biofiles volume 5 number 4 - cryopreservation
TRANSCRIPT
Volume 5, Number 4
New Products
Cell Freezing Media and Reagents
ECACC Cell Lines
Protocol for the Cryopreseravtion of Cell Lines
Cryoware
Cryopreservation
Biofiles
Biofilescontents
Introduction 3
New Products 4CryoStor™ 4HypoThermosol® 5Sericin 6
Cell Freezing Media and Reagents 8
ECACC Cell Lines 11
Protocol for the Cryopreservation of Cell Lines 14
Cryoware 16
Cryovial Color Coders 16Cryo Containers 16Cryo Storage Racks 17Cryo Vials 18Benchtop Coolers 19
Cryo Accessories 19Aldrich® wide-mouth dewar flasks 19Barnstead/Thermolyne® Dewars, Biocane™ Series 20Barnstead/Thermolyne® Dewars, Locator® Series 21 Nalgene® Dewars 21Taylor-Wharton Dewars 21Taylor-Wharton Dewar Accessories 22Wide-mouth Dewars 22Wide-mouth Dewars with Silvered Glass Liner 22
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Technical content: Don Finley, M. Sc.
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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
IntroductionDon FinleyMarket Segment Manager, Cell [email protected]
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) Revival of spermatozoa after vitrification and
dehydration at low temperatures. C. Polge, et al., Nature 164 (1949) 666–667.
(2) Prevention 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
New ProductsCryoStor™
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.
Liver Cells (Hepatocytes & C3A) •Cardiomyocytes
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•
CryoStor is ideal for cryopreservation applications that must be serum free and where high recovery is critical.
CryoStor™ cell cryopreservation media
CryoStor, a series of cell-specific, optimized preservation media, is uniquely formulated to address the molecular and biological aspects of cells during the cryopreservation process thereby directly reducing the level of Cryopreservation-Induced Delayed-Onset Cell Death and improving post-thaw cell viability and function.store at: 2–8 °C
CS2CryoStor CS2 is formulated to contain 2% dimethyl sulfoxide (DMSO). Suggested when reducing DMSO is of primary concern.
C3124-100ML 100 mL
CS5Cryostor CS5 is formulated to contain 5% dimethyl sulfoxide (DMSO). Recommended for cryopreservation of most cell types.
C2999-100ML 100 mL
CS10CryoStor™ CS10 is a uniquely formulated cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). Recommended for the preservation of hepatocytes, tissue samples and other extremely sensitive cell types.
C2874-100ML 100 mL
0
20
40
60
80
100
120
Control(37 °C)
2–8 °C Hold Ice-Nucleation
LN2Storage
2–8 °C Hold Ice-Nucleation
LN2Storage
Media + 5% DMSO CryoStor™ CS5
Effect of Cryopreservation Vehicle Solution on Cell Viability
Cell
Viab
ility
(Rel
ativ
e to
37C
Con
trol
)
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2% D
MSO
5% D
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10%
DM
SO
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MSO
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MSO
10%
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MSO
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MSO
10%
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SO
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MSO
5% D
MSO
10%
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SO
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MSO
5% D
MSO
10%
DM
SO
Culture Media CryoStor™ Culture Media CryoStor™ Culture Media CryoStor™
NHDF mDC CHO
E�ect of DMSO Concentration on Post-Thaw Cell Viability
Cell
Viab
ility
(Rel
ativ
e to
37C
Con
trol
)
Figure 1. Viability of NHDF cells following exposure to cell culture media + 5% DMSO or CryoStor + 5% DMSO (CS5). Solution efficacy was investigat-ed following a 10 minute hold at 2–8 °C , following ice-nucleation, and after LN2 storage. For each of the test conditions, samples were removed, thawed, diluted with media and plated. Relative cell viability was determined 1 day post-thaw and overall viability was compared to 37 °C non-cryopreserved control cultures.
Figure 2. Post-thaw viability of cells following cryopreservation with varying concentrations of DMSO in either cell culture media or CryoStor. Relative cell viability was determined 1 day post-thaw as described in the methods and overall viability was compared to 37 °C non-cryopreserved control cultures. DMSO concentration and vehicle solution impact cell viability post-thaw.
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HypoThermosol®
HypoThermosol®-FRSPreservation Solution
HypoThermosol is an optimized hypothermic (2–8 °C) preservation media that enables improved and extended preservation of cells, tissues and organs. HypoThermosol-FRS is uniquely formulated to address the molecular-biological response of cells during the hypothermic preservation process.
HypoThermosol-FRS includes key ions at concentrations that balance the intracellular state at hypothermic temperatures. Additional components include pH buffers, energy substrates, free radical scavengers, and osmotic/oncotic stabilizers.store at 2–8 °C
H4416-100ML 100 mL
Figures 1 and 2 clearly show that HypoThermosol outperforms commonly used hypothermic preservation media on Human Mesenchymal Stem Cells (hMSC) and on Human Dermal Fibroblasts. In addition to these cell lines, hypothermosol has been tested on the following cell lines, Tissues and organs:
Liver Cells (Hepatocytes & C3A) •Cardiomyocytes
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•
Recovery of Human Mesenchymal Stem CellsFollowing Hypothermic Storage at 2–8 °C for either 1 day or 5 days
Cell
Viab
ility
(Rel
ativ
e to
Con
trol
)
100
80
70
60
50
40
30
20
10
0
90
HTS-FRS Celsior Viaspan HTS-FRS Celsior Viaspan
1 Day Storage 5 Day Storage
Figure 1. Cells were assayed for metabolic activity following 24 hours recovery post-preservation.
Figure 2. Cells were assayed for metabolic activity following 24 hours recovery post-preservation.
