这项工作由意大利卫生部、Ricerca Corrente Cardiologico Monzino IRCCS 中心提供资金。

这一做法符合《赫尔辛基宣言》, 并通过 "Cardiologico Monzino IRCCS" 道德委员会批准了 (07/06/2012) 的右心室样本的收集。
1. 解决方案
<ol><li>用 Iscove 修饰 Dulbecco 培养基 (IMDM) 制作巴掌就培养基, 辅以20% 胎牛血清 (血清)、10 ng/毫升碱性成纤维细胞生长因子、1万毫升青霉素、1万µg/毫升链霉素、0.02 米谷氨酰胺。使用0.22 µm 无菌过滤装置过滤巴掌就介质, 并贮存在4摄氏度。</li>
	<li>为了制备胶原酶溶液, 并用重悬胶原酶 NB4 混合在 IMDM 培养基中, 以获得3毫克/毫升浓度的最终溶液。涡旋轻轻地溶解粉末, 并用0.2 µm 注射器过滤器过滤胶原酶溶液。整除1毫升体积成2毫升不育管和存储在-20 °c, 直到需要使用。</li>
	<li>制备 ADIPO 培养基, 使脂肪培养基与 IMDM 培养基辅以10% 血清, 0.5 毫米 3-异丁基1甲基黄嘌呤 (预稀释的二甲基亚砜 (亚砜) 在浓度0.5 米和保持在-20 °c, 直到使用), 1 µM 氢化 (预稀释在亚砜在100毫米和进一步稀释到10毫米在无菌蒸馏水和保持在-20 °c 直到用途) 和0.1 毫米消炎痛 (预先稀释在亚砜在浓度 0.5 M 并且保持在-20 °c 直到使用)。使用0.22 µm 无菌过滤装置过滤 ADIPO 培养基, 并贮存在4摄氏度。</li>
	<li>准备洗涤缓冲液, 由磷酸盐缓冲盐水0.0067 米 PO4 (PBS)、乙二胺四乙酸 (EDTA) 5 毫米和5% 牛血清白蛋白组成, 并贮存在4摄氏度。</li>
	<li>在60% 异丙醇中溶出1% 的未溶解粉末, 混合2小时, 用0.22 µm 过滤装置过滤, 以去除沉淀, 制备油红 O (破产) 工作液。在室温下 (RT) 存储工作溶液。</li>
</ol>2. 心脏间充质基质细胞的分离
<ol><li>
心脏活检的收集和处理 注:开始前, 请务必有胶原酶溶液和巴掌就培养基。<ol>
<li>将心内膜心肌活检 (通常约5毫克的组织) 在无菌管填充巴掌就和运输到实验室。 注意:根据以前的报告10, 在电生理学手术室采集了该活检。简要地, 电子解剖制图和心内超声心动图在右心室的整合 (请参见视频 1) 用于指导心内膜心肌活检 (参见视频2, 透视), 以对应的边界区域有病的心肌。健康控制 (HC) 样品由组织生物银行提供, 从尸体捐献者的右心室游离壁获得24小时死亡 (意外死亡, 健康的对象)。在运输和解剖之前, 将活检保存在巴掌就4摄氏度。限制活检收集和组织处理之间的时间 (最大24小时)。</li>
		<li>建立具有必要解决方案的生物安全柜, 完成酶消化过程。</li>
		<li>将灭菌器置于罩下, 在使用前打开仪器15分钟, 直到温度升高 200-250 摄氏度。</li>
		<li>十年代消毒剪刀和镊子. 在使用前要确保灭菌的仪器是冷的。准备一次性无菌手术刀。</li>
		<li>把试管和活检放在消毒罩里。打开管子, 将活检转移到不育的盘子里。</li>
		<li>用3毫升的无菌 PBS 冲洗两次右心室活检。如果你想在显微镜下拍一张活检的照片。</li>
		<li>用无菌镊子将右心室活检转移到含有胶原酶溶液1毫升的无菌2毫升管中。</li>
		<li>用无菌剪刀将样品切成 0.5-1 毫米3片。</li>
		<li>在连续旋转搅拌下, 在37摄氏度孵育1.5 小时。</li>
		<li>将消化液在 400 x g 上离心, 在 RT 上10分钟。</li>
		<li>取出上清液, 加入1毫升的无菌 PBS, 清洗颗粒。</li>
		<li>离心机在 400 x g 10 分钟的 RT。</li>
		<li>取出上清液, 并用重悬1毫升巴掌就培养基中的颗粒。</li>
		<li>将所获得的悬浮液在60毫米无菌的组织培养板上, 加入巴掌就培养基到最终体积3毫升。在37°c 的细胞培养孵化器中孵化出 5% CO2的板块。 注意:较高的培养基量可能会阻止消化活检片段的正确黏附。</li>
		<li>24小时后, 丢弃介质, 除去不附着的细胞和碎片。 注意:单个分离的间充质细胞可附着于塑料表面, 产生无性系;此外, 未消化的小活检碎片可以附着在塑料盘子上, 并允许细胞发芽。</li>
		<li>用5毫升的无菌 PBS 洗两次盘子, 加入3毫升新鲜的巴掌就培养基。</li>
		<li>每周更换巴掌就培养基三次, 直到细胞80% 汇合。 注意:附着细胞的数量取决于质量、组织数量和消化效率。</li>
	</ol>
</li>
</ol>3. 细胞扩张
注意:在开始之前, 确保有巴掌就介质准备就绪。
<ol><li>当细胞80% 汇合, 将被消化的小活检样品转移到一个新的不育的60毫米组织培养处理的板材, 并且增加3毫升新鲜巴掌就培养基。 注意:活检样品可以重新用于进一步的 C MSC 隔离。可以重复此过程, 直到有大量的孤立单元格。</li>
	<li>用3毫升的无菌 PBS 冲洗所附细胞。加入胰蛋白酶-EDTA 溶液 (0.5 毫升的60毫米菜), 并孵化37°c 3-5 分钟, 以允许细胞脱离。</li>
	<li>当细胞从板块表面分离出来时, 加入0.5 毫升不育的血清, 使胰蛋白酶失效, 并将细胞收集到新的50毫升无菌管中。</li>
	<li>用5毫升的无菌 PBS 洗两次盘子, 将剩余的细胞添加到同一管中。</li>
