Organoid Culture Handbook

Organoid Culture
Handbook
Matrices | Cells | Reagents
Table of Contents
Introduction to Organoid Culture ..................................................................................... 3
Organoid Models................................................................................................................ 4
Featured Products for Organoid Culture ........................................................................... 5
Extracellular Matrices................................................................................................ 5
BME (Basement Membrane Extract) for Organoid Culture............................. 5
Collagen I ......................................................................................................... 7
Reagents.................................................................................................................... 7
R-spondin (RSPO1) Expressing Cell Line.......................................................... 7
Proteins and Antibodies ................................................................................. 8
R-spondin (RSPO1) Purified Proteins ....................................................... 8
Wnt (Wnt3A) Proteins .............................................................................. 9
Lgr5 (Gastric Stem Cell Marker) Antibodies............................................. 10
Organoid Harvesting Solution ........................................................................ 10
Organoid Culture Protocols .............................................................................................. 11
General Submerged Method for Organoid Culture ................................................ 11
Crypt organoid culture techniques.......................................................................... 11
Air Liquid interface (ALI) Method for Organoid Culture ........................................ 12
Clonal Organoids from lgr5+ cells ............................................................................ 13
Brain and Retina Organoid Formation...................................................................... 14
Organoid Culture Examples............................................................................................... 15
Liver Organoid Culture............................................................................................. 15
Human Liver..................................................................................................... 15
Hepatocellular Carcinoma (HCC) .................................................................... 16
Mouse Liver...................................................................................................... 17
Gastrointestinal Organoid Culture........................................................................... 17
Small Intestine Organoids................................................................................ 17
Mouse Small Intestine ..................................................................................... 18
Human Colorectal ............................................................................................ 19
Mouse Adenoma ............................................................................................. 20
Mouse Intestinal- Colorectal Cancer Model ................................................... 20
Transgenic Mouse ............................................................................................ 20
Esophageal and Stomach Organoid Culture ..................... ...................................... 21
Esophageal Tumor ........................................................................................... 21
Esophageal Barrett’s Epithelial ........................................................................ 21
Esophageal Normal ......................................................................................... 22
Breast Organoid Culture........................................................................................... 23
Prostate Organoid Culture ....................................................................................... 23
Metastatic Prostate Cancer-Derived Organoids............................................... 23
Harvested Organoids ....................................................................................... 24
Organoids from Mouse Prostate Stem Cells.................................................... 24
Pancreas Organoid Culture....................................................................................... 25
Female Reproductive System Organoid Culture...................................................... 25
Fallopian Tube ................................................................................................ 25
Ovary .............................................................................................................. 25
Uterus (Endometrium) ................................................................................... 26
Brain Organoids ....................................................................................................... 27
Retina Organoids ..................................................................................................... 27
Experience Summary ....................................................................................................... 28
Organoid Citations ........................................................................................................... 29
Page| 2 Organoid Culture Handbook
Introduction to Organoid Culture
Organoids are organ-like structures that can be formed by 3D cell culture and differentiation of stem cells or
organ progenitors; and are capable of recapitulating aspects of organ function in vitro. Research & therapeutic
potential of organoids includes:
99 Organogenesis Models
99 Drug Testing
99 Tumor, Disease and Infection Models
99 Toxicity Screening
99 Personalized Medicine
99 Regenerative Medicine / Organ Replacement
Preclinical models are falling short in predicting biological responses because plastic is not a natural
component and mouse models are not representative of the human body. These models fail to
recreate the complexity and the specificity of living tissues.
To recreate In vivo structure and function the following factors are necessary to consider:
In vivo structure and function: Factors to consider Organoid Culture Recreates In Vivo Structure and Function
Tissue Resident Cells (Types, Quantities, Organoid Progenitor Cells (Differentiate into tissue-specific
Organization) cells)
Extracellular Matrix (Composition, Extracellular Matrix (Basement Membrane Extract (BME))

Organization, Compliance)
Soluble Factors (Growth Factors, Cytokines) Soluble Factors (Wnt, Noggin, EGF, R-Spondin-1, Tissue-
Specific Factors)
www.amsbio.com | info@amsbio.com Page | 3

Organoid Models
Organ Images and Results Citations and

Protocols (pg 28, 29)
Brain Pg 27 19, 20, 22, 23
Retina Pg 27 21
Esophagus Pg 21, 22 7
Lungs Successfully grown in
BME 2 RGF & BME R1
Breast Pg 23 14
Liver Pg 15, 16, 17 3, 8, 11
Pancreas Pg 25 3, 7, 10, 15, 16
Stomach Pg 22 15
Small Intestine Pg 17, 18 1, 2, 6, 9, 11, 15, 18
Colon Pg 19, 20 4, 6, 7, 12, 13, 17
Fallopian Tubes Pg 25
Ovary Pg 25
Uterus (Endometrium) Pg 26 5
Prostate Pg 23, 24
Featured Products for Organoid Culture
Extracellular Matrices
BME (Basement Membrane Extract) for Organoid Culture
99 Soluble form of basement membrane from Engelbreth-Holm-Swarm (EHS) tumor.
99 Major components include Laminin I, Collagen IV, Entactin & Heparan Sulfate Proteoglycan.
99 Gels at 37°C to form a reconstituted basement membrane
99 Higher concentration (14 -16 mg/ml)
99 Bigger Batches
99 Formulated for the Specific Task
99 Organoid qualified Matrix
BME can be used in a multiple applications, under a variety of cell culture conditions, for maintaining growth
or promoting differentiation of primary endothelial, epithelial, smooth muscle and stem cells. BME can also
be utilized in cell attachment, neurite outgrowth, angiogenesis, in vitro cell invasion and in vivo tumorigenicity
assays.
Name Catalog Number Pack size Buffer Tensile Recomended Applications

