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) are 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 in the alternatives are often lacking. Here, we have 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 media, we systematically analyzed the cultures in 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 detected. With the exception of K-562, FBS-free cultures showed a reduced proliferation rate and, in some conditions, slight morphological alterations in comparison to FBS cultures. 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 even long-established cancer models. Plain language summaryDespite existing alternatives and known drawbacks, fetal bovine serum (FBS) is still commonly used for culturing human cancer cell lines. So far, FBS-free alternatives are rarely used, as it is often unclear whether they alter the cellular phenotype. To close this gap, we adapted four widely used human cancer cell lines to different FBS-free media and then systematically analyzed recovery after cryopreservation, markers for genetic drift, proliferation, morphology and differences in global gene expression. While recovery after freezing and genetic stability markers remained unaffected, cells often grew more slowly in FBS-free media. In all cell lines culture media influenced genes involved in cholesterol synthesis and uptake, but, importantly, only few phenotypic and differentiation marker genes were affected by the media. Overall, the main characteristics of the cancer cells remained unchanged supporting the suitability of the chosen FBS-free media. Therefore, this study provides strong evidence for the transition to FBS-free media.