Isolation of Neonatal Extrahepatic Cholangiocytes

Summary

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

Abstract

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.

Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established^1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.

This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

Introduction

The origin and development of intra and extrahepatic cholangiocytes are markedly different. The liver develops from a diverticulum of the ventral foregut endoderm⁵. The caudal region of the diverticulum forms the extrahepatic biliary tree, while the cranial region generates the intrahepatic biliary tree⁵. Intrahepatic duct cholangiocytes are derived from progenitor cells around the ductal plate in the peri portal regions⁶. These cells have the ability to differentiate into either hepatocytes or cholangiocytes⁶. This has significant clinical implications given that cholangiopathies may specifically target one category of chol....

Protocol

The entire procedure is carried out at room temperature unless otherwise specified. All animal work should be carried out under humane conditions under a protocol approved by the local Institutional Animal Care and Use Committee (IACUC).

1. Preparation of Equipment and Solutions

1. Set up the mouse surgical table in close proximity to the tissue culture hood and incubator (Figure 1A). Note: Required equipment includes a dissecting microscope, a light source, 12.5 cm long straight iris scissors, 6 inch non serrated curved forceps with fine tips, and 4 inch serrated forceps with curved tips (Figure 1B

Results

The use of this protocol results in the isolation of a population of neonatal mouse extrahepatic cholangiocytes with excellent purity, as demonstrated by K19 immunofluorescence staining (Figures 2A and 2B); we achieve similar results isolating cells from adult mice. We have observed that it takes 3 weeks for cells from freshly isolated bile ducts to form monolayers on thick collagen gels. The cholangiocytes grow in a linear fashion throughout the thick collagen, forming sheets of cells from the isolated .......

Discussion

Described here is a technique to isolate pure cholangiocytes from the extrahepatic bile ducts of mice of mice of all ages, including neonates. The technique offers the advantage that extrahepatic cholangiocytes can be studied separately from intrahepatic cholangiocytes, and may facilitate studies to identify key differences between these populations of cells. We recently published a study demonstrating decreased cilia in extrahepatic cholangiocytes isolated by this method and infected with rhesus rotavirus⁸. D.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/F12 (1:1)	Gibco/ Life technologies	11320-033	500 ml, used in BEC media
FBS	Atlanta Biologicals	S11150	25 ml, used in BEC media
MEM with non-essential amino acids	Gibco/ Life technologies	11140-019	5 ml, used in BEC media
Insulin-transferrin-selenium	Gibco/ Life technologies	51300-044	5 ml, used in BEC media
Na Pyruvate	Cellgro	25-000-CL	5 ml, used in BEC media
Chemically-defined lipid concentrate	Gibco/ Life technologies	11905-031	5 ml, used in BEC media
Penicillin-Streptomycin	Cellgro	30-002-CI	5 ml, used in BEC media and 500 μl in the isolation
Gentamicin	Gibco/ Life technologies	15750-060	0.2 ml, used in BEC media and 500 μl in the isolation
Ethanolamine	Sigma Aldrich	E9508-100ml	0.13 ml, used in BEC media
MEM vitamin solution	Gibco/ Life technologies	11120-052	5 ml, used in BEC media
Soybean trypsin inhibitor	Biowhittaker	17-605E	5 ml, used in BEC media. Solvent is PBS, mix to 5 mg/ml stock concentration
L-glutamine	Cellgro	25-005-CL	5 ml, used in BEC media
Bovine pituitary extract	Gemini	500-102	1.1 ml, used in BEC media
Dexamethasone	Sigma Aldrich	D4902	0.5 ml, used in BEC media, Stock conc 393 μg/ml dilute with ethanol
3 3',5-triiodo-L-thyronine	Sigma Aldrich	T6397	0.5 ml, used in BEC media, Stock conc 3.4 mg/ml dilute with ethanol
Epidermal growth factor	Millipore	01-101	0.5 ml, used in BEC media, 25 μg/ml dilute with DMEM F12+1%BSA
Forskolin	Sigma Aldrich	F6886	5 ml, used in BEC media, use at stock concentration of 0.411 mg/ml and dilute with DMSO
Fungizone	Gibco/ Life technologies	15290-018	1 ml, used in BEC media and 500 μl in the isolation
Rat-tail collagen	BD Biosciences	354236	variable depending on concentration of collagen
PBS 10x	USB Corporation	75889	use at 10x, sterlie, used to make collagen, amount used depends on collagen concentration
dH2O	N/A	N/A	sterile, used to make collagen, amount used depends on collagen concentration
NaOH 10 N	Fischer Scientific	ss255-1	Dilute to 1 N, sterile, used to make collagen, amount used depends on collagen concentration
collagenase type XI from Clostridium histolyticum	Sigma Aldrich	C7657	dilute in DMEM and sterile filter before use
trypsin-EDTA (1x) 0.25%	Gibco/ Life technologies	25200-056	3 ml, incubate max 10 min
trypsin-EDTA (10x) 0.5%	Gibco/ Life technologies	15400-054	3 ml, incubate max 10 min
Dissecting microscope	Nikon	SMZ645	Other models acceptable
Light source (fiberoptic illuminator)	Schott-Fostec	Ace EKE LR 92240	Other models acceptable
12.5 cm straight iris scissors	Kent Scientific		Other models acceptable
6" non-serrated curved forceps with fine tips	Electron Microscopy Sciences		Other models acceptable
4" serrated stainless forceps with fine tips	Electron Microscopy Sciences		Other models acceptable