Generation of Organoids from Mouse Extrahepatic Bile Ducts

Summary

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

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.

Introduction

Cholangiopathies are incurable chronic progressive disorders that affect biliary cells located in intra- and extrahepatic biliary ducts (EHBDs)¹. Some cholangiopathies, like primary sclerosing cholangitis, cholangiocarcinoma, biliary atresia, and choledochal cysts, predominantly affect EHBDs. Development of therapies for cholangiopathies is restricted by the limited availability of preclinical models. In addition, previous studies focused on cholangiopathies grouped together: liver, intra-, and EHBDs. However, intra- and EHBDs have a distinct embryonic origin and, thus, should be considered as distinct molecular pathologies. Intrahepatic bile d....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Michigan.

1. Preparation of Equipment and Materials for Mouse EHBD Isolation

1. Prepare seeding medium and washing buffer (Table of Materials) in 50 mL conical tubes and keep them at 4 °C or on ice until use.
2. Set up a surgical table (Figure 1B). Prepare sterilized surgical instruments (

Discussion

This work describes the generation of an organotypic 3-dimensional model of mouse EHBD cholangiocytes. Important steps in EHBDO culture generation include meticulous EHBD dissection to avoid pancreas cell contamination, maintenance of sterile conditions to prevent bacterial and fungal contamination, and careful manipulation after centrifugation to avoid the loss of cellular material. A close adherence to described temperature conditions is required. There are some limitations to the technique. EHBDs of adult mice are sma.......

Materials

Name	Company	Catalog Number	Comments
L-WRN cell culture medium
Advanced DMEM/F12		Life Technologies	12634-010
Fetal Bovine Serum (FBS)	1%	Life Technologies	10437-028
Penicillin-Streptomycin	100 U/mL	Life Technologies	15140-122
Washing buffer
Phosphate Buffered Saline (PBS)	50 mL	Life Technologies	10010-023
Penicillin-Streptomycin	125 U/mL	Life Technologies	15140-122
Amphotericin B	6.25 µg/mL	Life Technologies	15290-018
Organoid culture medium
L-WRN Conditioned medium	1:1	ATCC	CRL-3276
Advanced DMEM/F12	1:1	Life Technologies	12634-010
Penicillin-Streptomycin	100 U/mL	Life Technologies	15140-122
N-Glutamine	10 µl/mL	Life Technologies	35050-061
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, HEPES	10 mM	Life Technologies	15630-080
B27	10 µl/mL	Gibco	17504-044
N2	10 µl/mL	Gibco	17502-048
Organoid seeding medium
Organoid culture medium
Epidermal growth factor (EGF)	50 ng/mL	Invitrogen	PMG8041
Fibroblast growth factor-10 (FGF10)	100 ng/mL	PeproTech	100-26
Primary antibodies
Anti-Cytokeratin 19 (CK19) antibody, Rabbit	1:250	Abcam	ab53119
Sex-Determining Region Y-Box 9 (SOX9) antibody, Rabbit	1:200	Santa Cruz	sc-20095
Pancreatic Duodenal Homeobox 1 (PDX1) antibody, Rabbit	1:2000	DSRB	F109-D12
E-cadherin antibody, Goat	1:500	Santa Cruz	sc-31020
Acetylated α-tubulin antibody, Mouse	1:500	Sigma-Aldrich	T6793
Secondary antibodies
488 labeled anti-rabbit, Donkey IgG	1:1000	Invitrogen	A-21206
594 labeled anti-goat, Donkey IgG	1:1000	Invitrogen	A-11058
568 labeled anti-mouse, Goat IgG2b	1:500	Invitrogen	A-21144
TopFlash Wnt reporter assay
TopFlash HEK293 cell line		ATCC	CRL-1573
Luciferase Assay Kit		Biotium	30003-2
0.05% Trypsin-EDTA		Life Technologies	25300054
0.4% Trypan Blue Solution		Life Technologies	15250061
Additional materials and reagents
Basement matrix, phenol free Matrigel		CORNING	356237
Dissociation buffer, Accutase		Gibco	A1110501
Cell culture freezing medium, Recovery		Life Technologies	12648010
Cell strainer (70 µm, steriled)		Fisherbrand	22363548
Guanidinium thiocyanate-phenol RNA extraction, TRIzol		Invitrogen	15596026
Specimen processing gel, HistoGel		Thermo Fisher Scientific	HG-4000-012
Universal mycoplasma detection kit		ATCC	30-1012K
1.5 mL microcentrifuge tube		Fisherbrand	05-408-129
24 well plate		USA Scientific	CC7682-7524
50 mL conical centrifuge tube		Fisher scientific	14-432-22
Fluorescence microscope		Nikon	Eclipse E800
Inverted microscope		Biotium	30003-2
Necropsy tray		Fisherbrand	13-814-61