Recovery of Human Dermal Fibroblasts FollowingHypothermic Storage at 2–8 °C for either 1 day or 3 days
Cell
Viab
ility
(Rel
ativ
e to
Con
trol
)
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100
HTS-FRS Celsior Viaspan HTS-FRS Celsior Viaspan
1 Day Storage 3 Day Storage
6
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 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 medium1. FBS supplemented with 10% DMSO; freezing medium2. PBS supplemented with 1% sericin, 0.5% maltose, 0.3% proline, 0.3% glutamine and
10% DMSO; freezing medium3. Competitor A4. Competitor B5. Competitor C6. PBS supplemented with 10% DMSO. The error bars indicate S.D. (n=3). *P<0.05.
0
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Freezing media
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Freezing media
*****
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(c)
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Freezing media
**
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ility
(%)
E�ect of Sericin on Cell Cryopreservation Figure 1. P3U1 myeloma cells (1×106) in 1 mL of freezing medium were frozen at –80 °C for 1 day. The cryovials were then thawed and the viability was determined. Freezing medium1. FBS supplemented with 10%
DMSO; freezing medium2. Freezing medium 3
supplemented with 1% sericin; freezing medium
3. PBS supplemented with 0.5% maltose, 0.3% proline, 0.3% glutamine and 10% DMSO; freezing medium
4. PBS supplemented with 10% DMSO. The error bars indicate S.D. (n=3). *P<0.05.
SericinSericin is a protein derived from the silkworm cocoon that has a number of beneficial characteristics for culturing mammalian cells. It has cryoprotective properties that allow for replacement of FBS in cryopreservation media1. Figures 1, 2 and 3 clearly show that 1% sericin (w/v) along with 0.5% (w/v) maltose, 0.3% (w/v) proline, 0.3% (w/v) glutamine and 10% DMSO is comparable to 90% FBS and 10% DMSO. Furthermore, sericin can act as a FBS replacement in cell culture and stimulate 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
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40
70
100
PC-12 NHDF HEK Sf -9
Viab
ility
(%)
Figure 3. PC12, NHDF, HEK or insect Sf-9 cells (1×106) in 1 ml of freezing medium were frozen at –80 °C for 1 day. The vials were then thawed and the viability of the cells was determined. Freezing media: FBS supplemented with 10% DMSO (red bars); PBS supplemented with 1% sericin, 0.5% maltose, 0.3% pro-line, 0.3% glutamine and 10% DMSO (tan bars); and PBS supplemented with 10% DMSO (gray bars). The error bars indicate S.D. (n=3). *P<0.05.
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References
(1) Development of a novel serum-free freezing medium for mammalian cells using the silk protein sericin. Sasaki, M., et al., Biotechnol. Appl. Biochem. 42, 183–188 (2005).
(2) Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines includ-ing a hybridoma. Terada, S., et al., Cytotechnology. Nov; 40(1–3):3–12, (2002).
(3) Sericin enhances attachment of cultured human skin fibroblasts. Tsubouchi, K., et al., Biosci. Biotechnol. Biochem., Feb; 69(2):403–5, (2005).
(4) The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture. Takahashi, M., Biotechnol. Lett., Nov; 25(21): 1805–9, (2003).
Effect of Freezing Media on Various Cells
8
Cell Freezing Media and Reagents
Cell Freezing Media and Reagents
Dimethyl sulf oxideDMSO; Methyl sulf oxide [67-68-5] (CH3)2SO FW 78.13 H3C
SCH3
O
mp .................................................................................................... 16 to 19 °Cvd .........................................................................................................2.7 (vs air)bp ................................................................................................................189 °Cvp ....................................................................................0.42 mmHg (20 °C)density ............................................................................................. 1.10 g/mLait ................................................................................................................. 573 °Fn20/D .......................................................................................................... 1.479lel(63 °F) ........................................................................................................42%
≥99.7%, Hybri-Max™, sterile-filtered, hybridoma tested
DMSO is a polar aprotic solvent used in chemical reactions, in polymerase chain reactions (PCR) and as a cryoprotectant vitrification agent for the preservation of cells, tissues and organs. DMSO is used in cell freezing media to protect cells from ice crystal induced mechanical injury. It is used for frozen storage of primary, sub-cultured, and recombinant heteroploid and hybridoma cell lines; embryonic stem cells (ESC), and hematopoietic stem cells. DMSO is frequently used in the combinations with BSA or fetal bovine serum (FBS).
5 mL and 10 mL in flame sealed ampules, 100 mL in amber bottleendotoxin ............................................................................................... tested
D2650-5X5ML 5 × 5 mLD2650-5X10ML 5 × 10 mLD2650-100ML 100 mL
Biotechnology Performance Certified, sterile-filtered, hybridoma tested, meets EP, USP testing specificationsHuman and animal cell lines grown in culture are generally stored frozen. Freezing protects the cell line from changes due to genetic drift and minimizes risk of contamination. Liquid nitrogen used in conjunction with a cryoprotective agent such as DMSO is a widely used method for preserving cells. Without the presence of a cryoprotective agent, freezing is lethal to most mammalian cells. Damage is caused by mechanical injury by ice crystals, concentration
of electrolytes, pH changes, and denaturation of proteins. These lethal effects are minimized by adding a cryoprotective agent which lowers the freezing point and allows for a slower cooling rate. BPC-tested DMSO also meets the requirements of biotechnology and tissue engineering applications.
