
of primary antibody solution per full 3D culture will be required whereas for 15 mm discs use
at least 75 L. In order to reduce antibody solution volumes, the 3D culture can be reduced
in size by cutting the AlvetexScaffold disc into pieces (e.g. quarters).
2.9. Place the 3D culture facing upwards so as to maintain orientation, onto a dry slide. Using
a PAP or DAKO pen, draw a desired area around your tissue section. This allows the applied
solution to be localised on AlvetexScaffold throughout the application time period.
2.10. Pipette the primary antibody solution onto the 3D culture on the slide inside a humidified
chamber.
Note: If the antibody is already conjugated to a fluorophore or a fluorescent dye is used, also
protect from light.

2.12. At the end of the primary antibody incubation period, return the 3D culture facing
upwards to a new plate and wash the 3D culture three times in the PBS/BSA wash solution.
(see 2.3 for volumes required).
2.13. Place the 3D culture facing upwards onto a new, dry glass slide. Circle with a PAP or
DAKO pen as previously (step 2.9.).
2.14. Pipette the secondary antibody solution onto the 3D culture inside a humidified chamber.
Protect from light and leave for 1h at room temperature (or as required by the manufacturers
instructions). In the meantime, label microscope slides.
2.15. At the end of the secondary antibody incubation period, return the 3D culture facing
upwards to a new plate and wash the 3D culture three times in the PBS/BSA washing solution
(see step 2.12.).
2.16. Put a drop of mounting medium on a microscope slide. One drop will be required for each
3D culture. Place the 3D culture facing upwards on the microscope slide and add one further
drop of mounting medium on top of the 3D culture. Cover the 3D culture with a coverglass
while taking care not to create air bubbles, then seal with nail varnish or tape. If using nail
varnish, leave to dry for 10-30 min at room temperature.
2.17. The sample is now ready for imaging by microscopy. If imaging is to be done at a later
date, store the prepared microscope slides in the dark at 40C.
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2.11. Leave the 3D culture in the primary antibody solution for the desired time at the required
temperature. This is usually 1-2 hours at room temperature or overnight at 2-8oC. In the
meantime, dilute the secondary antibodies or dyes in the PBS/BSA wash solution at the
working concentrations as recommended by the manufacturer.

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3.0 Special Considerations for the Imaging of Cell Cultures Grown in AlvetexScaffold
by Confocal Microscopy
The material used to manufacture AlvetexScaffold will produce minimal autofluorescence and
should not result in background signal when using confocal microscopy. Efficient washing and
blocking steps will prevent any significant binding of secondary antibodies to AlvetexScaffold.
However, lipophilic dyes, such as Nile Red (see below), will bind strongly to AlvetexScaffold.
This feature can be used to conveniently visualise the scaffold within the cell culture.
AlvetexScaffold discs are supplied at a thickness of 200 m, which might exceed the Z-stack
capacity of some models of confocal microscopes. The exact depth of sample that can be
successfully imaged will vary according to the density of the cell culture and the fluorophore
used, so that depths of more than 100 m can be imaged from a cell-free AlvetexScaffold disc,
while depths of between 50 and 100 m can be expected from a cell-seeded AlvetexScaffold
disc. As high-density cell cultures grown in AlvetexScaffold approximate the complexity and
structure of in vivo tissues, fluorophores specifically developed for in vivo deep imaging can be
used to improve performance if needed.
4.0. Example pictures
4.1. AlvetexScaffold stained with Nile Red
A working solution was prepared by diluting stock solution of Nile Red (Sigma, N3013) 1 in
1000. The stock solution was initially made up in methanol at 1 mg/ml, kept in a fridge and
protected from light. AlvetexScaffold was incubated 10 minutes at room temperature with
the working solution in the dark, washed three times in PBS and mounted.
Note: Nile Red staining is best carried out after the secondary antibody stage following the
PBS wash. Adhere strictly to incubation times and concentrations as higher concentrations of
the working solution will produce strong staining in the red channel and a signal in the green
channel.
Figure 1. Depth colour-coded Z stack of cell-free

AlvetexScaffold stained with Nile Red. Picture
taken on a Zeiss LSM 510 confocal microscope.
Note the depth of the Z-stack exceeds 150 m.
Scale bar 50 m.

Figure 2. HepG2 cells grown for 3 days in AlvetexScaffold 12-well plate format (AVP002).
Cells were stained with Hoechst 33342 (blue), cytokeratin 8 (green) and Nile Red (red).
Pictures were taken on a Zeiss LSM 510 confocal microscope. Note the background signal
from AlvetexScaffold in the blue and green channels is very low. Scale bar 10 m.
20
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Protocol Booklet
4.2. Triple staining of HepG2 cells grown in 3D on AlvetexScaffold

HepG2 cells were cultured for 3 days in AlvetexScaffold 12-well plate format and stained as
described above:
Primary antibody: mouse anti-cytokeratin 8, clone M20 (Sigma, C5301) applied 1 in 200
dilution for one hour at RT.
Secondary antibody: goat anti-mouse IgG (Invitrogen, A11001) applied 1 in 600 dilution for
one hour at RT.
Nuclear counterstain: Hoechst 33342 (Molecular Probes, H-3570) was applied at the same
time as the secondary antibody.
Nile Red: applied 1 in 1000 of a 1mg/ml working solution for 10 minutes at RT after the
secondary antibody.
AlamarBlue Cell Viability Assay of

upcyte hepatocyte Cultures Grown on AlvetexScaffold in 3D
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Protocol Booklet
Introduction:
The alamarBlue assay is based on the biological reduction of resazurin to the fluorescent
molecule, resorufin. Resazurin is nontoxic and will readily penetrate into viable cells
whereupon it is metabolised. The read out options for the assay are absorbance or
fluorescence, the latter being the more sensitive. The resazurin substrate is blue in colour and
virtually non-fluorescent, resulting in very low background values in the fluorescent read out.
The amount of resorufin product increases with time and deviation from linearity can be
anticipated at later time points. Therefore, it is recommended that for each cell type the linear
relationship between both cell number and time, and signal produced should be first
established in order to investigate the limits of the assay.
Methods:
These are example protocols which may require further optimisation depending on the rate
of cell growth of a particular cell type on AlvetexScaffold. The protocols describe cell viability
analysis of upcyte hepatocytes (Medicyte, www.medicyte.com) using the alamarBlue
Assay (Invitrogen, DAL1010, 1025, 1100). Upcyte cells were thawed, immediately seeded
onto the AlvetexScaffolds and medium added after a 3 hour attachment period. At various
time-points, the growth and viability of the upcyte hepatocytes were monitored as
described below.
Method 1: Performing the alamarBlue assay on dismantled AlvetexScaffold discs:
HPM/01/5/2012
1.
Prepare sufficient alamarBlue solution for the experiment, including controls,

by diluting 1/10 in appropriate phenol red-free medium.
2.
Remove the AlvetexScaffold disc from the culture vessel and dismantle inserts
where applicable. For 22 mm AlvetexScaffold discs, transfer to the wells of a
clean 12-well plate. For 15 mm AlvetexScaffold discs, transfer to the wells of a
clean 24-well plate.
3.
For 22 mm AlvetexScaffold discs add 0.8 ml of diluted alamarBlue solution

to each well. Also add 0.8 ml diluted alamarBlue solution to empty control
wells containing no cells. For 15 mm AlvetexScaffold discs add 0.4 ml of diluted
alamarBlue solution to each well. Also add 0.4 ml diluted alamarBlue solution
to empty control wells containing no cells.
4.
Incubate samples at 37 C and 5 % CO2 for 1 hour.
5.
Transfer 100 l aliquots of each sample to replicate wells of a black 96-well plate.
6.
Measure fluorescence at 590 nm, using an excitation wavelength of 530-560 nm.

Method 2: Performing the alamarBlue assay on AlvetexScaffold in situ in well inserts:

Prepare sufficient alamarBlue solution for the experiment, including controls,
by diluting 1/10 in appropriate phenol red-free medium.
2.
Remove the AlvetexScaffold insert from the culture vessel and transfer to a
clean plate. For 22 mm AlvetexScaffold inserts, transfer to a clean 6-well plate.
For 15 mm AlvetexScaffold inserts, transfer to a clean 12-well plate.
3.
For 22 mm AlvetexScaffold inserts add 7 ml of diluted alamarBlue solution to

each well. For 15 mm AlvetexScaffold inserts add 3 ml of diluted alamarBlue
solution to each well. Add the same corresponding volume of diluted alamarBlue
solution to empty control wells containing no cells.
4.
Incubate samples at 37 C and 5 % CO2 for 3 hours.
5.
Transfer 100 l aliquots of each sample to replicate wells of a black 96-well plate.
6.
Measure fluorescence at 590 nm, using an excitation wavelength of 530-560 nm.
Example Data Set:

The example data shown below was obtained from cutures of upcyte hepatocytes
(Medicyte) grown in 15 mm AlvetexScaffold presented in 12-well insert format.
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Protocol Booklet
1.
22

23
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Protocol Booklet

of cell viability using the
alamarBlue assay according
to Method 2, above. Data from
3 sample replicates of upcyte
hepatocytes cultured in
AlvetexScaffold in 12-well insert
format are shown (n=3, mean
SD). Note good reproducibility
between samples.
Figure 2. alamarBlue assay of

upcyte hepatocytes cultured for
5 hours in AlvetexScaffold discs
presented in 12-well insert
format (n=3, mean SD).
Correlation is shown between
cell number and fluorescent
signal at seeding densities
of 1.0, 2.0, 3.0 and 4.0 x 105
cells per disc (R2>0.71).
Figure 3. alamarBlue assay of

upcyte hepatocytes cultured for
5 hours in AlvetexScaffold discs
format (n=3, mean SD). Data
normalised to protein extracted
from AlvetexScaffold discs
seeded at 1.0, 2.0, 3.0 and 4.0 x
105 cells. Good linearity was
observed (R2=0.98) between
cellular protein and alamarBlue
fluorescence reading.
Determination of Cell Number and Growth Characteristics

within AlvetexScaffold using the PicoGreen Assay.
Method:
1.
To create a cell number standard curve, harvest cells from 2D cultures using an
appropriate detachment method.
2.
Count cells and re-suspend appropriate cell number ranges in 1 ml lysis buffer
(10 mM Tris pH 8, 1 mM EDTA and 0.2 % (v/v) Triton X-100). Note: Always work
RNase and DNase free where possible.
3.
Vortex samples for 10 seconds every five minutes for half an hour, keeping on ice
throughout. Note: At this point samples can be stored at -80 C until the assay is
ready to be performed.
4.
Thaw on ice, if required, and homogenise samples 10-15 times using a

21-guage needle.
5.
Prepare 1 x TE buffer (10 mM Tris-HCl, 1 mM EDTA) from 20 x stock (200 mM

Tris-HCl, 20 mM EDTA, pH 7.5) (Quant-iTPicoGreen dsDNA reagent kit,
Invitrogen) and 1 x PicoGreen reagent from 200 x stock in 1 x TE buffer.
6.
Dilute samples 1 in 10 in 1 x TE buffer by placing 90 l of 1 x TE buffer and 10 l

of sample into the well of a black bottom 96-well plate.
7.
Add 100 l of PicoGreen reagent, mix and incubate at room temperature for
5 minutes wrapped in aluminum foil.
8.
Read fluorescence measured at excitation and emission wavelengths of 460 nm

and 540 nm respectively, using a Biotek FLx800 fluorescence micro-plate reader,
or equivalent.
9.
For cells cultured in AlvetexScaffold, wash in PBS and remove disc from culture
format used using forceps and place in a 1.5 ml tube
10. Lyse cells with 1 ml of lysis buffer as described in step 3.

11. Repeat step 4 keeping the AlvetexScaffold disc within the 1.5 ml tube when
homogenising.
12. Place cell lysate in fresh 1.5 ml tube.
13. Repeat steps 5-8 for analysis.
HPM/01/5/2012
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Introduction:
The following protocol outlines a method for determining cell numbers within
AlvetexScaffold using the PicoGreen Assay. PicoGreen is an ultrasensitive ( 50 cells)
fluorescent nucleic acid dye which provides linear results over multiple orders of magnitude
with a single dye concentration. It is therefore the dye of choice over Hoescht and DAPI for
this type of quantitative assay. Example data was obtained using this protocol with HepG2
hepatocytes cultured in AlvetexScaffold for 7 days.

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Protocol Booklet
Example Data Set:

Cell Number Determination and Growth Curve of HepG2 Cells Cultured in 22 mm
AlvetexScaffold Discs in 6-Well Insert Format (AVP004).
Cell Culture:
For the generation of a standard curve for cell number determination; cells retrieved from
conventional 2D culture flasks were prepared as a series: 0.1, 0.2, 0.5, 1.0, 1.5 and 2.0 x106
and analysed as described above. Figure 1 shows the linear response of PicoGreen
fluorescence to cell number between 0.1 x106 and 2.0 x106 cells.
HepG2 cells were routinely maintained in T75 flasks. HepG2 complete media consisted of
MEM media (Gibco, 21090) containing 10 % v/v FBS, 2 mM L-glutamine and 100 U/ml
Penicillin/streptomycin. Cells were harvested by trypsinisation and centrifuged at 1000 rpm
for 5 minutes. Cells were seeded onto 22 mm AlvetexScaffold discs in 6-well insert format
at a cell density of 8 x 106 cells / ml (1 x 106 cells / 125 l). Cell aliquots were allowed to settle
for 30 minutes prior to flooding the wells with 10 ml of complete HepG2 culture medium.
Media changes were performed every day for up to 7 days.
Results:
Figure 1. HepG2 cell number

standard curve determined
using the PicoGreen assay
(Quant-iTPicoGreen dsDNA
reagent kit, Invitrogen), HepG2
cells were harvested from
conventional 2D T75 culture
flasks and counted using a
haemocytometer and Trypan Blue
dye exclusion. Serial dilutions
were made to give appropriate
cell, seeding numbers.
(Error bars +/- SE, n=3)

26
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Protocol Booklet
Figure 2. HepG2 cell number in

22mm AlvetexScaffold discs in
6-well insert format (AVP004).
Cell numbers were calculated
from the cell number standard
curve using the PicoGreen
assay (Quant-iTPicoGreen
dsDNA reagent kit, Invitrogen).
HepG2 cells were cultured for
1,2,3,4 and 7 days. (Error bars +/SE, n=6 for fluorescence output)

27
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Protocol Booklet
Figure 3. Log phase region of

HepG2 growth curve between
1-4 days in culture.
Conclusions:
The PicoGreen assay can be used to evaluate the growth characteristics of HepG2 cells
cultured on 22 mm AlvetexScaffold discs in 6-well insert format. HepG2 cells show an initial
lag phase for 24 hours before moving into log phase growth, with a population doubling time
of just over 48 hours (from gradient evaluation, 56 hours). After 4 days of culture HepG2
cell growth slows with a plateau region showing no increase in cell population between 4 and
7 days.
This method is a useful approach to determine cell number within a 3D culture.
MTS Cell Viability Assay of upcyte hepatocyte

Cultures Grown on AlvetexScaffold in 3D
Methods:
These are example protocols which may require further optimisation depending on the rate
of cell growth of a particular cell type on AlvetexScaffold. The protocols describe cell viability
analysis of upcyte hepatocytes (Medicyte) using the CellTiter 96 AQueous Non-Radioactive
Cell Proliferation Assay (Promega). Upcyte cells were thawed, immediately seeded onto
the AlvetexScaffolds and medium added after a 3 hour attachment period. At various timepoints, the growth and viability of the upcyte hepatocytes were monitored as described below.
Method 1: Performing the MTS assay on dismantled AlvetexScaffold discs:
HPM/01/5/2012
1.
Prepare sufficient MTS/PMS solution for the experiment, including controls, by

mixing the PMS solution with the MTS solution according to the manufacturers
instructions (Promega, G5421). Briefly this involves mixing 1 ml PMS solution per
20 ml MTS solution. The resulting mixture can be aliquoted and stored at -20 C
before use.
2.
Dilute the MTS/PMS solution 1:5 in appropriate diluent (e.g. PBS, or phenol red-free
culture medium).
3.
Remove the AlvetexScaffold disc from the culture vessel and dismantle
inserts where applicable. For 22 mm AlvetexScaffold discs, transfer to the wells
of a clean 12-well plate. For 15 mm AlvetexScaffold discs, transfer to the wells
of a clean 24-well plate.
4.
For 22 mm AlvetexScaffold discs add 0.8 ml of diluted MTS/PMS solution

to each well. Also add 0.8 ml diluted MTS/PMS solution to empty control wells
containing no cells. For 15 mm AlvetexScaffold discs add 0.4 ml of diluted
MTS/PMS solution to each well. Also add 0.4 ml diluted MTS/PMS solution
to empty control wells containing no cells.
28
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Protocol Booklet
Introduction:
This chromogenic assay involves the biological reduction by viable cells of the tetrazolium
compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (or MTS). The MTS assay reagent is composed of MTS and the electron coupling
agent phenazine methosulfate (PMS). The formazan product of MTS reduction is soluble in
tissue culture medium. This reaction only takes place when mitochondrial reductase enzymes
are active, and therefore the conversion can be directly related to the viability of cells in
culture. The MTS reagent alone results in very low background absorbance values in the
absence of cells. It is recommended that for each cell type the linear relationship between
cell number and signal produced should be first established in order to investigate the limits
of the assay.

