Isolation and Culture of Primary Cholangiocytes from Mice with Polycystic Liver Disease Using a Two-step Digestion Method

Summary

This protocol describes an optimized two-step digestion method for isolating high-purity and high-viability primary cholangiocytes from wild-type mice and mice with polycystic liver disease.

Abstract

In this protocol, we optimized a two-step digestion method to isolate high-purity and high-viability primary cholangiocytes from wild-type mice and mice with polycystic liver disease (PLD). After anesthetizing the mice, the livers were perfused through the inferior vena cava with 50 mL of Solution A, followed by 30 mL of Solution B at 37 °C to enzymatically digest the liver tissue. Mechanical dissociation, shaking, and microdissection were performed to remove adherent parenchymal cells, leaving an intact biliary tree. The biliary tree was then finely minced and digested with shaking for 60 min at 37 °C. The resulting single-cell suspension was collected using a 70 µm cell strainer. Cholangiocytes were purified using immunomagnetic isolation. The cell suspension was incubated with an anti-EpCAM antibody under rotation for 45 min at 4 °C, followed by the addition of Protein G beads and further rotation for another 45 min at 4 °C. After three washes with PBS, the cholangiocytes were collected using a magnetic separator. The purified primary cholangiocytes were resuspended in Cholangiocyte Culture Medium and seeded onto cell culture dishes coated with 1 mg/mL type I rat tail collagen. The purity of the cholangiocytes was confirmed by immunostaining for the cholangiocyte-specific marker cytokeratin-19 (CK19). Although this study focused on isolating primary cholangiocytes from wild-type and PLD mice, we are confident that the protocol can be applied to other disease mouse models as well. This detailed two-step digestion method facilitates in vitro studies of cholangiopathies and the development of targeted therapies.

Introduction

Cholangiocytes, the epithelial cells lining the intrahepatic biliary tree, form a monolayer and constitute approximately 3-5% of the liver's total cell population¹. These cells interconnect within the liver to create a complex three-dimensional ductal network². Under normal conditions, cholangiocytes perform vital functions, including secretion, absorption, injury repair, and serving as an immune barrier, thereby playing a critical role in liver physiology and pathology. However, cholangiocyte dysfunction can lead to various diseases, including PLD, primary sclerosing cholangitis, cholangiocarcinoma, and cholestatic ....

Protocol

All mouse care and experimental protocols were approved by the Ethical Committee of Tianjin Medical University (Doc. No: TMUa-MEC 2022016).

1. Preparation of equipment and solutions

1. Sterilize surgical instruments (scissors, forceps, and scalpel handles) by autoclaving at least 1 day before cell isolation.
2. At least 1 day prior to cell isolation, prepare the Cholangiocyte Culture Medium using the reagents listed in Table 1. Sterilize the Cholangiocyte Culture Medium by filtering it through a 0.22 µm filter.
3. At least 1 day prior to cell isolation, prepare Solution A and Sol....

Results

The workflow diagram for the two-step digestion process used to isolate cholangiocytes is shown in Figure 1. The entire procedure takes ~5 h. First, the liver is perfused with Solution A through the inferior vena cava to remove blood, as indicated by the liver turning pale. The liver is then perfused with Solution B containing collagenase II to initiate tissue digestion. This initial digestion step is time-sensitive, and successful digestion is evidenced by t.......

Discussion

This protocol provides a detailed method for isolating high-purity primary cholangiocytes from mice using a two-step digestion process, enabling the study of the molecular mechanisms underlying cholangiopathies. Several critical steps are essential to ensure the successful isolation of cholangiocytes.

The first critical step is ensuring effective perfusion and digestion using Solution A and Solution B. Successful perfusion with Solution A was confirmed by the liver turning pale, and successful.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm filter	PALL	4612
0.25% Trypsin	Gibco	25200-056
10 mL syringe	KONSMED	10 mL 1.2*30TWLB
20 mL syringe	KONSMED	20 mL 1.2*30TWLB
24 G intravenous catheter	WEGO	24GX19  mm/Y-G
3,3',5-triiodo-L-thyronine	Sigma	T5516	1.7 mg/mL stock
4% paraformaldehyde	Solarbio	P1110
5 mL syringe	KONSMED	5 mL 0.7*30TWLB
6 cm dishes	Thermo Scientific	150462
70 µm cell strainer	Corning	352350
anti-CK19 antibody	DSHB	TROMA-III
anti-EpCAM antibody	DSHB	G8.8
BSA	Solarbio	A8020
CaCl2	Sangon Biotech	A501330	2 M stock
Chemically-defined lipid concentrate (100x)	Gibco	11905-031
Collagenase II	Worthington	LS004176
Collagenase XI	Sigma	C7657	3.2 mg/mL stock
Dexamethasone	Sigma	D1756	10 mg/mL stock
Dissecting Microscope	Leica	EZ4
DMEM/F12 (1:1)	VivaCell biosciences	C3130-0500
DMSO	Sigma	D2650
DNase I	Sigma	D4513	10 U/μL stock
Donkey anti-rat Alexa Fluor 488 secondary antibody	Invitrogen	A21208
EGTA	Solarbio	E8050	50 mM (PH = 8) stock
Epidermal growth factor (1 mg/mL)	Sigma	SRP3196
Ethanolamine	Sigma	E9508
Fetal Bovine Serum	VivaCell biosciences	C04001-500
Forskolin	Sigma	F3917	20 mM stock
Gentamicin/amphotericin solution (500x)	Gibco	R01510
Hemocytometer	QIUJING	XB.K.25.
HEPES	Sigma	H4034
Hyaluronidase	Sigma	H3506	10 mg/mL stock
Insulin-transferrin-selenium (100x)	Gibco	41400045
Isoflurane	RWD	R510-22-10
KCl	Sangon Biotech	A100395
L-Glutamine (200 mM)	Sigma	G7513
Magnetic separator	Promega	Z5342
MEM non-essential amino acids (100x)	Gibco	11140050
MEM vitamin solution (100x)	Gibco	11120052
MgCl2	Sangon Biotech	A100288	1.5 M stock
Na Pyruvate  (100 mM)	Gibco	11360070
Na2HPO4	Sangon Biotech	A600487
NaCl	Sangon Biotech	A610476
NaOH	Sangon Biotech	A620617	1 N stock
PBS	VivaCell biosciences	C3580-0500
PBS	Solarbio	P1010
Penicillin-Streptomycin (100x)	Gibco	15140-122
Peristaltic pump	BaodingRongbai	YZ1515PPS
Protein G beads	Invitrogen	10004D
Rotator	Kylin-Bell	QB-528
RPMI	VivaCell biosciences	C3010-0500
Soybean trypsin inhibitor	Sigma	T6522	10 mg/mL stock
Swaged needle	Jinhuan Medical	HM601
Triton X-100	Solarbio	T8200
Trypan blue	Sigma	T6146	10 mg/mL stock
Type I rat tail collagen	BD	354236