Name: isolation of primary rat hepatocytes with multiparameter perfusion control
Uploaded: 2021-04-05T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control at JoVE.com

This work is supported in part by MOE ARC (MOE2017-T2-1-149); NUHS Innovation Seed Grant 2017 (NUHSRO/2017/051/InnovSeed/02); Mechanobiology Institute of Singapore (R-714-106-004-135); and Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR) (Project Numbers IAF-PP H18/01/a0/014, IAF-PP H18/01/a0/K14 and MedCaP-LOA-18-02) funding to Hanry Yu. Ng Chan Way is a research scholar of the National University of Singapore. We would like to thank Confocal Microscopy Unit &#38; Flow Cytometry Unit of the National University of Singapore for help and advice in hepatocyte purity analysis.

All procedures and animal housing were carried out under protocol numbers R15-0027 and R19-0669 in accordance with the requirements of the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore.
1. Preparation of solutions and surgical instruments
<ol>
	<li>Prepare buffers and cell culture media in Table 1 using ultrapure water.</li>
	<li>Pre-warm the calcium-free buffer and collagenase buffer to 37 &#176;C in a water bath before use.</li...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constrained+spheroids+for+prolonged+hepatocyte+culture.">Constrained spheroids for prolonged hepatocyte culture.</a> Biomaterials. 80, 106-120 (2016).</li><li>Xia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar-flow+immediate-overlay+hepatocyte+sandwich+perfusion+system+for+drug+hepatotoxicity+testing.">Laminar-flow immediate-overlay hepatocyte sandwich perfusion system for drug hepatotoxicity testing.</a> Biomaterials. 30 (30), 5927-5936 (2009).</li><li>Gupta, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+canaliculi+contract+autonomously+by+releasing+calcium+into+hepatocytes+via+mechanosensitive+calcium+channel.">Bile canaliculi contract autonomously by releasing calcium into hepatocytes via mechanosensitive calcium channel.</a> Biomaterials. 259, 120283 (2020).</li><li>Horner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Percoll+purification+on+isolation+of+primary+human+hepatocytes.">Impact of Percoll purification on isolation of primary human hepatocytes.</a> Scientific Reports. 9 (1), 6542 (2019).</li><li>Osypiw, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subpopulations+of+rat+hepatocytes+separated+by+Percoll+density-gradient+centrifugation+show+characteristics+consistent+with+different+acinar+locations.">Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations.</a> The Biochemical Journal. 304, 617-624 (1994).</li><li>Beal, E. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+animal+model+of+ex+vivo+normothermic+liver+perfusion.">A small animal model of ex vivo normothermic liver perfusion.</a> Journal of Visualized Experiments: JoVE. (136), e57541 (2018).</li><li>Hillebrandt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+decellularization+of+rat+livers+in+an+oscillating-pressure+perfusion+device.">Procedure for decellularization of rat livers in an oscillating-pressure perfusion device.</a> Journal of Visualized Experiments: JoVE. (102), e53029 (2015).</li></ol>

There are a few points that are particularly important to observe for two-step collagenase perfusion procedure in general. Firstly, special care must be given when resecting the liver. Ensure that the gastrointestinal tract is not damaged as leakage of the contents will result in bacterial contamination. In addition, avoid damaging the Glisson&#39;s capsule, which covers the surface of the liver during the animal procedure. If the tear is large enough, it might allow premature release of disassociated hepatocytes into th...

