作者感谢以下工作人员和研究人员: Andrée Gauthier-费希尔, 马修 Librach, 坦尼娅先生, Tharsan Velauthapillai 和萨拉 Laronde。

所有涉及动物的研究都是按照到达指南 29 进行的。所有研究都是通过机构研究伦理委员会的批准 (REB 号 4276) 进行的。所有动物程序均由大学卫生网络 (加拿大多伦多) 动物保育委员会批准, 所有动物都接受了符合《 动物保育和使用指南》的人道护理 , 8 th 版 (国立卫生研究院 2011). 
 1. 主动脉环试验装置 <ol> <li> 将大鼠主动脉分离。
	<ol> <li> 安乐 8-10 周老大鼠使用 co 2 窒息: 设置 co 2 室到20% 气体置换 (流速 = 0.2 x 室容积/分钟)。确认由触摸胸部没有捏反射和心脏跳动. </li>
		<li> 用70% 乙醇消毒纱布在胸部区域湿润毛皮。使用灭菌钳和剪刀, 使胸部中线切开, 并通过皮肤和肌肉层. </li>
		<li> 一旦肋骨笼暴露, 削减两侧的肋骨笼和折叠向上约5毫米从胸骨两侧。新鲜尸体上的肋间动脉可能有出血. </li>
		<li> 将肺组织和心脏推到解剖右侧以暴露主动脉。主动脉是位于脊柱附近的白色彩色血管. </li>
		<li> 用镊子夹住主动脉弓后的主动脉, 用手术剪刀做第一个切口, 同时用镊子抓住主动脉。在切割主动脉时, 胸腔很快就会充满血液, 因此在凝血前快速获得主动脉是至关重要的. </li>
		<li> 使用镊子握住主动脉, 将主动脉向下轻轻剥离, 远离脊柱。这种拉力将会切断肋间动脉而不会进一步切口。获得大约10厘米的组织和/或前主动脉分为两个分支在腹腔和削减主动脉的胴体。如果需要, 将膜片推到尾侧方向, 以获得对主动脉下部部分的访问. 
		注意: 轻轻地剥开主动脉, 因为强烈的力量会导致它撕裂。如果主动脉流泪, 允许血液凝固几秒钟, 用纸巾除去凝结的血液, 从而使余下的主动脉可见. </li>
		<li> 将主动脉置于15毫升管中, 10 毫升的 ice-cold 汉克和 #39 的平衡盐溶液 (HBSS) 补充1% 的青霉素/链霉素 (P/s)。把管子放在冰上. 
		注: 主动脉组织可以在冰上持续1-2 小时, 但最好尽快处理. </li>
	</ol> </li> <li> 在无菌生物安全柜 (BSC) 中准备主动脉环试验培养基。
	<ol> <li> 通过使用过滤单元来筛选内皮生长培养基 (华), 以过滤500毫升的内皮基底培养基 (循证医学), 辅以2% 胎牛血清 (FBS)、1% 庆大霉素和生长因子 (见 材料表 )。50毫升的过滤介质成珠或水浴在37和 #176; C. </li>
		<li> 使用另一个过滤单元来过滤100毫升的循证医学, 补充 2% FBS 和1% 米/秒, 以准备循证医学 2% FBS (循证医学-FBS), 并存储在4和 #176; C. </li>
	</ol> </li> <li> 在冰上工作时, 用基底膜萃取物 (BME) 将12层组织培养皿涂上。
	<ol> <li> 在冷表面 (大冰包或托盘) 上放置一个12井板。添加200和 #181; 使用冷冻吸管提示和旋流 BME 的新鲜解冻 BME, 确保均匀涂层。工作迅速, 避免了 BME 的不均匀聚合. 
		注: 一个典型的主动脉切除产生20主动脉环。为了解释主动脉环检测的变异性, 每个治疗组至少设置三主动脉环化验, 并包括一个控制。如果源许可证, 建议每治疗组6环. </li>
		<li> 将涂层的板置于湿恒温箱中 (95% 相对湿度, 37 和 #176; C, 5% CO 2 ; 这些条件适用于以下所有加湿孵化器步骤) 30 min. 1000 和 #181; L 吸管提示回到-20 和 #176; C 用于步骤 1.5.3. 
		注: 当基膜萃取物用于储存浓度时, 其稠度和刚度严格受聚合时间的控制。确保所有的平行和独立的实验都保持完全相同的聚合时间. </li>
	</ol> </li> <li> 将主动脉分为均匀环。
	<ol> <li> 从15毫升的管中取主动脉, 并将其放入10厘米的盘中, 10 毫升的新鲜 HBSS 补充1% 的 P/秒. </li>
		<li> 使用两套镊子仔细分离多余的结缔组织, 脂肪组织, 并使用手术刀, 其余分支的肋间动脉连接到主动脉。这减少了基于不同水平的残余组织的环单元之间的变异性。从主动脉内取出任何残余凝结的血液. </li>
		<li> 在 10 cm 盘下放置一个标尺或网格, 用无菌手术刀和镊子精确切割 1-2 毫米宽的主动脉部分。尽可能精确地剪切剖面, 以减少单位间的可变性. </li>
	</ol> </li> <li> 将主动脉环嵌入 BME. 
	注意: 如果在 BME 聚合孵化前未完成主动脉环的切片, 则从湿化恒温箱中取出涂层板, 并添加100和 #181; 每一个井都要终止聚合。确切的时机是至关重要的, 因为 over-polymerization 的 BME 可能会干扰内皮网络的发展和细胞迁移。一旦准备好主动脉环, 从井中移除循证医学, 并继续1.5.2。
	<ol> <li> 从湿润的恒温箱中取出 BME 涂层的井, 并返回到 BSC. </li>
		<li> 小心地拿起单独的主动脉环与钳子和安置一个在每个 BME 涂好的中间。对余下的主动脉环重复. 
		注: 与主动脉环一起传输的介质应保持最小, 以避免聚合不规则. </li>
		<li> 使用冷却 (-20 和 #176; C) 1000 和 #181; l 吸管提示添加300和 #181; l BME 在每个主动脉环的顶部, 并均匀分布 BME 周围的井. </li>
		<li> 将嵌入的主动脉环返回到湿式恒温箱30分钟. </li>
		<li> 将带有嵌入的主动脉环的板从加湿的恒温箱中取出, 然后缓慢地添加1000和 #181; 华 (在步骤1.2.1 中准备和加热), 将吸管尖端放在文化壁上. 
		注: 直接移介质上的 BME 会扰乱聚合 BME, 阻碍连续的内皮网络发育。如果可用, 请使用适当的多通道吸管. </li>
		<li> 24 小时后, 移除1000和 #181; 华, 替换为1000和 #181; 循证医学 FBS 的 1.2.2, 又慢慢地移在文化的侧面. </li>
	</ol> </li> <li> 通过替换500和 #181 来保持主动脉环检测; 循证医学 FBS 与500和 #181; 新鲜循证医学 FBS (37 和 #176; C) 每 48 h. 
	注意: 见 sec2开始 MSC 文化扩展与主动脉环化验建立同一天。这将确保 MSCs 可以在血管内皮网络准备就绪时进行共培养. </li>
	<li> 使用明亮的现场显微术跟踪主动脉环检测血管内皮网络的发育 (参见 图 1 作为最佳网络开发的参考)。一旦血管内皮网络方面如 图 2 所示, 继续建立主动脉环试验理学硕士 co-cultures (第3节)。有关血管内皮网络成像的进一步细节, 请参阅步骤 4.1. </li>
</ol> 2。组织培养 <ol> <li> 准备组织培养介质的库存解决方案。
	<ol> <li> 区域性 FTM HUCPVCs (以前已建立, n 和 #8805; 3 独立行) 30 和商业上可用的骨髓基质细胞在α-记忆补充 10% FBS 和1% 的 P/秒。
		<li> 使用0.2 和 #181 对介质进行消毒; m 孔径过滤瓶. </li>
		<li> 将准备好的介质解决方案存储在4和 #176; C 最多3周. </li>
	</ol> </li> <li> 在 70-80% 70-100 中保持 FTM HUCPVC 和骨髓的培养, 通过相衬显微镜确定。使用适当的培养基体积大小的组织培养皿使用 ( 即 10 毫升在一个10厘米的菜肴)。使用这些区域性条件来维护和传代 MSCs. </li>
	<li> co-cultures 使用离解酶溶液 (4 mL/10 cm 盘) 和在加湿的恒温箱中孵育3分钟, 将 msc 单分子膜传代或主动脉环试验分离出来. 确保细胞脱离使用明亮的野外显微镜; 孵化额外的 1-2 分钟, 如果需要. </li>
	<li> 将离解的单元格转换为15毫升的管, 并在 400 x g 处离心5分钟. </li>
