Isolation and Culturing Primary Cholangiocytes from Mouse Liver

Materials and Reagents

1. 100 µm nylon mesh (Greiner Bio-One, catalog number: 542000)

2. 50 ml syringe (BD, Catalog number: 309653)

3. 50 ml tubes (Greiner Bio-One, catalog number: 227270)

4. 15 ml tubes (Greiner Bio-One, catalog number: 188261)

5. 5 ml pipette (Greiner Bio-One, catalog number: 606180)

6. 48-well plates (Fisher Scientific, catalog number: 08-772-1C)

7. T-25 and T-75 flasks (Greiner Bio-One, catalog numbers: 1186Q51 and 658175)

8. Pasteur pipet (Fisher, catalog number: 1367820D)

9. Dexamethasone (400 μg/ml) (Sigma, catalog number: D4902)

10. 3,3’,5-Triiodo-L-thyronine (3.4 mg/ml) (Sigma, catalog number: T6397)

11. Forskolin (0.411 mg/ml) (Sigma, catalog number: F3917)

12. mEGF (10μg/ml) (Peprotech, catalog number: 315-09)

13. ITS (100×) (Corning Cellgro ITS, catalog number: MT25800CR)

14. L-glutamine (100×) (Gibco Life Technologies, catalog number: 102374)

15. Pen/Strep (100×) (Thermo Fisher Scientific, catalog number: 15070063)

16. MEM-NEAA (100×) (Gibco, catalog number: 11140050)

17. EpCam G8.8 Antibody (University of Iowa Hybridoma bank)

18. Percoll (GE Healthcare, catalog number: 17089101)

19. PBS (Fisher, catalog number: 14190250)

20. Hyaluronidase (Sigma-Aldrich, catalog number: H3506)

21. Collagenase D (Sigma-Aldrich, catalog number: 11088882001)

22. EpCam (murine) (CD326, G8.8; DSHB, https://dshb.biology.uiowa.edu/G8-8)

23. Sheep Anti-Rat IgG Dynabeads (Thermo Fisher Scientific, catalog number: 11035)

24. DMEM w/ F12 (Gibco, catalog number: 11320033)

25. MEM-NEAA (Gibco, catalog number: 11140050)

26. Triiodothyronine (Sigma-Aldrich, catalog number: T6397)

27. Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23227)

28. Rat anti-mouse CK19 IgG, Troma III (CK19) (DSHB, https://dshb.biology.uiowa.edu/TROMA-III)

29. Goat anti Rat antibody (Invitrogen, Fisher Scientific, catalog number: PI31470)

30. Donkey anti-Mouse IgG Secondary Antibody IRDye 800CW LI-COR (Biosciences, catalog number: 926-32212)

31. DNase I (Roche, catalog number: 4716728001)

32. Release Buffer (Thermo Fisher Scientific)

33. EasySep^TM Release Mouse PE Positive Selection Kit (Stem Cell Technology, catalog number: 17656)

34. PE anti-mouse CD326 (Ep-CAM), Antibody (Biolegend, catalog number: 118206)

35. DMEM w/ F1 (Gibco, catalog number: 11320033)

36. FBS (Thermo Fisher Scientific, catalog number: A4766801)

37. Chemically defined Lipid Concentrate (100×) (Gibco, catalog number: 11905031)

38. Gentamicin (50 mg/ml) (Gibco, catalog number: 15750-060)

39. MEM Vitamin Solution (100×) (Gibco, catalog number: 11120052)

40. Glacial acetic acid (Fisher Scientific, catalog number: A38S-500)

41. Pierce BCA Protein Assay Kit (Thermo Scientific, catalog number: PI23235)

42. β-actin (Sigma-Aldrich, catalog number: A5441)

