Name: incorporating pericytes into an endothelial cell bead sprouting assay
Uploaded: 2018-02-16T15:00:00.000+00:00
Duration: 6 min 21 s
Description: Watch this Scientific Journal Video about Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay at JoVE.com

我们感谢 Drs. 维多利亚 Bautch 和约书亚为帮助讨论和建议优化标准珠发芽试验条件和发芽化验染色协议。S.H.A. 得到了国家普通医学研究所授予 5T32 GM007092 奖的部分支持。

1天:
1. 血管内皮细胞的瞬态转染
<ol><li>如果需要, 使用基因调节寡核苷酸 (例如微 rna 或小干扰 RNA (siRNA)) 和适当的脂质体试剂, 对人脐静脉内皮细胞 (HUVEC) 进行瞬态转染, 并根据制造商的说明。 注: 该协议具有很大的成功执行反向 transfections 与定制 siRNA 序列和商业转染试剂 (见材料表)。作者没有测试其他转染的协议和试剂与此检测。使用所列试剂进行反向转染的步骤如下:</li>
	<li>重组冻干 siRNAs 或 microRNAs 浓度为20µM 在核酸酶自由水。</li>
	<li>以以下比率进行转染解决方案: 任何血清游离培养基的1毫升, siRNA 或 microRNA 的9µL。</li>
	<li>搅动溶液, 让它在室温下孵化5分钟。</li>
	<li>添加18µL 的转染试剂到溶液中搅拌。</li>
	<li>允许溶液在室温下孵育20-40 分钟, 在孵化期间每10到15分钟搅拌溶液。</li>
	<li>当转染解决方案正在孵化时, 使用细胞脱离解决方案 (例如、Accutase) 分离 HUVEC。对于 T175 瓶, 用10毫升磷酸盐缓冲盐水 (PBS) 冲洗烧瓶, 然后加入5毫升的细胞脱离溶液, 并在37摄氏度下孵化出10分钟的烧瓶。</li>
	<li>将5毫升完整的介质添加到 T175 瓶中, 从烧瓶中取出电池悬浮液, 离心机在1200转每分钟3分钟。</li>
	<li>使用真空吸引器取出上清, 并在 EGM2 中每毫升 1 X 106细胞的密度并用重悬细胞颗粒。 注: 本议定书取得了很大的成功, 使用内皮细胞生长培养基 (EGM2) 包含标准生长因子子弹袋, 以及额外的10% 血清。</li>
	<li>一旦转染溶液完成孵化, 板1毫升溶液在10厘米板, 并增加1毫升的 HUVEC 悬浮在上面。</li>
	<li>添加一个额外的7毫升完整的介质, 使最终体积在10厘米的板块到9毫升。</li>
	<li>为每个转染条件至少组成两个10厘米的细胞, 以确保在化验中有足够的细胞供以后的步骤使用。 注意: 这些条件导致基因调控寡核苷酸的最终工作浓度为 20 nM。作者发现这些条件导致60-80% 击倒基因表达为他们的兴趣目标。其他研究人员可能需要根据实验需要优化转染条件。</li>
	<li>交换媒体 4-6 h 后添加 siRNA 配合物与新鲜完整的媒体。 注意: 该协议是使用 1-2 HUVEC 的通道开发的。</li>
</ol>2天
2. 内皮细胞载体珠的涂敷
<ol><li>重建载体珠 (例如, Cytodex 3) 在 PBS 和高压釜集中6万珠/毫升。 注: 载体珠的浓度约为每克 3 x 106珠子。根据 PBS 每毫升20毫克珠子的比例重建珠子, 以获得大约6万珠/毫升的珠密度。</li>
	<li>整除1毫升完整的介质进入所需数量的圆底荧光活化细胞分拣 (资产管制局) 管。</li>
