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 lineReference 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.