Toward clinically relevant automated corneal biomanufacturing with human-derived FBS alternatives

The replacement of animal-derived components from cell culture protocols is essential for the development of human-compatible and clinically translatable systems. Fetal bovine serum (FBS) is still widely used for cell expansion, although its xenogeneic origin and batch variability limit regulatory compliance. In this study, human platelet lysate (hPL) and human serum (HS) were assessed as low-serum (2%) alternatives to FBS during the subculture phase of bone marrow-derived mesenchymal stromal cells (BM-MSC) prior to keratocyte differentiation for corneal tissue engineering. BM-MSC were gradually adapted over four days to 2% FBS, hPL, or HS, maintained for seven days, and then transferred to serum-free keratocyte differentiation medium for 14 days in either two-dimensional (2D) or three-dimensional (3D) cultures in 30 wt% riboflavin-arginine-triggered gelatin methacryloyl (RA-GelMA) hydrogels crosslinked under visible blue light. To demonstrate the applicability of this approach in corneal tissue engineering, differentiation was evaluated by immunofluorescence and quantitative PCR in 2D cultures, and by immunofluorescence in 3D cultures. Both hPL and HS maintained metabolic activity, supported keratocyte-associated marker expression, and suppressed α-SMA, performing comparably to FBS, thereby supporting their use as clinically compliant, human-derived alternatives for xeno-reduced corneal biomanufacturing.