	<li> 吸上清液而不干扰细胞颗粒, 重1毫升的适当培养基中的细胞 (α-传代细胞的完整培养基或循证医学-FBS 主动脉环试验/MSC co-cultures) 用于计数使用细胞计数器. </li>
</ol> 3。主动脉环试验/msc 共培养物的制备 <ol> <li> 种子 10 4 MSCs 在每个嵌入的主动脉环上 (9000 单元/cm 2 /12-井板)。根据主动脉环检测的数量, pre-stain 了具有可行的不可荧光染料的 MSCs。在对照组中维持至少三口不含 MSCs 的主动脉环。
	<ol> <li> 以着色 10 5 MSCs/mL 循证医学 FBS 介质, 将2.5 和 #181; 可转让的荧光染料/ml 介质 (5-#181; M) 添加到15毫升的管中, 并在湿化培养箱中放置30分钟. 轻轻地将试管混合在孵化的中途. </li>
		<li> 在30分钟孵化后, 在染色细胞中加入1体积的新循证医学 FBS, 并在 400 x g 处旋转5分钟. </li>
		<li> 在1毫升的循证医学 FBS 介质中去除上清和 re-suspend 细胞颗粒。使用荧光显微镜确认骨髓间充质干细胞的成功染色. </li>
	</ol> </li> <li> 从湿化培养箱中取出主动脉环板, 并移除500和 #181; FBS。
	<li> 小心种子 10 4 在500和 #181 中的 MSCs; 循证医学 FBS 对每个嵌入的主动脉环进行了均匀移周围的内皮网络。通过轻轻摇动培养基板确保均匀分布。
	<ol> <li> 添加额外的循证医学 FBS, 以确保主动脉环检测/MSC co-cultures 的介质总容量为1000和 #181; L. </li>
	</ol> </li> <li> 使用荧光显微镜来可视化主动脉环检测中的荧光标记的 MSCs. </li> </ol>
 4. 显微镜 <ol> <li> 在主动脉环检测/MSC co-cultures 基线 (天 0) 测量内皮网络属性 ( (如 、网络) 之前, 使用明亮场显微镜对大鼠内皮网络进行图像成像。长度, 网络循环)。图像4象限每井从主动脉环到最远的部分的内皮网络在该象限。
	<ol> <li> 图像 co-cultures 在3、5和7天的主动脉环检测/MSC 后主动脉环网络。使用这些图像来量化 MSC 对内皮网络发育的影响. </li>
	</ol> </li> <li> 使用荧光显微镜对主动脉环检测中的 pre-labelled MSCs 进行图像处理。
	<ol> <li> 在 24 h 主动脉环试验/msc co-cultures 后, 使用荧光显微镜来可视化 msc 的归巢、伸长和与内皮网络的融合。将 MSCs 的荧光图像与内皮细胞的明亮场图像重叠, 以观察两种细胞类型的定位. </li>
		<li> 将 mscs 定位在主动脉环组织近端的发达内皮网络内, 并将 mscs 定位在主动脉环组织远端的新发展的网络中 ( 图 2 ). </li>
	</ol> </li> </ol> 5。流式细胞仪和 qPCR <ol> <li> 前一日游离主动脉环内皮网络, 解冻冰冻中性在4和 #176; C O/n 段5.3 对于流式细胞术和 qPCR 是相同的. </li>
	<li> 暖50毫升的中性和50毫升0.5% 胰蛋白酶在37和 #176; C 珠或水浴. </li>
	<li> 从主动脉环检测/MSC co-cultures 中检索细胞进行分析。
	<ol> <li> 在1周主动脉环检测/MSC 共培养后, 移除培养基, 并将1毫升磷酸缓冲盐 (PBS) 缓慢地添加到每一个井中3分钟并取出。重复这洗两次. </li>
		<li> 添加800和 #181; 5ml 中性每个井, 并在湿化孵化器中孵育15分钟. </li>
		<li> 从湿化恒温箱中取出板, 并将移 (5-10x) 暂停中性, 以分解 BME, 并将每个井的内容转换为单独的15毫升管. </li>
		<li> 重复步骤5.3.3 再次与新鲜5ml 中性, 直到没有残留的 BME 在文化中观察良好。添加1体积 PBS 补充与 3% FBS 中性细胞悬液, 使中性。移除在细胞悬浮漂浮的主动脉环使用吸管提示. </li>
		<li> 将电池悬浮在 400 x g 处旋转5分钟, 小心取出上清, 在 15 ml 管中留下3毫升的细胞悬浮液. 
		注意: 一个明显的, 固体颗粒可能不可见, 但细胞和 BME 存在. </li>
		<li> 添加3毫升的 prewarmed 0.5% 胰蛋白酶到细胞悬浮和大力重 (5-10x 的移) 细胞。在湿润恒温箱中孵育 trypsinized 细胞溶液10分钟. </li>
		<li> 将 trypsinized 细胞悬浮液从加湿的恒温箱中取出, 并添加6毫升 PBS 补充 3% FBS 和重几个 tim达累斯萨拉姆.旋转细胞悬浮在 400 x g 为 5 min. </li>
		<li> 小心移除所有上清液, 将细胞颗粒留在试管中, 并在1毫升 PBS 重细胞中补充 3% FBS. </li>
		<li> 使用70和 #181 对 1 mL 细胞悬浮液进行过滤, 以去除细胞悬浮液中的聚集细胞和残余 BME。使用单元计数器对1毫升的细胞悬浮细胞进行计数. </li>
	</ol> </li> <li> 为流式细胞术准备细胞。
	<ol> <li> 在200和 #181 中孵化单元悬浮 (10 5 单元格; PBS 补充 3% FBS) 与荧光共轭 (FITC 或 APC) 主要抗体 (TRA-1-85, CD146, CD31) 浓度 = 1:40) 在4和 #176; C 为30分钟, 保护免受光. 
		注: al. 的流式细胞仪是由红 et , 2013 28 优化的。准确读数需要至少1万细胞. </li>
		<li> 30 分钟孵育后, 重2毫升 PBS 补充 3% FBS 和离心机 (400 x g, 5 min) 的细胞. </li>
		<li> 将单元格保持在4和 #176; C 保护不受光照, 直到流式细胞仪 (至少 1 x 10 4 事件) 在 1 h 内进行分析。有关浇口策略, 请参见 补充图 1 。分类人 MSCs 使用 anti-TRA-1-85 (APC) (浓度: 1:40 和 0.5% FBS/PBS) 和排序细胞成350和 #181; 细胞裂解缓冲 qPCR 分析. 
		注: 裂解细胞可存储在-80 和 #176; C 为未来 qPCR 分析3月. </li>
	</ol> </li> <li> 为 qPCR 分析准备单元格。
	<ol> <li> 使用列 rna 隔离套件将 rna 从裂解细胞中分离出来, 并确定 rna 的浓度和质量. </li>
		<li> 制备5和 #181 的 cDNA; 每100和 #181 的 RNA g; 反转录酶反应。如果 RNA 产量低 (#60; 10 ng/和 #181; L), 执行放大步骤。使用少于 100 ng 的 RNA 将导致高率的假阴性. 
		注: cDNA 模板可存储在-20 和 #176; C 用于进一步分析, 可长达4月. </li>
		<li> 使用血管生成表达式探查器数组执行 qPCR, 以检测基因表达的变化。每个反应使用 5 ng (40 循环, 60 和 #176; C 退火/延长温度)。与未分化 MSC 衍生的 cDNA 样本相比, 表达式的折叠变化。运行适当的负控制 (主动脉环没有人 MSCs). </li>
	</ol> </li> </ol> 6。主动脉环检测/MSC 联合培养后的网络量化 <ol> <li> 下载图像处理软件 (如 ImageJ) 以及血管生成分析仪插件 31 。
	<li> 使用该软件导入和打开的内皮网络图像. </li>
	<li> 根据所用显微镜的规格转换图像刻度. </li>
	<li> 使用 straight-line 工具来测量径向网络增长. </li>
	<li> 使用单元计数器工具计数内皮网络循环 (至少有4边的循环应该是量化的) </li>
	<li> 对于网络属性的其他量化, 请使用血管生成分析器插件或其他类似插件来分析主线段、总段长度、总网络区域和连接数。使用模糊蒙版工具来模糊主动脉环组织和空空间, 以避免假阳性计算. </li>
	<li> 将值转换为统计程序, 并在主动脉环检测/MSC co-cultures 后绘制内皮网络属性. </li>
</ol>

