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

间充质干细胞(MSCs)来源的细胞外小泡(EV)具有免疫调节和促进再生的功能，是治疗一系列临床疾病的有效药物。电动汽车生物制造的一个主要瓶颈是在产品开发期间的可扩展性，以实现临床相关的剂量。目前的做法依赖于对细胞和培养条件的侵入性操作，这可能会对最终产品产生有害影响，并可能导致高成本和小规模操作，使它们不太适合最佳实践制造。该项目将通过向在二维和三维系统中培养的骨髓间充质干细胞传递低幅度的机械信号，通过对骨髓间充质干细胞的非侵入性机械刺激来提高电动汽车的产量。该项目将确定细胞对生物物理调节的反应，并评估对整体分泌体、EV生产和功能的改善。该项目不仅将增加关于电动汽车释放背后的MSCs机械转导的新的基础知识，而且还将为克服电动汽车生产中的大规模制造挑战提供功能证据。来自代表性不足人群的学生将从事研究；这将有助于培养和发展一支强大和多样化的未来生物制造劳动力，共同对教育、科学和工程以及美国经济产生广泛影响。这一未来制造奖由以下部门/计划和办公室支持：ENG/CBET、MPS/CHE、MPS/DMR、EHR/DUE和OIA/EPSCoR。改善电动汽车生产的策略包括低氧培养条件、血清饥饿和骨髓间充质干细胞永生化-所有这些都对细胞完整性和功能具有有害影响，并以小规模运行，因此需要开发可行的方法来增强制造。该项目是基于MSCs是对机械刺激做出反应的机械敏感细胞这一科学前提。核心假设是，以低幅度机械刺激(LMM)的形式向MSCs传递机械信号/力可以影响(I)调节肌动蛋白动力学和囊泡运输的细胞膜整合素-细胞骨架相互作用，以及(Ii)改变细胞内钙水平从而导致EV的释放。该项目利用外力通过整合素/talin对肌动蛋白的机械传递，以及随后对EV机制的影响，从而全面改善MSCs的EV分泌。基于机械信号增加整体MSC分泌组和EV浓度的初步证据，这个未来制造项目将研究机械触发EV释放背后的机制，并将确定EV在响应机械刺激时分泌的质量和功能改善。该项目将建立一种最低限度操控的生物物理方法，通过提供低幅度的机械振动来扩大从MSCs制造电动汽车的规模。三个主要目标是(1)评估基于LMMS的生物物理细胞调制系统对EV释放的影响，并揭示响应LMMS的MSCs潜在的机械转导变化；(2)在临界大小的颅骨缺损模型中评估LMMS诱导的EVS在免疫抑制(体外)和组织再生(体内)中的功能效力；以及(3)通过吸引本科生、研究生和中学生参与生物制造教育和研究，利用研究成果支持阿肯色州的教育和熟练技术劳动力的发展。该项目将惠及生命科学、制造业和生物工程教育和研究领域，有助于美国在电动汽车生物制造研究和治疗方面的持续竞争力。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.