Name: isolation and culture of primary endothelial cells from canine arteries and veins
Uploaded: 2016-11-18T15:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

伦理声明:在本研究中所用的血管收获是从新鲜尸体犬获得的剩余材料（N = 4）安乐死其他无关的研究（大学3R政策）健康的狗。异常血管（内和肝外门体分流术，N = 1个）收获从提交给大学医院的乌得勒支大学的伴侣动物狗的业主知情同意后验尸。 1.隔离主犬内皮细胞的培养和<ol><li>用2ml /孔的0.5％w / v的明胶和预涂层6孔板，在37℃用5％CO 2的潮湿气氛离开2小时在恒温箱。播种原代细胞之前，除去多余的明胶溶液。 </li><li>无菌删除从新鲜尸体犬（ 图1A）血管（S）的利益（ 例如，主动脉，下腔静脉，肝门静脉）。维持血管系统的解剖结构通过将直镊子感兴趣的容器的两端。避免与镊子的组织的不必要的操作，以防止对血管内皮细胞的损伤。 <ol><li>切断与手术剪两端夹紧容器，并从尸体除去。运输中Hank氏冰上平衡盐溶液（HBSS）中。 注:大约5厘米的容器长度是优选的此过程，但那些测量1厘米还将向培养提供足够的细胞。 </li></ol></li><li>血管转移至培养皿填充用冰冷的HBSS。用手术剪，从容器的外侧除去任何粘附的组织和脂肪，保持容器本身完整（ 图1B）。确保切割时船舶在任何时候都清晰可见:拉拨周围组织用夹子或镊子最佳视图。 <ol><li>关闭船只的任何分支机构，连字（ 例如 ，聚乳糖3-0），随后将其删除与外科剪刀或手术刀。 </li></ol></li><li>小心进入具有弯曲霍尔斯特德蚊子镊子的容器端，在容器的另一端夹住钳子的前端，然后缩回，慢慢倒转容器中，直至其完全内向外（ 图1C-G）。外部现在由内皮细胞层的。如果任何困难是在反相容器中遇到，淹没在HBSS再次以减少摩擦。 </li><li>放置荷包缝合在容器的两端完全关闭它，并防止任何非内皮血管组织的消化过程（ 图1H）的曝光。使用绑带末端操纵倒置容器中。 </li><li>如果消化和文化稍后进行冷冻保存前的消化协议（步骤1.7）倒船。 <ol><li>将倒船只混和，细胞培养冻存液填充。冻结下降到-80℃，用冷冻合作ntainer。储存于-80℃，如果船只要在一周内使用。对于长期储存，在-180℃的冷冻管到位。当执行欧共体隔离，迅速解冻冷冻管在水浴（37℃），并立即放置在冰冷的HBSS。请按照1.7所示。 注意:考虑到该内皮细胞的分离后的总产率将降低，由于在冷冻过程中丧失活力。 </li></ol></li><li>容器转移至50ml管中，并在HBSS冲洗两次以除去红细胞（或剩余冷冻介质在解冻的情况下）（ 图1I）。 </li><li>消化在50ml管中在容器用30 ml胶原酶II型（0.15单位/ ml）和分散酶（0.15单位/毫升）的犬内皮细胞生长培养基（CECGM）1小时，在37℃，间歇温和溶液搅动。 </li><li>从管中取出容器，并在250离心细胞悬浮液5分钟×g下。 </li><li>重悬在CECGM一个细胞沉淀第二，每孔种子2毫升细胞悬液（1 - 3井取决于容器的大小）。培养在5％的CO 2在空气中的潮湿气氛中的细胞在37℃，并改变它的培养基每周两次。 </li><li>通道中的细胞70汇合时 - 在达到80％以上。 <ol><li>清洗细胞与预热HBSS去除死或非贴壁细胞。加入200μl重组细胞解离酶（1倍），以每个孔，并放回培养箱中5分钟或直到所有的细胞分离。 </li><li>转移细胞到15毫升管中并通过加入10ml培养基（CEGM）含10％胎牛血清（FCS）的停止胰蛋白酶消化。离心机在250×g下5分钟。继续培养明胶预涂板或瓶。 </li></ol></li></ol> 2.表征<ol><li>培养犬主动脉内皮细胞（CnAOECs）并比较初级和商业细胞系用显微镜每周两次的形态。 注:t他典型的成长内皮细胞的图案补丁是达到&gt; 70％汇合时，最能观察到。 <ol><li>培养CnAOECs在CECGM上的0.5％重量/预涂-V明胶T75烧瓶中。传代细胞每周一次当汇合为70％ - 80％。 <ol><li>为传代，用预温热的HBSS洗涤细胞一次，并加入1 ml重组细胞解离酶（1×）。放置烧瓶放回培养箱中5分钟或直到所有的细胞分离。通过加入10ml培养基（CEGM）含10％FCS的灭活胰蛋白酶。离心细胞悬浮液5分钟，在250 xg离心，弃去上清，重悬在1ml培养基中的细胞。 </li><li>取细胞悬浮液的10微升等分试样和稀释1:1 0.4％台盼蓝。计数使用自动细胞计数器的细胞，并在新的T75烧瓶积垢4.0×10 5个活细胞。在37℃下在5％的CO 2的潮湿气氛中加入10 mL预热CECGM的烧瓶中并培养。 </li></ol></li></ol></li><li>从培养CaPECs和CnAOECs的通道1分离RNA。 <ol><li>收集至少为1×10 3个细胞作为沉淀，加入20微升样品制备试剂（SPR）的孵育1分钟以裂解细胞。培养后，小心收集含在-70℃下的总RNA和存储的细胞裂解物。 </li></ol></li><li>使用按照制造商的指示的cDNA合成试剂盒（ 例如，的iScript）转换的mRNA合成cDNA。在4℃长达一个星期，或者在-20℃储存的cDNA长期直至准备执行的qPCR。 </li><li>测量血管内皮标记CD31基因的表达，以确认内皮细胞来源。执行实验装置和量化使用MIQE简要记录准则10。对于标准化，测量参考基因GAPDH，RPS19和B2MG 11的表达。 <ol><li>在无核酸酶的水制备得到的cDNA的10倍稀释。 </li><li>准备从合并的cDNA样品的4倍稀释的标准线，并使用不含核酸酶的水作为非模板对照。稀释样本五次以达到定量PCR反应的50倍的总稀释度。一式两份移取10微升的反应中使用4μl的cDNA和6微升反向和正向引物（ 表1）的20皮摩尔混合荧光团的一个384孔格式。 </li><li>程序设定为95℃，在95℃5分钟为Taq聚合酶活化，然后用10秒的39个循环变性，和在Tm 30秒退火和延伸。上述Tm为每个引物组示于表1中 。 </li><li>进行熔解曲线分析下列每一次运行，以确保只有一个产品被放大。正常化使用参考基因​​的平均相对量的样品的表达水平，并计算ΔCT如果反应效率为95％和105％之间。 </li></ol></li><li>与ANGI评估EC功能ogenesis检测。 <ol><li>添加10微升的细胞外基质的一预冷却的血管发生滑动的各孔，并用移液管尖端扩散到覆盖的表面良好。保持在冰上的细胞外基质和避免引入气泡进入凝胶。通过用水浸湿的纸巾在37℃，5％的CO 2将所述滑动在培养皿在培养箱中30分钟固化的凝胶。 </li><li>添加1.0×10 4个主 内皮在50μl内皮生长培养基每孔。孵育6小时，在37℃，5％的CO 2的幻灯片。 </li><li> 6小时后拍照用放大20倍，并确保包括整个很好的形象。 </li></ol></li></ol>