<ol>
	<li>Potente, M., Gerhardt, H., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+and+therapeutic+aspects+of+angiogenesis.">Basic and therapeutic aspects of angiogenesis.</a> Cell. 146 (6), 873-887 (2011).</li><li>Hoeben, A., Landuyt, B., Highley, M. S., Wildiers, H., Van Oosterom, A. T., De Bruijn, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+and+angiogenesis.">Vascular endothelial growth factor and angiogenesis.</a> Pharmacol Rev. 56 (4), 549-580 (2004).</li><li>Khan, T. A., Sellke, F. W., Laham, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+therapeutic+angiogenesis+for+limb+and+myocardial+ischemia.">Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia.</a> Gene Ther. 10 (4), 285-291 (2003).</li><li>Gupta, R., Tongers, J., Losordo, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+studies+of+angiogenic+gene+therapy.">Human studies of angiogenic gene therapy.</a> Circ Res. 105 (8), 724-736 (2009).</li><li>Chu, H., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+angiogenesis:+controlled+delivery+of+angiogenic+factors.">Therapeutic angiogenesis: controlled delivery of angiogenic factors.</a> Ther Deliv. 3 (6), 693-714 (2012).</li><li>Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+angiogenesis+for+ischemic+disorders:+what+is+missing+for+clinical+benefits?.">Therapeutic angiogenesis for ischemic disorders: what is missing for clinical benefits?.</a> Discov Med. 9 (46), 179-184 (2010).</li><li>Said, S. S., Pickering, J. G., Mequanint, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+growth+factor+delivery+for+therapeutic+angiogenesis.">Advances in growth factor delivery for therapeutic angiogenesis.</a> J Vasc Res. 50 (1), 35-35 (2013).</li><li>Bergers, G., Song, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pericytes+in+blood-vessel+formation+and+maintenance.">The role of pericytes in blood-vessel formation and maintenance.</a> Neuro Oncol. 7 (4), 452-464 (2005).</li><li>Leeper, N. J., Hunter, A. L., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapy+for+vascular+regeneration:+adult,+embryonic,+and+induced+pluripotent+stem+cells.">Stem cell therapy for vascular regeneration: adult, embryonic, and induced pluripotent stem cells.</a> Circulation. 122 (5), 517-526 (2010).</li><li>Sieveking, D. P., Ng, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapies+for+therapeutic+angiogenesis:+back+to+the+bench.">Cell therapies for therapeutic angiogenesis: back to the bench.</a> Vasc Med. 14 (2), 153-166 (2009).</li><li>Staton, C. A., Reed, M. W., Brown, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90 (3), 195-221 (2009).</li><li>Auerbach, R., Lewis, R., Shinners, B., Kubai, L., Akhtar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+assays:+a+critical+overview.">Angiogenesis assays: a critical overview.</a> Clin Chem. 49 (1), 32-40 (2003).</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>Arnaoutova, I., George, J., Kleinman, H. K., Benton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+cell+tube+formation+assay+on+basement+membrane+turns+20:+state+of+the+science+and+the+art.">The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art.</a> Angiogenesis. 12 (3), 267-274 (2009).</li><li>Arnaoutova, I., Kleinman, H. K. <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+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nat Protoc. 5 (4), 628-635 (2010).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J Cell Mol Med. 10 (3), 588-612 (2006).</li><li>Nicosia, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aortic+ring+model+of+angiogenesis:+a+quarter+century+of+search+and+discovery.">The aortic ring model of angiogenesis: a quarter century of search and discovery.</a> J Cell Mol Med. 13 (10), 4113-4136 (2009).</li><li>Baker, M., 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=Use+of+the+mouse+aortic+ring+assay+to+study+angiogenesis.">Use of the mouse aortic ring assay to study angiogenesis.</a> Nat Protoc. 7 (1), 89-104 (2011).</li><li>Guo, J., 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=A+secreted+protein+(Canopy+2,+CNPY2)+enhances+angiogenesis+and+promotes+smooth+muscle+cell+migration+and+proliferation.">A secreted protein (Canopy 2, CNPY2) enhances angiogenesis and promotes smooth muscle cell migration and proliferation.</a> Cardiovasc Res. 105 (3), 383-393 (2015).</li><li>Wittig, C., Scheuer, C., Parakenings, J., Menger, M. D., Laschke, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geraniol+Suppresses+Angiogenesis+by+Downregulating+Vascular+Endothelial+Growth+Factor+(VEGF)/VEGFR-2+Signaling.">Geraniol Suppresses Angiogenesis by Downregulating Vascular Endothelial Growth Factor (VEGF)/VEGFR-2 Signaling.</a> PLoS One. 10 (7), e0131946 (2015).</li><li>Masson, V. V., 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=Mouse+Aortic+Ring+Assay:+A+New+Approach+of+the+Molecular+Genetics+of+Angiogenesis.">Mouse Aortic Ring Assay: A New Approach of the Molecular Genetics of Angiogenesis.</a> Biol Proced Online. 4, 24-31 (2002).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells+for+tissue+engineering+versus+regenerative+medicine.">Adult mesenchymal stem cells for tissue engineering versus regenerative medicine.</a> J Cell Physiol. 213 (2), 341-347 (2007).</li><li>Caplan, A. I., Dennis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+trophic+mediators.">Mesenchymal stem cells as trophic mediators.</a> J Cell Biochem. 98 (5), 1076-1084 (2006).</li><li>Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+new+directions.">Mesenchymal stromal cells: new directions.</a> Cell Stem Cell. 10 (6), 709-716 (2012).</li><li>Kilroy, G. E., 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=Cytokine+profile+of+human+adipose-derived+stem+cells:+expression+of+angiogenic,+hematopoietic,+and+pro-inflammatory+factors.">Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors.</a> J Cell Physiol. 212 (3), 702-709 (2007).</li><li>Tang, Y. L., 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=Paracrine+action+enhances+the+effects+of+autologous+mesenchymal+stem+cell+transplantation+on+vascular+regeneration+in+rat+model+of+myocardial+infarction.">Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction.</a> Ann Thorac Surg. 80 (1), 229-236 (2005).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+MSCs+are+pericytes?.">All MSCs are pericytes?.</a> Cell Stem Cell. 3 (3), 229-230 (2008).</li><li>Hong, S. H., 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=Ontogeny+of+human+umbilical+cord+perivascular+cells:+molecular+and+fate+potential+changes+during+gestation.">Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation.</a> Stem Cells Dev. 22 (17), 2425-2439 (2013).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+The+ARRIVE+guidelines+for+reporting+animal+research.">Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research.</a> J Pharmacol Pharmacother. 1 (2), 94-99 (2010).</li><li>Sarugaser, R., Ennis, J., Stanford, W. L., Davies, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+propagation,+and+characterization+of+human+umbilical+cord+perivascular+cells+(HUCPVCs).">Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs).</a> Methods Mol Biol. 482, 269-279 (2009).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Gelati, M., Aplin, A. C., Fogel, E., Smith, K. D., Nicosia, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+angiogenic+response+of+the+aorta+to+injury+and+inflammatory+cytokines+requires+macrophages.">The angiogenic response of the aorta to injury and inflammatory cytokines requires macrophages.</a> J Immunol. 181 (8), 5711-5719 (2008).</li></ol>

