Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

Summary

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

Abstract

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen ¹.

Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77±10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

Introduction

A liver cell suspension can be prepared from the liver by mechanical or enzymatic methods. Mechanical methods used to prepare whole liver cells include forcing the liver through cheesecloth ², shaking a liver piece with glass beads in a Kahn shaker ³, using glass homogenizers with loose pestles ^4,5 etc. Over the years, mechanical methods have fallen out of favor due to the damage to cell membranes and the loss of function of the isolated hepatocytes ^6,7. Consequently, the use of an enzymatic method is currently the main method for isolation of hepatocytes.

Isolation of hepatocytes using an enzymatic method was greatly improved when Berry and Friend ⁸ perfused collagenase and hyaluronidase through the liver via the portal vein in rats. This perfusion process utilized the vasculature to allow the enzymes to come into close contact with the majority of the cells, leading to a 6-fold increase in yield of hepatocytes ⁸. Further, this method yielded cells that retained their structural integrity, with virtually no transformation of endoplasmic reticulum into isolated vesicles and no mitochondrial damage ⁸.

This method was modified by Seglen ¹, who pioneered a two-step perfusion procedure for liver cell isolation. In this procedure, the rat liver is perfused with a Ca²⁺ free buffer followed by perfusion with a collagenase buffer containing Ca^{2+ 1}. The removal of Ca²⁺ in the first step helps to disrupt desmosomes, while the addition of Ca²⁺ in the second step is required for optimum collagenase activity ^1,9.

Given that the published work described above has been performed in rats, this article aims to demonstrate a modified procedure that can be used for isolation of hepatocytes with high viability from human livers. The use of human hepatocytes remains important for translational research and for validating experiments using animal models. The human liver pieces used in this study were acquired with consent for governance through the Human Tissue and Cell Research Foundation, a state-controlled non-profit foundation ¹⁰. After a pathologist removed what was required for diagnosis, liver pieces were collected from the remaining tissue. The tissue sectioned off by the pathologist was morphologically healthy tissue obtained from resection margins after liver resection.

Protocol

1. Preparation of Perfusion and Isolation Solutions

1. Prepare the solutions required for the perfusion of the liver piece and the isolation of hepatocytes according to Table 1. Solutions can be stored at 4 °C until use.
2. Sterile filter all solutions using a 0.22 μm filter.
3. All solutions that come into contact with the liver should be sterile.

2. Preparation of Perfusion Equipment and Solutions

1. The equipment for the perfusion of the liver piece should be set up as shown in Figure 1.
2. The water bath should be set at an appropriate temperature, which is different in each particular experimental set-up, such that the solutions are at the temperature of 37 °C when they reach the liver piece. In this case, the water bath is set at 41 °C to warm the Solutions 1, 2 and 3 and the jacketed glass condenser. Solution 4 should be warmed up to 37 °C in a separate water bath for use to reduce the loss of collagenase activity.
3. Shortly before liver perfusion, turn on the regulator of the gas tank containing 95% O₂/5% CO₂ to gas the oxygenation apparatus (Figure 1E).

3. Perfusion of the Liver

1. A liver piece with as much intact Glisson's capsule as possible and ideally with only 1 cut surface should be obtained from a pathologist for perfusion.
2. Place this liver piece on the Büchner funnel that contains a perforated filter disc (Figure 1B).
3. The perfusion system should be primed with Solution 1.
4. With a low flow rate, curved irrigation cannulae with olive tips should be inserted into the larger blood vessels on the cut surface of the liver piece. As blood flushes out from the liver, the tissue becomes lighter in areas with good perfusion. The number of cannulae used for various sizes of livers is shown in Figure 2A. The gauge size chosen should result in a snug fit that will hold the cannulae in place. The smaller blood vessels should be left open for the perfusion buffer to drain out of the liver piece.
5. Increase the flow rate on the peristaltic pump to between 110-460 ml/min depending on the size of the liver (Figure 2B). This results in an average flow rate of 44±16 ml/min per cannula (Figure 2C). The speed chosen depends on the liver piece and should result in a slight plumping up of the liver piece. In some cases, it may be necessary to clamp shut some of the open vessels with micro vascular clamps to achieve the slight plumping mentioned above. A good perfusion can be observed when the liver piece is a lighter color throughout.
6. Keep the liver piece moist during perfusion by covering it with a piece of gauze soaked in saline.
7. Perfuse with 1 L of Solution 1 to flush out any remaining blood in the liver piece.
8. Change the perfusion fluid to Solution 2 and perfuse for 10 min.
9. Switch the perfusion fluid to Solution 3 and perfuse with 0.5 L.
10. Change the perfusion fluid to Solution 4, which contains 0.1-0.15% of collagenase (Table 2).
11. For this step, perfusion should be carried out in a recirculating manner for 9-12 min or until the liver is sufficiently digested; the liver tissue should appear to break apart slightly under the Glisson's capsule and feel softened when probed with the blunt side of a scalpel.

