Biofiles

Volume 5, Number 4

Cryopreservation
New Products

Cell Freezing Media and Reagents

ECACC Cell Lines

Protocol for the Cryopreseravtion of

Cell Lines

Cryoware
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Biofilescontents
Introduction 3
New Products 4
CryoStor™ 4
HypoThermosol® 5
Sericin 6
Cell Freezing Media and Reagents 8
ECACC Cell Lines 11
Protocol for the Cryopreservation
of Cell Lines 14
Cryoware 16
Cryovial Color Coders 16
Cryo Containers 16
Cryo Storage Racks 17
Cryo Vials 18
Benchtop Coolers 19
Cryo Accessories 19
Aldrich® wide-mouth dewar flasks 19
Barnstead/Thermolyne® Dewars,
Biocane™ Series 20
Barnstead/Thermolyne® Dewars,
Locator® Series 21
Nalgene® Dewars 21
Taylor-Wharton Dewars 21
Taylor-Wharton Dewar Accessories 22
Wide-mouth Dewars 22
Wide-mouth Dewars with
Silvered Glass Liner 22
Technical content: Don Finley, M. Sc.
Order sigma.com/order Technical service sigma.com/techinfo
Introduction
Don Finley
Market Segment Manager, Cell Culture
don.finley@sial.com
Freezing living cells with the hope of full
recovery began with the freezing of sperm
in glycerol in 1949.1 Later DMSO was
shown to be a better preservative as it can
better permeate cells like red blood cells.2
Cryopreservation for all practical purposes
is limited to individual cells or small clumps
of cells. So preserving mammalian cells,
plant cells, blood, sperm, and embryos is
routine practice for clinicians and researchers.
DMSO has proven to be the most robust
cryopreservative agent and the most widely
used. DMSO has some drawbacks in that
it is cytotoxic and given that it permeates
cells, it is difficult to completely eliminate.
There are a number of non-permeating
cryoprotectants that have also proven
effective such as hydroxyethyl starch,
PVP and CMC. Typically non-permeating
cryoprotectants are not as efficient at
preserving cell viability and are often used
in concert with DMSO to enhance the
efficiency of cryoprotection.
There are two common methods of
cryopreservation, controlled rate freezing and
vitrification. Controlled rate freezing typically
involves placing the cells in a cryoprotectant
media and cooling at a rate of –1 to –3 °C
per minute, to about –80 °C and finally
storing at –196 °C in liquid nitrogen. In this
method, ice crystals form outside of cells
first, and the higher concentrations of solutes
that are excluded from ice crystals draw
water from the cells and prevent or minimize
freezing inside the cells. Controlled rate
freezing is the most commonly practiced
sigma.com/lifescience 3
method, as it has proven to be robust and
widely applicable. However, the osmotic
changes and ice crystals formed using this
technique can lead to reduced recovery and
cryopreservation-induced delayed-onset cell
death. Vitrification involves rapid freezing in
higher concentration of cryoprotectants. The
higher concentrations of cryoprotectants
and rapid freezing prevent the formation of
crystalline ice and promote the formation
of an amorphous ice or glass. Unfortunately,
vitrification is not without some problems
as near toxic concentrations of DMSO are
often required to promote the formation of a
glass. Furthermore at this time the conditions
required to achieve vitrification vary from
application to application.
Sigma offers a wide selection of equipment
and reagents for your cryopreservation
needs that include DMSO, prepared
freezing media and Mr. Frosty Freezers. In
addition, we recenty introduced CryoStor™
a completely optimizable serum free
cryopreservation system, HypoThermosol®,
a highly protective media for storage of
cells and tissues at 2–8 °C and will soon
offer sericin, a protein from silkworm
that has proved to be a valuable FBS
replacement in cryopreservation.
References
(1) R
 evival of spermatozoa after vitrification and
dehydration at low temperatures. C. Polge, et al.,
Nature 164 (1949) 666–667.
(2) P
 revention of Freezing Damage to Living Cells by
Dimethyl Sulphoxide. Lovelock, J. E. and Bishop, M. W.
H. Nature, 183: 1394–1395 (1959).
4

New Products
CryoStor™

New Products
CryoStor™
The CryoStor line of serum free
cryoprotective products allow higher
recovery rates than using media with 10%
FBS and DMSO (Figure 1). CryoStor allows
researchers to optimize, and if necessary,
minimize the amount of DMSO needed
(Figure 2). The CryoStor line is serum-free so
concerns of viruses, BSE or other FBS/protein-
based contaminations are eliminated. In
addition, CryoStor is robust and has been
tested with the following cell types.
 CS5
CryoStor™ cell cryopreservation media
Cryostor CS5 is formulated to contain 5%
CryoStor, a series of cell-specific, optimized dimethyl sulfoxide (DMSO). Recommended for
preservation media, is uniquely formulated to cryopreservation of most cell types.
address the molecular and biological aspects of
cells during the cryopreservation process thereby C2999-100ML 100 mL
directly reducing the level of Cryopreservation-
 CS10
Induced Delayed-Onset Cell Death and improving
post-thaw cell viability and function. CryoStor™ CS10 is a uniquely formulated
store at: 2–8 °C
cryopreservation medium containing 10%
dimethyl sulfoxide (DMSO). Recommended for the
 CS2 preservation of hepatocytes, tissue samples and
CryoStor CS2 is formulated to contain 2% dimethyl other extremely sensitive cell types.
sulfoxide (DMSO). Suggested when reducing
DMSO is of primary concern. C2874-100ML 100 mL
C3124-100ML 100 mL

• Liver Cells (Hepatocytes & C3A)

Cardiomyocytes

• Stem Cells Hybridomas Effect of Cryopreservation Vehicle Solution on Cell Viability Figure 1. Viability of NHDF
cells following exposure to cell
• PBMC Pancreatic Islets
120
culture media + 5% DMSO or
Cell Viability (Relative to 37C Control)

CryoStor + 5% DMSO (CS5).

• Cord Blood Skeletal Muscle Cells (SKMC)
100
Solution efficacy was investigat-
80 ed following a 10 minute hold at
• T-Cells Smooth Muscle Cells (CASMC) 60
2–8 °C , following ice-nucleation,
and after LN2 storage. For each
• Lymphocytes PBSC 40
of the test conditions, samples
were removed, thawed, diluted
• Coronary Artery Endothelial Cells (CAEC) 20
with media and plated. Relative
cell viability was determined
Chondrocytes 1 day post-thaw and overall
0

• Cancer Cells (PC3, LNCaP) CHO Control

(37 °C)
2–8 °C Hold Ice-
Nucleation
LN2
Storage
2–8 °C Hold Ice-
Nucleation
LN2
Storage
viability was compared to
37 °C non-cryopreserved

• Skeletal Myoblasts C2C12 Media + 5% DMSO CryoStor™ CS5 control cultures.

• Neuronal Cells Ocular (BCE, HCE)

• Keratinocytes (A431) Engineered Tissue Effect of DMSO Concentration on Post-Thaw Cell Viability Figure 2. Post-thaw viability of
• Renal Cells (RPTEC, MDCK) Organ / Tissue 100 cells following cryopreservation
Cell Viability (Relative to 37C Control)

with varying concentrations

• Fibroblasts (NHDF, 3T3) Vascular Tissue 80 of DMSO in either cell culture
media or CryoStor. Relative
• Transfected Cells Hair Follicle 60 cell viability was determined
1 day post-thaw as described
• HEK Dendritic Cells 40 in the methods and overall
viability was compared to 37 °C
20 non-cryopreserved control
CryoStor is ideal for 0
cultures. DMSO concentration
and vehicle solution impact cell
cryopreservation applications
5% DMSO

5% DMSO

5% DMSO

5% DMSO

5% DMSO

5% DMSO
10% DMSO

10% DMSO

10% DMSO

10% DMSO

10% DMSO

10% DMSO
2% DMSO

2% DMSO

2% DMSO

2% DMSO

2% DMSO

2% DMSO

viability post-thaw.

that must be serum free and Culture Media CryoStor™ Culture Media CryoStor™ Culture Media CryoStor™

where high recovery is critical. NHDF mDC CHO

 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 5

HypoThermosol®
Recovery of Human Mesenchymal Stem Cells Figure 1. Cells were assayed
Following Hypothermic Storage at 2–8 °C for either 1 day or 5 days for metabolic activity
HypoThermosol®-FRS 100 following 24 hours recovery
Preservation Solution 90 post-preservation.

