We would like to thank Jia Li Liu for their support in creation of Figure 1. This study was supported by the German Federal Ministry of Education and Research (BMBF) project Virtual Liver: 0315741.

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 Charit&#233; - Universit&#228;tsmedizin Berlin.
1. Preparation of Materials and Solutions
<ol>
	<li>Sterilize all instruments and the materials in advance to avoid bacterial contamination during the isolation process.</li>
	<li>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 &#176;C and it is recommended to use them within 4 weeks after preparation.</li>
	<li>Sterilize all solutions using a 0.22 &#181;m bottle top filter.</li>
</ol>
2. Preparation of Perfusion Equipment
<ol>
	<li>Set up the equipment for the perfusion and digestion of the liver tissue sample as shown in Figure 1A.</li>
	<li>Adjust the water bath temperature to 39 &#176;C to ensure optimal collagenase P activity during the perfusion and digestion.</li>
</ol>
3. Perfusion and Digestion of the Liver Tissue Sample (1.5 hr)
<ol>
	<li>Select a tissue sample with an intact Glisson&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the cannulated liver tissue sample into the B&#252;chner funnel on its perforated filter disc (Figure 1A).</li>
	<li>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.</li>
	<li>Change the perfusion fluid to Digestion-Solution containing collagenase P (Table 1).</li>
	<li>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.</li>
</ol>
4. Isolation of Hepatocytes (1 hr)
<ol>
	<li>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&#180;s capsule stays intact.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge the cell suspension at 50 x g, 5 min, 4 &#176;C. Collect the supernatant for later non-parenchymal cell isolation. Wash the cell pellet with PBS (Figure 2A).</li>
	<li>Centrifuge the cell suspension again at 50 x g, 5 min, 4 &#176;C. Collect the supernatant and re-suspend the pellet in Hepatocyte Incubation medium (Table 2, Figure 2B).</li>
	<li>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)</li>
</ol>
5. Purification of Hepatocytes (1 hr)
Note: This purification step is recommended, if the viability is lower than 70%.
<ol>
	<li>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.</li>
	<li>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 &#176;C without brake (Figure 2D).</li>
	<li>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.</li>
	<li>Re-suspend the hepatocyte pellets with PBS and centrifuge again at 50 x g, 5 min, 4 &#176;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.</li>
</ol>
6. Cultivation of Hepatocytes
<ol>
	<li>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</li>
	<li>Dilute the rat tail collagen stock solution 1:200 in PBS. Transfer 100 &#181;l/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.</li>
	<li>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 &#176;C, 5% CO2 for at least 4 hr. After 4 hr the hepatocytes have adhered and the medium can be changed.</li>
	<li>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.</li>
</ol>
7. Isolation of Non-parenchymal Liver cells (1.5-2 hr)
<ol>
	<li>Centrifuge the collected supernatant (step 4.5 and 4.6) at 72 x g, 5 min, 4 &#176;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 &#176;C for the sedimentation of HSC, LEC and partly KC and 650 x g, 7 min, 4 &#176;C for sedimentation of the remaining KC.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge the cell suspension on the density gradient at 1,800 x g, 20 min, 4 &#176;C without brake (Figure 2.2).</li>
	<li>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.).</li>
</ol>
8. Separation of Kupffer Cells (Adherence Separation Step) (1 hr)
<ol>
	<li>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).</li>
	<li>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 &#176;C, 5% CO2. Primary KC adhere on cell culture plastics within a short period of time (Figure 2.3).</li>
	<li>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 &#176;C, 5% CO2 in a humidified incubator.</li>
</ol>
9. Separation of Endothelial Cells (1.5 hr)
<ol>
	<li>Centrifuge the collected supernatant (step 8.5.) at 300 x g, 5 min, 4 &#176;C. Wash the pellet with PBS. After centrifugation at 300 x g, 5 min, 4 &#176;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.</li>
	<li>Re-suspend 1 x 107 Mio cells in 1 ml Stellate Cell/Endothelial Cell separation medium, add 20 &#181;l Blocking Solution from the MACS-KIT and 20 &#181;l of the CD31 Micro Beads for immunolabeling and incubate the resulting suspension for 15 min at 4 &#176;C temperature (Figure 2.4).</li>
	<li>Separate LEC from HSC as described in the manufacturer&#180;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).</li>
	<li>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 &#176;C, 5% CO2 in a humidified incubator.</li>
</ol>
10. Separation of Stellate Cells (0.5 hr)
<ol>
	<li>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.</li>
	<li>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 &#176;C, 5% CO2 in a humidified incubator.</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/53069/53069table1.jpg" /> 
Table 1: Perfusion and isolation solution.
<img alt="Table 2" src="/files/ftp_upload/53069/53069table2.jpg" /> 
Table 2: Culture and isolation media.

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The authors declare that they have no competing interests.

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 populations18-20 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 ...

