Primary Human Intrahepatic Biliary Epithelial Cells (Cholangiocytes) Culture Protocol

• What are Primary Human Cholangiocytes?
• Primary Human Cholangiocytes Culture Materials
• Human Cholangiocyte Culture Method

What are Primary Human Cholangiocytes?

Primary human Cholangiocytes, also known as human intrahepatic biliary epithelial cells, are epithelial cells that line the intrahepatic bile ducts. Cholangiocytes are important for ductal bile modification and are targeted in multiple liver diseases, such as primary biliary cirrhosis, cholangiocarcinoma and sclerosing cholangitis.

Our frozen human Cholangiocytes cells are derived from the human liver, with each donor providing documented consent for research use of non-transplantable organs or tissues. Following the primary culture, these cells are cryopreserved. Each lot undergoes testing for specific cell markers and comes with a guarantee of ≥ 70% post-thaw viability. This protocol provides a step-by-step demonstration of how to thaw, plate, and cultivate primary human Cholangiocytes.

Primary Human Cholangiocytes Culture Materials

• Normal Human Intrahepatic Biliary Epithelial Cells (Cholangiocytes)
  Note: Upon receipt, immediately store cryovial(s) in vapor phase liquid nitrogen.
• Collagen Type I, Rat Tail
• Tissue culture treated multiwell plates
• Dulbecco’s PBS, without Calcium & Magnesium (DPBS)
• Trypsin

Preparing Primary Human Cholangiocyte Medium

Formulations for human hepatic stellate cell media are readily available from literature. Below is an example media from one of the publications.¹

Components	Catalog Number	Working Stock	Final Dilution	Final Conc.	Final Volume (mL)
DMEM/F-12 mix	D6421-500ML			1x	439
FBS	ES-009-M			10%	50
Hydrocortisone	H0888-1G	1mg/mL**	500x	2µg/mL	1
Insulin	I9278-5ML	10mg/mL	2,326x	4.3µg/mL	0.215
Cholera Toxin	C8052-0.5MG	0.1mg/mL	10,000x	10ng/mL	0.05
Triiodo-L-thyronine	T6397-100MG	20ug/mL (30uM)***	15,000x	2nM	0.033
Human EGF	E5036-200UG	0.1mg/mL	10,000	10ng/mL	0.05
L-glutamine	TMS-002-C	200mM	100x	2mM, 1x	5
Pen/Strep	P7539	100x	100x	1x	5
				Total volume	500

**Dissolve in 100% EtOH
***To prepare 20 µg/ml stock solution: add 1ml 1N NaOH per mg 3,3', 5-triiodo-L-thyronine; gently swirl to dissolve. To this, add 49 ml sterile medium per ml 1N NaOH added.

Human Cholangiocyte Culture Method

Unless otherwise specified, this protocol was performed in a Class II laminar flow biohood with an aspirator. Incubators are set to 37°C and 5% CO₂ and researchers should wear PPE such as lab coats, gloves, and safety glasses.

Thawing and Plating Human Cholangiocytes

1. Place vial in a 37°C water bath, holding and rotating the vial gently until the contents are completely thawed. Remove the vial from the water bath immediately, wipe dry, rinse the vial with 70% ethanol and transfer to a sterile field. Remove the cap, being careful not to touch the interior threads with fingers.
2. Using a pipette, gently transfer contents of vial to a 15 ml conical tube containing 5 mL of Cholangiocyte medium. Wash vial with 1 mL of the medium and add the wash to the same conical tube.
3. Centrifuge tube at 300xg for 5 minutes. After centrifugation, aspirate medium and re-suspend the contents in medium.
4. For expansion, seed the cells at a density of 5,000 cells/cm² on collagen I coated plates.
5. For best results, do not disturb the culture for at least 12 hours after seeding. Change the medium the next day to remove any residual DMSO or unattached cells.
6. Feed cells fresh Cholangiocyte medium every other day until ready for assay or expansion.

Sub-Culturing Human Cholangiocytes

1. Subculture cells when they have reached 70 - 80% confluency. Cells can be monitored with cell imaging instruments such as the Millicell^® DCI Digital Cell Imager.
2. Warm Cholangiocytes medium in a 37˚C water bath.
   Note: Make sure the 0.25% trypsin solution and the Dulbecco’s Phosphate Buffered Saline without Calcium & Magnesium (DPBS) are at room temperature.
3. Aspirate the medium and rinse cells with DPBS. Add trypsin solution into flask and incubate in a 37˚C incubator for 3-5 minutes or until the cells detach.
4. As soon as the cells detach, wash cells from flask using 2X the volume with Cholangiocyte medium. Transfer to centrifuge tube and centrifuge at 250xg for 5 minutes. After centrifugation aspirate the medium, re-suspend the cells, and count for seeding.
5. Seed the cells at a density of 5,000 cells/cm² in collagen I coated plates.

References

1. Heydtmann M, Lalor PF, Eksteen JA, Hübscher SG, Briskin M, Adams DH. 2005. CXC Chemokine Ligand 16 Promotes Integrin-Mediated Adhesion of Liver-Infiltrating Lymphocytes to Cholangiocytes and Hepatocytes within the Inflamed Human Liver. 174(2):1055-1062. https://doi.org/10.4049/jimmunol.174.2.1055