	<li>离心细胞在 400 x g 10 分钟的 RT。</li>
	<li>取出上清液, 并用重悬1毫升巴掌就培养基中的颗粒。</li>
	<li>在细胞计数室中加载10µL 细胞悬浮液。 注意:如果细胞过于集中, 稀释初始悬浮1:10 在 PBS 和负载10µL 稀释细胞在细胞计数室。</li>
	<li>显微镜下, 在3个大正方形和两个两侧的线上计数细胞, 并计算平均细胞数。 注意:为了获得细胞数/mL, 计数细胞的平均值乘以 104, 即细胞计数室的稀释因子。如果细胞被进一步稀释, 乘以稀释因子。细胞数与种子之间的比值和细胞浓度 (细胞数/ml) 对应于为下一步 (点 3.9) 电镀的初始悬浮量 (毫升)。</li>
	<li>将泥沙板上的无菌100毫米组织培养处理后的板材最终浓度为 5000-1万细胞/厘米2 , 并将巴掌就培养基添加到最终体积8毫升。在细胞培养孵化器中孵化板块。 注意:重复细胞扩张过程时, 细胞是 70-80% 汇合。在每一通道 (P), 建议 cryopreserve 部分细胞和板块的剩余量。展开细胞直到 P3 或 P4, 并用于荧光活化细胞分选器 () 分析 (4 节) 和脂肪分化 (5 节)。</li>
	<li>对于分离后的超低温保存, 将细胞在 400 x g 的室温下以10分钟的 RT 进行离心。</li>
	<li>按描述的单元格计数 (步骤 3.7-3.8) 和并用重悬 1 x10 6 单元格 (900 µL 无菌血清)。转移到一个无菌的冷冻瓶和添加100µL 不育亚砜和混合。</li>
	<li>将冷冻瓶快速贮存在-80 摄氏度的冷冻容器中, 至少2天, 然后将瓶子转移到液氮中。</li>
</ol>4. 流式细胞术对心间质基质细胞的表征
注意:在开始之前, 一定要有细胞离解试剂, 洗涤缓冲液和特定的抗体制备。
<ol><li>当单元格数达到至少 3 x 106 (按 3.7-3.8 中所述的单元格计数) 时, 用100毫升的无菌 PBS 洗涤两次10毫米的盘子。</li>
	<li>添加5毫升的细胞离解试剂和等待 7-10 分钟在 RT, 以允许细胞脱离。</li>
	<li>当细胞从板块表面分离出来时, 加入15毫升的无菌洗涤缓冲液, 并将悬浮液收集到新的50毫升无菌管中。</li>
	<li>用10毫升的洗涤缓冲器将盘子洗两次, 并将剩余的细胞添加到同一管中。</li>
	<li>离心细胞在 400 x g 10 分钟在 RT 和并用重悬颗粒在1毫升的洗涤缓冲。</li>
	<li>按所述计算单元数 (步骤 3.7-3.8), 并将 3 x 106单元格转换为新的无菌管, 并将洗涤缓冲器添加到最终体积1.5 毫升。 注意:总的单元格数和洗涤缓冲器的总容量取决于用于分析的选定标记的数量 (每个µL 的聚苯乙烯管的100个 3 x 105细胞)。</li>
	<li>在12种不同的分散剂聚苯乙烯管中分布100µL 细胞悬浮液, 并在产品数据表中标明的浓度添加特定抗体。 注意:用于 C MSC 表征的抗体有 CD34、CD105、CD45、CD29、CD90、CD44、CD31、CD14 和 HLA-DR (表 1)。重要的是要准备一个控制样本组成的100µL 悬浮细胞染色的同种控制, 以检查信号的特异性。</li>
	<li>在黑暗中孵化样品15分钟。</li>
	<li>将1毫升无菌洗涤缓冲液添加到每管, 以停止反应。</li>
	<li>离心细胞在 400 x g 10 分钟的 RT。</li>
	<li>取出上清液, 并用重悬250µL 洗涤缓冲器中的颗粒。</li>
	<li>用流式细胞仪进行 C MSC 的表征。</li>
</ol>5. 心间质基质细胞的脂肪分化
注意:在开始之前, 一定要准备好 ADIPO 介质和破产处理解决方案。
<ol><li>
ADIPO 培养基中的文化
	<ol>
<li>按照第3节中的说明分离和计数单元格。</li>
		<li>板 3 x 105细胞到一个无菌的6井组织培养的每个井在2毫升 ADIPO 培养基治疗板材。</li>
		<li>让 C-MSC 区分在 ADIPO 培养基72小时或1周, 改变培养基每 2-3 天。</li>
	</ol>
</li>
	<li>
油红色 O 染色 注:使用油红 O (破产) 染色检测 C MSC 脂质积累。<ol>
<li>将6井组织培养 plateunder 油烟机。</li>
		<li>取出脂肪培养基, 用2毫升 PBS 冲洗两次细胞。</li>
		<li>添加足够数量的4% 多聚甲醛 (粉煤灰), 以覆盖每一个井。将细胞固定在 RT 上5分钟。</li>
		<li>用2毫升的 PBS 丢弃粉煤灰并清洗两次细胞。</li>
		<li>用1小时的 RT 工作溶液孵化固定细胞, 用足够的容积覆盖每个井。 注意:在破产孵化期间, 请勿将6井组织培养板放在通风罩下, 以避免破产的工作液蒸发。</li>
		<li>拆卸所有的工作解决方案, 并清洗2毫升 PBS 3-5 次, 直到板完全清洗;放弃洗涤。当 PBS 使用时, 停止洗涤, 当你看不到的时候, 显微镜下, 不特异的染色细胞。</li>
		<li>使用倒置组织培养相衬显微镜, 以20X 倍的比例捕捉20张图像。</li>
		<li>用图像处理程序打开图片, 量化细胞破产的累积。</li>
		<li>通过 "分割通道" 功能分隔不同的颜色通道, 以便仅量化255红色通道中的亮度。</li>
		<li>通过计数原子核来计算每张图片的单元格数。使破产的信号强度正常化的细胞数。</li>
		<li>计算为同一样本的每张图片获得的结果的平均值。</li>
	</ol>
</li>
</ol>