Features and Benefits
Ready-to-use sterile filtered product conveniently packaged septum screw-capped amber bottles. Meets USP and EP testing requirements
(USP XXIV)
D2438-5X10ML 5 × 10 mLD2438-50ML 50 mL
Gly cerol
1,2,3-Propane triol; Gly cer in[56-81-5] HOCH2CH(OH)CH2OH FW 92.09
HO OHOH
Glycerol is used both in sample preparation and gel formation for polyacrylamide gel electrophoresis. Glycerol (5–10%) increases the density of a sample so that the sample will layer at the bottom of a gel’s sample well. Glycerol is also used to aid in casting gradient gels and as a protein stabilizer and storage buffer component.mp ................................................................................................................. 20 °Cvd .........................................................................................................3.1 (vs air)bp ...................................................................................... 182 °C/20 mmHgvp .......................................................................................<1 mmHg (20 °C)density ............................................................................................. 1.25 g/mLait ................................................................................................................. 698 °Fn20/D .......................................................................................................... 1.474
cell culture tested, insect cell culture tested, ~99% (GC)
G2025-100ML 100 mLG2025-500ML 500 mL
D-(+)-Trehalose dihydrate
α-D-Gluco pyran osyl-α-D-gluco pyrano side; α,α-Trehalose [6138-23-4] C12H22O11 · 2H2O FW 378.33
O
OH
OH
OH
O
OH
OH
OHO
• 2H2O
HO
HO
cell culture tested, insect cell culture tested, ≥99%
Use as a cryoprotectant in a variety of cell freezing media.
T0167-10G 10 gT0167-25G 25 gT0167-100G 100 g
Sucroseα-D-Gluco pyran osyl β-D-fructo furan o side; α-D-Glc-(1→2)-β-D-Fru; D(+)-Saccharose; Sugar; β-D-Fructo furan osyl-α-D-gluco pyrano side [57-50-1] C12H22O11 FW 342.30
O
HO
HO
OHO
O
OH
HO
OH
OH HO
mp ..............................................................................................185 to 187 °C
cell culture tested, insect cell culture tested, ≥99.5%Use to create sucrose gradients for purification of viruses and proteins.
S1888-500G 500 gS1888-1KG 1 kgS1888-5KG 5 kg
meets USP testing specifications
S3929-1KG 1 kgS3929-5KG 5 kgS3929-10KG 10 kg
insect cell culture tested
S9031
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d-Sorbitol
d-Glucitol[50-70-4] C6H14O6 FW 182.17 OH
H
OH
OH
H
HO
H
H
OH
OH
May be used for washing spheroplasts1 and in isoelectric focusing to minimize endoosmotic flow in agarose gels2. May be used to induce osmotic stress.mp ................................................................................................. 98 to 100 °Cvp ................................................................................... <0.1 mmHg (25 °C)vd .........................................................................................................<1 (vs air)
≥99.5% (HPLC)Allelic variation of the Tas1r3 gene affects behavioral taste responses to this sugar alcohol, suggesting that it is a T1R3 receptor ligand.
A plasmolyticum that reversibly inhibits the phenylalanine ammonia-lyase activity of protoplasts at concentrations exceeding 50 mM1; Synchronization of Plasmodium falciparum erythrocytic stages in culture2; Increases fungal α-amylase thermostability3.insoluble matter ...........................................................passes filter test
Lit cited: 1. B. Fritig, Phytochemistry 16, 895 (1977); 2. C. Lambros, J.P. Vanderberg, J. Parisitol. 65, 418 (1979); 3. M. Graber, D. Combes, Enzyme Microb. Technol. 11, 673 (1989)
85529-250G 250 g85529-1KG 1 kg85529-5KG 5 kg
d-Lactose monohydrate
β-D-Gal-(1→4)-D-Glc; Milk sugar; 4-O-β-D-Galacto pyran osyl-D-glucose[64044-51-5] C12H22O11 · H2O FW 360.31
OHOOH
OH
HO
• H2O
OOH
OH
HO
OHO
≥99.5% (HPLC)Sales restrictions may apply
Component of the liquid stationary phase in the GLC separation of nitroxylene and xylenol isomers1
insoluble matter ...........................................................passes filter test
Lit cited: 1. A. Ono, J. Chromatogr. 197, 251 (1980);
61339-25G 25 g61339-100G 100 g61339-250G 250 g
d-(+)-Malto se monohydrate
Malto biose; 4-O-α-D-Gluco pyran osyl-d-glucose [6363-53-7] C12H22O11 · H2O FW 360.31
O
OH
OH
OH
HO
O
O
OH
OH
HO
OH • H2O
cell culture tested, insect cell culture tested, ≥98%Glucose ........................................................................................≤0.3 mol %Maltotriose ................................................................................................≤1%
M5895-500G 500 gM5895-1KG 1 kg
Hydroxy ethyl starch
[9005-27-0]
OOR
ORO
OR
n
R = H or *OH
Degree of substitution; Average of 1 -OH group per 10 glucose units converted to -OCH2CH2OH.
Prepared from corn starch
H6382-250G 250 gH6382-1KG 1 kg
Dextran from Leuconostoc spp.[9004-54-0] (C6H10O5)n
Mr ~6,000
31388-25G 25 g31388-100G 100 g31388-500G 500 g
DEXTRAN 40 Mr ~40,000
31389-25G 25 g31389-100G 100 g31389-500G 500 g
DEXTRAN 70 Mr ~70,000Enhances immunoprecipitation sensitivity1
Lit cited: 1. M.-F. Dumon et al., Electrophoresis 11, 846 (1990)
31390-25G 25 g31390-100G 100 g31390-500G 500 g
Mr ~100,000May contain greater parts of lower molecular weight.
