Scalable processes are requisite for the robust biomanufacturing of human pluripotent stem cell (hPSC)‐derived therapeutics. Toward this end, we demonstrate the xeno‐free expansion and directed differentiation of human embryonic and induced pluripotent stem cells to definitive endoderm (DE) in a controlled stirred suspension bioreactor (SSB). Based on previous work on converting hPSCs to insulin‐producing progeny, differentiation of two hPSC lines was optimized in planar cultures yielding up to 87% FOXA2⁺/SOX17⁺ cells. Next, hPSCs were propagated in an SSB with controlled pH and dissolved oxygen. Cultures displayed a 10‐ to 12‐fold increase in cell number over 5–6 days with the maintenance of pluripotency (>85% OCT4⁺) and viability (>85%). For differentiation, SSB cultures yielded up to 89% FOXA2⁺/SOX17⁺ cells or ~ 8 DE cells per seeded hPSC. Specification to DE cell fate was consistently more efficient in the bioreactor compared to planar cultures. Hence, a tunable strategy is established that is suitable for the xeno‐free manufacturing of DE cells from different hPSC lines in scalable SSBs. This study advances bioprocess development for producing a wide gamut of human DE cell‐derived therapeutics.

中文翻译：

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自动化生物反应器中人多能干细胞的非异种扩增和定形内胚层分化

可扩展的过程是人类多能干细胞 (hPSC) 衍生疗法的稳健生物制造所必需的。为此，我们展示了在受控搅拌悬浮生物反应器 (SSB) 中，人类胚胎和诱导多能干细胞向定形内胚层 (DE) 的无异种扩增和定向分化。基于先前将 hPSC 转化为产生胰岛素的后代的工作，在平面培养物中优化了两种 hPSC 系的分化，产生高达 87% 的 FOXA2 ⁺ /SOX17 ⁺细胞。接下来，hPSC 在具有受控 pH 值和溶解氧的 SSB 中繁殖。在保持多能性的情况下，培养物在 5-6 天内细胞数量增加了 10-12 倍（>85% OCT4 ⁺) 和生存力 (>85%)。对于分化，SSB 培养物产生高达 89% FOXA2 ⁺ /SOX17 ⁺细胞或每个接种的 hPSC 约 8 个 DE 细胞。与平面培养物相比，生物反应器中对 DE 细胞命运的规范始终更有效。因此，建立了一种可调策略，适用于可扩展 SSB 中来自不同 hPSC 系的 DE 细胞的无异种制造。这项研究推进了生物过程开发，以生产广泛的人类 DE 细胞衍生疗法。