Name: generation and culture of blood outgrowth endothelial cells from human peripheral blood
Uploaded: 2015-12-23T14:00:00
Duration: 11 min
Description: Watch this Scientific Journal Video about Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood at JoVE.com

This work was supported by grants funded by the British Heart Foundation (BHF), Dinosaur Trust, McAlpine Foundation, Fondation Leducq, Fight for Sight, the Cambridge Biomedical Research Centre, National Institute of Health Research including (i) the BHF Oxbridge Centre of Regenerative Medicine [RM/13/3/30159], (ii) the BHF Cambridge Centre of Research Excellence, (iii) Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust and (iv) Papworth Hospital NHS Foundation Trust, and supported the Cambridge NIHR BRC Cell Phenotyping Hub. MLO is funded by a BHF Intermediate Fellowship. FNK is funded by a BHF PhD Studentship.

所述BOEC生成协议的示意图示于图1。 1.采血车和密度梯度离心<ol><li>对于每一个捐赠者，加3毫升柠檬酸钠每两个50毫升锥形离心管中。收集将60ml静脉抽血，并加30毫升每管。反转轻轻2-3次混合。使用少到40个毫升血液中的协议，与相应的调整柠檬酸卷。 注：重要的是血液样品2小时内收集的处理。延迟显着减少生长菌落产量血结果的处理。 </li><li>制备含有密度梯度离心介质通过首先颠倒瓶子几次以混合新锥形管。在无菌罩，每50 ml的新增15 ml的密度梯度离心介质的锥形管（1管每10毫升血液，每个供体6管）。 </li><li>稀释血液1:1用Dulbecco磷酸盐缓冲盐水（DPBS）。倾斜含有密度梯度离心介质为20°的工作表面的角度的管。使用移液管助剂和25毫升血清吸管，缓缓通过逐渐加入血液向下倾斜管壁层上的介质的顶部21毫升稀释血。 注意：这样做将减少血液与密度梯度层的混合和将产生更紧密的血沉棕黄层，以及增强的产量。 </li><li>离心样品在400×g离心35分钟，在室温与加速器和制动器关闭。 </li><li>在此离心期间，开始胶原蛋白涂层步骤详见2.1 - 2.4。确保在进行步骤3.1之前，这些步骤已经完成。 </li></ol>的T-75烧瓶和培养介质的制备2胶原涂层注：执行在细胞培养罩以下步骤。 <ol><li>通过稀释股票泰准备50微克/毫升胶原溶液PE I型胶原在10ml 0.02M乙酸实施例的：对胶原的库存为4.05毫克/ ml的浓度，添加123.5微升胶原至10毫升的0.02M乙酸。无菌过滤器胶原悬浮液在使用之前。 </li><li>使用血清吸管，加7.5毫升的胶原溶液为T-75细胞培养瓶。本卷给出了5微克胶原/ 平方厘米（或375微克/瓶）。外套烧瓶1小时，在室温。 </li><li>准备通过补充内皮基础培养基与由制造商提供的生长因子补充剂用于BOEC代内皮生长培养基中。添加所有的补充，但不添加血清。