Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Podsumowanie

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Streszczenie

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine¹. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.

Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.

Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

Wprowadzenie

Human liver tissue is highly complex and consists of two different cell entities, parenchymal cells and non-parenchymal cells (NPC). Parenchymal liver cells include hepatocytes and cholangiocytes. Hepatocytes represent 60 to 70% of total liver cells and account for most of the metabolic liver functions, e.g., bile acid and complement factor synthesis, biotransformation and energy metabolism^2,3.

The smaller NPC fraction constitutes 30-40% of total liver cells. NPC include different cell populations, namely Kupffer cells (KC), liver endothelial cells (LEC) and the hepatic stellate cells (HSC). This heterogenic cell fraction plays a central role in physiological processes of the liver. Additionally, NPC participate in mediating acute liver damage, e.g., drug-induced liver injury (DILI) as well as in chronic liver injuries, such as cirrhosis⁴.

In recent years, human liver cells have become more and more essential in research and development of drug testing, drug development and identification of new biochemical pathways in liver diseases. For in vitro testing PHH monocultures are still considered as the "gold standard"⁵. The main limitation of current homotypic liver models is dedifferentiation and loss of function of the hepatocytes within a few days⁴. The establishment of 3-dimensional (3D) culture techniques has shown that these limitations can be compensated^4,6. However, even modern 3D culture techniques are not able to display all hepatotoxic modes of actions⁷. Missing NPC populations in the existing in vitro models are discussed as a possible reason for this discrepancy to the in vivo situation. It has been shown that the cell-cell communication between the different liver cell populations plays a central role in physiological homeostasis but also in pathophysiologic processes⁸. Therefore the scientific attention focuses more and more on NPC and their cell-cell interactions. Their purposeful use in co-culture and tissue engineered systems could be a solution for the high demand of in vitro liver models^8,9 which are as close to the in vivo situation as possible.

Currently the main challenge is the development of a standardized human liver co-culture model, which contains clearly defined portions of PHH and NPC. In consequence, isolation techniques for the very heterogenic liver cells are needed and those have to be optimized to gain pure cell populations. While standardized protocols for PHH isolation exist¹⁰, the standardized isolation of human NPC is still under development. Most published NPC isolation protocols are based on experiments with non-human cells^11,12. Only a few publications describe the isolation process of human NPC and most cover only methods for the isolation of a single cell type^11-16. The most important cell characteristics that have been harnessed for cell separation are size, density, attachment behavior, and the expression of surface proteins. On the basis of these characteristics we developed a simplified protocol to isolate PHH, KC, LEC and HSC, which was published previously in Experimental Biology and Medicine¹. Because of the broad interest in this technique, the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow reproducing the technique more easily. The protocol also includes quality control methods for evaluation of yield and viability as well as for identification and purity evaluation using specific immunostainings.

Protokół

Note: All cells were isolated from resected non-tumorous human liver tissue, which remained after partial liver resection with primary or secondary liver tumors. Informed consent of the patients was obtained according to the ethical guidelines of the Charité - Universitätsmedizin Berlin.

1. Preparation of Materials and Solutions

1. Sterilize all instruments and the materials in advance to avoid bacterial contamination during the isolation process.
2. Prepare the solutions required for the perfusion of the liver tissue sample, the isolation process of hepatocytes and non-parenchymal liver cells and the cultivation of primary human liver cells according to the Tables 1 and 2, with exception of Digestion-Solution which is prepared freshly prior to use. All solutions can be stored at 4 °C and it is recommended to use them within 4 weeks after preparation.
3. Sterilize all solutions using a 0.22 µm bottle top filter.

2. Preparation of Perfusion Equipment

1. Set up the equipment for the perfusion and digestion of the liver tissue sample as shown in Figure 1A.
2. Adjust the water bath temperature to 39 °C to ensure optimal collagenase P activity during the perfusion and digestion.