29
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Protocol Booklet
5.
Incubate samples at 37 C and 5 % CO2 for 1 hour.
6.
Transfer 100 l aliquots of each sample to replicate wells of a clear 96-well plate.
7.
Measure the absorbance of the samples and controls at 490 nm.
Method 2: Performing the MTS assay on AlvetexScaffold in situ in well inserts:

1.
Prepare sufficient MTS/PMS solution for the experiment, including controls, by

mixing the PMS solution with the MTS solution according to the manufacturers
instructions (Promega, G5421). Briefly this involves mixing 1 ml PMS solution per
20 ml MTS solution. The resulting mixture can be aliquoted and stored at -20 C
before use.
2.
Dilute the MTS/PMS solution 1:5 in appropriate diluent (e.g. PBS, or phenol red-free
culture medium).
3.
Remove the AlvetexScaffold insert from the culture vessel and transfer to a
clean plate. For 22 mm AlvetexScaffold inserts, transfer to a clean 6-well plate.
For 15 mm AlvetexScaffold inserts, transfer to a clean 12-well plate.
4.
For 22 mm AlvetexScaffold inserts add 7 ml of diluted MTS/PMS solution to

each well. For 15 mm AlvetexScaffold inserts add 3 ml of diluted MTS/PMS
solution to each well. Add the same corresponding volume of diluted MTS/PMS
solution to empty control wells containing no cells.
5.
Incubate samples at 37 C and 5 % CO2 for 3 hours.
6.
Transfer 100 l aliquots of each sample to replicate wells of a clear 96-well plate.
7.
Measure the absorbance of the samples and controls at 490 nm.
Example Data Set:

The example data shown below was obtained from cutures of upcyte hepatocytes
(Medicyte, www.medicyte.com) grown in 15 mm AlvetexScaffold presented in 12-well
insert format.

Figure 2. MTS assay of upcyte

hepatocytes cultured for 5 hours
in AlvetexScaffold discs
format (n=3, mean SD). Good
linearity was observed (R2>0.9)
between cell number and signal
produced when cells were
seeded at 1.0, 2.0, 3.0 and
4.0 x 105 cells per disc.
Figure 3. MTS assay of upcyte

hepatocytes cultured for 5 hours
in AlvetexScaffold discs
format (n=3, mean SD).
Data normalised to protein
extracted from AlvetexScaffold
discs seeded at 1.0, 2.0, 3.0
and 4.0 x 105 cells. Good linearity
was observed (R2=0.9) between
cellular protein and MTS
viability reading.
30
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MTS assay according to Method
2, above. Data from 3 sample
replicates of upcyte
hepatocytes cultured in
AlvetexScaffold in 12-well
insert format are shown
(n=3, mean SD). Note good
reproducibility between samples.
01
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Protocol Booklet
Histology of Cell Cultures

Grown on AlvetexScaffold
(Series Parts 1 5):
Histology Series Part 1.

Choosing the Right Fixative to Preserve 3D Cell Cultures
Fixation methods
Fixation is performed in order to preserve the tissue or culture from the putrefaction
(destruction by micro-organisms), autolysis (self-digestion by lysosomal enzymes) and
abnormal metabolism of the isolated tissues deprived of oxygen and nutrients. Fixation of 3D
cultures in AlvetexScaffold can be achieved by chemical fixation. The use of uncontrolled
high heat by flame is not recommended.
Fixation is usually the first stage in a multistep process to prepare a sample for microscopy
or other analysis. So, when choosing a fixative, the final purpose of the section must be
considered and matched by the requirements for the analytical technique. Some fixatives
are suitable for general structure analysis, others for evaluating cytological detail and some
for histochemistry. Survival of tissue antigens for immunochemical staining depends on the
type and concentration of fixative, on fixation time, and on the size of the tissue specimen
to be fixed. Given the thin nature of AlvetexScaffold, fixation of cells is rapid, uniform and
efficient, preserving the 3D culture in a life-like condition.
Recommended chemical fixatives for tissue structures grown in AlvetexScaffold are:

Bouins Fixative:
Is an excellent fixative for use when samples are to be paraffin embedded,sectioned and
stained for general histology (see Figure 1.), especially for the trichrome stains. Bouins
fixation is not recommended for immuno-detection. Do not use Bouins fixative before
in situ hybridisation as it will prevent RNA from being detected.
Method
1. Working in a fume cupboard wearing appropriate Personal Protective
Equipment (PPE) measure out saturated picric acid (750 ml), formaldehyde
(250 ml) and glacial acetic acid (50 ml).
2. Mix well in a 1 litre Duran bottle. Fixative is stable for 1 year at room temperature.
Safety precautions
CARCINOGENIC, IRRITANT, CORROSIVE and TOXIC.
02
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Protocol Booklet
Introduction
These histology protocols contain a series of detailed methods that will allow the user to
examine the morphology of their cultured cells subsequent to 3D growth. These are not in
any way exhaustive and there is scope to include additional methods as the user deems
appropriate. Users should also perform their own risk assessments as safety evaluations
contained in these protocols are for information only.

03
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Protocol Booklet
Paraformaldehyde (4 % PFA):
Suitable for paraffin embedding and sectioning. It is the fixative of choice for immunocytochemical analysis. Samples can also be stained for general histology but the degree
of fixation is less vigorous than Bouins so the quality of the morphology obtained will
be less. This fixative allows for subsequent immuno-detection of certain antigens and
should therefore be used when the objective is to study morphology and protein
expression simultaneously. (See Figure 2.)
Method:
1. Working in a fume cupboard wearing appropriate PPE heat up 1 litre
of Phosphate Buffered Saline (PBS) to 60 C on a hotplate stirrer.
2. Add 40 g of paraformaldehyde (PFA) powder to the warm PBS. Use a magnetic
stirrer and hotplate to dissolve PFA.
3. Slowly add drops of 1 M sodium hydroxide solution until the solution is clear.
4. Filter the fixative and adjust pH to 7.3-7.4.
5. Allow fixative to cool to 53 C before use.
6. Aliquots can be stored at 20 C with a shelf life of 6 months.
Safety precautions:
CARCINOGENIC, IRRITANT, CORROSIVE, and TOXIC.
Karnovskys fixative:
Is a mixture of paraformaldehyde and glutaraldehyde. It is suitable for use when preparing
samples for light microscopy in resin embedding and sectioning, and for electron
microscopy. This fixative should always be prepared fresh. (See Figures 3 and 4
respectively).
Method:
Karnovskys fixative is 2 % paraformaldehyde, 2.5 % glutaraldehyde in 0.1 M
phosphate buffer pH 7.4.
1.
Prepare 8 % PFA:
1.1. Working in a fume cupboard wearing appropriate PPE heat up 90 ml of water

to 65 C on a hotplate stirrer.
1.2. Add 8 g of paraformaldehyde (PFA) powder to the warm water. Use a magnetic
stirrer and hotplate to dissolve PFA (approximately 15-20 minutes).
1.3. Slowly add drops of 1 M sodium hydroxide solution until the solution is clear.
Make up to 100 ml with additional water.
1.4. Filter the fixative and adjust pH to 7.3-7.4.

1.5. Allow fixative to cool to room temperature. Store at -20 C in aliquots.

Frozen aliquots are stable for up to 6 months.
2.
Prepare 0.2 M phosphate buffer
2.1. Weigh out 35.61 g of disodium monohydrogen phosphate (Na2HPO4.2H2O)

make up to 1000 ml with distilled H2O, stir until dissolved (Solution X).
1 year shelf life at room temperature.
2.2. Weigh out 27.6 g of sodium dihydrogen phosphate (NaH2PO4.H2O) make up
to 1000 ml with distilled H2O, stir until dissolved (Solution Y). 1 year shelf life
at room temperature.
2.3. Add 40.5 ml of Solution X to 9.5 ml of Solution Y to give 50 ml 0.2 M phosphate
buffer and adjust to pH 7.4. This is to be used fresh.
3.
Prepare Karnovskys Fixative (for 100 ml add the following together)
3.1. Mix together 8 % paraformaldehyde (25 ml), 25 % glutaraldehyde (Fluka

49362 or equivalent, 10 ml) and 0.2 M phosphate buffer (50 ml). Make up
to 100 ml with distilled water.
3.2. This is to be used fresh.
Safety precautions
Buffered Osmium Tetroxide Fixative:

Is suitable as a second-stage fixative when preparing samples for electron microscopy. It is also
used when samples are to be resin embedded and sectioned. This fixative increases the
samples bulk conductivity, which is required for scanning electron microscopy. (See Figures 3
and 4 respectively).
Method
1. Working in a fume hood throughout and wearing appropriate PPE, combine equal
amounts of the 2 % osmium tetroxide solution and 0.2 M phosphate
buffer (for recipe see above) in a clean 15 ml centrifuge tube.
2. Vortex gently.
3. Store in a fume hood at room temperature.
Consolidated
Protocol Booklet
1.6. Dispense 25 ml for use in fixative.
04

05
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Protocol Booklet
4. Final concentrations: 1 % osmium tetroxide in 0.1 M phosphate buffer.

Cacodylate buffer may be used to replace the phosphate buffer.
Note: Osmium tetroxide is highly toxic and volatile and should be used and
stored only in a chemical fume hood.
Safety precautions:
Examples
Below are examples of how the same 3D culture of human keratinocytes (HaCaT) can be
processed and subsequently imaged in alternative ways:
Figure 1. Human keratinocyte
cell line (HaCaT) grown in
AlvetexScaffold (7 days air
exposure). The subsequent
culture was fixed in Bouins fixative
and processed for paraffin wax
embedding and sectioning. The
sectioned materials (10 m) were
stained with Haematoxylin and
Eosin for morphological analysis.

culture was fixed in 4 %
paraformaldehyde and processed
for paraffin wax embedding and
immunohistochemical analysis by
fluorescent microscopy. The three
images from the same region show;
phase (top), blue fluorescent
Hoescht 33258 nuclei stain (middle)
and Ki67 staining (bottom).


culture was primarily fixed in
Karnovskys fixative followed
by a secondary fixative osmium
tetroxide and processed for
structural analysis by scanning
electron microscopy. For scale
see bar inserts.
06
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Protocol Booklet

culture was primarily fixed in
Karnovskys fixative followed by
a secondary fixative osmium
tetroxide and processed for
resin embedding (L R White
resin). Resin sections (1 m)
were stained with Toluidene
Blue for structural analysis by
light microscopy.

Processing Alvetex Scaffold 3D Cultures for PFA Fixation and
Paraffin Wax Embedding for Immunofluorescence
07
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Protocol Booklet
Introduction
Immunofluorescence uses the recognition of cellular targets by fluorescent dyes or antigenspecific antibodies coupled to fluorophores. Depending on the antibody or dye used,
proteins, lipids and DNA can be visualised within individual cells and tissues. AlvetexScaffold
can easily be processed like a standard tissue sample, allowing established
immunofluorescence methods to be followed with excellent results. An example protocol is
outlined below.
Materials and Methods

1.0 Materials Required for Fixing, Embedding and Sectioning
4 % Paraformaldehyde (PFA) fixative (for full method refer to Histology
Series Part 1. Choosing the Right Fixative to Preserve 3D Cell Cultures located
at www.reinnervate.com/alvetex/protocols).
Dehydration ethanols (30 %, 50 %, 70 %, 80 %, 90 %, 95 %, 100 %)
Histoclear (Note: the use of Histoclear is recommended over the use of Xylene).
Paraffin wax
Basic equipment: spade forceps to handle 3D AlvetexScaffold cultures,
scalpel (to trim / cut scaffold if required), disposable pipettes, embedding
moulds, convection oven, microtome.
2.0 Materials Required for Immunofluorescence

1x citrate buffer pH 6.0
Blocking buffer (5 % Normal goat serum; 1 % bovine serum albumin; 0.2 %
Triton X-100 in PBS)
Permeabilisation solution (1 % w/v Triton X-100 in PBS)
Primary and secondary antibodies (specific to experimental analysis)
Microwave oven (if antigen retrieval is required)
Humidified chamber
Vectashield/DAPI mountant

3.0 4 % PFA Fixation of Scaffolds
3.2. If using well inserts in Petri dishes, remove inserts from holder and place in
a conventional 6-well plate. To fix, add in 5 ml 4 % PFA fixative at 4 C. Fix
specimens at 4 C for a minimum of 12 hours but no longer than 24 hours.
3.3. Aspirate off the fixative, wash 3 times using 5 ml PBS to thoroughly remove
excess fixative, discarding the waste liquid.
3.4. If using inserts, remove AlvetexScaffold from the well insert either by
separating the two parts of the device or using a scalpel to cut out the scaffold.
At this time samples can be transferred to a tissue processing cassette to
minimize direct handling and damage to the 3D culture.
3.5. Aspirate off the PBS and add 5 ml of 30 % ethanol. Leave to equilibrate for
at least 15 minutes. Aspirate off the ethanol and discard.
3.6. Repeat with 50 %, 70 %, 80 %, 90 % and then 95 % ethanol. A gradual
dehydration of the sample will result in less tissue shrinkage. Material can
be stored in 95 % ethanol prior to paraffin embedding.
4.0 Paraffin Embedding and Sectioning

4.1. Fully dehydrate specimens stored in 95 % ethanol by replacing with 100 %
absolute ethanol for at least 30 minutes.
4.2. Aspirate off the ethanol and replace with Histoclear for at least 30 minutes.
(Note: Histoclear will dissolve certain types of plastic, therefore it is best to
process samples in glass tissue processing cassettes).
4.3. Replace the Histoclear with a 50:50 solution of Histoclear and molten paraffin
wax (60 C) mix and incubate in a convection oven at 60 C for 30 minutes.
4.4. Replace the Histoclear:wax mix with 100 % molten wax and incubate at 60 C
for a further 60 minutes.
4.5. Transfer the scaffold to plastic embedding moulds and orientate into the required
embedding position, with plane of section in mind. Embed in molten wax.
4.6. Allow wax to cool and set at room temperature for 1-2 hours or overnight.
4.7. Once the wax has hardened, remove the wax embedded block from the plastic
mould. The sample is now ready for sectioning on a suitable microtome
(e.g. Leica RM2125).
Consolidated
Protocol Booklet
3.1. Aspirate off the medium and carefully wash the 3D culture in PBS twice.
08

09
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Protocol Booklet
4.8. Following the microtome manufacturers instructions throughout, align the

block correctly with the microtome blade and proceed to cut 10 m sections
of the sample block.
4.9. Transfer sections to a slide water bath (40 C), floating them on the surface
of the water to enable them to flatten out.
4.10. Transfer selected sections to slides by flotation. Superfrost Plus slides
(Thermo, 4951PLUS4) are recommended since sections adhere to these
slides well.
4.11. Place on a slide drier and leave overnight. The sections should now be ready
for immunofluorescence.
5.0 Immunofluorescent Analysis

5.1.
Deparaffinise sections in Histoclear for 10 minutes. Handle samples

carefully to avoid loss of section from slide.
5.2. Hydrate specimen through a graded series of ethanols (100 %, 90 %, 70 %)

with 5 minutes incubation in each solution.
5.3. If antigen retrieval is required, place slides in 1x citrate buffer (pH 6) and
microwave (800 W) for 6 minutes. If antigen retrieval is not required go to
point 5.6.
5.4. Leave to stand outside microwave for 1 minute then microwave for a further
3 minutes at 800 W.
5.5. Allow to cool for 20 minutes and wash in PBS for 10 minutes. Repeat three
times.
5.6. Treat cells with permeabilisation solution for 15 minutes.
5.7.
Aspirate off permeabilisation solution, replace with blocking buffer and

incubate for 15 minutes.
5.8. Incubate with primary antibody diluted in blocking buffer (this will be
specific to the antigen chosen and will be used at a pre-determined
concentration) overnight at 4 C in a humidified chamber, or as stated in
the datasheet for the specific antibody used.

5.9. Wash 3 times for 10 minutes in PBS.
10
5.11. Wash 3 times for 10 minutes with PBS.

5.12. Mount in Vectashield/DAPI. This solution simultaneously mounts the
specimen and stains cell nuclei.
5.13. Seal the perimeter of the cover slip with nail varnish and dry in the dark.
5.14. Store slides in the dark at 4 C until ready for inspection using
a fluorescence microscope equipped with the appropriate filters.
Example results
Figure 1. below shows an example of a 3D AlvetexScaffold culture fixed with 4 % PFA,
embedded in paraffin wax, sectioned and probed with Ki67 antibody using
immunocytochemistry and standard fluorescence microscopy.
Figure 1 Protein expression in 3D

cell cultures can be localised using
immunocytochemistry and
fluorescence microscopy. Three
corresponding images from the
same region of AlvetexScaffold
show: (A) cell morphology by
phase microscopy; (B) expression
of cell nucleus marker DAPI; (C)
expression of cell proliferation
marker Ki67.
Consolidated
Protocol Booklet
5.10. Incubate with secondary antibody diluted in blocking buffer (this will be
concentration) for 2 hours in the dark at room temperature.

