我们想感谢Andrea波尔特布里的，批判性阅读的手稿，显微镜服务UNC援助与时间推移成像实验室，以及美国国立卫生研究院（授＃R01HL061656）的资金支持。

1。准备抗体共轭珠<ol><li>清扫前一天，选择的抗体相结合，用适当的磁珠（ 如鼠IgG抗体的磁珠大鼠，4×10 8珠/毫升），每制造商的指示。对于BD的大鼠抗CD31抗体（0.5微克/微升，用于分离内皮细胞），每25微升的有孔玻璃珠加入3微升的抗体，1.5微克的抗CD31抗体的终浓度为1×10 7的有孔玻璃珠/ 25μl的缓冲液中。 </li><li>在4°C孵育过夜珠和抗体</li></ol> 2。准备胶原板块的<ol><li>分离的细胞形成小管更迅速地生长时，在胶原凝胶，而不是仅仅是一种胶原包被的板。因此，胶原凝胶夹层的前一天晚上，6。冰在组织培养罩，结合下列试剂（够19井）：206.4微升无菌H &lt;子&gt; 140.4微升2 O，I型胶原（4.08毫克/毫升），38.4微升10X M199和1.15微升5 M氢氧化钠。 </li><li>吸取20微升到384-孔组织培养板的孔中的骨胶原凝胶。将板在37℃组织培养孵化30分钟。 </li><li>加入80微升的DMEM，每孔孵育15分钟，在37°C。取出培养基中，重复两次以上。 </li><li>最后冲洗后，加入80微升10％FBS / DMEM中的每个孔，孵育过夜，在37℃下取出培养基，在此之前，加入分离的细胞。 </li></ol> 3。消费税和消化的心<ol><li>安乐死定时孕鼠在使用经批准的安乐死技术所需的胚胎一天。所有的实验都是在北卡罗莱纳大学教堂山分校的机构动物护理和使用委员会批准。 </li><li>用鼠标在仰卧位，从优喷女性腹侧前用70％的乙醇清扫。使用镊子，抬起远离下腹部皮肤和肌肉内脏，剖开女性的下腹腔内脏可视化。 </li><li>宫颈钳，小心地剪尾钳，从腹腔开始解除子宫角。虽然解除子宫角，剪去任何地方结缔组织持有的号角。要完成切除子宫角，与输卵管子宫角切结释放子宫角。 </li><li>子宫角放置在培养皿中含有冷的1x磷酸盐缓冲盐水（PBS）漂洗根据需要。剖开子宫角，通过中线暴露所附的胚胎囊。 </li><li>剖开交界处的胎盘和胚胎的胚囊，避免意外切割胚胎和胚胎检索。将胚胎在第二个培养皿用冷PBS。返回培养皿子宫角冰。 </li><li> ðecapitate胚胎提高访问胸壁。如果基因分型是必要的，割尾巴，将其删除，并保存在一个Eppendorf管置于冰上。 </li><li>随着胚胎在它的后面，切开胸壁心脏和肺的可视化。为了避免切割心，使垂直切割沿侧的肋笼，附近的前肢，然后由水平切在底部的肋，然后，轻轻拉胸壁和出的方式。通过约胚胎13.5天，胸壁是透明的，可视化的帮助。 </li><li>使用镊子小心地抬起心脏和削减以下的船只。然后，切成以上的大血管，释放心。如果不切肺血管可能会被删除，肺与心脏。因此，去除多余的组织，如肺部，如果必要的。 </li><li>分离和丢弃从心室心房和大血管。用剪刀，肉切成小块（约1毫米3心室&lt;/支持&gt;），并在约500μl的胶原酶（1毫克/毫升的无菌PBS中）。保持样品置于冰上，直到所有的心室已剁碎，并放置在胶原酶。 </li><li>重复前面的步骤，直至心室已切除所有的胚胎，收集每一个心脏，在一个单独的Eppendorf管中。如果所有的胚胎具有相同的基因型，心中可能会集中在一个单一的Eppendorf管中。处理的样本的数目在一个单一的时间可能会受到限制，基于磁铁用于隔离; DynaMag（123-21D）这里使用的最多可容纳16 Eppendorf管中。 </li><li>将肉末心室在37℃45分钟摇动。每15分钟，轻轻取出样品和吸管向上和向下机械分离细胞。 </li></ol> 4。隔离的内皮细胞<ol><li>通过脑室胶原酶溶液通过70微米的过滤器，以去除剩余的细胞团块。 </li><li>离心细胞，在400×g离心10分钟。丢弃上清，更换约200微升10％FBS的DMEM，吸管向上和向下游离细胞。 </li><li>一次重复前面的步骤。离心机的细胞在400×g离心10分钟后，第二个10％FBS / DMEM培养液洗涤。在50μl的10％FBS / DMEM培养液中重悬细胞。 </li><li>在最后的离心步骤，准备珠。 <ol><li>将Eppendorf管中含有抗体和有孔玻璃珠上的磁铁，持续1分钟。 </li><li>磁铁与管子的地方，去除上清。从磁铁取出试管，并添加约100微升10％FBS / DMEM。 </li><li>管放置在磁体上，持续1分钟。除去上清液，并含10％FBS / DMEM培养液洗一遍。共洗3次重复上述步骤。 </li><li>最后一次洗涤后，悬浮在10％FBS / DMEM培养液的抗体共轭的有孔玻璃珠（每个样品取5μl）。 </li></ol></li><li>到每个细胞样品，添加5μl的抗体共轭的有孔玻璃珠。将磁珠的细胞悬浮液在4℃下与摆动FOR 30分钟。 </li><li>在层流罩中，将含有细胞和抗体结合的有孔玻璃珠上的磁铁1分钟的Eppendorf管中。取出上清液，从磁铁取出离心管，用约100微升10％FBS / DMEM。重复洗涤5次。 </li><li>最后一次洗涤后，将管在磁体上，持续1分钟，除去上清液，并删除从磁体的管。新增约100微升DMEM。 </li><li>将反应管在磁体上，持续1分钟，除去上清液，并取出离心管，从磁体。添加100-200微升0.25％胰蛋白酶-EDTA预热。孵育样品在37℃和5％CO 2中5分钟。 </li><li>将反应管2分钟，在磁体上。含有细胞的上清小心转移到新鲜的Eppendorf管中。以400×g离心5分钟。 </li><li>去除上清。重悬细胞在80-100微升生长培养基和板在96 - 或384以及处理过的培养皿中，根据预期收益率（约450-600细胞E14.5-E16.5胚胎脑室）。对于内皮细胞，使用内皮细胞生长培养基（定义​​见下文）。 </li></ol>