<ol><li>Brooke, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+applications+of+mesenchymal+stromal+cells%5BTitle%5D)+AND+%22Semin+Cell+Dev+Biol%22%5BJournal%5D)">Therapeutic applications of mesenchymal stromal cells.</a> Semin Cell Dev Biol. 18 (6), 846-858 (2007).</li>
<li>Rossini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+cardiac+and+bone+marrow+stromal+cells+exhibit+distinctive+properties+related+to+their+origin%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">Human cardiac and bone marrow stromal cells exhibit distinctive properties related to their origin.</a> Cardiovasc Res. 89 (3), 650-660 (2010).</li>
<li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li>
<li>Doppler, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+fibroblasts%3A+more+than+mechanical+support%5BTitle%5D)+AND+%22J+Thorac+Dis%22%5BJournal%5D)">Cardiac fibroblasts: more than mechanical support.</a> J Thorac Dis. 9, Suppl 1 36-51 (2017).</li>
<li>Moore-Morris, T., Guimaraes-Camboa, N., Yutzey, K. E., Puceat, M., Evans, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+fibroblasts%3A+from+development+to+heart+failure%5BTitle%5D)+AND+%22J+Mol+Med+%28Berl%29%22%5BJournal%5D)">Cardiac fibroblasts: from development to heart failure.</a> J Mol Med (Berl). 93 (8), 823-830 (2015).</li>
<li>Jugdutt, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ventricular+remodeling+after+infarction+and+the+extracellular+collagen+matrix%3A+when+is+enough+enough%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough.</a> Circulation. 108 (11), 1395-1403 (2003).</li>
<li>Brown, R. D., Ambler, S. K., Mitchell, M. D., Long, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cardiac+fibroblast%3A+therapeutic+target+in+myocardial+remodeling+and+failure%5BTitle%5D)+AND+%22Annu+Rev+Pharmacol+Toxicol%22%5BJournal%5D)">The cardiac fibroblast: therapeutic target in myocardial remodeling and failure.</a> Annu Rev Pharmacol Toxicol. 45, 657-687 (2005).</li>
<li>Sommariva, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+mesenchymal+stromal+cells+are+a+source+of+adipocytes+in+arrhythmogenic+cardiomyopathy%5BTitle%5D)+AND+%22Eur+Heart+J%22%5BJournal%5D)">Cardiac mesenchymal stromal cells are a source of adipocytes in arrhythmogenic cardiomyopathy.</a> Eur Heart J. 37 (23), 1835-1846 (2016).</li>
<li>Sommariva, E., Stadiotti, I., Perrucci, G. L., Tondo, C., Pompilio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+models+of+arrhythmogenic+cardiomyopathy%3A+advances+and+opportunities%5BTitle%5D)+AND+%22Dis+Model+Mech%22%5BJournal%5D)">Cell models of arrhythmogenic cardiomyopathy: advances and opportunities.</a> Dis Model Mech. 10 (7), 823-835 (2017).</li>
<li>Casella, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroanatomical+mapping+systems+and+intracardiac+echo+integration+for+guided+endomyocardial+biopsy%5BTitle%5D)+AND+%22Expert+Rev+Med+Devices%22%5BJournal%5D)">Electroanatomical mapping systems and intracardiac echo integration for guided endomyocardial biopsy.</a> Expert Rev Med Devices. 14 (8), 609-619 (2017).</li>
<li>Beltrami, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multipotent+cells+can+be+generated+in+vitro+from+several+adult+human+organs+%28heart%2C+liver%2C+and+bone+marrow%29%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow).</a> Blood. 110 (9), 3438-3446 (2007).</li>
<li>da Silva Meirelles, L., Chagastelles, P. C., Nardi, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+reside+in+virtually+all+post-natal+organs+and+tissues%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Mesenchymal stem cells reside in virtually all post-natal organs and tissues.</a> J Cell Sci. 119, Pt 11 2204-2213 (2006).</li>
<li>Cencioni, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+double+life+of+cardiac+mesenchymal+cells%3A+Epimetabolic+sensors+and+therapeutic+assets+for+heart+regeneration%5BTitle%5D)+AND+%22Pharmacol+Ther%22%5BJournal%5D)">The double life of cardiac mesenchymal cells: Epimetabolic sensors and therapeutic assets for heart regeneration.</a> Pharmacol Ther. 171, 43-55 (2017).</li>
<li>Caplan, A. I. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/1870029">Mesenchymal stem cells.</a> J Orthop Res. 9 (5), 641-650 (1991).</li>
<li>Germani, A., Foglio, E., Capogrossi, M. C., Russo, M. A., Limana, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+cardiac+progenitor+cells+through+epicardial+to+mesenchymal+transition%5BTitle%5D)+AND+%22J+Mol+Med+%28Berl%29%22%5BJournal%5D)">Generation of cardiac progenitor cells through epicardial to mesenchymal transition.</a> J Mol Med (Berl). 93 (7), 735-748 (2015).</li>
<li>Souders, C. A., Bowers, S. L., Baudino, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+fibroblast%3A+the+renaissance+cell%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Cardiac fibroblast: the renaissance cell.</a> Circ Res. 105 (12), 1164-1176 (2009).</li>
<li>Di Maggio, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-oxidizable+HMGB1+induces+cardiac+fibroblasts+migration+via+CXCR4+in+a+CXCL12-independent+manner+and+worsens+tissue+remodeling+after+myocardial+infarction%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Non-oxidizable HMGB1 induces cardiac fibroblasts migration via CXCR4 in a CXCL12-independent manner and worsens tissue remodeling after myocardial infarction.</a> Biochim Biophys Acta. , (2017).</li>
<li>Czapla, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Cardiac+Mesenchymal+Stromal+Cells+with+CD105%2BCD34-+Phenotype+Enhance+the+Function+of+Post-Infarction+Heart+in+Mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.</a> PLoS One. 11 (7), 0158745(2016).</li>