	<li>吸管20µL 载体珠悬浮到每管 (注意确保珠溶液是良好的悬浮之前, 吹打)。 注: 在给定的载体珠悬浮液中, 珠子的最终工作浓度可能会发生剧烈的变化。珠子可以很容易地附着在塑料和玻璃容器的两侧, 这可以极大地改变溶液中的珠浓度。每批载体珠悬浮液中添加到流化管中的最合适的珠溶液体积将需要由调查员以经验主义的方式确定。这里提出的数字, 是为了作为一个出发点。理想情况下, 条件应该优化, 所以有10-20 珠嵌入在纤维蛋白凝胶每井在24井板在化验结束。请参阅下面的讨论部分中关于优化检测的珠子数的进一步讨论。</li>
	<li>分离转染 HUVEC 24 h 后, 使用细胞脱离解决方案, 如下所示:<ol>
<li>用5毫升 PBS 冲洗每10厘米的细胞, 然后加入2毫升的细胞脱离溶液, 并在37摄氏度内孵化出10分钟的细胞。</li>
		<li>并用重悬在完全介质中的浓度为 1 X10 6 细胞/mL 的单元格。</li>
	</ol>
</li>
	<li>将1毫升的细胞悬浮液 (即1 X 106 HUVEC) 添加到每个含有珠子的流化管中。</li>
	<li>在37摄氏度 (如果继续步骤 3.4) 或2.5 到 3 h (如果继续步骤 3.2), 每隔20分钟轻轻地搅动一次流化管, 时间为4小时。 注意: 这一持续时间和躁动的频率已经由以前的研究人员进行了经验性的分析, 他们已经优化了基本的珠发芽试验。4作者还没有对其它搅拌频率和珠涂层步骤的持续时间进行测试。</li>
</ol>3. HUVEC 涂覆载体珠上毛细血管的序贯涂层
<ol><li>使用细胞脱离溶液 (胰蛋白酶) 开始分离 10T1/2 细胞 (毛细血管), 如下所示:<ol>
<li>用10毫升的 PBS 冲洗一个 T175 烧瓶, 然后加入5毫升的胰蛋白酶, 在37摄氏度处孵育10分钟。</li>
		<li>并用重悬 10T1/2 浓度为 2 X10 5 单元格/0.5 mL 在完全介质中。 注意: 细胞是从美国的类型文化集合 (ATCC) 购买的。 注: 培养 10T1/2 细胞的最低基本培养基 (记忆) 补充10% 血清和1% 青霉素链霉素。</li>
	</ol>
</li>
	<li>经过2.5 到3小时的搅拌珠 + HUVEC 解决方案, 等待5分钟, 让珠和自由浮动的 HUVEC, 以解决到底部的管。</li>
	<li>使用 P1000 吸管从每管中取出0.5 毫升完整的介质, 并在每一个流量为2毫升的介质中添加 2 x 105 10T1/0.5。搅动细胞和珠溶液, 然后继续孵化37摄氏度。继续搅拌每20分钟, 直到珠子已经搅拌了总共4小时。 注: 5 HUVEC: 1 周细胞对珠子的比例是维持船只适当周细胞覆盖的必要条件。 注意: 在协议的这一点上可以遵循以下替代步骤:</li>
	<li>搅动珠子和 HUVEC 仅为4小时。</li>
	<li>一旦载体珠的 4 h HUVEC 涂层完成后, 最后一次搅动管子, 等待30-60 秒, 然后立即从流化管中取出 1.5-1.75 毫升溶液。 注意: 这应该允许足够的时间, 大部分的珠子解决, 而许多自由浮动的 HUVEC 在媒体将被删除。<ol>
<li>在完整的介质中添加 2 X 105 10T1/2, 并将该流化管的体积增加到2毫升。</li>
		<li>每隔20分钟轻轻地搅动一小点, 再加一小时。</li>
	</ol>
</li>
	<li>一旦搅拌完成, 轻轻地搅动管子最后一次, 立即删除整个解决方案 (2 毫升), 并添加到一个6厘米的板块。增加一个额外的3毫升的完整的媒体, 每一个板块, 并把板块在37°c 孵化器过夜。</li>
</ol>3天
4. 纤维蛋白凝胶溶液的制备
<ol><li>在50单位/毫升的 PBS 和凝血酶中, 为1毫克/毫升浓度的抑肽酶制备工作溶液。 注: 这些解决方案中的一大股可以在4摄氏度至少一年内被制造和储存。</li>
	<li>在2毫克/毫升的浓度下, 做一个新的纤维蛋白原溶液。使足够的解决方案有2.5 毫升的解决方案每一个激动的流化管。<ol>
<li>在37摄氏度的水浴中加热溶液10分钟, 让所有的纤维蛋白原进入溶液中。</li>
		<li>添加37.5 µL 的抑肽酶溶液每1毫升的纤维蛋白原溶液。</li>
