这种材料是根据国家科学基金会资助的 No. 的工作。CMMI-1452943 和阿肯色大学荣誉学院。我们还要感谢阿肯色州脐带血库为我们提供脐血单位。

这项研究是在阿肯色大学机构审查委员会的批准下进行的 (批准编号 16-04-722)。在阿肯色州脐血库的柠檬酸盐磷酸葡萄糖 (方案) 溶液中收集脐血单位, 而不符合储存要求的单位则捐给研究。在环境温度下, 脐带血单位在收集24小时内快递到实验室. 
 1. 从脐血中分离内皮祖细胞 <ol> <li> 试剂的制备。
	<ol> <li> 通过将内皮基底培养基 (循证医学) 添加到10% 胎牛血清 (FBS), 并辅以 20 ng/毫升胰岛素样生长因子 (igf-i), 以制备华, 1 和 #181; g/毫升抗坏血酸, 5 ng/毫升重组人表皮生长因子 (肽), 22.5 和 #181; g/毫升肝素, 0.5 ng/毫升血管内皮生长因子 (VEGF), 以及 10 ng/毫升重组人成纤维细胞生长因子 b (rhFGF b), 0.2 和 #181; g/毫升氢化可的松, 2% 青霉素-链霉素-谷氨酰胺。用 0.2 #181 砜 (PES) 膜真空过滤器对培养基进行杀菌. </li>
		<li> 制备大鼠尾 i 型胶原溶液, 浓度为50和 #181; 使用0.02 的乙酸。使用0.2 和 #181; m 注射器过滤器对此解决方案进行消毒. </li>
		<li> 预涂 6-井板2毫升制备的大鼠尾 i 胶原溶液为每一个井。将它们放置在 5% CO 2 和37和 #176 的恒温箱中, 在播种前至少1小时. </li>
		<li> 用汉克斯平衡盐溶液 (HBSS) 在1:1 浓度下稀释大约25毫升脐血. </li>
		<li> 使用150毫米氯化钠、1% 海卫 X-100、0.5% 钠胆酸、1毫米和 #946、甘油、0.1% SDS 和 50 mm 三 (pH 8.0) 制备无线电免疫沉淀试验 (帕) 缓冲液. </li>
	</ol> </li> <li> 隔离内皮祖细胞。
	<ol> <li> 将所有准备好的试剂放在37和 #176 的水浴中预热; 隔离之前的 C. </li>
		<li> 在50毫升离心管中加入20毫升密度梯度介质试剂. </li>
		<li> 小心地在含有密度梯度介质的离心管中20毫升稀释的脐带血, 而不打破这一层, 目的是将吸管对准管子的侧壁. </li>
		<li> 分离血液的组分根据他们的密度 ( 图 1 ) 由离心在 800 x g 为30分钟在室温下, 与离心机转子的闸。请勿干扰不同的层. </li>
		<li> 通过小心地将等离子层移到管外, 而不干扰其他细胞层, 直到吸管尖端能够到达跨国公司层 (请参见 图 1 )。丢弃已移除的等离子体. 
		注: 在使用吸管提示去除等离子体的同时, 在跨国公司层 ( 图 1 ) 的上方留出一小层等离子, 以减少干扰. </li>
		<li> 使用装有18口径针头的注射器并将其转移到一个新的离心管上, 收集包含跨国公司的巴菲外衣. </li>
		<li> 向所收集的跨国公司添加同等数量的循证医学. 离心这些管在 500 x g 为10分钟在4和 #176; 放弃上清. 
		注: 细胞团将出现红色, 因为存在红细胞/红细胞. </li>
		<li> 添加5毫升氯化铵溶液, 以溶解红血球。在冰上孵育这管 5-10 分钟, 偶尔晃动. 
		注意: 在这一步中应谨慎行事, 不要超过时间期限, 因为这可能对跨国公司有害. </li>
		<li> 离心管 500 x g 为10分钟, 在4和 #176; C 和丢弃上清。如果红细胞坚持, 重复步骤1.2.8 和 1.2.9, 直到没有看到红色的颗粒. </li>
		<li> 在准备好的华中重颗粒, 并使用例来计算跨国公司的数量. </li>
		<li> 吸入鼠尾 i 胶原蛋白涂层 6-井板 (从步骤 1.1.3) 和洗涤井三次与1x 磷酸盐缓冲盐水 (PBS). </li>
		<li> 种子的跨国公司颗粒悬浮在华到胶原板的浓度为 1 x 10 7 跨国公司在每个井。将它放在37和 #176; C, 5% CO 2 孵化器. </li>
		<li> 24 小时后, 吸气培养基, 用华冲洗一次。添加3毫升的华介质到每一个井, 并把它放回37和 #176; C, 5% CO 2 孵化器. </li>
		<li> 每天7天用新鲜华培养基补充板材。7天后, 每隔一天更换华介质. </li>
		<li> 使用明亮的野外显微镜, 每天拍摄6孔板的图像, 以追踪内皮细胞的进展情况。标记的板块, 殖民地出现, 以保持他们的成长轨迹. 
