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

Abstract

AIMTo investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODSCrypts 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.RESULTSWe 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.CONCLUSIONThe 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.

Interleukin‐13 and its signaling pathway is associated with obesity‐related colorectal tumorigenesis

“…Our experiments using cancer cell lines and colonic organoid culture further showed that treatment with IL‐13 enhanced cell proliferation and induced morphological changes in a dose‐dependent manner. Although the influence of these morphological changes in organoids is not clear at present, a previous report indicated that morphological alterations in organoids represent possible distinct molecular and cellular differences in their tissue of origin . Interleukin‐13 regulates normal physiological processes, including inflammation and tissue reconstruction .…”

“…GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-γ, γ-interferon; KC, keratinocyte chemoattractant; MCP-1, monocyte chemotactic protein-1; TNF-α, tumor necrosis factor-α cell proliferation and induced morphological changes in a dose-dependent manner. Although the influence of these morphological changes in organoids is not clear at present, a previous report indicated that morphological alterations in organoids represent possible distinct molecular and cellular differences in their tissue of origin 36.…”

Adult Canine Intestinal Derived Organoids: A Novel In Vitro System for Translational Research in Comparative Gastroenterology

“…The concentration of these growth factors and the requirement of Wnt3a was optimized in our pilot experiments (see Supplementary materials section). The CMGF+ medium with ROCK and GSK3β inhibitors was used for the first 2 days of ISC culture to enhance ISC survival and prevent apoptosis [68]. The enteroid and colonoid cultures were replenished with CMGF + medium every 2 days.…”

Establishing a cryopreservation protocol for patient‐derived xenografts of prostate cancer

“…The most common method of cryopreservation, including for cultured prostate cancer cells, is slow freezing using the traditional cryoprotectant dimethyl sulfoxide (DMSO). This method may be improved by the addition of the Rho‐associated kinase (ROCK) inhibitor Y‐27632, as adding it to cryopreservation media during or after cryopreservation improves the recovery of embryonic stem cells, breast cancer cells and intestinal organoids, likely by preventing anoikis . Another method of cryopreservation is vitrification, where tissues are briefly immersed in high concentrations of cryoprotectants before being rapidly frozen in liquid nitrogen.…”

“…This method may be improved by the addition of the Rho-associated kinase (ROCK) inhibitor Y-27632, as adding it to cryopreservation media during or after cryopreservation improves the recovery of embryonic stem cells, breast cancer cells and intestinal organoids, likely by preventing anoikis. [10][11][12][13][14][15] Another method of cryopreservation is vitrification, where tissues are briefly immersed in high concentrations of cryoprotectants before being rapidly frozen in liquid nitrogen. Vitrification maintains the viability and proliferative capacity of ovarian and testicular tissue, [16][17][18] so may be another approach for cryopreserving prostate cancer PDXs.…”