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

来自间充质干细胞（MSCs）的细胞外囊泡（EVs）由于其免疫调节和促进再生功能而成为一系列临床疾病的有希望的治疗药物。EV生物制造的一个主要瓶颈是产品开发过程中的可扩展性，以达到临床相关剂量。目前的做法依赖于对细胞和培养条件的侵入性操作，这可能对最终产品产生有害影响，并可能产生高成本和小规模操作，使其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MSCs传递低强度机械信号，对MSCs进行无创机械刺激，从而提高EV的产生。该项目将确定细胞对生物物理调节的反应，并评估整体分泌组、EV生产和功能的改进。该项目不仅将增加关于电动汽车释放背后MSCs机械转导的新基础知识，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。来自代表性不足群体的学生将从事研究；这将有助于培养和发展一支强大而多样化的未来生物制造劳动力队伍，共同对教育、科学和工程以及美国经济产生广泛的影响。该未来制造奖由以下部门/项目和办公室提供支持：ENG/CBET， MPS/CHE, MPS/DMR， EHR/DUE和OIA/EPSCoR。提高EV生产的策略包括缺氧培养条件、血清饥饿和MSCs的永生化——所有这些都对细胞的完整性和功能有有害影响，并且在小范围内运作，因此需要开发可行的方法来提高生产。该项目基于MSCs是对机械刺激反应的机械敏感细胞这一科学前提。核心假设是，以低强度机械刺激（LMMS）的形式向间充质干细胞传递机械信号/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，导致相应的ev释放。该项目利用外力通过整合素/talin对肌动蛋白的机械转导，以及随后对EV机制的影响，以全面改善msc的EV分泌。基于机械信号增加MSC分泌组和EV浓度的初步证据，该未来制造项目将研究机械触发EV释放背后的机制，并将确定响应机械刺激分泌的EV的质量和功能改善。该项目将建立一种最低限度操作的生物物理方法，通过传递低幅度的机械振动来扩大MSCs的电动汽车生产规模。三个主要目标是：(1)评估基于LMMS的生物物理细胞调节系统对EV释放的影响，揭示MSCs对LMMS的潜在机械转导变化；(2)评估lmms诱导的体外免疫抑制和体内组织再生在临界尺寸颅骨缺损模型中的功能效力；(3)通过让本科生、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熟练技术劳动力的教育和发展。该项目将有利于生命科学、制造和生物工程教育与研究领域，为美国在电动汽车生物制造研究和治疗领域的持续竞争力做出贡献。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.