Strength
BME 2 Reduced Growth Factor (Organoid Matrix)
Cultrex® BME 2 (Reduced 3533-001-02 1ml DMEM High Reduced growth factor
Growth Factor Basement format optimized for robust
Membrane Extract, Type 2, 3533-005-02 5ml Organoid growth
Pathclear®)
3533-010-02 2 x 5ml
BME R1 Reduced Growth Factor
Cultrex® BME R1 (Reduced 3433-005-R1 5ml DMEM Higher Organoid qualified (batch
Growth Factor Basement tested for organoids)
Membrane Extract, Type suggested to use for difficult
R1) to grow Organoid cultures
BME 2 provides a proprietary formulation that is higher in tensile strength when compared to our original BME,
making it more physiologically aligned with the in vivo tumor environment. It is difficult to know which matrix
formulation is best suited for any specific organoids, cell type, or application. However in competitive beta
tests, BME 2 consistently outperforms competitor products. Each BME lot is qualified on Human Small Intestine
Organoids.
The Growth Factor Reduced format (BME 2 RGF “organoid matrix”) has been shown to work well for growing
organoids, especially using techniques based on the LGR5 stem cell marker / Wnt signalling system, pioneered by
Hans Clevers and co-workers.
• Each lot is validated for use in organoid culture

• Used extensively for organoid culture
• Reduced lot to lot variability
• Easy to use

Table
Recently of Contents
we have developed an additional formulation of Cultrex® BME known as Cultrex® BME R1. This matrix
provides a proprietary formulation that has higher tensile strength when compared to our other products Cultrex®
BME, Cultrex® BME 2 and Cultrex® BME 3. It has a higher concentration of entactin, one of the BME components
that connects laminins and collagens reinforcing the hydrogel structure. Cultrex® BME R1 has been specifically
designed to culture tissue organoids and is recommended for “difficult to grow” Organoid cultures.
BME-R1 Improves Take for Difficult to Culture Organoids
BME-2 BME-R1 BME-R1

BME-R1
Human Gastric
Organoids p9.
BME-R1 is Enriched in Entactin
BME-R1 BME-2
Batch 1
Batch 2
Batch 3
Batch 1
Batch 2
Batch 3
kDa
10
210
9
Percent Entactin (Densitometry)
8
111 7
6
5
4
3
71 2
1
0
55
BME-R1
Entactin Rich BME BME-2
41
Data provided by Organoid Resource Lab (ORL), Trevigen, Inc

Collagen I
Type I collagen is the major structural component of extracellular matrices found in connective tissue and
internal organs, but is most prevalent in dermis, tendons, and bone. It promotes cell attachment, proliferation,
differentiation, migration, and tissue morphogenesis. Cultrex® Rat Collagen I, Lower Viscosity is diluted to a lower
concentration (3 mg/ml); therefore it is less viscous and easier to handle.
Name Catalog Number Pack size

3443-003-01 1 ml @ 3 mg/ml
Cultrex® Rat Collagen I, Lower Viscosity
3443-100-01 35 ml @ 3 mg/ml
Cultrex® 3-D Collagen I (From Rat Tail Tendons) 3447-020-01 20 ml at 5mg/ml
Our 5 mg/ml rat collagen I (eg cat# 3447-020-01) is not pepsin treated, so it has intact telopeptides and is highly
viscous. It is provided in acetic acid and requires pH neutralization prior to use. Do not to attempt to pipet this
material using a graduated 1 ml pipette or narrow bore tip. Rather, we recommend using blue pipette tips (200-
1000uL capacity) that are clipped with sterile scissors to create a wide bore. Also, please consult the provided
product data sheet for pH neutralization instructions. We also offer 3 mg/ml Cultrex® Rat Collagen I, Lower
Viscosity (cat#s 3443-003-01 & 3443-100-01).
Our 3D Collagen has been tested extensively for the ability to promote growth and differentiation of cell types,
visualized by morphology in three dimensions in vitro (however, addition of laminin may be required as shown,
for MCF-10A cells, in the product data sheet).
Reagents
R-spondin (RSPO1) Expressing Cell Line
Roof plate-specific Spondin-1 (R-Spondin 1 or RSPO1), also known as CRISTIN3, is a 27 kDa secreted activator
protein that belongs to the R-Spondin family. R-Spondins positively regulate Wnt/beta-catenin signaling, most
likely by acting as a ligand for LGR4-6 receptors and an inhibitor for ZNRF3. R-Spondin-1 induces proliferation of
intestinal crypt epithelial cells, increases intestinal epithelial healing, and supports intestinal epithelial stem cell
renewal. The 293T cell line is stably transfected to express murine Rspo1 with an N-terminal HA epitope tag and
fused to a C-terminal murine IgG2a Fc fragment. This cell line is used to produce either purified Rspo1 or Rspo1
conditioned media. The murine Rspo1 protein has been used extensively in organoid culture to maintain Lgr5+
stem cells, and the FC and HA tags make it easy to purify or characterize.
Images provided by Organoid Resource Lab (ORL), Trevigen, Inc
Production of R-Spondin1 for organoid culture. The 293T cell line is stably transfected to express murine Rspo1
with an N-terminal HA epitope tag and fused to a C-terminal murine IgG2a Fc fragment. A) The HA-R-Spondin1-
Fc 293T cell line is cultured with zeocin to select for stably transfected cells. B) Production of HA-R-Spondin1-Fc
is characterized using Western Blot for R-Spondin1 protein (arrow). C) HA-R-Spondin1-Fc induces activation of
Wnt/ß-catenin response when evaluated using the Top-Flash Luciferase assay.
BENEFITS
• Cell line expresses recombinant mouse RSPO1 protein
• Positively regulates Wnt/beta-catenin signaling
• Essential medium component for most organoid culture models
• Purified protein and conditioned medium from our cell line has been used for culturing both human and mouse
organoids.
Name Catalog Number size

