title: Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Sung-Hoon
authors.initials: SH
authors.lastname: Han
authors.affiliation: 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.
authors.forename: Sehwan
authors.initials: S
authors.lastname: Shim
authors.affiliation: 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.
authors.forename: Min-Jung
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: 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.
authors.forename: Hye-Yun
authors.initials: HY
authors.lastname: Shin
authors.affiliation: 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.
authors.forename: Won-Suk
authors.initials: WS
authors.lastname: Jang
authors.affiliation: 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.
authors.forename: Sun-Joo
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: 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.
authors.forename: Young-Woo
authors.initials: YW
authors.lastname: Jin
authors.affiliation: 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.
authors.forename: Seung-Sook
authors.initials: SS
authors.lastname: Lee
authors.affiliation: 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.
authors.forename: Seung Bum
authors.initials: SB
authors.lastname: Lee
authors.affiliation: 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.
authors.forename: Sunhoo
authors.initials: S
authors.lastname: Park
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000818:Animals; D001691:Biological Therapy / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000166:cytology*; D007421:Intestine, Small / Q000166:cytology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009940:Organoids / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology*; D013997:Time Factors; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000577:Amides; D004791:Enzyme Inhibitors; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases
keywords: Rho kinase inhibitor; cryopreservation; intestinal organoid; long-term culture; three-dimensional culture