		<li>消毒-过滤溶液, 并在室温下预留。</li>
	</ol>
</li>
</ol>5. 准备用于凝胶植入的珠子
<ol><li>在显微镜下使用20X 目标检查含有载体涂层珠子的盘子, 以确保所有的珠子都被内皮细胞充分涂敷。</li>
	<li>大力吸出细胞的电镀溶液, 将它们从6厘米的盘子中分离出来, 然后将溶液放入管子中。用完整的介质冲洗盘子2X 次, 然后转移到管子上, 收集所有可能粘附在盘子上的残余珠子。<ol>
<li>避免引入气泡, 并使用 p1000 吸管减少内皮细胞涂层珠的剪应力。</li>
		<li>如果需要评估 siRNA 在内皮细胞中的击倒效率, 在步骤2中, 一定要有一盘 HUVEC 的包覆的珠子。然后在这一步骤 (5.2), 分离珠和收集残余 HUVEC 附着在板上的 RNA 分离和 qPCR 分析。</li>
	</ol>
</li>
	<li>等待3分钟, 让溶液中的珠子沉淀到管子的底部。</li>
	<li>清除尽可能多的上清, 而不干扰珠丸使用 p1000 吸管提示 (不要使用真空吸)。</li>
	<li>添加1毫升的完整的介质, 每管和并用重悬珠。</li>
	<li>等待30-60 秒, 让珠子沉淀到底部。然后用 P1000 吸管去除尽可能多的上清液, 而不干扰珠粒。不要使用真空吸尘器, 因为意外去除珠粒的可能性大大增加。</li>
	<li>重复步骤5.5 和5.6 另外两次。其目的是去除尽可能多的自由浮动的 HUVEC, 可能已经从 6 cm 板块分离, 而不是删除太多的 HUVEC 涂珠, 这应该解决在流化管比自由浮动细胞更快。</li>
	<li>将1毫升完整的介质添加到珠子上。</li>
</ol>6. 在纤维蛋白凝胶中嵌入涂层珠子
<ol><li>去除尽可能多的介质, 而不干扰每管的珠粒在每个应用 P1000 吸管。</li>
	<li>并用重悬2.5 毫升的纤维蛋白原溶液中的珠子。</li>
	<li>将13µL 的凝血酶溶液注入24井玻璃底板的每个理想井中。在玻璃底板上电镀对使芽苗的最佳成像至关重要。 注: 在下一步之前, 不要让凝血酶在井里坐5-10 分钟以上。</li>
	<li>每井加0.5 毫升的珠/纤维蛋白原溶液。<ol>
<li>在吹打解决方案之前, 一定要并用重悬珠溶液。</li>
		<li>小心地将吸管直接注入井中已经存在的凝血酶中。慢慢地用吸管上下2-3 次, 彻底混合溶液, 小心不要引入任何气泡。</li>
		<li>一定要改变水井之间的吸管尖端。</li>
		<li>在最初的纤维蛋白凝固过程中, 注意不要移动或扰乱盘子, 因为任何运动都可能扰乱凝胶的形成。</li>
	</ol>
</li>
	<li>在室温下, 把盘子放在引擎盖里30分钟。</li>
	<li>小心地将盘子转移到一个37摄氏度的孵化器中, 增加 1.5-2 小时, 让凝胶完全凝固</li>
</ol>7. 纤维蛋白凝胶上的电镀成纤维细胞
<ol><li>在凝胶凝固的最后30分钟期间, 分离正常人肺成纤维细胞 (NHLF) 如下:<ol>
<li>用10毫升的 PBS 冲洗一 T175 瓶, 然后加入5毫升的细胞脱离溶液, 在37摄氏度处孵育10分钟。</li>
		<li>并用重悬 NHLF 在2万细胞/毫升的浓度在完全培养基。 注: NHLF 细胞可培养与 Dulbecco 修饰鹰培养基 (DMEM) 补充10% 血清和1% 青霉素链霉素。</li>
	</ol>
</li>
	<li>板1毫升的 NHLF 细胞悬浮在每个井的凝胶和地方回到孵化器。</li>
	<li>在检测期间每隔一天更改完整的介质。</li>
</ol>8. 观察纤维蛋白凝胶中珠植入后2-7 天的发芽过程。发芽所需的时间长短将取决于 HUVEC 的通道和数量。
9. 发芽试验的固定
<ol><li>一旦出现足够的发芽, 能够确定治疗组之间的表型差异, 一次用2毫升的 PBS 冲洗所有水井。</li>
