Name: selective harvesting of marginating-hepatic leukocytes
Uploaded: 2016-07-21T13:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about Selective Harvesting of Marginating-hepatic Leukocytes at JoVE.com

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

Ethikerklärung: Verfahren unter Verwendung tierischer Probanden wurden von der Institutional Animal Care und Use Committee (IACUC) an der Universität Tel Aviv genehmigt. 1. Ratten-Protokoll <ol><li> Die Vorbereitungen <ol><li> Bereiten Sie heparinisiertem PBS (30 Einheiten / ml) Lösung verwendet werden, bei Raumtemperatur (RT) durch Zugabe von 30 Einheiten frei von Konservierungsstoffen Heparin pro ml Phosphat-gepufferte Salzlösung (PBS) 1x Lösung. Berechnen Sie 35 ml pro Tier. </li><li> Pass heparinisiertem PBS durch die peristaltische Pumpe Leitungen und den Schmetterling Nadeln Luftblasen zu beseitigen. </li><li> Sterilisieren chirurgische Instrumente - 2 Paar Scheren, abgestumpft kantigen Zange, 2 hemostats und Zahngewebe Zange (Autoklav bei 121 ° C für mindestens 30 min auf die Schwerkraft (trocken) Einstellung). </li></ol></li><li> Perfusion der Leber und Sammlung von MH-Leukozyten <ol><li> Euthanize das Tier durch eine Überdosis von 8% Isofluran. Als Vorsichtsmaßnahme ein 50-ml-Tube eine Isofluran enthält-soaked Polster um die Ratte den Kopf, bis seine Brusthöhle zu öffnen. </li><li> Öffnen Sie die Peritonealdialyse und Brusthöhlen, die Leber und die Herz-Lungen-Komplex unmittelbar nach Beendigung der Atmung aussetzen. Vermeiden Sie durch diesen Prozess Blutungen. <ol><li> Genauer gesagt, starten Sie den Schnitt an der unteren Bauchmittellinie Punkt, ohne Beschädigung der inneren Organe und des Fortschritts zu beiden Seiten schräg zu den Rippen, um durch sie zu oberen Brusthöhle Ebenen schneiden. </li></ol></li><li> Innerhalb von etwa einer Minute, zu sammeln, so viel Blut wie möglich (~ 6 ml einer 250 g Tier) aus dem rechten Ventrikel in eine Spritze. </li><li> Klemmen Sie die Schwanz Hohlvene mit einem hemostat so nah wie möglich an das Herz, die Sammlung des Perfusat zu ermöglichen. </li><li> Ziehen Sie die Eingeweide heraus und außerhalb des Tieres in die rechte Ecke des Experimentators, die Pfortader zu belichten. </li><li> Legen Sie eine 25 G IV-Katheter, der mit einer Schlauchpumpe in die Pfortader, wie Schwanz- wie möglich,aber rostral der Milzvene. </li><li> Legen Sie eine 25 G Butterfly-Nadel, die mit einer 5-ml-Spritze, in die untere Hohlvene, kaudal der hemostat. </li><li> Schalten Sie die peristaltische Pumpe bei einer Geschwindigkeit von etwa 3 ml / min und sanft die ersten ml Perfusat sammeln, die mit Blut in die Spritze kontaminiert sind. Fortfahren, bis das Perfusat Farbe erblasst rot (ca. 3-5 ml). Beachten Sie, dass die Farbe der Leber verändert sich in Richtung hellbraun. </li><li> Ohne die peristaltische Pumpe zu stoppen, ersetzen Sie die 5 ml Ernte Spritze mit einer 20 ml Ernte Spritze und sammeln mindestens 20 ml Leber Perfusat eine höhere Geschwindigkeit der Perfusion verwendet wird (bis zu 4 ml / min). Kontinuierlich überwacht die Perfusat Strom in die Sammel Spritze, und vermeiden Sie Vakuum, das zu stark ist (was die Nadel durch das Zusammenlegen der Hohlvene Wände verstopfen können). </li><li> Terminate Perfusion, wenn die Farbe der Leber bis hellbraun dreht. </li></ol></li><li> Leukozyten-Extraktion <ol&gt; <li> Zentrifugieren Sie die Perfusat für 10 Minuten bei 400 · g, 24 ° C. </li><li> Den Überstand aspirieren </li><li> 10 ml PBS, Zentrifugation bei 400 xg für 10 min und der Überstand absaugen. Basierend auf Studienziele, verwenden Vorbereitung wie es ist, oder zusätzliche Reinigungen durchführen (zB Dichtegradienttrennmittel). <ol><li> Insbesondere Schicht 