Dr. 克利福德 l. Librach 是专利的联合持有者:分离和使用从早期的脐带组织中提取的细胞的方法。在加拿大和澳大利亚被授予。

在建立一个成功的主动脉环试验 MSC 共培养实验的几个关键阶段。首先, 分离和剖胸主动脉的最重要步骤是: 1) 完全获得主动脉的胸椎段;2) 仔细去除分支血管、结缔组织和脂肪组织;3) 切割甚至部分主动脉 (〜 1 mm), 以限制每一个化验之间的变异性。第二, 成功地将主动脉环植入 BME 是该方法的关键。如果 BME 没有完全聚合或聚合不均匀, BME 中嵌入的主动脉环将无法启动内皮发芽, 或者它们可能发育不连续的内皮网络。如果 BME 聚合不充分, 则定量分析不可靠。第三, 完整的因子丰富的内皮细胞是必要的, 以提供营养的主动脉环开始发芽。一旦主动脉环启动血管发芽, 接下来的关键步骤将包括要测试的细胞类型。为此, MSCs 需要成功 pre-stained 和管理的萌芽主动脉环。本节有两个关键步骤。1) 选择合适的播种日: 网络应处于发展阶段, 不能很好地达到文化的高度覆盖率。如果没有及时地管理 mscs, 那么量化 mscs 对网络开发的净影响就变得具有挑战性。2) Prestaining 骨髓间充质干细胞在添加前: 如果 mscs 是 over-stained 或保持在颗粒中很长一段时间, 结块将发生损害归巢和整合内内皮网络。最后, 如果准备适当的控制, 可以很容易地实现对网络的量化。没有 mscs 的主动脉环将发展为血管内皮网络, 因为主动脉本身支持发芽, 但 mscs 可以促进不同网络结构的网络发展, 如本手稿所述。血管内皮网络的定量化应在建立 co-cultures 后至少 5 d。由于闭合系统, 血管生成反应是瞬变的, 内皮细胞网络开始退化, 大约 7 d。如果观察的目的是评估细胞类型对血管内皮网络发育的影响, 则应在大块的第一周内获得图像。
对目前的检测方法的一种可能的修改可以是应用初级人体组织。作为一个1毫米的大鼠主动脉的一部分, 使一个单位的共培养, 人类脉可以提供高数量的部分高度一致的测量。尽管可存活的人类原发性组织的高度限制, 但高产量表明潜在的可行性。在进行大规模评价的分析时, 预分析和储存其成分, 直到可以考虑用于测试的细胞。BME 涂层可以在组织培养皿上制备, 并以冷冻脱水的形式存放较长时间。此外, 正如我们在我们的实验室测试, 大鼠主动脉组织可以冷冻和储存以备以后使用, 从而使工作与最关键的元素的化验更方便。
尽管主动脉环试验有许多优点, 但还是有一些局限性需要列出。首先, 建立化验文化是有挑战性的。在目前的形式, 常规的应用需要足够的手工技能。操作员错误可以引入可变性。它可以反映在网络的增长, 使它难以可靠地评估的影响, 管理细胞和因此重要的血管生成的性质。然而, 如果与其他可用的方法相比, 这些挑战是相似的, 而且所需的培训期可以方便地缩短, 经济上与在体内实验相反。其次, 在植入前体主动脉组织与初始网络发展之间的滞后期, 这标志着细胞管理的时间需要由用户来解释。第三, 对血管内皮网络属性的量化可以是费时的, 但可以通过分配标志参数, 包括径向增长, 管长和网络网格尺寸来解决。这可以通过使用计算机辅助的图像分析 (图像 J) 来完成, 这可以显著减少一致性和可靠的量化所需的时间。第四, 由于动物组织来源的轻微不一致和操作者的操作, 每种化验结果之间的变异性都可能发生。我们发现, 这一挑战可以有效地克服和量化成为统计可靠通过建立 triplicates 为每个治疗组。最后, 提取和分类细胞的人和大鼠来源的 post-assay 分析可能是挑战。然而, 具体的蛋白酶, 包括中性和细胞恢复解决方案可用于恢复细胞型或基因表达分析。
与现有的方法, 无论是培养一个单一的内皮细胞类型或活动物系统, 这提出的化验使用的组合,前体主动脉组织和 BME。此设置使用户能够获得定性和定量的数据, 阐明细胞治疗候选的血管生成特性。结合 BME 和多的前体组织提供的性质密切模仿的微环境, 治疗细胞类型可能会遇到的地方管理。由于可能有大量的平行和对文化条件的严密控制, 操作员提供了一个系统, 其中包含了可靠评估所需的要素, 同时消除了变量和不一致动物模型。此外, 评估更可行, 因为它降低了成本和重复的动物程序的需要。单细胞分析和大多数动物模型缺乏的因素和变量引入的免疫反应, 否则将发生在临床应用的细胞治疗候选。炎症反应改变血管生成, 因此, 植入的物质或细胞的治疗效果不能精确量化。主动脉环试验排除许多炎症成分, 会扰乱化验测量。然而, 它包含的驻留巨噬细胞是重要的参与者在微血管发展32。通过简单地识别内皮网络和 prestained 人细胞, MSC 分泌炎症细胞因子的作用, 甚至细胞元素的免疫系统, 可以很容易地观察使用这种方法。
高控制由用户在测试条件和可再生性实验的设置允许对系统的进一步元素的介绍。首先, 主动脉环法可作为损伤模型。随着主动脉环和内皮细胞萌出的嵌入, 可以对缺血性损伤、炎症因子 (TNF-α) 或细菌性脂多糖 (LPS) 进行分析, 然后再进行 MSC, 以确定可能的血管生成的抢救受伤后的反应。其次, 阐明 MSCs 对血管生成反应的可能机制, neutrali可以利用细胞抗体来对潜在的受体进行沉默。最后, 为了研究骨髓间充质干细胞的分泌特性, 可以在 transwell 系统中建立主动脉环试验, 以排除血管生成反应细胞相互作用的影响, 而专注于分泌特性。
总之, 主动脉环法可以评估细胞治疗候选者的能力和效力, 调解 ECM 处理, 迁移到血管生成的区域, 并通过身体接触促进血管的发育。主动脉环体外血管生成试验可以作为一种有价值的、定量的筛选工具, 用于候选细胞系的再生治疗.