<li>Gambini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C-kit%2B+cardiac+progenitors+exhibit+mesenchymal+markers+and+preferential+cardiovascular+commitment%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">C-kit+ cardiac progenitors exhibit mesenchymal markers and preferential cardiovascular commitment.</a> Cardiovasc Res. 89 (2), 362-373 (2010).</li>
<li>Gourdie, R. G., Dimmeler, S., Kohl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+therapeutic+strategies+targeting+fibroblasts+and+fibrosis+in+heart+disease%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease.</a> Nat Rev Drug Discov. 15 (9), 620-638 (2016).</li>
<li>Le Blanc, K., Tammik, C., Rosendahl, K., Zetterberg, E., Ringden, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HLA+expression+and+immunologic+properties+of+differentiated+and+undifferentiated+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Exp+Hematol%22%5BJournal%5D)">HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells.</a> Exp Hematol. 31 (10), 890-896 (2003).</li>
<li>Detert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Atrial+Appendage+as+a+Suitable+Source+to+Generate+Cardiac-derived+Adherent+Proliferating+Cells+for+Regenerative+Cell-based+Therapies%5BTitle%5D)+AND+%22J+Tissue+Eng+Regen+Med%22%5BJournal%5D)">The Atrial Appendage as a Suitable Source to Generate Cardiac-derived Adherent Proliferating Cells for Regenerative Cell-based Therapies.</a> J Tissue Eng Regen Med. , (2017).</li>
<li>Miteva, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+cardiac-derived+adherent+proliferating+cells+reduce+murine+acute+Coxsackievirus+B3-induced+myocarditis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Human cardiac-derived adherent proliferating cells reduce murine acute Coxsackievirus B3-induced myocarditis.</a> PLoS One. 6 (12), 28513(2011).</li>
<li>Bassetti, B., Capogrossi, M. C., Pompilio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Power+Is+Nothing+Without+Control%3A+The+Enduring+Search+for+the+Best+Cell+in+Cardiac+Cell+Therapy+at+a+Crossroads%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Power Is Nothing Without Control: The Enduring Search for the Best Cell in Cardiac Cell Therapy at a Crossroads.</a> Circ Res. 119 (9), 988-991 (2016).</li>
<li>Nigro, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+therapy+for+heart+disease+after+15+years%3A+Unmet+expectations%5BTitle%5D)+AND+%22Pharmacol+Res%22%5BJournal%5D)">Cell therapy for heart disease after 15 years: Unmet expectations.</a> Pharmacol Res. , (2017).</li>
<li>Ikebe, C., Suzuki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+for+regenerative+therapy%3A+optimization+of+cell+preparation+protocols%5BTitle%5D)+AND+%22Biomed+Res+Int%22%5BJournal%5D)">Mesenchymal stem cells for regenerative therapy: optimization of cell preparation protocols.</a> Biomed Res Int. 2014, 951512(2014).</li>
<li>Marcus, F. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+of+arrhythmogenic+right+ventricular+cardiomyopathy%2Fdysplasia%3A+proposed+modification+of+the+task+force+criteria%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the task force criteria.</a> Circulation. 121 (13), 1533-1541 (2010).</li>
<li>Pieroni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+prevalence+of+myocarditis+mimicking+arrhythmogenic+right+ventricular+cardiomyopathy+differential+diagnosis+by+electroanatomic+mapping-guided+endomyocardial+biopsy%5BTitle%5D)+AND+%22J+Am+Coll+Cardiol%22%5BJournal%5D)">High prevalence of myocarditis mimicking arrhythmogenic right ventricular cardiomyopathy differential diagnosis by electroanatomic mapping-guided endomyocardial biopsy.</a> J Am Coll Cardiol. 53 (8), 681-689 (2009).</li>
<li>Casella, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Feasibility+of+combined+unipolar+and+bipolar+voltage+maps+to+improve+sensitivity+of+endomyocardial+biopsy%5BTitle%5D)+AND+%22Circ+Arrhythm+Electrophysiol%22%5BJournal%5D)">Feasibility of combined unipolar and bipolar voltage maps to improve sensitivity of endomyocardial biopsy.</a> Circ Arrhythm Electrophysiol. 8 (3), 625-632 (2015).</li>
<li>Cooper, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+endomyocardial+biopsy+in+the+management+of+cardiovascular+disease%3A+a+scientific+statement+from+the+American+Heart+Association%2C+the+American+College+of+Cardiology%2C+and+the+European+Society+of+Cardiology+Endorsed+by+the+Heart+Failure+Society+of+America+and+the+Heart+Failure+Association+of+the+European+Society+of+Cardiology%5BTitle%5D)+AND+%22Eur+Heart+J%22%5BJournal%5D)">The role of endomyocardial biopsy in the management of cardiovascular disease: a scientific statement from the American Heart Association, the American College of Cardiology, and the European Society of Cardiology Endorsed by the Heart Failure Society of America and the Heart Failure Association of the European Society of Cardiology.</a> Eur Heart J. 28 (24), 3076-3093 (2007).</li>
<li>Leone, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((2011+consensus+statement+on+endomyocardial+biopsy+from+the+Association+for+European+Cardiovascular+Pathology+and+the+Society+for+Cardiovascular+Pathology%5BTitle%5D)+AND+%22Cardiovasc+Pathol%22%5BJournal%5D)">2011 consensus statement on endomyocardial biopsy from the Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology.</a> Cardiovasc Pathol. 21 (4), 245-274 (2011).</li>
<li>Monsanto, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concurrent+Isolation+of+3+Distinct+Cardiac+Stem+Cell+Populations+From+a+Single+Human+Heart+Biopsy%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.</a> Circ Res. 121 (2), 113-124 (2017).</li>
<li>From, A. M., Maleszewski, J. J., Rihal, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+status+of+endomyocardial+biopsy%5BTitle%5D)+AND+%22Mayo+Clin+Proc%22%5BJournal%5D)">Current status of endomyocardial biopsy.</a> Mayo Clin Proc. 86 (11), 1095-1102 (2011).</li>
</ol>

