Media matters: phenol red and fetal bovine serum estrogen in traditional cell culture media influence human mesenchymal stromal cell (hMSC) processes and differentiation in a sex-biased manner.

Estrogens are global regulators of cellular signaling pathways, impacting fundamental processes and phenotypes that are essential for tissue remodeling and homeostasis. Traditional cell culture media contains estrogen-mimetic compounds, including phenol red and endogenous estrogen in fetal bovine serum (FBS). However, the potential of these compounds to bias in vitro studies, particularly when considering sex as a biological variable, remains unclear. This gap in understanding critically impacts the culture of human mesenchymal stromal cells (hMSCs), whose basic functions and differentiation potential, central to cell therapy and tissue engineering, are sensitive to perturbations in the culture conditions. Despite this, the effect of estrogens from cell culture media on male and female hMSCs is not currently considered in cell processing for clinical trials. As such, a baseline understanding of these estrogen-mimetic media influences on hMSCs is critical for clinical efficacy and adequate study design in research. To this end, we investigated the effects of phenol red and fetal bovine serum on the proliferation, metabolism, senescence, and differentiation capacity of male and female hMSCs. Phenol red, FBS, donor sex, and 17β-estradiol (E2) supplementation all had significant impacts on hMSC health and differentiation potential in culture. Notably, dosing with estrogen at the levels found in FBS did not recover most of the hMSC metrics tested. The notable outcomes that were significantly impacted by sex for the overall analysis include proliferation, metabolism, senescence, osteogenic differentiation, adipogenic differentiation, and gene expression for Col1a1, ESR1, Col10a1, RUNX2, FABP4, and PPARγ. Donor variability was a main driver in all the outcomes tested, highlighting the need for personalized medicine in research investigating cellular response to hormone signaling. Overall, these findings reveal the sex-biased effects of estrogen and estrogen-mimetic compounds in traditional culture media, underscoring a current gap in considering sex as a biological variable in cell therapy and tissue engineering research and manufacturing.