Table 2_GMP-compliant, serum-free cultures preserve therapeutic potential of extracellular vesicles from human mesenchymal stromal cells.xlsx

The therapeutic potential of extracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) is limited by the lack of standardized, Good Manufacturing Practice (GMP)-compliant production protocols. This study investigates the effects of MSC-Brew, a commercially available GMP-grade medium, on MSC-derived EVs in comparison to those produced in conventional cultures with DMEM supplemented with 10% fetal bovine serum (FBS). MSCs from adult dermis were successfully isolated and expanded in Brew medium while retaining their characteristic surface marker expression. MSC-EVs derived from Brew cultures met the Minimal Information for Studies of Extracellular Vesicles (MISEV) criteria, including particle size, concentration, marker expression, and minimal inflammatory cytokine content. Notably, Brew-EVs exhibited a significantly higher particle-to-protein ratio compared to EVs produced in FBS-containing cultures, indicating improved purity. Proteomic analysis revealed a largely conserved composition between Brew-EVs and conventionally produced EVs, and microRNA (miRNA) profiling identified only four differentially expressed miRNAs. Brew-EVs were enriched in anti-fibrotic miRNAs and effectively reduced collagen secretion in transforming growth factor (TGF)-β1-activated LX-2 cells, a human hepatic stellate cell line used as a model of liver fibrosis. These findings support MSC-Brew medium as a standardized, serum-free platform for the consistent production of high-quality EVs suitable for therapeutic applications.

人源性间充质基质细胞（mesenchymal stromal cells, MSCs）衍生的细胞外囊泡（extracellular vesicles, EVs）的治疗潜力，受限于缺乏标准化且符合药品生产质量管理规范（Good Manufacturing Practice, GMP）的生产工艺。本研究针对市售GMP级培养基MSC-Brew展开探究，对比其与添加10%胎牛血清（fetal bovine serum, FBS）的传统DMEM培养体系，对间充质基质细胞衍生细胞外囊泡的影响。研究成功从成人真皮中分离出间充质基质细胞，并在Brew培养基中完成扩增，同时保留了其标志性的表面标志物表达特征。由Brew培养基培养得到的MSC-EVs符合《细胞外囊泡研究最低信息标准》（Minimal Information for Studies of Extracellular Vesicles, MISEV）的各项要求，涵盖粒子粒径、浓度、标志物表达以及极低的炎症细胞因子含量。值得注意的是，与添加FBS的传统培养体系所得EVs相比，Brew-EVs的粒子-蛋白比显著更高，提示其纯度得到提升。蛋白质组学分析显示，Brew-EVs与传统方法制备的EVs在组成上整体保守性良好；微小RNA（microRNA, miRNA）谱分析仅检测到4种差异表达的miRNA。Brew-EVs富含抗纤维化相关miRNA，并可有效降低转化生长因子（transforming growth factor, TGF）-β1激活的LX-2细胞（一种用作肝纤维化模型的人肝星状细胞系）的胶原分泌量。上述研究结果表明，MSC-Brew培养基可作为一种标准化无血清平台，能够稳定制备适用于治疗用途的高质量细胞外囊泡。