Background Since their inception, human induced pluripotent stem cells (hiPSCs) have held much promise for pharmacological applications and cell-based therapies. However, their potential can only be realised if large numbers of cells can be produced reproducibly on-demand. While bioreactors are ideal systems for this task, due to providing agitation and control of the culture parameters, the common impeller geometries were not designed for the expansion of mammalian cells, potentially leading to sub-optimal results. Results This work reports for the first time the usage of the novel Vertical-Wheel single-use bioreactors for the expansion of hiPSCs as floating aggregates. Cultures were performed in the PBS MINI 0.1 bioreactor with 60 mL of working volume. Two different culture media were tested, mTeSR1 and mTeSR3D, in a repeated batch or fed-batch mode, respectively, as well as dextran sulfate (DS) supplementation. mTeSR3D was shown to sustain hiPSC expansion, although with lower maximum cell density than mTeSR1. Dextran sulfate supplementation led to an increase in 97 and 106% in maximum cell number when using mTeSR1 or mTeSR3D, respectively. For supplemented media, mTeSR1 + DS allowed for a higher cell density to be obtained with one less day of culture. A maximum cell density of (2.3 ± 0.2) × 106 cells∙mL- 1 and a volumetric productivity of (4.6 ± 0.3) × 105 cells∙mL- 1∙d- 1 were obtained after 5 days with mTeSR1 + DS, resulting in aggregates with an average diameter of 346 ± 11 μm. The generated hiPSCs were analysed by flow cytometry and qRT-PCR and their differentiation potential was assayed, revealing the maintenance of their pluripotency after expansion. Conclusions The results here described present the Vertical-Wheel bioreactor as a promising technology for hiPSC bioprocessing. The specific characteristics of this bioreactor, namely in terms of the innovative agitation mechanism, can make it an important system in the development of hiPSC-derived products under current Good Manufacturing Practices.

中文翻译：

---

在一次性 Vertical-Wheel™ 生物反应器中作为聚集体扩增人类诱导多能干细胞的策略。

背景自问世以来，人类诱导的多能干细胞 (hiPSC) 在药理学应用和基于细胞的治疗方面具有很大的前景。然而，它们的潜力只有在可以按需重复生产大量细胞的情况下才能发挥出来。虽然生物反应器是完成这项任务的理想系统，但由于提供了对培养参数的搅拌和控制，常见的叶轮几何形状不是为哺乳动物细胞的扩增而设计的，可能会导致次优结果。结果这项工作首次报告了使用新型垂直轮一次性生物反应器来扩展 hiPSC 作为浮动聚集体。在具有 60 mL 工作体积的 PBS MINI 0.1 生物反应器中进行培养。测试了两种不同的培养基，mTeSR1 和 mTeSR3D，分别以重复分批或补料分批模式，以及硫酸葡聚糖 (DS) 补充。mTeSR3D 被证明可以维持 hiPSC 扩增，尽管最大细胞密度低于 mTeSR1。当使用 mTeSR1 或 mTeSR3D 时，硫酸葡聚糖补充剂分别导致最大细胞数增加 97% 和 106%。对于补充培养基，mTeSR1 + DS 允许用更少的培养天数获得更高的细胞密度。使用 mTeSR1 + DS 5 天后，获得了 (2.3 ± 0.2) × 106 个细胞∙mL-1 的最大细胞密度和 (4.6 ± 0.3) × 105 个细胞∙mL-1∙d-1 的体积生产力，导致平均直径为 346 ± 11 μm 的聚集体。通过流式细胞术和qRT-PCR分析产生的hiPSC，并测定其分化潜能，揭示了它们在扩展后保持多能性。结论 这里描述的结果表明垂直轮生物反应器是一种很有前途的 hiPSC 生物加工技术。该生物反应器的特殊特性，即创新的搅拌机制，使其成为当前良好生产规范下开发 hiPSC 衍生产品的重要系统。