Cell Viability (Relative to Control)

HypoThermosol is an optimized hypothermic 80

(2–8 °C) preservation media that enables improved 70

and extended preservation of cells, tissues and 60

organs. HypoThermosol-FRS is uniquely formulated 50

to address the molecular-biological response of 40

cells during the hypothermic preservation process. 30

HypoThermosol-FRS includes key ions at 20

concentrations that balance the intracellular 10

state at hypothermic temperatures. Additional 0

HTS-FRS Celsior Viaspan HTS-FRS Celsior Viaspan
components include pH buffers, energy substrates,
1 Day Storage 5 Day Storage
free radical scavengers, and osmotic/oncotic
stabilizers.
store at 2–8 °C
H4416-100ML 100 mL
Recovery of Human Dermal Fibroblasts Following Figure 2. Cells were assayed
Hypothermic Storage at 2–8 °C for either 1 day or 3 days for metabolic activity
Figures 1 and 2 clearly show that 110
following 24 hours
HypoThermosol outperforms commonly 100
recovery post-preservation.
Cell Viability (Relative to Control)

90
used hypothermic preservation media on 80

Human Mesenchymal Stem Cells (hMSC) and 70

on Human Dermal Fibroblasts. In addition 60

50
to these cell lines, hypothermosol has been 40

tested on the following cell lines, Tissues 30

20
and organs: 10

• Liver Cells (Hepatocytes & C3A) 0

HTS-FRS Celsior Viaspan HTS-FRS Celsior Viaspan

Cardiomyocytes 1 Day Storage 3 Day Storage

• Stem Cells Hybridomas

• PBMC Pancreatic Islets
• Cord Blood Skeletal Muscle Cells (SKMC)
• T-Cells Smooth Muscle Cells (CASMC)
• Lymphocytes PBSC
• Coronary Artery Endothelial Cells (CAEC)
Chondrocytes
• Cancer Cells (PC3, LNCaP) CHO
• Skeletal Myoblasts C2C12
• Neuronal Cells Ocular (BCE, HCE)
• Keratinocytes (A431) Engineered tissue
• Renal Cells (RPTEC, MDCK) Organ / Tissue
• Fibroblasts (NHDF, 3T3) Vascular Tissue
• Transfected Cells Hair Follicle
• HEK Dendritic Cells
6

Sericin Effect of Sericin on Cell Cryopreservation Figure 1. P3U1 myeloma cells

100
Sericin is a protein derived from the (1×106) in 1 mL of freezing
medium were frozen at –80 °C
silkworm cocoon that has a number of * for 1 day. The cryovials were then
beneficial characteristics for culturing 75
* thawed and the viability was
determined. Freezing medium
mammalian cells. It has cryoprotective 1. FBS supplemented with 10%

Viability(%)
properties that allow for replacement of DMSO; freezing medium
FBS in cryopreservation media1. Figures 1, 50 2. Freezing medium 3
supplemented with 1%
2 and 3 clearly show that 1% sericin (w/v) sericin; freezing medium
along with 0.5% (w/v) maltose, 0.3% (w/v) 3. PBS supplemented with 0.5%
25
maltose, 0.3% proline, 0.3%
proline, 0.3% (w/v) glutamine and 10% glutamine and 10% DMSO;
DMSO is comparable to 90% FBS and 10% freezing medium
0 4. PBS supplemented with 10%
DMSO. Furthermore, sericin can act as a FBS 1 2 3 4 DMSO. The error bars indicate
replacement in cell culture and stimulate Freezing media S.D. (n=3). *P<0.05.
cell growth2,3 and in certain situations4,
inhibit cell death.
Sericin Sigma Cat. No. S5201 Coming Soon!
Check sigma-aldrich.com for availability.

Comparison of Freezing Media

(a) Figure 2. P3U1 myeloma (a), hybridoma (b) or CHO (c) cells (1 × 106) in 1 mL of
freezing medium were frozen at –80 °C for 1 day. The vials were then thawed and the
100 viability of the cells was either determined immediately (a, c) or the vials were frozen
* and thawed again before the viability was determined (b). Freezing medium
* 1. FBS supplemented with 10% DMSO; freezing medium
75 * 2. PBS supplemented with 1% sericin, 0.5% maltose, 0.3% proline, 0.3% glutamine and
10% DMSO; freezing medium
3. Competitor A
Viability(%

4. Competitor B
50 5. Competitor C
6. PBS supplemented with 10% DMSO. The error bars indicate S.D. (n=3). *P<0.05.

25

0
1 2 3 4 5 6
Freezing media
(c)
(b)
*
100
*
*
* 100
* * *
75
Viability(%)

75
Viability(%)

50
50

25
25

0 0
1 2 3 4 5 6 1 2 3 4 5 6
Freezing media Freezing media
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 7

References
Effect of Freezing Media on Various Cells
(1) Development of a novel serum-free freezing medium
for mammalian cells using the silk protein sericin. Figure 3. PC12, NHDF, HEK or insect
Sasaki, M., et al., Biotechnol. Appl. Biochem. 42, 100 Sf-9 cells (1×106) in 1 ml of freezing
183–188 (2005). medium were frozen at –80 °C for
(2) Sericin, a protein derived from silkworms, accelerates 1 day. The vials were then thawed

Viability (%)
the proliferation of several mammalian cell lines includ- and the viability of the cells was
ing a hybridoma. Terada, S., et al., Cytotechnology. Nov; determined. Freezing media: FBS
40(1–3):3–12, (2002). supplemented with 10% DMSO
70 (red bars); PBS supplemented with
(3) Sericin enhances attachment of cultured human
skin fibroblasts. Tsubouchi, K., et al., Biosci. Biotechnol. 1% sericin, 0.5% maltose, 0.3% pro-
Biochem., Feb; 69(2):403–5, (2005). line, 0.3% glutamine and 10% DMSO
(4) The silk protein, sericin, protects against cell death (tan bars); and PBS supplemented
caused by acute serum deprivation in insect cell with 10% DMSO (gray bars).
culture. Takahashi, M., Biotechnol. Lett., Nov; 25(21): 40 The error bars indicate S.D. (n=3).
1805–9, (2003).
PC-12 NHDF HEK Sf -9 *P<0.05.

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8

Cell Freezing Media and Reagents

Cell Freezing Media and Reagents

Dimethyl sulf­oxide of electrolytes, pH changes, and denaturation of D-(+)-Trehalose dihydrate
DMSO; Methyl sulf­oxide proteins. These lethal effects are minimized by
O α-D-Gluco­pyran­osyl- HO
[67‑68‑5] (CH3)2SO FW 78.13 S adding a cryoprotective agent which lowers the
H3C CH3 α-D-gluco­pyrano­side;
freezing point and allows for a slower cooling rate. α,α-Trehalose [6138‑23‑4] OH
O

mp..................................................................................................... 16 to 19 °C BPC-tested DMSO also meets the requirements C12H22O11 · 2H2O FW 378.33 OH OH • 2H2O
vd..........................................................................................................2.7 (vs air) of biotechnology and tissue engineering HO

bp.................................................................................................................189 °C applications. O
OH
vp.....................................................................................0.42 mmHg (20 °C)
Features and Benefits OH
O
density.............................................................................................. 1.10 g/mL OH

ait.................................................................................................................. 573 °F
Ready-to-use sterile filtered product conveniently
n20/D........................................................................................................... 1.479
packaged septum screw-capped amber bottles.  cell culture tested, insect cell culture tested, ≥99%
Meets USP and EP testing requirements
lel(63 °F).........................................................................................................42% Use as a cryoprotectant in a variety of cell freezing
(USP XXIV) media.
 ≥99.7%, Hybri-Max™, sterile-filtered,
hybridoma tested T0167-10G
D2438-5X10ML 5 × 10 mL 10 g
DMSO is a polar aprotic solvent used in chemical D2438-50ML 50 mL T0167-25G 25 g
reactions, in polymerase chain reactions (PCR) T0167-100G 100 g
and as a cryoprotectant vitrification agent for the Gly­cerol
preservation of cells, tissues and organs. DMSO is Sucrose
1,2,3-Propane­triol; Gly­cer­in OH
used in cell freezing media to protect cells from [56‑81‑5] HOCH2CH(OH)CH2OH HO OH α-D-Gluco­pyran­osyl HO
HO
ice crystal induced mechanical injury. It is used FW 92.09 β-D-fructo­furan­o­side; O O
OH HO
for frozen storage of primary, sub-cultured, and α-D-Glc-(1→2)-β-D-Fru;
Glycerol is used both in sample preparation HO O
recombinant heteroploid and hybridoma cell lines; D(+)-Saccharose; Sugar; OH OH OH
and gel formation for polyacrylamide gel
embryonic stem cells (ESC), and hematopoietic β-D-Fructo­furan­osyl-α-D-
electrophoresis. Glycerol (5–10%) increases the gluco­pyrano­side [57‑50‑1]
stem cells. DMSO is frequently used in the density of a sample so that the sample will layer
combinations with BSA or fetal bovine serum (FBS). C12H22O11 FW 342.30
at the bottom of a gel’s sample well. Glycerol is
5 mL and 10 mL in flame sealed ampules, 100 mL also used to aid in casting gradient gels and as a mp...............................................................................................185 to 187 °C
in amber bottle protein stabilizer and storage buffer component.  cell culture tested, insect cell culture tested,
endotoxin................................................................................................tested mp.................................................................................................................. 20 °C ≥99.5%
vd..........................................................................................................3.1 (vs air) Use to create sucrose gradients for purification of
D2650-5X5ML 5 × 5 mL bp....................................................................................... 182 °C/20 mmHg viruses and proteins.
D2650-5X10ML 5 × 10 mL vp........................................................................................<1 mmHg (20 °C)
D2650-100ML 100 mL density.............................................................................................. 1.25 g/mL S1888-500G 500 g
ait.................................................................................................................. 698 °F S1888-1KG 1 kg
 Biotechnology Performance Certified, sterile-
filtered, hybridoma tested, meets EP, USP testing n20/D........................................................................................................... 1.474 S1888-5KG 5 kg
specifications  cell culture tested, insect cell culture tested,  meets USP testing specifications
Human and animal cell lines grown in culture ~99% (GC)
are generally stored frozen. Freezing protects the G2025-100ML 100 mL S3929-1KG 1 kg
cell line from changes due to genetic drift and G2025-500ML 500 mL S3929-5KG 5 kg
minimizes risk of contamination. Liquid nitrogen S3929-10KG 10 kg
used in conjunction with a cryoprotective
 insect cell culture tested
agent such as DMSO is a widely used method
for preserving cells. Without the presence of S9031
a cryoprotective agent, freezing is lethal to
most mammalian cells. Damage is caused by
mechanical injury by ice crystals, concentration
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 9