Processing Alvetex Scaffold 3D Cultures for Bouins Fixation and
Paraffin Wax Embedding for Haematoxylin and Eosin Staining
11
Consolidated
Protocol Booklet
Introduction
Following fixation a variety of different cytological stains can be used to visualise cell components
in detail. Here we describe the use of Haematoxylin and Eosin, a general structural stain, one of
the most commonly used stains throughout histology. The hemalum component of haematoxylin
stains cell nuclei blue. Counterstaining of samples with an alcoholic eosin Y solution stains other
eosinophilic structures in various shades of red, pink and orange. Most of the cell cytoplasm is
eosinophilic and therefore stains pink. AlvetexScaffold can easily be processed like a standard
tissue sample, allowing established histology methods to be followed with excellent results.
An example protocol is outlined below.
Method
1.0 Materials Required
Bouins fixative (for full recipe see Histology Series Part 1. Choosing
the Right Fixative to Preserve 3D Cell Cultures at
www.reinnervate.com/alvetex/protocols)
Dehydration ethanols (30 %, 50 %, 70 %, 80 %, 90 %, 95 %, 100 %)
Histoclear (Note: the use of Histoclear is recommended over the use of Xylene).
Paraffin wax
Mayers Haematoxylin solution
Eosin solution (2 g eosin in 1000 ml 95 % alcohol)
Alkaline alcohol
DPX mountant
Basic equipment: spade forceps to handle 3D cultures, scalpel (to trim/cut
AlvetexScaffold cultures if required), disposable pipettes, convection oven,
embedding moulds, microtome.
2.0 Bouins Fixation of Scaffolds

2.1. Aspirate off the medium and carefully wash the 3D culture in PBS twice.
2.2. If using well inserts, remove inserts from cradle and place in a conventional
6-well plate. To fix, add 5 ml Bouins fixative at room temperature. Fix specimens
at room temperature for a minimum of 12 hours but no longer than 36 hours.
2.3. Aspirate off the fixative, wash three times using 5 ml PBS to thoroughly
remove excess fixative, discarding the waste liquid.

2.5. Aspirate off the PBS and add 5 ml of 30 % ethanol. Leave to equilibrate for
at least 15 minutes. Aspirate off the ethanol and discard.
2.6. Repeat with 50 %, 70 %, 80 %, 90 % and then 95 % ethanol. A gradual
dehydration of the sample will result in less tissue shrinkage. Material can
be stored in 95 % ethanol prior to paraffin embedding.
3.0 Paraffin Embedding and Sectioning
3.1. Fully
dehydrate
3.0 Paraffin
Embedding
andspecimens
Sectioningstored in 95 % ethanol by replacing with 100 %
absolute ethanol for at least 30 minutes.
3.1. Fully dehydrate specimens stored in 95 % ethanol by replacing with 100 %
ethanol
for at least
30 minutes.
3.2. absolute
Aspirate off
the ethanol
and replace
with Histoclear for at least 30 minutes.
Histoclear
will dissolve
certainwith
types
of plastic,
it is
best
3.2. (Note:
Aspirate
off the ethanol
and replace
Histoclear
fortherefore
at least 30
minutes.
to process samples in glass tissue processing cassettes).
(Note: Histoclear will dissolve certain types of plastic, therefore it is best
to process
in glass
3.3. Replace
thesamples
Histoclear
with atissue
50:50processing
solution of cassettes).
Histoclear and molten paraffin
wax (60 C) mix and incubate in a convection oven at 60 C for 30 minutes.
3.3. Replace the Histoclear with a 50:50 solution of Histoclear and molten paraffin
wax Replace
(60 C) mix
incubate in amix
convection
at 60 wax
C forand
30incubate
minutes.at
3.4.
the and
Histoclear:wax
with 100 oven
% molten
60 C for a further 60 minutes.
3.4. Replace the Histoclear:wax mix with 100 % molten wax and incubate at
C forthe
a further
60tominutes.
3.5. 60
Transfer
scaffold
plastic embedding moulds and orientate into the
required embedding position, with plane of section in mind. Embed in
3.5. Transfer the scaffold to plastic embedding moulds and orientate into the
molten wax.
required embedding position, with plane of section in mind. Embed in
wax.
3.6. molten
Allow wax
to cool and set at room temperature for 1-2 hours or overnight.
3.6. Once
Allow the
waxwax
to cool
set at room
temperature
for 1-2 hours
or from
overnight.
3.7.
has and
hardened,
remove
the wax embedded
block
the
plastic mould. The sample is now ready for sectioning on a suitable
3.7. Once the wax has hardened, remove the wax embedded block from the
microtome (e.g. Leica RM2125).
plastic mould. The sample is now ready for sectioning on a suitable
(e.g.
Leica RM2125).
3.8. microtome
Following the
microtome
manufacturers instructions throughout, align the
correctly
with the microtome
blade instructions
and proceedthroughout,
to cut 10 m
3.8. block
Following
the microtome
manufacturers
align the
sections of the sample block.
block correctly with the microtome blade and proceed to cut 10 m
of the sample
block.
3.9. sections
Transfer sections
to a slide
water bath (40 C), floating them on the surface
of the water to enable them to flatten out.
3.9. Transfer sections to a slide water bath (40 C), floating them on the surface
the water
to enable
themtotoslides
flattenbyout.
3.10.ofTransfer
selected
sections
flotation. Superfrost Plus slides
(Thermo, 4951PLUS4) are recommended.
3.10. Transfer selected sections to slides by flotation. Superfrost Plus slides
(Thermo,
areleave
recommended.
3.11. Place
on a4951PLUS4)
slide drier and
overnight. The sections
should now be readyfor staining.
3.11. Place on a slide drier and leave overnight. The sections
should now be readyfor staining.
12
Consolidated
Protocol Booklet
2.4. If using inserts, remove AlvetexScaffold from the well insert either by
separating the two parts of the device or using a scalpel to cut out the
scaffold. At this time samples can be transferred to a tissue processing
cassette to minimize direct handling and damage to the 3D culture.

13
4.0. Haematoxylin and Eosin staining
Consolidated
Protocol Booklet
4.1. Set up a series of slide chambers containing solutions as outlined in the

following steps and process the slides using slide holders according to the
indicated timings.
4.2. Deparaffinise in Histoclear
5 minutes
4.3. Transfer into absolute (100 %) alcohol
2 minutes
4.4. Rehydrate through 95 % alcohol
1 minute
4.5. 70 % alcohol
1 minute
4.6. Distilled water
1 minute
4.7. Stain in Mayers Haematoxylin
5 minutes
4.8. Wash in distilled water
30 seconds
4.9. Blue the nuclei in alkaline alcohol
30 seconds
4.10. Dehydrate in 70 % alcohol
30 seconds
4.11. 95 % alcohol
30 seconds
4.12. Stain in Eosin
1 minute
4.13. Dehydrate in 95 % alcohol
10 seconds
4.14. 95 % alcohol
10 seconds
4.15. Absolute alcohol
15 seconds
4.16. Absolute alcohol
30 seconds
4.17. Clear in Histoclear
3 minutes
4.18. Histoclear
3 minutes
4.19. Retain slides in Histoclear until ready to coverslip to prevent drying out.

5.0 Mounting and Cover-slipping
14
Example results
Figure 1. shows an example of paraffin embedded 3D AlvetexScaffold culture, sectioned
and stained with Hematoxylin and Eosin.
culture was fixed in Bouins
fixative and processed for
paraffin wax embedding and
sectioning. The sectioned
materials (10 m) were stained
with Haematoxylin and Eosin
for morphological analysis.
Consolidated
Protocol Booklet
5.1. Mount in DPX by spotting DPX around the section and carefully cover with
a coverslip ensuring no air bubbles are present. Allow to air dry for at least
one hour.

Processing Alvetex Scaffold 3D Cultures for Resin Embedding
and Toluidine Blue Staining for Light Microscopy
15
Consolidated
Protocol Booklet
Introduction
Following fixation, a variety of cytological stains can be used allowing different cell
components to be visualised in detail. When structural integrity of sample architecture is
paramount, samples are embedded in resin. Resin embedding allows ultrathin sections of
samples to be obtained with no loss of structural detail.
Here we describe the use of toluidine blue as a stain of sectioned material for light
microscopy. Toluidine blue is a general histology stain that, in basic solutions, binds to nucleic
acids and all proteins. It is also commonly used for staining thin sections (0.5-1 m)
of resin embedded tissues to aid with the orientation and visualisation of samples
subsequently prepared for electron microscopy. While everything in the sample stains
with the dye, structural details remain highly prominent due to the thinness of the sections.
AlvetexScaffold can easily be processed like a standard tissue sample, allowing
established histology methods to be followed with excellent results. An example protocol is
outlined below.
Method
1.0 Materials Required
Karnovskys fixative (2 % paraformaldehyde; 2.5 % glutaraldehyde in
0.1 M phosphate buffer pH 7.4. (for full recipe see Histology Series Part 1.
Choosing the Right Fixative to Preserve 3D Cell Cultures at
0.1 M phosphate buffer (for full recipe see Histology Series Part 1.
Choosing the Right Fixative to Preserve 3D Cell Cultures at
Dehydration ethanols (30 %, 50 %, 75 %, 95 %, 100 %)
1 % buffered osmium tetroxide (1 % osmium tetroxide solution
(Agar scientific Ltd), 0.1 M phosphate buffer (pH 7.4). (for full recipe detail
see Histology Series Part 1. Choosing the Right Fixative to Preserve 3D Cell
Cultures at www.reinnervate.com/alvetex/protocols)
L R White Resin (Agar Scientific Ltd)
1 % toluidine blue in 1 % sodium borate
DPX
Basic equipment: spade forceps to handle 3D cultures, scalpel (to trim/cut
AlvetexScaffold cultures if required), disposable pipettes, embedding
moulds, wire loop

2.0. Karnovskys and Osmium Fixation of Scaffolds
2.2. Remove well inserts from cradle and carefully cut small samples (2-3 mm
square) from the scaffold disc.
2.3. Immerse samples in Karnovskys fixative at 4 C for 90 minutes. The ratio
of fixative volume to specimen size should be approximately 20:1.
2.4. After fixation, aspirate the fixative into a waste container for disposal.
2.5. Wash the specimen twice for 2 minutes in 3 ml 0.1 M phosphate to remove
excess fixative, discarding waste liquid.
2.6. Add 20x the specimen volume of 30 % ethanol. Leave to equilibrate for
5 minutes preferably on a rotating wheel. Pour off the ethanol and discard.
Repeat twice.
2.7. Repeat step 2.6 with 50 %, 75 %, 95 % and then absolute ethanol.
The sample can be stored in absolute ethanol prior to resin embedding.
3.0. Resin Embedding and Sectioning

3.1. Prepare the resin/catalyst mix according to the manufacturers instructions.
3.2. Immerse samples in a 1:1 mix of absolute ethanol and L R White resin
(20x the specimen volume) and leave on a rotor for 1 hour at room
temperature.
3.3. Replace the resin/ethanol mix with 3 changes of resin alone, for at
least 60 minutes each. Samples can also be left in resin at room
temperature overnight.
3.4. Embed the specimen according the manufacturers instructions.
Specimens may be embedded in gelatin capsules or flat embedding moulds
made of PTFE.
3.5. It is possible to cut LR White resin blocks on a standard microtome
with a steel knife blade. The method of choice, however, is to use a
motorized ultra-microtome and glass knife. For Toluidine Blue staining
1.0 m sections should be cut.
Consolidated
Protocol Booklet
2.1. Aspirate off the medium and carefully wash the 3D culture twice with PBS.
16

17
Consolidated
Protocol Booklet
3.6. Place a drop of distilled water approximately 1 cm in diameter on a clean

glass slide. Pick the cut sections up with a wire loop and invert the loop
to place 2-4 sections into the drop of water on the slide.
3.7. Place the slide with the water droplet containing sections on a hot plate
adjusted to approximately 60-65 C and wait until all the water evaporates.
4.0 Toluidine Blue Staining
4.1. Locate the sections on the slide and circle the bottom of the slide with a
permanent marker to indicate their position.
4.2. Place 1-2 drops of 1 % toluidine blue in 1 % sodium borate onto the
sections and place the slide on the hot plate. Wait until the edges of the
drop of stain begin to turn golden (approximately 20-30 seconds).
4.3. Remove the slide and rinse the sections with a gentle stream of distilled
water from a wash bottle to wash off the excess stain.
4.4. Differentiate the sections by rinsing in a gentle stream of 50 % ethanol.
4.5. Replace the washed slide on the hot plate after gently drying the bottom
of the slide with a piece of paper towel and wait for the residual ethanol
to evaporate from the slide surface.
4.6. Remove the slide from the hot plate and add 1-2 drops of DPX
mounting medium to the top of the sections adhering to the slide.
Gently lower a coverslip onto the droplet of Polymount over the sections.
4.7. Allow to dry and store in a dust free location.

Figure 1 HaCaT cells grown in

AlvetexScaffold can be fixed
and processed for histological
analysis using standard
methods. Sample fixed in
Karnovskys, embedded in LR
White resin, processed and
counter stained with
Toluidine Blue.
18
Consolidated
Protocol Booklet
Example Results
Figure 1 shows an example of a resin embedded 3D AlvetexScaffold culture, sectioned
and stained with Toluidine Blue.

Processing Alvetex Scaffold 3D Cultures for
Scanning Electron Microscopy (SEM)
19
Consolidated
Protocol Booklet
Introduction
Scanning electron microscopy (SEM) is becoming a popular method for visualisation of
cultures grown in 3D. SEM is a form of electron microscopy where images are obtained by
scanning samples using a high-energy beam of electrons. As the samples are scanned the
electrons interact with atoms located on the sample surface, and it is these interactions that
are detected, and processed, leading to high-resolution images depicting the samples
topography and composition.
For conventional imaging, SEM samples must be electrically conductive and completely dry
(due to the specimen chamber being at high vacuum).To achieve this, samples usually
undergo chemical fixation to preserve and stabilise their structure. Standard SEM fixing
solutions are Karnovskys fixative followed by a second fixing in osmium tetroxide, which
increases the bulk conductivity of the sample. Samples are completely dried using a critical
point dryer, and finally, fixed samples are sputter coated with gold to further enhance
their conductivity.
AlvetexScaffold can easily be processed like a standard tissue sample, allowing established
methods to be followed with excellent results. An example protocol is outlined below.
Imaging of the specimens will need to be optimised according to the specifications of the
scanning electron microscope used.
Method
1.0 Materials required
Karnovskys fixative (2 % paraformaldehyde; 2.5 % glutaraldehyde in
0.1 M phosphate buffer pH 7.4. (for full recipe detail see Histology
Series Part 1. Choosing the Right Fixative to Preserve 3D Cell Cultures
at www.reinnervate.com/alvetex/protocols)
Dehydration ethanols (30 %, 50 %, 75 %, 95 %, 100 %)
1 % buffered osmium tetroxide (1 % osmium tetroxide solution; 0.1 M
phosphate buffer (pH 7.4). (For full recipe details see Histology Series
Part 1. Choosing the Right Fixative to Preserve 3D Cell Cultures at
0.1 M phosphate buffer pH 7.4
Basic equipment: spade forceps to handle 3D cultures, scalpel (to trim
AlvetexScaffold cultures if required), disposable pipettes.
Specialised equipment to prepare samples for SEM: critical point dryer,
sputter coater.

