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).

倫理文：動物を対象とする手順は、テルアビブ大学の施設内動物管理使用委員会（IACUC）によって承認されています。 1.ラットプロトコル<ol><li>準備<ol><li>ヘパリン化PBS（30単位/ ml）溶液を調製し、リン酸1ml当たり防腐剤フリーのヘパリン30単位を添加することにより、室温（RT）で使用されるように、生理食塩水（PBS）1×緩衝溶液。動物当たり35ミリリットルを計算します。 </li><li>気泡を除去するために蠕動ポンプラインと蝶の針を介して、ヘパリン化PBSを渡します。 </li><li> （重力（ドライ）設定で少なくとも30分間、121℃でオートクレーブ）はさみの2ペア、平滑化エッジの鉗子、2止血、および歯組織鉗子 - 手術器具を滅菌します。 </li></ol></li><li> MH-白血球の肝臓とコレクションの灌流<ol><li> 8％のイソフルランの過剰投与により動物を安楽死させます。念のため、イソフルランを含む50 mlチューブを配置その胸腔を開くまで、ラットの頭の周り-soakedパッド。 </li><li>呼吸の停止直後に肝臓や心肺複合体を露出させるために腹膜と胸の空洞を開きます。このプロセスを通じて出血は避けてください。 <ol><li>具体的には、内臓に損傷を与えることなく、下腹部正中ポイントで切開を開始し、斜めリブに向かって両側に進展、上胸部キャビティレベルにそれらを切断します。 </li></ol></li><li>約分以内に、注射器に右心室から（250グラムの動物から〜6ミリリットル）できるだけ多くの血液を採取。 </li><li>灌流液の収集を可能にするために、心臓にできるだけ近い止血剤と尾部大静脈をクランプします。 </li><li>門脈を露出させるために、実験者の権利に動物の外腸と場所を引き出します。 </li><li> 、できるだけ尾側として、門脈に、蠕動ポンプに接続された25 G IVカテーテルを挿入しかし、脾静脈の吻側。 </li><li>止血剤の尾、下大静脈に、5ミリリットルの注射器に接続された25 Gの翼状針を挿入します。 </li><li>約3ミリリットル/分の速度で蠕動ポンプの電源を入れ、ゆっくりとシリンジに血液で汚染された灌流液の最初のミリリットルを集めます。灌流液の色はターンが赤色（約3〜5 ml）を淡いなるまで続けます。肝臓の色は淡褐色に向かって変化していることに注意してください。 </li><li>蠕動ポンプを停止させることなく、20ミリリットル収穫シリンジで5ミリリットルの収穫シリンジを交換し、灌流の高速（最大4 ml /分）を用いた肝灌流液の少なくとも20ミリリットルを集めます。継続的に収集シリンジに灌流液の流れを監視し、（大静脈壁を崩壊を通して針を詰まらせる可能性がある）強すぎる真空を避けます。 </li><li>肝臓の色は淡褐色になると灌流を終了します。 </li></ol></li><li>白血球の抽出<oL&gt; <li> 、24 400×gで10分間の灌流液を遠心 °C。 </li><li>上清を吸引</li><li> 10 mlのPBS、10分間、400×gで遠心分離器を追加し、上清を吸引除去します。研究の目標に基づいて、あるとして準備を使用するか、または追加の精製（ 例えば 、密度勾配分離）を実施しています。 <ol><li>具体的には、層50 ccのポリプロピレン遠心分離管中の密度勾配分離の質量の4ミリリットルを超える灌流液の4ミリリットル。