4 ml Perfusat über 4 ml Dichtegradienttrennmittel Masse in einem 50 cc Polypropylen-Zentrifugenröhrchen. Drehen Sie die Röhrchen bei 754 xg, RT, 30 min mit der Pause ab. </li><li> Besorgen Sie sich die einkernigen Schichten an der Dichtegradienttrennmittel-Überstand-Schnittstelle durch manuelles Pipettieren, Waschen in PBS, und legen Sie in einem 5-ml-Tube. </li></ol></li></ol></li></ol> 2. Maus-Protokoll <ol><li> Die Vorbereitungen <ol><li> Bereiten Sie heparinisiertem PBS (30 Einheiten / ml) Lösung bei Raumtemperatur (RT) durch Zugabe von 30 Einheiten frei von Konservierungsstoffen Heparin pro ml Phosphat verwendet werden (PBS) 1x solution. Berechnen Sie 20 ml pro Tier. </li><li> Pass heparinisiertem PBS durch die peristaltische Pumpe Leitungen und den Schmetterling Nadeln Luftblasen zu beseitigen. </li><li> Sterilisieren chirurgische Instrumente - 2 Paar Scheren, abgestumpft kantigen Zange, 2 hemostats und Zahngewebe Zange (Autoklav bei 121 ° C für mindestens 30 min auf die Schwerkraft (trocken) Einstellung). </li></ol></li><li> Perfusion der Leber und Sammlung von MH-Leukozyten <ol><li> Euthanize das Tier durch eine Überdosis von 8% Isofluran. Als Vorsichtsmaßnahme, legen Sie ein 15-ml-Röhrchen eine Isofluran-getränkten Polster um die Maus Kopf bis zum Öffnen seiner Brusthöhle enthält. </li><li> Befestigen Sie die Schenkel der Maus auf ein Bett. </li><li> Öffnen Sie die Peritonealdialyse und Brusthöhlen, die Leber und die Herz-Lungen-Komplex unmittelbar nach Beendigung der Atmung aussetzen, intakt den unteren Aspekt der Membran zu halten (um eine Entleerung Brusthöhle Pool). Vermeiden Sie durch diesen Prozess Blutungen. <ol><li> Genauer gesagt, starten Sie den Einschnitt am unterenBauchmittellinie Punkt, ohne Beschädigung der inneren Organe und des Fortschritts zu den beiden diagonal gegenüber den Rippen Seiten, um durch sie zu oberen Brusthöhle Ebenen schneiden. </li></ol></li><li> Ausziehbares den Darm und außerhalb des Tieres in die rechte Ecke des Experimentators, die Pfortader zu belichten. </li><li> Legen Sie eine 30 g Nadel, verbunden mit einer Schlauchpumpe in die Pfortader rostral der Milzvene. </li><li> Schneiden Sie die untere Hohlvene oberhalb des Zwerchfells Entwässerungs- und Sammlung des Perfusat aus der Brusthöhle zu ermöglichen. </li><li> Schalten Sie die peristaltische Pumpe bei einer Geschwindigkeit von etwa 3 ml / min und Sammeln sanft die ersten 3 ml Perfusat, die mit Blut aus der Brusthöhle Pool kontaminiert sind. </li><li> Entsorgen Sie diesen Perfusat. Wiederholen solch ein Waschen wieder benötigt, wenn durch Kochsalzlösung verwendet, und erst dann starten Leber Perfusat gesammelt werden. </li><li> Wieder initiieren die Perfusion mit einer Geschwindigkeit von bis zu 4 ml / min, und sammeln 10 ml Perfusat aus der Brusthöhle unter Verwendung vonein Schmetterling mit einer Spritze verbunden. </li><li> Terminate Perfusion, wenn die Farbe der Leber bis hellbraun dreht. </li></ol></li><li> Leukozyten-Extraktion <ol><li> Zentrifuge das Perfusat 10 min bei 400 x g. </li><li> Den Überstand aspirieren. </li><li> In 10 ml PBS, Zentrifugation bei 400 × g für 10 min und den Überstand aspirieren Basierend auf Studienziele, verwenden Vorbereitung , wie oder führen zusätzliche Reinigungen (zB Dichtegradienttrennmittel). <ol><li> Insbesondere Schicht alle 4 ml Perfusat über 4 ml Dichtegradienttrennmittel Masse in einem 50 cc Polypropylen-Zentrifugenröhrchen. Drehen Sie die Röhrchen bei 754 xg, RT, 30 min mit der Pause ab. </li><li> Besorgen Sie sich die einkernigen Schichten an der Dichtegradienttrennmittel-Überstand-Schnittstelle durch manuelles Pipettieren, Waschen in PBS, und legen Sie in einem 5-ml-Tube. </li></ol></li></ol></li></ol>