		注意: 它通常需要5和9天的殖民地出现在文化中. </li>
	</ol> </li> <li> 扩展内皮祖细胞。
	<ol> <li> 将 T-25 培养瓶与鼠尾 i 型胶原蛋白 (50 和 #181; g/毫升), 并将其放置在37和 #176; C, 5% CO 2 孵化器至少60分钟的吸气和洗涤 T-25 细胞培养瓶三次与 1x PBS 之前播种的细胞. </li>
		<li> 处理的内皮细胞, 当他们达到约3毫米大小使用150和 #181; 每井0.05% 胰蛋白酶-EDTA 溶液。将6井板放回37和 #176; C, 5% CO 2 孵化器 2-3 min. </li>
		<li> 轻轻敲击6井板, 将所附的电池取出。立即添加2毫升的华, 并恢复15毫升离心管中的细胞. </li>
		<li> 使用例计数单元格并计算初始种子密度. </li>
		<li> 旋转15毫升离心管在 400 x g 重5分钟的细胞团在华和种子 T-25 烧瓶与〜50万细胞。将此烧瓶标记为通道 1, 并将 T-25 烧瓶放回37和 #176; C, 5% CO 2 孵化器. 
		注意: 细胞可以被镀在循证医学和 DMEM 来比较的增长与华. </li>
		<li> 对于进一步的通道, 当烧瓶达到90% 汇合, 按照步骤 1.3.1 1.3.5 和设定的细胞在 10 4 细胞/cm 2 T-75 或 T-175 细胞培养烧瓶。继续描述隔离单元格 (步骤2和 3). </li>
	</ol> </li> </ol> 2。利用西印迹的分离细胞的特性 <ol> <li> 添加 50-100 和 #181; 在每个通道中, 当按照扩展步骤来描述孤立细胞的特征时, 会溶解缓冲液。收集大约50万或更多细胞的蛋白质. </li>
	<li> 吸管向上和向下用力, 以破裂的细胞和释放的蛋白质。将缓冲区收集并传输到离心管. </li>
	<li> 旋转离心管在 500 x g 和小心地转移上清到一个新的离心管。标签和存储的管在-80 和 #176; C 长期存储. </li>
	<li> 量化每管中的蛋白质数量使用布拉德福德和 #39; s 或分析分析 27 , 28 , 29 . </li>
	<li> 使用标准程序执行西文印迹 27 , 28 , 29 。分析5和 #181; CD31、CD34、VEGFR2 和 #945 表达的总蛋白 g;-SMA。使用和 #945;-蛋白用于对数据进行规范化. </li>
</ol> 3。间接免疫荧光 <ol> <li> 几种18毫米玻璃片在50% 乙醇和干燥他们。将清洁的片放在一个12井的盘子里过夜, 在生物安全罩里, 用紫外线灯消毒. </li>
	<li> 用50和 #18 涂片1、大鼠尾 i 型胶原蛋白, 并将板材转移至37和 #176; C、5% CO 2 孵化器至少 60 min. </li>
	<li> 吸出胶原蛋白, 并将1毫升的 1x PBS 添加到 12-井板上以清洗片。吸气 1x PBS 并重复两次. </li>
	<li> 种子25万培养内皮在每个井并且安置他们回到37和 #176; C, 5% CO 2 孵化器在执行免疫之前至少3天. </li>
	<li> 用 1x PBS 清洗盘3次。在每个井中加入1毫升的 ice-cold 甲醇, 在室温下孵育15分钟来固定细胞. </li>
	<li> 执行免疫使用标准协议 27 、 28 、 29 。在适当的稀释上使用抗体 ( 例如, CD31 (1:20), CD34 (1:50), 和 #945;-SMA (1:100) 和 VEGFR2 (1:50)). </li>
	<li> 使用荧光显微镜拍摄 immunostained 片的图像. </li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Human+CD133++Non-adherent+Endothelial+Forming+Cells.">Isolation and Culture of Human CD133+ Non-adherent Endothelial Forming Cells.</a> Bio-Protocol. 6 (7), (2016).</li></ol>

作者没有什么可透露的。

如前所述, 附着内皮具有鹅卵石形态。我们的孤立的跨国公司从一个纺锤形的细胞殖民地 (图 2A-2D) 发展到一个鹅卵石殖民地 (图 2E-2F), 在文化中的十天内。内皮的标记不同的研究组, 即晚期内皮祖细胞10, 血管内皮细胞的形成,5, 或内皮祖细胞12。应该指出的是, 这些细胞在功?...