ブレークオフで30分間、754×gで、室温でチューブをスピン。 </li><li> 、手動ピペッティングにより密度勾配分離上清界面での単核層を得るPBSで洗浄し、5ミリリットルチューブに挿入します。 </li></ol></li></ol></li></ol> 2.マウスプロトコル<ol><li>準備<ol><li>調製ヘパリン化PBS（30単位/ ml）リン酸1ml当たり防腐剤フリーのヘパリン30単位を添加することにより、室温（RT）で使用する溶液は、緩衝生理食塩水（PBS）1X solution個。動物当たり20ミリリットルを計算します。 </li><li>気泡を除去するために蠕動ポンプラインと蝶の針を介して、ヘパリン化PBSを渡します。 </li><li> （重力（ドライ）設定で少なくとも30分間、121℃でオートクレーブ）はさみの2ペア、平滑化エッジの鉗子、2止血、および歯組織鉗子 - 手術器具を滅菌します。 </li></ol></li><li> MH-白血球の肝臓とコレクションの灌流<ol><li> 8％のイソフルランの過剰投与により動物を安楽死させます。念のため、その胸腔を開くまで、マウスの頭の周りイソフルランに浸したパッドを含む15mlチューブを配置します。 </li><li>ベッドにマウスの四肢を修正しました。 </li><li>無傷の横隔膜の下側面を維持し、肝臓や呼吸の停止時にすぐに心肺複合体を露出させるために腹膜と胸の空洞を開きます（排水胸腔プールを作成します）。このプロセスを通じて出血は避けてください。 <ol><li>具体的には、下で切開を開始腹部正中内臓に損傷を与えることなく、ポイント、および斜めリブに向かって両側に進展、上胸部キャビティレベルにそれらを切断します。 </li></ol></li><li>門脈を露出させるために、実験者の権利に動物の外腸と場所を撤退。 </li><li>脾静脈への門脈吻側に蠕動ポンプに接続された30グラムの針を挿入します。 </li><li>胸腔からの灌流液の排液および回収を可能にするために、振動板の上に下大静脈をカット。 </li><li>約3ミリリットル/分の速度で蠕動ポンプの電源を入れ、軽く胸腔プールからの血液で汚染された灌流液の最初の3ミリリットルを集めます。 </li><li>この灌流液を捨てます。必要に応じて生理食塩水を使用することにより、再びこのような洗浄を繰り返し、その後にのみ肝灌流液の収集を開始。 </li><li>再起動するまで4ミリリットル/分の速度で灌流し、そして使用して胸腔から灌流液の10ミリリットルを集めます注射器に接続されている蝶。 </li><li>肝臓の色は淡褐色になると灌流を終了します。 </li></ol></li><li>白血球の抽出<ol><li>遠心分離機400×gで10分間の灌流液。 </li><li>上清を吸引除去します。 </li><li> 10 mlのPBS、10分間、400×gで遠心分離器を追加し、学習目標に基づいて上清を吸引、あるとして準備を使用するか、または追加の精製（ 例えば 、密度勾配分離）を実施しています。 <ol><li>具体的には、層50 ccのポリプロピレン遠心分離管中の密度勾配分離の質量の4ミリリットルを超える灌流液のすべての4ミリリットル。ブレークオフで30分間、754×gで、室温でチューブをスピン。 </li><li> 、手動ピペッティングにより密度勾配分離上清界面での単核層を得るPBSで洗浄し、5ミリリットルチューブに挿入します。 </li></ol></li></ol></li></ol>

