The technique for producing organoids from standard, feeder-free PSC cultures, and infecting these organoids with ZIKV is presented in full. For guidance on the culturing methods needed for feeder-free PSCs, please refer to previous methods publications2526. Additionally, in order to apply consistent amounts of virus for multiple experiments, it is important to calculate the MOI ahead of time. This is done by conducting an infection of Vero cells, followed by treatment with overlay medium, incubation, and immunostaining. Descriptions and methods of this technique have been previously described1927.