d-Sorbitol d-(+)-Malto­se monohydrate  Mr ~100,000

d-Glucitol OH May contain greater parts of lower
Malto­biose; 4-O-α-D- HO HO
[50‑70‑4] C6H14O6 FW 182.17 molecular weight.
H OH Gluco­pyran­osyl-d-glucose O O OH • H2O
HO H OH 09184-10G-F 10 g
[6363‑53‑7] C12H22O11 · H2O OH
H OH
H OH
FW 360.31 OH OH
O
OH 09184-50G-F 50 g
09184-250G-F 250 g
OH
 cell culture tested, insect cell culture tested, ≥98%
Glucose.........................................................................................≤0.3 mol %  DEXTRAN 500
May be used for washing spheroplasts1 and in
isoelectric focusing to minimize endoosmotic flow Maltotriose.................................................................................................≤1% Mr ~500,000
in agarose gels2. May be used to induce osmotic M5895-500G 500 g 31392-10G 10 g
stress. M5895-1KG 1 kg 31392-50G 50 g
mp.................................................................................................. 98 to 100 °C 31392-250G 250 g
vp.................................................................................... <0.1 mmHg (25 °C) Hydroxy­ethyl starch
vd..........................................................................................................<1 (vs air) [9005‑27‑0]  Mr 15,000–25,000
OR
 ≥99.5% (HPLC) O R = H or *
OH 31387-25G 25 g
Allelic variation of the Tas1r3 gene affects OR 31387-100G 100 g
O
behavioral taste responses to this sugar alcohol, OR 31387-500G 500 g
n
suggesting that it is a T1R3 receptor ligand.
Degree of substitution; Average of 1 -OH group per  Mr ~40,000
A plasmolyticum that reversibly inhibits the 10 glucose units converted to -OCH2CH2OH.
phenylalanine ammonia-lyase activity of narrow molecular weight distribution
Prepared from corn starch
protoplasts at concentrations exceeding 50 mM1; 68084-25G 25 g
Synchronization of Plasmodium falciparum H6382-250G 250 g 68084-100G 100 g
erythrocytic stages in culture2; Increases fungal H6382-1KG 1 kg 68084-500G 500 g
α-amylase thermostability3.
insoluble matter............................................................passes filter test Dextran from Leuconostoc spp. Hydroxy­ectoine
Lit cited: 1. B. Fritig, Phytochemistry 16, 895 (1977); 2. C. [9004‑54‑0] (C6H10O5)n (4S,5S)-5-Hydroxy-2-methyl-1,4,5,6- O OH
Lambros, J.P. Vanderberg, J. Parisitol. 65, 418 (1979); 3. M. tetra­hydro­pyrimi­dine-4-carboxy­lic
 Mr ~6,000 HO
Graber, D. Combes, Enzyme Microb. Technol. 11, 673 (1989) acid; THP(A) N

31388-25G 25 g [165542‑15‑4] C6H10N2O3 FW 158.16 N

H
CH3
85529-250G 250 g 31388-100G 100 g
85529-1KG 1 kg 31388-500G 500 g  ≥98.0% (HPLC)
85529-5KG 5 kg
Stabilizing effect against various stress factors;
 DEXTRAN 40 used e.g. in the expression of proteins1,2
d-Lactose monohydrate
Mr ~40,000
Lit cited: 1. J.R. Blackwell, R. Hogan, FEBS Lett. 295,
β-D-Gal-(1→4)-D-Glc; Milk sugar; HO
31389-25G 25 g
10 (1991); 2. K. Lippert, E.A. Galinski, Appl. Microbiol.
4-O-β-D-Galacto­pyran­osyl-D- O Biotechnol. 37, 61 (1994)
HO OH 31389-100G 100 g
glucose
HO OO
OH 31389-500G 500 g 55243-100MG 100 mg
[64044‑51‑5] C12H22O11 · H2O OH
OH
FW 360.31 • H2O
OH  DEXTRAN 70  ≥95% (HPLC)
Mr ~70,000 Stabilizer for enzymes and biological
 ≥99.5% (HPLC)
Enhances immunoprecipitation sensitivity1 macromolecules1,2,3
Sales restrictions may apply
Lit cited: 1. M.-F. Dumon et al., Electrophoresis 11, Lit cited: 1. K. Lippert, E.A. Galinski, Appl. Microbiol.
Component of the liquid stationary phase in the Biotechnol. 37, 61 (1992); 2. T. Sauer et al., GIT Labor-
846 (1990)
GLC separation of nitroxylene and xylenol isomers1 Fachzeit. 39, 892, 894 (1995); 3. M.D. Vangala et al.,
insoluble matter............................................................passes filter test 31390-25G 25 g
Bioforum 23, 760 (2000)
31390-100G 100 g
Lit cited: 1. A. Ono, J. Chromatogr. 197, 251 (1980);
31390-500G 500 g 70709-1G-F 1 g
61339-25G 25 g
70709-10G-F 10 g
61339-100G 100 g
70709-100G-F 100 g
61339-250G 250 g
10

Ectoine Betaine Cell Freezing Medium-DMSO Serum free 1×

Thp(B); (S)-2-Methyl-1,4,5,6-tetra­ O OH Oxyneurine; (Carboxy­methyl) O  sterile-filtered, cell culture tested

CH3
hydro­pyrimi­dine-4-carboxy­lic acid tri­methyl­ammo­nium inner salt H3C N O- Complete ready-to-use medium containing
[96702‑03‑3] C6H10N2O2 FW 142.16 N [107‑43‑7] (CH3)3N+CH2COO- CH3 Minimum Essential Medium, dimethyl sulfoxide
N
H
CH3 FW 117.15 (8.7%) and methyl cellulose. Prepared according to
 anhydrous, ≥98.0% (NT) the formulation of Waymouth1.
An osmoprotectant in a wide variety of water...............................................................................................................≤1%
microorganisms including heterotrophic, halophilic Recommended for the cryopreservation of cells.
mp..................................................................................301 to 305 °C (dec.)
bacteria and non-halophilic bacteria such as endotoxin................................................................................................tested
Streptomyces species and E. coli. It also exerts 14290
Lit cited: 1. Waymouth, C. and Varnum, D., Simple
protective effects in E. coli during drying and Poly­vinyl­pyrrolidone freezing procedure for storage in serum-free media
storage. of cultured and tumor cells of mouse. TCA Manual
PVP; Poly­vidone; Povidone [9003‑39‑8]
E2271-25MG 25 mg (C6H9NO)n N O (1976), 2(1), 311–313
E2271-100MG 100 mg H
H store at −20 °C
n

Polyvinylpyrrolidone is a component of Denhardt′s C6295-50ML 50 mL

 ≥99.0% (HPLC)
Solution and is included at a concentration of 1%
44788
(w/v) in the standard 50X stock solution.
Cell Freezing Medium-Glycerol 1×
 ≥95.0% (HPLC)  mouse embryo tested
average mol. wt. 40,000  sterile-filtered, cell culture tested, endotoxin
Stabilizer for enzymes and biological tested
macromolecules1,2,3 P0930-50G 50 g
Complete ready-to-use medium. Proprietary
P0930-100G 100 g
Lit cited: 1. K. Lippert, E.A. Galinski, Appl. Microbiol. formulation containing Minimum Essential
Biotechnol. 37, 61 (1992); 2. T. Sauer et al., GIT Labor- Medium, glycerol, calf serum and fetal bovine
Fachzeit. 39, 892, 894 (1995); 3. M.D. Vangala et al., serum.
Cell Freezing Medium-DMSO 1×
Bioforum 23, 760 (2000) Recommended for the cryopreservation of cells.
81619-1G-F 1 g  sterile-filtered, cell culture tested store at −20 °C
81619-10G-F 10 g Complete ready-to-use medium. Proprietary
C6039-50ML 50 mL
81619-100G-F 100 g formulation with Minimum Essential Medium,
dimethyl sulfoxide, calf serum and fetal bovine Cell Freezing Medium-Serum-free 1×
serum.
Recommended for the cryopreservation of cells.  sterile-filtered, cell culture tested
endotoxin................................................................................................tested Ready-to-use formula that does not contain
store at −20°C dimethyl sulfoxide or serum.
C6164-50ML 50 mL For use with cells grown in serum-free conditions.
store at 2–8 °C
C6164-6X50ML 6 × 50 mL
C2639-50ML 50 mL
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 11

ECACC Cell Lines

ECACC Cell Lines

Sigma® and ECACC have partnered to bring expand the offering to up to 1500 cell lines and Canada. The following is a sampling of
you a great selection of high quality cell lines. later this year. Check our website often for the products that Sigma can offer
We are expanding our offering of stocked cell updates. Sigma now supplies ECACC cell lines from ECACC.
lines to the list below in the U.S. and plan to to Europe, China, Australia, New Zealand, U.S.

