Figure 5.

Wnt conditions optimized in mouse colonoids allow for the long-term survival of human colonoids. Survival of human colonoids derived from three healthy donors was compared in different culture conditions over five passages. Media culture conditions from the top: Wnt3a-Noggin-Rspondin conditioned media (WNR CM), Wnt3a-conditioned media supplemented with recombinant EGF, Noggin and R-spondin (Wnt3a CM + rENR), and media with recombinant Wnt3a, EGF, Noggin, and R-spondin (rWENR). (A) Representative bright field images for each growth condition medium at every passage from colonoids derived from Donor 1. Scale bars indicate 100 microns. (B) Viability was assessed before each passage using the Cell Titer-Glo 3D luminescent cell viability assay in colonoid cultures from all three donors (WNR CM as black lines, Wnt3a CM + rENR as red lines, and rWENR medium as blue line). For each donor and treatment at each timepoint, 3 wells of colonoids were quantified by Cell Titer-Glo 3D and error bars represent the average. Representative images and survival from two independent experiments is shown. (C) Impact of TGFb inhibitor and ROCK inhibitor on organoid viability and apoptosis during splitting were assessed by Cell Titer-Glo 3D and Caspase-Glo 3/7 Assay, respectively. Data from Donor 2 is shown and is representative of studies done is Donor 1 and Donor 2. Error bars are representative of the average of biological triplicates from two independent experiments, one in Donor 1 and one in Donor 2. Statistical significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.