2.0. Protocol
2.2. Remove well inserts from cradle and carefully cut small samples
(2- 3 mm square) from the scaffold disc.
2.3. Immerse samples in Karnovskys fixative at 4 C for 90 minutes. The ratio
of fixative volume to specimen size should be approximately 20:1.
2.4. After fixation, aspirate the fixative into a waste container for disposal.
2.5. Wash the specimen twice for 2 minutes in 3 ml 0.1 M phosphate to
remove excess fixative, discarding waste liquid.
2.6. Post fix samples in 1 % buffered osmium tetroxide for 90 minutes at
4 C, then remove the osmium tetroxide solution.
2.7. Add 20x the specimen volume of 30 % ethanol. Leave to equilibrate
for 5 minutes preferably on a rotating wheel. Pour off the ethanol and
discard. Repeat twice.
2.8. Repeat step 2.7 with 50 %, 75 %, 95 % and then absolute ethanol.
The sample can be stored in excess absolute ethanol.
2.9. Follow the manufacturers instructions to completely dry the specimen
using a critical point dryer.
2.10. Follow the manufacturers instructions to gold plate the sample using
a sputter coater. The gold coating will increase the samples
conductivity and consequently improve image quality.
2.11. The samples are now ready for imaging using a conventional scanning
electron microscope.
Consolidated
Protocol Booklet
2.1. Aspirate off the medium and carefully wash the 3D culture twice with PBS.
20

21
Consolidated
Protocol Booklet
Example Results
Figure 1 below shows an example of a 3D AlvetexScaffold culture imaged using the
SEM technique.
A
Figure 1. Detailed structure

of 3D cell cultures can be
visualised using scanning
electron microscopy. Inspection
of pieces of AlvetexScaffold
at low magnification shows
homogeneous coverage by
cultured cells (A). Higher
magnification imaging in this
transverse section reveals cells
growing throughout the scaffold
(B). For scale bar inserts.
Processing AlvetexScaffold
3D Cultures for Cryosectioning.
AlvetexScaffold 3D cell cultures can be easily processed for cryosectioning. This document
describes three separate methods and shows example data obtained from the culture and
processing of HaCaT and HepG2 cells.
Materials and Methods:

1.0 Materials Required for Fixing, Embedding and Sectioning:
OCT embedding matrix (Thermo Scientific, LAMB/OCT)
4 % Paraformaldehyde (PFA) fixative (for full method refer to Histology Series

Part 1. Choosing the Right Fixative to Preserve 3D Cell Cultures located at
www.reinnervate.com/alvetex/workflow)
Sucrose solutions (2 M, 15 % and 30 %)
Liquid nitrogen
Basic equipment: flat-ended forceps to handle 3D AlvetexScaffold cultures

scalpel (to trim / cut scaffold if required); surgical scissors or single edge razor
blade; disposable pipettes; embedding moulds; cryostat.
2.0 Materials Required for Histology and Immunocytochemistry:
HPM/01/5/2012
4 % Paraformaldehyde (PFA) fixative (for full method refer to Histology Series

Part 1. Choosing the Right Fixative to Preserve 3D Cell Cultures located
at www.reinnervate.com/alvetex/workflow)
Dehydration ethanols (70 % and 95 %)
Histoclear
Mayers Haematoxylin solution
Eosin Y solution (2 g eosin Y in 1000 ml 95 % alcohol)
Alkaline alcohol
DPX mountant
22
Consolidated
Protocol Booklet
Introduction:
Tissue freezing and sectioning is a rapid method of generating tissue samples (cryosections)
for histological analysis, and obviates the need for wax embedding. The method is popular
for preparing samples for immunostaining since the need for antigen retrieval is also
eliminated.
23
Consolidated
Protocol Booklet
Blocking buffer (5 % Normal goat serum; 1 % bovine serum albumin; 0.2 %

w/v Triton X-100 in PBS)
Permeabilisation solution (1 % w/v Triton X-100 in PBS)
Primary and secondary antibodies (specific to experimental analysis)
Humidified chamber
Vectashield/DAPI mountant
3.0 Freezing:
3.1
Aspirate culture medium and carefully wash the 3D culture in PBS twice.
3.2
If using well inserts, remove inserts from cradle and place in a conventional
6-well plate.
3.3
Bisect the membrane using surgical scissors or a single edge razor blade.
3.4
At this point one of the following three techniques can be chosen depending
upon personal preference and experience:
3.1 Method 1: Samples can be frozen directly

1. Transfer scaffold halves from each disc into a separate plastic embedding
mould containing OCT mounting medium, flat side down and orientated
longitudinal to the plane of sectioning. Allow the sample to soak for a few
minutes at room temperature.
2. Place a metal rack inside a Styrofoam box and fill with liquid nitrogen to a level
just below the top of the rack. Place the embedding moulds on top of the rack
ensuring that the scaffold pieces remain in an upright position. Let the sample
freeze gradually for 10 minutes.
3. Store at -80 C
3.2 Method 2: Samples can be soaked in sucrose before freezing to remove excess
water (more suitable for cells in collagen gels)
2. Transfer the scaffold to plastic embedding moulds containing OCT mounting

medium and orientate into the required embedding position, as described in
Method 1. Allow the sample to soak for a few minutes at room temperature.
4. Store at -80 C
3.3 Method 3: Samples can be fixed before freezing

1. Transfer the scaffold to 4 % PFA and store overnight at 4 C.
2. Remove the excess water gradually in two baths of sucrose, 15 % followed by
30 % (overnight at 4 C in each bath)
3. Transfer the scaffold to plastic embedding moulds containing OCT mounting
medium and orientate as described in Method 1. Allow the sample to soak for a few
minutes at room temperature.
5. Store at -80 C
Consolidated
Protocol Booklet
1. Transfer the scaffold to 2 M sucrose and store overnight at 4 C.
24
25
4.0 Sectioning
Consolidated
Protocol Booklet
4.1 At least twenty-four hours after freezing, the samples are ready for sectioning
on a suitable cryostat (e.g. Leica CM1850).
4.2 Let the sample sit in the cryostat for 20-30 minutes before sectioning to allow
the sample to adjust to the temperature.
4.3 Following the cryostat manufacturers instructions throughout, align the block
correctly with the cryostat blade and proceed to cut 5-10 m sections.
4.4 Transfer sections to microscope slides. Histobond slides are recommended
since sections adhere to these slides well.
4.5 Let the sections dry for at least 2 hours before processing. The sections should
now be ready for histological and/or immunocytochemical analysis.
5.0 Analysis
5.1 Histological analysis
Set up a series of slide baths and process sample slides using slide holders
in the following sequence of solutions, for the times indicated:
5.1.1 Rehydrate section in PBS.
10 minutes
5.1.2 Fix section in 4 % PFA at 4 C.
15 minutes
5.1.3 Wash in PBS. Repeat X3.
5 minutes
5.1.4 Wash in distilled water
1 minute
5.1.5 Stain in Mayers Haematoxylin
5 minutes
5.1.6 Wash in Distilled water
30 seconds
5.1.7 Blue the nuclei in Alkaline alcohol
30 seconds
5.1.8 Dehydrate in 70 % alcohol
30 seconds
5.1.9 95 % alcohol
30 seconds
5.1.10 Stain in Eosin
1 minute
5.1.11 Dehydrate in 95 % alcohol
10 seconds
5.1.12 95 % alcohol
10 seconds
5.1.13 Absolute alcohol
15 seconds
5.1.14 Absolute alcohol
30 seconds
3 minutes
5.1.16 Histoclear
3 minutes
5.1.17 Slides remain in Histoclear until ready to coverslip to prevent drying out.
5.1.18 Mount slides by spotting DPX solution around the section and cover with
a coverslip. Allow to air dry for at least one hour.
Figure 1 shows examples of frozen 3D AlvetexScaffold cultures, cryosectioned and stained
with Haematoxylin and Eosin.
5.2 Immunocytochemical Analysis

5.2.1 Rehydrate sections for 10 minutes in PBS.
5.2.2 Fix for 15 minutes in 4 % PFA at 4 C.
5.2.3 Wash in PBS for 5 minutes. Repeat PBS wash three times.
5.2.4 Treat cells with permeabilisation solution for 15 minutes at room temperature.
5.2.5 Aspirate permeabilisation solution, replace with blocking buffer and incubate
for 15 minutes at room temperature.
5.2.6 Incubate with primary antibody diluted in blocking buffer (this will be specific
to the antigen chosen and will be used at a pre-determined concentration)
overnight at 4 C in a humidified chamber, or as stated in the datasheet for
the specific antibody used.
5.2.7 Wash three times in PBS for 10 minutes each.
5.2.8 Incubate with secondary antibody diluted in blocking buffer (this will be
concentration) for 2 hours in the dark at room temperature.
5.2.9 Wash three times in PBS for 10 minutes each.
5.2.10 Mount in Vectashield/DAPI and place a cover slip onto the mountant.
This solution simultaneously mounts the specimen and stains cell nuclei.
5.2.11 Seal the perimeter of the cover slip with nail varnish and dry in the dark.
Store slides in the dark at 4 C until ready for inspection using a fluorescence microscope
equipped with the appropriate filters.
Figure 2 shows examples of a 3D AlvetexScaffold culture cryosectioned and stained for
immunofluorescence.
26
Consolidated
Protocol Booklet
5.1.15 Clear in Histoclear
27
Example Data:
Consolidated
Protocol Booklet
Figure 1: Haematoxylin and Eosin staining of AlvetexScaffold cultures of HaCaT and

HepG2 cells after cryosectioning and PFA fixation. Three different protocols have been
used to freeze the samples; samples have been frozen directly (Method 1, A), soaked
in sucrose before freezing (Method 2, B) or fixed before freezing (Method 3, C).
For all protocols used the integrity of the scaffold is maintained.
28
Consolidated
Protocol Booklet
Figure 2: Immunocytochemical staining of cryosectioned AlvetexScaffold 3D cell

cultures. Scaffolds were directly frozen after culture, according to Method 1. HaCaT cells
have been stained with an anti-keratin-10 antibody in red (A) and HepG2 cells have been
stained with an anti-keratin 8 antibody in red (B). Nuclei have been counterstained in blue
using a DAPI marker.
29
Consolidated
Protocol Booklet
01
Consolidated
Protocol Booklet
Methods for Molecular

Analysis of Cells Cultured
in AlvetexScaffold
Total Protein Extraction from Cells

Cultured in AlvetexScaffold in 3D
02
Consolidated
Protocol Booklet
Introduction
The following protocol outlines a method for the extraction of total protein from cells cultured
in AlvetexScaffold. Example data was obtained using this protocol to extract protein from
HepG2 hepatocytes cultured in AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in Well
Insert Holder in Deep Petri Dish (AVP015) format.
Method:
1. Prepare lysis buffer: 1 % v/v Igepal CA-630 (Sigma, Product No. I3021), 50 mM Tris-HCl
pH 8.0, 150 mM NaCl, 1 mM MgCl2, and Complete Mini Protease Inhibitor Cocktail (Roche
Diagnostics, 11 836 153 001).
Note: The Protease Inhibitor Cocktail is available in two forms, with or without EDTA lysate,
see manufacturer instructions for recommendation.
2. Remove AlvetexScaffold disc from culture format (e.g. well insert or 12-well multiwell
plate) and wash by gentle immersion in PBS (e.g. in a Petri dish). Transfer into a 1.5 ml tube.
3. Add 1 ml of lysis buffer and vortex for 10 seconds. Place on ice for 30 minutes with
vortexing (10 sec.) every 5 minutes.
4. Clarify lysates by centrifugation at approx 15,000 rpm for 3 minutes and place supernatant
into a fresh tube.
5. The samples can be frozen at this stage (e.g. at -80oC), for use at a later date.
6. Determine total protein content of samples using a protein determination assay (e.g. BioRad protein assay).
Example: Extraction of Total Protein from HepG2 Hepatocytes Cultured in AlvetexScaffold in 3D
Cell Culture:
and 100 U/ml Penicillin/Streptomycin. Cells were seeded onto AlvetexScaffold discs in 6-well
inserts (AVP004-3) in well insert holders in deep Petri dishes (Product No. AVP015), at a density
of 1 x 106 cells in 150 l media per insert. After settling for 3 hours in an incubator (5 % CO2,
37C) complete media was carefully added (70 ml per Petri dish). Cultures were maintained for
7 days, with a single media change on day 5. After 7 days, well inserts were dismantled and
cultures were processed according to the protocol described above.

Figure 1. Estimation of protein concentration (relative to BSA) in extracts from

AlvetexScaffold cultures of HepG2 hepatocytes. Extracts were assayed at a dilution of 1 in
100. Data from two biological replicate cultures are shown; each sample was read in triplicate.
Protein yield (mg/ml)
Sample 1
Sample 2
1.08 (0.01)
1.02 (0.01)
Table 1. Approximate protein yields in extracts from AlvetexScaffold cultures of HepG2

hepatocytes, using BSA as a standard reference. Values shown represent the mean of
triplicate readings per sample (SD).
03
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Protein analysis results:

Determination of relative protein concentration was made using Protein Assay Reagent (BioRad, 500-0006;). A dilution series of BSA was used for the generation of a standard curve
(Figure 1. inset). Approximately 1 milligram of protein was isolated per disc (Table 1).

04
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Protein extracts were also analysed by SDS-polyacrylamide gel electrophoresis and western
blotting (Figure 2). Protein extracts from two biological replicate cultures produced well-resolved
and highly reproducible SDS-PAGE profiles as determined via Coomassie Brilliant Blue R
staining. Western blotting demonstrated that the constitutive protein -actin was both intact
and equally loaded in both sample lanes.
Figure 2. SDS-polyacrylamide gel electrophoresis and western blot analysis of protein

extracts from AlvetexScaffold cultures of HepG2 hepatocytes. Polyacrylamide gels
(NuPAGE 10% Bis-Tris, Invitrogen, NP0302) were loaded with 20 g protein. Resolved gels
were stained with Commassie Brilliant Blue R (left) or blotted to PVDF membrane for
western analysis (right). The western blot was probed for the constitutive protein -actin
(arrow). Data from two biological replicate cultures are shown.
Extraction of RNA from Cells

Method:
1. Wash AlvetexScaffold culture by gentle immersion in PBS using flat-ended forceps and
transfer to a clean 12-well plate.
2. Lyse cells by adding 600 l Qiagen RNeasy kit lysis buffer RLT per well.
3. Place on a rotating platform (100 rpm) for 10 minutes at room temperature.
4. Homogenise the lysate by passage up and down through a 20-gauge needle 10 times
using a sterile plastic syringe.
5. Add an equal volume of 70 % ethanol to the homogenised lysate and pipette up and down
10 times to mix.
6. Transfer sample (up to 700 l at a time) to an RNeasy spin column in a 2 ml collection
tube. Close cap and microcentrifuge for 15 seconds at 8000 x g. Discard flow-through.
Repeat for remaining volume of lysate. Optional: On-column DNase digestion can be
performed at this point (steps 6 a-d). Alternatively proceed to step 7.
a. Add 350 l Buffer RW1 to the column. Close cap, and microcentrifuge for 15 seconds
at 8000 x g to wash the membrane. Discard flow-through.
b. Prepare DNase 1 reagent by adding 10 l DNase 1 stock solution (prepared according
to manufacturers instructions) to 70 l Buffer RDD. Mix by gentle pipetting or gentle
tube inversion.
c. Add the 80 l diluted DNase 1 reagent to the column membrane and incubate at room
temperature for 15 minutes.
d. Add 350 l Buffer RW1 to the column. Close cap and microcentrifuge for 15 seconds
at 8000 x g. Discard flow-through. Proceed to step 8.
7. Add 700 l Buffer RW1 to the column. Close cap and microcentrifuge for 15 seconds at
8000 x g, reusing the collection tube. Discard flow-through.
8. Add 500 l Buffer RPE (appropriately diluted with ethanol according to manufacturers
instructions) to the column. Close cap and microcentrifuge for 15 seconds at 8000 x g.
Discard flow-through.
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Introduction
The following protocol outlines the use of a commercial extraction kit (Qiagen RNeasy kit,
74104) for the isolation of total RNA from cells cultured in AlvetexScaffold. Example data was
obtained using this protocol to extract RNA from HepG2 hepatocytes cultured in
AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in Well Insert Holder in Deep Petri Dish
(AVP015) format.

06
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Add a further 500 l Buffer RPE to the column. Close cap and microcentrifuge for 2
minutes at 8000 x g. Discard flow-through.
10. Transfer column to a clean 2 ml collection tube. Close cap and microcentrifuge at full speed
for 1 minute.
11. Transfer column to a clean 1.5 ml collection tube and add 50 l RNase-free water onto the
column membrane. Close cap and leave at room temperature for 5 minutes.
Microcentrifuge for 1 minute at 8000 x g to elute RNA.
Example: Extraction of Total RNA from HepG2 Hepatocytes Cultured in AlvetexScaffold in 3D

and 100 U/ml Penicillin/Streptomycin. Cells were seeded onto AlvetexScaffold discs in 6-well
inserts (AVP004-3) in Well Insert Holders in Deep Petri dishes (AVP015), at a density of 1 x 106
cells in 150 l media suspension per insert. After settling for 3 hours in an incubator (5 % CO2,
37 C) complete media was carefully added (70 ml per Petri dish). Cultures were maintained
for 7 days, with a single media change on day 5. After 7 days, well inserts were dismantled and
cultures were processed according to the protocol described above.
RNA analysis results:
RNA samples were assessed for quantity and quality by spectrophotometric (Table 1) and
agarose gel (Figure 1) analyses.
Sample 1
Sample 2
RNA yield (g/ml)
185.1 (0.044)
182.7 (0.470)
A260/A280
2.123 (0.012)
2.117 (0.006)
Table 1. RNA yield and purity analysis. RNA samples from duplicate HepG2 AlvetexScaffold
cultures were diluted 5-fold in 10 mM Tris-HCl, pH 7.0. Values shown represent the mean of
triplicate readings per sample (SD), taken on a NanoDrop 1000 spectrophotometer (Thermo
Scientific).

