The authors and this work were supported by NIH/NCI grant # R01CA172138 (to SBE).

Ethics Statement: Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at Tel-Aviv University.
1. Rats Protocol
<ol>
	<li>Preparations
	<ol>
		<li>Prepare heparinized PBS (30 units/ml) solution to be used at room temperature (RT) by adding 30 units of preservative free heparin per ml of Phosphate buffered saline (PBS) 1x solution. Calculate 35 ml per animal.</li>
		<li>Pass heparinized PBS through the peristaltic pump lines and the butterfly needles to eliminate air bubbles.</li>
		<li>Sterilize surgical tools &#8211; 2 pairs of scissors, blunted-edged forceps, 2 hemostats, and tooth-tissue forceps (autoclave at 121 &#176;C for at least 30 min on gravity (dry) setting).</li>
	</ol>
	</li>
	<li>Perfusion of the Liver and Collection of MH-leukocytes
	<ol>
		<li>Euthanize the animal by an overdose of 8% isoflurane. As a precaution, place a 50 ml tube containing an isoflurane-soaked pad around the rat head until opening its chest cavity.</li>
		<li>Open the peritoneal and chest cavities to expose the liver and the cardiopulmonary complex immediately upon cessation of respiration. Avoid bleeding through this process.
		<ol>
			<li>Specifically, start the incision at the lower abdominal midline point, without damaging internal organs, and progress to both sides diagonally toward the ribs, cutting through them to upper chest-cavity levels.</li>
		</ol>
		</li>
		<li>Within approximately a min, collect as much blood as possible (~6 ml from a 250 g animal) from the right ventricle into a syringe.</li>
		<li>Clamp the caudal vena cava with a hemostat as close as possible to the heart, to enable the collection of the perfusate.</li>
		<li>Pull out the intestines and place outside the animal to the experimenter&#39;s right, to expose the portal vein.</li>
		<li>Insert a 25 G IV catheter, connected to a peristaltic pump, into the portal vein, as caudal as possible, but rostral to the splenic vein.</li>
		<li>Insert a 25 G butterfly needle, connected to a 5 ml syringe, into the inferior vena cava, caudal to the hemostat.</li>
		<li>Turn on the peristaltic pump at a speed of approximately 3 ml/min and gently collect the first ml of perfusate that are contaminated with blood into the syringe. Continue until the perfusate color is turns pale red (approximately 3-5 ml). Notice that the color of the liver is changing towards light brown.</li>
		<li>Without stopping the peristaltic pump, replace the 5 ml harvesting syringe with a 20 ml harvesting syringe and collect at least 20 ml of liver perfusate employing a higher speed of perfusion (up to 4 ml/min). Continuously monitor the perfusate flow into the collecting syringe, and avoid vacuum that is too strong (which may clog the needle through collapsing the vena cava walls).</li>
		<li>Terminate perfusion when the color of the liver turns to light brown.</li>
	</ol>
	</li>
	<li>Leukocytes Extraction
	<ol>
		<li>Centrifuge the perfusate for 10 min at 400 x g, 24 &#176;C.</li>
		<li>Aspirate the supernatant</li>
		<li>Add 10 ml PBS, centrifuge at 400 x g for 10 min, and aspirate the supernatant. Based on study goals, use preparation as is, or conduct additional purifications (e.g., density-gradient separation).
		<ol>
			<li>Specifically, layer 4 ml of perfusate over 4 ml of density-gradient separation mass in a 50 cc polypropylene centrifuge tube. Spin the tubes at 754 x g, RT, for 30 min with the break off.</li>
			<li>Obtain the mononuclear layers at the density-gradient separation-supernatant interface by manual pipetting, wash in PBS, and insert into a 5 ml tube.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Mouse Protocol
<ol>
	<li>Preparations
	<ol>
		<li>Prepare heparinized PBS (30 units/ml) solution to be used at room temperature (RT) by adding 30 units of preservative free heparin per ml of Phosphate buffered saline (PBS) 1x solution. Calculate 20 ml per animal.</li>