作者没有什么可透露的。

msc 和 C-msc:MSC 是居住在不同成体组织的基质含量的能细胞, 如骨髓, 脂肪组织, 软骨, 脑, 皮肤, 胎儿附件和心脏12。在基本和平移研究12、13中, 已经进行了不同的研究来分离和描述它们的潜在应用。
在健康的情况下, MSC 是静止的, 自我更新的低速率14。由于它们受到环境病理变化的影响, 它们通过直接转分化、基质沉积或分泌效应14来反应促进组织重塑。
心脏理学硕士 (C msc) 代表心脏4的一个大的非心肌细胞的人口。它们起源于心外膜, 并迁移到接受上皮间充质过渡过程的心肌中15。它们通过与心肌细胞和细胞外基质稳态的相互作用7,16, 在生理和病理状态下促进心脏结构的机械和电学完整性。然而, 广泛的 C MSC 函数仍未完全理解。在其分离后进行的体外研究可以促进对它们在生理和病理条件下的作用的更深认识。
从人类心脏的不同区域获得了 C MSC, 如心房附属物2,17和右心室18。
最近, 从人体右心室心内膜心肌活检样本中提取的 C-MSC 已经获得了 8, 表明源组织可能只有3-5 毫克.
可能的应用程序:本手稿中概述的方法允许获得少量简单通道的细胞, 如从非常小的心脏标本中消化和选择塑料附着。
C MSC 可以被认为是细胞模型, 因为它们易于放大和维持体外, 并且能够分化成间充质谱系 (内皮、骨和脂肪细胞)。此外, 从患者直接获得细胞的可能性构成了一个伟大的体外工具, 用于机械研究的背景下的个性化/精确医学。事实上, 这些细胞携带的基因背景和最终的特定突变的捐赠者, 并受特定的病人的特点, 如临床条件, 年龄, 性别, 生活方式和药物。此外, 对不同标记进行排序的可能性可能允许对特定的 C MSC 子集19进行研究。
众所周知, 理学硕士是不同心血管疾病的活跃参与者, 主要表现为心脏的不良重塑。因此, 它们代表了用于对抗心脏病的新治疗策略的候选目标8,20。
茎样性质及其缺乏显著的免疫原性表明了它们在心脏再生医学细胞治疗中的潜在应用。的确, 像骨髓或其他来源的 msc 一样, C-msc 可能在自体和同种异体环境中使用, 而不需要在捐助者和收件人21之间进行匹配。
此外, 从心脏组织直接分离的 C-MSC 具有由心脏微环境和表观遗传特征预处理的优点。在心脏再生医学方面, 这可能是特别重要的获得成功的结果。
到目前为止, 再生医学的前期研究发现了有用的治疗潜力在 C MSC 和他们的分泌活动18,22,23。重要的是, 临床试验中的细胞源是心脏正在进行的 cardiosfere 衍生细胞或与亚群的 C MSC13,24,25。然而, 对于骨髓衍生的 MSC, 可能需要不同的协议, 以获得临床等级 C msc26。
在 ACM 中的 C MSC:本协议主要适用于心内膜活检的病理学研究。为诊断目的, 活检的病人接受了27的治疗程序。他们的心肌逐渐被疤痕组织取代, 这是由脂肪细胞和纤维化组成的电子惰性组织。为了指导活检取样到疤痕区, 其中诊断产量最大, 心内膜心肌映射使用 10, 28, 29.本协议中使用的样本是在患病心肌的边界区。
Sommariva et . 最近定义了 c msc 在 acm 的发病机制8中的关键作用, 表明 c-msc 是在成脂心脏的活跃参与者, 因为前脂肪细胞在那些心脏是间充质来源。此外, 与本协议隔离的 C MSC 患者的活检显示, 脂肪积累和成脂的倾向比对照组多。因此, 这些细胞可以被用来确认一些分子机制的 ACM, 证明它们适合作为细胞模型的机械研究9。
限制和关键步骤:尽管直接从患者那里获得 C MSC 的好处 (见 "可能的应用" 一节), 本议定书受到不同的限制。
首先, 心脏活检程序是侵入性的, 并且经常避免, 如果不是严密地必要的。事实上, 取样心脏组织在伦理上和技术上都是有问题的。进行心脏活检的原因可能是在心肌的鉴别诊断, 监测心脏移植的状态, 或确定心脏肿瘤的存在的情况下明确诊断30。因此, 只有以协商一致声明表示为心内膜心肌活检的患者才可以注册为 C MSC 的研究.( 31)。此外, 心脏活检程序可能有临床并发症, 尤其是在型心肌病心脏。因此, electrophysiologist 的抽样总是谨慎的, 活检样本可能非常小, 危及细胞的隔离。未来的实验可以通过调整胶原酶浓度或消化时间来克服这个问题。
