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 °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 Some Repositories Prefer Glass
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 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