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 liver injury^3,4,5.

Among cholangiopathy-related diseases, PLD stands out as a hereditary disorder marked by the formation of numerous fluid-filled cysts originating from cholangiocytes. The progressive enlargement of these cysts significantly diminishes patients' quality of life⁶. Current treatment options for PLD remain insufficient, offering limited efficacy while often being associated with high recurrence rates and complications⁷. This highlights the urgent need to develop safe and effective therapeutic strategies to address the unmet clinical demands in PLD management.

Research into the mechanisms of cholangiopathies, including PLD, has been significantly hindered by the lack of suitable cell lines. To address this limitation, the use of disease mouse models and the isolation of primary cholangiocytes from these models for in vitro experiments have proven invaluable for uncovering the molecular mechanisms underlying cholangiopathies and identifying potential therapeutic strategies.

In our recent study⁸, we successfully established a PLD mouse model using Pkd1 conditional knockout (KO) mice. Liver cysts were observed as early as 1 month after Pkd1 deletion and progressively increased in size over time. While protocols for isolating cholangiocytes from rats and humans are well-documented^9,10, isolating primary cholangiocytes from mice remains particularly challenging due to their small size and the intricate architecture of the portal vein and biliary system. Existing methods face significant limitations, including low cell purity, poor viability, complex procedures, and high costs^11,12.

This manuscript presents a detailed protocol for isolating high-purity primary cholangiocytes from mice using a two-step digestion method. This optimized approach aims to support in vitro studies, facilitating the investigation of molecular mechanisms underlying cholangiopathies and advancing the development of novel therapeutic strategies.

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 Solution B using the reagents listed in Table 2 for mouse liver perfusion. Sterilize Solution A and Solution B by autoclaving or filtering through a 0.22 µm filter and adjust their pH to 7.35 using sterile NaOH under gentle stirring.
   NOTE: Cholangiocyte Culture Medium, Solution A, and Solution B can be stored at 4°C for at least one month.
4. Before starting the experiment, add collagenase II powder to Solution B and mix gently to achieve a final concentration of 0.5 mg/mL. Prewarm both Solution A and Solution B in a 37 °C water bath before use.
5. Immediately before starting the experiment, prepare 6 cm dishes filled with sterile PBS and place them on ice to keep them cold during the procedure.

2. Liver tissue digestion perfusion

1. Anesthetize the mouse using an overdose of isoflurane inhalation. Place the anesthetized mouse in a supine position and secure it with adhesive tape to facilitate the procedure.
2. Open the abdominal cavity of the mouse after sterilizing the abdomen with 75% ethanol and then, tie a slipknot around the inferior vena cava using a swaged needle. Rinse a 24 G intravenous catheter with Solution A using a 20 mL syringe.
3. Insert the intravenous catheter into the inferior vena cava below the slipknot, secure the slipknot, and fix the catheter in place with adhesive tape.
4. Perfuse the liver with 50 mL of Solution A through the inferior vena cava using a 20 mL syringe, and incise the hepatic portal vein to drain the perfusion solution. Maintain the perfusion rate at 10 mL/min.
5. Continue perfusion with 30 mL of Solution B containing collagenase II at a flow rate of 3.5 mL/min using a peristaltic pump.
   NOTE: This step is intended for the digestion of liver tissue.
6. Remove the successfully perfused liver tissue and place it in a 6 cm dish filled with cold sterile PBS.

3. Cholangiocytes isolation

NOTE: As the number of cholangiocytes in normal mice is relatively low, it is recommended to use a group of 2-6 mice for the procedure. However, for mouse disease models with abnormal cholangiocyte proliferation, such as PLD, the cholangiocytes from a single mouse are sufficient. Unless otherwise specified, samples and reagents need always be kept on ice, and all procedures should be performed in a biological safety cabinet.

