Once the PSC culture reaches the target confluence of 50%-70%, the cells are then dissociated and aggregated into ultra-low attachment 96-well plates. The cells are maintained for 3 days in xeno-free stem cell maintenance media (SCMM), and then converted to a neural induction media for the remainder of the culture. Once the organoids have differentiated for 3 weeks, the infection can be conducted. By routinely taking images during the week following infection, researchers will observe progressive cell death and disruption of the organoid. Researchers may also dissociate the organoids at this time to conduct transcriptional or proteomic profiling. Cryosectioning and lightsheet methods are recommended for imaging, and researchers can expect to see high levels of infection and viral replication particularly within the neural progenitor cell (NPC) populations in the organoids. Ultimately, this technique allows researchers to rapidly examine the mechanisms of viral infection of the human brain with low cost and limited equipment.
