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

Etik Beyanı: Hayvan denekleri Prosedürleri Tel-Aviv Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. 1. Sıçanlar Protokol <ol><li> hazırlıklar <ol><li> heparinize PBS (30 birim / ml) çözeltisi hazırlayın fosfat ml&#39;si başına koruyucu serbest heparinin 30 adet ekleyerek, oda sıcaklığında (RT) kullanılmak üzere tuzlu su (PBS) ile 1 x çözeltisi tamponlu. hayvan başına 35 ml hesaplayın. </li><li> Hava kabarcıklarını yok etmek için peristaltik pompa hatları ve kelebek iğne yoluyla heparinize PBS geçirin. </li><li> (Yerçekimi (kuru) ayarında en az 30 dakika süreyle 121 ° C&#39;de otoklav) makas 2 çift, körelmiş kenarlı forseps, 2 hemostat ve diş doku forseps - cerrahi aletler sterilize edin. </li></ol></li><li> MH-lökositlerin Karaciğer ve Toplama perfüzyon <ol><li> % 8 izofluran aşırı dozda ile hayvan Euthanize. Önlem olarak, bir izofluran içeren 50 ml&#39;lik bir tüp yerleştirmekonun göğüs boşluğunu açarak kadar sıçan başının etrafında -soaked ped. </li><li> Solunumun durması üzerine hemen karaciğer ve kardiyopulmoner kompleksi ortaya çıkarmak için periton ve göğüs boşlukları açın. Bu süreçte kanama kaçının. <ol><li> Özellikle, üst göğüs boşluğu seviyelere içlerinden kesme, kaburga doğru çapraz her iki taraf için zarar iç organlara olmadan alt karın orta hat noktasında kesi, ve ilerleme başlayın. </li></ol></li><li> yaklaşık dakika içinde, (~ 250 g hayvan 6 mi) mümkün olduğu kadar kan toplamak bir şırınga içine sağ ventrikül. </li><li> perfüzat toplanmasını sağlamak için, kalp mümkün olduğunca yakın bir hemostatla kaudal vena kava kelepçe. </li><li> portal ven ortaya çıkarmak için, deneycinin sağa hayvan dışında bağırsak ve yeri çekin. </li><li> mümkün olduğunca kaudal olarak, portal ven içine peristaltik pompaya bağlı 25 G IV kateter, insert,ancak dalak ven rostralinde. </li><li> Hemostata kaudal vena kava inferior içine 5 ml şırınga bağlı 25 G kelebek iğne,, yerleştirin. </li><li> yaklaşık olarak 3 ml / dakika bir hızda peristaltik pompa açın ve yavaşça şırıngaya kan ile kirlenmiş olan perfüzat ilk ml toplar. perfüzat renk dönüşler (yaklaşık 3-5 ml) soluk kırmızı olana kadar devam edin. Karaciğer rengi açık kahverengi doğru değişiyor dikkat edin. </li><li> peristaltik pompa durmadan 20 mi hasat şırınga ile 5 mi hasat şırınga yerine ve perfüzyon daha yüksek bir hızda (kadar 4 mL / dak) kullanılarak karaciğer perfüzat en az 20 ml toplar. Sürekli toplama şırıngaya perfüzyon akışını izlemek, ve (vena kava duvarları çöken yoluyla iğne tıkayabilir olan) çok güçlü vakum kaçının. </li><li> Karaciğer rengi açık kahverengi döndüğünde perfüzyon sonlandırın. </li></ol></li><li> lökositler Ekstraksiyon <ol&gt; <li> 24 400 x g&#39;de 10 dakika boyunca santrifüj perfüzat ° C. </li><li> süpernatantı aspire </li><li> 10 dakika boyunca 400 x g&#39;de 10 ml PBS, santrifüj ekleyin ve süpernatant aspire. Çalışma hedeflerinize dayalı olarak, olduğu gibi hazırlık kullanın veya ek saflaştırma (örneğin, yoğunluk-gradyan ayırma) yürütmek. <ol><li> Spesifik olarak, tabaka, 50 cc&#39;lik bir polipropilen santrifüj tüpüne yoğunluk gradyanlı ayrıştırma kütlesinin 4 ml üzerinde perfüzat 4 mi. Devre dışı ile 30 dakika boyunca, 754 x g&#39;de oda sıcaklığında tüpler