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BME PhD Thesis Defense| Generation and Dissemination of a Mesenchymal Stem Cell-Derived Functional Smooth Muscle Tissue by Jonian Grosha| Via Zoom

Wednesday, July 28, 2021
1:00 pm to 2:00 pm

Abstract:

Cardiovascular disease is the main cause of death worldwide with 17.7 million annual deaths. There are varying patient responses to current treatments, thus, there is a need to develop new therapies. Tissue engineered models can be used screen candidate drugs prior to clinical studies and predict their effectiveness. There are currently several available models, but most require large numbers of primary cells, which have limited population doublings and limited proliferation potential. Thus, there is a need to generate an engineered vascular tissue model from an available cell source that overcomes the limitations of primary smooth muscle cells. Our proposed model follows a self-assembly approach, where the cells are seeded without an external scaffold. This allows for the morphology, mechanical, and functional properties of the tissue to be governed by the cells, cell-derived extracellular matrix (ECM) and the culture conditions. We investigated human mesenchymal stem cells (hMSCs) as a readily available patient-specific source, with high proliferation rates, and myogenic differentiation potential. We hypothesized that a self-assembled hMSC tissue ring would differentiate into a SMC tissue when stimulated with TGF-β1 and BMP4. We concluded that the addition of BMP-4 did not cause a significant difference in the differentiation of the tissues, and TGF-β1 was sufficient to differentiate the tissues towards a contractile SMC phenotype. Engineered tissue rings differentiated with TGF-β1 expressed early, mid, and late-stage protein and gene markers of smooth muscle differentiation, and ultimately responded to tissue agonists. In summary, we generated an hMSC derived functional SMC tissue that expresses SMC gene and protein markers along with a contractile phenotype. These engineered SMC tissue rings could be used as screening tools for the development of new drug therapies. 

Since we created a tissue engineered product, the next step in the manufacturing pipeline would be to store or disseminate it. Tissue storage has been identified as a major bottleneck of the tissue engineering field. Cryopreservation is the primary mode of storage for cells and tissues. There are cryopreserving agents (CPAs) designed for cell cryopreservation, but there are no CPAs designed for tissue cryopreservation. In addition, freezing parameters such as freezing rate, freezing point and storage temperature, are cell type specific. Thus, there is a need for a high-throughput test platform that uses a standard tissue where multiple cryopreservation parameters and CPAs can be tested and quantitatively evaluated. To achieve this, we used our engineered tissue rings as standard test units. To develop this platform, we re-designed the agarose wells for cell seeding and tissue ring self-assembly, in order to fit in a 48-well plate and also fit in standard ryovials. Using this method, we cryopreserved tissues with 10% DMSO and a DMSO-free CPA. Tissues preserved with DMSO recovered their metabolic recovery of more than 100% but 62% of cells within tissue had DNA damage, while tissues frozen with DMSO-free CPA they had a lower recovery at 72% but only 12% cells with apoptosis or necrosis. This highlights the complex trade-off between viability and metabolic recovery and the need to look at multiple outcomes to decide the optimal cryopreservation conditions. We also discovered that a freezing rate of 2°C/min might be more appropriate for engineered vascular tissues compared to conventional 1°C/min. In summary, we created a novel high-throughput platform to thoroughly quantify the effects of cryopreservation that uses a uniform tissue. 

Defense Committee

Marsha Rolle, PhD, Professor, Biomedical Engineering, WPI (Thesis Advisor)
Kristen Billiar, PhD, Professor, Biomedical Engineering, WPI (Chair)
Jeannine Coburn, PhD, Assistant Professor, Biomedical Engineering, WPI
Suzanne Scarlata, PhD, Professor, Chemistry & Biochemistry, WPI
Peter Kilbride, PhD, Senior Research Scientist, Cytiva 

 

Please contact igjencaj@wpi.edu for a zoom link to this event.

DEPARTMENT(S): 
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Department of Biomedical Engineering
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