Animal component-free differentiation of human pluripotent stem cells into macrophages

• Robust xeno-free generation of precursor macrophages from hPSCs. • Macrophages polarized on recombinant human substrates show immune functionality. • Single-cell seeding improves macrophage differentiation on laminin-521. Macrophages are one of the key regulators of innate immunity and tissue homeostasis. Human pluripotent stem cell (hPSC)-derived macrophages offer a potential alternative to primary cells by addressing donor variability, limited availability, and scalability for in vitro applications. However, many differentiation protocols still depend on animal-derived components such as foetal bovine serum (FBS) and Matrigel, which introduce batch-to-batch variability and compromise reproducibility. In line with the 3R principles (Replacement, Reduction, and Refinement), we evaluated whether these components could be replaced by recombinant human vitronectin or laminin-521. Using defined xeno-free conditions, we demonstrate efficient generation of precursor macrophages on both substrates, with subsequent polarization into pro-inflammatory and anti-inflammatory subtypes. These macrophages exhibited expected surface marker expression, cytokine and chemokine secretion profiles, and phagocytic activity. To improve reproducibility even further, we replaced aggregate-based seeding with single-cell seeding on laminin-521. This yielded macrophages with similar phenotypic and functional characteristics. Overall, this study establishes a fully animal component-free protocol for generating immunocompetent myeloid cells from hPSCs that is robust across multiple independent cell lines. This supports the development of standardized in vitro models for immunological research and drug testing consistent with the 3R principles.