3. Perfusion and Digestion of the Liver Tissue Sample (1.5 hr)

1. Select a tissue sample with an intact Glisson's capsule from the resected liver tissue. When cutting the tissue sample, try to obtain a small cutting surface with good visible vessels. Avoid warm ischemia times by transporting and handling the liver tissue sample on ice until perfusion.
2. Take the tissue weight under sterile conditions and place the liver tissue sample in a Petri dish in the laminar air flow. Clean the surface of the tissue sample with a sterile compress from remaining blood and flush the cannula set using 1x Perfusion-Solution I to ensure that all cannula were permeable.
3. Use tissue glue to fix the olives of the cannulas in some larger blood vessels. Depending on the size of the liver tissue sample and the number of the vessels on the surface, use a cannula set with 3 to 8 cannulas. Test the perfusion and check for leakages. Close all blood vessels, which leak clear 1x Perfusion-Solution I, with tissue glue.
4. Place the cannulated liver tissue sample into the Büchner funnel on its perforated filter disc (Figure 1A).
5. Set the flow rate of the peristaltic pump between 7.5 ml/min and 14.6 ml/min depending on the number of cannulas used and on the resistance of the liver tissue. Adjust the flow rate each time to ensure that there is a current but slow perfusion. Perfuse the tissue until the whole blood is flushed out but at least 20 min. Observe the tissue become brighter in areas with good perfusion.
   Note: In some cases, it may be necessary to clamp one of the cannulas with plastic clamps or to increase the inner pressure of an area by pushing softly with a spatula against the liver capsule, to optimize the perfusion. A complete color change to a light yellow to light brownish color indicates a good perfusion.
6. Change the perfusion fluid to Digestion-Solution containing collagenase P (Table 1).
7. Rearrange the setup (Figure 1A) for the digestion step. Therefore perform a circular flow of Digestion-Solution according to Figure 1B for up to 15 min.
   Note: It is critical to stop the perfusion immediately when the liver tissue sample is sufficiently digested. A good digestion can be observed, when the tissue shows no sign of elasticity as assessed by maintenance of capsule deformations, when it is pushed with a spatula.

4. Isolation of Hepatocytes (1 hr)

1. Turn the peristaltic pump off and place the liver tissue sample in a glass dish. Rinse the outside of the tissue sample with ice cold Stop-Solution (Table 1). Remove the cannulas from the liver tissue sample. Use a scalpel to open the liver tissue sample, by incising in the middle of the area where the cannulas were attached. Keep care that the Glisson´s capsule stays intact.
2. Rinse the inside of the tissue sample and then cover the whole tissue sample with ice cold Stop-Solution. Shake the tissue gently to release the cells out of the tissue.
3. Collect the cell suspension and filter it through a gaze funnel (plastic funnel lined with gauze compress) into 50 ml plastic tubes. Add more Stop-Solution to the liver tissue sample until a final volume of 500 ml is consumed.
4. Centrifuge the cell suspension at 50 x g, 5 min, 4 °C. Collect the supernatant for later non-parenchymal cell isolation. Wash the cell pellet with PBS (Figure 2A).
5. Centrifuge the cell suspension again at 50 x g, 5 min, 4 °C. Collect the supernatant and re-suspend the pellet in Hepatocyte Incubation medium (Table 2, Figure 2B).
6. Determine the cell number and viability in the resulting cell suspension using trypan blue staining. Count the living and dead cells in a Neubauer counting chamber. Calculate the cell number, viability and yield of PHH using the formulas below.
   
   yield (counted cells) = counted cells x dilution factor x volume of cell suspension (ml) x 10,000
   
   yield (hepatocytes/(g liver tissue sample)) = (yield (hepatocytes/(ml media)) x Volume of cell suspension (ml))/(weight of liver tissue sample (g))
   
   viability (%) = 100% x (number of live cells)/(total cell number)

5. Purification of Hepatocytes (1 hr)

Note: This purification step is recommended, if the viability is lower than 70%.

1. Perform all steps on ice. Prepare a 25% density gradient by mixing 5 ml density gradient solution and 15 ml PBS for density gradient centrifugation.
2. Put a maximum of 50 Mio cells in total out of the hepatocyte rich cell suspension carefully and slowly on top of the 25% density gradient layer to ensure that a clear separation of both layers is achieved (Figure 2C). Put the tubes carefully into the centrifuge and centrifuge at 1,250 x g, 20 min, 4 °C without brake (Figure 2D).
3. Aspirate the remaining cell suspension and the dead cells in the interphase. Depending on the fat content one might also aspirate the density gradient-solution.
   Note: PHH with low lipid content form a dense pellet and the density gradient can be aspirated completely. PHH with high lipid content form a more diffuse pellet and a lot of viable cells may remain in the density gradient solution above the pellet.
4. Re-suspend the hepatocyte pellets with PBS and centrifuge again at 50 x g, 5 min, 4 °C. Pool the pellets, wash again with PBS and re-suspend purified PHH in Hepatocyte Incubation medium. Perform cell counting as described in step 4.6.

6. Cultivation of Hepatocytes

1. Prepare the cell culture dishes for seeding of PHH by coating them with an extracellular matrix, for example rat tail collagen (collagen type I). Prepare the rat tail collagen according to the protocol established by Rajan et al.¹⁷
2. Dilute the rat tail collagen stock solution 1:200 in PBS. Transfer 100 µl/cm² rat tail collagen solution into the culture dishes, taking care that the whole surface is covered. Incubate the cell culture plastics for 20 min at room temperature. Aspirate the remaining rat tail collagen solution.
3. Seed 15 x 10⁴ hepatocytes/cm² in Hepatocyte Incubation medium on culture dishes coated with rat tail collagen. Cultivate the cells in a humidified incubator at 37 °C, 5% CO₂ for at least 4 hr. After 4 hr the hepatocytes have adhered and the medium can be changed.
4. Perform investigations depending on the experimental setup. A culture time of 48 hr is recommended to allow the cells to recover from the isolation process.

