cWEHI-YH2 CM stimulates colon crypt formation maximally.

<p>Different proportions (2.5% to 50%, v/v) of ten-fold concentrated YH2CM conditioned media (cYH2CM, using the Vivaflow 50R) was used to grow colon crypts in culture. Corresponding co-cultures with WEHI-YH2 cells and without cYH2CM were used as positive and negative control respectively. Whole well image stacks of the cultures were acquired on days 0, 2, 4 and 6. Images were quantitated for average counts, sizes, crypts per organoid as well as the efficiency for formation of Colonosphere (A and C) and colonoid (B, D-F). For colonosphere formation, 12.5% of cYH2CM is sufficient to match the effect of YH2-CM co-culture in maintaining spheroid counts (A) and promoting increase in spheroid size (C). For forming colonoids, 25% cYH2CM was needed to match the effect of the WEHI-YH2 feeder layers in terms of counts (B) and proliferation (D). (E) When used at ≥12.5% (v/v) the cYH2CM improves the average crypt budding rate per colonoid significantly (compared to the co-cultures). (F) The colonosphere and colonoid formation efficiencies at Day 6 (compared to Day 0) using ≥25% cYH2CM (v/v) is comparable to the WEHI-YH2 co-cultures. Error bars: SEM, n = 3 experiments of duplicates, 2-way ANOVA multiple comparisons (A, B) Dunnett test, (C,D,E) Tukey’s test.