为了制备15毫升BOEC代培养基中，添加2.5 ml的限定的胎牛血清（FBS）的12.5毫升含有生长因子补充剂内皮生长培养基。 </li><li>继续执行在第3节中列出的1小时涂层期后的步骤，吸出胶原溶液洗去残留乙酸吹打在10ml DPBS。吸落DPBS并重复此清洗步骤一次。如果在步骤3.3中得到的细胞悬浮液是没有准备好，此时电镀，BOEC生成介质的添加5毫升的烧瓶中，以保持从干燥出胶原涂层。 </li></ol> 3.收集和外周血单个核细胞的电镀<ol><li>以下，在步骤1.4中概述的密度梯度离心，小心收集使用无菌塑料移液管血沉棕黄层层。收集的棕黄色大衣应该从每管产生约20 mL细胞悬液和血浆。避免传输密度梯度​​介质。 </li><li>稀释单个核细胞悬液1:1 DPBS并颠倒混合。离心在RT 20分钟，在300×g离心以制动器和加速器最大。 </li><li>离心后，通过加入1ml BOEC生成介质的与每个粒料和吹打上下反复吸出上清，重悬细胞。游泳池细胞悬液的ð充值总额10毫升，其余网上平台。 </li><li>为了获得总细胞数，样品10微升细胞悬浮液的估计和稀释50倍与490微升土耳其人的解决方案，这将溶解红细胞。计数使用血球稀释细胞悬浮液的10微升样品。 注：血液60毫升应为电镀收率约100-150×10 6个白细胞。 </li><li>板整个细胞悬浮液到一个单一的，胶原包被的T-75烧瓶中，补足培养基体积到15毫升/烧瓶中并培养在37℃下在含有5％ 的 CO 2的潮湿空气中。细胞铺板在这个时候表示通道0。 注意：可以将冷冻的外周血单个核细胞分离之前BOEC隔离以导出BOECs在稍后的时间或在为了运输的外周血单个核细胞，以不同的实验室可以在其中生成BOECs。然而，值得注意的是，冷冻外周血单个核细胞可以减少BOEC隔离的效率是重要的。小号下面EE第8条的方法。 </li></ol> 4.长期细胞培养<ol><li>通过加入15毫升新鲜BOEC代介质改变每2天介质（5：1的混合物内皮细胞生长培养基中，并限定FBS）中。在周末，介质改变可以延迟到每3天。 注：生长菌落7至14天的培养出现。 </li><li>监测7-14天的生长菌落外观BOEC培养瓶中。确定殖民地的细胞表现出典型的内皮细胞的鹅卵石形态圆形群体。 </li><li>一旦一个群体或多个群体中的烧瓶中确定，继续用中的改变，允许菌落传代才增长到大约1000至2000个细胞每殖民地。通过粗略目测估计确定每个细胞集落细胞数。 注：作为指南，这是习惯通过初始生长的菌落一个星期后的第一个产物殖民地的身份。 </li> &lt;LI&gt;传代细胞冲洗两次瓶10毫升的DPBS。加入5 ml的1X胰蛋白酶-EDTA的孵育在37℃的培养箱中培养5分钟。 5分钟后，中和胰蛋白酶，用10毫升（含FBS）培养基中，并把细胞悬浮进反复移液。 </li><li>离心悬浮液在300×g离心5分钟，悬浮在15毫升新鲜BOEC代介质和板整个细胞悬液到一个新的T-75烧瓶（表示通道1中，P1）。没有胶原涂层是必需的。 </li><li>继续如上所述直到细胞汇合（每瓶约3-5×10 6细胞）的培养基的变化。一旦汇合，代细胞如步骤4.3中所述。 注意：从第2代开始，细胞不再需要BOEC生成介质，并且可以是在内皮细胞生长培养基和10％的标准热灭活FBS中培养。胶原涂层可以作为一个可选步骤前进，因为有证据表明，胶原蛋白的存在可以增强BOEC prolifer通货膨胀率。 </li><li>对于连续传代，盘不下75万细胞每T-75瓶低细胞密度会导致BOECs停止增殖。 </li></ol> 5.冻融BOEC文化<ol><li> 4.3节和离心机在300 XG描述5分钟Trypsinize细胞。吸出上清液，重悬在10毫升培养基。这并不需要内皮细胞生长培养基;含10％FBS的标准将是足够的。收集的细胞悬浮液10微升样品使用血球执行手动细胞计数。 </li><li>离心再次重悬在2×10 6个细胞/在冰冷的冻存培养基（40％DMEM，50％FBS和10％DMSO）中毫升。