Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury
Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury
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DOI：
10.1186/s13287-020-01719-2
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发表时间：
2020-05-27
影响因子：
7.5
通讯作者：
Liu, Bicheng
中科院分区：
文献类型：
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作者：
Cao, Jingyuan;Wang, Bin;Liu, Bicheng
背景来源于间充质干细胞的外泌体（MSC-exos）因其内在的运载功能在治疗包括急性肾损伤（AKI）在内的多种人类疾病方面具有巨大的潜力。然而，存在低产率和缺乏有效生产MSC-exos的既定生物制造平台的主要挑战，从而限制了其治疗应用。在这里，我们的目的是建立一种新的策略，以生产MSC-外的中空纤维生物反应器为基础的三维（3D）培养系统和评估的治疗效果的三维外体（3D-外）AKI. MethodsMesenchymal Stem Cells（MSCs）从新鲜的人脐带中分离和培养在二维（2D）烧瓶。将2x10（8）个MSC接种到中空纤维生物反应器中进行3D培养。每1或2天收集培养物上清液用于分离外来体。来自2D（2D-exos）和3D培养物的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蛋白质印迹分析来表征。基于蛋白质定量，比较来自2D和3D培养系统中接种的2x10（8）MSC的外来体产量。结果与2D-exos相比，3D-exos对骨髓间充质干细胞的形态、大小及外泌体标记均无明显改变。与传统的2D培养相比，3D培养系统使总外泌体产量增加了19.4倍。3D-exos在收获的上清液中比2D-exos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均显著减轻顺铂诱导的小鼠AKI，表现为肾功能改善、肾小管病理改变减轻、炎症因子减少以及T细胞和巨噬细胞浸润抑制。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）的效率提高，从而表现出上级抗炎作用和提高TECs的活力在vitro. ConclusionsIn总结，我们的研究结果表明，中空纤维三维培养系统提供了一个有效的策略，连续生产的MSC-exos具有增强的治疗潜力顺铂诱导的AKI。
BackgroundExosomes 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.MethodsMesenchymal stem cells (MSCs) were isolated from fresh human umbilical cord and cultured in two-dimensional (2D) flasks. 2x10(8) 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 2x10(8) 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.Results3D 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.ConclusionsIn 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.