ECACC Cell Lines

Cat. No. Package Size Cell Line Name Cell Line Description Species
01051619 1 vial 1301 Human T-cell leukemia Human
86030402 1 vial 1321N1 Human brain astrocytoma Human
85120602 1 vial 293 (also known as HEK 293) Human Embryo Kidney Human
86110401 1 vial 3T3 clone A31 Mouse BALB/c embryo Mouse
86052701 1 vial 3T3 L1 Mouse Embryo Mouse
93112519 1 vial A2780 Human ovarian carcinoma Human
93112520 1 vial A2780ADR Human Caucasian ovarian carcinoma Human
93112517 1 vial A2780cis Human ovarian carcinoma Human
88113005 1 vial A375 Human malignant melanoma Human
97011320 1 vial A375-C5 Human malignant melanoma IL-1 insensitive Human
85090402 1 vial A431 Human squamous carcinoma Human
86012804 1 vial A549 Human Caucasian lung carcinoma Human
86050803 1 vial A7r5 Rat BDIX smooth muscle embryonic aorta Rat
89090402 1 vial AGS Human Caucasian gastric adenocarcinoma Human
94042254 1 vial B16 melanoma 4A5 Mouse melanoma, melanin producing Mouse
92101204 1 vial B16-F0 Mouse melanoma, producing melanin Mouse
92101203 1 vial B16-F1 Mouse melanoma, producing melanin Mouse
96080128 1 vial B9 Mouse B cell hybridoma Mouse
85011419 1 vial B95-8 Monkey Marmoset peripheral blood lymphocyte Monkey
95102433 1 vial BEAS-2B Human bronchial epithelium, normal Human
86082803 1 vial BeWo Human choriocarcinoma Human
85011433 1 vial BHK 21 (clone 13) Hamster Syrian kidney Hamster
93120816 1 vial BXPC-3 Human primary pancreatic adenocarcinoma Human
91031101 1 vial C2C12 Mouse C3H muscle myoblast Human
92090409 1 vial C6 Rat glial tumor Rat
86010202 1 vial CACO-2 Human Caucasian colon adenocarcinoma Human
85050302 1 vial CHO Hamster Chinese ovary Hamster
00102307 1 vial CHO (PROTEIN FREE) Chinese hamster ovary Hamster
85051005 1 vial CHO-K1 Hamster Chinese ovary Hamster
12

Cat. No. Package Size Cell Line Name Cell Line Description Species
87061208 1 vial COLO 205 Human Caucasian colon adenocarcinoma Human
85011437 1 vial DAUDI Human African American Burkitt’s lymphoma Human
85023105 1 vial EL4 Mouse ascites lymphoma lymphoblast Mouse
89042701 1 vial Fao Rat hepatoma Rat
94060901 1 vial FTC-133 Human follicular thyroid carcinoma Human
88092904 1 vial H9c2 (2-1) Rat BDIX heart myoblast Rat
91091005 1 vial HCT 116 Human colon carcinoma Human
93021013 1 vial HeLa Human African American cervix epitheloid carcinoma Human
86062703 1 vial Hep 3B Human African American hepatocyte carcinoma Human
85011430 1 vial Hep G2 Human Caucasian hepatocyte carcinoma Human
86030501 1 vial Hep2 (HeLa derivative) Human African American cervix carcinoma Human
85020207 1 vial Hep-2C (HeLa derivative) Human African American cervix carcinoma Human
98070106 1 vial HL60 Human Caucasian promyelocytic leukemia Human
91072201 1 vial HT29 Human Caucasian colon adenocarcinoma Human
85061105 1 vial HT55 Human colon carcinoma Human
01042712 1 vial Huh-7D 12 Human hepatocellular carcinoma Human
85020204 1 vial IMR-90 Human Caucasian fetal lung fibroblast Human
99040201 1 vial ISHIKAWA Human Asian endometrial adenocarcinoma Human
85011428 1 vial J774.2 Mouse BALB/c monocyte macrophage Mouse
91051511 1 vial J774A.1 Mouse BALB/c monocyte macrophage Mouse
88042803 1 vial Jurkat E6.1 Human leukemic T cell lymphoblast Human
92030501 1 vial K1 Human thyroid carcinoma Human
89121407 1 vial K562 Human Caucasian chronic myelogenous leukemia Human
92110411 1 vial KELLY Human neuroblastoma Human
85011425 1 vial L929 Mouse C3H/An connective tissue Mouse
89110211 1 vial LNCaP.FGC Human Caucasian prostate carcinoma Human
87060101 1 vial LoVo Human colon adenocarcinoma Human
99072810 1 vial MC3T3-E1 Mouse C57BL/6 calvaria Mouse
86012803 1 vial MCF7 Human Caucasian breast adenocarcinoma Human
92020424 1 vial MDA-MB-231 Human Caucasian breast adenocarcinoma Human
84121903 1 vial MDCK Canine Cocker Spaniel Kidney Dog
00062107 1 vial MDCK-II Canine Cocker Spaniel Kidney Dog
05071502 1 vial MDCK-SIAT1 Canine Cocker Spaniel Kidney Sialic Acid Over Expression Dog
86051601 1 vial MG-63 Human osteosarcoma Human
05081101 1 vial MRC-5 (PD 25) Human fetal lung Human
05090501 1 vial MRC-5 (PD 30) Human fetal lung Human
05072101 1 vial MRC-5 PD 19 Human fetal lung Human
84101801 1 vial MRC-5 pd30 Human fetal lung Human
97041101 1 vial NB2-11 Rat lymphoma Rat
95111734 1 vial NCI-H322 Human Caucasian bronchioalveolar carcinoma Human
92090903 1 vial ND7/23 Mouse neuroblastoma × Rat neurone hybrid Mouse
92090904 1 vial ND8/34 Mouse neuroblastoma × Rat neurone hybrid Mouse
89121404 1 vial Neuro 2a Mouse Albino neuroblastoma Mouse
93061524 1 vial NIH 3T3 Mouse Swiss NIH embryo Mouse
86032002 1 vial NRK Rat kidney fibroblast Rat
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 13

