Multiparametric evaluation of different FBS-free replacement media for widely used human cancer cell lines

Despite ethical concerns and scientific drawbacks, fetal bovine serum (FBS) remains a common supplement of culture media for continuous cancer cell lines. Although FBS alternatives like human platelet lysate (hPL) and animal component-free, chemically defined media (CDM) have been com­mercially available for many years, acceptance of alternative media is limited, as data verifying the stability of the phenotype and function of a cell line cultured using alternative media are often lacking. Here, we adapted four widely used human cancer cell lines (HELA, HL-60, JIMT-1, K-562) to hPL-supplemented media and different CDM. To evaluate the FBS-free replacements in com­parison to FBS-containing media, we systematically analyzed the cultures with respect to recovery after cryopreservation, short tandem repeat (STR) profile stability, proliferation, morphology, and transcriptomic alterations. Neither changes in STR profiles nor difficulties after cryopreservation were found. Except for K-562, FBS-free cultures showed a reduced proliferation rate and, in some conditions, slight morphological alterations in comparison to cultures with FBS. In all cell lines, gene set enrichment analyses revealed that culture media mainly affected expression of cholesterol homeostasis genes. In HELA and JIMT-1, media also influenced genes of epithelial-mesenchymal transition; nevertheless, their overall phenotypic hallmarks remained stable. Only a few differen­tiation markers were among the differentially expressed genes of HL-60 and K-562 cultures, while their main phenotypes remained unchanged. This was further confirmed by successful induction of differentiation of HL-60 in FBS-free media. In conclusion, our multiparametric approach provides strong evidence supporting the transition to FBS-free media for long-established cancer cell lines.