Brian Fuchs Research Mentor: Dr. Adam Higgins

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Presentation transcript:

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

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)

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

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

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.

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

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.

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

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

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

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

COOLING RATE SURVIVAL Solution Effects IIF

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 5 ºC/min. CRF process is ready for use on cultured neurons.

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