During the day of organoid formation, SC cultures should be in the log-growth phase, in which they are 50%-70% confluent, as shown in Figure 1(A-C). Cultures should form rounded colonies with distinct edges, and the wells should be clear of differentiation Figure 1(D-E). If differentiation is occurring, it is recommended to conduct a pick-to-remove to clean the affected areas within the plates at least one day prior to creating organoids. If a large portion of the culture is differentiated, it may be necessary to conduct an extra passage, thaw a new vial, or alter stem cell culture techniques to ensure that the organoids are being formed by pure stem cell cultures.
