Name: assessment of the anticoagulant and anti-inflammatory properties of endothelial cells using 3d cell culture and non-anticoagulated whole blood
Uploaded: 2017-09-05T13:00:00.000+00:00
Duration: 7 min 8 s
Description: Watch this Scientific Journal Video about Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood at JoVE.com

这项研究得到了瑞士国家科学基金会 (SNSF, 格兰特 No. 320030_156193) 的支持。作者感谢 Dr. Benoît Werlen 提供 Biosilon 载体珠。我们还感谢 Prof. 和 Prof. 安德烈 Haeberli 帮助建立载体珠模型。

德国长白猪 (野生型养殖在当地的农舍和转基因育种研究所的分子动物繁殖和生物技术学院, 德国慕尼黑, 路德维希-马西米兰大学, 重量介于30公斤到40公斤之间, 被用于这项研究。所有的动物都被安置在标准条件下的水和食物 ad lib 。所有动物实验都是按照《英国动物法》 (科学程序) 和《 NIH 指南》对实验动物的照顾和使用以及瑞士动物保护法进行的。所有动物研究符合到达的指南。县兽医服务的动物实验委员会 (瑞士伯尔尼的小行政区) 批准了所有动物规程, 允许没有。BE70/14实验规程根据3R 原则被提炼, 并且最先进的麻醉和痛苦管理被用于减少动物的数量和减少动物的曝光在实验期间的重音和痛苦. 
 血液是根据瑞士司法管辖和伯尔尼大学医院的道德准则, 通过闭合系统静脉穿刺从健康人身上抽取的。词组和 #34; non-anticoagulated 血液和 #34; 意味着血液没有被抗凝治疗. 
 以下步骤在无菌条件下执行。熟悉碱性细胞培养无菌技术是必需的. 
 1. PAEC 的隔离 注意: 6 至10厘米的胸主动脉段是从3至6月的安乐死的德国长白猪获得的 (用于其他 体内 实验), 并立即转移到一个500毫升的玻璃瓶含有运输介质 (DMEM + 1% 青霉素/链霉素). 
<ol> <li> 预涂一个6井板, 纤维连接蛋白12.5 和 #181; PBS 1x 中的 g/毫升, 并把它放在一个孵化器在37和 #176; C 为 1 h. </li>
	<li> 预热无菌 PBS 1x 和细胞培养基 (DMEM). </li>
	<li> 从传输介质中取出猪主动脉. </li>
	<li> 将主动脉置于聚苯乙烯板上. </li>
	<li> 事先轻轻地用暖 PBS 冲洗. </li>
	<li> 纵向切开主动脉并用针固定. </li>
	<li> 在内容器表面添加暖细胞培养基. </li>
	<li> 吸入纤维连接蛋白-细胞培养基和添加新鲜细胞培养基 (DMEM 补充 10% FBS, 1% 青霉素/链霉素和0.4% 的内皮细胞生长培养基补充剂). </li>
	<li> 在细胞培养基中浸泡一棉签。在内容器表面的顶端轻轻地和缓慢地在同一方向上擦拭棉毛芽. </li>
	<li> 将6个井板中的单元格旋转一圈. </li>
	<li> 对其余的油井也这样做. </li>
	<li> 检查显微镜下的细胞, 将6井板放在孵化箱中37和 #176; C, 5% CO 2 . </li>
	<li> 在第二天更改媒体, 每 2-3 天更改一次. </li>
	<li> 当细胞要汇合时, 处理细胞并将它们种子成 T75 烧瓶 (PAEC P1). </li>
</ol> 2。PAEC 特性 <ol> <li> 前涂层 8 chamberslide 与纤维连接蛋白12.5 和 #181; PBS 1x 中的 g/毫升, 并把它放在一个孵化器在37和 #176; C 为 1 h. </li>
