1. Brian Fuchs Research Mentor: Dr. Adam Higgins
Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation.
Brian Fuchs
Research Mentor: Dr. Adam Higgins

2. Cryopreservation
Long-term storage of living material at extremely low temperatures
Cryopreservation is currently implemented in:
Artificial insemination
Storage of certain types of cells (e.g. blood cells)

3. Future Applications Future applications of cryopreservation are:
Long term storage of tissues
Long term storage of organs
Use in cell-based biosensors

4. Problems with Freezing Process
2 main types of cellular damage:
Intracellular ice formation (IIF)
Damages membranes and cell structure
Cellular dehydration and solution effects
3rd type of damage is extracellular ice formation.
Typically is significant only in tissue freezing

5. Vitrification
Vitrification is the process of freezing a substance to a point where it becomes a glass like amorphous solid
Prevents death due to IIF.

6. 2 Treatments 2 ways being investigated to prevent cell damage:
Addition of cryoprotection agents (CPA)
Adjustment of cooling rates

7. CPA CPA’s are chemicals that are permeable to cellular membrane
Help to depress freezing point and prevent ice crystal formation
Some examples are glycerol and DMSO.

8. Cooling Rate Goal: determine cooling rate for optimal cell viability.
High cooling rate  intracellular ice formation (IIF)
Low cooling rate  cellular dehydration and solution effects
COOLING RATE
SURVIVAL
Solution Effects
IIF

9. Hypothesis
The optimum cooling rate for maximal endothelial cell viability is about 5 ºC/min.

10. Process Culture cells on a slide Add CPA
Run controlled rate freezing process
Thaw cells
Perform live-dead staining

11. Live/Dead Stain Controls
Live cells stained with ethidium homodimer
Live cells stained with calcein-AM
Dead cells stained with calcein-AM
Dead cells stained with ethidium homodimer

12. COOLING RATE
SURVIVAL
Solution Effects
IIF

13. Conclusion
There is a significant correlation between cooling rate and cell viability.
Of the experiments performed, cooling rates of 5 ºC/min provided maximum cell recovery.
More experiments are needed to determine if cell viability decreases at cooling rates lower than ºC/min.
CRF process is ready for use on cultured neurons.

14. Acknowledgements Dr. Adam Higgins Allyson Fry
Nadeem Houran, Austin Rondema, Ingemar Hudspeth
Dr. Kevin Ahern
Howard Hughes Medical Institute