MSTP SURF Presentation
-
Upload
sarah-albrecht -
Category
Documents
-
view
228 -
download
3
Transcript of MSTP SURF Presentation
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
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
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
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
Extracting Myocardial Decellularized ECM
Can be separated into soluble and insoluble components
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
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
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
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
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
HUVEC Viability 4 hours at 50,000 cells
Results:◦ SF higher than SS◦ Viable cells at higher H2O2
concentrations
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
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
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
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
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