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The authors have no competing financial interests.

在研究中注重对犬内皮细胞的CnAOEC主线路用于狗3，12，13的内皮细胞谱系的模型。在人类的研究中，HUVEC文化仍然被认为是黄金标准。显然，仅仅着眼于从脐带来源的内皮细胞是心血管研究的坚定限制。内皮细胞具有特定的基因表达模式确定动规范。为了占产后血管这些差异，我们提出了一种基于血管内皮细胞的特定解剖位置这种新的隔离法。通常用于主乳油分离方法被冲洗用酶溶液的容?...

狗被用作大型动物模型为心血管疾病的研究和也可以从天生（基因）血管异常1,2受到影响。为了研究这些疾病的商业内皮细胞系通常用于评估内皮细胞（EC）的功能。狗有可用（CnAOEC）一家商业内皮细胞线，从犬主动脉的。该细胞系在研究，作为控制正常内皮细胞3-5个最常用的。在人类心血管疾病研究中最常用的内皮细胞系分别来自人脐静脉和动脉，派生人脐静脉内皮细胞（HUVEC）和人脐动脉内皮细胞（HUAECs）。内皮细胞已被用来作为自1980年代以来6在血管研究的金标准。它们被认为是研究内皮功能和疾病适应的经典模型系统。从不同的血管分离的内皮细胞的变化appearance和由于遗传背景和暴露于微环境7的功能。另外，内皮细胞和HUAECs从脐带衍生的，可能不会完全模拟成人血管相对于该条件下，它们被暴露于并响应于疾病发展的血管结构。因此，在一般翻译内皮细胞中发现的结果和HUAECs心血管疾病是不够的。 当学习适应和成年内皮细胞的行为，从感兴趣的容器主内皮应作为一个更直接的方法。为了分离这些细胞，已报道的几种方法。一种广泛描述的方法，它也用于内皮细胞，用酶消化溶液8冲洗容器。这通常会导致与非内皮细胞污染如平滑肌细胞和成纤维9。为隔离另一种常用的方法是剁碎血管组织的酶消化，随后通过荧光 -激活细胞根据分化（CD）的31 7，8。FACS分选的内皮细胞标记物群集和随后的细胞培养分选（FACS）需要相对大量的细胞的，因此不适合于内皮细胞的小血管的隔离。因此，我们旨在开发用于从各种犬血管具有高纯度的分离出纯的内皮细胞群的新的鲁棒方法。为了测试新的隔离方法的有效性，我们分离并从不同的犬动脉和静脉，大型和小型纯获得主犬内皮细胞（CAPEC）文化。这种方法还可以使从患病和/或异常血管始发如先天内或肝外门体分流，犬2的常见疾病的内皮细胞的培养。该方法允许额外的相关细胞类型相同的容器，如血管平滑肌细胞的分离，因为大多数的船只保持INT在操作过程中起作用。

