mstp surf presentation
TRANSCRIPT
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Behavior of Cardiac Cells When Exposed to Different Formulations of Decellularized Extracellular MatrixMSTP-SURF 2015Sarah Albrecht, University of Washington-Seatt leUCSD Department of Bioengineering, Christman Lab
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Myocardial Infarction (MI) Cardiovascular disease is the leading cause of death in the United States killing an estimated 600,000 people each year.¹
Left Ventricle (LV) remodeling post-MI◦ Decrease in cardiomyocytes ◦ Degradation of the extracellular matrix of LV◦ Thinning of the LV wall◦ Eventually causes heart failure in the future
Only therapy for heart failure is a heart transplant
Infarcted heart and LV hours after MI
Infarcted heart and LV days after MI
1. Heron, M. Natl. Vital Stat. 2013
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Therapies for Post-MI Ventricular Constraint
◦ Mechanical device or synthetic material that encompasses the heart to limit ventricular dilation
Injectable Biomaterial◦ Injecting a biomaterial gel in
the infarct area with specific cells or biomolecules
Cardiac Patch◦ Placement of a biomaterial
sheet seeded either with specific cells or biomolecules over the infarct area
Rane, A. a. & Christman, K. L. J. Am. Coll. Cardiol. 2011
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Extracting Myocardial Decellularized ECM
Wang, R. M. & Christman, K. L. Adv. Drug Deliv. Rev. 2015
Reduced negative LV remodeling and improved heart function¹
Decellularized ECM has been shown to be biocompatible²
1. Sonya B. Seif-Naraghi et al. Science Translational Medicine. 20132. Jennifer M. Singelyn et al. Biomaterials. 2009
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Extracting Myocardial Decellularized ECM
Can be separated into soluble and insoluble components
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Project Overview Research Goal:
◦ Improve our understand of how the soluble component of the ECM hydrogel affects cell viability and migration.
Grow CPCs and HUVECs to a desired cell confluences
Isolate soluble ECM component
Plate cells with soluble ECM for
Viability and Migration assay
Analyze both cell lines appropriately to determine cell
survival and movement
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1. Plate CPCs and HUVECs 2. Prepare various H2O2 concentrations (n=4 or 6)
3. Add Alamar Blue in wells and incubate for 4
hours
4. Record the fluorescence of each well with plate reader
Viability Assay
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Migration Assay
1. Serum starve CPCs for 24 hours
2. Fluorescently label cells
3. Add media and cell solution into transwell of a 24-well plate
4. Count fluorescent cells to determine how many migrated
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CPC Viability 4 hours at 50,000 cells
Results:◦ SS and soluble ECM have
higher viability than SF◦ Survival of CPCs at high
H2O2 concentrations
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CPC Viability 8 hours at 25,000 cells
Parameters Changed:◦ Longer incubation time◦ Lower amount of cells per well
Results:◦ No viable cells at higher H2O2
◦ No significance between SS and SF
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HUVEC Viability 4 hours at 50,000 cells
Results:◦ SF higher than SS◦ Viable cells at higher H2O2
concentrations
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HUVEC Viability 8 hours at 25,000 cells
Parameters Changed:◦ Incubation time◦ Cell concentration◦ Media
Results:◦ No viable cells at higher
H2O2
◦ Significance between SS and SF no HE
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Migration Assay with CPCSS SF
6 mg/mL 24 mg/mL
Parameters:◦ Cells counted twice to reduce error◦ Images captured at 6 hours after initial plating◦ n=3
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Conclusion Viability Assay
◦ Increasing the incubation time, decreasing the cell concentration and using media without added molecules such as Heparin and E.C.G. affected cell survival
◦ Soluble ECM appears to not be detrimental to CPCs and HUVECs
Migration Assay◦ Soluble ECM encourages cell migration however it is not as affective as the SS media◦ At a higher concentration of the soluble ECM, more cell movement was observed compared to lower
concentrations of the ECM
The ECM can be a suitable injectable biomaterial therapy to treat the infarct region
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Future Directions Soluble ECM
◦ Continue with cell studies for ECM hydrogel◦ Preform an angiogenesis assay to observe vessel formation
and cell-cell interactions
Insoluble ECM◦ Perform more cell studies on different
formulations of the insoluble ECM◦ Pellet form vs ECM hydrogel
Control Sol ECM
Images captured by JG Wang, R. M. & Christman, K. L. Adv. Drug Deliv. Rev. 2015
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Acknowledgements Christman Lab
◦ Dr. Karen Christman◦ Dr. Mary Nguyen◦ Dr. Roberto Gaetani◦ Masaki Fujita◦ Rebecca Barden◦ Jessica Ungerleider◦ Melissa Hernandez◦ Raymond Wang◦ Heinz Strassle◦ Hillary Lam◦ Cori Espelien◦ Julian Garcia
MSTP-SURF Program◦ Dr. Robert Ross◦ Ryan Moore◦ Mary Alice Kiisel◦ Jennifer Dumdie◦ Aran Groves◦ Cindy Liu◦ Eulanca Liu
MSTP-SURF Participants◦ Pamela Capellan◦ Angel Diaz◦ Luis Gasca◦ Maryam Ige
◦ Kamren Livingston◦ Mauricio Mrquez
Palencia◦ Isaac Padilla◦ Assata Pyatt◦ Jasmine Rice◦ Ceciley Scarbrough