Cultrex® R-spondin1 (RSPO1) Cells 3710-001-01 1 vial (0.5 ml), 1x106 cells
Proteins and Antibodies

The Wnt family of secreted glycoproteins is involved in several important cell functions such as cell proliferation,
migration, polarity, survival, self renwal and cell fate. Among the most studied is a canonical Wnt signaling pathway
that regulates quantity of transcriptional co-activator β-catenin and in turn β-catenin then regulates expression
of the key developmental genes. In adition Wnt signals play an important role in the ability of organoids to
expand. In particular Wnt3a and RSPO1 (which acts as an Lgr receptor agonist) are two important factors used for
organoid expansion. AMSBIO offers a range of RSPO1 and Wnt3a recombinant human and mouse proteins. These
human recombinant proteins are purified from HEK293 cells while the mouse proteins are expressed in CHO cells.
Both are suitable for various cell assays and treatments.
R-spondin (RSPO1) Purified Proteins

Human R-Spondin 1 / RSPO1 Protein AMS.RS1-H4221-50UG 50 μg
Human R-Spondin 1 / RSPO1 Protein AMS.RS1-H4221-1MG 1 mg
Mouse R-Spondin 1 / RSPO1 Protein AMS.RS1-M5220-50UG 50 μg
Mouse R-Spondin 1 / RSPO1 Protein AMS.RS1-M5220-1MG 1 mg
R-spondin Comparative Results
300
Rspo (Peprotech)
Rspo (Amsbio)
No. of organoids
200
Organoid counts of small intestine and
gastric (pyloric and corpus) organoids
100
at 4 days in culture using RSPO1 from
AMSBIO and competitor.
0
-1 -1 -1 -1 -1 -1
small pylorus corpus
intestine
Data courtesy of Dr Nick

Barker, A*STAR Institute of
Medical Biology, Singapore

Wnt (Wnt3A) Proteins
Human Recombinant Wnt3a (75% purity) AMS.rhW3aL-002 2 μg
Human Recombinant Wnt3a (75% purity) AMS.rhW3aL-010 10 μg
Human Recombinant Wnt3a (85-90% purity) AMS.rhW3aH-002 2 μg
Human Recombinant Wnt3a (85-90% purity) AMS.rhW3aH-010 10 μg
Mouse Recombinant Wnt3a (75% purity) AMS.rmW3aL-002 2 μg
Mouse Recombinant Wnt3a (75% purity) AMS.rmW3aL-010 10 μg
Mouse Recombinant Wnt3a (85-90% purity) AMS.rmW3aH-002 2 μg
Mouse Recombinant Wnt3a (85-90% purity) AMS.rmW3aH-010 10 μg
Titration of Wnt Conditioned Medium with the TOPFlash assay.

A) Transfected HEK293 cells were exposed to different concentrations of recombinant murine Wnt3a (Time
Bioscience), from 100 to 0 ng/ml diluted in Advanced DMEM/F12 with Glutamine. B) Two different preparations
of L Wnt3a Conditioned Medium (produced with ATCC® CRL- 2647™) were diluted 1 to 2 in Advanced DMEM/
F12 with Glutamine. The dose curve presented in panel A serves as a reference to compare the activity of Wnt3A
conditioned media.
A Purified mWnt3A B L Wnt3A Conditioned Medium

35 35
Relative Luciferase Units
Relative Luciferase Units

30 30
25 25
20 20
15 15
10 10
5 5
0 0
0 1 5 25 50 100 0 Lot# 1 Lot# 2
[mWnt3A] ng/ml
Data provided by Organoid Resource Lab (ORL), Trevigen, Inc
Lipidure®-Coated Plates
Lipidure®-Coated plates are a top of the range solution for spheroid and embryoid body formation. Lipidure®-
coating of U or V-shaped wells provides a superior low-attachment surface for the formation of a single spheroid
or embryoid body in each well. Embryoid bodies can then be differentiated into organoids, see protocol on pg 14
and citations 19-22 (pg 30)
Name Catalog Number

Lipidure-Coat Low Adhesion Plate A-U96 (96 well U-bottom plate) AMS.LCP-A-U96
Lipidure-Coat Low Adhesion Plate A-V96 (96 well V-bottom plate) AMS.LCP-A-V96

