Name: isolation and characterization of mouse primary liver sinusoidal endothelial cells
Uploaded: 2021-12-16T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells at JoVE.com

这项工作得到了NIH国家糖尿病，消化和肾脏疾病研究所（1RO1DK122948至SHI）和NIH Silvio O. Conte消化系统疾病研究核心中心P30资助机制（DK084567）的支持。日本科学促进会（JSPS）海外研究奖学金也向钦哲基金会提供了支持。我们还要感谢Gregory J. Gores博士和Steven Bronk博士对胶原酶灌注装置的原创设计和优化。

动物协议是经梅奥诊所的机构动物护理和使用委员会（IACUC）批准的。从杰克逊实验室购买了八周龄的C57BL / 6J雄性小鼠。将小鼠饲养在温度控制的12：12-h明暗循环设施中，并可自由获得饮食。
1.胶原包被培养皿或培养盘的制备
<ol><li>要制成50 mL 0.02 mol/L乙酸，在49.4 mL H2O中加入0.6 mL冰醋酸。</li>
	<li>在0.02摩尔/升乙酸中制作50μg/ mL I型胶原蛋白。稀释取决于...

<ol>
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H., Bronk, S. F., Yagita, H., Gores, G. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+liver+sinusoidal+endothelial+cell+phenotype+is+maintained+by+paracrine+and+autocrine+regulation.">Rat liver sinusoidal endothelial cell phenotype is maintained by paracrine and autocrine regulation.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 287 (4), 757-763 (2004).</li><li>Cabral, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+hepatocytes+and+sinusoidal+endothelial+cells+from+mouse+liver+perfusion.">Purification of hepatocytes and sinusoidal endothelial cells from mouse liver perfusion.</a> Journal of Visualized Experiments: JoVE. (132), e56993 (2018).</li><li>Ishiguro, K., Yan, I. K., Patel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+tissue+extracellular+vesicles+from+the+liver.">Isolation of tissue extracellular vesicles from the liver.</a> Journal of Visualized Experiments: JoVE. (150), e58649 (2019).</li><li>Topp, S. A., Upadhya, G. A., Strasberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+preservation+of+isolated+sinusoidal+endothelial+cells+in+MMP+9+knockout+mice:+effect+on+morphology+and+platelet+adhesion.">Cold preservation of isolated sinusoidal endothelial cells in MMP 9 knockout mice: effect on morphology and platelet adhesion.</a> Liver Transplantation. 10 (8), 1041-1048 (2004).</li><li>Katz, S. C., Pillarisetty, V. G., Bleier, J. I., Shah, A. B., DeMatteo, R. 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在目前的手稿中，我们描述了一种从小鼠肝脏中分离LSEC的方案，包括两步胶原酶灌注和随后的磁活化细胞分选（MACS）。该方案包括以下三个步骤：（1）用无钙缓冲液灌注PV，然后用含胶原酶的缓冲液实现肝细胞分散;（2）低速离心排除肝细胞;和（3）使用抗CD146磁珠从非实质细胞（NPC）中选择性基于MACS的LSECs阳性选择。整个过程可以在3小时内完成。此外，该程序的成本（包括所有耗材）约为每只?...

