There are several caveats to consider when utilizing human cerebral organoids to investigate ZIKV infection. One important consideration is that organoid formation and structure is highly dependent on the stem cell line from which they are formed. Take care when comparing between cell lines, including isogenic engineered lines; it is best to make conclusions using multiple subclones, and ideally multiple stem cell lines. Additionally due to this difference in cell lines, pilot experiments to ensure that proper differentiation is occurring in the organoids is recommended. While the neuroepithelial structures are visible by brightfield microscopy, it is also suggested to conduct qRT-PCR or cryosectioning experiments to look for neural differentiation markers such as PAX6 or phospho-vimentin.
