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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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, 允许最大暴露的珠表面的内皮细胞, 但不那么积极, 它导致内皮细胞已经附着成脱离珠。
将毛细血管加入化验时, 必须维持5内皮细胞与1周细胞的比例。过量的毛细血管导致他们在检测期间 overgrow 和超过内皮细胞的数量, 而毛细血管的不足会导致血管覆盖率低下。如果保持适当的细胞密度, 但仍观察到血管的周细胞覆盖率较差, 则可能需要修改与毛细血管的搅拌时间。不建议更改单元格编号。另一种方法可能是只用内皮细胞涂上4小时的珠子, 然后通过 resuspending 珠和细胞溶液来去除介质中的内皮细胞, 并在细胞沉淀后取出介质。, 然后添加在毛细血管和搅拌每20分钟一个额外的小时。
当将包覆的珠子嵌入纤维蛋白凝块中时, 在凝块形成过程中, 如果不破坏凝胶的完整性, 也至关重要。破坏基质形成可能导致异常发芽。还必须确保在凝胶中保持适当的珠密度。如果珠子的数量太稠密, 那么靠近彼此的珠子就会影响到相邻珠子的发芽, 并且会开始向彼此吻合。根据研究人员的终极目标和对船只与船只相互作用特征的兴趣, 可能需要修改珠密度。然而, 在凝胶内彼此接近的单独的珠子和珠子的异质种群可能给出很大的可变发芽表型。通过改变在该协议的搅拌步骤中添加到流化管中的珠溶液体积, 或者改变用于在凝胶植入前并用重悬珠子的纤维蛋白溶液体积, 可以调整珠密度。
同样重要的是, 每毫升2万细胞密度的健康 NHLFs 在每一个纤维蛋白凝块的顶端被镀。请注意, 持续供应的生长因子提供的积极分裂 NHLFs 是必要的, 以强健发芽在试验期间。NHLF 条件的 EGM2 不是一个足够的替代品。13如果在检测过程中观察到发芽不良, 建议使用一瓶新鲜的完整培养基和新的、健康的 NHLF 细胞通道。
虽然这种检测开始以更大的分辨率来解决血管生成的细胞和表型复杂性, 但它仍然表示相对于真实的在体内上下文中的较简单化的系统。此外, 这种检测中出现的发芽血管生成更类似于一种健康的生理环境, 而不是像肿瘤血管生成这样的病理环境。这种方法的未来应用可能包括引入额外的细胞类型 (如调节血管生成反应的免疫细胞群), 以进一步重述这一复杂过程中所涉及的异型细胞相互作用。外部压力源的引入--如与纤维蛋白凝胶中的发芽血管相近的肿瘤--模拟病理血管生成在体外也可用于允许更彻底、更具机械性地描述分子通路在病理环境中激活。这些进展对于提高研究人员在更受控制的环境中进行血管生成的复杂阶段的能力是重要的, 最终能够发现和更深入地描述新的治疗途径以定位病理血管生成。

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。使用本协议的研究者们被鼓励进行标准的内皮细胞发芽试验, 平行于周细胞涂层发芽试验, 以更彻底地研究毛细血管在血管功能中的独特贡献。和表型研究。
在本协议中描述的条件是非常重要的, 以获得足够的涂层珠 (图 2A, B), 将能够在纤维蛋白凝胶的强健发芽。作者发现过量的毛细血管 (如毛细血管涂在 1 10T1/2 至 1 HUVEC) 导致整个油井周细胞过度生长, 随后无法辨别井内不同的血管结构。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57309/57309fig1.jpg"> 图1。毛细血管在珠发芽试验中与内皮血管紧密联系.(A) 标准发芽试验, 仅 HUVEC 涂珠。(B) 周细胞发芽试验的共焦显微图像表明, 毛细血管在发芽试验中环绕血管内皮血管, 但不会阻碍其发芽。蓝色是赫斯特 (核污点);红色是 CD31 (内皮染色), 绿色是 Desmin (周细胞染色)。所有刻度条, 50 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57309/57309fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57309/57309fig2.jpg"> 图2。涂层珠子有一个粗糙的, 高尔夫球般的外观。裸载体珠 (a) 有一个完全平滑的表面, 而适当涂敷的珠子为纤维蛋白凝胶植入 (B) 有一个粗糙的, 高尔夫球样的外观。缩放条, 50 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57309/57309fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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 ...

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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

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

内皮细胞迁移评估暴露于有毒化学品后，

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

科研

发育生物学

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, ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

从小鼠骨骼肌分离I型和II型周细胞

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

无珠光高音喇叭在食子胚胎中的细胞力学探测

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

微流控法评价人诱导的多能干细胞内皮细胞 (hipsc-ec) 对白细胞粘附的评价

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.