Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 14, 2017; 23(6): 964-975
Published online Feb 14, 2017. doi: 10.3748/wjg.v23.i6.964
Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor
Sung-Hoon Han, Sehwan Shim, Min-Jung Kim, Hye-Yun Shin, Won-Suk Jang, Sun-Joo Lee, Young-Woo Jin, Seung-Sook Lee, Seung Bum Lee, Sunhoo Park
Sung-Hoon Han, Sehwan Shim, Min-Jung Kim, Hye-Yun Shin, Won-Suk Jang, Sun-Joo Lee, Young-Woo Jin, Seung-Sook Lee, Seung Bum Lee, Sunhoo Park, Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Science, Seoul 01812, South Korea
Author contributions: Lee SB and Park S contributed equally to this work; Han SH performed the majority of experiments and analyzed the data; Shim S, Kim MJ, Shin HY and Jang WS participated in care/treatment of animals and performed the molecular biochemical investigations; Lee SJ, Jin YW and Lee SS designed and coordinated the research; Lee SB and Park S analyzed the data and wrote paper; all the authors contributed to this manuscript.
Supported by a grant of the Korea Institute of Radiological and Medical Sciences, funded by Ministry of Science, ICT and Future Planning, South Korea, No. 1711031810/50586-2016 and No. 1711031808/50581-2016.
Institutional review board statement: The study was revised and approved by the Korea Institute of Radiological and Medical Sciences Institutional Review Board.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Korea Institute of Radiological and Medical Sciences in Korea (IACUC protocol number: kirams2016-0043).
Conflict-of-interest statement: The authors declare that they have no potential conflicts of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Sunhoo Park, MD, PhD, Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Science, 75 Nowon-ro, Nowon-gu, Seoul 01812, South Korea. sunhoo@kirams.re.kr
Telephone: +82-2-9701274 Fax: +82-2-9702430
Received: September 5, 2016
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
Abstract

AIM

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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.

Keywords: intestinal organoid, Rho kinase inhibitor, three-dimensional culture, cryopreservation, long-term culture

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.