43. Pierce Fast Western Blot Kit, ECL Substrate (Thermo Scientific, catalog number: 35055)

44. Donkey anti-mouse IgG IRDye 800CW (LI-COR Biosciences, catalog number: 926-32212)

45. Horseradish peroxidase (Fisher Scientific, catalog number: PI31470)

46. Odyssey system (LI-COR) (https://www.licor.com/bio/odyssey-clx/)

47. DPBS (Fisher, catalog number: 14190 250)

48. Rat Tail Collagen Coating Solution (20 ml) (Cell Applications, catalog number: 122-20)

1. Perfusing media (see Recipes)

2. Digestion media (see Recipes)

3. Isolation buffer (see Recipes)

4. Dexamethasone (400 μg/ml) (see Recipes)

5. 3,3’,5-Triiodo-L-thyronine (3.4 mg/ml) (see Recipes)

6. Forskolin (0.411 mg/ml) (see Recipes)

7. mEGF (10 μg/ml) (see Recipes)

8. Collagen coating (see Recipes)

Equipment

1. Hemocytometer (Sigma, catalog number: Z359629-1EA)

2. Centrifuge (Eppendorf, catalog number: EP-5810S)

3. Rotator (Cambridge Scientific, catalog number: 17847)

4. Forceps and scalpel (Fine Science Tools, catalog number: 11075-00; VWR, catalog number: 4-422)

5. Shaker (Eppendorf, catalog number: M1352-0000)

6. BD Accuri C6 flow cytometer (BD Biosciences)

7. BD LSRFortessa Cell Analyzer (BD Biosciences, https://www.bdbiosciences.com/en-in/products/instruments/flow-cytometers/research-cell-analyzers/bd-lsrfortessa)

8. Bio-Rad ChemiDoc^TM MP Imaging System

Software

1. FlowJo software (version 10.7.1; Tree Star, Inc., Ashland, OR)

Procedure

1. Harvest and tissue dissociation

   1. Anesthetize mice using isoflurane.

   2. Open the abdominal cavity. Using 50 ml syringe, perfuse the liver with 30 ml of perfusing media through the inferior vena cava with a flow rate of 5 ml/min, and cut the portal vein to drain the perfusion media.

   3. Remove the liver and weigh it to correlate cell number with liver weight.

   4. Scrap the perfused liver with a scalpel; this will help to separate the bile ducts from the rest of the liver parenchyma. Mince the bile duct and left-over liver tissue mass into very small pieces using forceps and a scalpel.

   5. Place the minced pieces in 50 ml tubes containing 10 ml of digestion media.

   6. Place the 50 ml tubes in the shaker and shake at 280 rpm for 30 min at 37°C.

   7. After 30 min of constant shaking, remove the tubes from the shaker and add 5 ml (33%) of heat-inactivated FBS. The addition of FBS deactivates enzymatic activity.

   8. Pass the cell suspension through a 100 µm nylon mesh cell strainer into a 50 ml tube.

   9. Use a 3 ml syringe plunger to scrape and press tissue pieces through the strainer. Wash the strainer with 35 ml of isolation buffer and make up the volume to 50 ml.

   10. Centrifuge at 300 × g for 7 min at 4°C.

   11. Wash the cells two times with isolation buffer.




2. Cell isolation by gradient centrifugation

   1. Make percoll gradient solutions as shown in Table 1.

      
      

      Table 1. Concentration of percoll gradient solutions used for cell isolation

      Density	Vol. of percoll	PBS	Final vol.
      26.25%	6.3 ml	17.7 ml	24 ml
      41.5%	6.65 ml	9.35 ml	16 ml
      73.3%	8.8 ml	3.2 ml	12 ml

      
      

   2. The calculations shown in Table 1 are sufficient for four livers.

   3. Place a Pasteur pipet in the 15 ml tube.

   4. Resuspend the cells in 6 ml of 26.25% density gradient percoll and add into 15 ml tube with Pasteur pipet.

   5. Using a 5 ml pipette, add 4 ml of 41.5% density percoll through the Pasteur pipet and let it flow under the 26.25% gradient layer in the tube. Add 3 ml of 73.3% percoll solution through the Pasteur pipet in the tube. The 73.3% density solution will layer under the 41.5% density solution, as shown in Figure 2.