血管生成的复杂过程改善和维持组织灌注, 促进新的血管发育从预先存在的血管1。它是一个严密调控, 平衡的过程由 pro-angiogenic 和血管因素。该系统的任何缺陷都可能导致血管维持或生长不足, 导致严重的缺血性疾病, 包括心肌病、中风和神经退行性疾病。然而, 夸大的血管发育是典型的条件, 包括癌症和炎症性疾病2。
开发旨在控制血管生成以获得良好组织再生的治疗方法至关重要。尽管进行了广泛的前临床研究, 但使用 pro-angiogenic 因子和 rna 刺激血管生成的尝试未能达到预期的结果3,4,5。暂时性影响的可能原因包括: 血管生成蛋白和核酸的寿命有限, 以及靶生长因子的有限数量6,7。虽然可溶性血管生成因子对启动血管形成至关重要, 但血管的维持和功能依赖于支持的细胞类型, 包括周和平滑肌细胞8。pro-angiogenic 疗法的领域现在正在探索潜在的干细胞和祖细胞来源, 可能提供局部血管生成因子, 而物理支持新发展的血管, 更新甚至分化成内皮样细胞9,10。寻找能够满足这些功能要求的最佳血管生成细胞类型对缺血性组织再生有很大的希望。
为了成功地将可能的细胞疗法转化为临床试验, 前期研究需要证明它们的功效, 并强调潜在的血管生成机制。尽管已经建立了大量的血管生成试验, 但该领域缺乏一个 "金标准" 的体外检测方法, 可以可靠地评估潜在候选细胞类型的有效性11,12, 13. 大多数体外, 血管生成检测 (包括内皮细胞增殖、迁移和试管形成分析) 通常评估细胞或化合物对血管型变化或分化的影响管状和网络结构14,15。虽然这些功能对血管生成至关重要, 但 "可翻译" 的化验还应评估: 1) 增强或替换支持的细胞类型, 包括周或平滑肌细胞, 2) 处理 ECM 和/或基底膜, 和 3)促进功能性微血管形成的效率。在体内血管生成模型, 包括角膜检测和基质塞检测, 概括了独特的体内微环境, 但由于跟踪被管理的细胞观察物理相互作用的困难而面临挑战。此外, 在体内模型中, 异免疫排斥可能发生在测试潜在的异基因细胞治疗候选者16。前体血管生成模型, 特别是主动脉环试验可以提供: 1) 管状结构, 2) 辅助支持细胞, 3) 从主机和人工用品的 ECM, 4) 排斥炎症组件和 5) 快速且价格低廉的设置17,18。通常, 主动脉环试验可以检测小分泌蛋白、药理制剂和转基因啮齿动物模型的血管生成电位19,20,21。
骨髓间充质干细胞主要通过其分泌介导的效果22,23,24, 有望成为血管再生的候选对象。骨髓间充质干细胞已被证实能分泌血管内皮生长因子 (VEGF)、肝细胞生长因子 (HGF)、胰岛素样生长 Factor-1 (IGF-1)、碱性成纤维细胞生长因子 (bFGF) 和 angiopoeitin-1 (Ang-1) 的主要血管生成因子 (25 ,26。MSCs 也可以检测到缺血性或发炎的组织, 但确切的机制仍在调查中。越来越多的文献支持的假说, 大多数 MSCs 产生的血管细胞, co-express 细胞标记, 并可以表现出像周27。HUCPVCs 是人类脐带血管区的一个年轻的骨髓间充质干细胞来源。它们代表了具有细胞样性质的 MSCs 的种群, 并且从 FTM 和足月的脐带中都有特征。FTM HUCPVCs 展示了细胞标记的高表达, 包括 CD146 和 NG2, 高增殖和多向电位, 免疫特权属性, 并显示一个健壮的分泌配置文件28。FTM HUCPVCs 是一个理想的候选细胞类型, 通过促进新的血管, 通过其细胞的性质, 促进损伤组织再生。
为了测试人类骨髓间充质干细胞的血管生成潜能和细胞样的特性, 在积极的 angiotropic 迁移 (以下简称为 "归巢")、ECM 处理和发展物理在获得微血管发育的定量数据的同时, 可以研究细胞类型之间的相互作用。
在此, 我们提出了一个协议, 描述了一个新的应用的主动脉环试验。人骨髓间充质干细胞与开发大鼠主动脉内皮网络共, 以评估其对管的形成、成熟和稳态的贡献。这一版本的主动脉环试验评估细胞治疗候选者的能力和效力的地方, 血管生成, 执行和调解 ECM 处理, 并促进内皮管发展通过建立细胞样的物理相互.除了量化 mscs 对体外内皮网络形成和观察细胞间相互作用的净效应之外, 我们还优化了从 co-cultures 中分离 mscs 的协议。通过执行流式细胞仪和 qPCR, 可以表征 MSC 表型和基因表达在共培养后的变化。作为模型细胞类型, 我们比较了 ontogenetically 早期 (产前) 和晚期 (成人) 的人骨髓间充质干细胞的来源: FTM HUCPVCs 和人骨骨髓间充质干细胞 (骨髓), 分别在主动脉环试验。我们建议, 在研究血管生成再生应用时, 主动脉环法可用于分析任何物理支持细胞类型的血管生成潜能。