	<li> 种子 5 x 10 4 细胞/井和孵化过夜在孵化器在37和 #176; C. </li>
	<li> 使用 pbs 清洗两次电池 + + (pbs 补充 CaCl 2 和氯化镁 2 ), 300 和 #956; L/好。
	<li> 在室温、200和 #956 中用3.7% 甲醛固定电池10分钟; L/好. </li>
	<li> 使用 PBS 清洗电池3次 + + , 300 和 #956; L/好. </li>
	<li> 添加300和 #956; PBS 1 x-3% BSA (阻塞缓冲器), 室温下保持30分钟. </li>
	<li> 使用 PBS 1 x-1%BSA-0.05% 洗涤剂, 160 和 #956 中稀释的初级抗体 (抗 VE 钙黏附素, anti-CD31, anti-vWF); L/井, 室温下孵育1小时. </li>
	<li> 使用 PBS 洗涤3次 + + (200 和 #956; 我/好). </li>
	<li> 在 PBS 1 x 1%BSA-0.05% 洗涤剂、160和 #956 中应用二次抗体和 DAPI 稀释剂, 在室温下孵育1小时. </li>
	<li> 使用 PBS 洗涤3次 + + (200 和 #956; 我/好). </li>
	<li> 用甘油为基础的安装介质安装幻灯片, 并在荧光显微镜下验证内皮细胞标志物的表达. 
	注: 培养猪主动脉内皮细胞在 T175 烧瓶 (DMEM 低葡萄糖培养基 + 10% FBS, 1% 青霉素/链霉素和0.4% 的内皮细胞生长培养基补充剂) 直到90% 汇合到达。(播种密度 1 x 10 6 单元格, 90% 汇合大约对应于 5 x 10 6 单元格)。
</ol> 3。载体珠的涂层 <ol> <li> 将7毫升载体珠与42毫升的胶原蛋白溶液混合在一个50毫升的管中 (100 和 #956; 在0.2% 醋酸溶液中稀释), 在室温下孵育 1 h. </li>
	<li> 用25毫升 pbs pH 7.4 洗涤珠两次 (添加25毫升 pbs, 与吸管混合, 并等待, 直到珠被安顿下来, 然后丢弃上清和重复), 一次与25毫升的 DMEM 介质. </li>
	<li> 覆盖50毫升管中的珠子, 10mL 培养基199补充 10% FBS, 1% 青霉素/链霉素, 1% l-谷氨酰胺, 0.4% 的内皮细胞生长培养基补充剂和25和 #956; 肝素 (5000-毫升) 的 l, 并允许平衡10分钟在进一步使用之前. </li>
</ol> 4。收集单元格 <ol> <li> 从包含 PAEC 的 T175 烧瓶中删除细胞培养基, 并添加5毫升的 PBS pH 值 7.4. </li>
	<li> 从 T175 烧瓶中删除 PBS. </li>
	<li> 添加5毫升的 Trypsin-0.05%edta 和孵育 3-4 分钟, 在37和 #176; C. </li>
	<li> 通过用15毫升的细胞培养基冲洗烧瓶来收集细胞, 并在50毫升的管子中转移悬浮液. </li>
	<li> 在室温下离心 1200 x g 的细胞8分钟, 去除多余的培养基, 在5毫升的细胞培养基中重颗粒. </li>
</ol> 5。将细胞播种到搅拌器烧瓶中 <ol> <li> 将20毫升的细胞培养基添加到细胞悬浮和重. </li>
	<li> 将20毫升细胞培养基 (w/o 细胞) 加入到500毫升的磁力搅拌器烧瓶中. </li>
	<li> 将单元格添加到步骤3.3 中的洗涤载体珠, 并将其与25毫升的血清吸管进行仔细混合. </li>
	<li> 将珠子/电池混合物转到磁纺纱瓶中. </li>
	<li> 冲洗50毫升管与10毫升的细胞培养基收集剩余的细胞. </li>