09184-10G-F 10 g09184-50G-F 50 g09184-250G-F 250 g
DEXTRAN 500 Mr ~500,000
31392-10G 10 g31392-50G 50 g31392-250G 250 g
Mr 15,000–25,000
31387-25G 25 g31387-100G 100 g31387-500G 500 g
Mr ~40,000narrow molecular weight distribution
68084-25G 25 g68084-100G 100 g68084-500G 500 g
Hydroxy ectoine
(4S,5S)-5-Hydroxy-2-methyl-1,4,5,6-tetra hydro pyrimi dine-4-carboxy lic acid; THP(A)[165542-15-4] C6H10N2O3 FW 158.16
N
NH
O OH
HO
CH3
≥98.0% (HPLC)Stabilizing effect against various stress factors; used e.g. in the expression of proteins1,2
Lit cited: 1. J.R. Blackwell, R. Hogan, FEBS Lett. 295, 10 (1991); 2. K. Lippert, E.A. Galinski, Appl. Microbiol. Biotechnol. 37, 61 (1994)
55243-100MG 100 mg
≥95% (HPLC)Stabilizer for enzymes and biological macromolecules1,2,3
Lit cited: 1. K. Lippert, E.A. Galinski, Appl. Microbiol. Biotechnol. 37, 61 (1992); 2. T. Sauer et al., GIT Labor-Fachzeit. 39, 892, 894 (1995); 3. M.D. Vangala et al., Bioforum 23, 760 (2000)
70709-1G-F 1 g70709-10G-F 10 g70709-100G-F 100 g
10
Ectoine
Thp(B); (S)-2-Methyl-1,4,5,6-tetra-hydro pyrimi dine-4-carboxy lic acid [96702-03-3] C6H10N2O2 FW 142.16 N
NH
O OH
CH3
An osmoprotectant in a wide variety of microorganisms including heterotrophic, halophilic bacteria and non-halophilic bacteria such as Streptomyces species and E. coli. It also exerts protective effects in E. coli during drying and storage.
E2271-25MG 25 mgE2271-100MG 100 mg
≥99.0% (HPLC)
44788
≥95.0% (HPLC)Stabilizer for enzymes and biological macromolecules1,2,3
Lit cited: 1. K. Lippert, E.A. Galinski, Appl. Microbiol. Biotechnol. 37, 61 (1992); 2. T. Sauer et al., GIT Labor-Fachzeit. 39, 892, 894 (1995); 3. M.D. Vangala et al., Bioforum 23, 760 (2000)
81619-1G-F 1 g81619-10G-F 10 g81619-100G-F 100 g
Betaine
Oxyneurine; (Carboxy methyl)tri methyl ammo nium inner salt [107-43-7] (CH3)3N
+CH2COO- FW 117.15
O-
O
NCH3
CH3
H3C
anhydrous, ≥98.0% (NT)water ..............................................................................................................≤1%mp .................................................................................301 to 305 °C (dec.)
14290
Poly vinyl pyrrolidone
PVP; Poly vidone; Povidone [9003-39-8] (C6H9NO)n
NH
H
O
n
Polyvinylpyrrolidone is a component of Denhardt′s Solution and is included at a concentration of 1% (w/v) in the standard 50X stock solution.
mouse embryo testedaverage mol. wt. 40,000P0930-50G 50 gP0930-100G 100 g
Cell Freezing Medium-DMSO 1×
sterile-filtered, cell culture testedComplete ready-to-use medium. Proprietary formulation with Minimum Essential Medium, dimethyl sulfoxide, calf serum and fetal bovine serum.
Recommended for the cryopreservation of cells.endotoxin ............................................................................................... testedstore at −20°C
C6164-50ML 50 mLC6164-6X50ML 6 × 50 mL
Cell Freezing Medium-DMSO Serum free 1×
sterile-filtered, cell culture testedComplete ready-to-use medium containing Minimum Essential Medium, dimethyl sulfoxide (8.7%) and methyl cellulose. Prepared according to the formulation of Waymouth1.
Recommended for the cryopreservation of cells.endotoxin ............................................................................................... tested
Lit cited: 1. Waymouth, C. and Varnum, D., Simple freezing procedure for storage in serum-free media of cultured and tumor cells of mouse. TCA Manual (1976), 2(1), 311–313store at −20 °C
C6295-50ML 50 mL
Cell Freezing Medium-Glycerol 1×
sterile-filtered, cell culture tested, endotoxin testedComplete ready-to-use medium. Proprietary formulation containing Minimum Essential Medium, glycerol, calf serum and fetal bovine serum.
Recommended for the cryopreservation of cells.store at −20 °C
C6039-50ML 50 mL
Cell Freezing Medium-Serum-free 1×
sterile-filtered, cell culture testedReady-to-use formula that does not contain dimethyl sulfoxide or serum.
For use with cells grown in serum-free conditions.store at 2–8 °C
C2639-50ML 50 mL
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Sigma® and ECACC have partnered to bring you a great selection of high quality cell lines. We are expanding our offering of stocked cell lines to the list below in the U.S. and plan to
ECACC Cell Lines
ECACC Cell Lines
expand the offering to up to 1500 cell lines later this year. Check our website often for updates. Sigma now supplies ECACC cell lines to Europe, China, Australia, New Zealand, U.S.
and Canada. The following is a sampling of the products that Sigma can offer from ECACC.
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
ECACC Cell Lines
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 LinesAim
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.DCorning Incorporated, Life SciencesActon, 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;
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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.
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.
biomolecules
Bioselect.
wherebiobegins.com/biomolecules
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16
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
blue
C0812-100EA 100 ea
green
C0937-100EA 100 ea
red
C1062-100EA 100 ea
white
C1187-100EA 100 ea
yellow
C1312-100EA 100 ea
Cryo Containers
Nalgene® CryoBox™
For ultra-low temperature storage of cryogenic vials in mechanical or liquid nitrogen freezers. Usable temperature range: −196 to +121 °C. Numbers molded into grid system in box correspond to numbers printed on lid. Lid is keyed to box so markings will always match. Boxes accept writing with markers.
polycarbonate
5 × 5 array for 1.2 and 2.0 mL vialsL × W × H .....................................................................3 in. × 3 in. × 2 in.