<table><tbody><tr><td>Alpha-MEM</td><td>Gibco</td><td>12571071</td><td>For FTM HUCPVC and BMSC culture media.</td></tr><tr><td>APC-conjugated anti human TRA-1-85</td><td>R&amp;amp;D Systems</td><td>FAB3195A</td><td>Human-specific cell marker for flow cytometry and cell sorting</td></tr><tr><td>Basal membrane extract (BME) (Matrigel)</td><td>Corning</td><td>354234</td><td>For aorta embedding</td></tr><tr><td>Bullet-kit</td><td>Lonza</td><td>CC-3162</td><td>Includes: Gentamicin/Amphotericin-B&lt;br/&gt; (GA)human Epidermal&lt;br/&gt; Growth Factor (hEGF); Vascular&lt;br/&gt; Endothelial Growth Factor (VEGF); R3-&lt;br/&gt; Insulin-like Growth Factor-1 (R3-IGF-1);&lt;br/&gt; Ascorbic Acid; Hydrocortisone;&lt;br/&gt; human Fibroblast Growth Factor-Beta (hFGF-&amp;beta;); Heparin; Fetal Bovine Serum&lt;br/&gt; (FBS). Required to prepare EGM</td></tr><tr><td>CellTracker Green CMFDA Dye</td><td>Thermo-fisher</td><td>C2925</td><td>For staining MSCs, green is picked up optimally by MSCs</td></tr><tr><td>CKX53 Culture Microscope</td><td>Olympus</td><td></td><td>For bright-field imaging of endothelial network development</td></tr><tr><td>Countess automated cell counter</td><td>Invitrogen</td><td>C10227</td><td>Cell counting for MSC culture, flow cytometry and qPCR</td></tr><tr><td>Dispase</td><td>StemCell technologies</td><td>7923</td><td>For dissociating aortic ring-MSC co-cultures (pre-warm at 37 &amp;deg;C)</td></tr><tr><td>Disposable sterile scalpels</td><td>VWR</td><td>21909-654</td><td>For sectioning aorta</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>PBS. 1X, Without calcium chloride and magnesium chloride</td></tr><tr><td>Endothelial basal media (EBM)</td><td>Lonza</td><td>CC-3156</td><td>Basal media required for culturing aortic ring assay-MSC co-cultures (warm at 37 &amp;deg;C before use). Required for EGM and EBM-FBS</td></tr><tr><td>Ethanol, 70%, Biotechnology Grade</td><td>VWR</td><td>97064-768</td><td>To sterilize surfaces</td></tr><tr><td>EVOS</td><td>Life Technologies</td><td></td><td>In-house fluorescent microscope to track MSC migration and integration</td></tr><tr><td>Fetal bovine serum (FBS) (Hyclone)</td><td>GE Healthcare</td><td>SH3039603</td><td>Serum component of cell culture medium</td></tr><tr><td>FITC-conjugated anti-CD31 antibody</td><td>BD</td><td>558068</td><td>Human endothelial marker for flow cytometry</td></tr><tr><td>FITC-conjugated anti-CD146antibody</td><td>BD</td><td>560846</td><td>Human pericyte marker for flow cytometry</td></tr><tr><td>Forceps</td><td>Almedic</td><td>7727-A10-704</td><td>For handing rat tissue. Can use any similar forceps</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14175-094</td><td>1X Without calcium chloride and magnesium chloride</td></tr><tr><td>HERAcell 150i CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Fisher Scientific</td><td>51026410</td><td>For incubating cells</td></tr><tr><td>Human Angiogenesis RT2 profiler PCR array</td><td>Qiagen</td><td>PAHS-024Z</td><td>Human specific and includes primers for 84 genes involved in angiogenesis. Each well is 1 primer reaction</td></tr><tr><td>ImageJ</td><td></td><td></td><td>Open source image processing software. Require Angiogenesis analyzer plugin</td></tr><tr><td>LSR II</td><td>BD</td><td></td><td>UHN SickKids FC Facility. For flow cytometry.</td></tr><tr><td>MoFlo Astrios</td><td>Beckman Coulter</td><td></td><td>UHN SickKids FC Facility. For cell sorting.</td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>15140122</td><td>Antibiotic component to buffers and cell culture medium</td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td>For RNA purification. Includes cell lysis buffer</td></tr><tr><td>RT&lt;sup&gt;2&lt;/sup&gt;Easy First Strand Kit</td><td>Qiagen</td><td>330421</td><td>For preparation of cDNA for qPCR</td></tr><tr><td>RT&lt;sup&gt;2&lt;/sup&gt;PreAMP cDNA Synthesis Kit</td><td>Qiagen</td><td>330451</td><td>Pre-amplification of cDNA if low-yield RNA</td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>14059-11</td><td>For cutting skin, muscle and aorta</td></tr><tr><td>Sterile gauze</td><td>VWR</td><td>3084</td><td>To dampen and sterilize chest fur</td></tr><tr><td>TrypleE</td><td>Thermo Fisher Scientific</td><td>12605036</td><td>MSC dissociation enzyme pre-warm at 37 &amp;deg;C</td></tr><tr><td>0.2 &amp;mu;m pore filtration unit</td><td>Thermo Fisher Scientific</td><td>566-0020</td><td>To sterilize tissue culture media</td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Gibco</td><td>25200056</td><td>For cell dissociation, pre-warm at 37 &amp;deg;C</td></tr><tr><td>10 cm tissue culture dishes</td><td>Corning</td><td>25382-428</td><td>For cleaning and sectioning aorta and MSC cell culture</td></tr><tr><td>12 well-cell culture plates</td><td>Corning-Sigma Aldrich</td><td>CLS3513</td><td>For setting up aortic ring assay-MSC co-cultures</td></tr><tr><td>15 mL tube</td><td>BD Falcon</td><td>352096</td><td>For general tissue culture procedures</td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>Fisherbrand</td><td>22363548</td><td>To ensure a single cell suspension before flow cytometry or sorting</td></tr></tbody></table>

the aortic ring co-culture assay: a convenient tool to assess the angiogenic potential of mesenchymal stromal cells in vitro

血管生成是一个复杂的, 高度规范的过程, 负责提供和维持足够的组织灌注。血管维持不足和病理性畸形可能导致严重的缺血性疾病, 而过度丰富的心血管发育与癌症和炎症失调有关。pro-angiogenic 治疗的一种很有希望的形式是使用血管生成细胞源, 它可以为新发展的血管提供调节因子和物理支持。
骨髓间质细胞 (msc) 是广泛研究的候选血管再生由于其分泌的影响和能力, 以检测和家庭缺血性或发炎组织。特别是, 第一个四个月的人脐带血管细胞 (FTM HUCPVCs) 是一个非常有希望的候选者, 由于其细胞性质, 高增殖和多向电位, 免疫特权的属性, 和强健的分泌配置.为了有效地评估潜在的血管生成再生细胞, 这是一个必要的测试他们在可靠和 "可翻译的" 前临床化验。主动脉环试验是一种体外血管生成模型, 可以方便地量化管状内皮结构, 提供宿主的辅助支持细胞和胞内基质 (ECM), 不包括炎症成分, 并且快速和低廉的设置。这是有利的, 当与在体内模型 (如: 如, 角膜检测, 基质插头检测);主动脉环试验可以跟踪所管理的细胞, 观察细胞间的相互作用, 同时避免异免疫排斥反应。
我们提出了一个新的应用的主动脉环试验的协议, 其中包括人骨髓间充质干细胞在 co-cultures 发育大鼠主动脉内皮网络。这种方法可以分析 MSC 对导管的形成和发展的贡献, 通过物理细胞样的相互作用及其效力, 积极迁移到血管生成的部位, 并评估其执行和调解的能力ECM 处理。该协议提供了进一步的信息, 在 MSC 表型的变化和基因表达后共培养。