dönerler. </li><li> Manuel pipetleme yoğunluk-gradyan ayırma-süpernatant arayüzünde tek çekirdekli katmanları elde PBS içinde yıkayın ve 5 ml&#39;lik bir tüp içine yerleştirin. </li></ol></li></ol></li></ol> 2. Fare Protokolü <ol><li> hazırlıklar <ol><li> Fosfat ml&#39;si başına koruyucu serbest heparinin 30 adet ekleyerek, oda sıcaklığında (RT) kullanılmak üzere, heparinize PBS (30 birim / ml) çözeltisi hazırlayın tuz (PBS) 1 x eriyik tamponlun. hayvan başına 20 ml hesaplayın. </li><li> Hava kabarcıklarını yok etmek için peristaltik pompa hatları ve kelebek iğne yoluyla heparinize PBS geçirin. </li><li> (Yerçekimi (kuru) ayarında en az 30 dakika süreyle 121 ° C&#39;de otoklav) makas 2 çift, körelmiş kenarlı forseps, 2 hemostat ve diş doku forseps - cerrahi aletler sterilize edin. </li></ol></li><li> MH-lökosit Karaciğer ve Toplama perfüzyon <ol><li> % 8 izofluran aşırı dozda ile hayvan Euthanize. Önlem olarak, onun göğüs boşluğunu açarak kadar fare başının etrafında bir izofluran batırılmış pedi içeren 15 ml tüp yerleştirin. </li><li> Bir yatağa fare uzuvlarını sabitleyin. </li><li> bozulmamış diyaframın alt yönü tutarak, karaciğer ve solunum kesilmesi üzerine hemen kardiyopulmoner kompleksi ortaya çıkarmak için periton ve göğüs boşlukları açın (bir boşaltma göğüs boşluğu havuzu oluşturmak için). Bu süreçte kanama kaçının. <ol><li> Özellikle, düşük kesi başlatmakÜst göğüs boşluğu seviyelere içlerinden kesme çapraz kaburga doğru her iki taraf için karın orta hat zarar iç organları olmadan nokta, ve ilerleme. </li></ol></li><li> portal ven ortaya çıkarmak için, deneycinin sağa hayvan dışında bağırsak ve yeri çekilme. </li><li> dalak damara portal ven rostral bir peristaltik pompaya bağlı bir 30 g iğne, yerleştirin. </li><li> göğüs boşluğundan perfüzat drenaj ve toplama izin diyafram yukarıda inferior vena kava kesti. </li><li> yaklaşık olarak 3 ml / dakika bir hızda peristaltik pompa açın ve yavaşça göğüs boşluğu havuzundan kan ile kirlenmiş olan perfüzat ilk 3 ml toplar. </li><li> Bu perfüzat atın. Gerekirse tuz kullanılarak, yine böyle bir yıkama tekrarlayın ve ancak ondan sonra karaciğer perfüzyon toplamaya başlar. </li><li> Yeniden başlatma kadar ml / dakika ila 4 arasında bir hızla perfüzyon ve kullanılarak göğüs boşluğundan perfüzat 10 ml toplarbir şırıngaya bağlanmış bir kelebek. </li><li> Karaciğer rengi açık kahverengi döndüğünde perfüzyon sonlandırın. </li></ol></li><li> lökositler Ekstraksiyon <ol><li> Santrifüj 400 x g&#39;de 10 dakika boyunca perfüze. </li><li> süpernatant aspire. </li><li> 10 dakika boyunca 400 x g&#39;de 10 ml PBS, santrifüj ekleyin ve çalışma hedefleri dayanarak süpernatant aspire, olduğu gibi hazırlık kullanın veya ek saflaştırma (örneğin, yoğunluk-gradyan ayırma) yürütmek. <ol><li> Spesifik olarak, tabaka, 50 cc&#39;lik bir polipropilen santrifüj tüpüne yoğunluk gradyanlı ayrıştırma kütlesinin 4 ml üzerinde perfüzat her 4 mi. Devre dışı ile 30 dakika boyunca, 754 x g&#39;de oda sıcaklığında tüpler dönerler. </li><li> Manuel pipetleme yoğunluk-gradyan ayırma-süpernatant arayüzünde tek çekirdekli katmanları elde PBS içinde yıkayın ve 5 ml&#39;lik bir tüp içine yerleştirin. </li></ol></li></ol></li></ol>

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Yazarlar ifşa hiçbir şey yok.

