Fetal Bovine Serum Modulates Primary Human Cell Phenotypes, Endothelial Barrier Function, Vasculogenesis, and Angiogenesis in a Sex-Specific Manner.

Purpose: Sex differences in cellular biology significantly influence cell responses in culture. Yet, the sex-specific effects of culture reagents such as fetal bovine serum (FBS) remain understudied. Increased adoption of cell-based models such as microphysiological systems (MPS) as replacements for animal models demands a greater understanding sex-specific responses to common media formulations. This study examined the effects of FBS and hormone-free charcoal-stripped serum (CSS) on male (XY) and female (XX) cells in 2D and 3D MPS culture models to demonstrate profound sex-specificity in bioassays and inform the development of future sex-specific cell culture protocols and methods. Methods: Primary human endothelial cells and fibroblasts from multiple organ sources were cultured in 2D and in 3D MPS models. Cells were cultured with either FBS or CSS. Endothelial specific gene expression, cytoskeletal spreading, and cell cycle status were analyzed in 2D culture. Vascular network formation, macromolecular leakage, and directional angiogenic sprouting were assessed in 3D MPS models. Results: FBS promoted significant upregulation of genes associated with endothelial function in XX endothelial cells, but the same gene clusters were downregulated in XY cells. FBS increased cytoskeletal spreading and cell cycle participation of XX endothelial cells and fibroblasts relative to culture with CSS. Conversely, culture with CSS increased these 2D metrics in XY cells. Measurement of 40 kDa FITC-dextran leakage in a single vessel MPS model revealed that culture with FBS significantly decreased XX endothelial barrier permeability relative to culture with CSS. In line with 2D assays, CSS conversely enhanced XY endothelial barrier permeability relative to culture with FBS. Culture with FBS increased metrics of vasculogenesis in XX tissues relative to CSS cultures, whereas prolonged cultured in CSS supported vasculogenesis in XY models. MPS angiogenesis assays revealed increased sprouting in XX tissues cultured with FBS, while only minimal sprouting was observed in all other conditions. Conclusions: FBS imparted significant sex-specific effects on the gene expression patterns, morphology, and cell cycle status of human endothelial cells and fibroblasts in 2D culture. Sex-specific effects measured in 2D culture assays carried over to 3D MPS assays of endothelial barrier function, vasculogenesis, and angiogenesis. Notably, FBS significantly enhanced XX cell functions relative to XY cells in all 2D and 3D MPS assays. Thus, accounting for the sex-specific effects of culture media components will be imperative to improve reproducibility and translational relevance of MPS in preclinical research. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-025-00860-3.