Macrophages are crucial in immune responses, tissue repair and homeostasis, making them prime candidates for translational applications. Induced pluripotent stem cell (iPS cell)-derived macrophages hold considerable promise for regenerative medicine, cancer therapy, inflammatory disease treatment and in vitro bioassays. However, cost-effective, standardized intermediate-scale bioreactor systems tailored for early-stage research and drug discovery in academia remain limited. Here, we present an extension of our previously published protocol that is feeder free, semi-defined and user friendly, enabling the standardized production of iPS cell-derived macrophages in an intermediate (10-50 mL)-scale benchtop bioreactor. This Protocol can be implemented by users with basic iPS cell culture experience without requiring advanced bioprocessing expertise. This method consists of two primary endpoints: the generation of mesoderm-primed aggregates with hematopoietic potential, termed hemanoids, and the standardized production of iPS cell-derived macrophages that are ready for downstream applications. This Protocol enables continuous macrophage generation in long-term cultures, with a minimum of five consecutive collections, yielding an average of 2-3 × 10(7) cells per collection per vessel. Four vessels operate independently, each with a maximum culture volume of up to 50 mL, while critical process parameters (CO(2), temperature and pH) are monitored. This semi-automated platform and in-process monitoring improve process control, leading to higher yields, reproducibility and cell quality compared with other systems. The simplified process spans 24 d, starting from single-cell iPS cells to ready-to-use macrophages. By bridging the gap between small- and large-scale systems, this approach provides scalable, standardized manufacturing of iPS cell-derived macrophages, making it a valuable tool for academics focused on human immune cells such as macrophages.