1. Mechanically dissociate and shake the liver tissue using curved forceps. Subsequently, perform microdissection under a dissecting microscope to remove adherent parenchymal cells, leaving an intact biliary tree. Remove the gallbladder tissue prior to proceeding to the next step.
   Wash the tissue 2x with PBS.
2. Finely mince the biliary tree using a surgical blade, and digest it in 3 mL of Digestion Solution (Table 3) per mouse with shaking (90 rpm) at 37 °C for 60 min. At the 30 min mark, gently pipette the mixture using a pipette tip to mix thoroughly.
   NOTE: The Digestion Solution should be freshly prepared before use.
3. Pass the cell suspension through a 70 µm cell strainer. Use the plunger of a 5 mL or 10 mL syringe to gently press the cell suspension through the strainer. Wash the strainer with PBS.
4. Centrifuge the cell suspension at 500 x g for 5 min at 4 °C. Wash the cells 3x with RPMI medium.
5. Centrifuge again at 500 x g for 5 min at 4 °C.
6. Discard the supernatant and resuspend the cells in 900 µL of RPMI medium containing 40 U/mL DNase I. Add 2 µg of anti-EpCAM antibody and incubate the suspension on a rotator (20 rpm) at 4 °C for 45 min.
7. Centrifuge at 500 x g for 5 min at 4 °C.
8. Discard the supernatant and resuspend the cells in 1 mL of RPMI medium containing 40 U/mL DNase I. Prewash Protein G beads with 1 mL of RPMI medium. Add 20 µL of Protein G beads to the cell suspension and incubate with rotation (20 rpm) at 4 °C for another 45 min.
   NOTE: The anti-EpCAM antibody can also be preincubated with Protein G beads to create a bead-antibody complex prior to introducing the cell suspension.
9. Wash the cells 3x with PBS using a magnetic separator.
10. Perform cell counting and cell viability assessment using Trypan Blue staining (0.04%, 3 min) on a hemocytometer.
    NOTE: There is no need to remove the beads from the cholangiocytes, they will detach naturally once culturing begins and will not interfere with cell culture.

4. Cholangiocytes culture

NOTE: Prepare fresh type I rat tail collagen-coated cell culture dishes prior to cholangiocytes culture. Prechill all reagents on ice.

1. Following the manufacturer's instructions, mix sterile ddH₂O, 10x PBS, 1 N NaOH, and type I rat tail collagen to achieve a final concentration of 1 mg/mL type I rat tail collagen in 1x PBS. Evenly coat the cell culture dishes with the mixture, and place the dishes in an incubator at 37 °C with 5% CO₂ for 30 min to allow the collagen to solidify.
2. Wash the type I rat tail collagen-coated cell culture dishes with prewarmed PBS.
3. Resuspend the cholangiocytes in prewarmed Cholangiocyte Culture Medium. Seed the cell suspension onto 1 mg/mL type I rat tail collagen-coated cell culture dishes and incubate at 37 °C with 5% CO₂. Replace the medium every 2 days.

5. Passaging and cryopreservation of cholangiocytes

1. Wash cholangiocytes twice with PBS, then incubate the cells in 0.25% trypsin at 37 °C for 5-10 min.
2. Add an equal volume of Cholangiocyte Culture Medium to neutralize the trypsin, then centrifuge at 500 x g for 5 min.
3. Prepare type I rat tail collagen-coated cell culture dishes as described in Step 4.1.
4. Follow steps 4.2 and 4.3.
5. If cryopreservation is required, resuspend the cholangiocytes in Storage Medium (9:1 FBS: DMSO) and store in a -80 °C freezer or liquid nitrogen. Thaw the cells as needed for future use.

6. Cholangiocyte validation

NOTE: The purity of cholangiocytes was assessed by staining for cholangiocyte marker CK19.

1. Wash the cholangiocytes 2x with PBS, then fix the cells in 4% paraformaldehyde for 15 min.
2. Wash the cholangiocytes with PBS, then permeabilize the cells with 1% Triton X-100 for 8 min.
3. Wash the cholangiocytes with PBS, then block the cells in 5% BSA for 1 h.
4. Wash the cholangiocytes with PBS, then incubate the cells with the primary antibody against CK19 (dilution 1:12 with 2% BSA) overnight at 4 °C.
5. Wash the cholangiocytes 2x with PBS, then incubate the cells with donkey anti-rat Alexa Fluor 488 secondary antibody (dilution 1:1,000 with PBS) for 1 h at room temperature.
6. Wash the cholangiocytes with PBS, stain with 4',6-diamidino-2-phenylindole for 15 min, and observe under a microscope.

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

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