Improved serum-free culture conditions for the differentiation of human and murine fibrocytes

Abstract

CD14+ peripheral blood monocytes can differentiate into fibroblast-like cells called fibrocytes. Fibrocytes are associated with, and are at least partially responsible for, wound healing and fibrosis in multiple organ systems. In a variety of lesions in vivo, monocytes appear to differentiate into fibrocytes within days. However, in vitro culture conditions can take up to two weeks to generate fibrocytes. In this study, we describe enhanced serum-free conditions that support the rapid differentiation of human and murine fibrocytes. We compared the effect on fibrocyte differentiation of different anti-coagulants used when collecting blood, and for culturing cells, the effects of different commercial media formulations, the addition of a variety of supplements, cell density, conditioned medium, and glass and plastic substrates. We found that both heparin and EDTA were suitable anti-coagulants, but that blood treated with citrate–phosphate dextrose led to a reduced number of fibrocytes. Fibrocyte differentiation was enhanced when the serum-free medium was based on either FibroLife or StemPro formulations. We also found that only positively charged or hydrophilic glass and plastic surfaces provide adequate support for fibrocyte differentiation. Finally, the optimal cell density was 2.5 × 10 5 cells/ml (approximately 800 cells per mm 2). These results indicate that blood collection, substrates, media, and cell density all influence in vitro fibrocyte differentiation.