加入0.5毫升（10 6个细胞）到每个小瓶中，小瓶的地方，在冰冷的异丙醇冻存容器并将其放置在-80℃冷冻至少2小时之前转移到液氮中。不要让电池在-80℃下超过24小时。 </l我&gt; <li>解冻细胞，加入10 ml的预热介质到15ml锥形离心管中。 注意：当解冻出通道1细胞，解冻成补充有限定FBS的解冻后的第2天的20％的内皮细胞生长培养基。这导致更稳定的细胞分离前进。 </li><li>除去从液氮和解冻在37℃水浴中，轻轻摇动细胞直到只有小冰晶留在小瓶中。的小瓶滴加的内容添加到所述锥形离心管和降速，在300×g离心5分钟。 </li><li>吸去上清液和重悬细胞沉淀在10ml EGM-2MV + 10％FBS。加入T-75瓶加满油中到15ml。 </li></ol> BOECs通过流式细胞仪6.表征<ol><li>曾在第4节中描述的BOEC殖民地被允许扩大，trypsinize细胞作为第4.4节，重悬在每毫升10 6细胞染色缓冲液的浓度描述（DPBS机智H 2％FBS和2mM EDTA）中。 </li><li>结合10 5个细胞与针对CD45，CD14，CD34，CD31（使用全部1:20稀释）或VEGFR2（稀释1:10）荧光标记的抗体（ 见表 1的全 ​​部细节）。在黑暗中温育30分钟，在4℃。 </li><li>洗涤细胞用1ml染色缓冲液和离心机在300×g离心5分钟。吸上清，重悬在分析400微升染色缓冲液。保持细胞在4℃下并避光到分析前保护。 </li><li>进行细胞计数分析在装有以检测测定中使用的荧光标记的抗体细胞仪。 </li><li>使用未染色BOEC样品通过调整前向和侧向散射的电压，以及用于FITC和APC通道的电压，以确定在流式细胞仪的细胞群。 </li><li>确定选通使用同型染色的细胞为每个荧光阈值。评估阳性基于所述presenc每个细胞表面标记物Ë峰值转变荧光强度与同种型对照的荧光染料被分析。 </li></ol> BOECs通过免疫荧光7.表征<ol><li>板BOECs在24孔，平底组织培养板，而不盖玻片以5×10 4个细胞的500微升内皮细胞生长培养基的密度+ 10％热灭活的每孔FBS和离开附着并生长在37 ℃，5％的CO 2，直至细胞覆盖每个的表面积的至少70％-80％以及（通过目测估计汇合下的光microsope）。 </li><li>在每5分钟1毫升的DPBS洗涤细胞良好。 </li><li>修复细胞O / N在4℃下用500微升的DPBS 4％多聚甲醛溶液。 </li><li>为3×5分钟1毫升每孔的DPBS洗涤细胞。添加和删​​除用移液管和吸气的DPBS，分别。 </li><li>通透细胞3×10分钟，0.2％聚山梨醇酯20中的DPBS。 </li&gt; <li>在封闭含有10％FBS的DPBS中缓冲孵育细胞1小时，在室温。 </li><li>在300微升抗体稀释缓冲液中含有针对CD34（10微克/毫升），CD144第一抗体（在DPBS中的0.1％牛血清白蛋白）染色的细胞在4℃CO / N（1：300）或vWF的（1：250） 。 请参阅表1的全 ​​部细节。 </li><li>为5×10分钟用DPBS冲洗掉未结合的初级抗体。 </li><li>孵育细胞1小时在室温用适当的荧光标记的第二抗体，如（1所有：200）详述于表1中。 </li><li>为5×10分钟用DPBS冲洗掉未结合的二抗。 </li><li>孵育细胞与1mg / ml的4&#39;，6-二脒基-2-苯基吲哚（DAPI）在DPBS在室温10分钟。 </li><li>另外3×5分钟洗涤细胞用DPBS。 </li><li>在放大100倍使用倒置显微镜配备有外部光源，用于荧光激发用附加图像的细胞，并捕获图像编数码相机进行后续分析离线。 </li></ol> 8.冻融外周血单个核细胞<ol><li>在阶段3.2的端部，以下离心，吸出介质并手动破坏细胞沉淀反复吹打上下使用P1000枪头。加入1毫升冷冻培养基，90％血清和10％DMSO的，确保轻轻混匀，以产生细胞悬浮液。细胞悬液转移到离心管和冻结和解冻的第5节。 </li></ol>

