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 • Instructions for combining neurons, astrocytes, and microglia for brain cell modeling • Stepwise protocol to generate and assemble human iPSC-derived tri-cultures • Optimized media support mono-, co-, and tri-culture formats • Adaptable timelines allow flexibility for different experimental needs Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.