Cat. No. Package Size Cell Line Name Cell Line Description Species
85110503 1 vial NS0 Mouse myeloma Mouse
03061601 1 vial NS0-Serum-Free Mouse myeloma, serum-free Mouse
01071221 1 vial NTERA-2 CLONE D1 Human Caucasian pluripotent embryonal carcinoma Human
90011609 1 vial Nthy-ori 3-1 Human thyroid follicular epithelial Human
85101601 1 vial OAW28 Human ovarian tumor epithelial Human
96071721 1 vial OE19 Human Caucasian oesophageal carcinoma Human
96062201 1 vial OE21 Human Caucasian oesophageal squamous cell carcinoma Human
96070808 1 vial OE33 Human Caucasian oesophageal carcinoma Human
96110601 1 vial OX-86 Recombinant mouse OX-40 antigen Rat × Mouse Hybridoma
95102107 1 vial P19 Mouse teratocarcinoma Mouse
85011420 1 vial P3X63Ag8.653 Mouse BALB/c myeloma Mouse
87092802 1 vial PANC-1 Human Caucasian pancreas Human
88022401 1 vial PC-12 Rat adrenal phaeochromocytoma Rat
90112714 1 vial PC-3 Human Caucasian prostate adenocarcinoma Human
98020308 1 vial PMK Primary rhesus macaque kidney cells, blood, serum Macaque
95012614 1 vial PNT1A Human post pubertal prostate normal, immortalized with SV40 Human
95012613 1 vial PNT2 Human prostate normal, immortalized with SV40 Human
85011429 1 vial RAJI Human African American Burkitt’s lymphoma Human
85030802 1 vial RAMOS Human Caucasian Burkitt’s lymphoma Human
85062803 1 vial RAW 264 Mouse leukemic monocyte-macrophage Mouse
91062702 1 vial RAW 264.7 Mouse monocyte macrophage Mouse
00021715 1 vial RK13 Rabbit kidney, BVDV negative Rabbit
95041316 1 vial RPMI 8866 Human B lymphocyte Human
89050205 1 vial Saos-2 Human primary osteogenic sarcoma Human
89070101 1 vial Sf9 Spodoptera frugiperda pupal ovarian tissue Insect
94030304 1 vial SH-SY5Y Human neuroblastoma Human
86012802 1 vial SK.N.SH Human Caucasian neuroblastoma Human
91091004 1 vial SK-OV-3 Human Caucasian ovary adenocarcinoma Human
07032801 1 vial SNL 76/7 Mouse SIM strain embryonic fibroblast Mouse
85072401 1 vial SP2/0-Ag14 Mouse × Mouse myeloma, non-producing Mouse
87051203 1 vial SW 620 Human Caucasian colon adenocarcinoma Human
87092801 1 vial SW480 Human colon adenocarcinoma Human
85102201 1 vial T47D Human breast tumor Human
88021101 1 vial T84 Human colon carcinoma Human
92090213 1 vial T98G Human Caucasian glioblastoma Human
93022307 1 vial TF1 Human erythroleukaemia Human
88081201 1 vial THP 1 Human monocytic leukemia Human
96121229 1 vial tsA201 Human embryonal kidney, SV40 transformed Human
92022711 1 vial U-2 OS Human Osteosarcoma Human
89081402 1 vial U-87 MG Human glioblastoma astrocytoma Human
85011440 1 vial U937 Human Caucasian histiocytic lymphoma Human
06020201 1 vial VCaP Human Prostate Cancer Metastasis Human
90020107 1 vial WI-38 Human Caucasian fetal lung Human
Contact your local Sigma-Aldrich® office for price and availability.
14
Protocol for the Cryopreservation
of Cell Lines
Aim
This protocol is recomended by the
European Collection of Cell Cultures (ECACC)
for the cryopreservation of its cell lines.
This protocol employs the use of passive
methods involving an electric –80 °C freezer
for the cryopreservation of cell cultures.
ECACC routinely uses a programmable rate
controlled freezer (Planer Series Two) from
Planer Products. This is the most reliable
and reproducible way to freeze cells but
as the cost of such equipment is beyond
the majority of research laboratories the
methods below are described in detail.
If large numbers of cell cultures are regularly
being frozen then a programmable rate
controlled freezer is recommended.
Materials
• Freeze medium (commonly 70% basal
medium, 20% FBS, 10% DMSO
[Cat. No. D2650] or glycerol, check
ECACC data sheets for details).
• 70% ethanol in water (Cat. No. R8382)
• PBS without Ca2+ Mg2+(Cat. No. D8537)
• 0.25% trypsin/EDTA in HBSS, without Ca2+/
Mg2+ (Cat. No. T4049)
• DMSO (Cat. No. D2650)
• Trypsin/EDTA (Cat. No. T4049)
• HL60 (Cat. No. 98070106-1v1)
Equipment
• Personal protective equipment
(sterile gloves, laboratory coat)
• Full-face protective mask/visor
• Waterbath set to 37 °C
• Microbiological safety cabinet at
appropriate containment level
• Centrifuge
• Haemocytometer (Sigma Bright-Line™
Cat. No. Z359629, Improved
Neubauer-Camlab CCH.AC1)
• Pre labeled ampules/cryotubes
• Cell Freezing Device (e.g. Nalgene®
Mr. Frosty Cat. No. C1562)
Procedure
View cultures using an inverted microscope
to assess the degree of cell density and
confirm the absence of bacterial and
fungal contaminants.
Bring adherent and semi adherent cells
into suspension using trypsin/EDTA
(Cat. No. T4049) as above and re-suspend in
a volume of fresh medium at least equivalent
to the volume of trypsin. Suspension cell
lines can be used directly. Remove a small
aliquot of cells (100–200 uL) and perform
a cell count. Ideally the cell viability should
be in excess of 90% in order to achieve a
good recovery after freezing. Centrifuge the
remaining culture at 150 g for 5 minutes.
Re-suspend cells at a concentration of
2-4x106 cells per ml in freeze medium.
Pipette 1ml aliquots of cells into
cyroprotective ampules that have been
labeled with the cell line name, passage
number, cell concentration and date.
Place ampules inside a passive freezer e.g.
Nalgene Mr. Frosty (Cat. No. C1562). Fill
freezer with isopropyl alcohol and place at
–80 °C overnight. Frozen ampules should
be transferred to the vapor phase of a
liquid nitrogen storage vessel and the
locations recorded.
Key Points
The most commonly used cryoprotectant is
dimethyl sulphoxide (DMSO Cat. No. D2650),
however, this is not appropriate for all cell lines
e.g. HL60 (Cat. No. 98070106-1vl) where
DMSO is used to induce differentiation.
In such cases an alternative such as glycerol
should be used. ECACC freeze medium
recommended above has been shown
to be a good universal medium for most
cell types. Another commonly used freeze
medium formulation is 70% basal medium,
20% FBS, 10% DMSO but this may not be
suitable for all cell types. Check if it works
for your cells before using on a regular basis
(Cat. No. C6164). It is essential that cultures
are healthy and in the log phase of growth.
This can be achieved by using pre-confluent
cultures (cultures that are below their
maximum cell density) and by changing the
culture medium 24 hours before freezing.
The rate of cooling may vary but as a general
guide a rate of between –1 °C and –3 °C per
minute will prove suitable for the majority of
cell cultures.
An alternative to the Mr. Frosty system is
the Taylor Wharton passive freezer where
ampules are held in liquid nitrogen vapor
in the neck of Dewar. The system allows the
ampules to be gradually lowered thereby
reducing the temperature. Rate controlled
freezers are also available and are particularly
useful if large numbers of ampules are
frozen on a regular basis. As a last resort if no
other devices are available ampules may be
placed inside a well insulated box (such as a
polystyrene box with sides that are at least
1cm thick) and placed at –80 °C overnight. It
is important to ensure that the box remains
upright throughout the freezing process.
Once frozen, ampules should be transferred
to the vapor phase of a liquid nitrogen
storage vessel and the locations recorded.
If using a freezing method involving a –80 °C
freezer it is important to have an allocated
section for cell line freezing so that samples
are not inadvertently removed. If this
happens at a crucial part of the freezing, cell
viability will be compromised.
Problem Solving Suggestions
John Ryan Ph.D
Corning Incorporated, Life Sciences
Acton, MA 01720
Viability problems associated with cryogenic
storage are usually noticed soon after cultures
are thawed and plated. There are four major
areas where problems occur:
1. During harvesting and processing of the
cells. Problems may be caused by excessive
exposure of the cells to dissociating agents;
using a cryoprotective agent that is toxic;
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 15

or allowing high density cell suspensions
to remain too long at room temperature
or at a pH that is too basic.
2. During the cooling (freezing) process.
Excessive cell damage and reduced
culture viability often result from using a
cooling rate that is too fast or too slow,
or when the cooling process is temporarily
interrupted. Not using a suitable
cryoprotective agent at an appropriate
concentration will also result in
viability problems.
3. During cryogenic storage. Culture viability
is often reduced when vials are allowed
to warm up during transfer to the freezer,
or if the repository temperature is not
consistently maintained at appropriate
cryogenic temperatures.
4. During thawing and recovery. Problems
arise when the thawing process is
too slow or the cryoprotectants are
improperly removed.
These viability problems can often be
corrected by using the following technique
to identify the stage in the freezing
process where the problem originates.
Harvest enough cells to prepare at least
four vials. Then remove a sample of cell
suspension, equivalent in cell number to
that which will be placed into the vials, and
immediately place it into a culture vessel
with an appropriate amount of medium
and incubate. This culture will be used as a
control to compare with the cultures set up
in the remaining steps.
Next, add the cryoprotective agent to the
remaining cells and divide among three
vials. Place one vial at 4 °C for one hour.
Then remove the cells from the vial, process
as though they had just been thawed
from the freezer, and plate in medium as
above. This culture will be compared with
the control culture to determine if there
are any problems associated with the
cryoprotective agent.
Meanwhile, process the remaining vials
through the slow cooling process as usual.
One vial is then immediately thawed
and processed as above. This culture will
be compared with the control culture
to determine if there are any problems
associated with the slow cooling process.
The remaining vial is then transferred to
the cryogenic freezer and stored overnight
before being thawed and processed as
above. This culture will be compared with
the control culture to determine if there are
any problems associated with the cryogenic
storage conditions. If additional vials of
cells are available, several different recovery
techniques should be used to determine if
the recovery technique is the source of
the problem.
By comparing all of the cultures to the
original culture, it should then be possible
to determine at which stage of the freezing
process the problem occurred. Once this is
known, the information presented in this
guide and its references should be enough
to eliminate the problem.