07
DNA
28 S
18 S
Figure 1. Agarose gel RNA profile from duplicate HepG2 AlvetexScaffold 3D cultures
(Samples 1 and 2). 28S and 18S rRNA bands are clearly visible confirming RNA integrity
(1% agarose gel; visualisation via ethidium bromide staining; 1 kb molecular weight ladder:
Promega, G571A).
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Sample
1
2
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Retrieval of Cells from

AlvetexScaffold
Methods:
1.0. Standard Enzymatic Method
1.1. Unclip inserts and carefully remove the AlvetexScaffold discs using
flat-ended forceps.
1.2. Gently wash each disc in PBS, and transfer to a new 6-well plate.
1.3. 3 ml of 0.25 % Trypsin-EDTA (Sigma, T4049).
1.4. Incubate plate at 37 C, 5 % CO2 on a shaking platform set to 100 rpm,
for 15 minutes.
1.5. Transfer the resulting cell suspension to a 15 ml centrifuge tube.
1.6. Add 3 ml medium to the well containing the AlvetexScaffold membrane
and gently triturate to remove residual detached cells.
1.7. Combine the two solutions in the 15 ml centrifuge tube to neutralise
the trypsin.
1.8. Centrifuge at 1000 rpm for 5 minutes, to pellet the cells.
1.9. Resuspend the cell pellet in an appropriate volume of medium for
downstream processes.
2.0. Enzyme and Syringe Method
2.1. Unclip inserts and carefully remove the AlvetexScaffold discs using
flat-ended forceps.
2.2. Gently wash each disc in PBS, and transfer to a new 6-well plate.
2.3. 3 ml of 0.25 % Trypsin-EDTA (Sigma, T4049).
2.4. Incubate plate at 37 C, 5 % CO2 on a reciprocating shaker set to 100
strokes/min, for 15 minutes.
02
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Protocol Booklet
Introduction:
The following protocol outlines a method for the retrieval of cells cultured in AlvetexScaffold.
Example data were obtained using this protocol to extract HepG2 hepatocyte cells
cultured in AlvetexScaffold for 7 days in 6-well inserts (AVP004-3) in the Well Insert Holder
in a Deep Petri Dish (AVP015) format. Two methods of cell extraction may be employed;
a standard method and a syringe-based method which produces higher average cell yields.
Both methods demonstrate that partial retrieval of cells from AlvetexScaffold is possible.
03
2.5. Transfer the resulting cell suspension to a 15 ml centrifuge tube.
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Protocol Booklet
2.6. Transfer the AlvetexScaffold membrane to the bottom of a 30 ml plastic

syringe barrel. Add 3 ml medium and depress the plunger slowly to force
residual detached/loose cells from the scaffold.
Note: Care should be taken not to force the solution through the membrane
too quickly as this is liable to induce considerable shear stress upon the cells.
2.7. Combine the two solutions in the 15 ml centrifuge tube to neutralise
the Typsin.
2.8. Centrifuge at 1000 rpm for 5 minutes, to collect the cells.
2.9. Resuspend the cell pellet in an appropriate volume of medium required
for the downstream processes.
Example: HepG2 cells grown for 7 days in AlvetexScaffold 6-well insert in a Petri dish format.
HepG2 cells (ATCC, HB-8065) were routinely maintained in T-75 flasks. HepG2 complete
media consisted of: MEM media (Gibco, 21090) supplemented with 10 % v/v FBS, 2 mM Lglutamine and 100 U/ml Penicillin/Streptomycin. Cells were seeded onto AlvetexScaffold
discs in 6-well inserts (AVP004-3) in Well Insert Holders in Deep Petri dishes (AVP015), at a
density of 1x 106 cells in 100 l media per insert. After settling for 1 hour in an incubator
(5 % CO2, 37 C), complete media was carefully added (70 ml per Petri dish). Cultures were
maintained for 7 days, with media changes on days 2 and 4. After 7 days, cultures were
processed according to the protocol described above.
The total number, and number of viable cells retrieved were counted using Trypan
Blue exclusion method. AlvetexScaffold discs were analysed post-retrieval by MTT assay
(Figure 1.) and histological staining to estimate cell numbers remaining in the 3D scaffold
(Figure 2.).
On the basis of residual in-scaffold MTT assay data, 12 % of cells were retrieved (compared
to untreated control discs) using the standard protocol described above. When the syringebased method was employed, cell recovery increased to 24 %. Cell viability post-retrieval
was 99 % for both methods, as determined by cell counting in conjunction with Trypan
Blue exclusion (data not shown).
04
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cell viability using a standard MTT
assay on AlvetexScaffold discs
post-cell extraction using trypsinEDTA. Data from 3 sample
replicates of HepG2 cultures
are shown (n=3, mean SE).
05
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Protocol Booklet
Figure 2. Bright field micrographs showing the morphology of HepG2 cells cultured for 7 days on
AlvetexScaffold in 6-well insert within a Petri dish format (x10 and x40 objective). A: untreated HepG2 cells
and B: HepG2 cells after Trypsin-EDTA treatment for 15 minutes. Cells were fixed, embedded in paraffin
wax, sectioned (10 m) and counterstained with Haematoxylin and Eosin. The effect of Trypsin action can
clearly be seen on residual cells which lose contact with each other and adopt a rounded morphology.
01
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Specialised Applications
using AlvetexScaffold
02
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03
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04
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Example Protocols for the

Growth of Specific Cell
Types on AlvetexScaffold
Example protocol for the culture of the 3T3 cell line on

Alvetex Scaffold (22 mm disc in 12-well plate format, AVP002).
02
Preparation for 3D cell culture on AlvetexScaffold
Consolidated
Protocol Booklet
1.
3T3 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of 3T3 cells grown in conventional 2D culture plates.
Images show cells at low (left) and high (right) confluency. Scale bars: 200 m.
2.
Complete media consisted of: Dulbeccos Modified Eagles Medium (DMEM)

supplemented with 10% v/v FBS, 2 mM L-glutamine and 100 U/ml
Penicillin/Streptomycin.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm). The
supernatant was discarded and the cell pellet was re-suspended in an appropriate
volume of media for cell counting by Trypan Blue.
4.
Cells were re-suspended at a concentration of 8x106 cells/ml for seeding.
5.
AlvetexScaffold 12-well plates were prepared for seeding with 70% ethanol (2 ml per
well) and media washes (twice with 3 ml of media each) as described in the product
information leaflet.
6.
125 l of the cell suspension was added to the centre of the AlvetexScaffold disc, which
was equivalent to 1x106 cells per well.
7.
The plate was incubated 3 hours at 37 C with 5% CO2 to allow the cells to settle into
the scaffold.
8.
4 ml of media was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete media exchange after every
1-2 days.

Alvetex Scaffold (22 mm disc in 12-well plate format, AVP002).
Histology
03

replicates of 3T3 cells are
shown. Each well was sampled
in duplicate with mean value
shown. Cells were cultured for 3
days on 22 mm AlvetexScaffold
discs presented in the 12-well
plate format.
Consolidated
Protocol Booklet
Figure 2. Brightfield
micrographs showing the
structure of 3T3 cells cultured
for 7 days on 22 mm diameter
AlvetexScaffold discs presented
in 2-well plate format. Cells were
fixed, embedded in paraffin wax,
sectioned (10 m) and
counterstained with

Alvetex Scaffold (22 mm disc in 6-well insert format, AVP004-03).
04
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Protocol Booklet
1.
3T3 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of 3T3 cells grown in conventional 2D culture plates.
Images show cells at low (left) and high (right) confluency. Scale bars: 200 m.
2.

supplemented with 10% v/v FBS, 2 mM L-glutamine and 100 U/ml
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000 rpm).
The supernatant was discarded and the cell pellet was re-suspended in an appropriate
4.
Cells were re-suspended at a concentration of 8x10 6 cells/ml for seeding.
5.
AlvetexScaffold 6-well inserts were prepared for seeding by dipping in 70% ethanol
and washing twice with 7 ml of media per well.
6.
7.
The plate was incubated 60 min at 37 C with 5% CO2 to allow the cells to settle into the
scaffold.
8.
AlvetexScaffold.
9.
2-3 days.

Alvetex Scaffold (22 mm disc in 6-well insert format, AVP004-03).
Histology
05

replicates of 3T3 cells are
in duplicate with mean value
shown. Cells were cultured for 3
inserts in 6-well plates.
Consolidated
Protocol Booklet
Figure 2. Brightfield micrographs

showing the structure of 3T3
cells cultured for 7 days on 22
mm diameter AlvetexScaffold
discs presented in 6-well inserts
in 6-well plates. Cells were fixed,
embedded in paraffin wax,
counterstained with
Example protocol for the culture of the TERA2.cl.SP12 cell line on

AlvetexScaffold (22 mm disc in 12-well plate format, AVP002).
06
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Protocol Booklet

Preparation for 3D cell culture on Alvetex Scaffold
1. TERA2.cl.SP12 cells1,2
were routinely maintained in T-75 flasks.
1. TERA2.cl.SP12 cells1,2 were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of TERA2.cl.SP12 cells grown in conventional 2D

Figure plates.
1. Phase
contrast
of TERA2.cl.SP12
grown Scale
in conventional
2D
culture
Images
showmicrographs
cells at low (left)
and high (right)cells
confluency.
bars: 100 m.
culture plates. Images show cells at low (left) and high (right) confluency. Scale bars: 100 m.
2.
2.
3.
3.
4.
4.
5.
5.
6.
6.
7.
7.
8.
8.
9.
9.
1
1
2
2

Complete media
Dulbeccos
Modified
Eagles Medium
(DMEM)
supplemented
withconsisted
10% v/v of:
heat-treated
FBS,
2 mM L-glutamine
and 100
U/ml
supplemented
with 10% v/v heat-treated FBS, 2 mM L-glutamine and 100 U/ml
Cells were harvested
by trypsinisation
andpellet
centrifuged
for 5 minutesin(1000
rpm). The
supernatant
was discarded
and the cell
was re-suspended
an appropriate
supernatant
was for
discarded
and the
cell pellet
volume
of media
cell counting
by Trypan
Blue.was re-suspended in an appropriate
AlvetexScaffold 12-well plates were prepared for seeding with 70% ethanol (2 ml per well)
Scaffold
12-well
plateswith
were3prepared
for seeding
ethanolin(2the
ml per
well)
Alvetex
and media
washes
(twice
ml of media
each)with
as 70%
described
product
and
media leaflet.
washes (twice with 3 ml of media each) as described in the product
information
125 l
of the celltosuspension
wasper
added
to the centre of the Alvetex Scaffold disc, which
was
equivalent
500,000 cells
well.
was equivalent to 500,000 cells per well.
The scaffold.
plate was incubated 3 hours at 37 C with 5% CO2 to allow the cells to settle into
the
the scaffold.
4 ml ofScaffold.
media was added to each well taking care not to dislodge cells from
Alvetex
AlvetexScaffold.
Plates
were re-incubated and maintained by complete media exchange after every
1-2
days.
1-2 days.
Przyborski S.A. (2001). Isolation of human embryonal carcinoma stem cells by immuno-magnetic sorting.
Przyborski
Isolation of human embryonal carcinoma stem cells by immuno-magnetic sorting.
Stem
Cells,S.A.
19, (2001).
500-504.
Stem Cells, 19, 500-504.
Stewart R., Christie V. & Przyborski S.A. (2003). Manipulation of human pluripotent
Stewart R.,carcinoma
Christie V.stem
& Przyborski
S.A.development
(2003). Manipulation
of human Stem
pluripotent
embryonal
cells and the
of neural subtypes.
Cells,
embryonal carcinoma stem cells and the development of neural subtypes. Stem Cells,


replicates of TERA2.cl.SP12
cells are shown (n=3, mean
SD). Cells were cultured for 3
plate format.
07
Consolidated
Protocol Booklet

showing the structure of
TERA2.cl.SP12 cells cultured for
7 days on 22 mm diameter
AlvetexScaffold discs presented
in the 12-well plate format. Cells
were fixed, embedded in paraffin
wax, sectioned (10 m) and
counterstained with

AlvetexScaffold (22 mm disc in 6-well insert format, AVP004-03)
08
Consolidated
Protocol Booklet
TERA2.cl.SP12 cells1,2 were routinely maintained in T-75 flasks.
1.
Figure 1. Phase contrast micrographs of TERA2.cl.SP12 cells grown in conventional 2D

culture plates. Images show cells at low (left) and high (right) confluency. Scale bars: 100 m
2.

supplemented with 10% v/v heat-treated FBS, 2 mM L-glutamine and 100 U/ml
3.
4.
5.
and washed twice with 7 ml of media per well.
6.
125 l of the cell suspension was added to the centre of the AlvetexScaffold disc,
which was equivalent to 500,000 cells per well.
7.
scaffold.
8.
AlvetexScaffold.
9.
2-3 days.
Przyborski S.A. (2001). Isolation of human embryonal carcinoma stem cells by immuno-magnetic sorting.
Stem Cells, 19, 500-504.
Stewart R., Christie V. & Przyborski S.A. (2003). Manipulation of human pluripotent embryonal carcinoma
stem cells and the development of neural subtypes. Stem Cells, 21, 248-256.

AlvetexScaffold (22 mm disc in 6-well insert format, AVP004-03)

replicates of TERA2.cl.SP12
cells are shown (n=3, mean
SD). Cells were cultured for 3
inserts in 6-well-plate format.
09
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Protocol Booklet
structure of TERA2.cl.SP12
cells cultured for 7 days on 22
mm diameter AlvetexScaffold
discs presented in 6-well
inserts in 6-well plates.
Cells were fixed, embedded in
paraffin wax, sectioned (10 m)
and counterstained with
Example Preparation of Epidermal

Equivalent Using AlvetexScaffold
10
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Introduction:
HaCaT is an immortalised human adult keratinocyte cell line. This cell line has been known
to retain a capacity for normal differentiation up to multiple passages similar to normal human
epidermal keratinocytes (NHEK), and hence offers a suitable model for keratinisation studies.
However, even with their highly preserved differentiation capacity, terminal differentiation
can remain incomplete under airexposed conditions. This protocol describes a method for
differentiation of HaCaT cells under air-exposed conditions for up to 21 days.
Method:
1.
HaCaT cells were routinely maintained in T-75 flasks in 2D using complete media
(Dulbeccos Modified Eagles Medium supplemented with 10 % v/v FBS , 2 mM
L-glutamine and 100 U/ml Penicillin/Streptomycin).
Figure 1. Phase contrast micrographs of HaCaT cells grown in conventional 2D

culture flasks. Images show cells at low (left) and high (right) confluency. Scale
bars: 200 m.
HPM/01/5/2012
2.
The supernatant was discarded and the cell pellet re-suspended in an appropriate
volume of media for dilution for cell counting by Trypan Blue.
3.
Cells were re-suspended at a concentration of 20 x 106 cells/ml in Clonetics

Keratinocyte Growth Medium-2 (KGM-2; Lonza, CC-3107) for seeding.
4.
In the meantime AlvetexScaffold 12-well inserts (AVP005) were prepared for

seeding as described in the product information booklet. Briefly, well-inserts
were dipped into a beaker containing 70 % ethanol before placing into 6-well
plates after gentle tapping to release any excess ethanol. Well inserts
were immediately washed with media (7 ml/well).

11
5.
The media was aspirated and replaced with a second media wash. This was
aspirated just before application of the cells: 1 x 106 cells in 50 l were added
to the middle of each well insert.
Note: If preparation of cell suspension is delayed, incubate plate with medium
at 37 C with 5 % CO2 until further use.
6.
The plate was incubated for 30 minutes at 37 C with 5 % CO2 to allow the cells
to settle, wells were then carefully topped up with 10 ml of KGM-2 and cultured
for 3 days under submerged conditions. Culture media was fully changed after
2 days.
7.
Well inserts were then moved to Reinnervates custom-made Well insert holder
in a deep Petri dish (AVP015) at medium setting, and maintained at the air/liquid
interface (37 ml/per Petri dish) for up to 21 days to promote stratification. Culture
media was refreshed by partial media exchange (50:50) every 2 days.
8.
Cultures were examined histologically at the end of each week. 3D cultures were
processed in either Bouins fixative or 4 % paraformaldehyde, wax embedded,
sectioned (10 m) and analysed by haematoxylin and eosin staining or
immunohistochemistry. For full experimental details please refer to specific
histology protocols located at www.reinnervate.com/alvetex/workflow.
Example Data Set:

Between 7 and 14 days the HaCaT cells colonised 100 % of the AlvetexScaffold disc (Figure
2), with the majority of cells still mitotic in nature as indicated by ki67 antibody staining (Figure
3). After 21 days, cell differentiation is noticeable within the construct, indicated by intense
pink staining of the cell cytoplasm due to increased keratin production, anucleation and
flattening of cell morphology. This is further supported by the expression of late differentiation
markers (keratin 10 and involucrin) which occur towards the air exposed surface of the culture
with increasing expression from 7-21 days (Figure 3).
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Protocol Booklet
Note: If preferred, ethanol treatment can also be done in situ in the plates by
dispensing sufficient ethanol solution to cover the well inserts and
AlvetexScaffold disc.

12
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Figure 2. Brightfield micrographs showing the structure of HaCaT cells cultured on

AlvetexScaffold in 12-well inserts (AVP005) by haematoxylin and eosin staining of
Bouins fixed, paraffin embedded sections. HaCaT cells were initially cultured for 3
days under submerged conditions prior to air exposure. The time points referred to
in the figures represent the number of days cultures subsequently spent at the
air/liquid interface.

13
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Figure 3. Keratin 10 (mouse monoclonal), Ki67 (rabbit polyclonal) and involucrin

(mouse monoclonal) expression of HaCaT cells cultured on AlvetexScaffold in
12-well inserts (AVP005) by immunohistochemical analysis of 4 % formaldehyde
fixed, paraffin embedded sections (secondary antibodies, Alexa Fluor 488 donkey,
anti-mouse and Alexa Fluor 488 goat, anti-rabbit). HaCaT cells were initially cultured
for 3 days under submerged conditions prior to air exposure. The time points referred
to in the figures represent the number of days cultures subsequently spent at the
air/liquid interface (scale bars 100 m).