      
      

      
      Figure 2. Overlaying of percoll gradient solution

   
   

   6. Centrifuge at 796 × g for 30 min at 4°C without breaks.

   7. Remove the 15 ml tubes from the centrifuge.

   8. Aspirate the upper layer of hepatocytes and collect to 3 ml of interphase containing cholangiocytes; place it in the 50 ml tube.

   9. Wash the cells using 50 ml of isolation buffer by centrifuging at 300 × g for 7 min at 4°C. Repeat another time; this will help remove the percoll.

   10. Remove the supernatant and resuspend the cell pellet in 1 ml of isolation buffer.

   11. Assess the fraction of Epcam positive cells using the Epcam PeCy7 labeled flow antibody on a BD Accuri C6 flow cytometer and BD LSRFortessa Cell Analyzer (Figure 3).

   12. Count the cells using a hemocytometer.

       
       

       
       Figure 3. Fraction of Epcam+ cholangiocytes isolated with the gradient centrifugation method




3. Purification/enrichment of cholangiocytes using the Dynabead cell isolation kit

   1. Wash the Dynabeads before proceeding to cell isolation.

   2. Add desired volume (based on cell number) of beads to a tube. Wash the Dynabeads using 1 ml of isolation buffer.

   3. Place the tube in a magnet for 2 min and discard the supernatant.

   4. Remove the 15 ml tube from the magnet and resuspend the beads in the 1 ml of isolation buffer.

   5. Use approximately 10 μg of EpCAM G8.8 primary antibody (rat IgG) per 10⁷ cells, mix well, and incubate at 4°C on a rotator for 20 min.

   6. Wash the cells by adding 2 ml of isolation buffer and centrifuge at 300 × g for 7 min. Discard the supernatant.

   7. Resuspend the cells in isolation buffer. Add 20 μl of anti-rat IgG Dynabeads to 15 ml tube with cells. Incubate at 4°C on a rotator for 20 min.

   8. After incubating with Dynabeads, add 1 ml of isolation buffer, pipet up and down 2-3 times, and place the tube in a magnet for 4 min.

   9. For positive isolation, while the tube is still in the magnet, carefully remove and discard the supernatant containing unbound cells. Repeat Step C8 one more time to remove unbound cells; this will increase the purity of isolated cells.

   10. Resuspend the cell pellet in isolation buffer and wash the cells by centrifuging at 300 × g for 7 min at 4°C. Remove the supernatant and proceed.




4. Releasing bead-bound cells

   1. Resuspend the bead-bound cells in 1 ml of release buffer.

   2. Incubate for 15 min with rolling and tilting at room temperature, and then mix by pipetting up and down 10 times, preventing the formation of air bubbles. This will help in efficiently separating the cells from beads.

   3. Place the tube in a magnet for 4 min.

   4. Transfer the supernatant containing the bead-free cholangiocytes into a new 15 ml tube.

   5. Add Isolation buffer to 15 ml and centrifuge at 300 × g for 7 min. Discard the supernatant and resuspend the cell pellet in 1ml of cholangiocyte culture media. Count the cells using a hemocytometer.

   6. Keep the cells from 2°C to 8°C until further use in downstream applications.

   7. Cells isolated from neonatal cholangiocytes are expanded as described below.

      Note: In addition to the above-discussed method. Epcam positive cells can be purified using the PE anti-mouse CD326 (Ep-CAM) Antibody and EasySep^TM Release Mouse PE Positive Selection Kit. This method can be used after counting cells with a hemocytometer in Procedure B.




5. Expansion of cholangiocytes

   Media: To make a 500 ml stock, add the following reagents in the appropriate concentrations (Table 2).

   
   

   Table 2. List of reagents used to prepare 500 ml of stock media

   	Reagents	Volume
   1	DMEM w/ F12	450 ml
   2	FBS	50 ml
   3	L-glutamine (100×)	5 ml
   4	Pen/Strep (100×)	5 ml
   5	MEM-NEAA (100×)	5 ml
   6	Chemically defined        Lipid Concentrate (100×)	5 ml
   7	Gentamicin (50 mg/ml)	1 ml
   	MEM Vitamin Solution (100×)	5 ml

   
   

   Note: To make complete media, add the following supplements in Table 3 on the day staring cell culture.

