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.
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 established1-4. 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.
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 cholangiocytes. For example, biliary atresia initially affects the extrahepatic ducts of the neonate, while Alagille Syndrome affects intrahepatic ducts.
There are multiple descriptions of the isolation of intrahepatic cholangiocytes and bile duct units from mice and rats. Investigators have isolated single cells by duct isolation and subsequent outgrowth, or by liver digestion and antibody pull down of cholangiocytes expressing specific cell surface markers1-4. Functional and polarized bile duct units have been isolated by liver digestion and size filtration7. Bile duct units are capable of responding to secretory stimuli and demonstrate fluid secretion7, while isolated cholangiocytes have been shown to develop ductular structures in vitro1,2. Limitations of these methods include the need for specialized technical expertise and special equipment for liver perfusion with digestive enzymes. In addition, there is a risk of contamination with mesenchymal cells3.
A method to isolate extrahepatic bile duct cholangiocytes, specifically from neonatal extrahepatic bile ducts, has not been previously described. This paper outlines a simplified technique to isolate neonatal as well as adult extrahepatic bile duct cells at high levels of purity. This technique will facilitate the study of differences between intra and extrahepatic cholangiocytes and research into mechanisms of diseases like biliary atresia that involve the extrahepatic ducts.
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
2. Bile Duct Isolation and Culture
3. Isolating Primary Cholangiocytes from Thick Collagen Gels
Note: within 3 weeks, sheets of cholangiocytes (cells with large nuclei extending directly from embedded ducts) will be visible in the thick collagen. If there are large numbers of fibroblasts in the dish, it should be discarded. If a few regions of the dish have fibroblast growth, these can be cut out with a scalpel and removed prior to splitting the cholangiocytes.
4. Passage and Storage of Cells
Note: cells on thin collagen gels can be split as needed.
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 ducts. Cells should be split and re-plated on thin collagen gels before monolayers become overgrown, since holes appear in the sheets of cells if cultures are not split before 3 weeks. For optimal cell growth and to minimize fibroblast contamination, it is important to remove the connective tissue surrounding the delicate extrahepatic ducts carefully and meticulously during the dissection and cleaning steps (2.3, 2.4). If there is growth of fibroblasts, removal of the affected area of thick collagen with a scalpel or gentle suctioning can reduce the transfer of fibroblasts prior to splitting cells. These cells divide rapidly during the first three passages but at higher passage points, the growth rate slows, and the cells become larger and vacuolated. We have successfully frozen samples of cholangiocytes, and re-plated them at later times with excellent viability; thawed cholangiocytes, however, do not replicate as fast as freshly isolated and split cells.
Figure 1. Surgical equipment should be set up in proximity to tissue culture apparatus. (A) Light source is placed behind the dissecting microscope. (B) Surgical equipment includes sharp scissors, hemostat, a curved serrated tweezers, and a curved un-serrated tweezers. (C); Size of a 3 day old mouse used for isolations . (D) Tweezers are holding the common bile duct in the neonatal mouse.
Figure 2. Pure populations of extrahepatic cholangiocytes are isolated with minimal contamination with mesenchymal cells. Cholangiocytes were stained with K19 (green) with DAPI nuclear imaging (blue). Scale bars, 25 µm.
Media | Component | Amount |
Isolation Media | Gentamicin | 0.5 ml |
Penicillin-Streptomycin | 0.5 ml | |
Fungizone | 0.5 ml | |
DMEM/F12 (1:1) | 5 ml | |
Biliary Epithelial Cell (BEC) Media | FBS | 25 ml |
DMEM/F12 (1:1) | 500 ml | |
MEM non-essential amino acids | 5 ml | |
Insulin-transferrin-selenium | 5 ml | |
Na Pyruvate | 5 ml | |
Chemically-defined lipid concentrate | 5 ml | |
Penicillin-Streptomycin | 5 ml | |
Gentamicin | 0.2 ml | |
Ethanolamine | 0.13 ml | |
MEM vitamin solution | 5 ml | |
Soybean trypsin inhibitor* | 5 ml | |
L-glutamine | 5 ml | |
Bovine pituitary extract | 1.1 ml | |
Dexamethasone* | 0.5 ml | |
3,3',5-triiodo-L-thyronine* | 0.5 ml | |
Epidermal growth factor* | 0.5 ml | |
Forskolin* | 5 ml | |
Fungizone | 1 ml | |
Storage Media (for freezing cells) | Biliary Epithelial Cell (BEC) media | 7 ml |
DMSO | 1 ml | |
Sterile FBS | 2 ml | |
*Ingredients need to be prepared to appropriate concentrations before adding into media. Refer to material list for further instruction. | ||
**After ingredients are mixed, sterile filter media and aliquot. Media can be frozen up to 6 months in – 20˚C. |
Table 1. Media components.