	<li>增加0.5 毫升的细胞脱离解决方案, 每一个井, 并将该板块在37摄氏度孵化器5分钟。</li>
	<li>将盘子从孵化器中取出, 然后轻敲盘子的侧面。 注意: 这个步骤的目的是删除尽可能多的 NHLF 细胞生长在纤维蛋白凝胶尽可能促进抗体渗透到凝胶和成像的凝胶。细胞脱离液的总潜伏期可能需要调整以优化 NHLF 的去除。研究人员被告诫不要在过多的时间内孵化凝胶, 因为细胞脱离溶液可能渗入到凝胶中, 并开始扰乱体内的内皮结构。</li>
	<li>一旦有足够数量的 NHLF 细胞被移除, 用1毫升完整的培养基淬火细胞脱离溶液。</li>
	<li>用吸尘器吸入分离的 NHLF 和介质, 用1毫升的 PBS 冲洗一次水井。</li>
	<li>在 PBS 中添加300µL 2% 多聚甲醛溶液, 并在室温下孵育10分钟。</li>
	<li>去除2% 多聚甲醛溶液, 用1毫升的 PBS 在室温下清洗每井3次, 以3分钟。 注: 请按照《环境健康与安全 (EHS) 指南》适当处理2% 多聚甲醛解决方案。<ol>
<li>增加1毫升的 PBS, 每一个井和存储在4°c, 直到准备染色或图像的芽。</li>
	</ol>
</li>
</ol>10. 发芽试验染色
<ol><li>在 PBS 中添加300µL 0.5% 的海卫 x 100, 在室温下孵化30到45分钟。</li>
	<li>使用真空吸尘器, 去除 Triton-X100, 并在 PBS 中清洗3次, 每5分钟。</li>
	<li>添加500µL 的阻断溶液的每一个凝胶和孵化过夜在4摄氏度。 注: 阻断解决方案由以下组成部分组成: 1% 牛血清白蛋白 (BSA), 5% 血清, 0.1% tween-20 和1% 抗血清 (使用相同的物种, 你的第二抗体是在 PBS)。</li>
	<li>从每个井中移除阻塞溶液, 并添加300µL 的染色溶液, 其中含有主要抗体。 注: 染色液与阻断溶液相同, 但不存在抗血清。作者已经成功孵化与主要抗体在稀释1:200 到1:400。</li>
	<li>在4摄氏度的24小时内孵化出染色液中的主要抗体。</li>
	<li>吸入原发性抗体溶液, 在室温下以温和摇摆的方式, 在0.5% 补间内冲洗3倍于20分钟。第三次清洗后, 再次更换新的洗涤缓冲, 并在4摄氏度隔夜储存。</li>
	<li>在室温下, 用荧光二次抗体在染色液中稀释 1:1000, 在2小时内孵化。</li>
	<li>去除二次抗体染色液, 每20分钟洗5次, 室温下用含0.5% 补剂的 tb 进行轻柔摇摆。</li>
	<li>在 PBS 稀释核污渍, 在4摄氏度过夜。 注: 该协议已取得了巨大的成功与赫斯特稀释1:10000 在 PBS。 注: 在成像前无需清除核染色液。</li>
	<li>图像在几天内完成染色, 捕捉最佳荧光信号。</li>
</ol>11. 发芽试验量化
<ol><li>在芽苗被映像后, 将图像文件导入 ImageJ 并使用 count 工具计算芽数, 或测量芽长长度的直线工具。 注: 芽被定义为内皮突起, 长度大于或等于其发芽的珠子直径。11发芽长度可以测量为从芽开始凸出到芽尖的点的距离。每个珠的平均发芽长度和每根珠子的平均芽数是评估基因沉默在发芽血管生成中的表型效应的有用指标。每个治疗组至少含有一根芽的珠子的平均数量也可以用作衡量发芽的健壮性的指标。</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pericyte--a+review.">The pericyte--a review.</a> Tissue Cell. 18 (2), 153-174 (1986).</li><li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+developmental,+physiological,+and+pathological+perspectives,+problems,+and+promises.">Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.