R0763-8EA 8 ea
CryoCane™ alu mi num canes
Firmly holds 1.2 or 2.0 mL Nalgene cryogenic vials for storage in Dewar-type, liquid nitrogen freezers.
capacity: 5 vialsProjections support base of each vial.overall L............................................................................................... 290 mm
C9811-12EA 12 ea
Cryoware
CryowareCryovial Color Coders
9 × 9 Array For 1.2 and 2.0 mL vialsL × W × H ........................................................ 5 1/4 in. × 5 1/4 in. × 2 in.
R0888-4EA 4 ea
9 × 9 Array For 5.0 mL vialsL × W × H .................................................. 5 1/4 in. × 5 1/4 in. × 3 3/4 in.
R1013-4EA 4 ea
CryoBox 100, 10 × 10 arrayFor ultra-compact storage of 1.0 mL and 1.5 mL System 100 vials or other similar-sized vials. Box fits most existing vertical racks with 2 in. shelf spacing.L × W × H .................................................5 1/4 in. × 5 1/4 in. × 2 1/16 in.
Z359017-10EA 10 ea
CryoCane™ alu mi num canes
Firmly holds 1.2 or 2.0 mL Nalgene cryogenic vials for storage in Dewar-type, liquid nitrogen freezers. capacity: 6 vialsoverall L............................................................................................... 300 mm
C9936-12EA 12 ea
CryoSleeve™ clear PVC cane sleeve
Will not become brittle when frozen.
Encloses a Nalgene CryoCane™ for extra security during handling and storage. Easy to install and remove.
C1437-100EA 100 ea
Freezing container, Nalgene® Mr. Frosty
Polycarbonate container, blue high-density polyethylene closure, white high-density polyethylene vial holder, and foam insert.
Provides the critical, repeatable, 1 °C/min cooling rate required for successful cryopreservation of cells. Easy to use in any mechanical freezer. Container is imprinted with graphic instructions. Requires only isopropyl alcohol. Rigid vial holder keeps vials from contacting alcohol, preventing contamination by wicking. Vial holder floats to allow thawing in a water bath. Numbers molded for identification. All components withstand repeated freeze/thaw cycles. Stackable.
Holds 18 1.2 and 2.0 mL cryovialsH × diam .....................................................................86 mm × 117 mm
C1562-1EA 1 ea
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Online Metabolomics Resource Use the IUBMB–Sigma-Aldrich Interactive Metabolic Pathway Chart to find the Metabolite Standards you need.
Cryo Storage Racks
Cryogenic storage rack, stainless steel
Racks hold CryoBoxes securely but separately, allowing fast retrieval and immediate return to freezer. The vertical racks are best for use in chest freezers, horizontal racks for upright freezers.
Horizontal; 12-shelf for 5 1/4 × 5 1/4 × 2 in. boxesoverall H ................................................................................................8 7/8 in.
R1388-2EA 2 ea
Rack for Nalgene® cryogenic vials
Accept all Nalgene cryogenic vials. Bottom of each well interlocks with vial, allowing one-hand manipulation.
polycarbonateautoclavable ................................................................................................Yes
Holds 50 vials in a 5 × 10 arrayNon-skid rubber feetcolor ............................................................................................................ white
R0638-1EA 1 ea
Horizontal; 16-shelf for 5 1/4 × 5 1/4 × 2 in. boxesoverall H .................................................................................................8 7/8 in.
R1513-2EA 2 ea
Vertical; 2-shelf for 5 1/4 × 5 1/4 × 3 3/4 in. boxesoverall H .................................................................................................8 1/4 in.
R2888
Holds 25 vials in a staggered arrayWider spacing for easier manipulation color ...............................................................................................................blue
Z359041-4EA 4 ea
Vertical, 4-shelf for 3 × 3 × 2 in. boxesoverall H .................................................................................................8 3/4 in.
R1638-1EA 1 ea
Vertical, 4-shelf for 5 1/4 × 5 1/4 × 2 in. boxesoverall H..............................8 3/4 in.
R1763-1EA 1 ea
Vertical; 4-shelf for 5 1/4 × 5 1/4 × 33/4in. boxesoverall H ................................................................................................... 16 in.
R3388-1EA 1 ea
Vertical; 7-shelf for 5 1/4 × 5 1/4 × 3 3/4 in. boxesoverall H .............................................................................................27 3/4 in.
R3138-1EA 1 ea
Vertical, 9-shelf for 3 × 3 × 2 in. boxesoverall H ..............................................................................................19 3/4 in.
R1888-1EA 1 ea
Vertical, 9-shelf for 5 1/4 × 5 1/4 × 2 in. boxesoverall H ..............................................................................................19 3/4 in.
R2013-1EA 1 ea
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
Note: Do not use cryogenic vials for storage in the liquid phase of liquid nitrogen unless correctly sealed in Nunc CryoFlex™ tubing. Such use may cause entrapment of liquefied nitrogen inside the vial and lead to pressure build-up resulting in possible explosion or biohazard release.
polypropylene vials
high-density polypropylene closures
1.2 mL, sterilepackage = 25 vials 20
V4757-500EA 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 vialspolypropylene capssilicone 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 vialssilicone gasketpolypropylene caps
capacity 1.5 mL
Z359033-500EA 500 ea
Nalgene® specimen vial
capacity 15 mLVials 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
1.2 mL, non-sterileNon-sterile vials and closures are packed separately. Vials have no printing or graduations.
V4507-1000EA 1000 ea
2.0 mL, non-sterileNon-sterile vials and closures are packed separately. Vials have no printing or graduations.
V4632-1000EA 1000 ea
Rack for Nalgene® micro centrifuge tubes
Bottom of white polycarbonate, lid of clear polycarbonate, permanent foam insert.