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The authors have nothing to disclose.

We present a detailed protocol that allows for the robust and efficient derivation of BOECs from adult peripheral blood mononuclear cells (PBMNCs). Our protocol includes two important refinements that represent advances on previous methods of BOEC isolation.14-16 These include the absence of heparin in the initial PBMNC culture medium and the use of defined, embryonic stem cell-qualified serum. This latter refinement is of particular importance. Embryonic stem cell (ESC)-qualified serum is a more consistent grade of serum and, although it is not known yet what component(s) are enriched in the serum that benefit BOEC isolation, the impact of this defined serum on the efficiency of BOEC generation is clear in our hands. In addition, we have also had success in isolating BOECs using human serum, thereby allowing for the generation of BOECs for clinical translation. In our hands, this refined protocol results in the successful isolation of stable BOEC cultures from greater than 90% of donors, making it one of the most reliable BOEC generation methods reported thus far. Although the use of particular sera is critical to BOEC generation, it also represents a primary limitation of the current protocol. Future improvements to the technique could include the generation of these cells in serum-free, defined culture conditions.
Critical Steps in the protocol include processing blood samples as soon as possible after collection, complete harvesting of the buffy coat cells after density gradient centrifugation and the timely passaging of initial colonies from P0 to P1. This passaging step is critical to establishment of a stable isolation. Like other endothelial cells, BOECs appear to be very sensitive to plating density. If the plating density after passaging is too low, the BOECs will not proliferate. Conversely, if the colonies are allowed to become overconfluent before passaging, the cells will also cease to proliferate and have the tendency to convert into an elongated, mesenchymal cell phenotype. If few colonies appear from days 7 to 14, or if the colonies are small in size, troubleshooting can include increasing cell density by passaging P0 colonies into a T-25 flask instead of a T-75.
Once the technique is mastered, the resultant BOECs can be used in several applications, including in vitro studies of endothelial cell biology, disease modeling and drug screening, as well as in vivo cell transplantation therapies. An important consideration for the development of any cell therapy process is to use cells that are free from pathogenic mutations. We have previously shown that BOECs isolated using our protocol possess genomes that are free from copy number variations and are thus representative of the individual from which they were collected. In addition, we have also demonstrated that the majority of BOEC-derived iPSC lines are free from copy number variations.13 This contrasts with previous reports of copy number variation in fibroblast-derived iPSCs. To date, these cells remain the only iPSCs for which this degree of genomic fidelity has been reported. This feature is important for the field of iPSC biology and the use of iPSCs in disease modeling, drug screening and future cell transplantation therapies.

