Poster M19 - Engineering extracellular vesicles of human mesenchymal stem cell aggregates in a Vertical Wheel Bioreactor

Introduction: Human Mesenchymal Stem Cells (hMSCs) have a high potential for therapeutic treatments in regenerative medicine due to the anti-inflammatory and pro-angiogenic secretome. However, due to the safety and efficacy concerns with stem cell therapy, cell-free therapy utilizing hMSC-derived extracellular vesicles (EVs) has been a promising approach to treatments of various neurological disorders. However, low yield production limits EVs in clinical applications. Through the utilization of the PBS Vertical-Wheel bioreactor (VWBR), the influence of biochemical cues and shear stress on the secretion of EVs and cargo expression toward nerve regeneration and neuropathic treatment. This project aims to advance the knowledge of dynamic aggregation and metabolic influence on EV production, and fundamentally improve the preconditioned techniques used to promote hMSC secretome production.

Materials and

Methods: EVs secreted by undifferentiated hMSCs as 3D aggregates in VWBRs were investigated. Two types of EV collection media, MEM/Fetal Bovine Serum (FBS) and DMEM/F12/Serum-free B27, exposed to three speeds of the bioreactor, at 25, 40, and 64 rpm, for three days were compared. The hMSCs were characterized by metabolite analysis and gene expression, focusing on EV ESCRT machinery markers. The isolated hMSC-EVs were characterized by nanoparticle tracking analysis and Western blot, necessary to identify EV biogenesis markers. In vitro analyses of cellular proliferation were also conducted.

Results: hMSCs exposed to 64 rpm within the VWBR, reflected the highest EV biogenesis and glycolytic gene expression compared to 25 rpm and 40 rpm. The cell number decreased within all three conditions over the course of the experiments due to typical behavior of hMSC 3D aggregates without the assistance of microcarriers. Once isolated, EV concentration increased with high agitation speed when exposed to MEM, however the concentration decreased with higher agitation speed withing DMEM media. EV size consistently decreased with increased agitation speed within all culture conditions.

Results, Conclusions, and Discussions: hMSCs cultured as 3D aggregates within the VWBR at 64 rpm, exposed to MEM media effectively produced a much higher yield of EVs compared to DMEM media and lower agitation speed. The system of increased yield and subsequentially, enhanced capability of the generated EVs, promotes clinical applications of hMSC-EVs toward regenerative medicine.

Acknowledgements (Optional): We would like to acknowledge PBS Biotech. DB was supported in part by FAMU Title III funding. This work is partially supported by the National Science Foundation (CBET-1917618).