作为所有主要的人类细胞, C MSC 在所有表型的不同学科中表现出很高的变异性。事实上, 来自不同学科的细胞不仅在基因上是不同的, 而且还受到环境变化的制约。具体地说, 在这个实验中, 观察到细胞分离、生长和脂肪分化的高变异性。
必须承认本议定书的关键步骤。如果活检样品包括毛细血管, 它们必须被移除, 以避免内皮细胞的平行隔离, 这可能会污染 C MSC 的文化, 并且可以通过对血管的分析 (阳性 CD31) 来证明。为了获得有效的脂肪分化, 细胞必须处于活跃的生长阶段。汇流的程度也会影响脂质的积累。
方法的意义:对于以往的间充质基质细胞的分离方法, 本文首次提出了直接从人心室活检样品中提取 C MSC 的描述。尽管这一方法被建议用于治疗医用的样本, 它可能适用于所有的病人, 其中心脏活检表明。
该协议代表了以前的方法的有用的实现, 这些细胞需要更大的心脏样本32, 这往往很难收集。
此外, 样本来源也构成了一个有趣的创新。虽然心室活检通常在隔膜33上进行, 但本协议考虑了从右心室游离壁获得的样本。来自病右心室区的细胞可能更能代表 RV 疾病的病理状态。
此外, 本协议中使用的某些试剂与其他 C MSC 隔离和分化方法32不同。例如, 在本手稿中提出的胶原酶的类型是一个混合的 I 类和 II 类胶原酶与蛋白水解活动的平衡比例。此外, 消化液由溶于同一基底培养基 (IMDM) 的胶原酶混合物组成, 用于制备 C-msc 培养基, 使分离的 c msc 适应未来的生长条件。
此外, 虽然排序程序可以标准化的细胞批次, 使用全 C MSC 人口, 仅通过这些细胞的塑性黏附性质隔离, 构成简化, 而不改变免疫表型C-MSC 的特点。本文提出的 ADIPO 的组成能够导致脂肪分化, 避免其他成分如胰岛素引起的代谢失调。
此外, 该方法的脂积累量化, 这是基于对破产评估的比色强度, 提供了更多的信息关于累积脂质的数量, 如果比较的方法仅基于百分比对破产的染色阳性的细胞。通常, 通过提取与异丙醇细胞结合的破产组织, 测量其吸光度, 就能量化脂质积累。然而, 这种方法需要更多的通道, 并因异丙醇蒸发而受到变异。

间充质基质细胞 (MSC) 是成人细胞, 在许多组织中具有重要的支持作用1。骨髓代表了 MSC 的历史来源, 但它们可以从不同的组织, 包括胎盘, 脂肪组织, 脐血, 肝脏和心脏1,2分离。
在 2006年, 国际细胞治疗学会 (ISCT) 第一次指定了定义人类 MSC3的最低标准。特别是, MSC 必须有能力坚持塑料。它们表达特定的表面抗原: CD44、CD105、CD29 和 CD90 间充质标志物的阳性, 以及 CD14、CD45、CD34、CD31 造血和内皮标记的阴性。由于 HLA-DR 缺乏表达, MSC 无法触发 alloreactivity。此外, 它们是有潜力分化为脂肪、软骨细胞和成骨细胞血统的有能力的干细胞1,3。
心脏间充质基质细胞 (C MSC) 集中于心脏细胞组成, 在正常的成人心脏4,5中是丰富的。它们在正常的心脏功能和病理状况中起着关键作用。生理学上, C-MSC 提供了一种微环境, 支持心肌的结构和功能完整性, 在心脏疾病中, 它们被激活, 以响应心脏损伤参与伤口愈合和纤维化重塑6 ,7。
最近, 致心律失常心肌病 (ACM) 脂肪替代的 C MSC 的参与已被证明为8。特别是, ACM 是一种遗传紊乱, 主要是由于 desmosomal 基因突变导致心肌纤维-脂肪替代, 主要在右心室9。这种替代, 从心外膜延伸到心内膜, 创造了一个非传导性基质, 引起渐进性心力衰竭和恶化室性心律失常, 这可能导致, 在严重的情况下, 突然死亡。Sommariva et 。证实了说明心脏切片前脂肪细胞存在的间充质来源。此外, 从心内膜心肌活检中分离出的 C-MSC 表达 desmosomal 基因, 因此可能受其突变的影响。特别是, 在脂肪条件下, 其比控制心脏的脂肪积累更多的脂质。这一证据得出结论, C-MSC 在疾病发病机制中都有积极作用, 并代表了一个有效的细胞模型来研究。
为便于今后在这方面的研究或其他心脏活检的心脏病, 提出了一个详细的协议, 以隔离心内膜心肌组织的小片段, 他们的扩张, 他们的免疫表型鉴定表征和脂肪分化。