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本明細書に提示肝灌流法は、肝白血球をmarginatingのユニークな人口の選択的ハーベスティングと勉強を可能にします。またピットセル3と呼ばれる肝臓のNK細胞は、肝臓の類洞に存在する独特のNK細胞集団を構成しています。それらは、ラット、マウス17およびヒト18,19に見出されます。単離された末梢NK細胞と比較して、ピット細胞は、LFA-1の12のように、より小...

肝臓の正弦波は、生物にとって重要な種々の免疫活動の多数の白血球サブタイプが含まれています。例えば、また、ピット細胞として知られている肝をmarginating（MH）ナチュラルキラー（NK）細胞は、特徴づけされているとして形態学的に大顆粒リンパ球（LGLS）と機能的に血液の確立に対する肝臓の抵抗を可能に自発的な細胞傷害能を有する白血球、など負担腫瘍転移。本明細書に提示された方法の目標は、この重要かつユニークな細胞集団（および免疫コンパートメント）を研究するため、およびこれらの特定の細胞上の各種操作（ 例えば免疫活性化）の影響を解明するために、MHの白血球の選択的ハーベスティングを可能にすることです。 現像原発腫瘍を除去する免疫不全にもかかわらず、癌患者および動物モデルにおける証拠は、免疫系が腫瘍細胞、微小転移、およびthrou残存疾患、循環制御できることを示していますGH細胞性免疫（CMI）。しかし、これらのインビボでの機能の間の証明ヒトおよび動物のin vitro研究で明らかな矛盾があるほとんどの自己腫瘍細胞は、1,2-循環または脾臓白血球による細胞毒性に対して耐性があること。この矛盾は、別個の白血球亜集団、すなわちmarginating、肝臓（正弦波）白血球および活性化NK細胞、すなわちピットセル3のそれらの亜集団のin vivoで存在する、少なくとも部分的に帰することができます。実際、我々の研究室からの未発表データは、F344ラットに同系腫瘍細胞循環および脾臓白血球に耐性が認められた（MADB106）は、MH-NK細胞4によって溶解させたことを示しました。したがって、循環白血球に伝えられるところでは、「NK抵抗性」である腫瘍細胞をMH-NK細胞によって制御されてもよいです。注目すべきは、マウスにおいて、MH-NK活性の増強は、免疫in vivoで次明らかです刺激（ 例えば 、ポリIの使用により：CまたはCpG-C）5。 肝臓特異的NK細胞（MH-NK）は、内皮細胞およびクッパー細胞に付着し、正弦波の内腔の内側に位置しています。 MH-NK細胞はもっぱら生理活性物質7,8としてリソソーム酵素、およびパーフォリン及びグランザイムなどの酸性ホスファターゼを含む球状密顆粒およびロッド芯小胞6、によって特徴付けられます。 NK細胞の循環と比較して、MH-NK細胞は、顆粒及び小胞9-11のより高い数と大きさを示します。 NK細胞の循環と比較して、炎症状態下で、MH-NK細胞は、LFA-1 12のより高い発現を示すことが示されました。この増強された発現は、MH-NK細胞はNK細胞13,14を循環するよりも、特定の腫瘍細胞に対してより細胞傷害性であることにより、機構を構成している可能性があります。 nterestingly、インターロイキン（IL）-2 でビトロインキュベーション後、MH-NK細胞が肥大、およびプロFiのルリンホカイ ​​ン活性化キラー（LAK）細胞15のと一致しているすべてのそれらの顆粒の増加の彼らの数とサイズ、。 アクティブMH白血球は、排他的に粉砕し、組織の生物学的分解に基づいており、標準的な肝白血球の収穫方法、を介して取得することはできません。本明細書中に記載の当社の灌流アプローチは、標準的なアプローチと比較して、2つの大きな利点があります。まず、灌流アプローチは、選択的に他の肝臓の区画から他の白血球による汚染を防止し、MHの白血球を収穫します。第二に、灌流技術は、より良い組織処理は、その損傷細胞に接近またはその形態を変化させるとは異なり、完全性、活性、およびMH白血球の生理的な環境を維持し、組織の損傷に起因する、著しく免疫調節種々の因子の放出を誘発しますアクティビティ。 肝臓は、癌転移aの主要な標的器官でありますさまざまな感染症の16回目。 MH電池は、ユニークな特徴を示すように、これらの病変に対して様々な条件の下で、この特定の集団を研究することが重要です。例えば、それは様々なのBRMによって、その全身の免疫活性化に注意することは価値がある（ 例えば、ポリI：CまたはCpG-C）は、白血球5を循環させるよりもMH-白血球より多くを活性化することが示されています。

<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ラットでは、肝組織の機械的粉砕以下全体の肝細胞集団の細胞毒性に（強制的な肝臓灌流による肝臓類洞から収集）MH-NK細胞の細胞毒性を比較して、循環白血球の細胞毒性です。すべての細胞調製物は、免疫学的アッセイにおけるルーチンとして、少なくとも3回洗浄し、そして標的細胞株として、我々は、同種異系YAC-1または同系MADB106標的細胞株を使用しました?...

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-肝白血球の選択的ハーベスティング

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 ビュー.  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.

ビデオ: 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.

成人肝細胞の混合初代培養からマクロファージを分離するためにシンプルかつ効率的な方法

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

免疫・感染学

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.

新規の表面マーカーの組み合わせで識別離および肝前駆サブセットの濃縮

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

発生生物学

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肺白血球の選択的ハーベスティング

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.

リンパ管内皮細胞のフローサイトのマウス肝臓の消化

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.

初代ヒト肝細胞と非実質肝細胞の対応する主な集団の単離のためのプロトコル

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

生物学

磁気細胞選択法を用いたIn vitro RHEC培養法の樹立

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.

著者スポットライト:軟髄膜リンパ内皮細胞の細胞機能と潜在的な臨床的意味を明らかにする 

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.

軟髄膜リンパ管内皮細胞の採取と初代培養

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

神経科学

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.

マウスにおける肝部分切除後の再生肝細胞の単離

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

医学

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.

マウス初代肝臓正弦波内皮細胞の単離と特性評価

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

Marginating-肝白血球の選択的ハーベスティング

要約

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成人肝細胞の混合初代培養からマクロファージを分離するためにシンプルかつ効率的な方法

新規の表面マーカーの組み合わせで識別離および肝前駆サブセットの濃縮

Marginating肺白血球の選択的ハーベスティング

リンパ管内皮細胞のフローサイトのマウス肝臓の消化

初代ヒト肝細胞と非実質肝細胞の対応する主な集団の単離のためのプロトコル

軟髄膜リンパ管内皮細胞の採取と初代培養

軟髄膜リンパ管内皮細胞の採取と初代培養

軟髄膜リンパ管内皮細胞の採取と初代培養

マウスにおける肝部分切除後の再生肝細胞の単離

マウス初代肝臓正弦波内皮細胞の単離と特性評価