		<li>Pass heparinized PBS through the peristaltic pump lines and the butterfly needles to eliminate air bubbles.</li>
		<li>Sterilize surgical tools &#8211; 2 pairs of scissors, blunted-edged forceps, 2 hemostats, and tooth-tissue forceps (autoclave at 121 &#176;C for at least 30 min on gravity (dry) setting).</li>
	</ol>
	</li>
	<li>Perfusion of the Liver and Collection of MH-Leukocytes
	<ol>
		<li>Euthanize the animal by an overdose of 8% isoflurane. As a precaution, place a 15 ml tube containing an isoflurane-soaked pad around the mouse head until opening its chest cavity.</li>
		<li>Fix the limbs of the mouse to a bed.</li>
		<li>Open the peritoneal and chest cavities to expose the liver and the cardiopulmonary complex immediately upon cessation of respiration, keeping the lower aspect of the diaphragm intact (to create a draining chest cavity pool). Avoid bleeding through this process.
		<ol>
			<li>Specifically, start the incision at the lower abdominal midline point, without damaging internal organs, and progress to both sides diagonally toward the ribs, cutting through them to upper chest-cavity levels.</li>
		</ol>
		</li>
		<li>Pullout the intestines and place outside the animal to the experimenter&#39;s right, to expose the portal vein.</li>
		<li>Insert a 30 g needle, connected to a peristaltic pump into the portal vein rostral to the splenic vein.</li>
		<li>Cut the inferior vena cava above the diaphragm to allow drainage and collection of the perfusate from the chest cavity.</li>
		<li>Turn on the peristaltic pump at a speed of approximately 3 ml/min and gently collect the first 3 ml of perfusate that are contaminated with blood from the chest cavity pool.</li>
		<li>Discard this perfusate. Repeat such a washing again if needed, by using saline, and only then start collecting liver perfusate.</li>
		<li>Re-initiate the perfusion at a speed of up to 4 ml/min, and collect 10 ml of perfusate from the chest cavity using a butterfly connected to a syringe.</li>
		<li>Terminate perfusion when the color of the liver turns to light brown.</li>
	</ol>
	</li>
	<li>Leukocytes Extraction
	<ol>
		<li>Centrifuge the perfusate for 10 min at 400 x g.</li>
		<li>Aspirate the supernatant.</li>
		<li>Add 10 ml PBS, centrifuge at 400 x g for 10 min, and aspirate the supernatant Based on study goals, use preparation as is, or conduct additional purifications (e.g., density-gradient separation).
		<ol>
			<li>Specifically, layer every 4 ml of perfusate over 4 ml of density-gradient separation mass in a 50 cc polypropylene centrifuge tube. Spin the tubes at 754 x g, RT, for 30 min with the break off.</li>
			<li>Obtain the mononuclear layers at the density-gradient separation-supernatant interface by manual pipetting, wash in PBS, and insert into a 5 ml tube.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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M., van der Meulen, J., Daems, W. 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R., Hengartner, H., Lichtenheld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+of+perforin+in+cytolysis?.">A central role of perforin in cytolysis?.</a> Annu Rev Immunol. 9, 129-157 (1991).</li><li>Kamada, M. M., 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=Identification+of+carboxypeptidase+and+tryptic+esterase+activities+that+are+complexed+to+proteoglycans+in+the+secretory+granules+of+human+cloned+natural+killer+cells.">Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells.</a> J Immunol. 142, 609-615 (1989).</li><li>Wisse, E., 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=On+the+function+of+pit+cells,+the+liver-specific+natural+killer+cells.">On the function of pit cells, the liver-specific natural killer cells.</a> Semin Liver Dis. 17, 265-286 (1997).