Understanding cell culture dynamics: a tool for defining protocol parameters for improved processes and efficient manufacturing using human embryonic stem cells.
Understanding cell culture dynamics: a tool for defining protocol parameters for improved processes and efficient manufacturing using human embryonic stem cells.
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DOI：
10.1080/21655979.2021.1902696
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发表时间：
2021-12
期刊：
影响因子：
4.9
通讯作者：
Wilson SL
中科院分区：
文献类型：
--
作者：
Kusena JWT;Shariatzadeh M;Thomas RJ;Wilson SL
标准化在培养包括人类胚胎干细胞（hESC）在内的细胞时至关重要，这对治疗开发和疾病建模很有价值。关于方案再现性的固有问题是有问题的，因为它们阻碍了转化为良好生产规范（GMP），从而降低了临床疗效和吸收。多能培养物需要标准化，以确保输入材料在分化前是一致的，因为输入细胞的不一致会产生最终产物的变化。为了改进方案，开发人员首先必须了解他们正在使用的细胞及其相关的培养动力学。与通常实施的通用方案相比，这项创新工作强调了优化且具有成本效益的生物过程所需的关键条件。这需要研究影响生长、代谢和表型动力学的条件，以确保细胞质量适合使用。结果显示，关键工艺参数（CPP）（包括补料方案和接种密度）影响关键质量属性（CQA），包括比代谢率（SMR）和比生长率（SGR）。这意味着工艺理解和控制对于保持关键单元特性、减少工艺变化和保持CQA至关重要。细胞动力学和CPP的检查允许形成用于培养H9 hESC的限定方案。作者建议，H9接种密度为20，000个细胞/cm 2，培养4天或从冷冻保存中恢复传代后培养3天，48小时后100%更换培养基是最佳的。这些参数在3天内的SGR为0.018 h −1 ± 1.5x10−3，细胞活力≥95%±0.4，同时产生高表达多能和增殖标记物Oct 3/4（>99%阳性）和Ki-67（>99%阳性）的细胞。
Standardization is crucial when culturing cells including human embryonic stem cells (hESCs) which are valuable for therapy development and disease modeling. Inherent issues regarding reproducibility of protocols are problematic as they hinder translation to good manufacturing practice (GMP), thus reducing clinical efficacy and uptake. Pluripotent cultures require standardization to ensure that input material is consistent prior to differentiation, as inconsistency of input cells creates end-product variation. To improve protocols, developers first must understand the cells they are working with and their related culture dynamics. This innovative work highlights key conditions required for optimized and cost-effective bioprocesses compared to generic protocols typically implemented. This entailed investigating conditions affecting growth, metabolism, and phenotype dynamics to ensure cell quality is appropriate for use. Results revealed critical process parameters (CPPs) including feeding regime and seeding density impact critical quality attributes (CQAs) including specific metabolic rate (SMR) and specific growth rate (SGR). This implied that process understanding, and control is essential to maintain key cell characteristics, reduce process variation and retain CQAs. Examination of cell dynamics and CPPs permitted the formation of a defined protocol for culturing H9 hESCs. The authors recommend that H9 seeding densities of 20,000 cells/cm2, four-day cultures or three-day cultures following a recovery passage from cryopreservation and 100% medium exchange after 48 hours are optimal. These parameters gave ~SGR of 0.018 hour−1 ± 1.5x10−3 over three days and cell viabilities ≥95%±0.4, while producing cells which highly expressed pluripotent and proliferation markers, Oct3/4 (>99% positive) and Ki-67 (>99% positive).
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