直到最近，产后代新血管被认为是通过被称为血管生成的方法，定义为从预先存在血管内皮细胞的新血管的萌发发生完全。1本过程形成鲜明对比的血管，或从头形成血管内皮前体细胞，这被认为胚胎发育过程中发生的专门2然而，最近的研究已经鉴定和分离在成人的外周血循环内皮祖细胞（EPCs）。这些细胞具有分化成成熟的内皮细胞在培养的能力，被认为参与产后血管。3,4- 协议用于分离和扩增这些内皮祖细胞通常涉及含有内皮细胞生长因子，包括vascul外周血单核细胞（外周血单个核细胞）中的媒体的培养芳内皮生长因子（VEGF）和成纤维细胞生长因子-2 5-8 EPC培养产生各种显着不同的细胞类型。初始培养物（&lt;7天）由单核细胞类型，在文献中被称为“早”内皮祖细胞主导。尽管他们的名字，这些细胞表达单核细胞标志物CD14，是阴性的祖标志物CD34和表达经典内皮细胞标志物CD31和血管内皮生长因子受体2（VEGFR2）的唯一的最低水平。5续文化产生了细胞的二次人口，称为晚期生长内皮祖细胞或血长出内皮细胞（BOECs），其中出现的内皮样细胞为谨慎菌落。不同的是单核细胞早期内皮祖细胞，BOECs，这也被称为内皮细胞集落形成细胞（ECFCs），生长内皮细胞或后期生长的内皮细胞，表现出鹅卵石形态即是典型的内皮细胞单层，并在表面标志高度相似5和基因表达9到成熟内皮细胞。 从外周血内皮样细胞的产生提供了几个优点，特别是对于的内皮细胞功能障碍与血管疾病例如肺动脉高压（PAH）10，或血管性血友病11之前BOECs的可用性，内皮相关的研究细胞只能来自于植器官的死亡或器官移植，或隔离的时间从脐静脉出生。这种减小的可用性是一个严重的限制，以从患者的心血管疾病，以及内皮细胞和任何血细胞或壁细胞之间的相互作用的理解内皮细胞生物学。此外，分离和这些来源的培养的内皮细胞的一个纯人口在技术上是挑战性和由这些方法获得的细胞具有仅一个LIMITEð增殖能力。因此BOECs提供有价值替代物源自患者的原代内皮细胞的分离和培养。 除了 ​​它们在体外应用 ，BOECs，可以在自体细胞移植疗法可能有用。这些应用包括内皮细胞移植，以促进新血管形成（见12和其中的参考文献），以及诱导的多能干细胞的产生（iPS细胞）。13 BOEC衍生的iPSC可用于疾病建模，并提供巨大潜力作为出发材料为自体细胞治疗。 BOECs重新编程更快，具有更高的效率比皮肤成纤维细胞。此外，BOECs还允许iPSCs的是自由核型异常，这是任何技术，就可以适于平移应用的基本的特征的产生。以产生从患者血液样品中的iPSCs的能力LSO省去了皮肤活检和皮肤成纤维细胞的产生，从而促进细胞的产生，从患者的伤口愈合障碍，或非常年轻。 下面详细说明该协议，批准，并按照国家研究伦理服务委员会（英格兰东部）的指导方针进行，提供了简单可靠的方法BOECs的一代从一个相对小的体积大于90％的效率（60外周血毫升）。这些细胞是高度增殖性和可反复传代，允许亿万细胞从单个血样的产生。