Lgr5
Table(Gastric Stem Cell Marker) Antibodies
of Contents
Name Species Reactivity Catalog Number size
LGR5 mouse monoclonal antibody,clone UMAB212 Human UM800104 100 μl
LGR5 mouse monoclonal antibody,clone UMAB210 Human, Mouse UM800102 100 μl
LGR5 mouse monoclonal antibody,clone UMAB211 Human UM800103 100 μl
LGR5 mouse monoclonal antibody, clone OTI2A2 Human, Mouse TA503316 100 μl
LGR5 mouse monoclonal antibody, clone OTI7F2
Human, Mouse TA808752 100 μl
(formerly 7F2)
LGR5 mouse monoclonal antibody, clone OTI3F1
Human, Mouse TA808748 100 μl
(formerly 3F1)
Organoid Harvesting Solution

Organoid cultures exhibit cellular behaviors and PROTOCOL
morphologies similar to those seen in vivo. However,
the adaptation of these models for studying
biochemical processes has been impeded by the
challenge of separating intact organoids from extra-
cellular proteins comprising the hydrogel. Commonly,
proteases are employed to degrade these extracellular
proteins, however, proteases also degrade proteins
on the cell surface and protease activity may carry
over into subsequent cultures or lysate preparations.
Trevigen’s Cultrex® Organoid Harvesting Solution
provides a ready-to-use, non-enzymatic method
for depolymerizing extracellular matrix proteins to
allow for harvesting of intact organoids for passaging,
cryopreservation, or biochemical analysis.
BENEFITS
• Ready-to-use
• Non-enzymatic chelating solution
• Depolymerizes basement membrane matrix for
harvesting organoids from culture.
• Gentle for cells: preserves original morphology
APPLICATIONS
• Organoid passaging
• Sample preparation (PCR, Western Blot, and
Immunohistochemistry)
RESULTS
• See example results on page 22.

Cultrex® Organoid Harvesting Solution 3700-100-01 100 ml

Organoid Culture Protocols
General Submerged Method for Organoid Culture

Developed by the Hans Clevers Lab, Hubrecht Institute, Netherlands
Citation:
Sato, T., R. G. Vries, et al. (2009). “Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal
niche.” Nature 459(7244): 262-265.
Add Culture Medium

Centrifuge cells. Containing:
Such as: Cultrex® Organoid HA-R-spondin1-FC (from
Progenitor Cells; Mouse Small 3710-001-01) Add cold (4 °C)
Intestine (3750-001-01) Organoid Harvesting
Solution (3700-100-01) –
30 min with gentle rocking
Add 50 µl to a 24 Organoid Aspirate BME

well plate growth Organoid depolymerizes
Culture Medium leaving intact
Re-suspend cells in and wash with organoids
organoid appropriate ECM: cold (4 °C) PBS
1- BME 2 RGF (3533-010-02)
2- BME R1 (3433-005-R1)
Crypt Organoid Culture Techniques

Crypt Isolation

Organoids from Crypts
Air Liquid interface (ALI) Method for Organoid Culture

Developed by the Calvin Kuo Lab, Stanford University, USA
Citation:
– Ootani, A., X. Li, et al. (2009). “Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell
niche.” Nat Med 15(6): 701-706.
Organoids from Tissue

ALI Method Introduction to Organoid Culture
Clonal Organoids from lgr5+ cells

Brain and Retina Organoid Formation

Organoid Culture Examples
Liver Organoid Culture
Human Liver
Marker Expression in Human Liver Organoids on
BME 2 RGF.
Image courtesy of Helmuth Gehart/Professor
Hans Clevers, Hubrecht Institute, Utrecht,
Netherlands
In their paper in Cell, Huch, Clevers et al (2015)

demonstrated that:
• Primary human bile duct cells can readily be
expanded into 3D liver organoids in vitro using
BME 2 RGF
• Adult liver stem cells maintain self-renewal
capacity, differentiate into functional hepatocytes
in vitro and generate bona fide hepatocytes upon
in vivo transplantation.
• Expanded cells preserve their genetic integrity
over months in culture (agreeing with the authors’
previous observations in a mouse model).
• Organoids derived from patients with genetic
disorders can be used to model liver disease in
vitro.
Differential interference contrast image of human organoids grown in BME 2 RGF and cultured for >2
months in human liver complete medium. Magnification, 4X.
Image courtesy of Meritxell Huch, Gurdon Institute, University of Cambridge, UK
Long-term culture of Human Liver Organoids on BME 2 RGF. (Clonal cultures obtained by seeding sorted cells at
one cell per well)
Image courtesy of Meritxell Huch, Gurdon Institute, University of Cambridge, UK

Marker Expression in Human Liver Organoids on BME 2 RGF.
Table of Contents Confocal image stained for ECAD (green) and the hepatocyte
marker HNF4 (red); nuclei counter-stained with Hoechst
(Blue).
Image courtesy of Dr. Meritxell Huch, Gurdon Institute,
University of Cambridge, UK
“We have obtained culture conditions that

allow us to long-term expand genetically
stable human donor liver cells in organoid
culture. One of the clues to this success
is the use of ECM that allows the cells to
grow in 3D. BME 2 has been our ECM of
choice for these experiments.”
Dr. Meritxell Huch (Gurdon Institute,
University of Cambridge, UK)
Differentiation of Organoids into

Hepatocytes on BME 2 RGF.
Expression of hepatocyte genes
in human liver organoid after 11
days on differentiation medium.
Immunofluorescence for albumin
(ALB, red) and ZO-1 (green); nuclei
counterstained with Hoechst (Blue)
Image courtesy of Dr. Meritxell
Huch, Gurdon Institute, University of
Cambridge, UK
Hepatocellular Carcinoma (HCC)
Hepatocellular carcinoma (HCC) organoid model,

grown in BME 2 RGF.
Image courtesy of Prof. Dr. med. Markus Heim,
Hepatology Group, Department of Biomedicine,
University Hospital Basel, Basel, Switzerland.

