Highly Efficient Methods to Culture Mouse Cholangiocytes and Small Intestine Organoids

• DOI: 10.3389/fmicb.2022.907901
• Publication Date: 2022-05-20T09:19:25Z
• Authors: Wenyi Chen; Qigu Yao; Ruo Wang; Bing Fen; Junyao Chen; Yanping Xu; Jiong Yu; Lanjuan Li; Hongcui Cao
• Keywords: Organoid; Cholangiocyte; Matrigel; Immunostaining

Background Organoids, which enable disease modeling and drug screening closer to an in vivo environment, can be isolated grown from organs such as the brain, small intestine, kidney, lungs, liver. To facilitate establishment of liver intestinal organoids, we developed efficient protocols for cholangiocytes intestine crypts collecting organoid culturing. Methods Cholangiocytes were collected intrahepatic bile ducts, gallbladder, by gravity settling multistep centrifugation methods. The cells embedded with Matrigel grew three-dimensional spheroids a suitable culture medium. stability was assessed subculture, cryopreservation, thawing. RNA DNA extraction well immunostaining procedure, also optimized. Hand-picking procedures performed ensure similar growth characteristics organoids. Results A large number under these protocols. into cyst-like structures after 3–4 days Matrigel. After 1–2 weeks cultivation, organoids (in-orgs) buds formed mature structure. Compared derived cholangiocyte (Cho-orgs) ducts more slowly but had longer term, expressed markers Krt19 Krt7, recapitulated tissue organization. Conclusions Our simplified cell collection procedure avoided possibility exposing tissue-derived stem mechanical damage or chemical injury centrifugation. In addition, our approach allowed different mammalian sources. protocol requires 2–4 establish stable system. Organoids could stably passaged, cryopreserved, recovered guidance. Besides, intestines retained their original characteristics, tissue-specific marker expression, prepares them further experiments preclinical vitro trials mechanism research studies.