<table>
	<tbody>
		<tr>
			<td colspan="4">For blood collection</td>
		</tr>
		<tr>
			<td>60 mL syringe with luer-lok tip</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>19G Surflo Winged Infusion Set</td>
			<td>Terumo</td>
			<td>SV-19BL</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tube</td>
			<td>StarLab</td>
			<td>E1450</td>
			<td>2 per donor</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Martindale Pharmaceuticals</td>
			<td>270541</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For buffy coat isolation</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (without Ca2+ and Mg2+)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile wrapped plastic transfer pipettes</td>
			<td>Appleton Woods</td>
			<td>KC231</td>
			<td></td>
		</tr>
		<tr>
			<td>Turk&#8217;s Solution</td>
			<td>Millipore</td>
			<td>1.093E+09</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For cell culture, passaging and freezing cells</td>
		</tr>
		<tr>
			<td>Type 1 Collagen (derived from rat tail)</td>
			<td>BD Biosciences</td>
			<td>35-4236</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (without Ca2+ and Mg2+)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.02M Acetic Acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td>prepared in reagent grade water</td>
		</tr>
		<tr>
			<td>Endothelial Growth Medium-2MV 
			(containing Bullet Kit, but not serum)</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>Note: It is essential that the medium does not contain heparin.&#160;&#160; 
			Do not use EGM-2.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (U.S.), Defined</td>
			<td>Hyclone</td>
			<td>SH30070</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Trypsin EDTA</td>
			<td>Gibco</td>
			<td>T4174</td>
			<td>Dilute to 1x in PBS prior to use</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty Freezing Container</td>
			<td>Sigma-Aldrich</td>
			<td>C1562</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For flow cytometric characterization</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD14</td>
			<td>BD Biosciences</td>
			<td>555397</td>
			<td>Mouse IgG1k, Clone: WM59 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD31</td>
			<td>BD Biosciences</td>
			<td>555445</td>
			<td>Mouse IgG1k, Clone: WM59 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti-human CD34</td>
			<td>BD Biosciences</td>
			<td>555824</td>
			<td>Mouse IgG1k, Clone: 581/CD34 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>560976</td>
			<td>Mouse IgG1k, Clone: HI30 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>&#160;APC-conjugated mouse anti-human VEGFR2</td>
			<td>R&#38;D Systems</td>
			<td>FAB357A</td>
			<td>Mouse IgG1, Clone: 89106 
			Dilution: 1:10</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse IgG1k isotype control</td>
			<td>BD Biosciences</td>
			<td>555748</td>
			<td>Clone: MOPC-21 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1k isotype control</td>
			<td>BD Biosciences</td>
			<td>555751</td>
			<td>Clone: MOPC-21 
			Dilution: 1:20</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1k isotype control</td>
			<td>R&#38;D Systems</td>
			<td>IC002A 
			Dilution: 1:10</td>
			<td>Clone: 11711</td>
		</tr>
		<tr>
			<td>EDTA, 0.5M solution</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">For immunofluorescent microscopy&#160;</td>
		</tr>
		<tr>
			<td>Corning Costar 24-well tissue culture plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human CD34 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB72271</td>
			<td>Clone 756510, IgG1, use at 10 &#956;g/ml</td>
		</tr>
		<tr>
			<td>Polyclonal goat anti-human VE-cadherin (CD144)</td>
			<td>R&#38;D Systems</td>
			<td>AF938</td>
			<td>Antigen affinity- purified IgG, use at 1:300</td>
		</tr>
		<tr>
			<td>Monoclonal rabbit anti-human Von Willebrand Factor (vWF)</td>
			<td>Abcam</td>
			<td>ab154193</td>
			<td>Clone EPSISR15, use at 1:250</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-21202</td>
			<td>Polyclonal, 2 mg/ml, use at 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11055</td>
			<td>Polyclonal, 2 mg/ml, 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 568 conjugate</td>
			<td>Life Technologies</td>
			<td>A-10042</td>
			<td>Polyclonal,&#160; 2 mg/ml, 1:200</td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>use at 1 &#956;g/ml</td>
		</tr>
	</tbody>
</table>

generation and culture of blood outgrowth endothelial cells from human peripheral blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

