1. Fall 2009 Junior Colloquium Team Members (in alphabetical order): Elizabeth Chen Charles Chiang Elyse Geibel Steven Geng Stevephen Hung Kathy Jee Angela Lee Christine Lim Sara Moghaddam-Taaheri Adam Pampori Kathy Tang Jessie Tsai Diana Zhong Team Mentor: John P. Fisher

2. The Organ Shortage  There are over 100,000 people are on organ donor lists  Only 77 of these patients receive transplants daily  Hearts are limited to 4 hours in storage Preservation-related injury

3. Organ Storage Today  Static Cold Storage University of Wisconsin Solution No significant improvements in last two decades  Continuous Perfusion Organ Care System Effective, but extremely expensive

4. Overall Objective  Idea: To modify clinical cold storage procedures in place using hydrogen sulfide (H 2 S)  H 2 S is a compound thought to induce suspended animation and prolong organ storage  NaHS reacts with water to form H 2 S

5. Hydrogen Sulfide  H 2 S is depleted from solution H 2 S metabolized by tissues Since H 2 S is a gas, it escapes from solution quickly Limited protection time due to this depletion  As a result, need to control delivery of H 2 S treatment

6. Controlled Drug Delivery  Hydrogels – polymer networks Gelatin  Crosslinking  Size of microspheres

7. Hypothesis Gelatin microspheres can sustain H 2 S levels in the heart and induce protective effects

8. Objective I H 2 S & Cells  Objective: To determine if heart cells metabolize H 2 S and how they are affected by H 2 S  Methods: After incubation, aqueous H 2 S levels will be measured using a Zinc Acetate assay Other assays to determine cell viability

9. Objective I NaHS Dosage Test  Objective: What is the most effective concentration of NaHS for storage solutions?  Methods: 0 to 100µM NaHS in UW solution Sample at 2, 4, 6, 8 hours ○ Cellular viability assays ○ Functional evaluation

10. Objective II Sustaining Release of H 2 S  Objective: To determine if gelatin microspheres can release H 2 S in a controlled fashion  Method: Vary crosslinkage Detect change in release rate using zinc acetate assay

11. NaHS in UW solution Rat heart submerged in NaHS loaded UW solution UW solution Rat heart submerged in UW solution Objective III Effects of Sustained Release  Objective: To determine the effect of sustained H 2 S on heart storage  Methods: H 2 S microspheres added to UW solution Stored at 4ºC for eight hours Biopsy at 2, 4, 6, 8 hours ○ Cell viability assays ○ Functional evaluation NaHS in UW solution Rat heart submerged in NaHS loaded UW solution and injected with NaHS loaded microspheres NaHS loaded microsphere PBS loaded microspheres Rat heart submerged in NaHS loaded UW solution and injected with PBS loaded microspheres NaHS in UW solution

12. Current Progress  Funding  Review Paper  IACUC application has been submitted to UMD School of Medicine  Cell Culture Obtained H9c2 cells, a rat heart cell line Cultured these cells into a suitable population Will start experiments shortly.

13. Microsphere Fabrication  Fabricated microspheres by spraying the gelatin solution into an oil/water emulsion  Currently capable of making microspheres at 4±2µm Outliers greater than 10µm are excluded by means of a filter

14. Crosslinking  Crosslinking by adding gluteraldehyde to the gelatin solution Crosslinking amount will be controlled by altering the gluteraldehyde concentration  Once crosslinked, the microspheres are loaded with the NaHS solution

15. H 2 S Release  H 2 S release will be measured via a zinc acetate assay 50uL 50uL PBS Repeat for each time point

16. Initial loading concentration of 6.7M NaHS, 1M gluteraldehyde Net H 2 S Released Over Time

17. In Conclusion  We have: Started fabrication of microspheres ○ Achieved desired sizes ○ Currently running H 2 S release tests Cultured the desired cells Submitted the IACUC proposal  We will: Establish the relationship between crosslinking amount and release rate Perform H 2 S experiments on the cell cultures Perform H 2 S experiments on whole rat hearts

18. Team Dynamics  Big group:13 people on the team Split into subgroups for efficiency  Rotate facilitators during meetings to divide leadership  Be realistic Learn to accept setbacks

19. THE END  Any questions?

20. Global Hypothesis (Detailed)  Gelatin microspheres used to administer controlled delivery of H2S to the heart will induce protective effects and cause a state of hibernation in order to prolong viability of the heart and reduce ischemia- reperfusion injury in transplants