	<li> 将额外的85毫升细胞培养基添加到微调器烧瓶中, 并在37和 #176 过夜放入恒温箱中; C 在振动筛上 (100 x g, 混合间隔: 3 分钟每45分钟). </li>
	<li> 添加50毫升的细胞培养基 (总体积200毫升), 并继续搅拌额外的24小时, 在37和 #176; C 在一个振动筛 (100 x g, 混合间隔: 3 分钟每45分钟). </li>
	<li> 添加无色 RPMI 培养基 (补充 10% FBS, 1% 青霉素/链霉素, 1% l-谷氨酰胺, 0.4% 的内皮细胞生长培养基补充剂和25和 #956; l 肝素) 直到320毫升的总体积达到. </li>
	<li> 每48小时更换培养基: 去除100毫升的旧培养基, 加入100毫升的新鲜补充无色 RPMI. </li>
	<li> 将单元格的区域性培养为5到7天。时间取决于细胞包裹的珠子的汇合状态. </li>
</ol> p 类 = "jove_title ">> 6。汇流验证 <ol> <li> 收集200和 #956; 使用吸管将细胞包裹的珠子移到聚丙烯管中. </li>
	<li> 3 次用600和 #956 清洗珠子; pbs 1x (添加 pbs, 倾斜管和轻轻混合, 以避免细胞脱落, 等待珠子安顿下来, 丢弃 pbs 和重复). </li>
	<li> 通过添加200和 #956 来固定10分钟的珠子; parapicric 酸的 L. </li>
	<li> 使用600和 #956 清洗3次; PBS 1x. </li>
	<li> 添加 DAPI 稀释在 PBS 1x 和孵化10分钟. </li>
	<li> 在玻璃滑梯上传送珠子, 用甘油为基础的安装介质应用片. </li>
	<li> 在共聚焦显微镜下可视化珠子. </li>
</ol> 7。实验过程 <ol> <li> 从磁性搅拌器烧瓶中取出电池包覆的珠子 (程序不必在无菌条件下进行), 用10毫升的血清学吸管将它们转移到12毫升的圆底聚丙烯管中. </li>
	<li> 让珠子安定下来 (大约 1-2 分钟) 并且去除多余的媒介. </li>
	<li> 添加更多的珠子到管, 直到每管包含整整2毫升的珠子. </li>
	<li> 在每根管子上加5毫升的透明 RPMI, 并使用10毫升的血清吸管进行仔细的混合. </li>
	<li> 让珠子沉淀下来, 去除多余的介质. </li>
	<li> 再重复一次洗涤过程与 RPMI 和去除所有多余的媒介. </li>
</ol> 8。非抗凝血的孵育 <ol> <li> 小心而缓慢地 (既不使用喷气机也不 vacutainers) 从一个健康的志愿者那里抽取血液并在9毫升的中性聚丙烯管中收集 (没有抗凝剂). </li>
	<li> 缓慢转移8毫升血液与10毫升血清学吸管到每一个聚丙烯管含有2毫升的细胞包覆珠 (总体积将是10毫升)。避免粗暴处理血液或珠子, 以避免过早的 EC 活化。该过程需要 1-2 min. </li>
	<li> 小心地倾斜血/珠混合物, 以确保相同的混合和密封的上限与石蜡膜. </li>
	<li> 将管子放在一个水平的倾摆台上 (仅有温和的倾斜设置) 在37和 #176 内; C 恒温箱和记录凝固时间。
	<ol> <li> 在设置的时间间隔内, 例如 在10、20、30、50、70、90分钟后, 删除至少 1.5-2 毫升的血珠混合物, 用于血清或血浆分析. 
		注: 对于6时间点, 我们建议在一组细胞内有超过3复制, 因为血液取样将在不同的管中进行. </li>
		<li> 收集血清, 让血液凝固。要收集血浆, 加入 EDTA 或柠檬酸盐到2毫升管前添加血液样本. </li>
		<li> 将管在冰上, 离心机在 2500 x g 为10分钟在4和 #176; c 和储存血清/血浆在80和 #176; c 直到使用. 
		注意: 材料的详细信息在 材料表 </li> </ol> </li> </ol> 中提供