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. Disadvantages include that the technique is labor intensive, and meticulous dissection is required to prevent fibroblast contamination; additionally, outgrowth and at least 3 weeks in culture are required to obtain sufficient cells for most experiments. Thus, these cells may reflect changes associated with two dimensional culture. It has not yet been determined whether the cell populations obtained include significant numbers of stem cells or cells from peribiliary glands9,10.
We attempted to use this method to isolate extrahepatic cholangiocytes from rats; however, cells failed to grow in culture. Whether a key growth factor is missing in the culture media or whether other conditions need to be altered is currently under investigation.
Ultimately, this method to isolate extrahepatic cholangiocytes may contribute to understanding differences in intra and extrahepatic forms of biliary fibrosis, as well as differences between neonatal and adult cells.
The authors have nothing to disclose.
The authors are grateful to the Molecular Pathology and Imaging Core of the UPenn NIDDK Center for Molecular Studies in Digestive and Liver Diseases (P30 DK50306) for assistance with imaging. This work was supported by grants from the National Institutes of Health (R01 DK-092111) and from the Fred and Suzanne Biesecker Pediatric Liver Center (to R.G.W.) and by a fellowship from the Childhood Liver Disease Research and Education Network (to S.K.).
Name of Material/Equipment | Company | Catalog Number | Comments/Description |
DMEM/F12 (1:1) | Gibco/ Life technologies | 11320-033 | 500ml, 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 | 5ml, used in BEC media |
Chemically-defined lipid concentrate | Gibco/ Life technologies | 11905-031 | 5ml, used in BEC media |
Penicillin-Streptomycin | Cellgro | 30-002-CI | 5ml, used in BEC media and 500 ul in the isolation |
Gentamicin | Gibco/ Life technologies | 15750-060 | 0.2ml, used in BEC media and 500 ul in the isolation |
Ethanolamine | Sigma Aldrich | E9508-100ml | 0.13ml, used in BEC media |
MEM vitamin solution | Gibco/ Life technologies | 11120-052 | 5ml, used in BEC media |
Soybean trypsin inhibitor | Biowhittaker | 17-605E | 5ml, used in BEC media. Solvent is PBS, mix to 5mg/ml stock concentration |
L-glutamine | Cellgro | 25-005-CL | 5ml, used in BEC media |
Bovine pituitary extract | Gemini | 500-102 | 1.1ml, used in BEC media |
Dexamethasone | Sigma Aldrich | D4902 | 0.5ml, used in BEC media, Stock conc 393ug/ml dilute with ethanol |
3 3',5-triiodo-L-thyronine | Sigma Aldrich | T6397 | 0.5ml, used in BEC media, Stock conc 3.4 mg/ml dilute with ethanol |
Epidermal growth factor | millipore | 01-101 | 0.5ml, used in BEC media, 25ug/ml dilute with DMEM F12+1%BSA |
Forskolin | Sigma Aldrich | F6886 | 5ml, used in BEC media, use at stock concentration of 0.411mg/ml and dilute with DMSO |
Fungizone | Gibco/ Life technologies | 15290-018 | 1ml, used in BEC media and 500 ul 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 10N | Fischer Scientific | ss255-1 | Dilute to 1N, 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 minutes |
trypsin-EDTA (10x) 0.5% | Gibco/ Life technologies | 15400-054 | 3 ml, incubate max 10 minutes |
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 |