</a> Dev Cell. 21 (2), 193-215 (2011).</li><li>Tattersall, I. W., 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=In+vitro+modeling+of+endothelial+interaction+with+macrophages+and+pericytes+demonstrates+Notch+signaling+function+in+the+vascular+microenvironment.">In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment.</a> Angiogenesis. 19 (2), 201-215 (2016).</li><li>Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+tumor+vasculature:+an+emerging+concept+in+antiangiogenic+therapy.">Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy.</a> Science. 307 (5706), 58-62 (2005).</li><li>Tahergorabi, Z., Khazaei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+angiogenesis+and+its+assays.">A review on angiogenesis and its assays.</a> Iran J Basic Med Sci. 15 (6), 1110-1126 (2012).</li><li>Popson, S. A., 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=Interferon-induced+transmembrane+protein+1+regulates+endothelial+lumen+formation+during+angiogenesis.">Interferon-induced transmembrane protein 1 regulates endothelial lumen formation during angiogenesis.</a> Arterioscler Thromb Vasc Biol. 34 (5), 1011-1019 (2014).</li><li>Coultas, L., Chawengsaksophak, K., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+VEGF+in+vascular+development.">Endothelial cells and VEGF in vascular development.</a> Nature. 438 (7070), 937-945 (2005).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+for+fibroblasts+in+angiogenesis:+fibroblast-derived+matrix+proteins+are+essential+for+endothelial+cell+lumen+formation.">The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell lumen formation.</a> Mol Biol Cell. 22 (20), 3791-3800 (2011).</li></ol>