   
   

   Table 3. List of reagents added to stock to make complete media for the expansion of cholangiocytes

   	Reagents	Volume
   1	DMEM w/ F12 with supplement (Table 2)	50 ml
   2	ITS (100×)	500 µl
   3	Dexamethasone (400 μg/ml)	50 µl
   4	Triiodothyronine (3.4 mg/ml)	50 µl
   5	mEGF (10 μg/ml)	125 µl
   6	Forskolin (0.411 mg/ml)	50 µl

   Note: The complete media containing ITS, dexamethasone, triiodothyronine, mEGF, and Forskolin is usable for 1 week. Gentamicin (100 µg/ml) is used for cholangiocyte culture.

   
   

6. Culturing/Passaging

   Note: For initial cultures, start with 48-well plates and move to 12-well plate once cells are confluent; then, transfer the cells to T-25 or T-75 flasks depending on requirements. Cell growth is very slow for the first few passages.

   1. T-25 flask

      To coat 1 T-25 flask, add 31 µl of collagen (4 mg/ml) + 3 ml of 0.1% acetic acid

   2. T-75 flask

      To coat 1 T-75, add 94 µl of collagen (4 mg/ml) + 5 ml of 0.1% acetic acid

   3. Incubate for 2 h at room temperature or overnight at 4°C.

   4. Remove coating solution by washing with 5 ml of sterile PBS (when using T-25 flask).

   5. Gently rock the flask with PBS; repeat washing three to four times to remove acetic acid.

   6. If passaging, dissociate the adhered cells using TrypLE. Prewarm TrypLE to 37°C.

   7. Wash cells with 5 ml of DPBS without Ca⁺⁺ and Mg⁺⁺. Aspirate and discard.

   8. Add an appropriate volume (2 ml in T-25, 2-5 ml in T-75 flask) of TrypLE to flask.

   9. Incubate at 37°C for 2 to 4 min until cells detach. Gently tap the flask to dislodge cells if necessary.

   10. Wash cells with 13 ml DPBS without Ca⁺⁺ and Mg⁺⁺. Transfer the cell suspension to a 15 ml conical tube.

   11. Centrifuge at 300 × g for 7 min 4°C. Discard the supernatant and resuspend the cell pellet in pre-warmed complete medium. Count the cells and divide them accordingly. After a few passages, cells grow fast; therefore, split the cells based on requirements.

   12. Check the purity of cultured cells using Epcam PeCy7 labeled flow antibody on the BD Accuri C6 flow cytometer (Figure 4).

       
       

       
       Figure 4. Fraction of Epcam+ cholangiocytes from passage 6

   
   

   13. Immunoblotting for CK19 and β-actin was performed on purified and in vitro expanded cholangiocytes. Protein was quantified using the Pierce^TM BCA Protein Assay Kit, and 35 μg of protein per lane was used.

   14. For detection of β-actin and CK19, membranes were incubated at 4°C overnight with primary antibody.

   15. On the next day, blots were washed and incubated with horseradish peroxidase secondary antibody. CK19 was detected using a Bio-Rad ChemiDoc^TM MP Imaging System using the ECL Substrate.

   16. β-actin, used as internal control, was detected with donkey anti-mouse IgG secondary antibody. Immunoblots were visualized using the Odyssey system (LI-COR) (Figure 5).