<table><tbody><tr><td>IMDM</td><td>Gibco by Life Technologies&amp;nbsp;</td><td>12440053</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>PENICILLIN STREPTOMYCIN</td><td>Life Technologies Italia</td><td>15140122</td><td></td></tr><tr><td>Collagenase NB4</td><td>Serva</td><td>17454.02</td><td></td></tr><tr><td>Basic fibroblast growth factor</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>L-glutammine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>17-516F</td><td></td></tr><tr><td>Tryple select 1X (cell dissociation reagent)</td><td>Gibco</td><td>12563029</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>Trypsin-EDTA solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6689</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A2058</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I5879</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H4001</td><td></td></tr><tr><td>Indomethacin</td><td>Sigma-Aldrich</td><td>I7378</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma-Aldrich</td><td>O0625</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>I9516</td><td></td></tr><tr><td>Paraformaldehyde (PFA) Solution 4% in PBS</td><td>D.b.a. Italia S.r.l.</td><td>sc-281692</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>276855</td><td></td></tr><tr><td>Antibody CD14-FITC (M&amp;phi;P9)</td><td>Becton Dickinson</td><td>345784</td><td></td></tr><tr><td>Antibody Integrin &amp;beta;1 (CD29)-PE (clone MAR4)</td><td>Becton Dickinson</td><td>561795</td><td></td></tr><tr><td>Antibody PECAM-1 (CD31)- APC&amp;nbsp; (clone 9G11)</td><td>R&amp;amp;D Systems</td><td>FAB3567A</td><td></td></tr><tr><td>Antibody Hematopoietic progenitor cell antigen (CD34)-APC (clone 581)</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>CD34-581-05</td><td></td></tr><tr><td>Antibody H-CAM (CD44)-PE&amp;nbsp; (clone G44-26)</td><td>Becton Dickinson</td><td>561858</td><td></td></tr><tr><td>Antibody L-CA (CD45)-APC (clone HI30)</td><td>Thermo Fisher Scientific</td><td>MHCD4505-4</td><td></td></tr><tr><td>Antibody THY1 (CD90)-FITC (clone 5E10)</td><td>Becton Dickinson</td><td>561969</td><td></td></tr><tr><td>Antibody Endoglin (CD105)-APC (clone 266)</td><td>Becton Dickinson</td><td>562408</td><td></td></tr><tr><td>Antibody HLA-DR-FITC (clone L243)</td><td>Becton Dickinson</td><td>&lt;br/&gt; 347400</td><td></td></tr><tr><td>Gima Quick Plus sterilizer</td><td>Gima&amp;nbsp;</td><td>35642</td><td></td></tr><tr><td>Bench centrifuge Sigma 3-16K</td><td>Sigma Centrifuges</td><td>10330</td><td></td></tr><tr><td>Mr. Frosty Freezing Container</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>5100-0001</td><td></td></tr><tr><td>AxioVert 200M microscope&amp;nbsp;</td><td>Zeiss</td><td>B 40-080 e 03/01</td><td></td></tr><tr><td>FACS Gallios</td><td>Beckman Coulter&amp;nbsp;</td><td>773231AF</td><td></td></tr><tr><td>ImageJ (image processing program)</td><td>NIH</td><td></td><td></td></tr><tr><td>Stericup filter units</td><td>Merck S.P.A.&amp;nbsp;</td><td>SCGPU05RE</td><td></td></tr><tr><td>Conical Tubes, 50 mL</td><td>Eppendorf</td><td>30122178</td><td></td></tr><tr><td>Conical Tubes, 15 mL</td><td>Eppendorf</td><td>30122151</td><td></td></tr><tr><td>Safe-Lock Tubes, 2 mL</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>60 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>6 well TC-Treated Multiple Well Plates</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Polystyrene Round-Bottom Tubes, 5 mL</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>BRAND&amp;nbsp;counting chamber BLAUBRAND B&amp;uuml;rker-T&amp;uuml;rk</td><td>Sigma-Aldrich</td><td>BR719520</td><td></td></tr></tbody></table>

isolation and characterization of cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients

正常的成人心脏由几种不同的细胞类型组成, 其中心间质基质细胞代表丰富的人群。这些细胞的分离提供了研究其参与心脏疾病的可能性, 此外, 还提供了一个有用的主要细胞模型来调查生物机制。
本文介绍了从致心律失常心肌病患者活检标本中分离 C MSC 的方法。心内膜心肌活检抽样是引导在右心室区域附近的疤痕可视化的电解剖制图。本文介绍了胶原酶活检的消化及其在培养基中的塑料碟上的电镀, 以允许 C MSC 的生长。分离的细胞可以被扩展在文化中为几个段落。为证实其间充质表型, 提供了免疫表型鉴定的描述。c-msc 能够区分成几个细胞类型, 如脂肪、软骨细胞和成骨细胞: 在脂肪的背景下, 以患者心脏脂肪细胞沉积为特征, c-msc 的分化和介绍了脂滴积累的表征。

