Cryopreservation

1. Cryopreservation

2. Benefits Of Freezing Cells • Benefits Of Freezing A Validated Stock Of Cells • Genotypic drift • Senescence leading to extinction of cell line • Transformation to tumor related properties • Contamination • Distribution to others • Saving reagents, time • Equipment failure such as incubator • Cross-contamination by other cell lines

3. Theoretical Background Of Cell Freezing • Optimal Cell Freezing Is Characterized By • Maximum number of viable cells upon thawing • Minimum intracellular crystal formation • Minimum formation of foci of high solute concentration • Optimal Freezing Is Accomplished By • Cooling slowly so water escapes • Cooling fast enough to avoid crystal formation • Use hydrophylic cryoprotectant • Storing at lowest possible temperatures • minimize negative effect of solute foci on proteins • Thaw rapidly • minimize crystal growth and solute gradients

4. Cell Concentration And Freezing Medium • High Cell Concentration Seems To Enhance Survival • Possibly due to “leakiness” effect from cryogenic damage • Centrifugation is avoided since dilution of cryoprotectant is high when reseeding • Ex. 1 mL of 1x107 cells diluted to 20 mL volume giving a 5x105 cells/mL. If cryoprotectant was 10% it will become 0.5% • Toxicity is unlikely at 0.5% • Residual cryoprotectant can be removed as soon as cells start growing • Freezing Medium • DMSO, Glycerol • DMSO used at 5-15%, 10% is common • DMSO should be stored in glass or polypropylene • Can dissolve rubber and some plastics leading to impurities • Many laboratories increase FBS concentration to 40, 50 or 90%

5. Cooling Rate • Optimal Cooling Rate: 1°C/minute • Compromise between fast freezing minimizing crystal formation and slow allowing for extracellular water migration • Cooling Curve Is Affected By • Ambient temperature • Insulation • Specific heat of ampoule contents, volume of ampoule • latent heat absorption during freezing

7. Insulation During Freezing • Use 2 Polystyrene Foam Boxes To Store Vials • This set up provides insulation for 1°C/minute cooling • Place In A –80°C Freezer • Transfer To Liquid Nitrogen After 24 Hrs Or -150°C Freezer • If At Kean Just Leave It In The -80 °C • Liquid Nitrogen Is Widely Used To Store Cells Long Term • -196 °C • Several types of liquid nitrogen cryofreezers are available

8. Ampoules • Use polypropylene cryovials • Resistant to cracking • Some Repositories Prefer Glass • Better properties for long term storage • Labeling Is Very Important • Stored cells can outlive you! Proper labeling is essential • In your label include • Cell type, date and cell number • Use an alcohol resistant marker

9. Thawing Stored Ampoules • Thawing Of Cells Should Be Rapid • Water bath @ 37°C • Reason: minimize crystal formation • Spray Vial With Alcohol To Avoid Contamination • Dilution Should Be Done Slowly • DMSO will cause severe osmotic damage if done fast • Most Cells Do Not Require Centrifugation • Some Do Require Centrifugation • Ex. suspension growing cells