14
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Figure 4. Scanning electron

micrograph (SEM) image
showing HaCaT cells grown
in AlvetexScaffold cultured
for 7 days at the air/liquid
interface (scale bar 50 microns).
Example protocol for the culture of the HepG2 cell line on

AlvetexScaffold (22 mm disc in 6-well insert format, AVP004-03).
Preparation for 3D cell culture on AlvetexScaffold:

HepG2 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of HepG2 cells grown in conventional 2D culture

plates. Images show cells at low (left) and high (right) confluency. Scale bars: 100 m.
2.
Complete media consisted of: MEM media (Gibco 21090) supplemented with 10% v/v
FBS, 2 mM L-glutamine and 100 U/ml Penicillin/Streptomycin.
3.
4.
Cells were re-suspended at a concentration of 1.6x10 7 cells/ml for seeding.
5.
and washed twice with 7 ml of media per well.
6.
7.
scaffold.
8.
AlvetexScaffold.
9.
2-3 days.
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Protocol Booklet
1.
15

AlvetexScaffold (22 mm disc in 6-well insert format, AVP004-03).
16
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Protocol Booklet
structure of HepG2 cells
cultured for 7 days on 22 mm
diameter AlvetexScaffold discs
format. Cells were fixed,
counterstained with

replicates of HepG2 cells are
shown (n=3, mean SD). Cells
were cultured for 3 days on 22
mm AlvetexScaffold discs
presented in the 6-well inserts
in 6-well plate format.


HepG2 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of HepG2 cells grown in conventional 2D culture

2.
Complete media consisted of: MEM media (Gibco 21090) supplemented with 10 % v/v
FBS, 2 mM L-glutamine and 100 U/ml Penicillin/Streptomycin.
3.
4.
Cells were re-suspended at a concentration of 1.6x10 7 cells/ml for seeding.
5.
AlvetexScaffold 12-well plates were prepared for seeding with 70% ethanol (2 ml per well)
and media washes (twice with 3 ml of media each) as described in the product
6.
7.
the scaffold.
8.
AlvetexScaffold.
9.
1-2 days.
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Protocol Booklet
1.
17

18
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Protocol Booklet
structure of HepG2 cells
cultured for 7 days on 22 mm
diameter AlvetexScaffold discs
format. Cells were fixed,
counterstained with

replicates of HepG2 cells are
shown (n=3, mean SD).
Cells were cultured for 3 days
on 22 mm AlvetexScaffold
plate format.
Example Protocol for the Culture of the CHO-K1 Cell Line on

AlvetexScaffold (22 mm Disc in 6-Well Inserts Format, AVP004-3)

CHO-K1 cells (ATCC, CCL-61) were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of CHO-K1 cells grown in conventional 2D culture

plates. Images show cells at low (left) and high (right) confluency. Scale bars: 100m.
2.
Complete medium consisted of: F-12K nutrient mixture (Kaighns modification; Gibco
21127022) supplemented with 10% v/v FBS and 100g/ml Penicillin and 10g/ml
Streptomycin. Note: Medium already contains L-glutamine.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000rpm).

The supernatant was discarded and the cell pellet was re-suspended in appropriate
4.
Cells were re-suspended at a concentration of 1x106 cells / 150l for seeding per well.
5.
AlvetexScaffold 6-well inserts fitted into 6-well plates were prepared for seeding by washing
in 70% ethanol and rinsing once with 10ml of medium per well.
6.
150l of the cell suspension was added to the centre of the AlvetexScaffold disc, which
7.
The plate was incubated 30 minutes at 37C with 5% CO2 to allow the cells to settle into
the scaffold.
8.
10ml of medium was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete medium exchange after every
2-3 days.
HPM/01/5/2012
Consolidated
Protocol Booklet
1.
19

AlvetexScaffold (22 mm Disc in 6-Well Inserts Format, AVP004-3)
20
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Protocol Booklet

showing the structure of CHO-K1
cells cultured for 3 days on
22mm diameter AlvetexScaffold
discs presented in 6-well inserts
fitted into 6-well plates.
paraffin wax, sectioned (10m)
Haematoxylin and Eosin.

cell viability using a standard MTT
assay. Data from 3 sample
replicates of CHO-K1 cells are
in triplicate with 1/10 dilution.
Mean values are shown.
Cells were cultured for 7 days
on 22mm AlvetexScaffold discs
presented in 6-well inserts fitted
into 6-well plates.

AlvetexScaffold (22 mm Disc in 12-Well Insert Format, AVP005-3)

CHO-K1 cells (ATCC, CCL-61) were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of CHO-K1 cells grown in conventional 2D culture

plates. Images show cells at low (left) and high (right) confluency. Scale bars: 100m.
2.
Complete medium consisted of: F-12K nutrient mixture (Kaighns modification; Gibco
21127022) supplemented with 10% v/v FBS and 100g/ml Penicillin and 10g/ml
Streptomycin. Note: Medium already contains L-glutamine.
3.
Cells were harvested by trypsinisation and centrifuged for 5 minutes (1000rpm).

The supernatant was discarded and the cell pellet was re-suspended in appropriate
4.
Cells were re-suspended at a concentration of 1x106 cells / 150l for seeding per well.
5.
AlvetexScaffold 12-well discs were prepared for seeding by washing in 70% ethanol and
rinsing once with 4ml of medium per well.
6.
150l of the cell suspension was added to the centre of the AlvetexScaffold disc, which
7.
The plate was incubated 30 minutes at 37C with 5% CO2 to allow the cells to settle into
the scaffold.
8.
4ml of medium was added to each well taking care not to dislodge cells from
AlvetexScaffold.
9.
Plates were re-incubated and maintained by complete medium exchange after every
1-2 days.
HPM/01/5/2012
Consolidated
Protocol Booklet
1.
21

AlvetexScaffold (22 mm Disc in 12-Well Insert Format, AVP005-3)
22
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Protocol Booklet

showing the structure of CHO-K1
cells cultured for 3 days on
22mm diameter AlvetexScaffold
discs presented in 12-well plates.
paraffin wax, sectioned (10m)
Haematoxylin and Eosin.

replicates of CHO-K1 cells are
shown. Each well was sampled in
triplicate. Mean values are shown.
Cells were cultured for 3 days on
22mm AlvetexScaffold discs
presented in 12-well plates.
Example Protocols for the Culture of the MCF-7 Cell Line on

AlvetexScaffold in Well Plate and Well Insert Formats
Methods:
1. MCF-7 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrograph of MCF-7 cells grown in conventional 2D culture

plates. Scale bar: 200 m.
2.

supplemented with 10 % v/v FBS, 2 mM L-glutamine and 100 U/ml
3.
HPM/22/8/2012
23
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Protocol Booklet
Introduction:
AlvetexScaffold is currently available in four different cell culture formats: 24-well plate
(AVP006), 12-well plate (AVP002), 6-well insert (AVP004), and 12-well insert (AVP005).
24-well and 12-well plates are suitable for shorter term cultures and for applications
where limited cell penetration into the scaffold is required. Well insert formats
generally support longer term cultures and deeper cell penetration into the scaffold.
They also provide for conveniently tailored media set ups (see Quick Start Protocol,
www.reinnervate.com/alvetex/workflow). 6-well inserts can be placed in conventional
6-well plates, while 12-well inserts can be placed in either 6-well plates or 12-well plates,
depending on media requirements. Alternatively, both insert types can be housed in the
dedicated Well Insert Holder in Deep Well Petri Dish (AVP015) to allow for increased media
volumes and prolonged cell culture. The availability of two different AlvetexScaffold sizes
enables choice on the basis of desired culture size and cell expenditure.

24
Follow one of the below methods according to your choice of AlvetexScaffold format.
4.
Consolidated
Protocol Booklet
6-well Insert format AVP004:

a.
Cells were re-suspended at a concentration of 10 x 106 cells/ml for seeding.
b.
AlvetexScaffold 6-well inserts were prepared for seeding with a 70 % ethanol

wash (7ml) and subsequent media washes (twice, with 7ml media each).
c.
100 l of the cell suspension was added to the centre of the AlvetexScaffold,
which was equivalent to 1 x 106 cells per well.
d.
to settle into the scaffold.
e.
10 ml of media was added to each well taking care not to dislodge cells from the
membrane.
f.
2-3 days.
Note: This method can be applied to the use of AlvetexScaffold in 12-well insert format,
AVP005. Adjust cell seeding and media volumes according to the guidelines provided in
the Quick Start Protocol.
24-well Plate Format, AVP006:

a.
Cells were re-suspended at a concentration of 4 x 106 cells/ml for seeding.
b.
AlvetexScaffold 24-well plate were prepared for seeding with a 70 % ethanol

wash (2 ml per well) and subsequent media washes (twice, with 2 ml media each).
c.
50 l of the cell suspension was added to the centre of the AlvetexScaffold,

which was equivalent to 0.2 x 106 cells per well.
d.
e.
2 ml of media was added to each well taking care not to dislodge cells from the
membrane.
f.
Plates were re-incubated and maintained by complete media exchange every 2-3
days for the first 10 days, then every day.
Note: This method can be applied to the use of AlvetexScaffold in 12-well plate format,

Example Data: 6-well Insert Format, AVP004:
The cells showed good linear growth and expansion in 3D which plateaued between 14
and 21 days. We would not recommend growing cultures of MCF-7 cells beyond 14-18
days as when they reach confluency in 3D they can become overcrowded and the quality
of the culture is reduced resulting in cell death.
Consolidated
Protocol Booklet
MCF-7 cells were maintained on AlvetexScaffold 6-well inserts for up to 21 days.

Cultures underwent complete media change every 2-3 days. At specific time points,
cultures were harvested and analysed by histological staining (Figure 2) and MTT viability
assay (Figure 3) to monitor cell survival and proliferation within the scaffold. For histology
and MTT assay protocols refer to www.reinnervate.com/alvetex/workflow.
25

26
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Protocol Booklet
Figure 2. Brightfield micrographs showing the structure of MCF-7 cells cultured for A) 3
days, B) 7 days, C) 10 days or D) 14 days on 22 mm diameter AlvetexScaffold presented
in 6-well inserts. Cells were fixed, embedded in paraffin wax, sectioned (10 m) and

27
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Protocol Booklet
Figure 3. Biochemical analysis of cell viability using a standard MTT assay. Data from 3
sample replicates of MCF-7 cells are shown (mean SD). Each well was sampled in
triplicate with mean value shown. Cells were cultured for up to 21 days on 22 mm
AlvetexScaffold presented in 6-well inserts.
Example Data: 24-well Plate Format, AVP006:

MCF-7 cells were maintained on AlvetexScaffold 24-well plates for up to 18 days.
Cultures underwent complete media change every 2-3 days until day 10, then every day.
At specific time points, cultures were harvested and analysed by histological staining
(Figure 2) and MTT viability assay (Figure 3) to monitor cell survival and proliferation
within the scaffold. For histology and MTT assay protocols refer to
www.reinnervate.com/alvetex/workflow.
The cultures showed good linear growth and expansion in 3D which plateaued between
10 and 18 days. If cells are grown longer than 10 days, we would recommend changing
the medium every day. When the cells reach confluency in 3D they become overcrowded
and the quality of the culture is reduced, resulting in cell death, therefore we would not
recommend culturing these cells for longer than 14 days in this culture format.

28
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Protocol Booklet
Figure 4. Brightfield micrographs showing the structure of MCF-7 cells cultured for A)
3 days, B) 7 days, C) 10 days or D) 14 days on 15 mm diameter AlvetexScaffold
presented in 24-well plates. Cells were fixed, embedded in paraffin wax, sectioned
(10 m) and counterstained with haematoxylin and eosin.

29
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Protocol Booklet
Figure 5. Biochemical analysis of cell viability using a standard MTT assay. Data from
3 sample replicates of MCF-7 cells are shown (mean SD). Each well was sampled
in triplicate with mean value shown. Cells were cultured for up to 18 days on 15 mm
AlvetexScaffold presented in 24-well plate format.
Example Protocol for the Culture of Primary Rat

MSCs on AlvetexScaffold in Well Insert Formats
30
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Protocol Booklet
Introduction:
(AVP006), 12-well plate (AVP002), 6-well insert (AVP004), and 12-well insert (AVP005). 24-well
and 12-well plates are suitable for shorter term cultures and for applications where limited
cell penetration into the scaffold is required. Well insert formats generally support longer
term cultures and deeper cell penetration into the scaffold. They also provide for conveniently
tailored media set ups (see Quick Start Protocol, www.reinnervate.com/alvetex/workflow).
6-well inserts can be placed in conventional 6-well plates, while 12-well inserts can be placed
in either 6-well plates or 12-well plates, depending on media requirements. Alternatively,
both insert types can be housed in the dedicated Well Insert Holder in Deep Well Petri Dish
(AVP015) to allow for increased media volumes and prolonged cell culture. The availability of
two different AlvetexScaffold sizes enables choice on the basis of desired culture size and
cell expenditure.
Methods:
Preparation for 3D Cell Culture on AlvetexScaffold:
1.
Primary rat MSCs were derived from the bone marrow of 6-8 week old male Wistar
rats (Harlan) using standard, previously published, methodology [1], and used at P1
for experiments. MSCs were routinely maintained in T-75 flasks.
2.
Complete growth media consisted of: DMEM High glucose supplemented with
10 % v/v heat inactivated FBS, 1X non-essential amino acids, 2 M L-glutamine
and 100 U/ml Penicillin/Streptomycin.
3.
4.
Cells were re-suspended at a concentration of 1.0 x107 cells/ml for seeding.
6-well Insert Format, AVP004:

a.
AlvetexScaffold 6-well inserts in 6-well plate format were prepared for seeding
by dipping in 70 % ethanol and washed twice with media (7 ml per well).
b.
which was equivalent to 1 x 106 cells per well.
c.
The plate was incubated for at least 15 minutes at 37 C with 5 % CO2 to allow the
cells to settle into the scaffold.
d.
10 ml of media was added to each well taking care not to

dislodge cells from AlvetexScaffold.
HPM/06/8/2012
Example Protocol for the Culture of Primary Rat

MSCs on AlvetexScaffold in Well Insert Formats
e.
2-3 days.
Example Data: 6-well Insert Format, AVP004:
Figure 1. Brightfield micrographs showing the structure of primary rat MSCs cultured for
14 days on 22 mm diameter AlvetexScaffold discs presented in 6-well insert in 6-well
plate format. Cells were fixed, embedded in paraffin wax, sectioned (10 m) and
counterstained with haematoxylin and eosin. Scale bar 100 m.
Figure 2. Biochemical analysis of cell viability using a standard MTT assay. For each
timepoint, data from 3 sample replicates of primary rat MSCs are shown (n=3, mean
SEM). Cells were cultured for 4, 7 and 14 days on 22 mm AlvetexScaffold discs
presented in 6-well insert in 6-well plate format.
1.
Croft, A.P. and S.A. Przyborski, Formation of neurons by non-neural adult stem cells:
potential mechanism implicates an artifact of growth in culture. Stem Cells, 2006. 24(8):
p. 1841-51.
Consolidated
Protocol Booklet
31
Example Protocol for the Culture of the MG63 Cell

Line on AlvetexScaffold in Well Insert Formats
32
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Protocol Booklet
Introduction:
cell expenditure.
Methods:
1.
MG63 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of MG63 cells grown in conventional 2D culture

2.
10 % v/v heat inactivated FBS, 1X non-essential amino acids, 2 M L-glutamine
and 100 U/ml Penicillin/Streptomycin.
3.
4. Cells were re-suspended at a concentration of 1.0 x107 cells/ml for seeding.
HPM/06/8/2012
Example Protocol for the Culture of the MG63 Cell

Line on AlvetexScaffold in Well Insert Formats

by dipping in 70 % ethanol and washed twice with media (7 ml per well).
b.
c.
5 ml of media was added to each well taking care not to allow media over the
windows of the insert, i.e. independent feeding from below. The plate was incubated
overnight at 37 C with 5 % CO2 to allow the cells to settle into the scaffold.
d.
A further 5 ml of media was added to each well the following morning taking care
not to dislodge cells from AlvetexScaffold.
e.
2-3 days.
Example Data: 12-well insert format, AVP005:
Figure 2. Brightfield micrographs showing the structure of MG63 cells cultured for 7 days
on 15 mm diameter AlvetexScaffold discs presented in 12-well insert in 6-well plate
format. Cells were fixed, embedded in paraffin wax, sectioned (10 m) and
counterstained with haematoxylin and eosin. Scale bar 100 m.
Consolidated
Protocol Booklet
a.
33
Example Protocol for the Culture of the SW620 Cell Line

on AlvetexScaffold in Well Insert and Well Plate Formats
34
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Protocol Booklet
Introduction:
cell expenditure.
Methods:
1.
SW620 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrograph of SW620 cells grown in conventional 2D culture

plates. Image shows cells at low (left) and high (right) confluency. Scale bars: 100 m.
2.
10 % v/v heat inactivated FBS, 2 M L-glutamine and 100 U/ml Penicillin/Streptomycin.
3.
4.
5.
Follow one of the below methods according to your choice

of AlvetexScaffold format.
HPM/14/9/2012


AlvetexScaffold 24-well plates were prepared for seeding with a 70 % ethanol
wash (2 ml per well) and subsequent media washes (twice with 2 ml of media each).
b.
c.
The plate was incubated for at least 1 hour at 37 C with 5 % CO2 to allow the cells
d.
AlvetexScaffold.
e.
1-2 days.

a.
by dipping in 70 % ethanol followed by media washes (twice, with 7 ml per well).
b.
Wells were filled from the outside of the insert with enough medium to allow it
to rise inside the insert and cover the substrate, but not to go over the sides of the
inserts, i.e. 7ml 1ml, as described for feeding above and below separately in the
Quick Start Protocol located at (www.reinnervate.com/alvetex/workflow).
c.
100 l of the cell suspension was added in droplets over the surface of
the AlvetexScaffold disc, which was equivalent to 1 x 106 cells per well.
d.
The plate was incubated overnight at 37 C with 5 % CO2 to allow the cells to settle
into the scaffold.
e.
Media was added to each well to a total volume of 10ml the following morning taking
care not to dislodge cells from AlvetexScaffold.
f.
2-3 days.
Consolidated
Protocol Booklet
a.
35

36
Consolidated
Protocol Booklet
a.
by dipping in 70 % ethanol followed by media washes (twice with 4 ml per well).
b.
to rise inside the insert and cover the substrate, but not to go over the sides of
the inserts, i.e. 2.4ml 0.2ml, as described for feeding above and below separately
in the Quick Start Protocol (www.reinnervate.com/alvetex/workflow).
c.
50 l of the cell suspension was added in droplets over the surface of the
AlvetexScaffold disc, which was equivalent to 0.5 x 106 cells per well.
d.
into the scaffold.
e.
f.
2-3 days.
Example Data: 24-well plate format, AVP006:
Figure 6. Brightfield micrographs showing the structure of SW620 cells cultured for
7 days on 15 mm diameter AlvetexScaffold discs presented in the 24-well plate format.
Cells were fixed, embedded in paraffin wax, sectioned (10 m) and counterstained with
haematoxylin and eosin. Scale bar: 300 m

37
Consolidated
Protocol Booklet
3 sample replicates of SW620 cells are shown (n=3, mean SD). Cells were cultured
for 3 days on 15 mm AlvetexScaffold discs presented in the 24-well plate format.
7 days on 22 mm diameter AlvetexScaffold discs presented in 6-well inserts in the
6-well plate format. Cells were fixed, embedded in paraffin wax, sectioned (10 m)
and counterstained with haematoxylin and eosin. Scale bar: 300 m.

