Neurodegenerative disorders present a broad group of neurological diseases and remain one of the greatest challenges and burdens to mankind. Maladies like amyotrophic lateral sclerosis, Alzheimer's disease, stroke or spinal cord injury commonly features astroglia involvement (astrogliosis) with signs of inflammation. Regenerative, paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) could target the above components, thus opening new therapeutic possibilities for regenerative medicine. A special interest should be given to hMSCs derived from the umbilical cord (UC) tissue, due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard operating and scale-up good manufacturing practice (GMP) protocols of UC-hMSCs isolation, characterization, expansion and comparison of cells' properties when harvested on T-flasks versus using a large-scale bioreactor system. Human UC-hMSCs, isolated by tissue explant culture technique from Wharton's jelly, were harvested after reaching 75% confluence and cultured using tissue culture flasks. Obtained UC-hMSCs prior/after the cryopreservation and after harvesting in a bioreactor, were fully characterized for "mesenchymness" immunomodulatory, tumorigenicity and genetic stability, senescence and cell-doubling properties, as well as gene expression features. Our study demonstrates an efficient and simple technique for large scale UC-hMSCs expansion. Harvesting of UC-hMSCs' using classic and large scale methods did not alter UC-hMSCs' senescence, genetic stability or in vitro tumorigenicity features. We observed comparable growth and immunomodulatory capacities of fresh, frozen and expanded UC-hMSCs. We found no difference in the ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages between classic and large scale UC-hMSCs expansion methods. Both, methods enabled derivation of genetically stabile cells with typical mesenchymal features. Interestingly, we found significantly increased mRNA expression levels of neural growth factor (NGF) and downregulated insulin growth factor (IGF) in UC-hMSCs cultured in bioreactor, while IL4, IL6, IL8, TGFb and VEGF expression levels remained at the similar levels. A culturing of UC-hMSCs using a large-scale automated closed bioreactor expansion system under the GMP conditions does not alter basic "mesenchymal" features and quality of the cells. Our study has been designed to pave a road toward translation of basic research data known about human UC-MSCs for the future clinical testing in patients with neurological and immunocompromised disorders. An industrial manufacturing of UC-hMSCs next will undergo regulatory approval following advanced therapy medicinal products (ATMP) criteria prior to clinical application and approval to be used in patients.

中文翻译：

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大规模的中空纤维生物反应器的脐带衍生人间质基质细胞的大规模自动化的神经系统疾病。

神经退行性疾病表现出广泛的神经系统疾病，并且仍然是人类最大的挑战和负担之一。肌萎缩性侧索硬化症，阿尔茨海默氏病，中风或脊髓损伤等疾病通常以星状胶质受累（星状胶质增生）为特征，并伴有炎症迹象。人间充质基质细胞（hMSCs）的再生，旁分泌和免疫调节特性可以靶向上述成分，从而为再生医学开辟了新的治疗可能性。源自脐带（UC）组织的hMSC，由于其来源，性质和缺乏道德范式，应给予特别关注。这项研究的目的是建立UC-hMSC的分离，表征，与使用大型生物反应器系统相比，在T瓶上收获时细胞的扩增和细胞特性比较。通过组织外植体培养技术从沃顿商学院的果冻中分离出的人UC-hMSC在达到75％汇合后收获，并使用组织培养瓶进行培养。冷冻保存之前/之后和在生物反应器中收获后获得的UC-hMSC，具有“同质性”免疫调节，致瘤性和遗传稳定性，衰老和细胞倍增特性以及基因表达特征的特征。我们的研究表明大规模UC-hMSCs扩展的有效和简单的技术。使用经典和大规模方法收获UC-hMSCs不会改变UC-hMSCs的衰老，遗传稳定性或体外致瘤性特征。我们观察到了新鲜，冷冻和扩增的UC-hMSC的可比生长和免疫调节能力。我们发现在经典和大规模的UC-hMSCs扩增方法之间，向成脂，成骨和成软骨谱系的分化能力没有差异。两种方法都能够衍生出具有典型间充质特征的遗传稳定细胞。有趣的是，我们发现在生物反应器中培养的UC-hMSC中神经生长因子（NGF）和胰岛素生长因子（IGF）的mRNA表达水平显着增加，而IL4，IL6，IL8，TGFb和VEGF的表达水平保持在相似的水平。在GMP条件下使用大规模自动封闭式生物反应器扩展系统培养UC-hMSC不会改变细胞的基本“间质”特征和质量。我们的研究旨在为翻译有关人UC-MSC的基础研究数据铺平道路，以便将来在神经系统疾病和免疫功能低下患者中进行临床测试。下一步，工业化生产的UC-hMSCs将在临床应用和批准用于患者之前，先按照高级治疗药物产品（ATMP）的标准接受监管部门的批准。