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.
Koelz et al. (2026) studied this question.