For safe storage of samples in microcentrifuge tubes from −135 °C to +121 °C. Not for use in liquid-phase liquid nitrogen. May be repeatedly autoclaved. Holes in foam are placed diagonally and spaced to avoid overlapping of tube closures. Lid accepts writing with marker.size × H ............................................................. 5 1/4 in. × 5 1/4 in. × 2 in.
capacity: 81 0.5-mL microcentrifuge tubes
R1138-4EA 4 ea
capacity: 64 1.5-mL microcentrifuge tubes
R1263-4EA 4 ea
Cryo Vials
Nalgene® cryogenic vials
Vials designed for exceptional ease of use and sample integrity in ultra-low temperature storage. Closure seals without gasket or O-ring, minimizing risk of contamination. The deep skirt of the closure allows single-handed aseptic technique without exposing fingers to vial contents. Ribs around the bottom of the vial interlock with wells in the Cryovial Racks so the vial will not turn when opening or closing. Bottoms of vials are skirted; vial stands without holder.
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.
1.2 mL, sterile, pkg = (Bulk packed)
V4882-1000EA 1000 ea
2.0 mL, sterilepackage = 25 vials 20
V5007-500EA 500 ea
2.0 mL, sterile, pkg = (Bulk packed)
V5132-1000EA 1000 ea
5.0 mL, sterilepackage = 10 vials 25
V5257-250EA 250 ea
Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 19
Quick Chill™ cooler
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.
Z359068-1EA 1 ea
Cryo Accessories
Dewar 35L accessories
aluminum cane without tube stops, diam. 1.2 cm × L 29 cmHolds tubes totalling approx. 28 cm in height.
C3296
Plastic storage boxRugged alternative to cardboard storage boxes; numbered grid printed on topsize ........................3 in. (7.6 cm) × 3 in. (7.6 cm) × 2 in. (5.1 cm)
S7397-8EA 8 ea
aluminum cane with tube stopsHolds 5 tubes, each up to approx. 5 cm in height.diam. × L..............................1.2 cm × 29 cm
C3171-50EA 50 ea
Cryoclaw
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
Aldrich® wide-mouth dewar flasks
Borosilicate glass with protective aluminum or plastic mesh housing and extended aluminum base weighted for stability.
capacity 350 mL, aluminum housingI.D. ...............................................................................................................7.0 cmD ............................................................................................................... 11.3 cm
Z120758-1EA 1 ea
capacity 665 mL, aluminum housingD ............................................................................................................... 19.5 cmI.D. ...............................................................................................................7.0 cm
Z120774-1EA 1 ea
capacity 1,000 mL, aluminum housingI.D. ...............................................................................................................7.0 cmD ............................................................................................................... 30.2 cm
Z120790-1EA 1 ea
Benchtop Coolers
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.
−20 °C Labtop cooler, gel-filled lidMaintains 32 microtubes below −15 °C for up to 2 hr at room temperature.
C2312-1EA 1 ea
0 °C Labtop cooler, gel-filled lidMaintains 32 microtubes below 1 °C for up to 5 hr at room temperature.
Z359084-1EA 1 ea
−20 °C Labtop cooler, Jr., transparent lidMaintains 12 microtubes below −15 °C for up to 1 hr at room temperature.
C2437-1EA 1 ea
0 °C Labtop cooler, Jr., transparent lidMaintains 12 microtubes below 1 °C for up to 3 1/2 hr at room temperature.
Z359076-1EA 1 ea
0 °C Labtop cooler, clear lidMaintains twelve 12–13 mm diameter tubes below 1 °C for up to 5 hr at room temperature.
Z370509-1EA 1 ea
0 °C Labtop cooler, clear lidMaintains twelve 16–17 mm diameter tubes below 2 °C for up to 4 hr at room temperature.
Z370517-1EA 1 ea
20
sigma-aldrich.com
capacity 4,300 mL, Plastic mesh housingD. .............................................................................................................. 30.0 cmI.D. ............................................................................................................ 14.3 cm
Z120782-1EA 1 ea
Barnstead/Thermolyne® Dewars, Biocane™ Series
Barnstead/Thermolyne® Dewars,Biocane™ series
A can and cane cryopreservation system with features to meet the requirements of almost any research lab: available in five sizes for small- to large-scale sample storage; unsurpassed temperature uniformity with low liquid nitrogen consumption rates; secure locking hasp prevents unauthorized access; each Biocane vessel includes six stainless steel canisters that accommodate Nalgene CryoCanes or similar storage systems. CryoCanes not included.
Biocane, 11product of Barnstead CK509X1diam. × H. ..................................10.2 in. (26 cm) × 21.6 in. (55 cm)capacity ........................................................................................................11 Lneck size .......................................................................................2 in. (5 cm)static liquid nitrogen .......0.14 L/day (average consumption)storage ................................................................................................180 vials
F1286-1EA 1 ea
Biocane, 20product of Barnstead CK509X2capacity ........................................................................................................20 Lneck size .......................................................................................2 in. (5 cm)diam. × H ................................14 1/2 in. (37 cm) × 25.7 in. (65 cm)static liquid nitrogen .......0.09 L/day (average consumption)storage ................................................................................................180 vials
F1411-1EA 1 ea
capacity 1,900 mL, aluminum housingI.D. ............................................................................................................ 12.0 cmD ............................................................................................................... 19.8 cm
Z120766-1EA 1 ea
capacity 4,300 mL, aluminum housingI.D. ............................................................................................................ 14.3 cmD ............................................................................................................... 30.0 cm
Z103128-1EA 1 ea
capacity 350 mL, Plastic mesh housingI.D. ...............................................................................................................7.0 cmD ............................................................................................................... 12.5 cm
Z120669-1EA 1 ea
capacity 665 mL, Plastic mesh housingI.D. ...............................................................................................................7.0 cmD ............................................................................................................... 19.5 cm
Z120693-1EA 1 ea
capacity 1,000 mL, Plastic mesh housingI.D. ...............................................................................................................7.0 cmD ............................................................................................................... 30.2 cm
Z120707-1EA 1 ea
capacity 1,900 mL, Plastic mesh housingD. .............................................................................................................. 19.8 cmI.D. ............................................................................................................ 12.0 cm
Z120677-1EA 1 ea
The new Corning Epic System
Corning® Cell CultureMaking your research possibilities real
HYPER• Flask® — Grow enough cells in one 1,720 cm2 HYPERFlask Cell Culture Vessel to seed 2,000 microplates.