<ol>
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Hemost. 36, 227-235 (2010).</li><li>Dong, Q. G., Bernasconi, S., 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=A+general+strategy+for+isolation+of+endothelial+cells+from+murine+tissues.+Characterization+of+two+endothelial+cell+lines+from+the+murine+lung+and+subcutaneous+sponge+implants.">A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 17, 1599-1604 (1997).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <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+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat. 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E., Sugimoto, H., Zeisberg, M., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+kidney+fibrosis+emerge+via+endothelial-to-mesenchymal+transition.">Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.</a> Journal of the American Society of Nephrology : JASN. 19, 2282-2287 (2008).</li><li>Chang, L., Noseda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+vascular+smooth+muscle+cells+from+local+precursors+during+embryonic+and+adult+arteriogenesis+requires+Notch+signaling.">Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling.</a> PNAS. 109, 5993-6998 (2012).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., DeLisser, H. M. <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+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> American Journal of Physiology: Lung Cellular and Molecular Physiology. 296, L1095-L1103 (2009).</li><li>Frauli, M., Ludwig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+fibroblast+proliferation+in+a+culture+of+human+endometrial+stromal+cells+using+a+medium+containing+D-valine.">Inhibition of fibroblast proliferation in a culture of human endometrial stromal cells using a medium containing D-valine.</a> Archives of Gynecology and Obstetrics. , 241-96 (1987).</li><li>Lazzaro, V. A., Walker, R. J., 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=Inhibition+of+fibroblast+proliferation+in+L-valine+reduced+selective+media.">Inhibition of fibroblast proliferation in L-valine reduced selective media.</a> Research communications in chemical pathology and pharmacology. 75, 39-48 (1992).</li><li>Arima, S., Nishiyama, K., 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=Angiogenic+morphogenesis+driven+by+dynamic+and+heterogeneous+collective+endothelial+cell+movement.">Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement.</a> Development. 138, 4763-4776 (2011).</li></ol>