在图 1中演示了建立主动脉环/MSC 共培养试验的示意图工作流程。主要步骤包括: 大鼠主动脉的分离、主动脉环的切片和嵌入、监测内皮的萌发和网络发育, 最后对 MSCs 进行标记和管理。内皮网分析的时间线概述了窗口为分析可行为每期间大块: 1 天, 5 和 #38; 7。附加注释由虚线框突出显示。
主动脉环内皮细胞培养物中结构上不同区域的鉴定是由光场显微镜, 3-5 d 在主动脉环嵌入 ECM (图 2) 后进行的。非结构化区域 (图 2A) 的特征是在主动脉组织的直接邻近区域中, 高细胞增殖, 但结构组织低。发达的内皮网络 (图 2B) 指的是具有高结构组织的区域, 在这里, 网络是完全建立的, 主要由闭环组成, 甚至在增加 MSCs 之前也是如此。发展中的网络 (图 2C) 位于内皮细胞培养的远端区域。这些都是内皮细胞迁移的部位, 延伸作为新的网络结构形式。上面提到的3区域在整个实验中都很容易识别, 并且在 co-cultures 中的 MSCs 家中引用时使用。当所有三网段都发育时, prelabelled 间充质干细胞应共主动脉环试验。在 MSC co-cultures 后的内皮网络的量化包括发达和发展网络的区域 (图 2, 白色框架)。
荧光显微镜允许定性测量 msc 迁移, 整合和形态学与发展内皮网络在主动脉环 msc 共培养试验 (图 3)。在管理后对 co-cultures 进行了24小时的细胞标记, 具有可行的细胞质荧光。FTM HUCPVCs 在发展的内皮网络的边缘被发现, 显示拉长的细胞形态, 并且促进了内皮网络的进一步发展 (图 3A, B)。相比之下, 骨髓基质干细胞则在发育的网络中, 与内皮细胞的相互作用较少 (图 3C, D)。高倍率图像在72小时显示, FTM HUCPVCs 保持高覆盖率和稳定的内皮网络, 而骨髓基质细胞在 co-cultures 呈现球形形态, 有限的整合到内皮网络 (图3E , F)。观察到的差异清楚地显示了人类 MSC 类型之间的定性、功能差异。具有显著的归巢和内皮细胞融合特性的治疗候选细胞类型 (图 3A、B 和 #38; E) 与具有有限内皮网络集成和增强的单元格类型相比突出功能 (图 3C, D 和 #38; F)。
为了进一步解剖管网中内皮细胞与 FTM HUCPVCs 的物理相互作用, 获得了高倍率荧光显微图像。pre-labelled FTM HUCPVCs (图 4A, 绿色) 显示为粘附不内皮细胞 (图 4A, 白色箭头). FTM HUCPVCs 被发现提供结构支持的内皮细胞, 作为轴和连接表面之间的节点。主动脉环内皮网络也可以 pre-labelled 使用一个可行的荧光类似 MSC 染色 (图 4B, 红色)。在双染色 co-cultures, 荧光显微镜揭示了两种细胞类型之间的拉长粘连, 加强观察合作和支持细胞的行为。
co-cultures (图 5) 的5天, 对内皮网络结构特性进行了量化。统一象限被定义为测量整个主动脉环内皮网络 (图 2, 白色框架)。平均网络增长计算为从第一个近端闭网环路的距离由主动脉环到最远的远端闭合环 (半径大小) (图 5A)。主动脉环-MSC co-cultures 的平均网络生长如下: FTM HUCPVCs (2.98 0.3 毫米) 和骨髓基质细胞 (1.5 0.15 毫米)。未经治疗的内皮细胞网络的平均半径为1.9 ±0.1mm。MSC 治疗组之间的统计学比较表明, 与骨髓基质干细胞和未经处理的网络 (p ≤0.001) 相比, FTM HUCPVCs 有助于显著提高网络的生长。未经治疗的内皮网络的发展明显超过骨髓含有 co-cultures (p ≤0.01) (图 5B)。
在每个象限中计算循环的总数, 并表示为平均总回路形成 (图 5C)。全闭环的平均数目如下: FTM HUCPVC 治疗 co-cultures (81 ±7), 骨髓基质细胞 (26 3) 和未处理的环 (55 ±7)。与骨髓基质细胞 (p ≤0.01) 和未经处理的网络 (p ≤0.01) 相比, FTM HUCPVCs 的内皮循环形成明显增加。与未经处理的网络 (p ≤0.01) 相比, 骨髓 co-cultures 导致了较少的内皮网循环。这一量化确定了一个细胞类型, 对内皮网络发育 (FTM HUCPVC) 和细胞类型有明显的积极影响, 甚至可能损害内皮网络。值得注意的是, 显著增加的网络发展被观察到与理学硕士覆盖的内皮网络有很好的关联。它表明, 直接相互作用对 MSCs 执行其支持功能至关重要。
主动脉环共培养机构的可能缺陷在图 6中表示。BME 聚合不足或不均匀是影响内皮细胞网络成功发展的一个问题。定时 BME 聚合整整30分钟, 并在不扰乱聚合物的情况下传输介质将确保一个完整的, 功能 BME 阶段 (图 6A)。对 mscs 进行长时间的潜伏期染色, 并允许 mscs 延长周期, 可能会影响 co-cultures 的细胞形态和表型。图 5B显示 FTM HUCPVCs 着色为1小时, 并允许在共培养前1小时的颗粒, 两者都是次优计时。FTM HUCPVCs 不显示首选的内皮网络的网站, 并分散在整个网络。将经过适当处理的单元 (图 3) 与不当的 FTM HUCPVCs 进行比较, 处理不当的单元格显示圆的细胞形态学和有限的细胞与内皮细胞的相互作用 (图 6B)。最后, 如果在解剖的当天不能建立主动脉环的检测, 脉可在-80 ° c 的华培养基中以10% 二甲基亚砜和 10% FBS 为补充。当应用解冻的主动脉环与新鲜组织相比, 血管内皮网络的发展可能需要 1-3 的时间, 但内皮网络对于 MSC 共培养的建立是足够的 (图 6C)。
作为主动脉环共培养评价的替代方法, 采用流式细胞术 (补充图 1) 提取和分析 BME 植入的主动脉环的细胞分数。人类特异性标记 TRA-1-85 (补充图 1A, B, y 轴) 阳性细胞数量从 FTM HUCPVC 含有 co-cultures 被确定为人骨髓基质。co-labelling 显示的内皮细胞标记 CD31 (补充图 1A) 的低表达, 细胞标记 CD146 (补充图 1B) 在人类特定标记阳性细胞群中的高表达。这种评价可以破译与内皮细胞相互作用诱导的骨髓间充质干细胞的最终型和谱系承诺的变化, 并提供有关被测细胞类型的进一步有价值的信息。为了获得足够数量的细胞进行 FC 分析, 同一实验组的平行井提取的细胞可以结合。重要的是要考虑使用无染色 MSC 包含的主动脉环的流式细胞仪分析, 使令人想起荧光的可行染料不干扰信号的抗体相关的荧光。反之, 负浇口应考虑 pre-stained 细胞荧光。
我们用人体细胞表面标记特异抗体 (TRA-1-85) 对人骨髓间充质干细胞提取形成主动脉环 co-cultures (共培养7天) 进行分类。从主动脉环试验中回收 FTM HUCPVCs 和骨髓基质细胞, 进行 qPCR 分析, 以检测血管生成基因的表达。使用商业上可用的 qPCR 阵列, 三主动脉环 co-cultures 提供了足够数量的遗传物质来量化84人生长因子基因的表达 (补充图 2A)。初步结果表明, FTM HUCPVCs 和骨髓基质细胞表达的关键分泌血管生成因子在可比水平 (补充图 2B)。在分析中, 除了比较不同细胞类型的表达水平外, 还应考虑将细胞转移到基于循证医学的培养基上。当使用更高的表型或遗传可塑性的细胞时, 在评价中应包括一个对照组的人骨髓间充质干细胞在无主动脉组织的循证医学媒介中。
骨髓间充质干细胞的血管生成因子表达的相似性表明, 其网络增强的显著差异不是不同分泌活动的结果。这与早先的观察结果是一致的, 即增加直接细胞间相互作用, 促进内皮细胞网络的发育。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56083/56083fig1.jpg"> 
图 1: 主动脉环试验装置的新应用的原理图。 主要步骤的设置和分析 MSC co-cultures 与主动脉环试验概述了固体盒和附加说明概述了虚线框。缩放栏 = 1000 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56083/56083fig1large.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/56083/56083fig2.jpg"> 
图 2: 主动脉环网络分析的代表性图像。 内皮细胞网络根据结构区别被划分成三同心区域: 非结构化的区域在接近与主动脉环组织 (A), 开发的或结构的内皮网络 (B), 和开发的网络位于前体内组织培养的外围 (C)。在发达的内皮网络 (B) 内定义了径向网络生长和循环计数的均匀象限。x: 闭合的内皮循环计数在一致的象限。缩放栏 = 250 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56083/56083fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56083/56083fig3.jpg"> 
图 3: 在24和 #38 后的主动脉环试验中, 网络区域依赖性的人骨髓基质的荧光成像; 72 小时 Prestained (绿色) FTM HUCPVCs 和骨髓基质干细胞被添加到发展主动脉环内皮管网络。荧光显微镜图像拍摄24小时后, 建立 MSC co-cultures 显示 FTM HUCPVCs, 迁移通过 ECM 和家庭的外围开发的内皮网络 (A)。高倍放大图像在与内皮网络 (B) 密切接触时显示 FTM HUCPVCs 的拉长形貌。更少的骨髓基质干细胞处理的 ECM 和家庭对内皮网络没有明显的偏好的外围开发网络 (C)。骨髓基质干细胞显示球形细胞形态 (D)。 大鼠主动脉环 prestained 间充质干细胞的高倍率荧光显微成像 (E、F)。FTM HUCPVCS 在网络节点和小管 (E) 中, 通过直接细胞与内皮细胞 (实心白色箭头) 的相互作用显示内皮覆盖, 从而呈现拉长的形态。骨髓基质干细胞保持球状细胞形态聚集在内皮网络节点 (F)。折断的箭头代表了主动脉环组织的内皮细胞网络的生长方向。A, C: 刻度栏 = 1000 µm B, D: 刻度酒吧 = 400 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56083/56083fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56083/56083fig4.jpg"> 
图 4: 高倍放大荧光显微镜图像的细胞-内皮细胞的物理相互作用。 不内皮细胞被发现相关的连续突起连接 pre-stained FTM HUCPVCs 在管网 (A)。荧光图像的 pre-stained 内皮网络 (EC, 红色) 与 pre-stained FTM HUCPVCs (FTM, 绿色) 演示相互作用综述内皮细胞, 细胞相互作用 (B)。A: 标尺 = 100 µm B: 刻度栏 = 200 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56083/56083fig4large.jpg" target="_blank">请点击这里查看这个数字的更大版本</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56083/56083fig5.jpg"> 
图 5: 在联合文化建立后, MSC 治疗组的网络增长的定量化 5 D。 低倍率相衬显微镜图像取自主动脉环治疗组, 以测量网络的生长和发育 (A)。散射箭头显示网络增长的方向。缩放条 = 250 µm. 显微镜图像被用来量化网络的性质, 包括平均网络的增长和平均网络环路形成在一个分析象限。与骨髓 co-cultures 和未经处理的网络 (p ≤0.001) (B) 相比, FTM HUCPVCs 有助于实现更大的网络增长。使用 one-way 方差分析将p值计算为p 和 #60;0.0001 使用 Tukey 的 Post 测试 (N = 3)。至少有四闭合边的网络环路是量化的 (C)。与骨髓 co-cultures (p ≤0.001) 和未经处理的网络 (p ≤0.01) 相比, FTM HUCPVC co-cultures 开发了更大的网络环路。使用 one-way 方差分析法计算p值为 p 和 #60; 0. 0001 使用 Tukey 的 Post 测试 (N = 3)。平均4领域的内皮网络的量化。缩放条 = 250 µm。对于两两比较, * = p ≤0.05, ** = p ≤0.01, *** = p ≤0.001。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56083/56083fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56083/56083fig6.jpg"> 
图 6: 主动脉环试验建立中可能出现的错误。 嵌在不完全 BME 聚合中的主动脉环可能导致不一致和破裂的内皮网络 (A)。FTM HUCPVCs 长期染色, 在染色和建立 co-cultures 之间有较大的时间间隔, 产生最小的归巢和内皮细胞相互作用 (B)。大鼠主动脉内皮网络在-80 ° c 下贮存, 并解冻用于主动脉环试验 (C)。A: 标尺 = 250 µm B, C: 标尺 = 400 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56083/56083fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图 1: 从主动脉环 Co 培养物中提取的人 FTM HUCPVCs 的流式细胞仪分析。 在流式细胞仪分析中分离和处理主动脉环培养的细胞组分。荧光共轭抗体抗人特异细胞表面标记 (TRA-1-85, APC) 的应用与内皮标记 (CD31, FITC, (A)) 或细胞标记 (CD146, FITC, (B)) 特异抗体。细胞数量阳性的人标记 (y 轴) 检测低阳性内皮标记 CD31 (a, Q2) 和高阳性细胞标记 CD146 (B, Q6)。这表明, FTM HUCPVCs 维持其血管细胞的性质在主动脉环 co-cultures, 并没有形成内皮表型。在地块上的象限被定义使用同种控制与应用的主要抗体相匹配。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56083/SuppFigures.pdf">请单击此处下载此文件.</a>
补充图 2: 关键血管生成基因的定量 PCR 分析。 FTM HUCPVCs 和骨髓基质干细胞在主动脉环试验中1周共培养后的基因表达有代表性的 CT 值显示在表 (B) 中。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56083/SuppFigures.pdf">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro at JoVE.com