Epic System• — The Corning Epic System, a new high-throughput, label-free detection technology, allows researchers to bring drugs to market faster.
CellBIND®• — Improve cell attachment and yield with the Corning CellBIND surface, a nonbiological surface with high oxygen levels.
Corning, CellBIND and HYPERFlask are registered trademarks of Corning.
For more information on our Corning product offerings, visit sigma-aldrich.com/corning
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Biocane, 34product of Barnstead CK509X3capacity ........................................................................................................34 Ldiam. × H ...................................18.2 in. (46 cm) × 26.6 in. (68 cm)neck size .................................................................................3 1/2 in. (9 cm)static liquid nitrogen .......0.17 L/day (average consumption)storage ................................................................................................720 vials
F1536-1EA 1 ea
Biocane, 47product of Barnstead CK509X4neck size ................................................................................ 5 in. (12.7 cm)capacity ........................................................................................................47 Ldiam. × H ....................................20 in. (51 cm) × 26 1/2 in. (67 cm)static liquid nitrogen .......0.37 L/day (average consumption)storage .............................................................................................1512 vials
F1661-1EA 1 ea
Cart for transporting Dewars safelyAccepts Biocane 20, 34, and 47
product of Barnstead AY509X9
Z363642
Barnstead/Thermolyne® Dewars, Locator® SeriesBarnstead/Thermolyne® Dewars,Locator® series
For ultra-cold storage of biological specimens in liquid nitrogen. Not to be used for transportation of samples.
Features: Three sizes fit the needs of almost any •research lab
Advanced vacuum insulation provides a •maximum −190 °C throughout flask with as little as 2 inches of liquid nitrogen, and delivers improved liquid nitrogen consumption rates
Secure locking clasp prevents unauthorized •access
Each Locator vessel includes stainless steel racks •that accommodate Nalgene Cryoboxes for 2 mL freezing vials. Cryoboxes not included
Locator Jr., 50L flaskproduct of Barnstead International CY50925diam. × H .......................................... 22 in. (56 cm) × 26 in. (66 cm)neck size. .........................................................................8 1/2 in. (21.6 cm)static liquid nitrogen ................................................ 0.65 L/day (avg.)storage .............................................................................................1296 vials
F0911-1EA 1 ea
Locator 4, 110L flaskproduct of Barnstead International CY50935neck size .......................................................................... 8 1/2 in. (21.6 cm)diam. × H .......................................... 22 in. (56 cm) × 36 in. (91 cm)static liquid nitrogen ................................................ 0.65 L/day (avg.)storage .............................................................................................2916 vials
F1036-1EA 1 ea
Locator 8, 110L flaskproduct of Barnstead International CY50945neck size ................................................................................. 6 in. (156 cm)diam. × H .......................................... 22 in. (56 cm) × 36 in. (91 cm)static liquid nitrogen ...................................................0.4 L/day (avg.)storage .............................................................................................1800 vials
F1161-1EA 1 ea
Four-wheel roller baseproduct of Barnstead International AY509X1
R9640-1EA 1 ea
Nalgene® DewarsNalgene® Dewars
Shatterproof. Safe for short-term storage of ice water, dry ice/solvent baths, and liquid nitrogen; can also be used as a hot bath. Temperature range: −196 to +100 °C. Cover is vented. Polyethylene-coated handle on 1, 2, and 4 L sizes only.
Style 4150.
Dimensions are inside depth × I. D.
urethane foam-filled high-density polyethylene
capacity 1 Lpolyethylene handlesize ..........................................7 5/8 in. (194 mm) × 3 3/4 in. (95 mm)
F9401-1EA 1 ea
capacity 2 Lpolyethylene handlesize ....................................... 8 7/8 in. (225 mm) × 4 3/4 in. (121 mm)
F9526-1EA 1 ea
capacity 4 Lpolyethylene handlesize .....................................11 7/8 in. (287 mm) × 5 3/4 in. (146 mm)
F9651-1EA 1 ea
capacity 10 Lno handlesize .....................................15 1/2 in. (394 mm) × 7 3/4 in. (197 mm)
F9776-1EA 1 ea
Taylor-Wharton Dewars
Taylor-Wharton Dewars
For storage of small quantities of liquid nitrogen. High-strength aluminum shell is both durable and light-weight. Super insulation provides advanced thermal protection. Neck tube joins inner and outer container, providing maximum durability and minimum nitrogen consumption. Cap and neck tube core provide positive closure. Accessories sold separately.
capacity 4 Lpitcher stylediam. × H ..................................19.3 cm (7.6 in.) × 43.2 cm (17 in.)neck I.D ................................................................................. 3.0 cm (1.2 in.)
F9018-1EA 1 ea
capacity 10 Lbeaker styleneck 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 25 Lbeaker stylediam. × H ...........................39.7 cm (15.6 in.) × 65.5 cm (25.8 in.)neck I.D ................................................................................. 6.4 cm (2.5 in.)
F4524-1EA 1 ea
22
capacity 35 Lcapacity to hold 6 × 11-inch canisters
beaker style
wide-mouthneck I.D ...............................................................................11.9 cm (4.7 in.)diam. × H ...........................47.8 cm (18.8 in.) × 64.8 cm (25.5 in.)