<table><tbody><tr><td>Collagenase type II</td><td>Life Technologies</td><td>17101-015</td><td></td></tr><tr><td>Dispase</td><td>Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>DMEM (1x) + GlutaMAX</td><td>Life Technologies</td><td>31966-021</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Life Technologies</td><td>14025-050</td><td></td></tr><tr><td>Canine Endothelial Cells Growth Medium&amp;nbsp;</td><td>Cell Applications</td><td>Cn211-500</td><td></td></tr><tr><td>CnAOECs</td><td>Cell Applications</td><td>Cn304-05</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)&amp;nbsp;</td><td>GE Healthcare</td><td>16000-044</td><td></td></tr><tr><td>TrypLE Express</td><td>Life Technologies</td><td>12604-013</td><td></td></tr><tr><td>SPR</td><td>Bio-Rad</td><td>170-8898</td><td></td></tr><tr><td>iScript synthesis kit</td><td>Bio-Rad</td><td>170-8891</td><td></td></tr><tr><td>SYBR green super mix</td><td>Bio-Rad</td><td>170-8886</td><td></td></tr><tr><td>Recovery Cell Freezing Medium</td><td>Gibco/Life Technologies</td><td>12648-010</td><td>Keep on ice prior to use</td></tr><tr><td>Freezing container, Nalgene Mr. Frosty</td><td>Sigma-Aldrich</td><td>C1562</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Surgical scissors (Mayo or Metzenbaum)</td><td>B. Braun Medical</td><td>BC555R</td><td></td></tr><tr><td>Mosquito forceps&amp;nbsp;</td><td>B. Braun Medical</td><td>FB440R</td><td></td></tr><tr><td>Mosquito forceps curved</td><td>B. Braun Medical</td><td>FB441R</td><td></td></tr><tr><td>polyglactin 3-0</td><td>Ethicon</td><td>VCP311H</td><td></td></tr><tr><td>Trypan blue</td><td>Bio-Rad</td><td>145-0013</td><td></td></tr><tr><td>Automated counting chamber</td><td>Bio-Rad</td><td>145-0102</td><td></td></tr><tr><td>Counting Slides, Dual Chamber</td><td>Bio-Rad</td><td>145-0011</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>BD356231</td><td>Slowly thaw on ice</td></tr><tr><td>&amp;micro;-Slide Angiogenesis</td><td>Ibidi</td><td>81501</td><td></td></tr><tr><td>Endothelial Growth Medium</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>EGM-2 SingleQuot Kit&amp;nbsp;</td><td>Lonza</td><td>CC-4176</td><td></td></tr></tbody></table>

isolation and culture of primary endothelial cells from canine arteries and veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

不同的血管被成功经受所描述的隔离协议（ 图2）。这是可能的剖析和反转主动脉，下腔静脉，肝门静脉和冠状动脉健康的狗（从每只犬的所有船只，N = 4）。用同样的方法进行内皮两个先天性门体分流术（肝和肝内，N = 1个）隔离。虽然主动脉很容易倒，胸主动脉段均高于腹主动脉更具挑战性。在胸段中的主动脉有许多肋间动脉从它的分支，这需要单独连接?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

isolation-culture-primary-endothelial-cells-from-canine-arteries

Research

JoVE Journal

Medicine

11.2K 次观看.  Utrecht University. Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

视频: Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

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 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 ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

从鼠标阻力动脉分离微血管内皮管

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

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 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.

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 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 ...

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

人隐静脉内皮细胞分离和暴露于受控水平的剪切应力和拉伸

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...

医学

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 Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

从不同的脂肪库分离和鉴定血管内皮细胞，用于下游应用

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination

Source: H&#248;rlyck, S. et al. <a href="https://www.jove.com/video/61253" target="_blank">Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.</a>&#160;J. Vis. Exp. (2020)
In this video, we demonstrate the procedure to isolate and culture pericytes from bovine brain capillaries without endothelial cell contamination.

原代脑周细胞的分离和培养:从牛脑毛细血管中分离和培养周细胞而不受内皮细胞污染的程序

Source: H&#248;rlyck, S. et al. Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.&#160;J. Vis. Exp. ...