</li><li>Bouwens, L., Remels, L., Baekeland, M., Van Bossuyt, H., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+granular+lymphocytes+or+&amp;#34;pit+cells&amp;#34;+from+rat+liver:+isolation,+ultrastructural+characterization+and+natural+killer+activity.">Large granular lymphocytes or &amp;#34;pit cells&amp;#34; from rat liver: isolation, ultrastructural characterization and natural killer activity.</a> Eur J Immunol. 17, 37-42 (1987).</li><li>Vanderkerken, 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=Origin+and+differentiation+of+hepatic+natural+killer+cells+(pit+cells).">Origin and differentiation of hepatic natural killer cells (pit cells).</a> Hepatology. 18, 919-925 (1993).</li><li>Luo, D., 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=The+role+of+adhesion+molecules+in+the+recruitment+of+hepatic+natural+killer+cells+(pit+cells)+in+rat+liver.">The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver.</a> Hepatology. 24, 1475-1480 (1996).</li><li>Shresta, S., MacIvor, D. M., Heusel, J. W., Russell, J. H., Ley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+and+lymphokine-activated+killer+cells+require+granzyme+B+for+the+rapid+induction+of+apoptosis+in+susceptible+target+cells.">Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells.</a> Proc Natl Acad Sci U S A. 92, 5679-5683 (1995).</li><li>Luo, D., 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=Involvement+of+LFA-1+in+hepatic+NK+cell+(pit+cell)-mediated+cytolysis+and+apoptosis+of+colon+carcinoma+cells.">Involvement of LFA-1 in hepatic NK cell (pit cell)-mediated cytolysis and apoptosis of colon carcinoma cells.</a> J Hepatol. 31, 110-116 (1999).</li><li>Wiltrout, R. 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=Augmentation+of+mouse+liver-associated+natural+killer+activity+by+biologic+response+modifiers+occurs+largely+via+rapid+recruitment+of+large+granular+lymphocytes+from+the+bone+marrow.">Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow.</a> J Immunol. 143, 372-378 (1989).</li><li>Schluter, 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=Organ-specific+metastatic+tumor+cell+adhesion+and+extravasation+of+colon+carcinoma+cells+with+different+metastatic+potential.">Organ-specific metastatic tumor cell adhesion and extravasation of colon carcinoma cells with different metastatic potential.</a> Am J Pathol. 169, 1064-1073 (2006).</li><li>Wiltrout, R. 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=Augmentation+of+organ-associated+natural+killer+activity+by+biological+response+modifiers.+Isolation+and+characterization+of+large+granular+lymphocytes+from+the+liver.">Augmentation of organ-associated natural killer activity by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver.</a> J Exp Med. 160, 1431-1449 (1984).</li><li>Winnock, M., 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=Functional+characterization+of+liver-associated+lymphocytes+in+patients+with+liver+metastasis.">Functional characterization of liver-associated lymphocytes in patients with liver metastasis.</a> Gastroenterology. 105, 1152-1158 (1993).</li><li>Hata, 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=Isolation,+phenotyping,+and+functional+analysis+of+lymphocytes+from+human+liver.">Isolation, phenotyping, and functional analysis of lymphocytes from human liver.</a> Clin Immunol Immunopathol. 56, 401-419 (1990).</li><li>Vanderkerken, K., Bouwens, L., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+phenotypically+and+functionally+distinct+subset+of+large+granular+lymphocytes+(pit+cells)+in+rat+liver+sinusoids.">Characterization of a phenotypically and functionally distinct subset of large granular lymphocytes (pit cells) in rat liver sinusoids.</a> Hepatology. 12, 70-75 (1990).</li><li>Sorski, L., Melamed, R., Lavon, H., Matzner, P., Rosenne, E., Ben-Eliyahu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2015).</li></ol>