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

在这里, 我们提出了一个新的应用的主动脉环法 prelabelled 间充质细胞共大鼠主动脉源性内皮网络。这种新的方法可以可视化间质基质细胞 (MSCs) 归巢和与内皮网络整合, 网络属性的量化, 并评价 MSC 免疫和基因表达。

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 ...

the-aortic-ring-co-culture-assay-convenient-tool-to-assess-angiogenic

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

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

10.9K 次观看.  Create Fertility Centre. 在这里, 我们提出了一个新的应用的主动脉环法 prelabelled 间充质细胞共大鼠主动脉源性内皮网络。这种新的方法可以可视化间质基质细胞 (MSCs) 归巢和与内皮网络整合, 网络属性的量化, 并评价 MSC 免疫和基因表达。

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

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

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

生物工程

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

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 ...

发育生物学

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

主动脉环含量

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

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 ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

工程血管移植缩放使用3D打印指南和环堆叠方法

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

医学

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

阻力动脉的细胞培养模型

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

3D 共培养检测:用于癌症药物筛选的血管化成骨生态位

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

作者聚焦: 在成像微孔板中使用预制聚乙二醇 （PEG） 水凝胶简单建立血管化成骨骨髓生态位  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

在成像微孔板中使用预制聚乙二醇（PEG）水凝胶简单建立血管化成骨骨髓壁龛

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...

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

摘要

探索更多视频

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

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

将毛细血管纳入内皮细胞珠发芽试验

主动脉环含量

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

工程血管移植缩放使用3D打印指南和环堆叠方法

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

阻力动脉的细胞培养模型

在成像微孔板中使用预制聚乙二醇（PEG）水凝胶简单建立血管化成骨骨髓壁龛

在成像微孔板中使用预制聚乙二醇（PEG）水凝胶简单建立血管化成骨骨髓壁龛