PriCells: Methods of Isolation and Culture of Human Hepatocytes                                           

Safety note. When working with human material, a solution of medical disinfectant, i.e., Virkon solution (2%), should always be available to disinfect spills. All waste media should be disinfected prior to disposal and any tissue remaining after the perfusion must be autoclaved before disposal. Local safety rules for the handling of human material need to be carefully observed.

Human hepatocytes are isolated from liver using a three-stage perfusion modified from the method, which was derived from the method. The major modifications were the substitution of EBSS for HEPES as the perfusion buffer and an additional washing step to remove residual EGTA prior to collagenase perfusion, adopted later by other workers.

1. Sterile techniques should be employed during the isolation of human hepatocytes for culture. All solutions should be either filtered (0.22 μm) or autoclaved and instruments autoclaved when possible. Human liver wedge biopsy (5–10 g) samples should be obtained with only one cut surface where possible. Polyethylene catheters (18 gauge) should be placed into the lumen of the exposed vessels. It is essential to use several catheters (preferably four) and obtain a good perfusion of the tissue sample.

2. The tissue sample should be perfused initially with the chelating solution containing EGTA (reservoir 1) at a flow rate of 8 mL/min/cannula for up to 8 min. This should result in rapid blanching of the liver sample. Areas not perfused will not yield isolated hepatocytes. The perfusate should be allowed to drain to waste.

3. The second step should be to perfuse with EBSS (reservoir2) for a further 10min to remove the chelating agent from the samples; EGTA inhibits the action of collagenase in the subsequent perfusion. The perfusate should be allowed to drain to waste.

4. The third stage should be to perfuse with enzyme solution (reservoir 3). The sample should be perfused with collagenase in the presence of calcium ions and trypsin inhibitor. Commercial collagenase preparations may be contaminated with trypsin, which if active during perfusion is thought to be detrimental to cell- surface receptors. The perfusion of the liver sample with collagenase solution should continue for up to 40 min or until the surface structure of the liver sample obviously changes owing to digestion of the tissue. This solution should be continuously recirculated. The perfusion buffers should be continuously gassed with oxygen, carbon dioxide (95%, 5% v/v), and the pH of each buffer monitored. Adjustments should be made to keep the pH constant as required. Per- fusion of the liver acidifies the buffers which will be observed by a change in the color of the phenol red indicator.

5. Following digestion, the liver sample should be removed carefully into ice-cold dispersal buffer containing DNase I (4 mg/100 mL). DNase helps prevent cell clumping by acting on nucleic acids released by damaged cells during the isolation procedure. Care should be taken not to rupture the capsule of the perfused sample during transfer to the dispersal buffer. Once in the buffer, the capsule surrounding the liver should be ruptured with forceps and the hepatocytes gently combed from the tissue mass. The cell suspension should be filtered through prewetted boulting cloth, and the filtrate gassed with oxygen and carbon dioxide (95%5% v/v). The cell suspension should be centrifuged at 50 g and 4°C for 2 min. Round-bottom tubes (20 mL) rather than conical shaped tubes should be used to minimize cell damage. The supernatant containing cell debris and nonviable cells should be removed by aspiration and the cell pellet resuspended in dispersal buffer containing DNase I by gentle swirling of the tube. The centrifugation procedure should be repeated. The third and final wash of the hepatocyte preparation should be repeated in the absence of DNase I. After centrifugation, resuspend the viable hepatocyte preparation in pregassed WME (30–100 mL) without phenol red and albumin, but containing L-glutamine and maintain at 4°C.

6. Plasma membrane integrity and hepatocyte number should be determined using trypan blue exclusion in the absence of supplemented protein. Ensure that no albumin is present at this stage. An aliquot of trypan blue solution (50 μL) should be mixed with the human hepatocyte suspension (250 μL) and allowed to stand for 2 min at room temperature. The cell suspension should then be analyzed using an improved Neubauer counting chamber. The sample should be mixed and applied to both grids using a Pasteur pipet. With the cover slip in the correct position, the chamber has a depth of 0.1 mm. The cells should be counted in the central grid in the squares surrounded by triple lines. The area of this grid is 1 mm2 and the volume is therefore 0.1 mL. The number of cells in 1 mL is therefore the number of cells counted in the grid × 10⁴. Cells will always be found overlapping the outer lines of the grids and the usual practice is to include the top and left-hand lines in the count and ignore those cells over lapping the bottom and right-hand lines. Any dilution used (e.g., the addition of trypan blue) needs to be taken into account.