Burada sunulan karaciğer perfüzyon yöntemi seçici bir hasat sağlar ve hepatik lökosit marginating benzersiz nüfusun incelenmesi. Ayrıca çukur hücreleri 3 olarak adlandırılan Karaciğer NK hücreleri, hepatik sinüzoitlerde ikamet farklı bir NK hücre popülasyonunu oluşturmaktadır. Bunlar, sıçanlar, fareler 17 ve insanlarda 18,19 bulunmaktadır. Izole edilmiş periferal NK hücreleri ile karşılaştırıldığında, çukur hücreleri küçük sitoplazmik granüllerin <s...

karaciğer sinüsoidlerinden organizma için önemli çeşitli immün faaliyetleri pek çok lökosit alt tipleri içerir. Örneğin, aynı zamanda çukur hücreleri olarak bilinen hepatik marginating (MH), doğal katil (NK) hücreler, özelliği, morfolojik olarak büyük granüler limfositler (LGLs) ve fonksiyonel olarak kan-kurulmasına karşı hepatik-direnç sağlayan kendiliğinden sitotoksik kapasiteli lökositler gibi kaynaklı tümör metastazları. Burada sunulan yöntemin amacı bu önemli ve eşsiz hücre popülasyonu (ve bağışıklık bölmesi) araştırmak için MH lökositlerin selektif hasat, etkinleştirmek için, ve bu spesifik hücreler üzerinde çeşitli manipülasyonlar etkisini (örn bağışıklık aktivasyon) aydınlatmak etmektir. Gelişmekte olan bir primer tümör ortadan kaldırmak için bağışıklık yetersizliği rağmen, kanser hastaları ve hayvan modellerinde kanıtlar bağışıklık sistemi tümör hücrelerini, mikro-metastazı ve Throu rezidüel hastalık sirkülasyon kontrol edilebileceğini gösterirgh hücre aracılı immünite (CMI). Bununla birlikte, in vivo özellikleri ve insanlarda in vitro çalışmalar, en otolog tümör hücrelerinin, dolaşımdaki veya dalak lökositleri 1,2 ile sitotoksisiteye karşı olduğunu göstermek hayvanlar, bu arasında açık bir tutarsızlık yoktur. Bu tutarsızlık, bir tat lökosit alt popülasyonuna, yani marginating-hepatik (sinüzoidler) lökosit ve aktive NK hücreleri, yani çukur hücre 3 kendi alt popülasyonunda in vivo varlığı, en azından kısmen, bağlı olabilir. Gerçekten de, laboratuar yayınlanmamış veriler F344 sıçanlarda singeneik dolaşan tümör hücrelerinin ve dalak lökositleri dirençli bulunmuştur (MADB106), MH-NK hücrelerinin 4 çözülmüştür göstermiştir. Bu nedenle, dolaşımdaki lökositlerin iddia edilen &quot;NK dirençli&quot; olarak tümör hücrelerinin MH NK hücreleri tarafından kontrol edilebilir. Dikkate değer olarak, farelerde MH-NK geliştirilmiş etkinliği sadece bağışıklık vivo aşağıdaki belirgindir(poli I kullanılarak, örneğin: Cı veya CpG-C) uyarma 5. Karaciğer spesifik NK hücrelerinin (MH-NK) endotel hücreleri ve Kupffer hücreleri bağlı kalarak, sinüzoidal lümen içinde yer almaktadır. MH-NK hücreleri, sadece biyolojik olarak aktif madde 7,8 olarak lizozomal enzimler ve perforin ve granzymes gibi asit fosfataz içeren küresel yoğun granüller ve çubuk özlü veziküllerin 6, ile karakterize edilir. NK hücrelerini dolaşımdaki ile karşılaştırıldığında, MH-NK hücreleri granüller ve veziküller 9-11 daha yüksek bir sayı ve boyut sergilerler. NK hücrelerini dolaşım ile karşılaştırıldığında, inflamatuar koşullar altında, MH-NK hücreleri, LFA-1 12 daha yüksek ifadesini gösterir gösterildi. Bu geliştirilmiş ekspresyon MH-NK hücreleri, NK hücreleri 13,14 dolaşımdaki daha bazı tümör hücrelerine karşı daha fazla sitotoksik olan bir mekanizma oluşturabilir. nterestingly, interlökin (IL) -2 ile in vitro inkübasyondan sonra MH-NK hücrelerinin büyütülmüş hale gelir vebunların sayısı ve granül boyutu pro fi lenfokin ile aktive edilen katil (LAK) hücreler, 15 ile uyumlu olan, her biri, artar. Aktif MH lökositler özel öğütme ve dokunun biyolojik bozunma göre standart karaciğer lökosit hasat yöntemleri ile elde edilemez. Burada tarif edilen Bizim perfüzyon yaklaşım standart yöntemlere göre iki önemli avantaja sahiptir. İlk olarak, perfüzyon yaklaşımı seçici diğer karaciğer bölmeleri diğer lökositler tarafından kirlenmeyi önleyerek, MH lökositleri hasadın. Doku işleme hasar hücreler yaklaşımların ya da morfolojisi değiştirmek ve doku hasarına bağlı, belirgin bağışıklık modüle çeşitli faktörlerin salınımını teşvik aksine İkincisi, daha iyi perfüzyon tekniği, bütünlük, aktivite ve MH lökositlerin fizyolojik ortamını korur aktivite. karaciğer kanseri metastazı a için önemli bir hedef organdırnd çeşitli enfeksiyonlara 16. MH hücreleri benzersiz özelliklere sergilerler olarak patolojiler açısından çeşitli koşullar altında bu özel nüfus incelemek önemlidir. Örneğin, çeşitli BRM göre sistemik immün aktivasyon dikkat çekicidir (örneğin poli I: C veya CpG-C) lökosit 5 dolaşımdaki daha MH-lökositler fazla aktif olduğu gösterilmiştir.