7. Isolation of Non-parenchymal Liver cells (1.5-2 hr)

1. Centrifuge the collected supernatant (step 4.5 and 4.6) at 72 x g, 5 min, 4 °C to eliminate the remaining erythrocytes and hepatocytes. Pool the supernatants and centrifuge them twice to gain two cell pellets: 300 x g, 5 min, 4 °C for the sedimentation of HSC, LEC and partly KC and 650 x g, 7 min, 4 °C for sedimentation of the remaining KC.
2. Pool both pellets and re-suspend them in HBSS. Prepare a 25% and a 50% density gradients by mixing density gradient solution and PBS for density gradient centrifugation (25% density gradient solution: 5 ml density gradient solution and 15 ml PBS, 50% density gradient solution: 10 ml density gradient solution and 10 ml PBS, see Figure 2). Place the 25% density gradient solution carefully on top of the 50% density gradient solution layer.
3. Put the NPC suspension carefully and slowly on top of the 25% density gradient solution layer in a way that a clear separation of both layers is achieved.
4. Centrifuge the cell suspension on the density gradient at 1,800 x g, 20 min, 4 °C without brake (Figure 2.2).
5. Aspirate dead cells and cell debris from the uppermost layer. The NPC are located in the interphase between the 25% and 50% density gradient layer (Figure 2). Collect NPC, wash them with HBSS and centrifuge the cell suspension applying the above described dual centrifugation step (step 7.2.).

8. Separation of Kupffer Cells (Adherence Separation Step) (1 hr)

1. Perform a cell count for the KC in the NPC fraction as described in step 4.6. (For appearance of KC in suspension see Figure 3B). Centrifuge the NPC fraction with the above described dual centrifugation step (step 7.2) and re-suspend the NPC in Kupffer Cell seeding medium (Table 2).
2. Seed the KC containing fraction on plastic cell culture vessels at a density of 5 x 10⁵ KC/cm². Incubate the KC cultures for 20 min in a humidified incubator at 37 °C, 5% CO₂. Primary KC adhere on cell culture plastics within a short period of time (Figure 2.3).
3. Collect the supernatant containing not adhered NPC, consisting mainly of LEC and HSC. Pool the supernatants for later separation of LEC (see section 9) and HSC (see section 10). Wash the adherent KC with HBSS and cultivate them in Kupffer Cell culture medium (Table 2) at 37 °C, 5% CO₂ in a humidified incubator.

9. Separation of Endothelial Cells (1.5 hr)

1. Centrifuge the collected supernatant (step 8.5.) at 300 x g, 5 min, 4 °C. Wash the pellet with PBS. After centrifugation at 300 x g, 5 min, 4 °C re-suspend the cells in Stellate Cell/Endothelial Cell separation medium and perform a cell count for all remaining cells as described in step 4.6.
2. Re-suspend 1 x 10⁷ Mio cells in 1 ml Stellate Cell/Endothelial Cell separation medium, add 20 µl Blocking Solution from the MACS-KIT and 20 µl of the CD31 Micro Beads for immunolabeling and incubate the resulting suspension for 15 min at 4 °C temperature (Figure 2.4).
3. Separate LEC from HSC as described in the manufacturer´s protocol for the magnetically activated cell sorting system MACS (Figure 2.5). Elute magnetically retained CD31-positive LEC and suspend them in Stellate Cell/Endothelial Cell culture medium (Table 2).
4. Perform cell counting for LEC as described in step 4.6. Seed LEC in a density of 1.25 x 10⁵ cells/cm² in cell culture vessels coated with rat tail collagen (see step 6.1). Cultivate the cells at 37 °C, 5% CO₂ in a humidified incubator.

10. Separation of Stellate Cells (0.5 hr)

1. Unlabeled HSC pass the separation column during the MACS procedure. Collect the HSC fraction (see step 9.5, Figure 2.5). Perform cell counting as described in step 4.6.
2. Seed HSC with a density of 5 x 10⁴ cells/cm² in cell culture vessels coated with rat tail collagen (see step 6.1) in Stellate Cell/Endothelial Cell culture medium (Table 2) and cultivate them at 37 °C, 5% CO₂ in a humidified incubator.

Table 1: Perfusion and isolation solution.

Table 2: Culture and isolation media.