一些调查人员研究了人类内皮的特征和潜能5,10,11,12,13。内皮可以被描述为在缺氧、缺血、损伤或肿瘤形成的部位有能力黏附于内皮组织的循环细胞, 并有助于形成新的血管结构4,14。他们观察到的参与新生血管, 以产后血管的形式, 导致了对这些细胞的病理生理学的理解和他们的用途在治疗应用4,15, 16. 一个人的内皮的数量已被证明与心血管病病理学相关9、15、161718 ,20。其他研究也将内皮分化成成纤维细胞样表型, 并建议这些细胞可用于组织工程心脏瓣膜7,21。
由于调查4之间的差异, 没有明确确定分离内皮所需的特定细胞表面分子。跨国公司对某一矩阵的附着力, 暴露于各种培养条件下, 已由几个组1、17、22、23进行, 建议假定内皮可能显示不同的表型属性。这些性质包括缺乏吞噬能力, 管形成在基质, 和摄取 Dil-乙酰低密度脂蛋白。高克隆和增殖电位是内皮可以排列5的两个属性。内皮也可以形成体外小管当共与人胎肺成纤维细胞的4。这些细胞是已知的表达内皮细胞表面标记和分享一些造血标记13,24,25。积极表达的标记, 被广泛接受的分内皮是 CD31, CD34, 血管内皮生长因子受体 2 (VEGFR2), 冯血友病因子 (vWF), CD133, c-试剂盒, 血管内皮钙黏蛋白 (VE 钙黏附素)4,18. co-express CD90、CD45、CD14、CD115 或α-平滑肌肌动蛋白 (α-SMA) 的细胞不被认为是内皮, 因为它们的增殖潜能有限, 吞噬细菌的能力, 以及无法形成de 从头人类容器在体内4,7。本文概述了一个改进的协议, 分离内皮祖细胞从人脐血不需要任何细胞排序协议。本文以 CD31、CD34 和 VEGFR2 为阳性标记, 用α-SMA 作为负性指标。
本文提出了一种利用生长因子 (华) 对脐血内皮祖细胞进行无细胞分类的分离培养方法。该华包含血管内皮生长因子 (VEGF) 和成纤维细胞生长因子 (增殖素), 它能增强内皮细胞膜的存活、增生和迁移26。它还包括抗坏血酸, 它负责维持细胞的鹅卵石形态;胰岛素样生长 factor-1 (IGF-1), 提供血管生成和迁移功能;和肝素, 在培养基26中导致生长因子的长期稳定性改善。其他生长因子添加到内皮细胞培养基包括补充表皮生长因子 (egf), 这有助于刺激细胞增殖和分化, 和氢化可的松, 其中使细胞的 egf26.我们表明, 这一特定生长培养基的使用产生的内皮比内皮基培养基 (循证医学) 或 Dulbecco 氏改良鹰培养基 (DMEM) 的数量更高。