F2344-1EA 1 ea
Wide-mouth Dewars
Wide-mouth Dewars
Detachable handle and lid (lids do not fasten to the body of the flask).
stainless steel
capacity 0.5 LDoes not have handle.inside dimensions D × H .......................2.5 in. (65 mm) × 7.1 in.(180 mm)outside dimensions D × H ........................3.4 in. (87 mm) × 8 in.(204 mm)
D3920-1EA 1 ea
capacity 1 Linside dimensions D × H .......................3.3 in. (85 mm) × 8.1 in.(206 mm)outside dimensions D × H .................4.2 in. (107 mm) × 9.1 in. (232 mm)
D4045-1EA 1 ea
1 Lextra wide mouthoutside dimensions D × H .....................4.8 in. (122 mm) × 7 in. (177 mm)inside dimensions D × H ........................4 in. (100 mm) × 6.2 in.(157 mm)
D4170-1EA 1 ea
capacity 2 Loutside dimensions D × H .............. 4.8 in. (116 mm) × 12.3 in. (228 mm)inside dimensions D × H .....................4 in. (100 mm) × 11.2 in. (285 mm)
D4295-1EA 1 ea
Wide-mouth Dewars with Silvered Glass LinerWide-mouth Dewars with silveredglass liner
Straight sides, a swing-over carrying handle, and no neck constriction; suitable for benchtop immersion applications. Vented insulating lids clamp to the body of the flask.
1 L, stainless steel jacket and vented capoutside 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
2 L, stainless steel jacket and vented capinside dimensions D × H ................... 4.2 in. (106 mm) × 8.1 in. (205 mm)outside dimensions D × H ............. 5.6 in. (142 mm) × 10.5 in. (268 mm)
D3295-1EA 1 ea
4.5 L, stainless steel jacket and vented capinside dimensions D × H .....................5.9 in. (150 mm) × 11 in. (280 mm)outside dimensions D × H .............. 7.2 in. (182 mm) × 10.5 in. (350 mm)
D3420-1EA 1 ea
1 L, Blue enamelled steel jacket and vented capoutside 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)
D3545-1EA 1 ea
2 L, Blue enamelled steel jacket and vented capinside dimensions D × H ................... 4.2 in. (106 mm) × 8.1 in. (205 mm)outside dimensions D × H ............. 5.6 in. (142 mm) × 10.5 in. (268 mm)
D3670-1EA 1 ea
4.5 L, Blue enamelled steel jacket and vented capinside dimensions D × H .................... 5.9 in. (150 mm) × 11 in. (280 mm)outside dimensions D × H ............. 7.2 in. (182 mm) × 10.5 in. (350 mm)
D3795-1EA 1 ea
Taylor-Wharton Dewar Accessories
Taylor-Wharton Dewar accessories
Cap for 4 L Dewar
C0671-1EA 1 ea
Cap/necktube core for 10 L Dewar
C0796-1EA 1 ea
Tipping stand for 25 L DewarThis stand supports the Dewar and allows convenient, safe pouring of liquid. Wheels add mobility and additional handling ease.
S8777-1EA 1 ea
Cap/necktube core for 25 L Dewar
C7431-1EA 1 ea
Cap/necktube core for 35 L Dewar
C0544-1EA 1 ea
Cast aluminum roller base with 5 wheels for 35 L Dewar
R0509-1EA 1 ea
Stainless steel canister for 35 L DewarFor storage and easy retrieval of vials in canes (canes not included).size ..................................................3.7 in. (9.4 cm) × 11 in. (27.9 cm)
C3421-1EA 1 ea
Measuring rod for 35 L DewarFor determination of liquid nitrogen level.
M9904-1EA 1 ea
Rack, 5-shelf for 35 L DewarEach shelf holds one 3 × 3 × 2 in. (7.6 × 7.6 × 5.1 cm) box with a capacity of 25 x 2mL vials. A spring-action retaining clip holds each box in place. Boxes not included.
R0384-1EA 1 ea
Cardboard storage box for 35 L Dewarsupplied with 25 position insert.size .........................3 in. (7.6 cm) × 3 in. (7.6 cm) × 2 in. (5.1 cm)
S7272-5EA 5 ea
sigma.com/cellculture
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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©2010 Sigma-Aldrich Co. All rights reserved. SIGMA, SAFC, SIGMA-ALDRICH, ALDRICH, FLUKA, and SUPELCO are trademarks belonging to Sigma-Aldrich Co. and its affiliate Sigma-Aldrich Biotechnology, L.P. Sigma brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip. Biocane is a trademark of Barnstead/Thermolyne Corp. Locator and Thermolyne are registered trademarks of Barnstead/Thermolyne Corp. Biomek is a registered trademark of Beckman Instruments, Inc. CryoStor is a trademark of BioLife Solutions, Inc. HypoThermosol is a registered trademark of BioLife Solutions, Inc. CellBIND, Corning and HYPER Flask are registered trademarks of Corning, Inc. Eppendorf is a registered trademark of Eppendorf-Netheler-Hinz GmbH. Cy, Sepharose are registered trademarks of GE Healthcare. nanoHA, Select-HA are trademarks of Hyalose LLC. Coomassie is a registered trademark of Imperial Chemical Industries Ltd. Alexa, SYBR, and Texas Red are registered trademarks of Molecular Probes, Inc. Nalgene, CryoBox, CryoCane, CryoFlex, CryoSleeve and Quick Chill are a registered trademarks of Nalge Nunc International. Nanomiser is a trademark of nGimat. Shimadzu is a registered trademark of Shimadzu Corporation. Aldrich, Prestige Antibodies, Ampliflu, EZBlue, GlycoProfile, HumanKine, Hybri-Max, Material Matters, ProteoMass, ProteoSilver, and Sigma are trademarks of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. Freedom Evo is a registered trademark of Tecan. Bright-Line is a trademark of Warner-Lambert Technologies, Inc.
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