       
       

       
       Figure 5. CK19 expression in cholangiocytes from passage 6




7. Summary

   Cholestasis is defined as an impairment in bile formation or flow, while the etiology of cholestasis differs, ranging from genetic mutation to autoimmunity. Bile is the digestive fluid produced in the liver by hepatocytes, and disorders in hepatocytes, biliary ducts/biliary cells, and pancreas can cause cholestasis causing retention of bile salts in the body. Bile acids precipitated during cholestasis mediate biliary injury and further cause obliteration of cholangiocytes, a cellular lining for bile ducts known as the biliary tree. Cholangiocyte damage causes obstruction of bile transport and bile modification. We identified a homozygous, nonsense pathogenic variant in ABCC12 encoding an adenosine 5′-triphosphate (ATP)-binding cassette (ABC)-transporter, multidrug resistance-associated protein 9 (MRP9) in a patient. These patients elicit bile duct paucity while showing lesser GGT levels. Expression of MRP9 transporters was restricted to cholangiocytes in the liver. So far, the role of this transporter has not been discussed in the liver. Therefore, we aimed to explore the role of ABCC12 and its biological function using primary cholangiocytes isolated from neonates and adult mice. Biliary epithelial cells were isolated from 2-day-old pups and from adult mice. In adult mice, the liver was perfused with DMEM+F12 media containing collagenase D. Liver tissue was diced into small pieces and further digested with Collagenase D, Hyaluronidase, and DNase I. Then, 5 ml of heat-inactivated FBS was added to digested tissue to inactivate enzymes. The digest was further washed with DMEM+F12 containing 10% FBS. Three different concentrations/densities of percoll were used to isolate cholangiocytes. Cells resuspended in percoll were centrifugated at 2,000 rpm for 30 min without brakes. The interface layer was collected, washed with DMEM+F12, and incubated with the EpCAM Ab to purify cholangiocytes from the rest of the liver cells. Cells were washed and incubated with Sheep Anti-Rat IgG Dynabeads, and the bead-bound Epcam positive cells were separated from other cells using magnetic columns. Epcam positive cells were used for different experiments or expanded in vitro to identify the role of ABCC12 in cholangiocytes. This protocol was used in our recent study published in Gastroenterology (Pham et al., 2021).

Recipes

1. Perfusing media

   50 ml of DMEM F12 + 500 µl (1 mg/ml) of Collagenase D + 1% FBS

2. Digestion media

   30 ml DMEM + 15 mg Collagenase D + 15 mg Hyaluronidase + 200 μl of 200 mM CaCl₂ + 200 µl of 200 mM MgSO₄, DNases I (10 U/ml).

3. Isolation Buffer

   500 ml of Ca²⁺ and Mg²⁺ free PBS supplemented with 0.1% BSA and 2 ml of 0.5 M; final concentration is 2 mM EDTA and pH 7.4.

4. Dexamethasone (400 μg/ml)

   1. Weigh 4 mg of dexamethasone.

   2. Use 2-4 ml of ethanol to dissolve.

   3. Add sterile media to 10 ml.

5. 3,3’,5-Triiodo-L-thyronine (3.4 mg/ml)

   1. Dissolve 100 mg of Triiodo-L-thyronine powder in 2 ml of 1 N NaOH.

   2. Place in 50 ml tube.

   3. Add 27.4 ml of sterile media for a total of 29.4 ml.

6. Forskolin (0.411 mg/ml)

   Dissolve 10 mg of Forskolin in 5% DMSO/H₂O (1.2 ml DMSO + 23.1 ml sterile H₂O).

7. mEGF (10 μg/ml)

   Dissolve 100 µg in 10 ml 1× PBS/1% BSA.

8. Collagen coating 4 mg/ml

   Make a 0.1% (~0.02 M) acetic acid solution: dilute glacial acetic acid (17.5 M) 1:1,000 in dH₂O. Calculate amount of collagen to coat dishes at 5 µg/cm². To coat one T-25 flask, add 31 µl of collagen (4 mg/ml) to 3 ml of 0.1% acetic acid.

Acknowledgments

The authors would like to acknowledge support for this work from the Center for Autoimmune Liver Disease at Cincinnati Children’s Hospital Medical Center and R01 DK095001 (A.M.).

Competing interests

All authors declare no conflict of interest.

Ethics

The Laboratory animal care and use committee of the Cincinnati Children’s Research Foundation (Cincinnati, OH) approved the work described here.

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