<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.

F344 sıçanlarda karaciğer dokusunun mekanik taşlama aşağıdaki tüm karaciğer hücre popülasyonunun sitotoksisite (zorla karaciğer perfüzyon ile karaciğer sinüsoidlerinden toplanan) MH-NK hücrelerinin sitotoksisitesini karşılaştırıldı ve lökosit dolaşan sitotoksisite. Tüm hücre preparatları imünolojik deneylerde rutin olarak, en az 3 kez yıkandı ve hedef hücre çizgileri gibi allojenik YAC-1 veya singeneik MADB106 hedef hücre çizgileri kullanılır. ...

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-hepatik lökosit seçici Hasat

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

Research

JoVE Journal

Immunology and Infection

5.7K Görüntüleme.  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.

Yetişkin Karaciğer Hücrelerinin Karma İlköğretim Kültürler Makrofajlar yalıtın Basit ve Etkin Yöntem

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

İmmünoloji ve Enfeksiyon

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.

İzolasyon ve Roman Yüzey Etiket Kombinasyon ile tanımlanır Karaciğer progenitör Alt Grupları zenginleştirilmesi

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

Gelişim Biyolojisi

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 pulmoner lökosit seçici Hasat

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.

Lenfatik endotel hücreleri akış sitometrik çözümlenmesi için fare karaciğer sindirim

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.

İlköğretim İnsan Hepatositlerin İzolasyonu ve Non-parankimal karaciğer hücrelerinin Binbaşı popülasyonları İlgili Protokolü

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

Biyoloji

Manyetik Hücre Seçimi Kullanılarak İn Vitro LLEC Kültürlerinin Oluşturulması

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.

Yazar Gündemi: Leptomeningeal Lenfatik Endotel Hücrelerinin Hücresel Fonksiyonlarının ve Potansiyel Klinik Etkilerinin Ortaya Çıkarılması 

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 Lenfatik Endotel Hücrelerinin Hasadı ve Primer Kültürü

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

Nörobilim

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.

Farelerde Parsiyel Hepatektomi Sonrası Rejenere Hepatositlerin İzolasyonu

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

Tıp

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.

Fare primer karaciğer sinüzoidal endotel hücrelerinin izolasyonu ve karakterizasyonu

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

Marginating-hepatik lökosit seçici Hasat

Özet

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Fare primer karaciğer sinüzoidal endotel hücrelerinin izolasyonu ve karakterizasyonu