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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+applications+of+mesenchymal+stromal+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cardiac+and+bone+marrow+stromal+cells+exhibit+distinctive+properties+related+to+their+origin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblasts:+more+than+mechanical+support.">Cardiac fibroblasts: more than mechanical support.</a> J Thorac Dis. 9, 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblasts:+from+development+to+heart+failure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+remodeling+after+infarction+and+the+extracellular+collagen+matrix:+when+is+enough+enough.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cardiac+fibroblast:+therapeutic+target+in+myocardial+remodeling+and+failure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mesenchymal+stromal+cells+are+a+source+of+adipocytes+in+arrhythmogenic+cardiomyopathy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+models+of+arrhythmogenic+cardiomyopathy:+advances+and+opportunities.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroanatomical+mapping+systems+and+intracardiac+echo+integration+for+guided+endomyocardial+biopsy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+cells+can+be+generated+in+vitro+from+several+adult+human+organs+(heart,+liver,+and+bone+marrow).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+reside+in+virtually+all+post-natal+organs+and+tissues.">Mesenchymal stem cells reside in virtually all post-natal organs and tissues.</a> J Cell Sci. 119, 2204-2213 (2006).</li><li>Cencioni, C., 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=The+double+life+of+cardiac+mesenchymal+cells:+Epimetabolic+sensors+and+therapeutic+assets+for+heart+regeneration.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cardiac+progenitor+cells+through+epicardial+to+mesenchymal+transition.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblast:+the+renaissance+cell.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-oxidizable+HMGB1+induces+cardiac+fibroblasts+migration+via+CXCR4+in+a+CXCL12-independent+manner+and+worsens+tissue+remodeling+after+myocardial+infarction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Cardiac+Mesenchymal+Stromal+Cells+with+CD105+CD34-+Phenotype+Enhance+the+Function+of+Post-Infarction+Heart+in+Mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-kit++cardiac+progenitors+exhibit+mesenchymal+markers+and+preferential+cardiovascular+commitment.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+therapeutic+strategies+targeting+fibroblasts+and+fibrosis+in+heart+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA+expression+and+immunologic+properties+of+differentiated+and+undifferentiated+mesenchymal+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Atrial+Appendage+as+a+Suitable+Source+to+Generate+Cardiac-derived+Adherent+Proliferating+Cells+for+Regenerative+Cell-based+Therapies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cardiac-derived+adherent+proliferating+cells+reduce+murine+acute+Coxsackievirus+B3-induced+myocarditis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Power+Is+Nothing+Without+Control:+The+Enduring+Search+for+the+Best+Cell+in+Cardiac+Cell+Therapy+at+a+Crossroads.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+for+heart+disease+after+15+years:+Unmet+expectations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+for+regenerative+therapy:+optimization+of+cell+preparation+protocols.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+arrhythmogenic+right+ventricular+cardiomyopathy/dysplasia:+proposed+modification+of+the+task+force+criteria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+myocarditis+mimicking+arrhythmogenic+right+ventricular+cardiomyopathy+differential+diagnosis+by+electroanatomic+mapping-guided+endomyocardial+biopsy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+combined+unipolar+and+bipolar+voltage+maps+to+improve+sensitivity+of+endomyocardial+biopsy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2011+consensus+statement+on+endomyocardial+biopsy+from+the+Association+for+European+Cardiovascular+Pathology+and+the+Society+for+Cardiovascular+Pathology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+Isolation+of+3+Distinct+Cardiac+Stem+Cell+Populations+From+a+Single+Human+Heart+Biopsy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+endomyocardial+biopsy.">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 border="0" cellpadding="0" cellspacing="0" style="width:804px;" width="804">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">IMDM</td>
			<td style="width:195px;">Gibco by Life Technologies&#160;</td>
			<td style="width:141px;">12440053</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">F2442</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">PENICILLIN STREPTOMYCIN</td>
			<td style="width:195px;">Life Technologies Italia</td>
			<td style="width:141px;">15140122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Collagenase NB4</td>
			<td style="width:195px;">Serva</td>
			<td style="width:141px;">17454.02</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Basic fibroblast growth factor</td>
			<td style="width:195px;">Peprotech</td>
			<td style="width:141px;">100-18B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">L-glutammine</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">G7513</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">PBS</td>
			<td style="width:195px;">Lonza</td>
			<td style="width:141px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Tryple select 1X (cell dissociation reagent)</td>
			<td style="width:195px;">Gibco</td>