从60毫升血液的血沉棕黄层的单核细胞的分离通常产生100-150x10 6总白血细胞。当镀成一个单一的T-75烧瓶中，将大量单元中未溶解的细胞悬浮液使得难以用明场显微术解决个别贴壁细胞。反复介质改变导致非粘附细胞的清除，并允许单核细胞的“早”内皮祖细胞粘附人口的可视化。在第7天，在T-75将包含约这些细长粘附细胞7×10 6。从第7天至14，BOECs的菌落出现的烧瓶（图2A）内。菌落第一显示为3至5个相邻的单元。长出菌落数可从每60毫升血液1至10个菌落而变化。通常，年轻供（即，20-25岁）趋向于产生更多数量的产物的菌落，这也较早在培养出现的。BOECs从细胞中的这个中心组增殖，形成圆形菌落由几百个细胞。这些殖民地内的细胞表现出典型的内皮鹅卵石形态。 优选通路初始菌落当它们包含每菌落约1000个细胞。生长的菌落通常其原貌在培养后7天达到这个规模。一旦原始菌落传代到新的T-75烧瓶中，它们应该增殖以形成汇合单层（每烧瓶3-5x10 6个细胞 ）在5天或更少（图2B）。在隔离的小百分比（&lt;10％），长出的菌落无法显示，或不增殖充分以下这个初始传代步骤。他们的第一次传代后表现出低增殖BOECs很少能够继续成为稳定BOEC隔离并经常阻止两个人到三段之内激增。单核细胞包含在BOEC培养物中早期的EPCs是非增殖性并且通常从培养清零1-2通道。这些早期的细胞间隙是由于细胞死亡，早期的细胞未能传代和稀释的非增殖早期内皮祖细胞反复传代后，再坚持。 通道1细胞既可以进一步在培养传代或冷冻向下供以后使用。一旦产生了稳定的隔离，细胞可以在1汇合烧瓶至3-5新的T-75烧瓶的速率进行传代，直到通路8或9成为静止之前。再次，细胞密度是在整个培养关键的。根据我们的经验，没有少于750000细胞应接种于T-75烧瓶中，作为低细胞密度可导致生长停滞。尽管BOECs的高增殖潜能，细胞应该也不会被允许成为overconfluent（即，&gt;每瓶5×10 6个细胞 ），因为这可能会导致convers<html> BOECs的离子到非增殖性表型。 我们建议，任何细胞被标记为BOEC应内皮标志显示适当的细胞表面和细胞内染色。 BOECs表征可以通过流式细胞术（图3）或荧光显微镜（图4）来实现，以下染色典型的内皮细胞标记物。 BOECs是积极为内皮细胞表面标志物CD31和VEGFR2和为负的单核细胞标志物CD14和泛白细胞标记CD45。 BOECs也posess维贝尔-Palade小体，从而表达血管性血友病因子（vWF）作为离散的，点状胞浆染色。不像其他的成熟内皮细胞类型，如肺动脉或主动脉内皮细胞，BOECs也表达在其表面上的祖细胞和活化标志物CD34。 ftp_upload / 53384 / 53384fig1.jpg“/&gt; 图1. BOEC生成协议的示意图。外周血单核细胞是从静脉血中分离通过密度梯度离心和培养在胶原包被的平板在内皮生长培养基含有限定FBS中。 BOEC殖民地在7-14天文化的出现。 <a href="https://www.jove.com/files/ftp_upload/53384/53384fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53384/53384fig2.jpg" /> 图代表BOEC培养物的明视2.图像。（A）中生长的菌落出现在本为内皮样细胞，其被布置在鹅卵石单层并增殖径向从一个中心点的集合天7和14之间的菌落培养物。围绕生长菌落附着的单细胞性细胞构成细胞的绝大多数早期文化。这些细胞，前面描述为“早”内皮祖细胞，具有纺锤状形态和表达单核细胞标志物CD14。 （B）按照传代，所述高度增生BOECs接管培养，作为非增殖性单核细胞或者死光或不传代后重新连接。比例尺为250μm。 <a href="https://www.jove.com/files/ftp_upload/53384/53384fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53384/53384fig3.jpg" /> 图BOECs的通过流式细胞术3.表征。BOECs用胰蛋白酶处理并用荧光标记的同种型对照抗体（灰色填充的峰），或直接抗特定表面标志物（红线）。表面标志物流式细胞仪鉴定包括造血标志物CD45和CD14，内皮标记CD31和VEGFR2和progentior和内皮细胞活化标志物CD34。 <a href="https://www.jove.com/files/ftp_upload/53384/53384fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53384/53384fig4.jpg" /> BOECs图4.免疫荧光染色为内皮细胞标记物。BOECs的代表性免疫荧光图像用针对内皮细胞表面标记物CD144的抗体免疫染色（VE-钙粘蛋白，右上图）和血液糖蛋白血管性血友病因子（vWF和右下面板） 。对应的面板表示核DAPI染色示到左侧。比例尺为50μm。为CD144。 <a href="https://www.jove.com/files/ftp_upload/53384/53384fig4large.jpg" target="_blank">请点击此处查看大图</a>版本这个数字。

Watch this Scientific Journal Video about Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood at JoVE.com

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

人外周血代文化血液的产物内皮细胞

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

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