38
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Protocol Booklet
for 3 days on 22 mm AlvetexScaffold discs presented in 6-well inserts in the 6-well
plate format.
Figure 4. Brightfield micrographs showing the structure of SW620 cells cultured for 7
days on 15 mm diameter AlvetexScaffold discs presented in 12-well insert in 12-well
counterstained with haematoxylin and eosin. Scale bar: 300 m

39
Consolidated
Protocol Booklet
Figure 5. Biochemical analysis of cell viability using a standard MTT assay. Data from 3
sample replicates of SW620 cells are shown (n=3, mean SD). Cells were cultured for 3
days on 15 mm AlvetexScaffold discs presented in 12-well insert in 12-well plate format.

40
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Protocol Booklet
Introduction:
cell expenditure.
Methods:
1.
SW480 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrograph of SW480 cells grown in conventional 2D culture

plates. Image shows cells at low (left) and high (right) confluency. Scale bars: 100 m.
2.
10 % v/v heat inactivated FBS, 2 M L-glutamine and 100 U/ml Penicillin/Streptomycin.
2.
3.
4.
Follow one of the below methods according to your choice

of AlvetexScaffold format.
HPM/14/9/2012


AlvetexScaffold 24-well plates were prepared for seeding with a 70 % ethanol
wash (2 ml per well) and subsequent media washes (twice with 2 ml of media each).
b.
c.
The plate was incubated for at least 1 hour at 37 C with 5 % CO2 to allow the cells
d.
AlvetexScaffold.
e.
1-2 days.

a.
by dipping in 70 % ethanol followed by media washes (twice with 4 ml per well).
b.
to rise inside the insert and cover the substrate, but not to go over the sides of
the inserts, i.e. 2.4ml 0.2ml, as described for feeding above and below separately
in the Quick Start Protocol (www.reinnervate.com/alvetex/workflow).
c.
50 l of the cell suspension was added in droplets over the surface of the
AlvetexScaffold disc, which was equivalent to 0.5 x 106 cells per well.
d.
into the scaffold.
e.
f.
2-3 days.
Consolidated
Protocol Booklet
a.
41

42
Example Data: 24-well plate format, AVP006:
Consolidated
Protocol Booklet
Figure 2. Brightfield micrographs showing the structure of SW480 cells cultured for 7
days on 15 mm diameter AlvetexScaffold discs presented in the 24-well plate format.
for 3 days on 15 mm AlvetexScaffold discs presented in the 24-well plate format.

43
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Protocol Booklet
7 days on 15 mm diameter AlvetexScaffold discs presented in 12-well insert in 12-well
for 3 days on 15 mm AlvetexScaffold discs presented in 12-well insert in 12-well
plate format.
Example Protocols for the Co-Culture of either the

SW620 or the SW480 Cell Line with the 3T3 Cell Line
on AlvetexScaffold in Well Insert Formats
44
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Protocol Booklet
Introduction:
cell expenditure.
Methods:
1.
SW480, SW620 and 3T3 cells were routinely maintained in T-75 flasks.
Figure 1. Phase contrast micrographs of SW480 cells (left), SW620 cells (middle)
and 3T3 cells (right) grown in conventional 2D culture plates. Images show cells at high
confluency. Scale bars: 200 m.
2.
For SW480 cells and SW620 cells, complete growth media consisted of: DMEM High
glucose supplemented with 10 % v/v heat inactivated FBS, 2 M L-glutamine and 100
U/ml Penicillin/Streptomycin.
3.
For 3T3 cells, complete growth media consisted of: DMEM High glucose supplemented
with 10 % v/v FBS, 2 M L-glutamine and 100 U/ml Penicillin/Streptomycin.
4.
5.
Cells were re-suspended at a concentration of 1.0 x107

cells/ml for seeding.
HPM/14/9/2012

Note: 3T3 cells are seeded on AlvetexScaffold 7 days prior to seeding SW480 cells
(or SW620 cells).
a.
AlvetexScaffold 6-well inserts in 6-well plate format were prepared for seeding by
dipping in 70 % ethanol and washed twice with media (7 ml per well).
b.
50 l of the 3T3 cell suspension was added in the centre of the AlvetexScaffold disc,
c.
The plate was incubated for a minimum of 15 minutes at 37 C with 5 % CO2 to allow
the cells to settle into the scaffold.
d.
3T3 media was added to each well to a total volume of 10 ml taking care not to
dislodge cells from AlvetexScaffold.
e.
Plates were re-incubated and maintained for a further 7 days by complete 3T3 media
exchange after every 2-3 days.
f.
After 7 days, inserts were transferred to deep well Petri dish holders in deep well Petri
dishes (AVP015) and dishes were filled from the outside of the insert with enough
SW480/SW620 medium to allow the medium to rise inside the insert and cover the
substrate, but not to go over the sides of the inserts, i.e. 50ml 5ml, as described for
feeding above and below separately in the Quick Start protocol.
g.
100 l of either the SW480 cell suspension or the SW620 cell suspension was added
in droplets over the surface of the AlvetexScaffold disc, which was equivalent to 1 x
106 cells per well.
h.
The Petri dish was incubated overnight at 37 C with 5 % CO2 to allow the cells to
settle on top of the scaffold.
i.
The following morning SW480/SW620 media was added to each Petri dish to a total
volume of 70 ml taking care not to dislodge cells from AlvetexScaffold.
j.
Petri dishes were re-incubated and maintained by complete SW480/SW620 media

exchange after every 2-3 days.
Consolidated
Protocol Booklet
45

46
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Protocol Booklet
Figure 2. Brightfield micrograph showing the structure of 3T3 cells cultured for 7 days on
22 mm diameter AlvetexScaffold discs presented in 6-well insert in 6-well plate format.
haematoxylin and eosin. Scale bar: 100 m.
Figure 3. Brightfield micrograph showing the structure of SW480 cells co-cultured for
7 days with established 3T3 cells on 22 mm diameter AlvetexScaffold discs presented in
6-well insert in 6-well plate format. Cells were fixed, embedded in paraffin wax, sectioned
(10 m) and counterstained with haematoxylin and eosin. Scale bar: 100 m.

47
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Protocol Booklet
Figure 4. Brightfield micrograph showing the structure of SW620 cells co-cultured for 7
days with established 3T3 cells on 22 mm diameter AlvetexScaffold discs presented in
6-well insert in 6-well plate format. Cells were fixed, embedded in paraffin wax, sectioned
(10 m) and counterstained with haematoxylin and eosin. Scale bar: 100 m.
01
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Protocol Booklet
White Papers
02
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Protocol Booklet
AlvetexScaffold
technology represents a
novel tool for the scientist
working in the cell culture
field by offering an
opportunity for major
advancements in cellular
organisation over
traditional 2D cultures.
Because cells in
AlvetexScaffold form
tissue-like structures,
sophisticated techniques
traditionally reserved for
the analysis of tissues
become more appropriate
for cell visualisation.
Legend: Scanning electron microscopy image of the structure AlvetexScaffold (main picture) and populated with
TERA2.cl.SP12 cells as visualised by histological staining (right)
A Review of Imaging Techniques Compatible

with Three Dimensional Culture of Cells
Grown in AlvetexScaffold
Introduction:
As scientists better understand the benefits of growing cells
in three dimensions (3D) and routinely adopt 3D culture
techniques, methods for visualising cells must also be
adapted and optimised.
The most common and routinely used technique for tracking
two dimensional (2D) cell cultures is light microscopy.
Traditional 2D monolayer cultures are highly transparent and
within a single optical plane. The minimal light diffraction and
diffusion presented by the plastic surface allows the
collection of focussed microscopic images. Cells cultured in
genuine 3D environments, such as in AlvetexScaffold
present some of the same constraints as tissue samples or
biopsies in that simple, live observation of cultures via phase
microscopy is not optimal.
There are however, other techniques that can be
implemented which will allow the user to monitor culture
progress easily and effectively in 3D; Simple dyes can be
used to identify culture confluence and viability. The variety
of end-point visualisation techniques available to those
culturing cells in 3D is extensive. Options include, but are
not limited to, live cell imaging, fluorescent marker analysis,
confocal analysis, histology using a range of cytological
stains and electron microscopy. All of these techniques have
been performed on cultures grown in AlvetexScaffold with
excellent results. Here we review common imaging methods
and outline their use and suitability for cultures grown in 3D
within the AlvetexScaffold.
Cell visualisation options within

AlvetexScaffold:
One of the simplest methods to visualise cultured cells is
phase contrast microscopy. This technique is routinely used
to observe cells grown in 2D. Cells do not have to be
sacrificed, therefore cultures can be visualised throughout
the experimental time course, allowing the scientist to
gauge culture quality and progress. This method of
visualisation, however, is not optimum for detailed analysis
of 3D cultures or sections of tissue.
Why cant I easily see cells in

AlvetexScaffold under the light
microscope?
AlvetexScaffold is a polystyrene scaffold in which voids
have an average diameter of 40m and interconnects of
average diameter 13m. AlvetexScaffold is supplied as a
200m thick membrane. It is this specific architecture that
allows extensive cell-cell contacts throughout the scaffold
and the formation of tissue-like structures. Cells within
AlvetexScaffold retain a more cuboidal morphology and 3D
cell structure particularly in the z-plane (Figure 1) and this in
turn has a significant impact on cell function.
Setting The Standard For REAL 3D Cell Culture
01
2D cell culture
03
3D cell culture
Consolidated
Protocol Booklet
Figure 1. An example of how cells retain a more in-vivo-like structure when cultured in 3D within AvetexScaffold.
It is the increased volume of cells in the z plane that hinders optimal in-focus visualisation of 3D cultured cells
under standard light micrcoscope.conditions. Data generated as part of a collaborative project between
Reinnervate and LGC Standards [1].
When viewing an unstained, unsectioned AlvetexScaffold

culture under a standard light microscope, the combined
density and thickness of the scaffold and the 3D culture
within it prevent the clear visualisation of individual cells, due
to light diffraction and its inability to penetrate into the 3D
structure. This is to be expected of true 3D cultures and
pieces of tissue, and is a well documented fact in recent
published reviews, for example see [2,3].
As a result, many groups are focussing on devising
alternative methods for cell visualisation within 3D scaffold
cultures and tissue-engineered materials (for a recent review
see [4]). Methods vary, ranging from the detection of
fluorescent labelling (for example see [5, 6]) or
autofluorescence [7], to the use of magnetic resonance
imaging to locate and track cells within the scaffold
architecture [8]. Many of these methods require expensive
equipment, although alternative methods exist to track 3D
culture progress easily and cheaply as outlined in this
document. A quick comparison of common techniques
available for imaging cells in 2D versus 3D cultures / tissues
is summarised in Table 1 (page 3).
Which techniques can be used to

visualise cells in AlvetexScaffold?
Choosing the most appropriate technique to visualise cells in
AlvetexScaffold depends on several factors. The following
sections will help in deciding which methods are best
adapted to the individual experimental situation.
If the aim is to simply check for and monitor the presence of
growing cells within AlvetexScaffold, then a number of
different techniques can be employed. Images obtained via
standard light microscopy can be enhanced by the addition
of a cellular dye which increases the contrast of cells over
the background.
Neutral Red Staining of Cultures for

Light Microscopy
Neutral Red is a common histological dye used for staining
cell nuclei (Figure 2.) It is also used widely as a cell vitality
stain. Results can be qualitative or quantitative depending
upon the method of analysis implemented.
To visualise cells cultured in AlvetexScaffold, a range of

methods are appropriate and available. These are discussed
in detail below.
SEM image of the

structure of
AlvetexScaffold (high
magnification).
SEM image showing

cells growing through
AlvetexScaffold so that
the scaffold itself is no
longer clearly visible.
AlvetexScaffold is a 200 thick

highly porous, inert polystyrene
scaffold that provides cultured
cells with an optimal
environment to grow in 3D. This
allows for the formation of 3D
niche microenvironments in
which cell-cell interaction and
communication networks occur.
The geometry and dimensions of
AlvetexScaffold have been
specifically designed to mimic
the in vivo cellular environment:
no cell is more than 100 m from
a source of nutrients and gasses.
This compares favourably to the
typical in vivo arrangement
where cells are generally no
more than 150-200 m away
from a capillary. Once seeded
onto the AlvetexScaffold,
typically cells easily invade the
scaffold and start to produce
genuine, homogeneous 3D
cellular structures that resemble
micro-slabs of tissue.
F igure 2. Cell cultures can be visualised on AlvetexScaffold by staining with the nontoxic dye Neutral Red. CHO-K1 cells were seeded onto AlvetexScaffold (AVP002) at
a density of 0.5 million cells per scaffold. After 2 days cells were exposed to 0.5 %
Neutral red dye solution (Sigma, N6264-50ML). For full experimental details refer to
Neutral red staining protocol located at www.reinnervate.com/alvetex/protocols
02
04
Comments
2D samples
3D & tissue samples
Brightfield / Phase
Contrast Microscopy
Commonly available
No cost
Easy-to-use
Routine
Best suited for 2D culture and tissue
sections
Unstained or stained samples

Live or fixed samples
Stained samples only (brightfield)

Fixed and sectioned samples
preferably
Live or un-sectioned samples may
be visible if the cells are at a
relatively low density (phase)
Standard Fluorescence
Microscopy
Generally available
Expensive
Moderate training required
Routine
Best suited for 2D culture and tissue
sections
Stained samples only


preferably
Not recommended for unsectioned samples
Confocal Laser
Scanning Microscopy
Less available
Expensive
Training required
Less routine
Suited for both 2D and 3D cultures and
tissues


obtain higher resolution images
Live or un-sectioned samples can
be visible (using reporters)
Reconstruction of 3D architecture
of thicker samples is feasible
Histology
Generally available
Inexpensive
Moderate training required
Routine
Ideally suited for 3D cultures and
tissues
Fixed and Stained samples only

Fixed samples only

Fixed and sectioned samples only
Transmission Electron
Microscopy (TEM)
Less available
Expensive
Substantial training required
Less routine
tissues
Fixed samples only
Fixed and sectioned samples only
Scanning Electron
Microscopy (SEM)
Less available
Expensive
Substantial training required
Less routine
tissues
Fixed samples only
Fixed and un-sectioned samples

only
Consolidated
Protocol Booklet
Method
Table 1. Comparison of common techniques available for imaging cells in 2D versus 3D cultures/tissues.
03
no cells
0.5 million cells
2 million cells
05
disc
Consolidated
Protocol Booklet
x4
x10
Visualisation of cells
growing in 3D is
enhanced by reagents
which produce a colour
contrast between the
cells and the scaffold:
Light Microscopy is thus
compatible with
AlvetexScaffold by
staining the cells. Cell
staining as the byproduct of a colorimetric
assay or fixation
procedure can also be
exploited for cell
visualisation and
monitoring the progress
of cultures.
x20
Figure 3. Cell cultures can be visualised on AlvetexScaffold by staining with the non-toxic dye Neutral Red. CHO-K1
cells were seeded onto AlvetexScaffold (AVP002) at a density of 0, 0.5 million and 2 million cells per scaffold. After 24
hours cells were exposed to 0.5 % Neutral Red dye solution (Sigma, N6264-50ML). Scaffolds were then transferred to a
glass microscopic slide, kept wet by adding a drop of PBS and imaged under an ICC50HD Leica microscope with LAS EZ
software (brightfield setting). For full experimental details refer to Simple staining methods for viewing cells on
AlvetexScaffold by light microscopy protocol located at www.reinnervate.com/alvetex/protocols.
The benefit of using non-toxic dyes are that they can be

administered at a range of time points throughout the
experiment, washed off and the scaffolds re-incubated with
culture media for further cell growth. (Please note; it is
recommended that if using dyes for the first time their effect
on culture growth and cell survival is checked for each cell
type used. Reinnervate recommends setting up extra
scaffolds for dye analysis). This allows users to monitor cell
survival and proliferation within the AlvetexScaffold over a
time course.
In this context, Neutral Red solution can be used as a very

quick and simple staining technique to follow culture growth
and survival within AlvetexScaffold. Note with increased cell
densities the staining intensity also increased both
macroscopically and microscopically (Figure 3).
04
Methylene Blue Staining of Cultures for