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,&#160;e.g., bile acid and complement factor synthesis, biotransformation and energy metabolism2,3.
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,&#160;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 &#34;gold standard&#34;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 compensated4,6. 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 models8,9 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 cells11,12. Only a few publications describe the isolation process of human NPC and most cover only methods for the isolation of a single cell type11-16. 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.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">General Equipment</td>
			<td style="width:147px;"></td>
			<td style="width:293px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PIPETBOY</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CO2-incubator</td>
			<td>Binder</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lamin Air</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge Varifuge 3.0R</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Urine Beaker</td>
			<td>Sarstedt</td>
			<td>2041101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">perfusor syringe 50 ml</td>
			<td>B.Braun</td>
			<td>12F0482022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bottle Top Filter</td>
			<td>Nalgene</td>
			<td>1058787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon 50 ml Polypropylene Conical Tube</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon 15 ml Polypropylene Conical Tube</td>
			<td>BD Biosciences</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Culture plate</td>
			<td>BD Biosciences</td>
			<td>533047</td>
			<td>24 well</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">serological pipettes</td>
			<td>BD Biosciences</td>
			<td>357525, 357551, 357543</td>
			<td>25 ml, 10 ml, 5 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pipette tips</td>
			<td>SARSTEDT</td>
			<td>0220/2278014, 0005/2242011, 0817/2222011</td>
			<td>100 &#181;l, 200 &#181;l, 1000 &#181;l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isolation Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">water bath</td>
			<td>Lauda</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">peristaltic pump</td>
			<td>Carl Roth</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">circulation thermostat</td>
			<td>Lauda</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH meter</td>
			<td>Schott</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fine scales</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">stand</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B&#252;chner funnel</td>
			<td>Haldenwanger</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plastic funnel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silicone tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cannulae with olive tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scalpel</td>
			<td>Feather</td>
			<td>12068760</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neubauer counting chamber</td>
			<td>Optic Labor</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cell lifter</td>
			<td>Costar</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Drape</td>
			<td>Charit&#233; Universit&#228;tsmedizin Berlin</td>
			<td>A2013027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">compress</td>
			<td>Fuhrmann</td>
			<td>40013331</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sterile surgical gloves</td>
			<td>Gammex PF</td>
			<td>1203441104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue glue</td>
			<td>B. Braun</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass bottle</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td>Roche</td>
			<td>13349524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Percoll Separating Solution</td>
			<td>Biochrom</td>
			<td>L6145</td>
			<td>Density 1.124 g/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s BSS</td>
			<td>PAA</td>
			<td>H00911-3938</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#8217;s PBS</td>
			<td>PAA</td>
			<td>H15 - 002</td>
			<td>without Mg/Ca</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampuwa</td>
			<td>Plastipur&#160;</td>
			<td>13CKP151&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Merck</td>
			<td>1,064,041,000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Merck</td>
			<td>49,361,000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Pufferan&#160;</td>
			<td>Roth</td>
			<td>133196836</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>E-5134</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Media Equipment&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>PAA</td>
			<td>E15-005</td>
			<td>Low Glucose (1 g/l) (without L-Glutamine)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffer Solution 1M</td>
			<td>GIBCO</td>
			<td>1135546</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>GIBCO</td>
			<td>25030-024</td>
			<td>200 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM NEAA</td>
			<td>GIBCO</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin/streptomycin</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>PAA</td>
			<td>E15 - 039</td>
			<td>without L-Glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Pyruvate</td>
			<td>GIBCO</td>
			<td>1137663</td>
			<td>100 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue Solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td>0.4%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">William&#8217;s E&#160;&#160;</td>
			<td>GIBCO</td>
			<td>32551-020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">with GlutaMAX&#8482;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>03780-50G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fortecortin</td>
			<td>Merck</td>
			<td>49367</td>
			<td>8 mg/2 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human-Insulin</td>
			<td>Lilly</td>
			<td>HI0210</td>
			<td>100 I.E./ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-Acetyl cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal calf serum (FCS)</td>
			<td>PAA</td>
			<td>A15-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment for Immunostainings</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD 68</td>
			<td>R&#38;D Systems, USA</td>
			<td></td>
			<td>monoclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CK 19</td>
			<td>Santa Cruz</td>
			<td>D2309</td>
			<td>polyclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CK18</td>
			<td>Santa Cruz</td>
			<td>K2105</td>
			<td>monoclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vimentin</td>
			<td>Santa Cruz</td>
			<td></td>
			<td>monoclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GFAP</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>monoclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>23.472-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Mouse IgG1-PE</td>
			<td>Santa Cruz</td>
			<td>C0712</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit IgG-FITC</td>
			<td>Santa Cruz</td>
			<td>L0412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>J.T.Baker</td>
			<td>1104509006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formaldehyde 4%</td>
			<td>Herbeta Arzneimittel</td>
			<td>200-001-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for isolation of primary human hepatocytes and corresponding major populations of non-parenchymal liver cells

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 &#34;gold standard&#34; 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.

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...

Watch this Scientific Journal Video about Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells at JoVE.com

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

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.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

protocol-for-isolation-primary-human-hepatocytes-corresponding-major

Research

JoVE Journal

Biology

36.8K Views.  Charité University Medicine Berlin. 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.

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

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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

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&plusmn;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.

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.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...