Name: isolation and primary culture of mouse aortic endothelial cells
Uploaded: 2016-12-19T13:00:00.000+00:00
Duration: 8 min 20 s
Description: Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

1.主动脉的小鼠中分离这里所描述的所有程序均通过的韦恩州立大学的机构动物护理和使用委员会批准。 <ol><li>把鼠标放到麻醉机的感应室。设置异氟烷流至4％结合25％的新鲜空气和75％ 的 O 2。 通过感应腔随后由专用的鼻锥异氟醚（诱导4％，±1.0％用于保养）用25％的新鲜空气和75％的O 2一起吸入麻醉鼠标。注:麻醉用异氟烷可以在75％氧气/ 25％空气中或在100％的氧气递送。 </li><li>检查鼠标每隔5分钟，直到达到麻醉的适当水平（缺乏撤退到脚趾捏）。 </li><li>取鼠标移出感应腔，并继续吸入异氟烷虽然连接到麻醉机专用的鼻锥。切换异氟烷流至1％，以保持麻醉。 </li><li>虽然这是麻醉下，把鼠标到手术垫，放置在它的后面。使用磁带的实验室，以确保四肢。 </li><li>使用加热灯，保持鼠标的温暖。保持该灯在适当的距离从鼠标，使得鼠标不会过热。 </li><li>喷用70％乙醇的胸部。 </li><li>用解剖剪来自中线打开腹部，并露出腹主动脉。打开胸腔暴露心脏和肺部。 </li><li>切腹主动脉与在解剖剪刀释放血液中的中间。 </li><li>填补了1毫升注射器（用25g针），用1毫升的PBS含1000 U / mL的肝素。注入含1000单位/毫升肝素到左心室的PBS中，灌注主动脉。推心脏和肺钳在大动脉到小鼠的右侧，露出胸主动脉。 </li><li>快速使用微迪取出胸主动脉ssection镊子，把它放在冰冷的1X PBS（无菌），那么运输容器放入一个层流罩。 </li><li>插入一个1毫升的注射器装配用25g针进入主动脉的一端，并轻轻冲洗以冰冷的PBS主动脉以除去血液。 </li><li>使用显微解剖钳去除尽可能多的附加脂肪组织的和小的横向容器越好。 </li><li>马上转移的主动脉内皮生长培养基。 </li><li>切主动脉成用无菌手术刀刀片1毫米环。收获大约8 - 每10主动脉环。 </li><li>打开使用一对显微解剖剪的每个主动脉环。 </li></ol> 2.种子矩阵的主动脉段<ol><li>在不使用时，保持生长因子减少基质中-20℃，以防止凝固。把生长因子减少基质中4℃至少过夜以允许完全解冻。 </li><li>预冷6孔板枪头到-20℃，至少10分钟。置于冰上并涂覆6孔板一个孔用1mL基质的板，而不会引入任何气泡。放置板在37℃培养箱中20分钟，以使基质固化。 </li><li>植入主动脉片上使用无菌显微解剖镊固化的基质。放置件内腔面朝下上的基质不接触内皮。放置3 - 4彼此接近的矩阵主动脉段。 </li><li>加入足够的血管内皮细胞生长培养基，以保持湿润段（约200微升）。 </li><li>孵育在37℃的板在5％CO 2的4至6小时。在一天结束时，加入足够的媒体覆盖主动脉段。 </li><li>观察主动脉段相差显微镜下周期性地发芽。检查中等水平和细胞生长的每一天。添加媒体如有必要，要盖上主动脉段。 </li><li>在第 4天，轻轻除去培养基并取出主动脉曲线段F使用无菌针而不中断生长的内皮细胞ROM基质。 </li><li>添加2mL的新内皮细胞生长培养基中，并允许内皮细胞继续增殖基质2 - 3天。 </li></ol> 3.小鼠主动脉内皮细胞的初始传代<ol><li>涂层用明胶（0.1％）的新T12.5烧瓶中，孵育在37℃下30分钟。 </li><li>用无菌37℃PBS仔细清洗矩阵板。 </li><li>加入2毫升中性蛋白酶（50U /毫升）到矩阵板中，在与偶尔摇动摇杆平台室温孵育。检查相衬显微镜下的细胞，以确保大部分细胞被分离。 </li><li>加入2毫升D-瓦尔的灭活中性蛋白酶。 </li><li>仔细收集上清在15毫升离心管中。用2mL的D-缬氨酸洗涤板，并收集上清。 </li><li>离心在900 xg离心所述细胞悬浮液在袋鼠5分钟米温度。 </li><li>重悬在涂有明胶的T12.5烧瓶在4mL内皮细胞生长介质和板的细胞沉淀。孵育细胞在37℃，5％CO 2的2小时。 </li><li>更换介质。孵育细胞在37℃，5％的CO 2，直到它们的85％ - 90％汇合。 </li></ol> 4.传代的小鼠主动脉内皮细胞的<ol><li>外套两个新T12.5烧瓶用明胶（0.1％），孵育在37℃下30分钟。预热胰蛋白酶-EDTA（0.25％胰蛋白酶，0.02 EDTA在PBS中）和无菌PBS在37℃下约15分钟。 </li><li>用无菌37℃PBS仔细洗涤细胞。 </li><li>添加0.5毫升胰蛋白酶-EDTA（0.25％胰蛋白酶，0.02 EDTA在PBS中）中，并在37℃孵育约1分钟，或直到大部分细胞转身。 </li><li>添加2mL的内皮细胞生长培养基以停止消化。吸取细胞悬液上下烧瓶几次内。如有必要，可使用电池刮板以收集细胞。 </li><li>收集在15毫升离心管中的细胞悬浮液。离心细胞悬浮液在900×g离心，在室温下5分钟。 </li><li>重悬在涂有明胶2 T12.5烧瓶在4mL内皮细胞生长介质和板的细胞沉淀。孵育细胞在37℃，5％CO 2的2小时。 </li><li>更换介质。孵育细胞在37℃，5％的CO 2，直到它们的85％ - 90％汇合。 </li><li>使用鼠标主动脉内皮细胞后的2 - 3代。 </li></ol> 5.表征小鼠主动脉内皮细胞的<ol><li>重新板上的细胞。 <ol><li>涂层用明胶（0.1％）的6孔板，孵育在37℃下30分钟。用PBS冲洗板。 </li><li>预加热胰蛋白酶-EDTA和无菌PBS中至37℃约15分钟。 </li><li>清洗细胞，用无菌PBS中的3倍。添加0.5胰蛋白酶毫升/ EDTA对细胞，孵育约1分钟，在37℃õ- [R，直到大部分细胞转身。 </li><li>添加2mL的内皮细胞生长培养基以停止消化。吸取细胞悬液上下烧瓶几次内。如有必要，使用细胞刮刀收集细胞。 </li><li>收集在15毫升离心管中的细胞悬浮液。离心细胞悬浮液在900×g离心，在室温下5分钟。 </li><li>弃去上清液，再悬浮在内皮细胞生长培养基中的细胞。种子涂有明胶的6孔板的细胞以3×10 5 /孔的密度。 </li><li>孵育细胞在37℃，5％的CO 2过夜以使细胞附着到培养表面。 </li></ol></li><li> DIL-AC-LDL摄取和荆豆 -lectin约束力。 <ol><li>存储1,1&#39;-双十八烷基-3,3,3&#39;，3&#39;- tetramethylindo-羰花青高氯酸盐标记在-20℃乙酰低密度脂蛋白（语-AC-LDL）。染色之前，解冻冰语-AC-LDL。该股语-AC-LDL SOLU灰是1毫克/毫升。储存在4℃的FITC标记的荆豆凝集素（ 荆豆 -Lectin，1微克/毫升）。股票荆豆 -Lectin溶液是1毫克/毫升。商店赫斯特33342在4℃。股票的Hoechst 33342溶液为100微克/毫升。 </li><li>添加的语-AC-LDL原液10微升至1毫升培养基中，以使10微克/毫升的最终浓度。 </li><li>染色前，删除旧的介质，并用新的培养基洗涤细胞每孔。 </li><li>取出新媒体，并添加语-AC-LDL染色网上平台。 </li><li>孵育细胞在37℃，5％CO 2的1小时。 </li><li>清洗细胞，用无菌1×PBS中的3倍。 </li><li>加入1毫升10％福尔马林固定细胞。放于室温下的板约30分钟。 </li><li>清洗细胞，用无菌1×PBS中的3倍。避免光线。 </li><li>加入1毫升无菌1X PBS包含荆 -Lectin的10微升。 </li><li>孵育在室温temperatu细胞重新在黑暗中保持1小时。 </li><li>通过用无菌1×PBS中3次洗涤细胞除去荆豆凝集素。 </li><li>添加的Hoechst 33342至1微克/毫升的最终浓度，孵育在黑暗中10分钟。 </li><li>查看语-AC-LDL（红色，576纳米的激发波长）， 荆豆 -Lectin（绿色，519 nm的激发波长）和赫斯特的荧光（蓝色，361 nm的激发波长），用倒置荧光显微镜。 </li></ol></li><li>荧光染色CD31，VEGFR2，eNOS的，VE-cadherin和钙调节蛋白。 <ol><li>商店FITC缀合的抗小鼠CD31抗体，eNOS的抗体，VE钙粘蛋白抗体，FITC缀合的抗兔IgG在4℃下在黑暗中。商店VEGFR2抗体在-20℃。 </li><li>清洗细胞，用无菌1×PBS中的3倍。 </li><li>加入1毫升10％福尔马林固定细胞。放于室温下的板约30分钟。 </li><li>清洗细胞，用无菌1×PBS中的3倍。 </li><li>染色CD31，加入1毫升无菌1X的PBS包含CD31-FITC抗体10μL的。染色要么VEGFR2，eNOS的，VE钙粘蛋白或调宁蛋白，添加包含4μLVEGFR2，eNOS的无菌1X PBS 1毫升，VE钙粘蛋白或调宁一抗，分别为。 </li><li>在冰上孵育细胞，在黑暗中1小时。 </li><li>通过用无菌PBS 3次洗涤细胞除去抗体。 </li><li>对于VEGFR2的染色，eNOS的，VE-钙粘蛋白或调宁蛋白，添加包含FITC缀合的抗兔IgG的10微升无菌1×PBS中的1毫升。在冰上孵育细胞，在黑暗中1小时。通过用无菌PBS 3次洗涤细胞除去IgG的。 </li><li>添加的Hoechst 33342至1微克/毫升的最终浓度，孵育在黑暗中10分钟。 </li><li>查看CD31，VEGFR2，eNOS的荧光，VE钙粘蛋白，调宁（绿色的518-535纳米的激发波长）和赫斯特（蓝色，361 nm的激发波长），用倒置荧光显微镜。 </li></ol></li></ol>

