Protocol for generating a human iPSC-derived tri-culture model to study interactions between neurons, astrocytes, and microglia

Human induced pluripotent stem cell (hiPSC) models enable disease modeling and drug screening, but standardized methods for multi-lineage co-culture remain limited. Here, we present a cryopreservation-compatible tri-culture system of neurons, astrocytes, and microglia. We describe steps for transducing induced pluripotent stem cells (iPSCs) with cell type-specific factors, generating intermediate cryopreserved stocks, differentiating each cell population, and assembling them into tri-culture. This protocol provides a reproducible platform to study dynamic interactions between human brain cells in a physiologically relevant environment. For complete details on the use and execution of this protocol, please refer to Lish et al.¹^,².