Wyniki

The separation into a parenchymal and non-parenchymal fraction, using density gradient centrifugation as a clean-up procedure combined with the use of adherence properties and MACS leads to successful PHH and NPC isolation. PHH and NPC can be isolated in high quality and quantity. Figure 1 shows the representative setup of the equipment for liver perfusion and digestion. 10% FCS was added to the collagenase P containing Perfusion - Solution II to reduce proteolytic activi...

Dyskusje

The published protocol describes a technique to isolate pure PHH and NPC, namely KC, HSC and LEC, simultaneously in high quality and purity from the same sample of human liver tissue. The majority of publications dealing with liver cell isolations cover only one of those cell populations^18-20 and isolation procedures performed with human tissue are rare (reviewed by Damm et al.)²¹. Adaption of methods established with animal tissue (e.g., rat liver) to human liver revealed several ...

Materiały

Name	Company	Catalog Number	Comments
General Equipment
PIPETBOY	Eppendorf
pipettes	Eppendorf
microscope	Carl Zeiss
microscope	Olympus
CO2-incubator	Binder
Lamin Air	Heraeus
Centrifuge Varifuge 3.0R	Heraeus
Urine Beaker	Sarstedt	2041101
perfusor syringe 50 ml	B.Braun	12F0482022
Bottle Top Filter	Nalgene	1058787
Falcon 50 ml Polypropylene Conical Tube	BD Biosciences	352070
Falcon 15 ml Polypropylene Conical Tube	BD Biosciences	352096
Tissue Culture plate	BD Biosciences	533047	24 well
serological pipettes	BD Biosciences	357525, 357551, 357543	25 ml, 10 ml, 5 ml
pipette tips	SARSTEDT	0220/2278014, 0005/2242011, 0817/2222011	100 µl, 200 µl, 1,000 µl
Name	Company	Catalog Number	Comments
Isolation Equipment
water bath	Lauda
peristaltic pump	Carl Roth
circulation thermostat	Lauda
pH meter	Schott
fine scales	Sartorius
stand
Büchner funnel	Haldenwanger
plastic funnel
silicone tube
cannulae with olive tips
glass dish
forceps
scalpel	Feather	12068760
Neubauer counting chamber	Optic Labor
cell lifter	Costar
Surgical Drape	Charité Universitätsmedizin Berlin	A2013027
compress	Fuhrmann	40013331
sterile surgical gloves	Gammex PF	1203441104
Tissue glue	B. Braun	1050052
glass bottle	VWR
Collagenase P	Roche	13349524
Percoll Separating Solution	Biochrom	L6145	Density 1.124 g/ml
Hank’s BSS	PAA	H00911-3938
Dulbecco’s PBS	PAA	H15 - 002	without Mg/Ca
Ampuwa	Plastipur	13CKP151
Albumin	Sigma-Aldrich	A7906
NaCl	Merck	1,064,041,000
KCl	Merck	49,361,000
Hepes Pufferan	Roth	133196836
EDTA	Sigma	E-5134
Name	Company	Catalog Number	Comments
Media Equipment
DMEM	PAA	E15-005	Low Glucose (1 g/L) (without L-Glutamine)
HEPES Buffer Solution 1 M	GIBCO	1135546
L-Glutamine	GIBCO	25030-024	200 mM
MEM NEAA	GIBCO	11140-035
penicillin/streptomycin	GIBCO	15140-122
RPMI 1640	PAA	E15 - 039	without L-Glutamine
Sodium Pyruvate	GIBCO	1137663	100 mM
Trypan Blue Solution	Sigma-Aldrich	T8154	0.4%
William’s E	GIBCO	32551-020
with GlutaMAX™
EGTA	Sigma-Aldrich	03780-50G
Fortecortin	Merck	49367	8 mg/2 ml
Human-Insulin	Lilly	HI0210	100 I.E./ml
N-Acetyl cysteine	Sigma-Aldrich	A9165-5G
Fetal calf serum (FCS)	PAA	A15-101
Name	Company	Catalog Number	Comments
Equipment for Immunostainings
CD 68	R&D Systems, USA		monoclonal
CK 19	Santa Cruz	D2309	polyclonal
CK18	Santa Cruz	K2105	monoclonal
Vimentin	Santa Cruz		monoclonal
GFAP	Sigma Aldrich		monoclonal
Triton X-100	Sigma Aldrich	23.472-9
Goat anti-Mouse IgG1-PE	Santa Cruz	C0712
Goat anti-rabbit IgG-FITC	Santa Cruz	L0412
Methanol	J.T.Baker	1104509006
Formaldehyde 4%	Herbeta Arzneimittel	200-001-8
Bovine serum albumin (BSA)	Sigma Aldrich	A7906-100G