E.A..B得到了美国国家卫生研究院（1F31HL140850）、ARCS基金会、圣地亚哥分会的支持，并且是SDSU心脏研究所的M.D.学者菲利普斯·高塞维茨的里斯-偷窃研究基金会。E.A..B和A.S.B得到了伊那莫里基金会的支持。CCG由（NIH）授予R01 HL135893，R01 HL141463和HL149931。

本文报告的所有对小鼠的研究都经过SDSU机构动物护理和使用委员会的审查和批准，并符合国家研究委员会发布的《实验室动物护理和使用指南》。
1. 准备隔离和文化媒体和电镀
<ol><li>在使用前准备 1 升心脏注入介质，将 Joklik 修改的最低基本介质 （MMEM） 添加到 1 升无菌水。将 pH 调整为 7.36，使用 10 N NaOH，然后通过 0.2 μm 过滤器进行过滤，并在 4 °C 下存储长达 2 周。 注:乔克利克MM由112毫米纳克、5.4米KCl、1米M MgCl2、9mNaH2PO4和11.1m D-葡萄糖组成。这补充了10米海佩斯（2.38克/升），30米陶林（3.75克/升），2米D-l-肉碱（0.4克/升），2米肌酸，和10米丁胺单一（1.01克/升）。</li>
	<li>在注入之前准备消化缓冲液，如下所示:补充50 mL的心脏注入介质与6.25μL的100 mM CaCl2（ 见步骤1.3）和拼贴酶2型（酶活性+310-320 U/mg dw）。 注意:在牺牲前称量动物。补充到消化缓冲的拼贴酶类型2的量是由动物的重量决定的（每1克体重2.25毫克拼贴酶类型2）。</li>
	<li>要准备100mM CaCl2，添加1.47克CaCl2 至100mL分子生物学级水。搅拌至溶解，然后通过 0.2 μm 过滤器，在室温下存放长达 2 个月。</li>
	<li>准备肌细胞停止缓冲区1通过补充90毫升的心脏灌注缓冲液与10毫升的胎儿牛血清（FBS）和125μL的100 mM CaCl2.通过 0.2 μm 过滤器，在 4 °C 下存储长达 2 周。</li>
	<li>准备肌细胞停止缓冲区2通过补充114毫升的心脏灌注缓冲液与6毫升的FBS和150μL的10 mM CaCl2.通过 0.2 μm 过滤器，在 4 °C 下存储长达 2 周。</li>
	<li>在灌注前准备心房肌细胞电镀介质，通过补充 95 mL 的 Dulbecco 改良鹰介质 （DMEM） 与 4 mL FBS， 1 mL 的 100x 笔 /链球菌 - 谷氨酰胺， 1 mL 的 100 倍胰岛素转移素 - 氦， 和 1 mL 的 10 μm 德萨梅松.通过 0.2 μm 过滤器，在 37 °C 下存储，直到电镀。</li>
	<li>通过补充 95 mL 的最低基本介质 （MEM） 与 4 mL FBS， 1 mL 的 100x 笔 /链球菌 - 谷氨酰胺， 10 mL 的 1 M HEPES 溶液， 1 mL 的 100 倍胰岛素转移素 - 氦， 和 0.1 克丁尼多尼单西梅， 准备心室肌细胞电镀介质。通过 0.2 μm 过滤器，在 4 °C 下存储长达 2 周。</li>
	<li>通过补充 99 mL 的 MEM，补充 1 mL 的 100 倍胰岛素转移素-氦、0.1 毫克/mL 牛血清白蛋白 （BSA）、1 M HEPES 溶液的 10 mL 和 1 mL 的 100x 笔/链球菌-谷氨酰胺，制备心室肌细胞维持介质。通过 0.2 μm 过滤器，在 4 °C 下存储长达 2 周。</li>
	<li>要准备层压涂层实验板和滑梯，解冻鼠标层压素库存溶液（1.19 毫克/mL）。将 10 μL 的层压素库存溶液添加到 DMEM 的每 1 mL 中，然后混合。涂上实验板和幻灯片均匀，并存放在 37 °C， 5% CO2 孵化器至少 1 小时之前灌注， 以允许平衡。 注:涂层实验板和幻灯片可在4°C下存储长达2天。</li>
</ol>2. 隔离装置
<ol><li>每次隔离之前，通过将气泡陷阱填充到顶部（关闭底部的塞子并保持顶部的塞子打开），用三次 70% EtOH 的完整洗涤清洁系统中的管子和其他组件。</li>
	<li>通过将气泡陷阱填充到顶部（关闭底部的塞子并保持顶部的塞子打开），用无菌 H 2 O 冲洗整个系统3倍。</li>
	<li>用心脏灌注缓冲液冲洗整个系统，用介质填充半个气泡陷阱。</li>
	<li>将循环水浴设置为 37 °C。</li>
	<li>将介质水浴设置为 37 °C。</li>
	<li>去除腹股油泵管中的任何气泡。</li>
	<li>将永久泵的流速调整为 3 mL/min。</li>
</ol>3. 外科手术（非生存）
<ol><li>将手术器械浸泡在 70% EtOH 中至少 5 分钟。</li>
	<li>将心脏注水介质、肌细胞停止缓冲液和消化缓冲液放置在37°C水浴中。</li>
	<li>在使用前将心室肌细胞电镀介质、心室肌细胞电镀介质和心室肌细胞维持介质放置在 37 °C、5% CO2 孵化器 1 小时之前，并松开盖子以实现平衡。</li>
	<li>用0.35 mL的肝素注射10周大的雄性或雌性C57b6/j小鼠，在磷酸盐缓冲盐水（PBS）中稀释至100 IU/mL。让药物生效约10分钟。 注:如果两颗心脏要接受这种隔离程序，第二只小鼠可以麻醉并在第一个程序的此时施用肝素。为了尽量减少对动物的压力，可以在密封的感应室中使用2%的异氟/氧混合物进行轻麻醉。</li>
