Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks.
Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks.
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DOI：
10.1007/s00253-023-12634-w
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发表时间：
2023-09
影响因子：
5
通讯作者：
Rafiq, Qasim A. A.
中科院分区：
文献类型：
--
作者：
Couto, Pedro Silva;Stibbs, Dale J. J.;Rotondi, Marco C. C.;Takeuchi, Yasuhiro;Rafiq, Qasim A. A.
由于其免疫调节特性和体外分化能力，人类间充质基质细胞 (hMSC) 在过去十年中已在 1000 多项临床试验中进行了研究。多项探索基因修饰 hMSC 产品开发的研究现已进入临床试验项目的早期阶段。从工程角度来看，挑战在于开发能够生产足够剂量的体外基因修饰 hMSC 以用于临床应用的制造方法。这项工作首次展示了使用微载体生物反应器系统扩展基因修饰 hMSC 的可扩展制造工艺。分离后，使用带有绿色荧光蛋白 (GFP) 或血管内皮生长因子 (VEGF) 转基因的慢病毒载体 (LV) 转导脐带组织间充质基质细胞 (UCT-hMSC)。然后使用 Spherecol 微载体将细胞接种到 100 mL 旋转烧瓶中并扩增 7 天。培养六天后，未转导和转导的细胞群都达到了相当的最大细胞浓度（约 1.8 × 105 个细胞/mL）。对培养物上清液的分析发现，第五天后细胞群中的葡萄糖完全耗尽。在整个培养物中观察到的乳酸浓度在第七天达到最大值 7.5 mM。免疫表型分析表明，转导后的扩增步骤并不导致用于鉴定 hMSC 的细胞表面受体的下调。未转导和转导细胞的CD73、CD90和CD105表达细胞的水平高于90%。此外，阴性标记物（CD11b、CD19、CD34、CD45和HLA-DR）的表达也低于5%，这与国际细胞与基因治疗学会（ISCT）制定的hMSCs标准一致。这项工作为基因修饰 hMSC 的可扩展生产奠定了基础，这将克服重大的转化和商业瓶颈。 • hMSC 被携带两种不同转基因的慢病毒载体成功转导：GFP 和 VEGF • 转导的 hMSC 在 7 天的时间内使用转瓶在微载体上成功扩增 • 遗传修饰步骤并未对 hMSC 免疫表型特征造成任何有害影响 在线版本包含可在以下网址获取的补充材料 10.1007/s00253-023-12634-w。
Due to their immunomodulatory properties and in vitro differentiation ability, human mesenchymal stromal cells (hMSCs) have been investigated in more than 1000 clinical trials over the last decade. Multiple studies that have explored the development of gene-modified hMSC-based products are now reaching early stages of clinical trial programmes. From an engineering perspective, the challenge lies in developing manufacturing methods capable of producing sufficient doses of ex vivo gene-modified hMSCs for clinical applications. This work demonstrates, for the first time, a scalable manufacturing process using a microcarrier-bioreactor system for the expansion of gene-modified hMSCs. Upon isolation, umbilical cord tissue mesenchymal stromal cells (UCT-hMSCs) were transduced using a lentiviral vector (LV) with green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) transgenes. The cells were then seeded in 100 mL spinner flasks using Spherecol microcarriers and expanded for seven days. After six days in culture, both non-transduced and transduced cell populations attained comparable maximum cell concentrations (≈1.8 × 105 cell/mL). Analysis of the culture supernatant identified that glucose was fully depleted after day five across the cell populations. Lactate concentrations observed throughout the culture reached a maximum of 7.5 mM on day seven. Immunophenotype analysis revealed that the transduction followed by an expansion step was not responsible for the downregulation of the cell surface receptors used to identify hMSCs. The levels of CD73, CD90, and CD105 expressing cells were above 90% for the non-transduced and transduced cells. In addition, the expression of negative markers (CD11b, CD19, CD34, CD45, and HLA-DR) was also shown to be below 5%, which is aligned with the criteria established for hMSCs by the International Society for Cell and Gene Therapy (ISCT). This work provides a foundation for the scalable manufacturing of gene-modified hMSCs which will overcome a significant translational and commercial bottleneck. • hMSCs were successfully transduced by lentiviral vectors carrying two different transgenes: GFP and VEGF • Transduced hMSCs were successfully expanded on microcarriers using spinner flasks during a period of 7 days • The genetic modification step did not cause any detrimental impact on the hMSC immunophenotype characteristics The online version contains supplementary material available at 10.1007/s00253-023-12634-w.
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影响因子：
23.9
作者：
Caplan AI;Correa D
通讯作者：
Correa D
逆转录病毒DNA整合：靶位点选择的病毒和细胞决定因素。
影响因子：
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作者：
Du WJ;Chi Y;Yang ZX;Li ZJ;Cui JJ;Song BQ;Li X;Yang SG;Han ZB;Han ZC
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作者：
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通讯作者：
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作者：
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通讯作者：
Cabral, Joaquim M. S.