Microglia-containing neural organoids as brain Microphysiologic Systems for long-term culture

Alexandra Rittenhouse¹Caroline Krall^1,2Jesse David Plotkin¹Dowlette Mary Alam El Din¹Breanne Kincaid¹Lena Smirnova^1*

• ¹Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States
• ²School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States

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 (immunecompetent 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.