Mouse Liver
GFP-labeled organoids, grown from mouse liver stem

cells. grown on BME 2 RGF.
The cells were infected with a lentivirus expressing GFP
Image courtesy of Dr. Derk ten Berge, Erasmus MC
Stem Cell Institute, Rotterdam, Netherlands
Intestinal Organoid Culture
Small Intestine Organoids
Small intestine organoids with DNA in blue

and structural proteins in red, grown on
Cultrex BME R1, reduced growth factor.
Images courtesy of J Wosen, E Mellins Lab,
Stanford University, USA.

Table of Contents
Differentiation of Mouse Small Intestine Organoids Cultured on RGF BME R1. During expansion, Mouse Small
Intestine Organoids grow as spherical structures (A), but under differentiating conditions, crypt-like structures
bud from the organoid (B), mimicking intestinal epithelium. Nuclei were stained using DAPI (blue), and E-cadherin
was visualized via immunofluorescence (green).
A B
Mouse Small Intestine Organoids cultured on RFG BME R1

Images provided by Organoid Resource Lab (ORL), Trevigen, Inc

Human Colorectal Introduction to Organoid Culture
Human Colorectal Cancer (CRC)

organoids grown from single cells
on BME 2 RGF.
Immunofluorescence for Phalloidin
(red) to mark actin filaments; and
E- cadherin (green) as epithelial
marker; nuclei counterstained with
DAPI (blue)
Images courtesy of the Batlle Lab,
IRB Barcelona, Spain
BF IF
Human Colorectal Cancer (CRC) organoids

grown for 7 days in BME 2 RGF.
Brightfield (BF) and Immunofluorescence
for Phalloidin (red) to mark actin filaments;
and laminin (green); nuclei counterstained
with DAPI (blue). Laminin stains basement
membrane and mediates the attachment,
migration and organization of cells.
Images courtesy of the Batlle Lab, IRB
Barcelona, Spain

Mouse
Table Adenoma
of Contents
Images courtesy of the Batlle Lab, IRB Barcelona, Spain
Mouse Intestine - Colorectal Cancer Model
WILD TYPE APC-DELETED

Intestinal organoids derived from normal and Apc-deleted mice. (Loss of the negative Wnt pathway regulator
APC occurs in the majority of colorectal cancers.)
Images courtesy of the Li Lab, Francis Crick Institute, London, UK
Transgenic Mouse
Organoids (passage 1) derived directly into BME 2 Existing organoid culture transferred to BME 2 RGF.
RGF. Organoids were split once (using mechanical Organoids were digested to single cells, which were
disruption) following derivation; and the image was plated onto BME 2 RGF. The image was taken 6 days
taken 3 days later. after plating.
Organoids prepared from the intestinal crypts of transgenic mice.
Images courtesy of the Sansom Lab, Beatson Institute of Cancer Research, Glasgow, UK
Esophageal & StomachIntroduction
OrganoidtoCulture
Organoid Culture
The images below show Organoids grown from tumors derived from fresh
tissue samples. These images are showing cells taken from the lowest part of
the esophagus almost into the stomach.
Esophageal Tumor
Images courtesy of Dr Mathew Garnett, Sanger Wellcome Trust Institute, Cambridge, UK
Esophageal Barrett’s Epithelial
Barrett’s esophagus is a precancerous condition where the esophageal lining becomes similar to the tissue
architecture of the intestine, it can develop following long term cases of gastro-esophageal reflux disease (GERD).

Table of Contents
Esophageal Normal
Stomach
Organoid counts of gastric corpus organoids at 4

days in culture in BME (BME 1, BME 2 and BME
R1 and competitor matrix).
Data courtesy of Dr Nick Barker, A*STAR Institute

of Medical Biology, Singapore

Breast Organoid Culture
Mammary cancer organoid from

mice, grown on / invading into BME
2 RGF.
Image courtesy of Tuula Kallunki,
Danish Cancer Society Research
Centre, Copenhagen, Denmark
Prostate Organoid Culture
Metastatic Prostate Cancer-Derived Organoids
BME 2 (RGF)
Confocal images of
metastatic prostate
cancer (mCRPC)-lymph
node biopsy grown
derived organoids grown
side-by-side on either
BME 2 (RGF) or BME R1
(RGF) for comparison.
Images courtesy of Dr BME R1 (RGF)

Veronica Gil, De Bono
Lab, ICR, Sutton, UK

Table of Contents
Harvested Organoids
These images on the left belong to same tumor biopsy-derived organoids

from the pictures above where growth and morphology was compared
between BME 2 and BME R1. These images are post-first passage using
Organoid Harvesting Solution and replating them.
The viability was very good and organoid proliferation rate has not been
affected by the solution and replating procedure, showing that the
reagent is effective.
Images courtesy of Dr Veronica Gil, De Bono Lab, ICR, Sutton, UK
Organoids from Mouse Prostate Stem Cells

Brightfield Alexa 488 (YFP)
Images courtesy of the Baena lab, Prostate Oncobiology, CRUK Manchester Institute, Manchester, UK
YFP-labeled organoids, grown from mouse prostate stem cells, grown on BME 2 RGF.
The prostate cells (from Rosa26 -LstopL-YFP mouse) were infected with Adenovirus expressing Cre to activate
YFP expression.

