The importance of cell culture parameter standardization: an assessment of the robustness of the 2102Ep reference cell line.
The importance of cell culture parameter standardization: an assessment of the robustness of the 2102Ep reference cell line.
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DOI：
10.1080/21655979.2020.1870074
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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;Studd AJ;James JR;Thomas RJ;Wilson SL
使用胚胎癌2102 Ep细胞系进行的工作强调了对稳健、良好表征和标准化方案的需求。利用“快速命中”实验的系统方法证明了由于培养条件的轻微变化而引入培养系统的可变性（途径A）。结果表明，以20，000 cells/cm ~ 2接种，48 h后换液，72 h后重新接种，第59代P59细胞的比生长率（SGR（途径B2）培养的P55细胞的SGR值平均略高于同等条件下培养的P55细胞（途径A1）;其中SGR值为（0.021±0.004）和（0.019±0.004）。与途径A1（83.3±8.8）相比，途径B2在10次传代中的活力更高，报告的平均活力为（86.3%±8.1）。代谢物数据表明，培养途径B1（P57细胞以66，667个细胞/cm 2接种）和B2对葡萄糖具有一致的特异性代谢率（SMR），但途径B1的SMR值始终低于途径B2（0.00001 mmol，cell-1.d-1和0.000025）。结果显示表型之间的相互作用，生长速率或观察到的形态可能无法准确反映SMR和补料方案。这意味着当前的方案控制方案不能充分考虑变异性，因为关键细胞特征，包括表型和SMR，无论标准化接种密度如何都会发生变化。这突出了需要通过规定的方案控制培养参数，用于涉及治疗用途培养、生物制剂生产和参考线的工艺。
Work undertaken using the embryonic carcinoma 2102Ep line, highlighted the requirement for robust, well-characterized and standardized protocols. A systematic approach utilizing ‘quick hit’ experiments demonstrated variability introduced into culture systems resulting from slight changes to culture conditions (route A). This formed the basis for longitudinal experiments investigating long-term effects of culture parameters including seeding density and feeding regime (route B).Results demonstrated that specific growth rates (SGR) of passage 59 (P59) cells seeded at 20,000 cells/cm2 and subjected to medium exchange after 48h prior to reseeding at 72h (route B2) on average was marginally higher than, P55 cells cultured under equivalent conditions (route A1); whereby SGR values were (0.021±0.004) and (0.019±0.004). Viability was higher in route B2 over 10 passages with average viability reported as (86.3%±8.1) compared to route A1 (83.3±8.8). The metabolite data demonstrated both culture route B1 (P57 cells seeded at 66,667 cells/cm2) and B2 had consistent-specific metabolite rates (SMR) for glucose, but SMR values of route B1 was consistently lower than route B2 (0.00001 mmol, cell-1.d-1 and 0.000025).Results revealed interactions between phenotype, SMR and feeding regime that may not be accurately reflected by growth rate or observed morphology. This implies that current schemes of protocol control do not adequately account for variability, since key cell characteristics, including phenotype and SMR, change regardless of standardized seeding densities. This highlights the need to control culture parameters through defined protocols, for processes that involve culture for therapeutic use, biologics production, and reference lines.
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