1 / 19

Cameron Glasscock

Determining cryoprotectant toxicity with adherent endothelial cells. http://www.pages.drexel.edu/~nb93/images/heart.gif. Source: http://www.2n2u.com/wp-content/uploads/2011/02/Vascular.jpg. Source: http://www.pages.drexel.edu/~nb93/images/heart.gif. Cameron Glasscock. Oregon State University

brian
Download Presentation

Cameron Glasscock

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. Determining cryoprotectant toxicity with adherent endothelial cells http://www.pages.drexel.edu/~nb93/images/heart.gif Source: http://www.2n2u.com/wp-content/uploads/2011/02/Vascular.jpg Source: http://www.pages.drexel.edu/~nb93/images/heart.gif Cameron Glasscock Oregon State University Chemical, Biological, and Environmental Engineering Mentor: Dr. Adam Higgins HHMI Summer 2011

  2. Cryopreservation • Storage of biological materials • Tissue engineering, transplantation medicine, and other cell-based therapies • The problem: Ice crystal formation causes damage Source: http://en.wikipedia.org/wiki/File:Iceman_(Bobby_Drake).png

  3. Cryoprotectant chemicals • Reduces damage caused by ice crystal formation • Vitrification • Addition and removal causes two types of damage • Osmotic damage • Toxicity damage Source: http://blog.bioethics.net/cryopreservation.jpg

  4. Project • Goal: Determine toxicity of cryoprotectant chemicals with adherent endothelial cells. • Hypothesis: Cryoprotectant type, concentration, temperature, and exposure time have an effect on cryoprotectant toxicity Glycerol Source: http://www.benbest.com/cryonics/DMSO.jpg Source: http://www.bmrb.wisc.edu/metabolomics/standards/glycerol/lit/3416.png

  5. Procedures • Endothelial cells seeded onto well plates • Exposure to cryoprotectant solutions Source http://www.porvair-sciences.com/acatalog/205003_1.jpg Source: http://us.123rf.com/400wm/400/400/phakimata/phakimata0806/phakimata080600061/3131934-blue-multi-channel-pipet-used-for-pipetting-a-96-well-plate-with-pink-solution-on-white.jpg

  6. Procedures (Continued…) • Toxicity damage needs to be isolated from osmotic damage • Multi-step addition/removal during cryoprotectant exposure • Predict procedures with permeability and osmotic tolerance limits data Source: http://www.ccs.k12.in.us/chsteachers/amayhew/Biology%20Notes/transport%20notes.htm

  7. Procedures (Continued…) • Toxicity measured using fluorescent cell viability assay PrestoBlue. • High fluorescence indicates more living cells Source: http://www.invitrogen.com/etc/medialib/en/images/ics_organized/applications/cell_tissue_analysis/popups.Par.16964.Image.-1.-1.1.gif

  8. Procedures (Continued…) • PrestoBlue measurements taken twice • Directly before solution exposure to give initial seeding density fluorescence • 24 hours after solution exposure to give fluorescence after treatment • Accounts for apoptosis 3) Read fluorescence 2) Incubate PrestoBlue reagent 1) Add reagent to cells

  9. Experimental Variables Propylene Glycol Ethylene Glycol DMSO Glycerol Cryoprotectant Type 3m 1m 5m 7m Concentration Exposure Time 60 min 40 min 20 min 10 min 5 min 0 min Temperature 21C 37C 4C

  10. Data Analysis • Represented on cell survival versus time plot • Fit to exponential regression of the form:

  11. Accounting for Multi-Step Add/Rem • Toxicity accumulated from lower concentrations • Accounted for with derived correction factor: 2-Step Add/Rem Procedure

  12. Toxicity Function • The toxicity rate k is then plotted against concentration • Regression gives toxicity as a function of concentration • Mathematical representation of toxicity • Next step: Create a 3D regression to represent toxicity as a function of both concentration and temperature

  13. Experimental Results • Initial Experiments • 1,3-molal Glycerol at 21C • Used 96-Well Plates • Results were highly variable • Possible Sources of Variability • Uneven seeding distribution • Cell loss during wash steps

  14. Investigating Seeding Distribution • Uneven seeding distribution caused by thermal gradients • Pre-incubation to reduce variability • Involves placing well plates with freshly seeded cells at room temperature for 1 hour before placing in 37C incubator Pre-Incubated 1,3-Glycerol Toxicity Data

  15. Investigating Cell Loss During Wash Steps • Experiment • Cells seeded onto 96-well plate • Wells were washed with a PBS buffer solution • PrestoBlue measurement taken after wash steps

  16. Revised Experiments • 24-well plates • Avoid cell loss during wash steps • Increased well size helps to reduce variability

  17. Experimental Conclusion • Initial experiments using 96-well plates yielded inconclusive data • Attempts to isolate cause of data variability • Seeding distribution • Cell loss due to wash steps • Experiment revised with some improvement using 24-well plates

  18. Future Work • Improve experimental method • Try different cell viability assays • Optimization of cryoprotectant addition/removal for vitrification using: • Mathematical function for toxicity • Osmotic tolerance limits • Cell permeability data

  19. Acknowledgements • HHMI • Kevin Ahern • Mentor: Dr. Adam Higgins • Allyson Fry • Ratih Lusianti • Kenneth Huang • Corey Lerch

More Related