1 / 15

Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation.

Brian Fuchs Research Mentor: Dr. Adam Higgins. Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation. . Cryopreservation. Long-term storage of living material at extremely low temperatures. Cryopreservation is currently implemented in: Artificial insemination

freya
Download Presentation

Effect of Cooling Rate on the Viability of Cultured Cells After Cryopreservation.

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  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 Solution Effects IIF SURVIVAL COOLING RATE

  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. Solution Effects IIF SURVIVAL COOLING RATE

  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 5 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

More Related