心脏基质细胞隔离:从心内膜心肌活检过程中分离出的 C MSC 是在图1 中总结的。
心间质细胞间充质特性:正如国际细胞治疗学会 (ISCT) 所建立的, 定义多能间充质间质细胞的最低标准包括其免疫表型特征3。特别是, 为了确认其间充质血统, 细胞用适当的 FITC/PE/APC 共轭抗体进行孵化, 并通过流式细胞仪进行分析。在 C MSC 特性中使用的所有抗体都列在材料表中。
如图 2所示, 从活检获得的 C MSC 对特定的间充质表面抗原 CD29、CD44 和 CD105 是阳性的。CD90 阳性单元格的百分比是可变的, 如以前报告的11所述。没有表达内皮 (CD31、CD34)、单核细胞/巨噬细胞 (CD14)、造血 (CD45) 标记和主要组织相容性复合体 (HLA-DR) (图 2)。
心间质基质细胞脂肪分化:为了促进其脂肪分化, 从患者中获得的 C-MSC 和 HC, 必须培养 ADIPO 培养基 (见解决方案部分)。细胞在培养中保持72小时或1周, 每周更换两次培养基。
细胞内脂滴的积累是由破产检测 (图 3) 证明的。不同的能力, 以及在分化程度上, 可以观察到的细胞之间从 ACM 和 HC。如代表性图像所示, 在脂肪培养基中 72 h 的培养基 (图 3) 后, ACM c-msc 积累的脂滴比 HC c msc 多。当单元格暴露在脂肪的差异条件 (1 周) (图 3) 时, 也会保持这些差异。
<img alt="Video 1" src="/files/ftp_upload/57263/57263video1.jpg"> 视频 1:electroanatomical 映射与心内超声心动图的集成.在接受心内膜心肌活检 (下板) 的患者中, 心内膜单极 electroanatomical 右心室 (左前斜和右前斜视图) 的地图。有限的低电压区域 (红色/绿色) 在顶点可见。心内膜心肌活检样本 (标记为圆圈) 是收集与患病的心肌和室隔膜。实时心内超声心动图允许检查 bioptome 在目标区域 (上部面板) 的正确位置。<a href="https://www.jove.com/files/ftp_upload/57263/Video_1.avi" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
<img alt="Video 2" src="/files/ftp_upload/57263/57263video2.jpg"> 视频 2: 透视视频右前斜视图.bioptome 通过长偏转弹头鞘插入并进入心室。仔细检查与心肌的 bioptome 接触后, 颌骨被打开, 然后紧紧地关闭收集样本。<a href="https://www.jove.com/files/ftp_upload/57263/Video_2.avi" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57263/57263fig1.jpg"> 图 1:过程草稿.心内膜心肌活检样品是收集使用一个 bioptome 导管, 一个小钳形切割仪器。获得的活检的重量约为5毫克 (A)。心内膜心肌活检样品被切碎成 0.5-1 毫米3片段, 用无菌剪刀 (B), 并添加胶原酶溶液. 该样本被放置在一个37°c 孵化器的旋转平台搅拌机为1.5 小时的消化 (C)。所消化的溶液在塑料盘子 (D) 中离心和镀巴掌就。由于其在单细胞或无性系中的塑性附着特性, 或从小的未消化的活检片段 (E) 中发芽, 因此获得了 C MSC。然后用流式细胞仪 (F) 来表征 C MSC。脂肪介质中的 C MSC 和脂滴积累通过油红色 O 染色 (G) 进行测试。刻度条指示50µm.<a href="//cloudflare.jove.com/files/ftp_upload/57263/57263fig1large.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/57263/57263fig2.jpg"> 图 2:代表细胞荧光光谱-理学硕士.每个直方图显示细胞计数与荧光强度在指示的渠道 (PE: 藻红蛋白;APC: 复方阿司匹林;FITC: 荧光素-isothyocyanate)。在每个图中, 同种控制 (白色) 和一个与特定细胞表面标记抗体 (红色) 共轭的样本被显示出来。C-MSC 是阳性的间充质表面抗原 CD29, CD44, CD105, 部分 CD90, 而他们不表达 CD31, CD34, CD14, CD45 和 HLA-DR.<a href="//cloudflare.jove.com/files/ftp_upload/57263/57263fig2large.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/57263/57263fig3.jpg"> 图 3:脂肪区分 C MSC。有代表性的图象和 HC C MSC, 培养在脂肪培养基72小时和1周, 染色的破产 (左图; n = 3)。在右图中, 报告了 255-红色通道染色亮度的量化: 在任意单位 (天文单位) 表示强度。比对照组积累更多的脂滴。学生的 T 测试被使用了, *: p < 0.05。刻度条指示50µm.<a href="//cloudflare.jove.com/files/ftp_upload/57263/57263fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients at JoVE.com

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

致心律失常心肌病患者心内膜心肌活检样间质基质细胞的分离与鉴定

本文提供了一种从致心律失常心肌病患者心内膜心肌活检样品中分离心间质基质细胞的方法。描述了它们的表征和提高脂肪分化的协议。

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

isolation-characterization-cardiac-mesenchymal-stromal-cells-from

Research

JoVE Journal

Developmental Biology

7.7K 次观看.  Centro Cardiologico Monzino-IRCCS. 本文提供了一种从致心律失常心肌病患者心内膜心肌活检样品中分离心间质基质细胞的方法。描述了它们的表征和提高脂肪分化的协议。

视频: Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

科研

发育生物学

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

医学

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

从人类和小鼠心肌标本中分离巨噬细胞子集和细胞位细胞

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

免疫与感染

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

代谢综合征心力衰竭模型大鼠心房心肌细胞的分离与保留射血分数的关系

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

生物学

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

人体心室心肌细胞与颤音-切切心肌切片分离

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

来自成人鼠心脏的心外肌细胞、心室肌细胞和非肌细胞的同步隔离和文化

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...