The authors have nothing to disclose.

The liver perfusion method presented herein enables a selective harvesting and studying of the unique population of marginating hepatic leukocytes. Hepatic NK cells, also called pit cells3, constitute a distinctive NK cell population that reside in the hepatic sinusoids. They are found in rats, mice17 and in humans18,19. Compared to isolated peripheral NK cells, pit cells demonstrated higher cytotoxicity against YAC-1 and CC531s target cell lines20, were shown to have a higher ...

The liver sinusoids contain numerous leukocyte subtypes of various immune activities critical to the organism. For example, marginating hepatic (MH) natural killer (NK) cells, also known as pit cells, are characterized morphologically as large granular lymphocytes (LGLs) and functionally as leukocytes with spontaneous cytotoxic capacity, enabling hepatic-resistance against the establishment of blood-borne tumor metastases. The goal of the method presented herein is to enable selective harvesting of MH leukocytes, in order to study this important and unique cell population (and immune compartment), and to elucidate the impact of various manipulations (e.g. immune activation) on these specific cells.
Despite the failure of immunity to eliminate a developing primary tumor, evidence in cancer patients and animal models indicate that the immune system can control circulating tumor cells, micrometastases, and residual disease through cell-mediated immunity (CMI). However, there is an apparent inconsistency between these in vivo capabilities and in vitro studies in humans and in animals, which demonstrate that most autologous tumor cells are resistant to cytotoxicity by circulating or splenic leukocytes1,2. This inconsistency may be ascribed, at least in part, to the in vivo existence of a distinct leukocyte subpopulation, namely the marginating-hepatic (sinusoids) leukocytes and their subpopulation of activated NK cells, namely pit cells3. Indeed, unpublished data from our laboratory indicated that in F344 rats syngeneic tumor cells (MADB106), which were found resistant to circulating and splenic leukocytes, were lysed by MH-NK cells4. Thus, tumor cells that are allegedly &#34;NK-resistant&#34; to circulating leukocytes may be controlled by MH-NK cells. Noteworthy, in mice the enhanced activity of MH-NK is evident only following in vivo immune stimulation (e.g. through the use of Poly I:C or CpG-C)5.
Liver-specific NK cells (MH-NK) are situated inside the sinusoidal lumen, adhering to the endothelial cells and Kupffer cells. MH-NK cells are exclusively characterized by spherical dense granules and rod-cored vesicles6, which contain acid phosphatase as lysosomal enzymes, and perforin and granzymes as bioactive substances7,8. Compared with circulating NK cells, MH-NK cells exhibit a higher number and size of granules and vesicles9-11. Under inflammatory conditions, MH-NK cells were shown to exhibit higher expression of LFA-112, as compared to circulating NK cells. This enhanced expression might constitute a mechanism by which MH-NK cells are more cytotoxic against certain tumor cells than circulating NK cells 13,14. nterestingly, following in vitro incubation with interleukin (IL)-2, MH-NK cells become enlarged, and their number and size of granules increase, all of which are consistent with a pro&#64257;le of lymphokine-activated killer (LAK) cells15.
Active MH leukocytes cannot be exclusively obtained through the standard liver leukocyte harvesting methods, which are based on grinding and biological degradation of the tissue. Our perfusion approach described herein has two major advantages compared to the standard approaches. First, the perfusion approach selectively harvests MH leukocytes, preventing contamination by other leukocytes from other liver compartments. Second, the perfusion technique better preserves the integrity, the activity, and the physiological milieu of MH leukocytes, unlike the tissue processing approaches that damage cells or alter their morphology, and due to tissue damage, induce the release of various factors that markedly modulate immune activity.
The liver is a major target organ for cancer metastasis and for various infections16. As MH cells exhibit unique characteristics it is important to study this specific population under various conditions with respect to these pathologies. For example, it is worthy to note that systemic immune activation by various BRMs (e.g. poly I:C or CpG-C) have been shown to activate MH-leukocytes more than circulating leukocytes5.

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26 G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21 G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>22 W Fluorescent Daylight Magnifier Lamp</td><td></td><td></td><td></td></tr></tbody></table>

selective harvesting of marginating-hepatic leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

In F344 rats we compared the cytotoxicity of MH-NK cells (collected from the liver sinusoids by forced liver perfusion) to cytotoxicity of the entire liver cell population following mechanical grinding of the liver tissue, and to the cytotoxicity of circulating leukocytes. All cell preparations were washed at least 3 times, as routine in immunological assays, and as target cell lines we used the allogenic YAC-1 or the syngeneic MADB106 target cell lines. As indicated in Figures 1<...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-hepatic Leukocytes at JoVE.com

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

selective-harvesting-of-marginating-hepatic-leukocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

5.6K Views.  Tel-Aviv University. Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Video: Selective Harvesting of Marginating-hepatic Leukocytes

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Developmental Biology

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

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 ...

Biology

Establishing In Vitro LLEC Cultures Using Magnetic Cell Selection

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

Neuroscience

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Medicine

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture

Source: Slone, W. L., et al. <a href="https://jove.unicartagenaproxy.elogim.com/t/53645">Modeling Chemotherapy Resistant Leukemia In Vitro.</a> J. Vis. Exp. (2016).
This video describes a method for spatial separation of three leukemic cell subpopulations: the suspended leukemic cells freely floating in the media, phase bright leukemic cells adhered to the surface of the mesenchymal stromal cell monolayer, and the phase dim leukemic cells that have migrated beneath the mesenchymal stromal cell monolayer from 2D co-culture.

Source: Slone, W. L., et al. Modeling Chemotherapy Resistant Leukemia In Vitro. J. Vis. Exp. (2016).
This video describes a method for spatial separation ...

JoVE Encyclopedia of Experiments

Cancer Research

Leukemia

Cell isolation & characterization

Isolation of Marginating Hepatic Leukocytes from a Mouse Liver

Source: Sorski, L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53918">Selective Harvesting of Marginating-hepatic Leukocytes.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of marginating hepatic leukocytes from mouse liver by forced perfusion.

Source: Sorski, L., et al. Selective Harvesting of Marginating-hepatic Leukocytes. J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of ...