Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, Jiangsu Province, China.
Department of Cardiothoracic Surgery, Zhongda Hospital, Southeast University School of Medicine, Nanjing, 210009, Jiangsu Province, China.
Stem Cell Res Ther. 2020 May 27;11(1):206. doi: 10.1186/s13287-020-01719-2.
Exosomes derived from mesenchymal stem cells (MSC-exos) have been demonstrated with great potential in the treatment of multiple human diseases including acute kidney injury (AKI) by virtue of their intrinsic cargoes. However, there are major challenges of low yield and the lack of an established biomanufacturing platform to efficiently produce MSC-exos, thereby limiting their therapeutic application. Here, we aimed to establish a novel strategy to produce MSC-exos with a hollow fiber bioreactor-based three-dimensional (3D) culture system and evaluate the therapeutic efficacy of 3D-exosomes (3D-exos) on AKI.
Mesenchymal stem cells (MSCs) were isolated from fresh human umbilical cord and cultured in two-dimensional (2D) flasks. 2 × 10 MSCs were inoculated into the hollow fiber bioreactor for 3D culture. The culture supernatants were collected every 1 or 2 days for isolating exosomes. Exosomes from 2D (2D-exos) and 3D cultures were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting analysis of exosome markers. The yield of exosomes from 2 × 10 MSCs seeded in 2D and 3D culture system was compared, based on protein quantification. The therapeutic efficacy of 2D-exos and 3D-exos was investigated in a murine model of cisplatin-induced AKI in vivo and in vitro.
3D culture did not significantly change the surface markers of MSCs, as well as the morphology, size, and exosomal markers of 3D-exos when compared to those of 2D-exos. Compared with conventional 2D culture, the 3D culture system increased total exosome production up to 19.4-fold. 3D-exos were more concentrated in the harvested supernatants (15.5-fold) than 2D-exos, which led to a higher exosome collection efficiency of 3D culture system. In vivo, both 2D-exos and 3D-exos significantly alleviated cisplatin-induced murine AKI evidenced by improved renal function, attenuated pathological changes of renal tubules, reduced inflammatory factors, and repressed T cell and macrophage infiltration. Impressively, 3D-exos were more effective than 2D-exos. Moreover, 3D-exos were taken up by tubular epithelial cells (TECs) with improved efficiency, thereby exhibiting superior anti-inflammatory effect and improved viability of TECs in vitro.
In summary, our findings demonstrate that the hollow fiber 3D culture system provides an efficient strategy for the continuous production of MSC-exos which has enhanced therapeutic potential for cisplatin-induced AKI.
间充质干细胞衍生的外泌体（MSC-exos）因其内在货物而在治疗多种人类疾病方面显示出巨大潜力，包括急性肾损伤（AKI）。然而，由于产量低以及缺乏成熟的生物制造平台来有效地生产 MSC-exos，因此存在主要挑战，从而限制了它们的治疗应用。在这里，我们旨在建立一种使用基于中空纤维生物反应器的三维（3D）培养系统生产 MSC-exos 的新策略，并评估 3D-外泌体（3D-exos）在 AKI 中的治疗效果。
从新鲜人脐带中分离间充质干细胞（MSCs）并在二维（2D）培养瓶中培养。将 2×10 MSCs 接种到中空纤维生物反应器中进行 3D 培养。每隔 1 或 2 天收集培养上清液以分离外泌体。通过透射电子显微镜、纳米颗粒跟踪分析和外泌体标志物的 Western 印迹分析来表征 2D（2D-exos）和 3D 培养的外泌体。根据蛋白质定量比较了在 2D 和 3D 培养系统中接种 2×10 MSCs 产生的外泌体的产量。在体内和体外 cisplatin 诱导的 AKI 小鼠模型中研究了 2D-exos 和 3D-exos 的治疗效果。
与 2D-exos 相比，3D 培养并未显着改变 MSC 的表面标志物，以及 3D-exos 的形态、大小和外泌体标志物。与传统的 2D 培养相比，3D 培养系统将总外泌体产量增加了 19.4 倍。3D-exos 在收获的上清液中更浓缩（15.5 倍），这导致 3D 培养系统的外泌体收集效率更高。在体内，2D-exos 和 3D-exos 均显着减轻顺铂诱导的 AKI，表现为肾功能改善、肾小管病理变化减轻、炎症因子减少以及 T 细胞和巨噬细胞浸润减少。令人印象深刻的是，3D-exos 比 2D-exos 更有效。此外，3D-exos 被肾小管上皮细胞（TECs）摄取的效率提高，从而表现出更好的抗炎作用，并提高了 TECs 的体外活力。
总之，我们的研究结果表明，中空纤维 3D 培养系统为连续生产 MSC-exos 提供了一种有效的策略，对顺铂诱导的 AKI 具有增强的治疗潜力。