<ol>
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作者没有什么可透露的。

该模型适用于凝血相关研究, 可以分析凝血的不同方面及其与 EC 的相互作用。8在异种移植的研究中, 用人血9对不同基因修饰的猪 ECs 的抗凝血性能进行检测是一个有用的系统。
该协议最关键的步骤是确保微在开始实验之前的完整的细胞覆盖率 (70-100), 以及那些关于仔细收集和处理 non-anticoagulated 全血以避免过早血小板活化由于高剪切应力, ?...

血管内皮由一单层内皮细胞 (EC) 组成, 它能使血管的腔管排成一线。在生理状态下, 静止 EC 负责维持一个抗凝和抗炎环境。1这是由 EC 表面的抗凝和抗炎蛋白的表达所介导的。例如, 由异移植器官的缺血/再灌注损伤或血管排斥引起的 EC 活化导致内皮表面从抗凝和抗炎状态转变为 pro-coagulant 和炎国家.1
为了研究内皮和凝血因子之间的奇妙和复杂的相互作用,在体外模型, 模仿尽可能密切的在体内情况是非常可取的。传统的体外凝血分析的一个共同的局限是使用抗凝血液, 使得凝血介导的作用复柠檬酸全血不能重现结果可获得新鲜 non-anticoagulated 血。2此外, 在传统的平板细胞培养系统中, 不可能利用内皮的抗凝血特性, 因为不能达到每个血液体积的足够的内皮细胞表面。该模型通过在球形载体珠表面培养 ec, 克服了这些局限性, 使 ec surface-to-blood 比 #62; 16 厘米2/毫升可以达到, 这是类似的情况下小动脉或静脉,被描述为足以使血液在 EC 表面进行 "自然" 抗凝。3,4全血可以在不添加抗凝剂的情况下使用。血液标本可在实验过程中采集, 细胞因子、凝血因子和可溶性补体活化标志物均可检测和量化。此外, 还可以通过共聚焦显微镜对补体和免疫球蛋白的沉积以及 ec 活化标记物的表达进行分析, 对 ec 包覆的载体珠进行研究。另一个有趣的应用包括测试药物, 这应该是防止内皮细胞活化, 从而, 凝血。5虽然这个模型不能完全取代动物实验, 它提供了一种方法来测试特定的功能假说,前体使用细胞, 从而减少了动物的数量, 用于基础研究的缺血/再灌注损伤或 (异) 移植。
用该模型模拟了猪主动脉内皮细胞 (PAEC) 在载体念珠上生长并与整个 non-anticoagulated 人血培养的异种移植环境。不同的转基因 PAEC, 携带了一些人类基因, 如 CD46 调节的补充系统和/或血栓调节蛋白 (hTBM) 的调节凝血系统, 分析了他们的抗凝血性能。内皮细胞活化、补体和凝血系统受到严密控制和相互连接。6因此, 重要的是要了解不同的转基因细胞在接触人体血液后的行为与黏附分子的表达和细胞因子的释放、糖的脱落以及抗凝蛋白的丧失有关。7

<table><tbody><tr><td>PAEC</td><td></td><td></td><td>Isolated from pig aorta in our Lab</td></tr><tr><td>Microcarrier beads (Biosilon)</td><td>Thermo Fisher Scientific</td><td>160-250</td><td></td></tr><tr><td>Collagen I, Bovine</td><td>Gibco</td><td>A10644-01</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>21885-025</td><td></td></tr><tr><td>Medium 199</td><td>Sigma</td><td>M7528</td><td></td></tr><tr><td>RPMI</td><td>Gibco</td><td>32404-014</td><td></td></tr><tr><td>Neutral tubes</td><td>Sarstedt</td><td>02.1726.001</td><td></td></tr><tr><td>Polypropylene tubes</td><td>Simport</td><td>T406-2A</td><td></td></tr><tr><td>Stirrer flasks</td><td>Tecnomara</td><td></td><td></td></tr><tr><td>Biological stirrer</td><td>Brouwer</td><td>MCS-104S</td><td></td></tr><tr><td>Rat anti CD31</td><td>R&amp;amp;D Systems</td><td>MAB33871</td><td>Dilution 1:100</td></tr><tr><td>Goat anti-rat IgG-Cy3</td><td>Jackson Immuno Research</td><td>112-166-003</td><td>Dilution 1:500</td></tr><tr><td>Goat anti VE-cadherin</td><td>Santa Cruz</td><td>sc-6458</td><td>Dilution 1:100</td></tr><tr><td>Rabbit anti vWF</td><td>DAKO</td><td>A0082</td><td>Dilution 1:100</td></tr><tr><td>Dk anti Gt IgG &amp;ndash; Alexa 488</td><td>Molecular Probes</td><td>A11055</td><td>Dilution 1:500</td></tr><tr><td>Goat anti Rabbit - FITC</td><td>Southern Biotech</td><td>4050-02</td><td>Dilution 1:500</td></tr><tr><td>DAPI</td><td>Sigma</td><td>32670-25MG-F</td><td>Dilution 1 &amp;micro;g/mL</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10270-106</td><td>Concentration: 10% in cell culture medium</td></tr><tr><td>Adapter</td><td>Sarstedt</td><td>14.1205</td><td></td></tr><tr><td>Confocal microscope</td><td>Carl Zeiss</td><td>LSM 710</td><td></td></tr><tr><td>Image analysis software</td><td>Image J</td><td></td><td>Available for free at: https://imagej.net</td></tr><tr><td>Endothelial Cell Growth Medium SupplementMix</td><td>PromoCell</td><td>C-39216</td><td>2 mL in 500 mL of DMEM</td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Concentration: 1% in cell culture medium</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-024</td><td>Concentration: 1% in cell culture medium</td></tr><tr><td>Heparinun natricum</td><td>Drossa Pharm AG</td><td></td><td>Stock concentration: 5000 U.I./ mL</td></tr><tr><td>Chamberslides</td><td>Bioswisstec AG</td><td>30108</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; x 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>2382.0500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2 &lt;/sub&gt;x6 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.5833.1000</td><td></td></tr><tr><td>Tween 20</td><td>Axon Lab AG</td><td>9005-64-5</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A7030</td><td></td></tr><tr><td>Glycergel mounting medium</td><td>Dako</td><td>C0563</td><td></td></tr></tbody></table>

