Gong Shixin, Li Nan, Peng Qinqing, Wang Feng, Du Rulong, Zhang Boyang, Wang Jian, Han Le, Zhang Yu, Ning Zemin, Tan Shengjiang, Gu Yuchun, Wu Lida
Allife Medicine Co., Ltd, No. 22, Jinyuan Road, Economic Development Zone, Daxing District, Beijing, 100053, China.
The Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK.
Stem Cell Res Ther. 2025 Aug 5;16(1):426. doi: 10.1186/s13287-025-04507-y.
Extracellular Vesicles (EVs) derived from mesenchymal stem cells (MSCs) have gained recognition as promising therapeutic and drug delivery agents in regenerative medicine. However, their clinical application is limited by donor variability, low scalability, and inconsistent therapeutic quality. To overcome these challenges, a robust and standardized production platform is urgently needed.
We developed a scalable biomanufacturing strategy by generating and expanding MSCs from extended pluripotent stem cells (EPSC) using a suspension bioreactor culture system. A fixed-bed bioreactor was integrated for automated, continuous expansion of iMSCs and downstream EV harvesting. EVs were isolated through a streamlined protocol and characterized for size, morphology, surface markers, and bioactivity. Therapeutic efficacy was assessed in a bleomycin-induced pulmonary fibrosis mouse model.
iMSC-derived EVs (iMSC-EVs) exhibited comparable characteristics to primary MSC-EVs, including a size distribution of 70-80 nm, cup-shaped morphology, and expression of canonical EV markers (CD63, CD81, TSG101). iMSCs were expanded for up to 20 days in 3D culture, yielding > 5 × 10⁸ cells per batch using a suspension bioreactor culture system and producing ~ 1.2 × 10¹³ EV particles/day in a fixed-bed bioreactor. In vivo, iMSC-EVs significantly reduced Ashcroft fibrosis scores and bronchoalveolar lavage fluid protein levels in bleomycin-injured lungs, with therapeutic efficacy comparable to primary MSC-EVs.
This study establishes a scalable and standardized platform for producing high-quality iMSC-EVs using bioreactor-based systems. Our approach addresses key limitations in traditional EV production and sets the stage for AI-integrated, fully automated, GMP-compliant manufacturing of therapeutic EVs suitable for clinical translation.
间充质干细胞（MSC）衍生的细胞外囊泡（EV）在再生医学中已被公认为是有前景的治疗和药物递送剂。然而，它们的临床应用受到供体变异性、低可扩展性和治疗质量不一致的限制。为了克服这些挑战，迫切需要一个强大且标准化的生产平台。
我们通过使用悬浮生物反应器培养系统从扩展多能干细胞（EPSC）生成并扩增MSC，开发了一种可扩展的生物制造策略。集成了固定床生物反应器用于诱导多能干细胞（iMSC）的自动化、连续扩增和下游EV收获。通过简化方案分离EV，并对其大小、形态、表面标志物和生物活性进行表征。在博来霉素诱导的肺纤维化小鼠模型中评估治疗效果。
iMSC衍生的EV（iMSC-EV）表现出与原代MSC-EV相当的特征，包括70-80nm的大小分布、杯状形态以及典型EV标志物（CD63、CD81、TSG101）的表达。iMSC在三维培养中扩增长达20天，使用悬浮生物反应器培养系统每批产生>5×10⁸个细胞，并在固定床生物反应器中每天产生约1.2×10¹³个EV颗粒。在体内，iMSC-EV显著降低了博来霉素损伤肺中的阿什克罗夫特纤维化评分和支气管肺泡灌洗液蛋白水平，治疗效果与原代MSC-EV相当。
本研究建立了一个使用基于生物反应器的系统生产高质量iMSC-EV的可扩展且标准化的平台。我们的方法解决了传统EV生产中的关键限制，并为适合临床转化的治疗性EV的人工智能集成、全自动、符合GMP的制造奠定了基础。
