Published online Feb 14, 2017. doi: 10.3748/wjg.v23.i6.964
Peer-review started: September 6, 2016
First decision: October 20, 2016
Revised: November 3, 2016
Accepted: November 23, 2016
Article in press: November 23, 2016
Published online: February 14, 2017
To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.
Crypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1 (ENR) or ENR/CHIR99021/VPA (ENR-CV). For subculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging.
We established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.
The maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.
Core tip: The phenotypes of intestinal organoids under epidermal growth factor/Noggin/R-spondin1 (ENR) medium were maintained over a long duration, whereas organoid under ENR/CHIR99021/VPA medium exhibited morphological change, reduced numbers of Lgr5+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types upon extended passages. We also demonstrated an efficacious long-term cryopreservation method for intestinal organoids through optimization of the organoid state and timing of treatment with the Rho kinase inhibitor Y-27632. Thus, the suitable long-term culture system and optimal cryopreservation of small intestinal organoid may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids and subsequent clinical applications of these cell sources.