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16

Cryoware
Cryovial Color Coders

Cryoware
Cryovial Color Coders
Color Coders for Nalgene® cryogenic vials
Colored disks fit into the recessed tops of Nalgene
cryogenic vials. The system allows color-coding
of vials for quick visual identification. Cut out top
facilitates handling of vials with forceps. Flat surface
permits writing.
polystyrene
CryoCane™ alu­mi­num canes
Firmly holds 1.2 or 2.0 mL Nalgene cryogenic vials
for storage in Dewar-type, liquid nitrogen freezers.
 capacity: 6 vials
overall L............................................................................................... 300 mm
C9936-12EA 12 ea
CryoSleeve™ clear PVC cane sleeve
 5 × 5 array for 1.2 and 2.0 mL vials Will not become brittle when frozen.
L × W × H......................................................................3 in. × 3 in. × 2 in. Encloses a Nalgene CryoCane™ for extra security
during handling and storage. Easy to install and
R0763-8EA 8 ea remove.
C1437-100EA 100 ea

 9 × 9 Array For 1.2 and 2.0 mL vials Freezing container, Nalgene® Mr. Frosty
L × W × H......................................................... 5 1/4 in. × 5 1/4 in. × 2 in.
Polycarbonate container, blue high-density
R0888-4EA 4 ea polyethylene closure, white high-density
polyethylene vial holder, and foam insert.
 blue  9 × 9 Array For 5.0 mL vials Provides the critical, repeatable, 1 °C/min cooling
L × W × H................................................... 5 1/4 in. × 5 1/4 in. × 3 3/4 in. rate required for successful cryopreservation
C0812-100EA 100 ea
of cells. Easy to use in any mechanical freezer.
R1013-4EA 4 ea
 green Container is imprinted with graphic instructions.
C0937-100EA 100 ea Requires only isopropyl alcohol. Rigid vial holder
 CryoBox 100, 10 × 10 array
keeps vials from contacting alcohol, preventing
For ultra-compact storage of 1.0 mL and 1.5 mL contamination by wicking. Vial holder floats to
 red
System 100 vials or other similar-sized vials. Box fits allow thawing in a water bath. Numbers molded
C1062-100EA 100 ea most existing vertical racks with 2 in. shelf spacing. for identification. All components withstand
L × W × H..................................................5 1/4 in. × 5 1/4 in. × 2 1/16 in. repeated freeze/thaw cycles. Stackable.
 white
Z359017-10EA 10 ea
Holds 18 1.2 and 2.0 mL cryovials
C1187-100EA 100 ea
H × diam......................................................................86 mm × 117 mm
 yellow CryoCane™ alu­mi­num canes
C1312-100EA 100 ea
Firmly holds 1.2 or 2.0 mL Nalgene cryogenic vials
for storage in Dewar-type, liquid nitrogen freezers.
Cryo Containers  capacity: 5 vials
Projections support base of each vial.
Nalgene® CryoBox™ overall L............................................................................................... 290 mm
For ultra-low temperature storage of cryogenic
vials in mechanical or liquid nitrogen freezers. C9811-12EA 12 ea
Usable temperature range: −196 to +121 °C.
Numbers molded into grid system in box
C1562-1EA 1 ea
correspond to numbers printed on lid. Lid is keyed
to box so markings will always match. Boxes
accept writing with markers.
polycarbonate
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 17

Cryo Storage Racks  Vertical, 4-shelf for 3 × 3 × 2 in. boxes Rack for Nalgene® cryogenic vials
overall H..................................................................................................8 3/4 in. Accept all Nalgene cryogenic vials. Bottom of
Cryogenic storage rack, stainless steel R1638-1EA 1 ea each well interlocks with vial, allowing one-hand
Racks hold CryoBoxes securely but separately, manipulation.
allowing fast retrieval and immediate return to  Vertical, 4-shelf for 5 1/4 × 5 1/4 × 2 in. boxes polycarbonate
freezer. The vertical racks are best for use in chest overall H..............................8 3/4 in.
autoclavable.................................................................................................Yes
freezers, horizontal racks for upright freezers. R1763-1EA 1 ea

 Vertical; 4-shelf for 5 1/4 × 5 1/4 × 33/4in. boxes

overall H.................................................................................................... 16 in.
R3388-1EA 1 ea

 Vertical; 7-shelf for 5 1/4 × 5 1/4 × 3 3/4 in. boxes

overall H..............................................................................................27 3/4 in.
R3138-1EA 1 ea

 Vertical, 9-shelf for 3 × 3 × 2 in. boxes  Holds 50 vials in a 5 × 10 array

 Horizontal; 12-shelf for 5 1/4 × 5 1/4 × 2 in. boxes
overall H...............................................................................................19 3/4 in. Non-skid rubber feet
overall H.................................................................................................8 /8 in.
7

R1888-1EA 1 ea color............................................................................................................. white

R1388-2EA 2 ea
R0638-1EA 1 ea
 Horizontal; 16-shelf for 5 1/4 × 5 1/4 × 2 in. boxes  Vertical, 9-shelf for 5 1/4 × 5 1/4 × 2 in. boxes
 Holds 25 vials in a staggered array
overall H..................................................................................................8 7/8 in. overall H...............................................................................................19 3/4 in.
Wider spacing for easier manipulation
R1513-2EA 2 ea R2013-1EA 1 ea
color................................................................................................................ blue
 Vertical; 2-shelf for 5 /4 × 5 /4 × 3 /4 in. boxes
1 1 3 Z359041-4EA 4 ea
overall H..................................................................................................8 1/4 in.
R2888

Online Metabolomics Resource

Use the IUBMB–Sigma-Aldrich Interactive
Metabolic Pathway Chart to find the
Metabolite Standards you need.
http://www.sigma-aldrich.com/ProductLookup.html?
ProdNo=C7352&Brand=SIGMA

The Metabolic Pathways Map contains over 500 hyperlinks to Sigma

product listings. Just click on the metabolite name or the enzyme’s
E.C. number to access product information.

You can access the chart at

sigma-aldrich.com/metpath
18
Rack for Nalgene® micro­centrifuge tubes Note: Do not use cryogenic vials for storage in
the liquid phase of liquid nitrogen unless correctly
Bottom of white polycarbonate, lid of clear
sealed in Nunc CryoFlex™ tubing. Such use may
polycarbonate, permanent foam insert.
cause entrapment of liquefied nitrogen inside
For safe storage of samples in microcentrifuge
the vial and lead to pressure build-up resulting in
tubes from −135 °C to +121 °C. Not for use in
possible explosion or biohazard release.
liquid-phase liquid nitrogen. May be repeatedly
polypropylene vials
autoclaved. Holes in foam are placed diagonally
and spaced to avoid overlapping of tube closures. high-density polypropylene closures
Lid accepts writing with marker.
size × H.............................................................. 5 1/4 in. × 5 1/4 in. × 2 in.
 1.2 mL, sterile
package = 25 vials 20
V4757-500EA 500 ea
 1.2 mL, sterile, pkg = (Bulk packed)
V4882-1000EA 1000 ea
 2.0 mL, sterile
 capacity: 81 0.5-mL microcentrifuge tubes package = 25 vials 20
V5007-500EA 500 ea
R1138-4EA 4 ea
 2.0 mL, sterile, pkg = (Bulk packed)
 capacity: 64 1.5-mL microcentrifuge tubes
V5132-1000EA 1000 ea
R1263-4EA 4 ea
 5.0 mL, sterile
Cryo Vials package = 10 vials 25
V5257-250EA 250 ea
Nalgene® cryogenic vials
 1.2 mL, non-sterile
Vials designed for exceptional ease of use and
sample integrity in ultra-low temperature storage. Non-sterile vials and closures are packed
Closure seals without gasket or O-ring, minimizing separately. Vials have no printing or graduations.
risk of contamination. The deep skirt of the closure V4507-1000EA 1000 ea
allows single-handed aseptic technique without
exposing fingers to vial contents. Ribs around  2.0 mL, non-sterile
the bottom of the vial interlock with wells in
Non-sterile vials and closures are packed
the Cryovial Racks so the vial will not turn when separately. Vials have no printing or graduations.
opening or closing. Bottoms of vials are skirted; vial
stands without holder. V4632-1000EA 1000 ea
Sterile vials and closures are pre-assembled and
radiation-sterilized. A white marking area, fill line
and graduations are printed on the side. Certified
sterile, non-cytotoxic and non-pyrogenic.
Nalgene® cryogenic vials, System 100
Innovative vials suitable for long-term sample
storage in vapor-phase liquid nitrogen freezers.
Gasket allows leakproof use in microcentrifuge up
to 8,000 × g. Vial is externally threaded and closure
has deep skirt to facilitate aseptic transfers. Vials
have slightly reduced diameter to fit ultra-compact
System100 CryoBox. Self-standing skirt at bottom
has grooves that mate with holder, facilitating
one-hand manipulation.
sterile (certified)
polypropylene vials
polypropylene caps
silicone gasket
 capacity 1.0 mL
Z359025-500EA 500 ea
Nalgene® cryogenic vials, System 100
Innovative vials suitable for long-term sample
storage in vapor-phase liquid nitrogen freezers.
Gasket allows leakproof use in microcentrifuge up
to 8,000 × g. Vial is externally threaded and closure
has deep skirt to facilitate aseptic transfers. Vials
have slightly reduced diameter to fit ultra-compact
System100 CryoBox. Self-standing skirt at bottom
has grooves that mate with holder, facilitating one-
hand manipulation.
sterile (certified)
polypropylene vials
silicone gasket
polypropylene caps
 capacity 1.5 mL
Z359033-500EA 500 ea
Nalgene® specimen vial
 capacity 15 mL
Vials of polypropylene, linerless screw closures of
high-density polyethylene. For storage of solid
specimens. Use in mechanical freezers only.
Wide-mouth, shoulderless vial allows easy access
to sample. Flat bottom permits self-standing on
flat surface. Certified sterile, non-cytotoxic and
non-pyrogenic.
sterile
75 vials per package
V5382-1PAK 1 pkg
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 19