Pancreas Organoid Culture
Human pancreatic organoids

growing in BME 2 matrix, 14 days
after the isolation of ducts from
the human pancreas.
Image courtesy of Dr. Meritxell
Huch, Gurdon Institute,
University of Cambridge
BME 2 (RGF)
Female Reproductive System Organoid Culture

Fallopian Tube
20 day culture of normal human fallopian

tube organoids, grown in BME R1
Image courtesy of Dr Debbie Sanders, Brenton lab,

CRUK Cambridge Institute, Cambridge, UK
Ovary
2 day culture of high grade serous ovarian cancer

(HGSOC) organoids, grown in BME R1
Image courtesy of Dr Maria Vias (Brenton lab,

CRUK Cambridge Institute, Cambridge, UK

Uterus
Table (Endometrium)
of Contents
Bright field images (left and middle) and DAPI stained (right) images of the organoids isolated from Type 1
endometrial cancer patient specimens grouwn on Cultrex BME 2 RGF (Organoid Matrix) Pathclear. Passage 2
Organoids were used in the study.
BME 2 (RGF)
Below are confocal images of the Organoids isolated from Type 1 endometrial cancer patient specimens
grown on Cultrex BME2 RGF (Organoid Matrix) Pathclear. These Organoids (passage 2) were treated with
either DMSO (control) or Verteporfin (VP) at 10nM for 3h and stained with YAP or Caspase-3 antibodies.
BME 2 (RGF)
Data courtesy of Radhika Gogoi Lab, Weis Center for Research: from Oncotarget paper Verteporfin exhibits
YAP-independent anti-proliferative and cytotoxic effects in endometrial cancer cells, reproduced under a
Creative Commons Attribution 3.0 License

Brain Organoid Culture
(A) Single iPSC-derived Embryoid Body (EB) generated in Lipidure-Coat Plate

A-U96 (96 well U-bottom plate).
(B) iPSC-derived EBs transferred from the Lipidure Plate after a 6 day culture
period in Lipidure-Coat Plate A-U96 as above.
A B
Images courtesy of Dr Julia Ladewig, Neural Development Group, Institute of Reconstructive Neurobiology,
LIFE & BRAIN Center, University of Bonn, Germany
Retina Organoid Culture

(A) Bright field image of retinal organoid taken at Day (D) 7 when the organoids
are still in the Lipidure-coated plate and eyefield induction has occurred. (B)
Immunohistochemistry evaluation of a mature D21 retina organoid section
showing the layered structure of the organoid. The sample was stained for
photoreceptor marker Recoverin (red), amacrine and ganglion cell marker Pax6
(green) and DAPI (blue).
Lipidure-coated 96-well U-bottom
Images courtesy of Manuela

Völkner and Dr. Mike
Karl, German Center for
Neurodegenerative Diseases
(DZNE), Dresden, Germany
A B
Experience Summary
Baena lab, Prostate Oncobiology
CRUK Manchester Institute, Manchester, UK ǀ pg 24
Batlle Lab
IRB Barcelona, Spain | pg 19, 20 | Citation 4
Nick Barker Lab

A*STAR Institute of Medical Biology, Singapore | pg 8
Brenton Lab
CRUK Cambridge Institute, Cambridge, UK | Dr Maria Vias pg 25| Dr Debbie Sanders pg 25
Professor Hans Clevers

Hubrecht Institute, Utrecht, Netherlands | Helmuth Gehart pg 15 | Citations 2, 6, 8, 9, 12, 13
De Bono Lab
ICR, Sutton, UK | Dr Veronica Gil pg 23, 24
Dr Mathew Garnett
Sanger Wellcome Trust Institute, Cambridge, UK | pg 21, 22 | Citations 7, 12
Radhika Gogoi Lab

Weis Center for Research, Geisinger Medical Center, Danville, PA, USA | pg 26 | Citation 5
Prof. Dr. med. Markus Heim

Hepatology Group, Department of Biomedicine, University Hospital Basel, Basel, Switzerland | pg 16
Dr. Meritxell Huch

Gurdon Institute, University of Cambridge, UK | pg 15, 16, 25 | Citations 3, 8
Tuula Kallunki
Danish Cancer Society Research Centre, Copenhagen, Denmark | pg 23
Dr Mike Karl
German Center for Neurodegenerative Diseases (DZNE), Dresden, Germany | Manuela Völkner pg 27 | Citation 21
Dr Julia Ladewig
Neural Development Group, Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn,
Germany | pg 27 | Citation 19
Li Lab,
Francis Crick Institute, London, UK | pg 20
E Mellins Lab
Stanford University, USA | J Wosen pg 17
Sansom Lab
Beatson Institute of Cancer Research, Glasgow, UK | pg 17
Dr. Derk ten Berge

