FMSG: Bio: Biophysical Modulation for Scalable Biomanufacturing of Stem Cell-Derived Therapeutics

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Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are promising therapeutic agents for a range of clinical disorders owing to their immunomodulatory and pro-regenerative functions. A major bottleneck in EV biomanufacturing is scalability during development of a product to achieve clinically relevant doses. Current practices rely on invasive manipulations of cells and culture conditions, which can have deleterious implications on the final product and can incur high cost and operate at small scales, making them less viable for best-practice manufacturing. This project will improve EV production through noninvasive mechanical stimulation of MSCs by delivering low magnitude mechanical signals to MSCs cultured in two-dimensional and three-dimensional systems. This project will identify cellular responses to biophysical modulations and evaluate improvements to overall secretome, EV production, and functionality. The project will not only add new fundamental knowledge on mechanotransduction in MSCs behind EV release, but also provide functional evidence for overcoming large-scale manufacturing challenges in EV production. Students from underrepresented populations will engage in research; this will help train and develop a strong and diverse future biomanufacturing workforce, together having wide-reaching impacts on education, science and engineering, and the U.S economy. This Future Manufacturing award is supported by the following Division/Programs and Offices: ENG/CBET, MPS/CHE, MPS/DMR, EHR/DUE, and OIA/EPSCoR.Strategies to improve EV production include hypoxic culture conditions, serum starvation, and immortalization of MSCs – all of which have deleterious implications in cell integrity and functionality and operate at small scales, thus necessitating development of feasible methods to enhance manufacturing. This project is based on the scientific premise that MSCs are mechanosensitive cells responding to mechanical stimuli. The central hypothesis is that delivery of mechanical cues/forces in the form of low-magnitude mechanical stimulation (LMMS) to MSCs can affect (i) cell membrane integrin-cytoskeletal interactions that regulate actin dynamics and vesicle transport, and (ii) alter intracellular calcium levels leading to consequential release of EVs. The project harnesses the mechanotransduction of external forces on actin via integrins/talin, and subsequent influence on EV machinery towards the overall improvement in EV secretion by MSCs. Based on preliminary evidence that mechanical signals increase overall MSC secretome and EV concentration, this Future Manufacturing project will investigate the mechanisms behind mechanically triggered EV release, and will determine qualitative and functional improvements in EVs secreted in response to mechanical stimulation. The project will establish a minimally manipulative biophysical method to scale-up manufacturing of EVs from MSCs through delivery of low magnitude mechanical vibrations. The three major objectives are (1) To evaluate the effects of LMMS-based biophysical cell modulation system on EV release and unravel underlying mechanotransduction changes in MSCs in response to LMMS; (2) To assess functional potency of LMMS-induced EVs for immunosuppression (in vitro) and tissue regeneration (in vivo) in a critical-sized calvarial defect model; and (3) Leverage research outcomes to support education and development of a skilled technical workforce in the state of Arkansas by engaging undergraduate, graduate, and middle school students in biomanufacturing education and research. The project will benefit the field of life sciences, manufacturing and bioengineering education and research, contributing to sustained U.S. competitiveness in EV biomanufacturing for research and therapy.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.