Isolation of Human Intrahepatic Biliary Epithelial Cells

Materials and Reagents

1.Autoclave sterilised equipment: 250 ml glass beakers, 0.5 mm mesh metal sieves (Sigma), glass (fine bore) Pasteur pipettes.

2. Tissue culture plastic ware:sterile large tissue culture dishes,10 ml and 15 ml conical base centrifuge tubes, 25 ml Universal centrifuge tubes, plastic pipettes, plastic (wide bore) Pasteur pipettes, 10 ml syringes, 0.2 um syringe filters, scalpels.

3. Stock solutions: autoclave sterilised phosphate-buffered saline (PBS – normal 1×and hypertonic 10×strengths prepared from commercially available PBS tablets).

4. Collagenase type 1A (Sigma) dissolved in normal PBS(10 mg/ml stock solution), stored as 5 ml aliquots at –20℃.

5. Normal PBS, 1% w/v bovine serum albumin (PBS, 1% BSA –pH 7.4, filter sterilised).

6. Normal PBS, 0.1% w/v bovine serum albumin (PBS, 0.1%BSA – pH 7.4, filter sterilised).

7. 0.2 M Tris, 0.1% BSA (0.2 M Tris-HCl, 0.1% BSA – pH 8.5,filter sterilised).

8. Percoll density gradient media (Pharmacia Amersham Biotech) prepared as follows: Percoll stock solution; 99 mlsPercoll combined with 11 mls hypertonic (10×) PBS storedat 4℃ in glass bottles; 33% Percoll solution (1.04 mg/ml); 33 mls percoll stock solution combined with 67 mls normal (1×) PBS; 77% Percoll solution (1.09 mg/ml); 77 mls of Percoll stock solution combined with 23 mls normal PBS.

9. Magnetic beads: HEA125-conjugated microbeads (Miltenyi);CD56 Dynabeads prepared by conjugation of Dynal M-450tosylactivated Dynabeads with anti-CD56 antibody (purchased from Dako – see Section C).

10. Magnetic particle concentrators: Magnetic particle concentrator (Dynal, UK); MACS separator and large cell separation columns (Mitenyi).

Procedure

All procedures are undertaken in a sterile laminar flow cabinet unless otherwise stated. All centrifugations are at 800×g for 10 min with 3 min break unless otherwise stated (standard centrifugation).

A.Preparation ofLiver Tissue Extract

1. A total of 30 g of liver tissue(Note: Although we routinely use 30 g approximately of liver tissue, it is possible to obtain adequate cell yield for further studies with as little as 5 g of tissue.) is finely diced (dice no less than 1 mm³) using a pair of scalpels and transferred to a 250 ml sterile glass beaker containing 45 mls of normal (1×) PBS.

2.A 5 ml aliquot of collagenase type 1A is sterilised by passing through a 0.2 um sterile syringe filter and added to the tissue dice. The dice is stirred to give a 2 mg/ml final collagenase concentration and incubated at 37℃ without stirring or shaking for 1–2 h (Note: The duration of enzymatic digestion depends of the degree of fibrosis in the starting tissue.A further incubation of 30–60 min in digestive enzyme is beneficial in releasing more cells depending on the degree of fibrotic tissue remaining after the first incubation. Incubation with collagenase beyond 3 h is damaging to cells and counterproductive in terms of achievable yield.).

3.The beaker is removed from the incubator to a laminar flow cabinet and the digest sieved through a sterile 0.5 mm mesh metal sieve. The liquor containing detached cells is collected into a clean 250 ml beaker. Tissue pieces remaining in the sieve are gently manipulated using the plunger of a 10 ml syringe to encourage further release of cells.

4.Residual undigested dice are transferred to a clean tissue culture dish and diced further with scalpels to encourage release of cells from the cut surfaces.The dice and sieved liquor are recombined and returned to the incubator for a further 30–60 min.

5.The contents of the beaker are strained through a clean sterile 0.5 mm mesh metal sieve and the liquor containing detached cells collected into a 250 ml clean sterile beaker. The undigested tissue pieces are gently manipulated using the plunger of a 10 ml syringe while adding normal PBS to enhance the sieving process and wash further cells through the sieve and into a total liquor volume of 200 mls.

