Generation of organoids from mouse extrahepatic bile ducts

Junya Shiota, Nureen H.Mohamad Zaki, Juanita L. Merchant, Linda C. Samuelson, Nataliya Razumilava

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

Original languageEnglish (US)
Article numbere59544
JournalJournal of Visualized Experiments
Volume2019
Issue number146
DOIs
StatePublished - Apr 2019
Externally publishedYes

Keywords

  • 3-dimensional culture system
  • Biliary progenitor cells
  • Cholangiocytes
  • Cholangiopathies
  • Developmental biology
  • Drug screening
  • Issue 146
  • Organoids

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Fingerprint

Dive into the research topics of 'Generation of organoids from mouse extrahepatic bile ducts'. Together they form a unique fingerprint.

Cite this