Primary hepatocytes are important tools for liver-related basic research, disease treatment, and application such as drug testing. The current gold standard for primary hepatocyte isolation is the two-step collagenase perfusion procedure1,2,3 introduced by Seglen in the 1970s4. However, this procedure is technically challenging and has a high failure rate when performed by novice surgeons. Even when a perfusion is considered successful, drastic differences in hepatocyte viability (typically 60%-95%) and yield (0.5-5 x 108 per 200-300 g rat) may be observed between isolations. This influences the quality and scale of downstream experiments. Apart from the technical procedure, the perfusion setup used for the isolation, either commercially available or custom built, is a contributing factor. Attention must be given to the assembly, optimization, and maintenance of the perfusion setup. The purpose of this protocol is to improve the success rate and stability between isolations of primary rat hepatocytes through multiparameter perfusion control of the technical procedure and perfusion setup of the two-step collagenase perfusion procedure.
From the technical aspect, the most difficult step in the procedure is the portal vein cannulation. As for the other steps, if good practice is observed and general precautions are taken, the stability of the isolation can be improved. Therefore, understanding of the reasoning for each step is important so that the surgeon could respond to various variables that may occur during the procedure.
Various protocols for the isolation of hepatocytes and liver non-parenchymal cells from rat and mouse have been published1,2,5,6,7,8,9. The perfusion setups used in these protocols had several disadvantages, which include the reuse of perfusion tubing, problems with temperature control, need for routine optimization of perfusion parameters, and/or usage of unsuitable type of intravenous (IV) catheter for portal vein cannulation. The reuse of perfusion tubing will increase the chances of contamination, especially if the tubing was not cleaned and disinfected properly. Reuse of tubing without routine replacement will also expose the perfusion setup to problems such as leaky tubing or connectors, clogged bubble trap and constricted tubing, all of which will substantially reduce the perfusate pressure and flow rate, thus, affecting liver digestion efficiency. Without a constant heat source in some setups for temperature control, pre-warmed buffers will cool down over time, leading to low collagenase activity and digestion. Although other setups utilize a jacketed glass condenser connected to a water circulator to warm the buffer, they are bulky and require careful cleaning. Temperature, pressure, and flow rate of buffer exiting the catheter must be measured and optimized before the start of isolation toensure stable perfusion condition. Even after optimization, the parameters could still change halfway during isolation due to the actions of the operator, thereby leading to suboptimal perfusion and digestion. Most types of IV catheter are not suitable for portal vein cannulation because they do not allow continuous perfusion during cannulation. They are unable to immediately inform the surgeon when the cannulation is successful. Furthermore, it is challenging to secure the portal vein on the soft catheter without deforming it.
Here, we address these problems using standardized disposable sterile tubing, a silicone heater jacket for precise and stable temperature control, real-time monitoring and alarm system with data storage and management and use of a special IV catheter, which allows continuous perfusion while puncturing portal vein during cannulation. To the best of our knowledge, we are the first group to combine all these features into an integrated perfusion system (IPS) that is compact, making it highly portable and able to be fit into a laminar flow hood to ensure aseptic operation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Nipro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27G needle</td>
			<td>Nipro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Black braided silk non-absorbable, non-sterile surgical suture</td>
			<td>Look</td>
			<td>SP117</td>
			<td></td>
		</tr>
		<tr>
			<td>Bochem 18/10 stainless steel forceps, sharp tip contain bent round tip</td>
			<td>Bochem</td>
			<td>10333511</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Perfusion Set</td>
			<td>Vasinfuse</td>
			<td>BPF-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Floating circular 1.5 mL microcentrifuge tube rack</td>
			<td>Sigma-Aldrich</td>
			<td>R3133</td>
			<td></td>
		</tr>
		<tr>
			<td>German Standard Tissue Forceps, Serrated / 1&#215;2 teeth , 14.5cm</td>
			<td>Walentech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Greiner Cellstar aspirating pipette</td>
			<td>Merck</td>
			<td>GN710183</td>
			<td></td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Integrated Perfusion System</td>
			<td>Vasinfuse</td>
			<td>IPS-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors curved, stainless, 11cm</td>
			<td>Optimal Medical Products Pte Ltd</td>
			<td>CVD</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope with 10X lens</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh Sheet 100&#181;M Nylon</td>
			<td>Spectra-Teknic(s) Pte Ltd</td>
			<td>06630-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, BL/BL, 13cm</td>
			<td>Optimal Medical Products Pte Ltd</td>
			<td>STR &#8211; BL/BL</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, SH/BL, 13cm</td>
			<td>Optimal Medical Products Pte Ltd</td>
			<td>STR &#8211; SH/BL</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse force hemostatic clip</td>
			<td>Shanghai Jin Zhong Pte Ltd</td>
			<td>XEC230</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents/Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9056</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Merck</td>
			<td>137101</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>TCI</td>
			<td>D1961</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>31600-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen</td>
			<td>11344-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>1-9278</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>VWRC26764.298</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr>
			<td>Linoleic acid</td>
			<td>Sigma-Aldrich</td>
			<td>L9530</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck</td>
			<td>106462</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Type I bovine collagen</td>
			<td>Advanced BioMatrix</td>
			<td>5005-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>William&#8217;s E Media</td>
			<td>Sigma-Aldrich</td>
			<td>W1878</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A surgeon could tell whether liver perfusion is going on smoothly by observing the outcome after certain steps. The first outcome can be observed upon cannulation, cutting of the infrahepatic IVC, and restoring the perfusion flow rate. The liver should have completely changed color from dark red to brown, while maintaining its volume. If the liver looks slightly deflated and has a reddish tint or blotches of red, it means that the perfusion flow rate was set wrongly (too low), or the portal vein was not cannulated correc...