作者没有什么可透露的。

该协议提出了一种方法, 以描述复杂的阶段和异型细胞相互作用的发芽血管生成通过使研究员使用遗传和成像方法进行彻底的机械调查。在进行检测时, 必须在珠搅拌步骤中进行有效的珠状血管内皮涂层。不良的内皮涂层将明显, 如果珠似乎没有像高尔夫球一样粗糙表面第二天早晨之前, 凝胶植入, 而不是完全平滑。注意确保在每一个搅拌步骤中的珠子充分悬浮, 通过大力搅拌管, 允许最大暴露的珠?...

血管生成对适当的胚胎发生和创面愈合至关重要, 在包括癌症进展1和冠状动脉疾病在内的众多疾病中也起着关键作用。2,3更好地了解在正常发育过程中血管生成的发生方式, 以及如何在病理环境中重新激活, 对于开发新颖有效的治疗方法至关重要。忠实的体外模型重述了血管生成体内中所涉及的重要阶段和细胞类型, 以使研究人员能够更好地描述推动血管生成的分子机制并作出新的发现。内皮细胞调节。
中津和休斯优化了发芽珠的检测, 他们已经证明经历了许多已知的阶段发芽血管生成。4,5本文提出的方法的目的是在中津和休斯通过将 perictyes 加入到实验中进行优化的分析, 从而将壁画细胞在内皮细胞发芽中的分泌和 juxtracrine 作用纳入新的血管生成研究。毛细血管是由它们的角色定义的壁画细胞, 因为它们被嵌入在血管基底膜中, 与内皮细胞保持紧密的物理接触。6毛细血管和内皮细胞通过信号通路进行复杂的交叉谈话, 包括凹口信号、Ang-Tie2、PDGFRβ、TGFRβ和许多其他。7,8小鼠模型在这些信号通路中缺乏, 表明在胚胎发生中发育血管的周细胞覆盖率较差, 导致血管重塑和功能失调。7此外, 毛细血管在病理血管生成中的作用是重要的, 但往往不受重视。例如, 肿瘤血管的一个独特的特点是, 血管是更不成熟, 漏水, 和功能失调, 由于不良的周细胞覆盖率。9已建议毛细血管的存在或缺失会显著影响肿瘤血管的表型, 是对血管生成和抗肿瘤治疗反应的重要调解人。9因此, 包括毛细血管在体外化验中的作用, 是更全面地捕捉内皮调节的重要机制的关键。
尽管目前有许多用于研究血管生成的体外和体内检测方法, 但在每个方面都存在一些不足之处。一些, 如内皮增生和内皮移植的检测, 是过度简化, 并集中在一个内皮功能的一个孤立的设置, 在组织培养塑料。10其他检测在3维 (3D) 设置中发生, 如胶管形成试验、10 , 但这些化验仍然过于简单化, 更注重内皮细胞迁移的能力, 并形成-新生血管结构, 而不是从预先存在的血管发芽。此外, 这些化验都没有纳入壁画细胞类型。有前体模型, 如环形主动脉检测, 包括毛细血管存在于宿主器官中, 但这些模型的遗传操作更具挑战性, 因为有必要产生挖空或转基因小鼠模型的感兴趣的途径。珠发芽法是理想的, 因为它模型内皮发芽, 增殖, 迁移, 甚至吻合和流明形成的3D 矩阵。4该检测忠实地允许对发芽的许多不同阶段进行机械性评估, 同时仍然允许在更受控制的环境中对内皮细胞或毛细血管进行直接的基因修饰。含有发芽珠的纤维蛋白凝块可以很容易地固定, 染色, 并在不同的发芽阶段成像;这些芽也可以放置在现场成像室, 以执行实时成像的发芽。本文提出的方法是研究血管生成的基本机制, 通过深入分型和深入分析血管生成过程中激活的通路。

<table><tbody><tr><td>Sterile Pipette tips</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipettors</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Complete EGM2 Media Bullet Kit</td><td>Lonza</td><td>CC-3162</td><td>HUVEC Media</td></tr><tr><td>MEM</td><td>Gibco</td><td>11095114</td><td>10T1/2 Media</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965118</td><td>NHLF Media</td></tr><tr><td>Tissue culture-grade PBS</td><td>Gibco</td><td>14190-144</td><td>Magnesium and calcium free</td></tr><tr><td>Accutase</td><td>Life Technologies</td><td>A1110501</td><td>For lifting HUVEC</td></tr><tr><td>Trypsin</td><td>Life Technologies</td><td>15050065</td><td>For lifting 10T 1/2 and NHLF</td></tr><tr><td>Customs siRNAs</td><td>Sigma</td><td></td><td></td></tr><tr><td>Lipofectamine RNAiMax</td><td>Life Technologies</td><td>13778150</td><td></td></tr><tr><td>HUVEC</td><td>Lonza</td><td>C2517A</td><td></td></tr><tr><td>10T 1/2</td><td>ATCC</td><td></td><td></td></tr><tr><td>NHLF</td><td>ATCC</td><td></td><td></td></tr><tr><td>Cytodex 3 microcarrier beads</td><td>Sigma</td><td>C3275</td><td></td></tr><tr><td>Tissue culture-coated 6 and 10 cm plates</td><td>Corning</td><td></td><td></td></tr><tr><td>Fibrinogen from bovine plasma</td><td>Sigma</td><td>F8630</td><td></td></tr><tr><td>Thrombin</td><td>Sigma</td><td>t9549</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>a3428</td><td></td></tr><tr><td>Falcon Round-Bottom Tubes</td><td>Corning</td><td></td><td></td></tr><tr><td>Tissue culture incubator and hood</td><td></td><td></td><td></td></tr><tr><td>24-well glass bottom plates</td><td>MatTek</td><td>P24G1.513F</td><td>Glass-bottom plates are needed only if the sprouts are going to be imaged. If not, tissue culture plastic is also acceptable.</td></tr><tr><td>Sterile Filtration Device</td><td></td><td></td><td></td></tr></tbody></table>