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The authors have nothing to disclose.

这项研究表明了一个简单的方法来分离，培养内皮细胞从鼠标主动脉细胞无任何特殊设备。免疫荧光染色显示，大部分细胞是第二通道后内皮细胞。有人建议，细胞在第二至第三通道适于在体外 和体内实验来研究内皮生物学。 从本议定书的重要注意事项 有在步骤五个关键点。首先，血管腔与含肝素的PBS冲洗，以尽量减少内皮?...

血管内皮不仅是分隔的血液和组织的屏障层，它被认为是一个巨大的内分泌腺，绵延在整个血管树具有400平方米1的表面积。内皮的福祉是血管稳态的关键。的内皮机能障碍参与各种心血管疾病，包括动脉粥样硬化，血管炎和缺血/再灌注损伤， 等 2-4。迄今为止，涉及这些疾病的设置特定的细胞和分子机制尚未很好地理解，由于内皮的漫射解剖性质。 鼠标是研究的重要模型，因为遗传操作技术在小鼠比较发达的比任何其他哺乳动物物种。然而，由于主动脉的小尺寸使得enzymat原代鼠主动脉内皮细胞的分离被认为是特别困难内皮不切实际的IC消化。一些报道的方法分离和纯化的EC要求5-7。 该协议的目标是用一个简单的方法来从鼠标主动脉隔离和扩大血管内皮细胞，而无需使用任何特殊的设备。在这个协议中，新鲜分离的主动脉切成小片段，并接种到朝下以允许内皮出芽内皮的基质。段被删除后，内皮细胞在血管内皮青睐中等扩大，准备好两三个代后实验。所描述的方法的优点是:1）相当高数量的内皮细胞来自单一主动脉收获的; 2）细胞存活率保存良好; 3）不需要特殊的设备或技术。它提供了鉴定内皮细胞病理生理学特异性细胞和分子机制的一种有效手段。对于那些有兴趣谁在学习公关从任一基因敲除小鼠imary培养的内皮细胞，基因敲除小鼠中，或鼠疾病模型中，该协议是非常有用和易于实践。

