College - Author 1

College of Engineering

Department - Author 1

Biomedical Engineering Department

Degree Name - Author 1

BS in Biomedical Engineering

Date

12-2025

Primary Advisor

Trevor Cardinal, College of Engineering, Biomedical Engineering Department

Abstract/Summary

Peripheral Artery Disease (PAD) affects an estimated 200 million people worldwide and is the leading cause of amputation in the United States. PAD is characterized by atherosclerotic narrowing of arteries, resulting in ischemia from reduced blood flow to distal tissues. Although surgical and percutaneous interventions can restore perfusion, fewer than 50% of patients experience sustained benefit, highlighting the need for alternative therapeutic strategies. One promising approach is promoting arteriogenesis, the growth and outward remodeling of pre-existing collateral vessels that improve blood flow around the occluded arteries. Arteriogenesis is regulated by regenerative, M2-polarized macrophages, making macrophage polarization a key therapeutic target. Myoblasts, which reside in close proximity to the vasculature, and which communicate with macrophages during muscle regeneration, represent a compelling candidate for promoting arteriogenesis.

Previous studies demonstrated that myoblasts enhance arteriogenesis in mice with diet-induced obesity, and that in vitro mouse myoblasts can polarize macrophages from an inflammatory M1 phenotype, which are prevalent in muscle tissue of mice with diet-induced obesity, to a regenerative M2 phenotype. To translate these findings to more clinically relevant human cells, we needed to implement standardized and reproducible culture protocols for primary human myoblasts. Primary human myoblast cultures were successfully initiated, maintained, and passed with consistent proliferation across multiple passages. Culture parameters including seeding density, extracellular matrix coating, and growth factor dosing were evaluated, and population doubling level correlated strongly with passage number. Additionally, cryopreservation was explored using cryopreservant formulations with high concentrations of fetal bovine serum, yielding moderate, albeit variable, recovery success.

Together, this work establishes reliable protocols for primary human myoblast culture, providing a critical methodological foundation for future studies investigating myoblast-mediated macrophage polarization in human cells, defining the paracrine mechanism of action using measurements of gene expression, and evaluating the therapeutic potential of myoblasts as a cell-based strategy for promoting arteriogenesis in patients with PAD.

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