Cryopreservation

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

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

• 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%

Cell Concentration And Freezing Medium

• 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

Cooling Rate

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

• 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

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

Thawing Stored Ampoules