作者宣称，他们有没有竞争经济利益。

初级胚胎干细胞工作，让新颖的实验来解决发展在体外关键的步骤。然而，分离过程中是不平凡的。隔离任何类型的细胞的关键步骤包括确保细胞分离后，胶原酶消化，然后，他们悬浮在洗涤步骤。机械清扫通过移液大大有助于细胞分离在胶原酶步骤，过滤步骤除去细胞团块。这些措施提高人口的纯度和产量。 电镀密度也是一个显着的关注，从一个小器官分离细胞时?...

永生细胞系的问世彻底改变基本细胞生物学1。当前可用的细胞系是来自于广泛的器官，包括所有的主要细胞类型。然而，建立的细胞系有一定的局限性。永生化的方法，明显改变细胞的行为，特别是相对于寿命和扩散，而且还可以影响意想不到的蛋白质的表达，如细 ​​胞骨架蛋白2。此外，虽然有许多不同的细胞系，甚至一个单一的细胞类型内的整个有机体之间的显着的多样性。内皮细胞在特定的显示出不同的行为根据他们是否行动脉或静脉，是什么样的流量目前在容器3。更为迫切，但是，从发展的角度来看是可用的细胞系中，绝大多数是来自成人组织（ 如：T他收集可通过ATCC）。这些成人细胞株可能不复述他们的胚胎前体的动态性质。迅速变化的时空在开发过程中观察到的基因表达模式也表明，在给定的发育阶段从​​一个器官的血管内皮细胞，可能无法从该相同的器官，血管内皮细胞在不同发育阶段的行为方式相同。 作为一种替代方法，使用市售的永生化细胞系，原代细胞可以分离出感兴趣的特定组织。在这种技术的优点，这些原代细胞可以分离出特定的器官，或什至在任何特定的发育阶段的器官的一部分。此外，这些原代细胞可以从可用的转基因动物的各种各样的分离， 从而允许该基因敲除和爆震的同时避免了其他问题，如转染效率的体外研究。毫不奇怪，许多技术已出版，详细介绍了如何分离特定的细胞类型4,5。一般情况下，这些技术涉及收集感兴趣的区域，解离的细胞，感兴趣的特定的细胞类型进行标记，并分离这些细胞进行进一步的分析。 要研究冠状动脉内皮细胞的早期胚胎人口，我们缩减先前公布的技术4，使用一个较小的器官。有了这个比例缩小的过程中，我们可以分离出冠状动脉内皮细胞从胚胎心脏特异性胚胎阶段。这些细胞可以被用在传统的内皮细胞的检测，如迁移分析。直至早期胚胎细胞系变得更加普遍，工作与原代细胞是一个非常宝贵的技术。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Timed-pregnant mice</td>
			<td></td>
			<td></td>
			<td>To be dissected at the embryonic stage of interest</td>
		 </tr>
		 <tr>
			<td>Anti-mouse CD31</td>
			<td>BD Bioscience</td>
			<td>553370</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat IgG Dynabeads</td>
			<td>Invitrogen</td>
			<td>110-35</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagen</td>
			<td>BD Bioscience</td>
			<td>354236</td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS (1x)</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagenase, type I</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Cellgro </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich </td>
			<td>F2442 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Endothelial cell growth medium</td>
			<td>(made in lab as described in notes)</td>
			<td></td>
			<td>DMEM containing 20% FBS, 5 &mu;M &beta;-mercaptoethanol, 50 &mu;g/ml ECG, and 1x penicillin/streptomycin</td>
		 </tr>
		 <tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>ECGS</td>
			<td>Biomedical Technologies, Inc.</td>
			<td>BT-203</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>384-well culture plate</td>
			<td>Greiner</td>
			<td>T-3037-6</td>
			<td>Plate was prepared with a thin collagen gel, as described in 6; working surface area 10 mm2</td>
		 </tr>
		 <tr>
			<td>Collagen type I</td>
			<td>BD</td>
			<td>354236</td>
			<td>Use at a final concentration of 1.5 mg/ml</td>
		 </tr>
		 <tr>
			<td>M199, 10x</td>
			<td>Invitrogen</td>
			<td>11825-015</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Syto-16</td>
			<td>Invitrogen </td>
			<td>S7578 </td>
			<td>Used as directed in 13 </td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Stereoscopic microscope</td>
			<td>Nikon </td>
			<td>SMZ645</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cell culture incubator</td>
			<td>Thermo </td>
			<td>3110 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>DynaMag</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Table-top centrifuge</td>
			<td>Thermo</td>
			<td>75002430</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation of embryonic ventricular endothelial cells

