Ma Shaoyang, Ene Justice, McGarraugh Colton, Ma Shaoxuan, Esmonde Colin, Liu Yuan, Li Yan
Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, 2525 Pottsdamer St., Tallahassee, FL 32310, USA.
Division of Biology and Medicine, Brown University, 69 Brown St., Providence, RI 02912, USA.
Bioengineering (Basel). 2025 Aug 29;12(9):933. doi: 10.3390/bioengineering12090933.
Human mesenchymal stem cells (hMSCs) and their secreted extracellular vesicles (EVs) are promising therapeutics to treat degenerative or inflammatory diseases such as ischemic stroke and Alzheimer's disease (AD). hMSC-EVs have the coveted ability to contain therapeutically relevant biomaterials; however, EV biogenesis is sensitive to the culture microenvironment in vitro. Recently, the demand for hMSC-EVs has increased dramatically, highlighting the need for scalable bioreactors for large-scale biomanufacturing. In this study, adipose-derived hMSCs were seeded in 2D plates, an ultralow-attachment (ULA) plates as static aggregates, a novel vertical wheel bioreactor (VWBR) as aggregates, and a spinner flask bioreactor (SFB). EV secretion was quantified and compared using ExtraPEG-based ultracentrifugation and nanoparticle tracking analysis. Compared to the 2D group, significantly higher total EV production and cell productivity in the bioreactors were observed, as well as the upregulation of EV biogenesis genes. Furthermore, there was increased EV production in the VWBR compared to the SFB and the static ULA control. Functional assessments demonstrated that EVs, when delivered via culture medium or hydrogel-based systems, significantly attenuated oxidative stress elevation, suppressed proinflammatory cytokine secretion (e.g., TNF-α) and gene expression, and inhibited nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activation and neurodegenerative markers across in vitro assays. These findings suggest EV-mediated mitigation of oxidative and inflammatory pathways, potentially through modulation of the NF-κB signaling cascade. This study shows the influence of bioreactor types and their microenvironments on EV secretion in hMSCs and their applications in hMSC-EV production and bioengineering.
人间充质干细胞（hMSCs）及其分泌的细胞外囊泡（EVs）是治疗诸如缺血性中风和阿尔茨海默病（AD）等退行性或炎症性疾病的有前景的治疗方法。hMSC-EVs具有包含治疗相关生物材料的令人垂涎的能力；然而，EV的生物发生在体外对培养微环境敏感。最近，对hMSC-EVs的需求急剧增加，凸显了对用于大规模生物制造的可扩展生物反应器的需求。在本研究中，将脂肪来源的hMSCs接种在二维平板、作为静态聚集体的超低附着（ULA）平板、作为聚集体的新型垂直轮生物反应器（VWBR）和转瓶生物反应器（SFB）中。使用基于ExtraPEG的超速离心和纳米颗粒跟踪分析对EV分泌进行定量和比较。与二维组相比，在生物反应器中观察到EV的总产量和细胞生产力显著更高，以及EV生物发生基因的上调。此外，与SFB和静态ULA对照相比，VWBR中的EV产量增加。功能评估表明，当通过培养基或基于水凝胶的系统递送时，EVs显著减轻氧化应激升高，抑制促炎细胞因子分泌（例如，TNF-α）和基因表达，并在体外试验中抑制活化B细胞的核因子κB轻链增强子（NF-κB）激活和神经退行性标记物。这些发现表明EV可能通过调节NF-κB信号级联介导氧化和炎症途径的减轻。本研究显示了生物反应器类型及其微环境对hMSCs中EV分泌的影响及其在hMSC-EV生产和生物工程中的应用。