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Die Methode Leberperfusion hier vorgestellten ermöglicht eine selektive Ernte und das Studium der einzigartigen Population von marginating Leber Leukozyten. Leber NK - Zellen, auch Grube Zellen 3 genannt, bilden eine unverwechselbare NK - Zellpopulation , die in den Leber sinusoids befinden. Sie werden bei Ratten, Mäusen 17 und beim Menschen 18,19 gefunden. Im Vergleich zu isolierten peripheren NK - Zellen zeigte pit Zellen höhere Zytotoxizität gegen YAC-1 und CC531s Zielzelllinien <...

Die Lebersinusoide enthalten zahlreiche Leukozytensubtypen verschiedener Immunaktivitäten entscheidend für den Organismus. Zum Beispiel marginating Leber (MH) natürliche Killerzellen (NK), die auch als Pit-Zellen bekannt sind, charakterisiert morphologisch als große granuläre Lymphozyten (LGLs) und funktionell als Leukozyten mit spontaner zytotoxische Kapazität, so dass Leber-Widerstand gegen die Errichtung von blut- getragen Tumormetastasen. Das Ziel des Verfahrens vorgestellt hierin selektive Ernte von MH Leukozyten zu aktivieren, um diese wichtige und einzigartige Zellpopulation zu untersuchen (und Immunraum), und die Auswirkungen der verschiedenen Manipulationen (beispielsweise Immunaktivierung) auf diesen spezifischen Zellen aufzuklären. Trotz des Ausfalls der Immunität eines Entwicklungsprimärtumor, Nachweis bei Krebspatienten und Tiermodelle zu eliminieren, zeigen an, dass das Immunsystem zirkulierende Tumorzellen steuern können, Mikrometastasen und residual disease through zellvermittelte Immunität (CMI). Es gibt jedoch eine scheinbare Unstimmigkeit zwischen diesen in vivo - Funktionen und in vitro - Studien in Menschen und in Tieren, die zeigen , dass die meisten autologe Tumorzellen - Zytotoxizität durch zirkulierende oder splenic Leukozyten 1,2 resistent sind. Diese Inkonsistenz kann zurückgeführt werden, zumindest teilweise auf die in vivo Existenz einer deutlichen Leukozytensubpopulation, nämlich die marginating-hepatische (Sinusoide) Leukozyten und deren Subpopulation von aktivierten NK - Zellen, nämlich pit Zellen 3. Tatsächlich unpublizierte Daten aus unserem Labor zeigten , daß in F344 Ratten syngene Tumorzellen (MADB106), das Zirkulieren und splenic Leukozyten resistent gefunden, 4 durch MH-NK Zellen lysiert wurden. Somit Tumorzellen, die angeblich &quot;NK-resistant&quot; auf zirkulierende Leukozyten sind, können durch MH-NK-Zellen gesteuert werden. Bemerkenswert ist die verstärkte Aktivität von MH-NK bei Mäusen in vivo nach nur offensichtlich immunStimulation (zB durch die Verwendung von Poly I: C oder CpG-C) 5. Leberspezifischen NK-Zellen (MH-NK) innerhalb der sinusförmigen Lumens befindet, an die Endothelzellen und Kupffer-Zellen anhaften. MH-NK - Zellen werden durch sphärische dichten Granula und stäbchen entkernt Vesikeln 6, die enthalten saure Phosphatase als lysosomale Enzyme und Perforin und Granzyme als bioaktive Substanzen 7,8 ausschließlich aus. Verglichen mit NK - Zellen zirkulierende weisen MH-NK - Zellen eine höhere Anzahl und Größe der Granula und Vesikel 9-11. Unter entzündlichen Bedingungen wurden MH-NK - Zellen gezeigt höhere Expression von LFA-1 12 aufweisen, im Vergleich zu den NK - Zellen zirkulieren. Diese erhöhte Expression könnte einen Mechanismus dar , mit dem MH-NK - Zellen mehr zytotoxischen gegen bestimmte Tumorzellen sind als NK - Zellen 13,14 zirkuliert. nterestingly nach in vitro - Inkubation mit Interleukin (IL) -2, werden MH-NK - Zellen vergrößert undihre Anzahl und Größe der Körner zu erhöhen, die alle mit einem Pro fi l von Lymphokin-aktivierten Killerzellen (LAK) 15 konsistent sind. Aktive MH Leukozyten kann nicht ausschließlich durch die Standard Leber Leukozyten Ernteverfahren erhalten werden, die auf Schleif- und biologischen Abbau des Gewebes beruhen. Unser Perfusion Ansatz hier beschriebenen hat zwei wesentliche Vorteile im Vergleich zu den Standard-Ansätzen. Erstens, erntet die Perfusion Ansatz selektiv MH Leukozyten, Verunreinigungen durch andere Leukozyten aus anderen Leberfächer zu verhindern. Zweitens behält die Perfusionstechnik besser, die Integrität, die Aktivität und die physiologischen Milieu MH Leukozyten, im Gegensatz zu der Gewebeverarbeitung dass Zellen schädigen nähert oder ihre Morphologie zu verändern, und aufgrund von Gewebeschäden induzieren die Freisetzung von verschiedenen Faktoren, die deutlich immune modulieren Aktivität. Die Leber ist ein wichtiges Zielorgan für die Krebsmetastasierung and für verschiedene Infektionen 16. Als MH-Zellen einzigartige Eigenschaften aufweisen, ist es wichtig, diese spezifische Population unter verschiedenen Bedingungen in bezug auf diese Pathologien zu studieren. Zum Beispiel ist es angemessen , dass die systemische Immunaktivierung durch verschiedene BRMs (zB Poly I: C oder CpG-C) zu beachten sind MH-Leukozyten gezeigt mehr zu aktivieren , als Leukozyten 5 zirkuliert.