	<li>用2%的异氟/氧混合物麻醉动物，然后注射五巴比妥（10毫克/mL库存0.3 mL），然后用70%EtOH擦拭来准备胸部。</li>
	<li>准备一个绑 5 - 0 丝绸缝合， 准备把心脏液液到灌装罐。手术显微镜旁边的坎努拉山。</li>
	<li>快速打开胸部，首先做一个中线皮肤切口，从中腹到下巴，然后进入腹膜与大剪刀，清除隔膜通过钝解剖。然后，用剪刀切掉肋骨笼，在两侧侧面的胸壁上切开。</li>
	<li>剪掉心脏和胸壁（包括胸腺）之间的纤维连接。然后，一起切掉肋骨笼。使用小钳子和剪刀，轻轻抬起心脏的顶点，露出心脏的后部。</li>
	<li>通过解剖立即低于上升主动脉上的无名动脉来切除心脏，并立即将心脏置于冰冷的PBS或冷心灌注介质中。随后，迅速解剖出冰冷心脏注水介质中从被切除的心脏中分离出的剩余组织，露出上升的主动脉。 注:用胸腺完好无损地切除心脏作为解剖地标是有用的。</li>
	<li>使用微解剖钳和剪刀清洁多余组织主动脉周围的区域。使用细尖钳将主动脉放置在管子上，并用 5-0 丝缝固定。 注意:最好的位置通常发生在主动脉延伸约2毫米的管子上。</li>
	<li>以 3 mL/min 的流速为 4 分钟注入心脏灌注介质。然后将介质从心脏注水介质切换到消化缓冲液，进行 15.0-17.5 分钟的注水（图 1A）。 注:冠状动脉流动率可能会增加，表明有效的组织消化（即苍白，肿胀的心脏）。</li>
	<li>在注水的最后几分钟收集 8 mL 的消化缓冲流，供第 5.4 步以后使用。</li>
	<li>从管子中取出心脏，放在60毫米的塑料培养皿上。取出多余的组织（即主动脉、静脉），将心脏浸入2.5 mL的消化缓冲液中，为机械分离做准备。 注:此时，对心室进行解剖，一名实验者应执行心室细胞隔离方案，而第二名实验者应进行心室细胞分离。</li>
</ol>4. 纹状细胞隔离与培养
<ol><li>解剖远离心脏的阿提亚，并将其放入一个30毫米的塑料培养盘。将其浸入0.75 mL的消化缓冲液中，用于机械分离。将心室保持在 60 毫米盘中（步骤 3.12），同时执行单独的隔离方法（第 5 节，图 1B）。 注:此时，如果需要分离左侧和右侧细胞，腹膜和心室可以进行进一步分离。</li>
	<li>开始切碎和取笑阿提亚分开， 最初用细尖手术剪刀， 然后细钳子进一步切碎。避免搅动组织，不要快速拉开肌肉纤维。</li>
	<li>使用无菌转移移液器尖端，继续轻轻地混合和分离组织15分钟。每5分钟观察一次在10倍目标光场显微镜下与组织分离的体外肌细胞分离。当组织进一步消化时，继续使用毛孔尺寸较小的无菌转移移液器尖端轻轻地混合和分离组织。</li>
	<li>将细胞悬架转移到2mL无菌微离心管中。用 37 °C 肌细胞停止缓冲区 1 的 0.75 mL 冲洗 30 mm 板，并与单元格悬架（端体积 = 1.5 mL）相结合。</li>
	<li>让心房肌细胞在室温下通过重力沉积10分钟。轻轻搅拌细胞悬浮物，让肌细胞沉积到圆锥管底部，形成可见的颗粒。</li>
	<li>离心细胞悬浮5分钟，在20 x g。小心地去除含有非肌细胞的超纳坦，并使用无菌移液器尖端转移到 15 mL 聚丙烯锥形管中，而不会干扰心房肌细胞的颗粒。</li>
	<li>离心非肌细胞分数为5分钟，在20，000 x g。吸附超纳坦，在10 mL的DMM中重新悬浮非肌细胞颗粒，并辅以10%的胎儿小腿血清（FCS）。</li>
	<li>使用血细胞计或其他方法计算非肌细胞，然后根据实验需要板，或通过荧光激活细胞分类进一步分离成单个特定细胞群（图1C）。</li>
	<li>在1mL的体外肌细胞电镀介质中从步骤4.6中重新悬浮分离的体外肌细胞颗粒，并将10μL的这种悬架涂抹在造细胞仪上。执行每个字段的杆形肌细胞的细胞计数。</li>
	<li>从预涂层实验板/滑梯中吸附层压素，并在适当体积的心外肌细胞电镀介质中重新悬生分离的心外肌细胞，并辅以25μM blebbistatin。每个实验需要所需的密度板（图 1C）。 注:此处描述的长期栽培的典型电镀密度为四室（1.7 厘米2）玻璃滑梯上的 5 x 105单元/室。</li>
</ol>5. 心室细胞隔离和文化
<ol><li>开始切碎和取笑心脏分开心室组织，首先用细尖手术剪刀，然后细钳进一步切碎（图1B）。避免通过快速拉开肌肉纤维来搅动组织。</li>
	<li>将电池悬架转移到 15 mL 聚丙烯锥形管中。用 2.5 mL 的 37 °C 肌细胞停止缓冲区 1 冲洗板，并结合细胞悬架（端体积 = 5 mL）。</li>
	<li>使用无菌转移移液器尖端，继续轻轻地混合和分离组织4分钟。将此细胞悬架的 10 μL 涂抹在滑梯上，可视化棒状肌细胞的存在，以确保隔离质量。</li>
	<li>通过 100μm 无菌尼龙过滤器将细胞悬架传递到 50 mL 聚丙烯锥形管中。使用第 3.12 步收集的 2 mL 消化缓冲液，从无菌尼龙过滤器中清洗任何剩余的细胞。</li>
	<li>让心室肌细胞在室温下通过重力沉积6分钟。轻轻搅拌过滤的细胞悬浮物，使肌细胞沉积在圆锥体底部，形成可见的颗粒。</li>
