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

@article{koelz2026multiparametric,
  title = {{Multiparametric evaluation of different FBS-free replacement media for widely used human cancer cell lines}},
  author = {Anne L. Koelz and Claudia Pommerenke and Pascal Woitschewski and Yvonne Merkhoffer and Wilhelm G. Dirks and Sonja Eberth},
  journal = {ALTEX: Alternatives to Animal Experimentation},
  year = {2026},
  month = jul,
  doi = {10.14573/altex.2512111},
  url = {https://doi.org/10.14573/altex.2512111},
  note = {CC BY},
}

TY  - JOUR
TI  - Multiparametric evaluation of different FBS-free replacement media for widely used human cancer cell lines
AU  - Anne L. Koelz
AU  - Claudia Pommerenke
AU  - Pascal Woitschewski
AU  - Yvonne Merkhoffer
AU  - Wilhelm G. Dirks
AU  - Sonja Eberth
PY  - 2026
DA  - 2026/07/01
JO  - ALTEX: Alternatives to Animal Experimentation
DO  - 10.14573/altex.2512111
UR  - https://doi.org/10.14573/altex.2512111
AB  - 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.  Plain language summary Despite existing alternatives and known drawbacks, fetal bovine serum (FBS), obtained from the blood of calf fetuses, is still commonly used for culturing human cancer cell lines. So far, FBS-free alternatives are rarely used, as it is often unknown whether they alter cellular properties. We adapted four widely used human cancer cell lines to different FBS-free media and systematically analyzed numerous parameters in comparison to cultures grown in FBS-containing media. While recovery after freezing and genetic stability markers were unaffected, cells often grew more slowly in FBS-free media. In all cell lines, the culture media influenced genes involved in cholesterol syn­thesis and uptake, but importantly, only few phenotypic and differentiation marker genes were affected. Overall, the main characteristics of the cancer cells remained unchanged, supporting the suitability of the chosen FBS-free media. This study provides strong evidence supporting the transition to FBS-free media.
SN  - 1868-596X
SN  - 1868-8551
ER  -