Fall 2009 Junior Colloquium

Team Members (in alphabetical order):

Elizabeth Chen Charles Chiang

Elyse GeibelSteven Geng

Stevephen HungKathy Jee

Angela Lee

Christine LimSara Moghaddam-

Taaheri Adam Pampori

Kathy TangJessie Tsai

Diana Zhong

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 storagePreservation-related injury

Organ Storage Today Static Cold Storage

University of Wisconsin SolutionNo significant improvements in last two

decades Continuous Perfusion

Organ Care SystemEffective, but extremely expensive

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

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

Controlled Drug Delivery Hydrogels – polymer networks

Gelatin Crosslinking Size of microspheres

Hypothesis

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

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 assayOther assays to determine cell viability

Objective INaHS Dosage Test

Objective: What is the most effective concentration of NaHS for storage solutions?

Methods:0 to 100µM NaHS in UW solutionSample at 2, 4, 6, 8 hours

○ Cellular viability assays○ Functional evaluation

Objective IISustaining Release of H2S Objective: To determine if gelatin

microspheres can release H2S in a controlled fashion

Method:Vary crosslinkageDetect change in release rate using zinc

acetate assay

NaHS in UW

solution

Rat heart submerged in

NaHS loaded UW solution

UW solution

Rat heart submerged in UW solution

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 hoursBiopsy 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 microspher

es

Rat heart submerged in NaHS loaded UW

solution and injected with PBS loaded

microspheres

NaHS in UW

solution

Current Progress Funding Review Paper IACUC application has been submitted

to UMD School of Medicine Cell Culture

Obtained H9c2 cells, a rat heart cell lineCultured these cells into a suitable

populationWill start experiments shortly.

Microsphere Fabrication Fabricated microspheres

by spraying the gelatin solution into an oil/water emulsion

Currently capable of making microspheres at 4±2µmOutliers greater than 10µm

are excluded by means of a filter

Crosslinking Crosslinking by adding gluteraldehyde

to the gelatin solutionCrosslinking amount will be controlled by

altering the gluteraldehyde concentration Once crosslinked, the microspheres are

loaded with the NaHS solution

H2S Release H2S release will be measured via a zinc

acetate assay

50uL

50uL PBS

Repeat for each time point

Initial loading concentration of 6.7M NaHS, 1M gluteraldehyde

Net H2S Released Over Time

In Conclusion

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

THE END Any questions?

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