细胞培养，大大提高了我们的能力，评估个人的无数文化条件下的细胞群体。永生化的细胞系提供了其易用性的显着的优点，这些细胞系对所有潜在的细胞类型是不可用的。隔离从一个特定的感兴趣区域的，特别是来自转基因小鼠的原代细胞，可以进行更细致的研究。的基本技术包括解剖的器官或部分感兴趣的器官（ 例如心脏或心脏的特定区域）和单细胞解离的器官。与磁珠结合到感兴趣的细胞类型的抗体，该抗体识别这些细胞，然后进行培养。感兴趣的细胞然后可以使用磁铁进行分离，解离的细胞从有孔玻璃珠和一个短的胰蛋白酶孵育。在这些分离的细胞可以被培养，分析的需要。这种技术最初是专为成年小鼠器官，但也可以很容易地缩小与胚胎器官的使用，本文所证明。因为我们的兴趣是在发展中国家的冠状动脉血管，我们想研究这个细胞群体，在特定的胚胎阶段。因此，原始的协议进行修改，以兼容胚胎脑室的小尺寸和低电位在这些发育阶段的血管内皮细胞的产率。利用这种按比例缩小的方法，我们已经评估冠状动脉丛转基因胚胎心室重构的内皮细胞。

使用内皮特定的抗体，该抗体识别CD31，分离内皮细胞，胚胎（E）13.5（ 图1A）和18.5（ 图1B-1D）的胚胎从心室。当未处理的培养皿上生长，这些细胞保持圆形，将形成一个鹅卵石图案，如果附近汇合（ 图1A）。稀释胶原蛋白也被用于涂覆的孔，而内皮细胞将坚持（ 图1B），它们形成较少的链比镀上的胶原蛋白凝胶中（ 图1C和1D）。内皮...

Watch this Scientific Journal Video about Isolation of Embryonic Ventricular Endothelial Cells at JoVE.com

Isolation of Embryonic Ventricular Endothelial Cells

原代细胞培养是一个非常有用的技术分析特定的细胞群体，在特定的发展阶段，特别是从转基因小鼠胚胎。这里，胚胎脑室解剖的和离解，抗体共轭的有孔玻璃珠的识别和分离出来的血管内皮细胞进行进一步的分析。

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-embryonic-ventricular-endothelial-cells

Research

JoVE Journal

Biology

9.3K 次观看.  University of North Carolina at Chapel Hill. 原代细胞培养是一个非常有用的技术分析特定的细胞群体，在特定的发展阶段，特别是从转基因小鼠胚胎。这里，胚胎脑室解剖的和离解，抗体共轭的有孔玻璃珠的识别和分离出来的血管内皮细胞进行进一步的分析。

视频: Isolation of Embryonic Ventricular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

分离人脐静脉内皮细胞（HUVEC）

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

科研

生物学

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

胚胎干细胞衍生的内皮细胞治疗肢体缺血

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

禽流感胚胎瓣膜祖细胞的分离和培养

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

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 Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

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 ...

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 ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...