6.A volume of 25 ml aliquots of liquor are decanted into eight universal centrifuge tubes and centrifuged at 800×g for 10 min, break 3 min (standard centrifugation).

7.Supernatants are decanted and the pellets resuspended thoroughly (Note: A large, thick and sometimes viscous pellet is formed at the bottom of the tubes with a cloudy supernatant. Such pellets can be difficult to disaggregate and cells may adhere to the bottom of the tube and require vigorous pipetting for adequate resuspension.) combing two pellets into a single tube (to generate four tubes) and standard centrifugation repeated.

8. Step 7 is repeated until all the pellets (liver extract pellet) are combined in one universal tube in a volume of 24 mls.

B. Semipurification of BEC onPercoll Density GradientMedia

1.3.5 ml of 33% Percoll is transferred into each of eight 10 ml centrifuge tubes.

2. 3.5 ml of 77% Percoll is added by inserting the pipette tip below the 33% Percoll and pipetting the 77% Percoll beneath it very slowly and gently to minimise turbulence and mixing of Percoll densities (Note: Good Percoll gradients can be achieved if 33% Percoll is underlayed by 77% so that the 33% Percoll layer floats on top of the 77% layer; attempts to overlay 77% Percoll on top of 33% generally lead to excessive mixing of the two concentrations.). This procedure results in a Percoll bi-layer with 33% Percoll floating on top of 77% Percoll.

3.Each of the eight Percoll gradients are gently overlayed with 3–4 ml liver extract using a plastic Pasteur pipette (Note: Liver extract should float on top of and not mix with the 33% Percoll layer. The gentler action of a wide bore plastic pipette minimises mixing of liver extract with 33% Percoll.).

4.The tubes are centrifuged at 800×g for 30 min with no break, resulting in several discernible layers beneath the supernatant, in descending order these being: (1) a viscous floating pellicle, partially characterised as containing hepatocytes, stellate cells, undetermined non-viable cells and sub-cellular debris; (2) mononuclear cells including iBEC; (3) a range of mononuclear cells partially characterised as containing endothelium, immune cells and BEC (approximately 10%); (4) 77% Percoll, cell content undetermined (no cells detected); and (5) erythrocye pellet.

5. From each Percoll gradient, the supernatant and layer 1 are gently removed using a plastic pipette and discarded to waste.

6. Layers 2 and 3 (approximately 3–4 ml) are collected from each gradient and transferred to a conical base tube thus yielding eight tubes containing approximately 3–4 ml each (Note: It is important that only layers 2 and 3 are harvested; although collection of some 77% Percoll (layer 4) may be unavoidable this must be minimised. Contribution of excessive volumes of 77% to the cell suspension at this stage will result in cells failing to pellet adequately at the subsequent centrifugation, leading to poor yield of cells.).

7. The volume in each conical tube is adjusted to 10 ml by the addition of normal PBS to dilute the Percoll and the cell suspension is then standard centrifuged.

8. Supernatants are decanted and the pellets resuspended in 0.5 ml normal PBS. The pellets are combined in a single tube and the original tubes washed thoroughly with normal PBS using a fine bore glass Pasteur pipette to detach any residual cells from the tube wall.

9. The volume of cell suspension is adjusted to 10 ml and standard centrifuged yielding a cell pellet containing BECs (pellet).

C. Preparation of CD56-Conjugated Dynabeads

1.At least 2 days in advance of cell isolations 4×10⁸ tosylactivated Dynabeads are washed 3× in normal PBS, harvesting after each wash by placing on a Dynal magnetic particle concentrator for 2 min.

2. After the final wash the beads are combined with 500 ul anti-CD56 antibody and rotated slowly for 10 min at 37℃ before addition of 50 ul of 1% PBS/BSA to give a final 0.1% BSA concentration.

3.  Incubation is continued for a further 24 h at 37℃ with slow magnetic stirring.

4. The labelled beadsare harvested on a Dynal magnetic particleconcentrator for 2 min and washed 4×; washes 1 and 2 in PBS/0.1% BSA, 5 min each at 4℃; wash thrice in Tris/0.1% BSA for 24 h at room temperature; wash four times in 0.1% PBS/BSA for 5 min at 4℃.

