Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor

Figure 4 Low recovery of cryopreserved intestinal organoids upon long-term cryopreservation. Cultured organoids (P3) under ENR medium were subjected to dissociation or were left undissociated, followed by cryopreservation. A: Representative morphology (left panel) and quantification of recovery (right panel) for organoids on day 7 cultured under ENR conditions after thawing organoid cryopreserved for 1 mo in the presence of 10% DMSO as a cryoprotectant. Scale bars: 200 μm. The data are shown as the means ± SDs of triplicate MTT assays (^bP < 0.01, Student’s t-tests). B: Quantification of recovery from cryopreserved organoid was performed using MTT assay. After 1 or 3 mo of cryopreservation with 10% DMSO or RCCFM (commercial freezing media), organoids were cultured for 7 d under ENR conditions. The data are shown as the mean ± SDs of triplicate experiments (^bP < 0.01, two-way analysis of variance with Dunnett’s T3 tests). MTT: Methyl thiazolyl tetrazolium; ENR: Epidermal growth factor/Noggin/R-spondin1.