Light Microscopy
Methylene Blue is a heterocyclic aromatic chemical
compound with the molecular formula C16H18N3SCl.
Following a very quick and easy staining procedure, the dye
stains cell nuclei a blue colour which are visible under
standard light microscopy (Figure 4). Methylene Blue dye is
toxic to cells, therefore once cells have been stained, the
culture is sacrificed.
Cultures can also be visualised as an extra step during

routine chromogenic cell viability assays such as MTT. This
assay involves the conversion of a yellow, water soluble
compound, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide), a tetrazole, to a purple,
insoluble formazan, which remains within the cell until the
membranes are lysed, releasing the dye for assay detection.
At the point before cell membranes are usually lysed,
images of the purple-stained cells can be obtained (Figure 5).
While this method provides a means for imaging cells, the
MTT reagent is cytotoxic and therefore can only be used at
the experiment end point, or on surplus scaffolds.
0.5 million cells
2 million cells
disc
Figure 4. Visualisation of cells on AlvetexScaffold with Methylene Blue solution. HepG2 cells were seeded onto
AlvetexScaffold (AVP002) at a density of 0, 0.5 million and 2 million cells per scaffold. After 24 hours cells were
exposed to 0.5 % Methylene Blue dye solution (Sigma, 03978-250ML). For full experimental details refer to Simple
staining methods for viewing cells on AlvetexScaffold by light microscopy protocol located at
www.reinnervate.com/alvetex/protocols.
Figure 5: The gross location of viable cells is clearly visible on the AlvetexScaffold disc after staining
with MTT cell viability reagent. HaCat cells were cultured on AlvetexScaffold in the 12-well plate format
(AVP002) for 4 days prior to analysis (for full experimental details see separate MTT protocol available at
05
06
Consolidated
Protocol Booklet
no cells
Visualisation as a By-product of an Assay
Visualisation as a by-product of Cell Fixation

Cell fixation methods which employ coloured fixatives are also able to provide a visual estimation of cell growth in
AlvetexScaffold. Bouins reagent colours areas of cell growth yellow during fixation, which enables visual comparison of cell
growth between samples and also cellular distribution within a single sample (Figure 6A). The Bouins stain remains visible
through wax embedding and is only lost during subsequent histological staining, for example using haematoxylin and eosin.
07
Consolidated
Protocol Booklet
Figure 6. Cells can be visualised on AlvetexScaffold following fixation with the yellow coloured Bouins fixative.
SW480 colon carcinoma cells were cultured on AlvetexScaffold and removed for fixation at 4, 7, 11, 14, 18, and 21
days after seeding. The increasing number of cells over time is reflected in increasing staining intensity. A. SW480
cultures in AlvetexScaffold discs following Bouins fixation and ethanol dehydration; B. Bouins fixed and wax
embedded SW480 AlvetexScaffold cultures; C. Slide mounted cross sections (10m slices) of Bouins fixed and wax
embedded SW480 AlvetexScaffold cultures stained with haematoxylin and eosin.
Live Cell Imaging in AlvetexScaffold Using Confocal Microscopy

For a more in-depth, single cell analysis, live cell imaging, and other more involved techniques can be implemented.
Traditionally, imaging of live cells allows migrating cells to mimic wound healing or substrate invasion in vitro. In more recent
years this technique has been implemented to follow live cell cultures grown in 3D scaffolds [9], and is providing information
regarding how cell cultures interact within the 3D niche microenvironment.
Figure 7: GFP-transfected CHO-K1 cells were cultured on AlvetexScaffold 6 well inserts (AVP004) for 6 days. Captured
images were obtained every 30 minutes using a Zeiss LSM 510 confocal microscope with heated stage (for full
experimental details refer to live-cell imaging protocol located at www.reinnervate.com). In this example, a series of
integrated z-stacks is presented which shows the behaviour of a single transfected CHO-K1 cell over a period of 3 hours.
06
Fixation of Cells Within AlvetexScaffold

and Subsequent Downstream Analysis
Fluorescence Microscopy:
Immunofluorescence uses the recognition of cellular targets
by fluorescent dyes or antigen-specific antibodies coupled to
fluorophores. Depending on the antibody or dye used,
proteins, lipids and DNA can be visualised within individual
cells and tissues. AlvetexScaffold can easily be processed
like a standard tissue sample, allowing established
immunocytochemical methods to be followed with excellent
results (Figure 8).
AlvetexScaffold cell
cultures are amenable to
highly sophisticated cell
imaging techniques such
as confocal imaging.
Confocal microscopy can
be used to visualise fixed
cells or to follow living
cultures in real time.
AlvetexScaffold cultures
can be regarded as in vitro
miniature tissues. As such
standard histological
techniques can be applied
to their analysis. These
methods include
immunohistochemistry,
histological staining,
confocal microscopy, and
electron microscopy.
Figure 8. Human keratinocyte cell line (HaCaT) grown in AlvetexScaffold (7 days air exposure). The
culture was fixed and processed for paraffin wax embedding and immunohistochemical analysis by
fluorescent microscopy. The three images from the same region show; phase contrast (top), blue
fluorescent Hoescht 33258 nuclei stain (middle) and Ki67 staining (bottom). For the full
experimental details refer to Immunocytochemistry protocol located at
08
Consolidated
Protocol Booklet
All of the techniques discussed below require fixation of

cells within the AlvetexScaffold. Once cells are fixed
numerous downstream analytical techniques can be
performed. The choice of the method will depend on the
specific data required. Fixation is achieved either by chemical
means or rapid freezing with the use of a tissue support
solution such as the cryoprotective embedding solution OCT
to keep the membrane in place. For full details of
AlvetexScaffold -compatible fixation protocols refer to
Histology Series Part 1. Choosing the Right Fixative to
Preserve 3D Cell Cultures found at
www.reinnervate.com/alvetex/protocols. Given the
relatively thin nature of AlvetexScaffold compared with
typical tissue samples, fixation of cells is rapid, uniform and
efficient, preserving the 3D culture in a life-like condition.
07
Confocal Laser Scanning Microscopy:
09
Consolidated
Protocol Booklet
Confocal microscopy relies on the combination of point

illumination and a pinhole to eliminate most of the out-offocus light signal and allows for reconstruction of 3D
volumes, making it ideal to image cultures grown in fullthickness AlvetexScaffold. It should be noted that lipophilic
dyes, such as Nile Red (Figure 9), will bind strongly to the
AlvetexScaffold. However, this feature can be used to
conveniently visualise the scaffold within the cell culture. As
high-density cell cultures grown in AlvetexScaffold
approximate the complexity and structure of in vivo tissues,
fluorophores specifically developed for in vivo deep imaging
can be used to improve performance if needed.
Figure 9. Depth colour-coded Z stack of cell-free

AlvetexScaffold stained with Nile Red. Picture taken on
a Zeiss LSM 510 confocal microscope. Note the depth of
the Z-stack exceeds 150m. Scale bar 50m.
Figure 10. HepG2 cells grown for 3 days in AlvetexScaffold 12-well plate format (AVP002). Cells were stained with
Hoechst 33342 (blue), cytokeratin 8 (green) and Nile Red (red). Pictures were taken on a Zeiss LSM 510 confocal
microscope. Note the background signal from AlvetexScaffold in the blue and green channels is very low. For full
experimental details refer to Confocal protocol located at www.reinnervate.com/alvetex/protocols.
08
Histology:
Unlike other 3D cell culture supports, AlvetexScaffold can

easily be processed like a standard tissue sample, allowing
established histology protocols to be followed with excellent
results as seen in Figure 11.
Figure 11: Cells grown in AlvetexScaffold can be fixed and processed for histological
analysis using standard methods. Both staining examples are carried out on human
keratinocytes (HaCaT) grown in AlvetexScaffold for 7 days. The sample on the top
shows a culture that was fixed and processed for resin embedding (L R White resin).
Resin sections (1 m) were stained with Toluidene Blue for structural analysis by light
microscopy. The sample on the bottom shows a culture that was fixed and processed for
paraffin embedding. Sections (10 m) were stained with Haematoxylin and Eosin for
morphological analysis by light microscopy. For full experimental details refer to the
Histology Series of protocols located at www.reinnervate.com/alvetex/protocols.
10
Consolidated
Protocol Booklet
Histology is seen as the gold standard of cell visualisation in

tissues, and therefore is very suitable for 3D cell culture.
Histology is essentially the art of observing a thin section of
fixed material under either a light microscope or electron
microscope. The ability to specifically identify cellular
components can be enhanced by the addition of histological
stains. Common stains include: Haematoxylin and Eosin,
which are generally used together to visualise gross cell
architecture; Toluidine Blue stain, which is also a general cell
stain, staining most proteins; and Massons trichrome which
is a three-colour staining protocol producing red keratin and

muscle fibers, blue or green collagen and bone, light red or
pink cytoplasm, and dark brown to black cell nuclei.
09
Electron Microscopy:
11
Consolidated
Protocol Booklet
Scanning electron microscopy (SEM) is becoming a popular method of visualisation of cultures grown in 3D. SEM is a form of
electron microscopy where images are obtained by scanning samples using a high-energy beam of electrons. As the samples
are scanned the electrons interact with the sample surface, and these interactions are detected and processed, leading to
high-resolution images depicting the sample topography and composition.
AlvetexScaffold can easily be processed like a standard tissue sample, allowing established methods to be followed with
excellent results (Figure 12).
Figure 12. Detailed structure of 3D cultures can be visualised using scanning electron microscopy. Inspection of pieces
of AlvetexScaffold at low magnification shows homogeneous coverage by cultured cells (A). Higher magnification
imaging in this transverse section reveals cells growing throughout the scaffold (B). Increasingly higher magnification
micrographs reveal how cells interact with each-other and the AlvetexScaffold (C & D). See scale bar inserts. For full
experimental details refer to SEM protocol located at www.reinnervate.com/alvetex/protocols.
10
Transmission electron microscopy (TEM) can also be

performed on cell cultures grown in AlvetexScaffold (Figure
13). TEM allows the ultrastructure of cells to be visualised
due to the extreme high magnification achieved. Samples
are processed in a similar way to SEM, however, instead of
being sputter coated in gold the samples are embedded in
resin and sectioned into ultrathin sections (1 m).

Samples are loaded into the TEM and a beam of electrons
is transmitted through the sections. The electron beam
interacts with the sample architecture and it is these
interactions which are detected, processed and images
are obtained.
12
Consolidated
Protocol Booklet
Figure 13. (A) TEM image of keratinocytes cultured at air-liquid interface without collagen or fibroblasts for 14 days. The
scaffold is indicated by a black arrow. (B) TEM image showing cells progressively flattening towards the upper surface of
the culture after 14 days at air-liquid interface. (C) High magnification image showing desmosomes, indicated with white
arrows. (D) High magnification image showing bundling of keratin filaments underneath cell membrane, indicated with
white arrows. (Scale bar 500 m) For full experimental details refer to TEM protocol located at
11
Conclusions:
13
Consolidated
Protocol Booklet
As outlined throughout the document, there are many imaging techniques which can
be implemented to visualise cultures and cells grown within AlvetexScaffold in For
following culture progress, dyes stain cells contrasting them against the scaffold
background allowing visualisation via light microscopy. For more in depth culture
analysis, a range of more complex techniques can be implemented similar to those
performed on tissue samples with excellent results.
References.
1. Schutte, M., et al., Rat primary hepatocytes show
enhanced performance and sensitivity to acetaminophen
during three-dimensional culture on a polystyrene
scaffold designed for routine use. Assay Drug Dev
Technol, 2011. 9(5): p. 475-86.
2. Smith, L.E., R. Smallwood, and S. Macneil, A comparison
of imaging methodologies for 3D tissue engineering.
Microsc Res Tech, 2010. 73(12): p. 1123-33.
6. Liu, E., et al., Quantitative biorelevant profiling of

material microstructure within 3D porous scaffolds via
multiphoton fluorescence microscopy. J Biomed Mater
Res B Appl Biomater, 2007. 82(2): p. 284-97.
7. Dittmar, R., et al., Assessment of cell viability in 3D
scaffolds using cellular auto-fluorescence. Tissue Eng
Part C Methods, 2011.
3. Graf, B.W. and S.A. Boppart, Imaging and analysis of

three-dimensional cell culture models. Methods Mol Biol,
2010. 591: p. 211-27.
8. Lalande, C., et al., Magnetic resonance imaging tracking

of human adipose derived stromal cells within threedimensional scaffolds for bone tissue engineering. Eur
Cell Mater, 2011. 21: p. 341-54.
4. Freimark, D., F. Ehlicke, and P. Czermak, The need for

imaging methods in bioengineering of three-dimensional
cell cultures. Int J Artif Organs, 2010. 33(4): p. 193-7.
9. Gantenbein-Ritter, B., et al., Confocal imaging protocols

for live/dead staining in three-dimensional carriers.
Methods Mol Biol, 2011. 740: p. 127-40.
5. Thevenot, P., et al., Method to analyze three-dimensional

cell distribution and infiltration in degradable scaffolds.
Tissue Eng Part C Methods, 2008. 14(4): p. 319-31.
149
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2. 3D Cell Culture Set Up

3. Use of Coating Reagents with AlvetexScaffold

4. Cell Visualisation & Monitoring Cell Attachment/Growth

5. Histology of Cell Cultures Grown on AlvetexScaffold

6. Methods for Molecular Analysis of Cells Cultured in AlvetexScaffold

8. Specialised Applications using AlvetexScaffold

9. Example Protocols for the Growth of Specific Cell Types on AlvetexScaffold

Retrieval of Cells from AlvetexScaffold

10. White Papers

Schutte et al (2011). Rat primary hepatocytes show enhanced performance and

Neofytou et al (2011). Adipose tissue-derived stem cells display a proangiogenic

Rajan et al (2011). Dysregulated TRK signalling is a therapeutic target in CYLD

Maltman and Przyborski (2010). Developments in three dimensional cell culture

Bokhari et al (2007). Culture of HepG2 liver cells on three-dimensional polystyrene

Bokhari et al (2007). Emulsion templated porous polymers as scaffolds for

Carnachan et al (2006). Tailoring the morphology of emulsion-templated porous

10. Barbetta et al (2005). Porous polymers by emulsion templating. Macromolecular

2 x Well Insert Holder

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Technology for Routine Three

What is 3D cell culture?

Choosing the right AlvetexScaffold format

AlvetexScaffold is available in a variety of formats, each specifically designed for different

Choosing the right AlvetexScaffold format

Histology (10 m sections)

Choosing the right AlvetexScaffold format

All procedures concerning the handling of AlvetexScaffold should be performed wearing

3D Cell Culture Set Up

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Use of AlvetexScaffold in 24-Well Plate Format (AVP006)

Figure 1. Presentation of AlvetexScaffold in 24-well plate format.

Add approximately 1 ml of 70 % EtOH to each well to pre-treat the

Similarly to 2D culture, if using serum-free medium, consider the use of

The 24-well plate format is a simple presentation of AlvetexScaffold

3D Cell Culture Using the AlvetexScaffold 24-Well Format:

3D cell culture is different to conventional 2D cell culture and as such requires

Use of AlvetexScaffold in 12-Well

Figure 1. Presentation of AlvetexScaffold in 12-well plate format.

AlvetexScaffold 12-well plate format

Use of AlvetexScaffold in 12-Well

Use of AlvetexScaffold in well insert formats

Figure 1. Presentation of AlvetexScaffold in 6- and 12-well formats.

AlvetexScaffold 6-well insert (AVP004-3) and 12-well insert (AVP005-3)

Use of AlvetexScaffold in well insert formats

ii. Media from

Figure 2. Media filling levels and well insert configurations.

Use of AlvetexScaffold in well insert formats

12-well insert in 6-well plate:

6-well insert in 6-well plate:

Use of AlvetexScaffold in well insert formats

6-well insert in a 6-well plate

3.5 0.5 ml/well

12-well insert in a 6 well plate

3.5 0.5 ml/well

12-well insert in a 12-well plate

1.6 0.2 ml/well

2.4 0.2 ml/well

3.8 0.2 ml/well

Use of Well Insert Holder in

Use of Well Insert Holder in