<table><tbody><tr><td>&lt;strong&gt;A) For isolation and culturing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EGM-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td>This product comes with all the growth factors needed to make the Endothelial Growth Medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Pencillin-Streptomycin-Glutamine (100x)</td><td>Thermofisher Scientific</td><td>10378016</td><td></td></tr><tr><td>Ficoll-Paque</td><td>GE Heatlhcare</td><td>17-1440-02</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermofisher Scientific</td><td>14170-112</td><td></td></tr><tr><td>Ammonium Chloride</td><td>Stem Cell Technologies</td><td>7850</td><td></td></tr><tr><td>1x Phosphate Buffer Saline</td><td>Thermofisher Scientific</td><td>14190250</td><td></td></tr><tr><td>Rat Tail I Collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Amresco</td><td>0714-500ML</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25300054</td><td></td></tr><tr><td>HEPES buffer</td><td>Thermofisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Thermofisher Scientific</td><td>10566-016</td><td></td></tr><tr><td>&lt;strong&gt;B) Antibodies and cell lysates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD31&amp;nbsp;</td><td>Abcam</td><td>ab28364</td><td>1:250 dilution&amp;nbsp; for Western blotting</td></tr><tr><td>CD34</td><td>Santa Cruz Biotechnology</td><td>sc-7045</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-SMA</td><td>abcam</td><td>ab5694</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-tubulin</td><td>abcam</td><td>ab7291</td><td>1:2,500 dilution for Western blotting</td></tr><tr><td>VEGFR2</td><td>abcam</td><td>sc504</td><td>1:100 dilution for Western blotting</td></tr><tr><td>Human umbilical vein endothelial cell lysate</td><td>Santa Cruz Biotechnology</td><td>sc24709&amp;nbsp;</td><td></td></tr><tr><td>Valve interstitial cell lysate</td><td></td><td></td><td>Primary cell line cultured from own lab and lysed with RIPA buffer</td></tr><tr><td>&lt;strong&gt;C) Western blotting and immunostaining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x Tris/Glycine/SDS buffer</td><td>Biorad</td><td>161-0772</td><td>Used as running buffer</td></tr><tr><td>10x Tris/Glycine buffer</td><td>Biorad</td><td>161-0771</td><td>Used as transfer buffer</td></tr><tr><td>Immobilon-FL transfer membrane</td><td>Merck Millipore</td><td>IPFL0010</td><td>This is a PVDF transfer membrane that has 45 &amp;micro;m pore size and is mentioned in the protocol as western blot membrane</td></tr><tr><td>4x Laemmli sample buffer</td><td>Biorad</td><td>161-0747</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Biorad</td><td>161-0710</td><td></td></tr><tr><td>10% Criterion TGX precast gel</td><td>Biorad</td><td>5671033</td><td></td></tr><tr><td>Prolong Gold antifade</td><td>Thermofisher Scientific</td><td>P36930</td><td>Used for mounting immunostained coverslips for long term storage</td></tr><tr><td>Methanol</td><td>VWR Analytical</td><td>BDH1135-4LP</td><td></td></tr></tbody></table>

isolation of endothelial progenitor cells from human umbilical cord blood

Ashara 和同事们首次报告了外周血中内皮祖细胞 (内皮) 的存在及其在血管中的参与情况1。后来, 其他人记录了来自骨髓的类似类型的内皮的存在2,3。最近, 奥约德和英格拉姆表明, 内皮从脐血获得了较高的增殖潜能与孤立的成人外周血4,5,6。除了参与产后血管, 内皮也显示了承诺作为一个细胞源创建组织工程血管和心脏瓣膜构建7,8。存在各种隔离协议, 其中一些涉及在内皮和造血标志物的帮助下从先前提到的来源获得的单个核细胞的细胞分类, 或培养这些具有特殊内皮生长的跨国公司中, 或这些技术的组合9。在这里, 我们提出了一个内皮的分离和培养的协议使用专门的内皮培养基补充生长因子, 不使用 immunosorting, 其次是对孤立细胞的表征使用西部印迹和免疫.

内皮祖细胞的分离和扩展: 提供了一个示意图 (图 1) 描述了整个协议。采用密度梯度介质对人脐血密度梯度离心法进行了不同的血成分层观察。在将跨国公司播种到胶原处理的盘子上时, 首先观察到5天和 7 (图 2A) 之间的菌落的生长。这些殖民地继续生长并且有纺锤形的细胞形态学 (图 2...

Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

该协议的目的是分离脐血内皮祖细胞。一些应用包括利用这些细胞作为生物标志物来识别心血管风险患者, 治疗缺血性疾病, 并创建组织工程的血管和心脏瓣膜结构。

人脐血内皮祖细胞的分离

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

isolation-endothelial-progenitor-cells-from-human-umbilical-cord

Research

JoVE Journal

Developmental Biology

11.0K 次观看.  University of Arkansas. 该协议的目的是分离脐血内皮祖细胞。一些应用包括利用这些细胞作为生物标志物来识别心血管风险患者, 治疗缺血性疾病, 并创建组织工程的血管和心脏瓣膜结构。

视频: Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

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 ...

生物学

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

免疫与感染

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

人脐带血衍生CD34的泛骨髓分化–造血干细胞和祖细胞

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

从健康志愿者和它们的迁徙可能通过血清中的影响心脏手术后的内皮祖细胞的分离

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

医学

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

来自人类脐带血内皮细胞集落形成细胞的表型和功能特性

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

丙戊酸在人脐带血源性 CD34 + 细胞造血干细胞体外扩增中的作用

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

人脐血内皮祖细胞的分离

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