Highly Efficient Methods to Culture Mouse Cholangiocytes and Small Intestine Organoids

Wenyi Chen; Qigu Yao; Ruo Wang; Bing Fen; Junyao Chen; Yanping Xu; Jiong Yu; Lanjuan Li; Hongcui Cao

doi:10.3389/fmicb.2022.907901

Highly Efficient Methods to Culture Mouse Cholangiocytes and Small Intestine Organoids

Front Microbiol. 2022 May 20:13:907901. doi: 10.3389/fmicb.2022.907901. eCollection 2022.

Authors

Wenyi Chen^{1

2}, Qigu Yao^{1

2}, Ruo Wang^{1

2}, Bing Fen^{1

2}, Junyao Chen^{1

2}, Yanping Xu^{1

2}, Jiong Yu^{1

2}, Lanjuan Li^{1

2}, Hongcui Cao^{1

2

3}

Affiliations

¹ State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
² National Clinical Research Center for Infectious Diseases, Hangzhou, China.
³ Zhejiang Provincial Key Laboratory for Diagnosis and Treatment of Aging and Physic-chemical Injury Diseases, Hangzhou, China.

Abstract

Background: Organoids, which enable disease modeling and drug screening closer to an in vivo environment, can be isolated and grown from organs such as the brain, small intestine, kidney, lungs, and liver. To facilitate the establishment of liver and small intestinal organoids, we developed efficient protocols for cholangiocytes and intestine crypts collecting and organoid culturing.

Methods: Cholangiocytes were collected from intrahepatic bile ducts, the gallbladder, and small intestine crypts by gravity settling and multistep centrifugation methods. The cells isolated were embedded with Matrigel and grew in three-dimensional spheroids in a suitable culture medium. The stability of organoid cells was assessed by subculture, cryopreservation, and thawing. RNA and DNA extraction of organoids, as well as immunostaining procedure, were also optimized. Hand-picking procedures were developed and performed to ensure similar growth characteristics of organoids.

Results: A large number of cholangiocytes and small intestine crypts were collected under these protocols. Cholangiocytes developed into cyst-like structures after 3-4 days in Matrigel. After 1-2 weeks of cultivation, small intestinal organoids (in-orgs) developed buds and formed a mature structure. Compared to organoids derived from the gallbladder, cholangiocyte organoids (Cho-orgs) from intrahepatic the bile ducts grew more slowly but had a longer culture term, expressed the cholangiocytes markers Krt19 and Krt7, and recapitulated in vivo tissue organization.

Conclusions: Our protocols simplified the cell collection procedure and avoided the possibility of exposing tissue-derived stem cells to mechanical damage or chemical injury by gravity settling and multistep centrifugation. In addition, our approach allowed similar growth characteristics of organoids from different mammalian tissue sources. The protocol requires 2-4 weeks to establish a stable organoid growth system. Organoids could be stably passaged, cryopreserved, and recovered under protocol guidance. Besides, the organoids of cholangiocytes and small intestines retained their original tissue characteristics, such as tissue-specific marker expression, which prepares them for further experiments such as preclinical in vitro trials and mechanism research studies.

Keywords: 3D culture; cholangiocytes; intestinal crypt; liver; organoids; small intestine.