5. After the final wash CD56-conjugated beads are stored in PBS/0.1% BSA at 4℃ (Note: CD56-conjugated Dynabeads can be stored in PBS/0.1% BSA for several months at 48C (we do not recommend freezing). After storage for more than 2 weeks we recommend two 5 min washes in PBS/0.1% BSA before final suspension in normal PBS and use in cell purification.).

6. Before use in cell separations an aliquot of the CD56-conjugated Dynabeads (10⁷ beads per cell preparation) is washed in normal PBS and resuspended in 0.5 ml normal PBS.

D. Separation ofMature and ImmatureHuman Intrahepatic BEC Populations

1.Purification of HEA125+ve cells. 40 ul HEA 125-labelled Miltenyi microbeads are added to the BEC pellet obtained from layers 2 and 3 following semi-purification by differential density centrifugation on Percoll (Section B, step 9, above).

2. After thorough but gentle mixing, cells and beads are incubated at 4℃ (or on ice) for 30 min (Note: Incubation with magnetic beads must be performed at 0–4℃ to prevent phagocytic activity, which can lead to the contamination of purified cells by phagocytes that ingest beads at physiological temperatures.).

3. During the incubation, two large cell separation columns are placed in the magnetic field of a MACS separator (Miltenyi) and conditioned by running 2×1 ml of normal PBS (the suspension buffer of the cells) through the column.

4.After incubation with HEA125 microbeads the volume of thecell per bead suspension is adjusted to 15 ml with normal PBS.

5. A volume of 7.5 ml of cell per bead suspension is added to eachof the two columns and cell separation is performed (Miltenyi MACS). HEA125–ve cells elute into collection tubes below the columns while HEA125+ve cells are retained by the magnet.

6. Columns on the magnetic cell separator are washed 3× with normal PBS, then removed and HEA125+ve cell per bead complexes eluted into collection tubes ensuring that all cells are eluted by using the column plunger to propel the cells down the column (Note: Thorough washing of the cell per bead complexes is required for with complete removal of all cells unbound to beads, thus minimising contamination of purified HEA125+ve cells with HEA125–ve cells. Such HEA125–ve cells can become trapped in bead per cell complexes and efficient washing is essential to release them and prevent reduced purity of the HEA125+ve isolate.).

7. The cell per bead complexes are resuspended in 30 ml normal PBS and distributed in 2×15 ml conical base centrifuge tubes and standard centrifuged for a final wash.

8. Purification of CD56+ve cells from the HEA125+ve fraction.Supernatants are decanted and the pellet containing cell per bead complexes resuspended in 0.5 ml normal PBS containing 10⁷ CD56-conjugated Dynabeads and incubated at 4℃ for 30 min with occasional agitation of the tube.

9. The cells and beads are washed in 5 ml of cold normal PBSand placed on a Dynal magnetic particle concentrator for 2 min after which the supernatant is gently decanted and retained. All residual supernatant is removed with a glass Pasteur pipette and retained with the decanted supernatant.

10. Normal PBS is added to the CD56+ve cells per dynabead and washing (Step 9) is repeated to a total of three repeats after which the CD56+ve per HEA125+ve cells (iBEC) are ready for subsequent application.

11. The decanted supernatants are standard centrifuged to yield a HEA125+ve per CD56–ve pellet (mBEC).

12. The cell per bead preparations is suspended in culture medium appropriate for subsequent application.

References

1.Joplin, R., Kachilele, S. (2009). Human Intrahepatic Biliary Epithelial Cell Lineages: Studies In Vitro. In: Dhawan, A., Hughes, R. (eds) Hepatocyte Transplantation. Methods in Molecular Biology, vol 481. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-201-4_16.

2. Fabris, L., Strazzabosco, M., Crosby, H., et al. (1995) Characterisation and isolation of immature atypical ductular cells coexpressing NCAM and Bcl-2 from primary cholangiopathies and ductal plate malformations. Am J Pathol 156, 1599–1612.

Manufacturer’s Link

Sigma-Aldrich: https://www.sigmaaldrich.com

Pharmacia Amersham Biotech: https://www.gehealthcare.com

Miltenyi Biotec: https://www.miltenyibiotec.com › US-en

Dako: https://www.agilent.com › Dako Products

Dynal: https://www.thermofisher.com › … › Brands

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