			<td style="width:141px;">12563029</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">EDTA</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">EDS</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Trypsin-EDTA solution&#160;</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">T6689</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Bovine Serum Albumin</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">A2058</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">I5879</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Hydrocortisone</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">H4001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Indomethacin</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">I7378</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Oil Red O</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">O0625</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Isopropanol</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">I9516</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Paraformaldehyde (PFA) Solution 4% in PBS</td>
			<td style="width:195px;">D.b.a. Italia S.r.l.</td>
			<td style="width:141px;">sc-281692</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">276855</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Antibody CD14-FITC (M&#966;P9)</td>
			<td style="width:195px;">Becton Dickinson</td>
			<td style="width:141px;">345784</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Antibody Integrin &#946;1 (CD29)-PE (clone MAR4)</td>
			<td style="width:195px;">Becton Dickinson</td>
			<td style="width:141px;">561795</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Antibody PECAM-1 (CD31)- APC&#160; (clone 9G11)</td>
			<td style="width:195px;">R&#38;D Systems</td>
			<td style="width:141px;">FAB3567A</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:404px;">Antibody Hematopoietic progenitor cell antigen (CD34)-APC (clone 581)</td>
			<td style="width:195px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:141px;">CD34-581-05</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Antibody H-CAM (CD44)-PE&#160; (clone G44-26)</td>
			<td style="width:195px;">Becton Dickinson</td>
			<td style="width:141px;">561858</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Antibody L-CA (CD45)-APC (clone HI30)</td>
			<td style="width:195px;">Thermo Fisher Scientific</td>
			<td style="width:141px;">MHCD4505-4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Antibody THY1 (CD90)-FITC (clone 5E10)</td>
			<td style="width:195px;">Becton Dickinson</td>
			<td style="width:141px;">561969</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Antibody Endoglin (CD105)-APC (clone 266)</td>
			<td style="width:195px;">Becton Dickinson</td>
			<td style="width:141px;">562408</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:404px;">Antibody HLA-DR-FITC (clone L243)</td>
			<td style="width:195px;">Becton Dickinson</td>
			<td style="width:141px;"> 
			347400</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">Gima Quick Plus sterilizer</td>
			<td style="width:195px;">Gima&#160;</td>
			<td style="width:141px;">35642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">Bench centrifuge Sigma 3-16K</td>
			<td style="width:195px;">Sigma Centrifuges</td>
			<td style="width:141px;">10330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">Mr. Frosty Freezing Container</td>
			<td style="width:195px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:141px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">AxioVert 200M microscope&#160;</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:141px;">B 40-080 e 03/01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">FACS Gallios</td>
			<td style="width:195px;">Beckman Coulter&#160;</td>
			<td style="width:141px;">773231AF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">ImageJ (image processing program)</td>
			<td style="width:195px;">NIH</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">Stericup filter units</td>
			<td style="width:195px;">Merck S.P.A.&#160;</td>
			<td style="width:141px;">SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">Conical Tubes, 50 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:141px;">30122178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">Conical Tubes, 15 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:141px;">30122151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">Safe-Lock Tubes, 2 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:141px;">30120094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">100 mm TC-Treated Cell Culture Dish</td>
			<td style="width:195px;">Corning</td>
			<td style="width:141px;">430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">60 mm TC-Treated Cell Culture Dish</td>
			<td style="width:195px;">Corning</td>
			<td style="width:141px;">430166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">6 well TC-Treated Multiple Well Plates</td>
			<td style="width:195px;">Corning</td>
			<td style="width:141px;">3516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">Polystyrene Round-Bottom Tubes, 5 mL</td>
			<td style="width:195px;">BD Falcon</td>
			<td style="width:141px;">352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:404px;">BRAND&#160;counting chamber BLAUBRAND B&#252;rker-T&#252;rk</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:141px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.6K 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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

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 ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...