Benchtop Coolers Quick Chill™ cooler Cryoclaw

Nalgene® Labtop cooler
Molded of durable polycarbonate and filled
with non-toxic freezable gel. A convenient
alternative to ice buckets for keeping reagents and
enzymes cool. Also protects critical samples from
temperature fluctuations in the freezer.
Holes accommodate tubes:
• 1.5 mL microtubes or cryogenic vials
(with adapters to hold 0.5 mL tubes)
• 12–13 mm diameter tubes
• 16–17 mm tubes
Domed lid permits storage of taller screw cap
microtubes. Lids have gridded write-on surfaces
for sample identification. Stackable. Usable to
−135 °C.
A reusable, economical alternative to dry ice-
solvent baths for quickly chilling specimens. The
unit is molded of durable polycarbonate and
filled with a non-toxic solution. After thorough
cooling in an ultra-cold freezer, unit can be used
on benchtop for up to 45 min to bring alcohol
solutions to –20 °C in 3 min or less; aqueous
solutions to 0 °C in 4 min or less. There is no
direct contact between sample tube and chilling
solution, so labels and markings stay on. Unit holds
12 x 1.5 mL microcentrifuge tubes; adapters are
included to modify holes for 0.5 mL tubes. Non-
skid rubber feet keep the unit from sliding on
the benchtop.
Remote manipulator designed specifically for
removing vials and boxes that have fallen to the
bottom of cryogenic vessels. Heavy-duty stainless
steel with rubber gripper cups, the Cryoclaw is
long enough to reach the bottom of any common
Dewar and narrow enough to work through a 2 in.
(5 cm) necktube.
Replacement parts sold separately.
product of Barnstead International AY509X18

C6816-1EA 1 ea

Aldrich® wide-mouth dewar flasks

Z359068-1EA 1 ea
Aldrich® wide-mouth dewar flasks
Borosilicate glass with protective aluminum or
Cryo Accessories plastic mesh housing and extended aluminum
 −20 °C Labtop cooler, gel-filled lid
base weighted for stability.
Maintains 32 microtubes below −15 °C for up to Dewar 35L accessories
2 hr at room temperature.
 aluminum cane without tube stops, diam. 1.2 cm
C2312-1EA 1 ea × L 29 cm
Holds tubes totalling approx. 28 cm in height.
 0 °C Labtop cooler, gel-filled lid
Maintains 32 microtubes below 1 °C for up to 5 hr
at room temperature.
Z359084-1EA 1 ea

 −20 °C Labtop cooler, Jr., transparent lid

Maintains 12 microtubes below −15 °C for up to
1 hr at room temperature.
C2437-1EA
 0 °C Labtop cooler, Jr., transparent lid
Maintains 12 microtubes below 1 °C for up to 3
1
/2 hr at room temperature.
Z359076-1EA
 0 °C Labtop cooler, clear lid
Maintains twelve 12–13 mm diameter tubes below
1 °C for up to 5 hr at room temperature.
Z370509-1EA
 0 °C Labtop cooler, clear lid
Maintains twelve 16–17 mm diameter tubes below
2 °C for up to 4 hr at room temperature.
C3296
 capacity 350 mL, aluminum housing
1 ea
 Plastic storage box I.D.................................................................................................................7.0 cm
Rugged alternative to cardboard storage boxes; D................................................................................................................ 11.3 cm
numbered grid printed on top Z120758-1EA 1 ea
size.........................3 in. (7.6 cm) × 3 in. (7.6 cm) × 2 in. (5.1 cm)
 capacity 665 mL, aluminum housing
1 ea S7397-8EA 8 ea D................................................................................................................ 19.5 cm
I.D.................................................................................................................7.0 cm
 aluminum cane with tube stops
Z120774-1EA 1 ea
Holds 5 tubes, each up to approx. 5 cm in height.
diam. × L..............................1.2 cm × 29 cm  capacity 1,000 mL, aluminum housing
1 ea I.D.................................................................................................................7.0 cm
C3171-50EA 50 ea D................................................................................................................ 30.2 cm
Z120790-1EA 1 ea

Z370517-1EA 1 ea
20

 capacity 1,900 mL, aluminum housing  capacity 4,300 mL, Plastic mesh housing

I.D.............................................................................................................. 12.0 cm D................................................................................................................ 30.0 cm
D................................................................................................................ 19.8 cm I.D.............................................................................................................. 14.3 cm
Z120766-1EA 1 ea Z120782-1EA 1 ea

 capacity 4,300 mL, aluminum housing

I.D.............................................................................................................. 14.3 cm Barnstead/Thermolyne® Dewars,
D................................................................................................................ 30.0 cm
Biocane™ Series
Z103128-1EA 1 ea

 capacity 350 mL, Plastic mesh housing Barnstead/Thermolyne® Dewars,  Biocane, 11

I.D.................................................................................................................7.0 cm Biocane™ series product of Barnstead CK509X1
D................................................................................................................ 12.5 cm A can and cane cryopreservation system with diam. × H....................................10.2 in. (26 cm) × 21.6 in. (55 cm)
Z120669-1EA 1 ea features to meet the requirements of almost capacity.........................................................................................................11 L
any research lab: available in five sizes for small- neck size........................................................................................2 in. (5 cm)
 capacity 665 mL, Plastic mesh housing
to large-scale sample storage; unsurpassed static liquid nitrogen........0.14 L/day (average consumption)
I.D.................................................................................................................7.0 cm temperature uniformity with low liquid nitrogen storage.................................................................................................180 vials
D................................................................................................................ 19.5 cm consumption rates; secure locking hasp prevents
Z120693-1EA 1 ea unauthorized access; each Biocane vessel includes F1286-1EA 1 ea
six stainless steel canisters that accommodate
 capacity 1,000 mL, Plastic mesh housing  Biocane, 20
Nalgene CryoCanes or similar storage systems.
I.D.................................................................................................................7.0 cm product of Barnstead CK509X2
CryoCanes not included.
D................................................................................................................ 30.2 cm
capacity.........................................................................................................20 L
Z120707-1EA 1 ea neck size........................................................................................2 in. (5 cm)
 capacity 1,900 mL, Plastic mesh housing diam. × H.................................14 1/2 in. (37 cm) × 25.7 in. (65 cm)
D................................................................................................................ 19.8 cm static liquid nitrogen........0.09 L/day (average consumption)
I.D.............................................................................................................. 12.0 cm storage.................................................................................................180 vials
Z120677-1EA 1 ea F1411-1EA 1 ea

Corning® Cell Culture

Making your research possibilities real
• HYPERFlask® — Grow enough cells in one • CellBIND® — Improve cell attachment and
1,720 cm2 HYPERFlask Cell Culture Vessel to yield with the Corning CellBIND surface, a
seed 2,000 ­microplates. nonbiological surface with high oxygen levels.
• Epic System — The Corning Epic System, The new Corning
a new high-throughput, label-free detection Epic System
technology, allows researchers to bring drugs to
market faster.
For more information on our Corning product offerings, visit sigma-aldrich.com/corning
Corning, CellBIND and HYPERFlask are registered trademarks of Corning.

