Developing a Serum-Free and Cytokine-Optimizing Induction Medium to Increase the Production of CD14 + CD16 + and CD14 + CD16 − Monocytes from Human CD133 + Hematopoietic Stem and Progenitor Cells

Immunotherapy utilizes immune cells to target cancer and improves treatment outcomes with few side effects. Despite the effectiveness of immunotherapy, the limited availability of monocytes, which are essential for the differentiation of antigen-presenting cells, remains a major challenge. In this study, we developed a technique for inducing monocytes from hematopoietic stem and progenitor cells by using a serum-free (SF) medium supplemented with optimal concentrations of serum substitutes and cytokines. Three key serum substitutes, namely lipids, ascorbic acid, and β-glycerophosphate, were identified through factorial design screening, with their concentrations optimized through steepest ascent path analysis. Iscove's modified Dulbecco's medium was identified as the optimal basal medium. Long-term culturing confirmed the successful induction of CD14⁺CD16⁺ and CD14⁺CD16^- monocytes. Functional assays validated the efficacy of this technique with comparable gene expression, cytokine secretion, phagocytosis ability, and T-cell stimulating ability between SF and serum-containing cultures. Under SF conditions, high expression levels of CD16 were detected, indicating the broad range of potential applications of CD16⁺ monocytes. Overall, this technique represents a feasible SF alternative for monocyte generation, with potential benefits for immunotherapy.