<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 Ratten verglichen wir die Zytotoxizität von NK-MH-Zellen (aus der Leber Sinusoide durch erzwungene Leberperfusion gesammelt) auf die Zytotoxizität der gesamten Leberzellpopulation folgende mechanische Mahlung des Lebergewebes und auf die Zytotoxizität von Leukozyten zirkulieren. Alle Zellpräparationen wurden mindestens 3 Mal, als Routine, die in immunologischen Assays und als Zielzelllinien verwendeten wir die allogenen YAC-1 oder die syngenen MADB106 Zielzelllinien gewaschen...

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.

Selektive Ernte von Marginating-Leber-Leukozyten

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

5.7K Aufrufe.  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.

Serie: 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.

Eine einfache und effiziente Methode, um Makrophagen aus Mixed primären Kulturen von adulten Leber Zellen zu isolieren

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

Forschung

Immunologie und Infektion

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.

Isolierung und Anreicherung von Progenitor-Subsets Leber identifiziert durch eine neuartige Oberflächenmarkerkombination

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

Entwicklungsbiologie

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.

Selektive Ernte von Marginating-Lungen-Leukozyten

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.

Verdauung der murinen Leber für eine durchflusszytometrischen Analyse der lymphatische Endothelial Zellen

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.

Protokoll für die Isolierung von primären humanen Hepatozyten und entsprechende Hauptpopulationen von Nicht-parenchymale Leberzellen

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

Biologie

Etablierung von In-vitro-LLEC-Kulturen mittels magnetischer Zellselektion

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.

Autor im Rampenlicht: Enthüllung zellulärer Funktionen und potenzieller klinischer Implikationen von leptomeningealen lymphatischen Endothelzellen 

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.

Gewinnung und Primärkultur von leptomeningealen lymphatischen Endothelzellen

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

Neurowissenschaften

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.

Isolierung regenerierender Hepatozyten nach partieller Hepatektomie bei Mäusen

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

Medizin

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.

Isolierung und Charakterisierung von primären Leber-Sinus-Endothelzellen der Maus

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

Selektive Ernte von Marginating-Leber-Leukozyten

Zusammenfassung

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Gewinnung und Primärkultur von leptomeningealen lymphatischen Endothelzellen

Isolierung regenerierender Hepatozyten nach partieller Hepatektomie bei Mäusen

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