<table><tbody><tr><td>4- or 6-week-old mice</td><td>Jackson Laboratory</td><td>#000664</td><td></td></tr><tr><td>Sterile 1x phosphate-buffered saline (PBS)</td><td>Gibco</td><td>#10010-023</td><td></td></tr><tr><td>Sterile 1x PBS containing 1,000 U/mL of heparin</td><td>Sigma Aldrich</td><td>H3149</td><td></td></tr><tr><td>Endothelial cell growth medium (Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium [DMEM] with 25&amp;nbsp;mM HEPES [Gibco, #12320-032], supplemented with 100&amp;nbsp;&amp;mu;g/mL endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma, #2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000&amp;nbsp;U/mL heparin [Sigma Aldrich, H3149], 10,000&amp;nbsp;U/mL penicillin and 10&amp;nbsp;mg/mL streptomycin [Gibco, #15140-122])</td><td></td><td></td><td></td></tr><tr><td>Growth factor reduced matrix</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>Neutral proteinase (Dispase, 1&amp;nbsp;U/mL, Fisher Scientific, #CB-40235) and D-Val medium (D-Valine, 0.034&amp;nbsp;g/L, Sigma, #1255), in Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium, low glucose (Gibco, #12320-032)</td><td></td><td></td><td></td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL)</td><td>Life Technologies</td><td>L3484</td><td></td></tr><tr><td>FITC-labeled &lt;em&gt;Ulex europaeus&lt;/em&gt; agglutinin (&lt;em&gt;Ulex&lt;/em&gt;-Lectin)</td><td>Sigma</td><td>L9006</td><td></td></tr><tr><td>Anti-mouse CD31-FITC conjugated antibody</td><td>BD Biosciences</td><td>553372</td><td></td></tr><tr><td>Anti-mouse vascular endothelial growth factor receptor 2 antibody</td><td>Cell Signaling</td><td>9698</td><td></td></tr><tr><td>Anti-mouse endothelial nitric oxide synthase</td><td>Abcam</td><td>ab5589</td><td></td></tr><tr><td>Anti-mouse vascular endothelium-cadherin</td><td>Abcam</td><td>33168</td><td></td></tr><tr><td>Anti-mouse calponin</td><td>Abcam</td><td>700</td><td></td></tr><tr><td>FITC-conjugated anti-rabbit IgG</td><td>Sigma</td><td>F6005</td><td></td></tr><tr><td>1 mL syringe fitted with 25-G needle</td><td>Fisher Scientific</td><td>50-900-04222</td><td></td></tr><tr><td>100 mm Peri dishes</td><td>Fisher Scientific</td><td>07-202-516</td><td></td></tr><tr><td>Six-well cell culture plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>T12.5 culture flask</td><td>Fisher Scientific</td><td>50-202-076</td><td></td></tr><tr><td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade</td><td>Fine Science Tools, Inc.</td><td></td><td></td></tr><tr><td>Anesthesia machine with isoflurane</td><td>Webster Veterianary Supply</td><td>07-806-3204</td><td></td></tr><tr><td>heating lamp</td><td></td><td></td><td></td></tr><tr><td>Centrifuge machine</td><td></td><td></td><td></td></tr><tr><td>Inverted phase-contrast microscope</td><td></td><td></td><td></td></tr><tr><td>inverted fluorescence microscope</td><td></td><td></td><td></td></tr></tbody></table>

isolation and primary culture of mouse aortic endothelial cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

内皮细胞发芽 自发的内皮细胞出芽来自小鼠主动脉段开始。鼠标主动脉段被允许在低生长因子基质生长然后在内皮细胞生长培养基中4天。内皮细胞出芽通常出现在2 - 4天。显微照片拍摄第4天（ 图1）。正如图片所示，无数的内皮细胞迁移从段路程。新成立的豆芽继续从段和分支延伸。 内皮细胞表型 <p class="jove_...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

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 ...

isolation-and-primary-culture-of-mouse-aortic-endothelial-cells

Research

JoVE Journal

Biology

30.5K 次观看.  Wayne State University. The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

视频: Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

原代鼠脑微血管内皮细胞的分离

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

科研

神经科学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

生物学

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

医学

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

原代小鼠骨骼肌微血管内皮细胞的分离

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

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 of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

小鼠原代肺内皮细胞的分离

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

免疫与感染

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

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 ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

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.

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. ...