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Team Organ Storage & Hibernation

Fall 2009 Junior Colloquium. Team Organ Storage & Hibernation. Team Mentor : John P. Fisher. 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.

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Team Organ Storage & Hibernation

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  1. Fall 2009 Junior Colloquium Team Organ Storage & Hibernation 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 (H2S) H2S is a compound thought to induce suspended animation and prolong organ storage NaHS reacts with water to form H2S

  5. Hydrogen Sulfide • H2S is depleted from solution • H2S metabolized by tissues • Since H2S is a gas, it escapes from solution quickly • Limited protection time due to this depletion • As a result, need to control delivery of H2S treatment

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

  7. Hypothesis Gelatin microspheres can sustain H2S levels in the heart and induce protective effects

  8. Objective IH2S & Cells • Objective: To determine if heart cells metabolize H2S and how they are affected by H2S • Methods: • After incubation, aqueous H2S levels will be measured using a Zinc Acetate assay • Other assays to determine cell viability

  9. Objective INaHS 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 IISustaining Release of H2S Objective: To determine if gelatin microspheres can release H2S in a controlled fashion Method: Vary crosslinkage Detect change in release rate using zinc acetate assay

  11. NaHS in UW solution UW solution NaHS in UW solution NaHS in UW solution PBS loaded microspheres NaHS loaded microsphere Rat heart submerged in UW solution Rat heart submerged in NaHS loaded UW solution Rat heart submerged in NaHS loaded UW solution and injected with NaHS loaded microspheres Rat heart submerged in NaHS loaded UW solution and injected with PBS loaded microspheres Objective IIIEffects of Sustained Release • Objective: To determine the effect of sustained H2S on heart storage • Methods: • H2S microspheres added to UW solution • Stored at 4ºC for eight hours • Biopsy at 2, 4, 6, 8 hours • Cell viability assays • Functional evaluation

  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. H2S Release 50uL PBS 50uL Repeat for each time point H2S release will be measured via a zinc acetate assay

  16. Net H2S Released Over Time Initial loading concentration of 6.7M NaHS, 1M gluteraldehyde

  17. In Conclusion • We have: • Started fabrication of microspheres • Achieved desired sizes • Currently running H2S release tests • Cultured the desired cells • Submitted the IACUC proposal • We will: • Establish the relationship between crosslinking amount and release rate • Perform H2S experiments on the cell cultures • Perform H2S 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

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