	<li>在不干扰心室肌细胞颗粒的情况下，小心地取出超纳坦（非肌细胞），使用无菌移液器尖端转移到 50 mL 聚丙烯锥形管中。离心非肌细胞分数为5分钟，在20，000 x g。吸气超纳坦和重新悬浮非肌细胞颗粒在10 mL的DMEM补充10%FCS。</li>
	<li>使用半球形计计算非肌细胞，并在适当的DMEM量中重新悬生，并辅以10%FCS。然后，根据实验需要板，或进一步隔离到个别特定的细胞群通过荧光激活细胞排序（图1C）。</li>
	<li>将分离的心室肌细胞在2 mL的肌细胞停止缓冲区2中重新悬浮，并将10μL的细胞悬浮物涂抹在血细胞计上。执行每个字段的杆形肌细胞的细胞计数。</li>
	<li>使用渐进式范式重新引入 Ca2+ 注:钙重新引入步骤是特定于心室肌细胞的，不应该为心室肌细胞执行，因为这将导致细胞死亡。<ol>
<li>在心室肌细胞悬浮液中添加 50 μL 的 10 mm CaCl2。 混合好，在室温下孵育4分钟。</li>
		<li>在心室肌细胞悬架中再添加 50 μL 的 10 mM CaCl2。 混合好，在室温下孵育4分钟。</li>
		<li>在心室肌细胞悬浮液中再添加 100 μL 的 10 mM CaCl2。 混合好，在室温下孵育4分钟。</li>
		<li>将 100 mM CaCl2 的 80 μL 添加到心室肌细胞悬架中。混合好，在室温下孵育4分钟。</li>
	</ol></li>
	<li>根据实验需要（图1C），从板或滑梯上取下层压涂层，将分离的心室肌细胞重新悬生在适量的心室肌细胞电镀介质中。 注:此处描述的长期栽培的典型电镀密度为四室（1.7 厘米2）玻璃滑梯上的 5 x 105单元/室。避免电镀的密度大于75%的汇合，因为细胞过密度促进细胞团块，从而抑制对板的依恋。此外，在第一次介质变化期间将丢失大量细胞。板状细胞在分离后迅速，因为细胞外Ca2+促进过度竞争和失去可行的肌细胞。</li>
	<li>允许心室肌细胞沉淀并粘附至少1小时。随后将介质改为心室肌细胞维持介质，辅以25μM布比斯塔汀。 注:在 μM 布利比斯塔汀中电镀后，心室肌细胞可培养至 96 小时。实验应在室外肌细胞维持介质中进行，而不要使用布比斯塔汀。</li>
</ol>

<ol>
	<li>Peter, A. K., Bjerke, M. A., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+the+cardiac+myocyte+in+heart+disease.">Biology of the cardiac myocyte in heart disease.</a> Molecular Biology of the Cell. 27 (14), 2149-2160 (2016).</li><li>Kruppenbacher, J. P., May, T., Eggers, H. J., Piper, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocytes+of+adult+mice+in+long-term+culture.">Cardiomyocytes of adult mice in long-term culture.</a> Naturwissenschaften. 80 (3), 132-134 (1993).</li><li>Ackers-Johnson, 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=A+Simplified,+Langendorff-Free+Method+for+Concomitant+Isolation+of+Viable+Cardiac+Myocytes+and+Nonmyocytes+From+the+Adult+Mouse+Heart.">A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart.</a> Circulation Research. 119 (8), 909-920 (2016).</li><li>Jin, J. 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=ATF6+decreases+myocardial+ischemia/reperfusion+damage+and+links+ER+stress+and+oxidative+stress+signaling+pathways+in+the+heart.">ATF6 decreases myocardial ischemia/reperfusion damage and links ER stress and oxidative stress signaling pathways in the heart.</a> Circulation Research. 120 (5), 862-875 (2017).