4. Isolation of Hepatocytes

1. Turn off the peristaltic pump and remove cannulae from the liver piece.
2. Place the liver piece in a crystallizing dish containing 100-200 ml of Solution 5.
3. Remove the Glisson's capsule carefully and gently shake out the cells. If there are regions that are not well perfused, a scalpel can be used to cut through these regions to release cells contained within. Add more Solution 5 as needed during the process.
4. Add more Solution 5 until a final volume of 500 ml is reached.
5. Filter cell suspension twice; first through a 210 μm nylon mesh followed by a 70 μm nylon mesh. Next, pour the cell suspension into 200 ml centrifuge tubes.
6. Centrifuge the cell suspension at 72 g for 5 min at 4 °C. Aspirate supernatant and gently resuspend cell pellet gently in 200 ml of Solution 5.
7. Repeat the washing step number 4.6 three times. On the final centrifuge step, resuspend cells in cold storage solution (see list of materials). Cells should be approximately 2-5 million hepatocytes per milliliter for assessment of yield and viability using a hemocytometer-based trypan blue exclusion assay.
8. To carry out a trypan blue exclusion assay, add 0.1 ml of appropriately diluted cells (≈2-5 million/ml) to a microfuge tube containing 0.5 ml of trypan blue solution (0.4% trypan blue dissolved in phosphate-buffered saline (PBS)) and 0.4 ml of PBS. After mixing the cell suspension thoroughly, load a hemocytometer with the suspension and examine under a microscope at 100X magnification. Under microscopy, the dead cells will be stained blue while the live cells appear unstained. Count the number of live and total cells in each of the 1 mm² grids marked on the hemocytometer. Viability (%), yield of live cells (million hepatocytes per ml cold storage solution (CSS) or million hepatocytes per g liver) can be calculated using the formulae below.

Note: In this case, the trypan blue dilution factor is 10 and the hemocytometer factor is 10,000.

Results

Perfusion Setup

The equipment required for liver perfusion should be set up according to Figure 1.

Viability and Yield of Isolated Human Hepatocytes

The average viability of isolated human hepatocytes was 77±10% and the average yield of hepatocytes was 13±11 million hepatocytes/g liver, with values expressed as means ± standard deviation. The number of hepatocyte isolations carried out to obtain these averages wa...

Discussion

This protocol results in the isolation of human hepatocytes with high viability and purity. In order to achieve these results, it is important to start with a suitable piece of liver. The piece of liver should have intact Glisson's capsule on all surfaces except for 1 cut surface. Another important factor is the particular batch of collagenase used, as different batches can result in marked differences in viabilities of hepatocytes after digestion ¹¹. Therefore, different batches of collagenase should be teste...

Materials

Name	Company	Catalog Number	Comments
Bubble trap	Gaßner Glastechnik
Glass jacketed condenser	Gaßner Glastechnik
41 °C Water bath	Julabo	35723-H24/EG
37 °C Water bath	GFL	1083
Compressed gas cylinder (95 % O2/5 % CO2)	Linde
Gas permeable tubing	Neolab	2-4440
Peristaltic pump	Ismatec	IP65
Scalpel	Feather	320010
Forceps	Omnilab	5171014
Conical flasks 1 L	Schott Duran	2121654
Conical flasks 5 L	Schott Duran	2121673
Beakers	Schott Duran	2110654
200 ml centrifuge tubes	Becton Dickinson	352075
Crystallizing dish	Omnilab	5144063
Curved irrigation cannulae with ball tips	Ernst Kratz GmbH	1464LL/ 1465LL A+B/ 1472LL
Micro vascular clamps	Ernst Kratz GmbH
Büchner funnel	Carl Roth	HT38.1
Nylon mesh 210 μm	Neolab	4-1413
Nylon mesh 70 μm	Neolab	4-1419
0.22 μm sterile filters	Peske	99505
500 ml bottles	Schott Duran	2180144
1 L bottles	Schott Duran	2180154
Hemocytometer	Peske	06-0001
1.5 ml tubes	Eppendorf	0030 120,086
50 ml conical tubes	BD Biosciences	352070
Ice bucket	Neolab	1508454
Sterile Pasteur pipettes	Brand	747715
Motorised pipette filler (Pipette boy acu)	Integra	155017
Refridgerated centrifuge	Eppendorf	5810R
Laminar flow	Kendro	Hera safe-KS9
Aspirator (Low-flow surgical suction pump)	Atmos	C361
Laboratory Gas Burner	Integra	Fire Boy eco
Disposable laboratory coat	Paperlynen GmbH	MD0202414
Surgical mask with visor	Kimberly-Clark	48247
Surgical hood	Barrier	42072
Latex gloves	Semper Care	CE0321
Collagenase (Batch number NB 4G)	Serva	17465
Calcium chloride dihydrate	Merck	2382
EGTA	Sigma	E4378
Sodium chloride	Roth	9265.2
Hepes	Roth	9105.3
Potassium chloride	Serva	26868
Albumin	Biomol	01400-2
Glucose	Serva	22700
Sodium hydrogen carbonate	Serva	30180
0.4% Trypan blue solution	Lonza	17-942E
Cold storage solution	Hepacult GmbH