Cellular yields should be in the order of 1 × 10⁷ cells/gm tissue. Viable cells are recognized by their ability to exclude trypan blue from the nucleus. Therefore, cell viability is determined by counting the number of stained and unstained cells. All counts should be completed in duplicate. The counting chambers should be viewed using a standard laboratory 16 binocular microscope (×400 magnification). Typically 70–90% of isolated human hepatocytes exclude trypan blue.

Suspensions of isolated human hepatocytes can be used for short-term experiments, i.e., up to 4 h. If longer term experiments are needed, the cells require culturing.

7. If viability has been established, only hepatocyte preparations of greater than 90% should be used for culture. The final cell suspension should be centrifuged and the medium aspirated. The isolated hepatocytes should then be resuspended in the attachment medium. The final cell suspension should be in the region of 5 × 10⁵ cells/mL.

8. For cell culture, all cell manipulations should be performed under sterile conditions in a class II laminar flow cabinet. All liquid handling should be performed under sterile conditions using a Pipettus system or similar equipment.

9. Isolated hepatocytes sediment rapidly on standing, therefore, to achieve a homogeneous cell suspension, the cell mixture should be swirled prior to pipeting. The hepatocyte suspension (2 mL) should be dispersed into the wells of the culture plates; either commercially precoated or “in house" prepared plates should be used. A seeding density of approx 10–15 × 10⁴ cells/cm² can be used as recommended by a number of workers. Depending on the size of culture plate used, the initial hepatocyte suspension can be diluted or concentrated to achieve the seeding density.

10. After plating with hepatocytes, the culture plates should be placed in a CO_² incubator at 37°C to allow hepatocytes to attach to the extracellular matrix. The incubator should contain a humidified atmosphere of 5% carbon dioxide in air. Attachment of human hepatocytes can take up to 16 h.

11. Culture plates containing hepatocytes should be viewed under the inverted microscope. Flattening of the hepatocytes should indicate cell attachment. Once hepatocyte attachment is complete, the media above the attached monolayer should be removed by aspiration using a sterile glass pipet or the Pipettus system. The culture plates should be tilted slightly and care must be taken not to damage the hepatocyte monolayer. The monolayer should then be covered with serum-free incubation media and the plates returned to the CO_² incubator. If the hepatocyte culture is to be kept longer than 24 h, the media should be replaced every 24 h. The morphology of the hepatocyte monolayers and the attachment index should be inspected and determined using an inverted microscope.

12. For xenobiotic metabolism or induction studies using cultured hepatocytes, compounds should be added to the media after cell attachment. Metabolism experiments are usually of up to 24 h duration. However, induction experiments can last significantly longer. On completion of the incubation the media should be removed and rapidly frozen. The monolayer should be washed with phosphate-buffered saline (PBS). The hepatocyte monolayer can then be disrupted by either a detergent solution (Triton X-100, 1% solution in PBS) or mechanical action. The supernatant containing the disrupted cell contents should also be rapidly frozen.

Notes
The preparation and culture of animal hepatocytes, particularly rat, has been extensively evaluated and refined. This evaluation and refinement has led to the development of standard protocols for the isolation and culture of rat hepatocytes. In contrast, the limited availability of human material for the preparation and culture of hepatocytes means that human systems are still under evaluation and, as such, the protocols used for the isolation and culture of human hepatocytes are diverse.

The protocol presented here is just one of many that have been reported for human hepatocytes. Different protocols may give different results as observed with rat hepatocytes, although these differences have not yet been fully evaluated for human hepatocytes. Alternative protocols and approaches are reviewed in the following sections.