Supplementary Information files for The importance of cell culture parameter standardization: an assessment of the robustness of the 2102Ep reference cell line

Supplementary Information files for The importance of cell culture parameter standardization: an assessment of the robustness of the 2102Ep reference cell line
Reference cell lines are often used for quality assessment; however, their effectiveness can be encumbered by the lack of standardized culture protocols. This results in variation to a supposed standard reference, inherently causing variation in measurement and analysis of the cells of interest. This is problematic when reference marker stability and characteristics are affected by use of non-standardized culture procedures. The overarching aim of this work was to apply defined parameter changes to an in-house protocol adapted from the National Institute for Biological Standards and Control. This was to investigate the impact of cell culture parameter changes on process output variation i.e. variability of growth rate and cell phenotype. The use of defined seeding densities and time-defined passage points sought to mitigate human-based sources of variation. Work was undertaken using the embryonic carcinoma 2102Ep reference line. highlighted the requirement for robust, well-characterised and standardized culture protocols. Initially, a systematic approach utilising "quick hit" experiments demonstrated significant variability introduced into culture systems resulting from slight changes to culture conditions (culture route A). This formed the basis for longitudinal experiments investigating long-term effects of culture parameters including seeding density and feeding regime (culture route B). Our results demonstrated that the specific growth rate (SGR) of passage 59 (P59) cells seeded at 20,000 cells/cm2 and subjected to a medium exchange after 48 hours prior to reseeding at 72 hours (route B2) on average was marginally higher than, P55 cells cultured under equivalent conditions (culture route A1); where the SGR values were (0.021 ±0.004) and (0.019 ±0.004) respectively. Cell viability was higher in route B2 over 10 passages with average cell viability reported as (86.3 % ±8.1) compared to route A1 (83.3 ±8.8). The metabolite data demonstrated that both culture route B1 (P57 cells seeded at 66,667 cells/cm2) and B2 had a consistent specific metabolite rate (SMR) for glucose metabolism over the 10 passages but glucose SMR values of route B1 was consistently lower than route B2 (0.00001 mmol. cell-1.d-1 and 0.000025 respectively). The present work noted that cell behaviour differences and characteristics are based not only on density, but also feeding regimes. Results revealed an interaction between phenotype, SMR and feeding regime that may not be accurately reflected by growth rate or observed morphology. This infers 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 cell culture for therapeutic use, biologics production, and reference lines. For the latter, this is imperative as operator-defined protocol changes result in cell characteristic variation, abating the effective use as reference lines for process control and product validation.

《细胞培养参数标准化的重要性：2102Ep参考细胞系稳健性评估》补充信息文件
参考细胞系常被用于质量评估，但由于缺乏标准化的培养方案，其应用效能往往受到限制。这会导致既定标准参考物出现偏差，进而不可避免地引发目标细胞的检测与分析结果产生波动。当非标准化培养操作影响参考标志物的稳定性与特性时，该问题将更为突出。
本研究的核心目标是，对源自国家生物标准与控制研究所的自研培养方案施加可控的参数调整，以此探究细胞培养参数变化对实验产出波动的影响，即生长速率与细胞表型的变异程度。研究采用可控的接种密度与时间限定的传代节点，以降低人为因素带来的误差，实验材料为胚胎癌2102Ep参考细胞系。
该研究凸显了建立稳健、经过充分表征且标准化的培养方案的必要性。初期，采用‘快速验证’实验的系统性研究方法显示，培养条件的细微调整（培养路径A）会给培养体系引入显著的波动。该结果为后续探究接种密度、补料策略等培养参数长期影响的纵向实验奠定了基础（培养路径B）。
本研究结果显示，以20000个细胞/平方厘米的密度接种、且在接种后48小时换液、72小时再次传代的第59代（P59）细胞（培养路径B2），其平均比生长速率（SGR）略高于同等培养条件下的第55代（P55）细胞（培养路径A1）；二者的SGR值分别为(0.021 ±0.004)与(0.019 ±0.004)。在10次传代过程中，培养路径B2的细胞存活率始终更高，平均存活率为(86.3% ±8.1)，而培养路径A1的平均存活率为(83.3 ±8.8)。
代谢组数据显示，在10次传代过程中，培养路径B1（接种密度为66667个细胞/平方厘米的第57代P57细胞）与B2的葡萄糖代谢比代谢速率（SMR）均保持稳定，但路径B1的葡萄糖SMR值始终低于路径B2，二者分别为0.00001 mmol·细胞^-1·天^-1与0.000025。
本研究发现，细胞行为差异与特性不仅取决于接种密度，还与补料策略密切相关。研究结果还揭示，细胞表型、比代谢速率与补料策略之间存在交互作用，而该作用无法通过生长速率或观测到的形态学特征准确体现。这表明当前的方案管控体系未能充分考虑变异来源，因为即便采用标准化的接种密度，包括表型与比代谢速率在内的关键细胞特性仍会发生变化。
这凸显了通过标准化方案管控培养参数的必要性，其应用场景涵盖治疗用细胞培养、生物制品生产以及参考细胞系相关流程。对于参考细胞系而言，该必要性更为迫切：操作人员自行调整培养方案会导致细胞特性发生变异，进而削弱其作为流程管控与产品验证参考系的有效应用价值。