肝正弦内皮细胞（LSECs）排列在肝窦壁上，是肝脏中最丰富的非实质细胞1。LSECs与身体其他毛细血管内皮细胞的区别在于存在开窗和缺乏经典的基底膜或隔膜2，3。因此，LSECs具有独特的表型和结构特征，可增强其通透性和内吞能力，以消除各种循环大分子，包括脂质和脂蛋白。LSECs在实质细胞和非实质细胞（如星状细胞和免疫细胞）之间的串扰中起着关键作用。LSECs通过保持星状细胞和Kupffer细胞处于静止状态来维持肝脏稳态的关键4。LSECs通过介导循环白细胞的粘附和经内皮迁移来调节肝免疫细胞群的组成5，6。在急性和慢性肝损伤7 期间，包括缺血再灌注损伤 （IRI）8、非酒精性脂肪性肝炎 （NASH）9 和肝细胞癌 （HCC），LSECs 会发生表型改变，称为毛细血管形成，其特征为防御和基底膜10 形成。LSECs 的这些表型变化与 LSECs 功能障碍以及获得血栓前、促炎和促纤维化特性有关。
已经开发了几种从小鼠肝脏中分离LSECs的方法11。一些技术依赖于分离非实质和实质细胞，然后进行密度梯度离心以从非实质部分纯化LSECs。该方法的局限性在于LSECs分离的最后步骤中存在污染巨噬细胞，这可能会影响分离的LSECs12的纯度。该方案基于小鼠肝脏的胶原酶灌注和CD146 + 磁珠阳性选择非实质细胞以纯化LSECs。使用这种方法分离的LSECs显示出高纯度和保留的形态和活力。这些 LSEC 是功能研究的最佳选择，包括渗透性和粘附测定，以及目标途径的下游研究。此外，随着人们对在临床研究和发现科学中生成大数据集的兴趣日益浓厚，这些从患有非酒精性脂肪性肝炎（NASH）或其他疾病的健康和患病肝脏中分离出来的高质量LSECs可以合并或单独使用，从而可以生成多组学数据并比较健康与疾病13，14.此外，分离的LSECs可用于开发二维和三维体外模型，如类器官，以破译LSECs中激活的信号通路及其在不同有害刺激下与其他肝细胞的细胞间通讯，并响应各种治疗干预。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0-inch 20 G Intra Venous (IV) catheter</td>
			<td>Terumo, SOmerset, NJ, USA</td>
			<td>SR-OX2051CA</td>
			<td></td>
		</tr>
		<tr>
			<td>2&#8211;3-inch perfuion tray with a hole in the center</td>
			<td></td>
			<td></td>
			<td>customized; made in house</td>
		</tr>
		<tr>
			<td>405/520 viability dye</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-110-205</td>
			<td></td>
		</tr>
		<tr>
			<td>4-inch regular curved dressing forceps</td>
			<td>Fisher Brand</td>
			<td>FS16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Perma-Hand silk suture</td>
			<td>Ethicon, Raritan, NJ, USA</td>
			<td>A182H</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-stabilin-2 (Mouse) mAb-Alexa Fluor&#195; 488</td>
			<td>MBL International, Woburn, MA, USA</td>
			<td>D317-A48</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA stock</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD146 (LSEC)-PE, anti-mouse</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-118-407</td>
			<td></td>
		</tr>
		<tr>
			<td>CD146 (LSEC) MicroBeads, mouse</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-092-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD45-Viogreen, anti-mouse</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-110-803</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen type I</td>
			<td>Corning, Corning, NY, USA</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Gibco, Waltham, MA, USA</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cells growth medium</td>
			<td>ScienCell Research Laboratories, Carlsbad, CA, USA</td>
			<td>211-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR blocking reagent, mouse</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software, version 10.6</td>
			<td>Becton, Dickinson and Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened Fine scissors</td>
			<td>F.S.T, Foster city, CA, USA</td>
			<td>14091-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated (37 &#176;C) and humidified recirculating perfusion apparatus equipped with Oxygen injection at a rate of 10psi.</td>
			<td></td>
			<td></td>
			<td>customized; made in house</td>
		</tr>
		<tr>
			<td>Hitachi S 4700 scanning electron microscope</td>
			<td>Hitachi Inc, Pleasanton, CA, USA</td>
			<td>SEM096</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS pre-separation filters (70 &#956;m)</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-095-823</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS rinsing buffer</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Smart Strainer (70 &#956;m)</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td>130-098-462</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQunt flow cytometer</td>
			<td>Miltenyi, Bergisch Gladbach, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Insert</td>
			<td>Millipore Sigma, Burlington, MA, USA</td>
			<td>PITP01250</td>
			<td></td>
		</tr>
		<tr>
			<td>Nexcelom cell counter</td>
			<td>Nexcelom bioscience, Lawrence, MA, USA</td>
			<td>Cellometer Auto T4 Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare, Chicago, IL, USA</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>F.S.T, Foster city, CA, USA</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Very small curved dressing forceps</td>
			<td>F.S.T, Foster city, CA, USA</td>
			<td>11063-07</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of mouse primary liver sinusoidal endothelial cells

