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

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).pnghttp://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.jpghttp://blog.bioethics.net/cryopreservation.jpg

4. Source: http://www.benbest.com/cryonics/DMSO.jpghttp://www.benbest.com/cryonics/DMSO.jpg Source: http://www.bmrb.wisc.edu/metabolomics/standards/glycerol/lit/3416.pnghttp://www.bmrb.wisc.edu/metabolomics/standards/glycerol/lit/3416.png 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

5. Procedures 1. Endothelial cells seeded onto well plates 2. 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/trans port%20notes.htm

7. Procedures (Continued…) 3. 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 PrestoBlue reagent 1) Add reagent to cells 2) Incubate 3) Read fluorescence

9. 1m Experimental Variables DMSO 60 min40 min20 min10 min5 min0 min 4C37C 21C Glycerol Ethylene Glycol Propylene Glycol Cryoprotectant Type Concentration Exposure Time Temperature 3m 5m 7m

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