sigma-aldrich.com
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 21

 Biocane, 34  Locator Jr., 50L flask  capacity 2 L

product of Barnstead CK509X3 product of Barnstead International CY50925 polyethylene handle
capacity.........................................................................................................34 L diam. × H........................................... 22 in. (56 cm) × 26 in. (66 cm) size........................................ 8 7/8 in. (225 mm) × 4 3/4 in. (121 mm)
diam. × H....................................18.2 in. (46 cm) × 26.6 in. (68 cm) neck size...........................................................................8 1/2 in. (21.6 cm) F9526-1EA 1 ea
neck size..................................................................................3 1/2 in. (9 cm) static liquid nitrogen................................................. 0.65 L/day (avg.)
static liquid nitrogen........0.17 L/day (average consumption) storage..............................................................................................1296 vials
 capacity 4 L
storage.................................................................................................720 vials polyethylene handle
F0911-1EA 1 ea
F1536-1EA 1 ea size......................................11 7/8 in. (287 mm) × 5 3/4 in. (146 mm)
 Locator 4, 110L flask F9651-1EA 1 ea
 Biocane, 47 product of Barnstead International CY50935
 capacity 10 L
product of Barnstead CK509X4 neck size........................................................................... 8 1/2 in. (21.6 cm)
no handle
neck size................................................................................. 5 in. (12.7 cm) diam. × H........................................... 22 in. (56 cm) × 36 in. (91 cm)
size......................................15 1/2 in. (394 mm) × 7 3/4 in. (197 mm)
capacity.........................................................................................................47 L static liquid nitrogen................................................. 0.65 L/day (avg.)
diam. × H.....................................20 in. (51 cm) × 26 1/2 in. (67 cm) storage..............................................................................................2916 vials F9776-1EA 1 ea
static liquid nitrogen........0.37 L/day (average consumption) F1036-1EA 1 ea
storage..............................................................................................1512 vials
 Locator 8, 110L flask
Taylor-Wharton Dewars
F1661-1EA 1 ea
product of Barnstead International CY50945 Taylor-Wharton Dewars
 Cart for transporting Dewars safely neck size.................................................................................. 6 in. (156 cm)
Accepts Biocane 20, 34, and 47 For storage of small quantities of liquid nitrogen.
diam. × H........................................... 22 in. (56 cm) × 36 in. (91 cm)
High-strength aluminum shell is both durable
product of Barnstead AY509X9 static liquid nitrogen....................................................0.4 L/day (avg.)
and light-weight. Super insulation provides
storage..............................................................................................1800 vials
Z363642 advanced thermal protection. Neck tube joins
F1161-1EA 1 ea inner and outer container, providing maximum
durability and minimum nitrogen consumption.
Barnstead/Thermolyne Dewars, ®
 Four-wheel roller base Cap and neck tube core provide positive closure.
Locator® Series product of Barnstead International AY509X1 Accessories sold separately.
R9640-1EA 1 ea
Barnstead/Thermolyne® Dewars,
Locator® series
For ultra-cold storage of biological specimens in Nalgene® Dewars
liquid nitrogen. Not to be used for transportation
of samples. Nalgene® Dewars
Features: Shatterproof. Safe for short-term storage of ice
• Three sizes fit the needs of almost any water, dry ice/solvent baths, and liquid nitrogen;
can also be used as a hot bath. Temperature range:
research lab
−196 to +100 °C. Cover is vented. Polyethylene-
• Advanced vacuum insulation provides a coated handle on 1, 2, and 4 L sizes only.
maximum −190 °C throughout flask with as
Style 4150.
little as 2 inches of liquid nitrogen, and delivers
improved liquid nitrogen consumption rates Dimensions are inside depth × I. D.
 capacity 4 L
• Secure locking clasp prevents unauthorized urethane foam-filled high-density polyethylene
pitcher style
access
diam. × H...................................19.3 cm (7.6 in.) × 43.2 cm (17 in.)
• Each Locator vessel includes stainless steel racks neck I.D.................................................................................. 3.0 cm (1.2 in.)
that accommodate Nalgene Cryoboxes for 2 mL F9018-1EA 1 ea
freezing vials. Cryoboxes not included
 capacity 10 L
beaker style
neck I.D...................................................................................... 5.1 cm (2 in.)
diam. × H............................29.0 cm (11.4 in.) × 59.7 cm (23.5 in.)

F9143-1EA 1 ea

 capacity 1 L  capacity 25 L

polyethylene handle beaker style
size...........................................7 5/8 in. (194 mm) × 3 3/4 in. (95 mm) diam. × H............................39.7 cm (15.6 in.) × 65.5 cm (25.8 in.)
neck I.D.................................................................................. 6.4 cm (2.5 in.)
F9401-1EA 1 ea
F4524-1EA 1 ea
22

 capacity 35 L  Rack, 5-shelf for 35 L Dewar Wide-mouth Dewars with Silvered
capacity to hold 6 × 11-inch canisters Each shelf holds one 3 × 3 × 2 in. (7.6 × 7.6 ×
5.1 cm) box with a capacity of 25 x 2mL vials.
Glass Liner
beaker style
A spring-action retaining clip holds each box in Wide-mouth Dewars with silvered
wide-mouth place. Boxes not included. glass liner
neck I.D................................................................................11.9 cm (4.7 in.)
R0384-1EA 1 ea Straight sides, a swing-over carrying handle,
diam. × H............................47.8 cm (18.8 in.) × 64.8 cm (25.5 in.)
and no neck constriction; suitable for benchtop
F2344-1EA 1 ea  Cardboard storage box for 35 L Dewar immersion applications. Vented insulating lids
supplied with 25 position insert. clamp to the body of the flask.
Taylor-Wharton size..........................3 in. (7.6 cm) × 3 in. (7.6 cm) × 2 in. (5.1 cm)

Dewar Accessories S7272-5EA 5 ea

Taylor-Wharton Dewar accessories

Wide-mouth Dewars
Wide-mouth Dewars
Detachable handle and lid (lids do not fasten to
the body of the flask).
stainless steel  1 L, stainless steel jacket and vented cap
outside dimensions D × H.....................4.6 in. (116 mm) × 9 in.
(228 mm)
inside dimensions D × H.......................3.3 in. (85 mm) × 7.3 in.
(185 mm)

D3170-1EA 1 ea
 Cap for 4 L Dewar
 2 L, stainless steel jacket and vented cap
C0671-1EA 1 ea
inside dimensions D × H.................... 4.2 in. (106 mm) × 8.1 in.
(205 mm)
 Cap/necktube core for 10 L Dewar
outside dimensions D × H.............. 5.6 in. (142 mm) × 10.5 in.
C0796-1EA 1 ea (268 mm)
 capacity 0.5 L
 Tipping stand for 25 L Dewar D3295-1EA 1 ea
Does not have handle.
This stand supports the Dewar and allows
convenient, safe pouring of liquid. Wheels add inside dimensions D × H........................2.5 in. (65 mm) × 7.1 in.  4.5 L, stainless steel jacket and vented cap
mobility and additional handling ease. (180 mm) inside dimensions D × H......................5.9 in. (150 mm) × 11 in.
outside dimensions D × H.........................3.4 in. (87 mm) × 8 in. (280 mm)
S8777-1EA 1 ea (204 mm) outside dimensions D × H............... 7.2 in. (182 mm) × 10.5 in.
D3920-1EA 1 ea (350 mm)
 Cap/necktube core for 25 L Dewar D3420-1EA 1 ea
 capacity 1 L
C7431-1EA 1 ea  1 L, Blue enamelled steel jacket and vented cap
inside dimensions D × H........................3.3 in. (85 mm) × 8.1 in.
(206 mm) outside dimensions D × H................................4.6 in. (116 mm) ×
 Cap/necktube core for 35 L Dewar 9 in. (228 mm)
outside dimensions D × H..................4.2 in. (107 mm) × 9.1 in.
C0544-1EA 1 ea inside dimensions D × H..............................................................................
(232 mm)
3.3 in. (85 mm) × 7.3 in. (185 mm)
 Cast aluminum roller base with 5 wheels for D4045-1EA 1 ea
D3545-1EA 1 ea
35 L Dewar
 1 L
R0509-1EA 1 ea  2 L, Blue enamelled steel jacket and vented cap
extra wide mouth
inside dimensions D × H.................... 4.2 in. (106 mm) × 8.1 in.
outside dimensions D × H......................4.8 in. (122 mm) × 7 in. .
 Stainless steel canister for 35 L Dewar (205 mm)
(177 mm)
For storage and easy retrieval of vials in canes outside dimensions D × H.............. 5.6 in. (142 mm) × 10.5 in.
inside dimensions D × H.........................4 in. (100 mm) × 6.2 in.
(canes not included). (268 mm)
(157 mm)
size...................................................3.7 in. (9.4 cm) × 11 in. (27.9 cm) D3670-1EA 1 ea
D4170-1EA 1 ea
C3421-1EA 1 ea
 4.5 L, Blue enamelled steel jacket and vented cap
 capacity 2 L inside dimensions D × H..................... 5.9 in. (150 mm) × 11 in.
 Measuring rod for 35 L Dewar outside dimensions D × H............... 4.8 in. (116 mm) × 12.3 in.
(280 mm)
For determination of liquid nitrogen level. (228 mm)
outside dimensions D × H.............. 7.2 in. (182 mm) × 10.5 in.
M9904-1EA 1 ea inside dimensions D × H......................4 in. (100 mm) × 11.2 in.
(350 mm)
(285 mm)
D3795-1EA 1 ea
D4295-1EA 1 ea
GREAT BREAKTHROUGH IN OVER-ENGINEERING: REVELATION #78:

Some things are great

just the way they are.

FIGURE 1: FIGURE 2:
Alternative Hamster Propulsion Engine
Energy Source

For over 25 years, Sigma Life Science has been a primary

manufacturer of quality cell culture media, sera and reagents.
During that time, a lot has changed. The breadth of application-
tested cell culture products we offer has increased substantially,
as have the speed and simplicity with which they’re delivered to
you. Some things, however, we’ve intentionally left alone. Like
our unwavering commitment
to dependability and quality,
and our dedication to delivering
world-class chemicals in that
square bottle you’ve come to
know and love. After all, it’s
what’s on the inside that counts.

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