Erasmus MC Stem Cell Institute, Rotterdam, Netherlands | pg 17 | Citation 11
Trevigen, Inc, Organoid Resource Lab (ORL)

Trevigen, Inc., Gaithersburg MD, USA | pg 6, 7, 9, 18

Organoid Citations
BME 2 RGF
1. Andersson-Rolf, A., Fink, J., Mustata, R. C., & Koo, B. K. (2014). A video protocol of retroviral infection in primary intestinal
organoid culture. Journal of visualized experiments: JoVE, 90, 51765.
2. Basak, O., Beumer, J., Wiebrands, K., Seno, H., van Oudenaarden, A., & Clevers, H. (2017). Induced Quiescence of Lgr5+
Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells. Cell Stem Cell, 20,
1-14.
3. Broutier, L., Andersson-Rolf, A., Hindley, C. J., Boj, S. F., Clevers, H., Koo, B. K., & Huch, M. (2016). Culture and
establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.
Nature Protocols, 11(9), 1724-1743.
4. Cortina, C., Turon, G., Stork, D., Hernando‐Momblona, X., Sevillano, M., Aguilera, M., ... & Batlle, E. (2017). A genome
editing approach to study cancer stem cells in human tumors. EMBO Molecular Medicine, e201707550.
5. Dasari, V. R., Mazack, V., Feng, W., Nash, J., Carey, D. J., & Gogoi, R. (2017). Verteporfin exhibits YAP-independent anti-
proliferative and cytotoxic effects in endometrial cancer cells. Oncotarget. Also cites 3D Culture Cell Harvesting Kit: 3448-020-K
6. Drost, J., Van Jaarsveld, R. H., Ponsioen, B., Zimberlin, C., Van Boxtel, R., Buijs, A., ... Korving, J. … & Clevers, H. (2015).
Sequential cancer mutations in cultured human intestinal stem cells. Nature, 521(7550), 43-47.
7. Francies, H. E., Barthorpe, A., McLaren-Douglas, A., Barendt, W. J., & Garnett, M. J. (2016). Drug Sensitivity Assays of
Human Cancer Organoid Cultures. Methods in molecular biology (Clifton, NJ)
8. Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M. M., ... & Clevers, H. (2014) Long-Term Culture
of Genome-Stable Bipotent Stem Cells from Adult Human Liver. Cell , 160, 299 – 312
9. Schwank, G., & Clevers, H. (2016). CRISPR/Cas9-Mediated Genome Editing of Mouse Small Intestinal Organoids.
Gastrointestinal Physiology and Diseases: Methods and Protocols, 3-11.
10. Steinhart, Z., Pavlovic, Z., Chandrashekhar, M., Hart, T., Wang, X., Zhang, X., ... Sidhu, S., Moffat, J., and Angers, S.
(2016). Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant
pancreatic tumors. Nature Medicine 23, 60–68 doi:10.1038/nm.4219doi:10.1038/nm.4219
11. Tüysüz, N., Van Bloois, L., Van Den Brink, S., Begthel, H., Verstegen, M. M., Cruz, L. J., ... & Braakman, E. (2017). Lipid-
mediated Wnt protein stabilization enables serum-free culture of human organ stem cells. Nature Communications, 8,
14578.
12. van de Wetering, M., Francies, H.E., Francis, J.M., Bounova, G., Iorio, F., Pronk, A., M., Garnett M.J., & Clevers, H. (2015)
Prospective derivation of a ‘Living Organoid Biobank’ of colorectal cancer patients. Cell 161(4), 933-945
13. Verissimo, C. S., Overmeer, R. M., Ponsioen, B., Drost, J., Mertens, S., Verlaan-Klink, I., ... Clevers, H., and Snippert, H.J.
(2016). Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening. eLife, 5,
e18489.
BME 1 RGF
14. Morrison, C. D., Allington, T. M., Thompson, C. L., Gilmore, H. L., Chang, J. C., Keri, R. A., & Schiemann, W. P. (2016). c-Abl
inhibits breast cancer tumorigenesis through reactivation of p53-mediated p21 expression. Oncotarget, 7(45), 72777.
Also cites 3D Culture Cell Harvesting Kit: 3448-020-K
Cultrex ® Rat Collagen I

15. Li, X., Ootani, A., & Kuo, C. (2016). An Air–Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary
Gastrointestinal Tissues. Gastrointestinal Physiology and Diseases: Methods and Protocols, 33-40. (Citing our 3443-003-01 -
Cultrex® Rat Collagen I, Lower Viscosity)
16. Panciera, T., Azzolin, L., Fujimura, A., Di Biagio, D., Frasson, C., Bresolin, S., ... & Piccolo, S. (2016). Induction of Expandable
Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ. Cell Stem Cell, 19(6), 725-737. (Citing our
3447-020-01 - 3-D Collagen I Rat Tail)
Organoid Citations
A-R-Spondin1-Fc Cell Line

17. Fan, Y. Y., Davidson, L. A., & Chapkin, R. S. (2016). Murine Colonic Organoid Culture System and Downstream Assay
Applications. Methods in molecular biology (Clifton, NJ).
Cultrex Organoid Harvesting Solution