肝正弦内皮细胞（LSECs）是位于循环和肝实质之间界面的特化内皮细胞。LSECs具有独特的形态，其特征在于存在开窗和没有基底膜。LSECs在肝脏的许多病理性疾病中起着至关重要的作用，包括代谢失调，炎症，纤维化，血管生成和癌变。然而，关于LSEC的分离和表征的出版物很少。在这里，该协议讨论了从健康和非酒精性脂肪性肝病（NAFLD）小鼠中分离LSAC。该方案基于小鼠肝脏的胶原酶灌注和磁珠阳性选择非实质细胞以纯化LSECs。本研究通过流式细胞术使用特异性标记物表征LSECs，并通过扫描电子显微镜鉴定特征性表型特征。按照该协议分离的LSECs可用于功能研究，包括粘附和渗透性测定，以及特定目标途径的下游研究。此外，这些LSECs可以合并或单独使用，允许多组学数据生成，包括RNA-seq本体或单细胞，蛋白质组学或磷酸 - 蛋白质组学，以及使用测序（ATAC-seq）进行转座酶可及染色质的测定等。该方案对于研究LSECs与其他肝细胞在健康和疾病中的通讯的研究者非常有用，并允许深入了解LSECs在急性和慢性肝损伤致病机制中的作用。

实验示意图及设备设置： 在该方案中，使用封闭的灌注回路消化小鼠肝脏，然后通过以50× g 低速离心2分钟分离非实质细胞和肝细胞。使用从非实质部分选择的CD146磁珠分离原代LSECs。实验原理图如图 1A所示。套管通过PV放置，而下腔静脉被绑起来以确保胶原酶通过肝脏的单向灌注（图1B）。内部灌注室配有加热和加湿系统，以确保...

Watch this Scientific Journal Video about Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells at JoVE.com

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

在这里，我们概述并演示了原代小鼠肝脏正弦内皮细胞（LSEC）分离方案。该方案基于肝胶原酶灌注、低速离心纯化非实质细胞和CD146磁珠选择。我们还使用流式细胞术和扫描电子显微镜对这些分离的LSECs进行表型和表征。

