Microglia-containing neural organoids as brain microphysiological systems for long-term culture

Microglia are cells essential for many aspects of brain development, homeostasis, and neuroinflammation that originate from the yolk sac during embryogenesis and migrate to the developing brain. Considering their developmental origin, many brain organoid models would naturally lack them and require a co-culture. We developed a microglia-integrated brain organoid model (immune-competent brain microphysiological system: µbMPS) using hiPSC-derived neural and microglia progenitors aggregated in U-bottom 96-well plates, enabling controlled and reproducible incorporation of microglia progenitors. We demonstrated that microglia integrated, matured, and survived long-term in the neural environment without costly exogenous microglia-specific growth factors and cytokines. We maintained microglia for over nine weeks, demonstrating functional activity, phagocytosis, and neuroinflammatory responses. µbMPS exhibited enhanced neuronal activity and maturity, offering a scalable, reproducible model for neurodevelopment, disease modeling, and neurotoxicology research. Overall design: Human iPSC derived neuronal progenitor cells were combined with human iPSC derived immature microglia to form immune competent brain microphysiologic systems, that will mature to include functioning neurons, astrocytes, microglia and myelinating oligodendrocytes. Following two weeks of maturation the bMPS were collected for RNA sequencing to analyze expression of microglia related marker genes.