18. Noel, G., Baetz, N. W., Staab, J. F., Donowitz, M., Kovbasnjuk, O., Pasetti, M. F., & Zachos, N. C. (2017). A primary human
macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions. Scientific
Reports, 7. doi: 10.1038/srep45270.
Lipidure-coated 96-well U-bottom

19. Iefremova, V., Manikakis, G., Krefft, O., Jabali, A., Weynans, K., Wilkens, R., ... & Ladewig, J. (2017). An Organoid-Based
Model of Cortical Development Identifies Non-Cell-Autonomous Defects in Wnt Signaling Contributing to Miller-Dieker
Syndrome. Cell Reports, 19(1), 50-59.
20. Nguyen, D. T. T., Richter, D., Michel, G., Mitschka, S., Kolanus, W., Cuevas, E., & Wulczyn, F. G. (2017). The ubiquitin
ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation. Cell
Death & Differentiation.
21. Völkner, M., Zschätzsch, M., Rostovskaya, M., Overall, R. W., Busskamp, V., Anastassiadis, K., & Karl, M. O. (2016).
Retinal organoids from pluripotent stem cells efficiently recapitulate retinogenesis. Stem cell reports, 6(4), 525-538.
Lipidure-coated 96-well V-bottom

22. Abud, E. M., Ramirez, R. N., Martinez, E. S., Healy, L. M., Nguyen, C. H., Newman, S. A., ... & Caraway, C. A. (2017). iPSC-
Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron, 94(2), 278-293.
Cell Banker 2 Cryopreservation Solution

23. Bagley, J. A., Reumann, D., Bian, S., Lévi-Strauss, J., & Knoblich, J. A. (2017). Fused cerebral organoids model interactions
between brain regions. Nature Methods.
Published Protocols that have been used successfully with

BME 2 RGF matrix for organoid growth
24. Procedure for subculturing normal human gastric organoids, derived from the submerged method as described in
Barker, N., et al Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro. Cell Stem
Cell 6(1), 25–36
25. Huch, M., Dorrell, C., Boj, S. F., van Es, J. H., Li, V. S., van de Wetering, M., ... & Haft, A. (2013) ). In vitro expansion of
single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature, 494(7436), 247-250
26. Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., ... & Clevers, H. (2009) Single Lgr5
stem cells build crypt villus structures in vitro without a mesenchymal niche. Nature, 459(7244), 262-265

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Extracellular Matrix (Composition, Extracellular Matrix (Basement Membrane Extract (BME))

www.amsbio.com | info@amsbio.com Page | 3

Organ Images and Results Citations and

Name Catalog Number Pack size Buffer Tensile Recomended Applications

• Each lot is validated for use in organoid culture

www.amsbio.com | info@amsbio.com Page | 5

BME-R1 Improves Take for Difficult to Culture Organoids

BME-2 BME-R1 BME-R1

BME-R1 is Enriched in Entactin

Data provided by Organoid Resource Lab (ORL), Trevigen, Inc

Page| 6 Organoid Culture Handbook

Name Catalog Number Pack size

Images provided by Organoid Resource Lab (ORL), Trevigen, Inc

Name Catalog Number size

Proteins and Antibodies

R-spondin (RSPO1) Purified Proteins

R-spondin Comparative Results

Data courtesy of Dr Nick

Page| 8 Organoid Culture Handbook

Titration of Wnt Conditioned Medium with the TOPFlash assay.

A Purified mWnt3A B L Wnt3A Conditioned Medium

Relative Luciferase Units

Data provided by Organoid Resource Lab (ORL), Trevigen, Inc

Name Catalog Number

www.amsbio.com | info@amsbio.com Page | 9

Organoid Harvesting Solution

Name Catalog Number size

Page| 10 Organoid Culture Handbook

General Submerged Method for Organoid Culture

Add Culture Medium

Add 50 µl to a 24 Organoid Aspirate BME

Crypt Organoid Culture Techniques

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Air Liquid interface (ALI) Method for Organoid Culture

Organoids from Tissue

Page| 12 Organoid Culture Handbook

Clonal Organoids from lgr5+ cells

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Page| 14 Organoid Culture Handbook

Liver Organoid Culture

In their paper in Cell, Huch, Clevers et al (2015)

www.amsbio.com | info@amsbio.com Page | 15

“We have obtained culture conditions that

Differentiation of Organoids into

Hepatocellular Carcinoma (HCC)

Hepatocellular carcinoma (HCC) organoid model,

Page| 16 Organoid Culture Handbook

GFP-labeled organoids, grown from mouse liver stem

Intestinal Organoid Culture

Small Intestine Organoids

Small intestine organoids with DNA in blue

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Mouse Small Intestine Organoids cultured on RFG BME R1

Page| 18 Organoid Culture Handbook

Human Colorectal Cancer (CRC)

Human Colorectal Cancer (CRC) organoids

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Images courtesy of the Batlle Lab, IRB Barcelona, Spain

Mouse Intestine - Colorectal Cancer Model

WILD TYPE APC-DELETED

Images courtesy of Dr Mathew Garnett, Sanger Wellcome Trust Institute, Cambridge, UK

Esophageal Barrett’s Epithelial

Images courtesy of Dr Mathew Garnett, Sanger Wellcome Trust Institute, Cambridge, UK

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