小鼠原发性肝正弦内皮细胞的分离与表征

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

在这里，我们展示了我们的原代小鼠肝脏正弦细胞分离或LSECs分离方案。该方案包括肝胶原酶灌注和用于非实质细胞纯化的低速超速离心和CD146磁珠选择。我们的LSEC分离方法非常高效，适用于健康和患病的小鼠肝脏。分离的LSECs具有高纯度，并保持了结构和功能。使用此协议分离的LSECs是功能和分子研究以及多组学数据生成的最佳选择。该方案将有助于研究肝脏中的细胞间通讯。首先在10厘米培养皿上涂上三毫升胶原蛋白溶液。然后，将培养皿在室温下孵育一小时。一小时后，从涂层表面除去多余的液体。用PBS清洗碗碟三次，让它们风干。设置加热加湿的再循环灌注装置。使用10%漂白剂冲洗灌注系统五分钟。然后，再次用无菌水冲洗系统10分钟。在用胶原酶溶液灌注之前，请尽可能多地沥干冲洗液。通过灌注系统注入胶原酶溶液，将其预热至37摄氏度。在速度控制拨盘上将泵速设置为速度 1。在整个过程中保持相同的速度。称量鼠标以进行该过程。然后将其固定在手术表面上。用70%乙醇喷洒小鼠腹部。使用手术剪刀，从腹部下部开始到xiphoid过程，做一个大约五厘米的切口。之后，在腹部两侧使用小虹膜剪刀进行两次横向切口，以完全暴露腹部器官。将静脉导管的鞘放在动物的背部下方，以抬起并平整腹部。使用常规的弯曲敷料镊子，轻轻地将肠道和胃拉到动物的左侧。然后，在下腔静脉或IVC下放置5-0的手术缝合线，就在暴露的左肾下方。在缝合线中系上松散的挂钩。接下来，在肝门静脉周围再放置一个5-0的手术缝合线。将缝合线放在肝门静脉的脾静脉分支点的正上方。再次，在缝合线中系上松散的挂钩。使用门静脉缝合线作为张力，将 20 号静脉导管插入肝门静脉以下 1 厘米处，该导管向右和左肝门静脉分支。然后，将导管向上滑动静脉，但将其保持在分支区域下方。让血液沿着导管向动，直到它开始滴出。用门静脉缝合线固定静脉导管。使用IV线将带有缓冲液A的IV瓶连接到导管上。然后，用这种溶液冲洗肝脏，同时避免空气进入系统。将肾下方IVC周围的缝合线绑起来。之后，将IVC切到缝合线下方，让动物流血。灌注后，切除附着在肝脏上的胃，肠，脾脏和其他内脏。然后，从胸腔切除隔膜和主要血管。从动物身上取出肝脏并将其放在灌注盘上。小心地取出静脉输液管线，并将胶原酶溶液连接到再循环室中。让肝脏灌注，直到胶囊变得模型化并呈现糊状。这通常需要 10 到 15 分钟。消化后，从腔室中取出肝脏并将其放在10厘米的培养皿上，其中含有约20毫升无血清DMEM。用几个移液器吸头轻轻挑开肝脏，丢弃胆道树。之后，通过70微米的细胞过滤器将肝脏悬浮液过滤到50毫升的锥形管中。在室温下以50倍G离心细胞悬浮液两分钟。然后，收集含有非实质肝细胞的上清液。将上清液在4摄氏度下以300倍G离心五分钟。之后，收集细胞沉淀并将其重悬于一毫升分离缓冲液中。按照制造商的说明使用自动细胞计数器确定细胞编号。接下来，离心细胞悬浮液。完全吸出上清液，并用每1000万个细胞90微升的分离缓冲液重悬沉淀。每1000万个细胞中加入10微升CD146微珠。充分混合，在4摄氏度下孵育15分钟。为了洗涤细胞，每1000万个细胞加入1至2毫升的分离缓冲液。将溶液以300倍G离心10分钟。之后，完全吸出上清液，并在500微升的分离缓冲液中重悬多达10亿个细胞。接下来，通过用三毫升分离缓冲液冲洗来制备分离柱。将细胞悬浮液涂在堆叠有70微米预分离过滤器的色谱柱上。用三毫升分离缓冲液洗涤色谱柱三次。之后，从分离器中取出色谱柱并将其放在15毫升离心管上。将五毫升分离缓冲液移液到色谱柱上。要恢复磁珠标记的细胞，请用力将柱塞推入色谱柱以冲洗出细胞。最后，在4摄氏度下以300倍G离心细胞5分钟。用流式细胞术评估分离的LSECs纯度和表面标志物。门控LSEC和单线的数据分析和门控策略基于前向散射和侧散射数据。用活性染料和CD45，CD146和稳定素-2抗体的组合染色分离的LSECs。对CD45阴性人群进行活LSECs门控，并分析CD146和稳定素-2的表达。从这些数据中可以证实，分离出的LSECs具有大于94%的生存能力和大于90%的纯度。使用光学显微镜和扫描电子显微镜进一步证实了分离细胞的LSAC特异性表型。分离的LSECs在完整的生长培养基中培养六小时，并通过光学显微镜检查。SEM图像中的白色箭头表示LSE开窗，这是LSEC的特征性形态特征。确保肝脏组织完全灌注的关键步骤是正确的导管胶带定位，避免在导管中引入空气，以及胶原酶消化的最佳时机。使用我们的方案获得的高质量LSEC将有助于鉴定LSEC功能的分子机制，并将提高我们对它们在肝脏，健康和疾病中细胞间通讯中的作用的理解。

Research

JoVE Journal

Biology

Isolation of Valvular Endothelial Cells

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

科研

生物学

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Isolation of Embryonic Ventricular Endothelial Cells

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Murine Valve Endothelial Cells

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

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

Isolation of Mouse Coronary Endothelial Cells

鼠标冠状动脉内皮细胞的分离

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...