incorporating pericytes into an endothelial cell bead sprouting assay

血管生成是新血管从预存在血管生长而来的, 是许多生物过程的重要组成部分, 包括胚胎发生和发育、伤口愈合、肿瘤生长和转移以及眼部和心血管疾病。有效的体外模型概括了血管生成的生物学, 需要适当地研究这一过程, 并确定可最终针对新的治疗策略的调控机制。微珠血管生成实验已被证明, 以概括的多阶段内皮发芽在体外.然而, 这一检测的局限性是缺乏内皮-壁画细胞相互作用,这是关键的分子和表型调节内皮细胞功能在体内。这里给出的协议提出了一个方法, 将壁画细胞纳入珠血管生成试验, 并表明在发芽体外的血管内皮细胞和毛细血管紧密联系。该议定书还详细说明了一种方法, 有效地沉默的目标基因使用 siRNA 在内皮细胞的机械学研究。总之, 本协议提供了一种体外分析方法, 可以更恰当地模拟发芽血管生成过程中所涉及的不同细胞类型, 并为治疗评估和新发现提供了一个更加生理学相关的平台。血管生成调控机制。

此协议允许两个单元格类型的紧密关联体外,和毛细血管的存在补充发芽的发生 (图 1A, B)。该协议还允许有效的沉默 (例如通过 RNA 干扰) 的基因感兴趣的细胞类型的兴趣 (如 VEGFA 特别是在内皮细胞或 PDGFRβ在毛细血管)7,12在检测期间, 这可转化为对发芽表型的抑制作用。使用本协议?...

Watch this Scientific Journal Video about Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay at JoVE.com

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

本协议提出了一种新的体外微珠分析方法, 更恰当地模拟体内的过程, 并结合毛细血管。这种修改使珠粒化验更忠实地重述内皮细胞和壁画细胞之间的异型细胞相互作用, 对血管生成至关重要。

将毛细血管纳入内皮细胞珠发芽试验

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

incorporating-pericytes-into-an-endothelial-cell-bead-sprouting-assay

Research

JoVE Journal

Developmental Biology

7.9K 次观看.  University of North Carolina at Chapel Hill. 本协议提出了一种新的体外微珠分析方法, 更恰当地模拟体内的过程, 并结合毛细血管。这种修改使珠粒化验更忠实地重述内皮细胞和壁画细胞之间的异型细胞相互作用, 对血管生成至关重要。

视频: Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

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, branching, 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 within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an 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. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

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

科研

生物学

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 ...

发育生物学

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

生物工程

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

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 ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

评估血脑屏障网状血管形成和破坏的体外测定

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

医学

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

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

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 ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

使用划痕伤口迁移和球状体发芽测定法研究血管生成

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

作者聚焦：通过检测开发中的挑战和创新研究血管生成

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

将毛细血管纳入内皮细胞珠发芽试验

摘要

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此视频中的章节

优化的纤维蛋白凝胶珠检测血管生成的研究

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

缩微和3D微血管大会

微流控装置中组织模型的 Perfusable 血管网

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

评估血脑屏障网状血管形成和破坏的体外测定

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

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成