AUTHOR=Perez-Diaz Noelia , Hoffman Ewelina , Clements Julie , Cruickshank Rebecca , Doherty Ann , Ebner Daniel , Elloway Joanne , Fu Jianan , Kelsall Joanne , Millar Val , Saib Ouarda , Scott Andrew , Woods Ian , Hutter Victoria 

TITLE=Longitudinal characterization of TK6 cells sequentially adapted to animal product-free, chemically defined culture medium: considerations for genotoxicity studies

JOURNAL=Frontiers in Toxicology

VOLUME=Volume 5 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/toxicology/articles/10.3389/ftox.2023.1177586

DOI=10.3389/ftox.2023.1177586

ISSN=2673-3080

ABSTRACT=In vitro approaches are an essential tool in screening for toxicity of new chemicals, products and therapeutics. Currently, FBS is routinely used as a supplement in cell culture medium, but batch-to-batch variability may introduce inconsistency in inter-and intra-lab assessments. Several chemically defined serum replacements (CDSR) have been developed to provide an alternative to FBS, but not every cell line adapts easily and successfully to CDSR-supplemented medium, and the long term effect on cell characteristics remains uncertain. 
The aim of this study was to adapt the TK6 cell line to animal-product free CDSR-supplemented medium and evaluate the long-term effects on cell health, growth, morphology, phenotype and function. Gradual adaptation and direct adaptation methodologies were compared. The metabolic activity and membrane integrity was assessed every 4-8 passages. A detailed morphology study by high content imaging was performed and the expression of cell surface markers (CD19 and CD20) was conducted via flow cytometry to assess the potential for phenotypic drift during longer term culture of TK6 in animal-free conditions. Finally, functionality of cells in the OECD TG 487 assay was evaluated.
The baseline characteristics of TK6 cells cultured in FBS-supplemented medium were established and variability among passages was used to set up acceptance criteria for CDSR adapted cells. The new animal cells showed comparable (p > 0.05) viability and cell size as the parent FBS-supplemented cells, with the exception of growth rate. The new animal free cells showed a lag phase double the length of the original cells. Cell morphology and phenotype were in line (p > 0.05) with the original cells.
TK6 cells were successfully transitioned to FBS-and animal product-free medium. The gradual transition methodology utilised in this study can also be applied to other cell lines of interest. Maintaining cells in CDSR-supplemented medium eliminates variability from FBS, which in turn is likely to increase the reproducibility of in vitro experiments. Furthermore, removal of animal derived products from cell culture techniques is likely to increase the human relevance of in vitro methodologies.