VCOM Research Day Program Book 2023

Medical Student Research Biomedical

11 The Effects of Neuronal Endothelial Cell Priming on the Uptake and Delivery Potential of Schwann Cell Exosomes

Dr. Kelly Roballo; Virginia “Grey” Fritz Corresponding author: vfritz@vcom.edu

Edward Via College of Osteopathic Medicine-Virginia Campus

Exosomes are small vesicles that carry waste, genetic material, lipids, and proteins out of cells. Historically, scientists believed exosomes only contained waste products, but in the last few years, researchers have discovered that exosomes can play a role in cell signaling, cell fusion, and peripheral nerve injury (PNI). Current PNI surgery involves using an allograft which provides a structure or nerve extracellular matrix; however, allografts undergo Wallerian degeneration due to the lack of regenerative cells, such as Schwann cells (SC). SCs secrete factors which create an environment to myelinate extracellular matrix during regeneration. Research suggests exosomes are involved in SC axonal regeneration. During regeneration, a large number of SC exosomes are in the microenvironment as the SC exosomes guide cells and regenerate axons along the distal segment of the injured nerve. Therefore, PNI research has shifted focus to investigate how exosomes can improve PNI outcomes. The main goal for my project was to analyze mechanisms that could enhance the regenerative capability of peripheral

nerve cells by testing how priming, cell quantity, exosome concentration, and timing affect the uptake of SC exosomes by endothelial cells. First, exosomes were isolated from SC cultures and labeled with a florescent tag. Next, endothelial cells were counted, plated, and allowed to grow overnight. Then, different concentrations of exosomes were added to each well of endothelial cells and allowed to prime for one day. After priming, the exosomes were isolated from the endothelial cells and added to a new culture of endothelial cells and the cells were incubated for 3 and 7 days. Slides were placed at the bottom of the wells during culturing, and after incubating, the cells in each well were fixed to the microscope slide and images were taken. The results of this experiment were primed SCs-derived small vesicles were taken up by neuronal endothelial cells. When different doses of primed SCs-derived small vesicles (5uL/mL, 10uL/ mL, 15uL/mL, 25uL/mL, 100uL/mL) were tested, the best dose response in 3 and 7 days of co-culture were 10uL/mL. With these results, we believe that primed SC-derived small vesicles can be taken up

by neuronal endothelial cells, and small doses of small vesicles had better uptake rate. At 10uL/mL, there was an upward trendline for the smaller and larger quantity of endothelial cells for 3 and 7 days, so the 10uL/mL was less toxic over time than other concentrations. For next steps, we can use this data to introduce other molecules, such as therapeutic agents, into SC exosomes and investigate the uptake of those vesicles into endothelial cells. Ultimately, exosome uptake research can be used to explore drug delivery capabilities of SC exosomes to improve the outcomes of peripheral nerve damage and transplant.

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