assessment of the anticoagulant and anti-inflammatory properties of endothelial cells using 3d cell culture and non-anticoagulated whole blood

在体内, 内皮细胞是血液循环的自然抗凝血的关键。因此, 内皮细胞活化导致凝血。这种现象在许多临床情况下被观察, 象器官移植在存在预先 anti-donor 抗体, 包括异种移植, 并且在缺血或再灌注损伤。为了减少动物实验根据3R 标准 (减少, 替换和提炼),在体外模型研究内皮细胞活化作用对凝血是非常可取的。然而, 普通的平板系统的内皮细胞培养提供了一个表面积的比例为 1-5 厘米2的内皮每毫升的血液, 这是不足够的自然, 内皮介导的抗凝。在载体珠上培养内皮细胞可使表面积率提高到 40-160 cm2/毫升。这种增加的比例足以确保 "自然" 的全血抗凝, 从而可以避免使用抗凝剂。本文介绍了一种体外microcarrier-based 系统, 研究了猪内皮细胞的遗传修饰对全 non-anticoagulated 人血液凝固的影响。在所述的测定方法中, 原发猪主动脉内皮细胞, 无论是野生型或转基因的人 CD46 和血栓调节蛋白, 都生长在载体珠上, 然后暴露在新绘制的 non-anticoagulated 人血中。该模型可以测量和量化的细胞因子释放, 以及活化标志的补充和凝聚在血浆中。此外, 通过共聚焦显微镜对活化的内皮细胞进行成像, 并在 endothelialized 珠上进行免疫球蛋白、补体和凝血蛋白的沉积。这种化验也可以用来测试药物, 这是应该防止内皮细胞活化, 从而, 凝血。在它的潜力, 以减少动物的数量用于此类调查, 所述的化验是容易执行和一贯重现性。

在 7-10 天的文化在微调瓶 (图 1) 的细胞是汇合的, 覆盖整个表面的载体珠 (图 2)。70-100 状态的验证是一个重要的步骤, 因为 non-confluent 单层的 EC 在微将导致明显减少凝血时间, 因为载体珠的表面强烈 pro-coagulant (凝固时间: 4 ± 1 min) (图 3)。
另一个需要注意的重要...

Watch this Scientific Journal Video about Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood at JoVE.com

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

我们提出了一个体外模型, 允许对整个 non-anticoagulated 血液中的凝血进行研究和分析。该系统的抗凝血依赖于健康的内皮细胞的天然抗凝作用, 内皮细胞活化会导致血栓形成。

3D 细胞培养和非抗凝全血对内皮细胞抗凝及抗炎作用的评价

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

assessment-anticoagulant-anti-inflammatory-properties-endothelial

Research

JoVE Journal

Medicine

8.3K 次观看.  University of Bern. 我们提出了一个体外模型, 允许对整个 non-anticoagulated 血液中的凝血进行研究和分析。该系统的抗凝血依赖于健康的内皮细胞的天然抗凝作用, 内皮细胞活化会导致血栓形成。

视频: Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

科研

生物工程

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

片上内皮炎症表型

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

发育生物学

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

免疫与感染

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

标准化和可扩展的测定，以研究iPSC衍生内皮细胞在Vitro的渗透3D血管生成发芽

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

在机械生物学研究的轨道摇床上的 6 井板中分段内皮细胞的生长

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

研究流动下血管药物靶向的体外3D细胞培养动脉模型

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

实时研究全血-内皮相互作用和血栓动力学的体外微流体疾病模型

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...