Watch this Scientific Journal Video about Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control at JoVE.com

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

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 ...

This rat hepatocytes isolation protocol is based on the liver resection prior to digestion approach. The protocol introduces a compact and portable integrated perfusion system for cell isolation. This technique involves collagenase recirculation which reduces the volume of collagenase use, and provides more flexibility in extension of digestion time while ensuring high gravity yield and hepatocytes specific function.Put the rat in the hood. After cutting the abdominal muscles, push the intestines to the right with the back of the curved forceps. Break the membrane underneath the bile duct with the tip of the curved forceps for looping the suture around the portal vein and the bile duct.Use a silk surgical suture to make a very loose ligature around the portal vein close to the liver, just before the vein branches left and right into different liver lobes. About two to three centimeters upstream of the first ligature, just before the gastric vein branches off from the portal vein, create a hole through the tissue underneath the portal vein with the tips of two pairs of curved forceps. Then stretch the hole to support the portal vein during cannulation.Prevent pressure buildup in the liver by reducing the pump speed to four RPM for a flow rate of approximately three milliliters per minute. And ensure that the outflow of the calcium free buffer from the intravenous catheter is reduced to a slow drip. While gently supporting the portal vein with forceps, hold the intravenous catheter with the bevel of the needle facing up.And insert the needle into the portal vein at a 10 to 20 degree angle. Then slowly advance until the whole bevel is inside the vein. Correct insertion should result in blanching of the liver color.Advance the cannula over the needle, and retract the needle behind the cannula by pulling back the wing with the index finger while holding the catheter with the thumb and middle finger. Secure the portal vein onto the catheter with a vein clip and clip below the portal vein to avoid perfusing flow disruption. Next, cut the infrahepatic inferior vena cava to prevent the pressure buildup in the liver, and observe for blood spurting impulses to ensure the correct cut.Increase the pump speed to 38 RPM for a flow rate of approximately 33 milliliters per minute. And flush the liver three times by closing the infrahepatic inferior vena cava with forceps for two to three seconds, and then reopening it. In order to perfuse all liver lobes, adjust the position of the cannula tip and make sure that it is placed before the portal vein branches into different liver lobes.Then, tighten the loose ligature around the portal vein, slightly upstream of the branching point, and secure the position of the cannula in the portal vein to prevent backflow of the buffer by making three knots at the point where the hard needle supports the cannula, in between the bevel and the side hole. After liver resection allow the liver to perfuse with the calcium free buffer for 12 minutes. Then, tighten the roller clamp of calcium free buffer and loosen the roller clamp of collagenase buffer to change the perfusion buffer.After the collagenase buffer reaches the liver, flush the liver three times by closing the suprahepatic inferior vena cava for two to three seconds and reopening it. Carefully move the liver to the stage on top of the beaker for recirculation of the collagenase buffer by holding the diaphragm with forceps while supporting the catheter, taking care not to pull the catheter to prevent accidental detachment. Digest the liver for 12 minutes, and stop the perfusion when the liver loses its smooth brown texture and becomes mushy.Extend the digestion time if necessary. After digestion of the liver cut the portal vein carefully remove the cannula. And then transfer the liver into cold DMEM to isolate the hepatocytes.Gently tap the liver using the backside of the curved forceps to break and peel off the glistens capsule. And release the liver cells by gently swaying the liver in DMEM until the cells are fully dissociated in the media. The viability of hepatocytes was between 88 to 94.8%as determined by trip and blue counting.The yield of hepatocytes from rats weighing 200 to 300 grams was up to five times 10 to the eighth cells per isolation. The representative images are showing unpurified liver cell suspension and purified hepatocyte suspension. The purity of the isolated hepatocytes was approximately 96.8%which was quantified by counting albumin positive fluorescent stained cells in relation to the total number of the cell nuclei.In the representative images shown in false color, green represents the albumin, and blue indicates the cell nuclei with albumin. Cell nuclei without albumin are highlighted red. The hepatocytes in sandwich culture formed distinct bile canaliculi, and had good cell cell contact.As shown by albumin, urea and cytochrome P450 assays, the hepatocytes exhibited high urea and albumin secretion. On day three, the hepatocytes exhibited high enzyme activities for CYP1A2, CYP2B1/2, and CYP3B2. Be mentally prepared before inserting the needle into the portal vein.Upon insertion, a surgeon will need to continue without pulse until the pump speed was increased after cutting the IVC.

isolation-primary-rat-hepatocytes-with-multiparameter-perfusion

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Biology

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

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. ...

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 ...

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.

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 ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...