PriCells: Materials of Isolation and Culture of Human Primary Hepatocytes              

1. Human liver samples can be used for hepatocyte isolation and culture following approval from the local Ethical Review Board. These samples are often obtained as surgical waste following partial hepatactomy. Where fresh tissue cannot be obtained, cryopreserved hepatocytes or hepatic cell lines may be considered.

2. A good supply of cell culture grade water should be used.

3. The following reagents are required for cell culture.  Bovine serumal bumin (BSA) (Fraction V, fatty-acid free), collagen type I (soluble rat tail or calf skin), collagenase (EC 3.4.24.3) from Clostridium histolyticum (specific activity, 0.17–0.44 Wunsch U/mg lyophilisate), deoxyribonucleate 5′ oligonucleotidohydrolase (DNase I; EC 3.2.21.1) from bovine pancreas, Earle’s balanced salt solution (EBSS; 100 mL 10X concentrate, calcium and magnesium free), ethylenediamine-tetraacetic acid (EDTA), ethylene glycol bis-(β-aminoethyl ether) N,N,N′,N′-tetra acetic acid (EGTA), 4-(2-hydroxyethyl) piperazine-2-ethanesulfonic acid (HEPES), L-glutamine (200 mM), insulin (from bovine pancreas), sodium bicarbonate solution (7.5%), trypsin inhibitor (from soyabean, Type II-S), Williams’ Medium E (WME) without phenol red or L-glutamine, trypan blue [4,4′-bis(8-amino-3, 6-disulpho-1-hydroxy-2-naphthalazo)3,3′-dimethylbiphenyl tetrasodium salt] for- mulated as a 0.5% w/v solution in 0.85% saline, penicillin/streptomycin (lyophilized; 10,000 U/10,000 μg/mL). All other chemicals and solvents should be of Analar grade or equivalent.

4. Perfusion buffer: EBSS (magnesium and calcium free); 117 mM NaCl (6.8 g/L), 5.4 mM KCl (0.4 g/L), 0.9 mM NaH_²PO_⁴•H_²O (0.125 g/L), 26.16 mM NaHCO_³ (2.2 g/L), 5.56 mM glucose (1 g/L), and 0.05 mM phenol red (0.02 g/L). Add EBSS (10X concentrate) to cell-culture grade water (870 mL). Sodium bicarbonate (7.5% solution; 30 mL) should be added to give a final concentration of 26 mM and the pH adjusted to 7.4. Commercial EBSS (10 ??concentrate) can be purchased and stored for up to 6 mo. Solutions should be prepared on the day of isolation.

5. Chelating solution: A stock solution of EGTA (25 mM) should be prepared in 0.1 M NaOH and the pH adjusted to 7.4. This stock can be stored at 4°C for up to 3 mo. An aliquot of the stock EGTA (25 mM) solution (10 mL) is then added to 490 mL EBSS solution (pH 7.4) to give a final concentration of 0.5 mM EGTA, prepare on the day of isolation.

6. Enzyme solution: Collagenase (0.24 U/mL) is dissolved in EBSS (pH 7.4) (100 mL). Standardization on a fixed number of units allows for the varying specific activities of different batches of enzyme. Trypsin inhibitor (10 mg) and calcium chloride (2 mM) are also added. Prepare on the day of isolation.

7. Hepatocyte dispersal buffer: HEPES (10 mM) containing NaCl (142 mM) and KCl (7 mM) should be adjusted to pH 7.4. BSA should be added slowly to give a 1% w/v solution. Stir slowly and continuously. Prepare on the day of isolation.

8. Culture medium: L-Glutamine (20 mM) should be added to WME to give a final concentration of 4 mM. It is recommended that the culture medium be purchased from a commercial supplier.

9. Attachment media: WME containing L-glutamine (4 mM), penicillin/streptomycin solution (100 U/100 μg/mL), insulin (0.25 U/mL), and 10% fetal calf serum (FCS). The penicillin/streptomycin, insulin are prepared as ?100 stock solutions in WME without L-glutamine and stored at –20°C in aliquots (1 mL). The stock solutions described above are added to WME in a final volume of 100 mL. A variety of attachment factors have been used to culture human hepatocytes and are discussed.

10. Incubation media is the same as the attachment media excluding FCS.

11. Culture plates can be purchased commercially pre-coated with collagen (0.4–1.4μg type I collagen/cm2). Alternatively, plastic culture plates or flasks can be coated with soluble collagen. A stock solution of collagen is prepared by dissolving either soluble rat tail or calf skin collagen type I in acetic acid (0.1 M). This solution is diluted with sterile water and 100 μg applied to a 35-mm culture well. This gives a coating of 5–10 μg collagen/cm2. Plates are air-dried in sterile conditions, preferably in a laminar flow cabinet. The dried plates are washed well with either culture quality water or media to remove the acetic acid.

12. Perfusion apparatus: The equipment used for the perfusion of liver samples to prepare isolated hepatocytes is shown. All perfusion equipment should either be autoclaved or washed with 70% ethanol and rinsed with sterile distilled water prior to use. The perfusion system consists of three reservoirs each of which contains one of the three solutions used to perfuse the liver biopsy sample. Reservoir 1 contained chelating solution [perfusate (EBSS, pH 7.4) with EGTA], reservoir 2 contained perfusate only, and reservoir 3 contained enzyme solution (collagenase and calcium ions). The reservoirs are linked via a three-way tap system. The flow of perfusate is maintained by a Watson-Marlow 502S peristaltic pump. The temperature of the perfusate is maintained at 37°C by passage through a heat exchanger. The water jacket around the heat exchanger is maintained at 40°C by a recirculating thermoregulator to accommodate a 3°C loss in perfusate temperature between the heat exchanger and the polyethylene catheters used to cannulate the liver sample. A bubble trap is placed between the heat exchanger and the cannulae. Silicon tubing (1.6 mm id and 1.6 mm wd) is used to connect the system.

All perfusion buffers are continually gassed with a mixture of oxygen and carbon dioxide (95%/5% v/v) and the pH of each solution is monitored through- out the perfusion using a pH stick electrode.

Similar types of apparatus allowing constant perfusion can be designed according to space limitations as long as the main components (water-jacketed temperature-controlled reservoirs, constant flow, nonpulsatile pump, and bubble trap) are present.

13. Other equipment requirements include 18-gauge catheters, boulting cloth (64 μm), refrigerated bench top centrifuge, Improved Neubauer counting chamber, a standard laboratory and an ID03 inverted laboratory binocular microscope (×400 and ×320 magnification), a thermostatically controlled CO_² incubator, an autoclave, a class II laminar flow cabinet and a sterile form of dispensing media, i.e., an automatic Pipettus system.