</li><li>Judd, J., Lovas, J., Huang, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Culture+and+Transduction+of+Adult+Mouse+Cardiomyocytes.">Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes.</a> Journal of Visualized Experiments. (114), e54012 (2016).</li><li>Li, D., Wu, J., Bai, Y., Zhao, X., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Adult+Mouse+Cardiomyocytes+for+Cell+Signaling+and+in+vitro+Cardiac+Hypertrophy.">Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy.</a> Journal of Visualized Experiments. (87), e51357 (2014).</li><li>O'Connell, T. D., Ni, Y. G., Lin, K. M., Han, H. P., Yan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+cardiac+myocytes+for+signaling+studies.">Isolation and culture of adult cardiac myocytes for signaling studies.</a> AfCS Research Reports. 1 (5), 1-9 (2003).</li><li>Pinz, I., Zhu, M., Mende, U., Ingwall, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improvised+isolation+procedure+for+adult+mouse+cardiomyocytes.">An improvised isolation procedure for adult mouse cardiomyocytes.</a> Cell Biochemistry and Biophysics. 61 (1), 93-101 (2011).</li><li>Piper, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+of+calcium+stable+adult+cardiac+myocytes.">Culturing of calcium stable adult cardiac myocytes.</a> Journal of Molecular and Cellular Cardiology. 14 (7), 397-412 (1982).</li><li>Shioya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+isolating+healthy+heart+cells+from+mouse+models.">A simple technique for isolating healthy heart cells from mouse models.</a> Journal of Physiological Sciences. 57 (6), 327-335 (2007).</li><li>Omatsu-Kanbe, M., Yoshioka, K., Fukunaga, R., Sagawa, H., Matsuura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+antegrade+perfusion+method+for+isolating+viable+single+cardiomyocytes+from+neonatal+and+aged+mice.">A simple antegrade perfusion method for isolating viable single cardiomyocytes from neonatal and aged mice.</a> Physiological Reports. 6 (9), 13688 (2018).</li><li>Jansen, H. J., Rose, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Atrial+Myocytes+from+Adult+Mice.">Isolation of Atrial Myocytes from Adult Mice.</a> Journal of Visualized Experiments. (149), e59588 (2019).</li><li>Yao, 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=Enhanced+Cardiomyocyte+NLRP3+Inflammasome+Signaling+Promotes+Atrial+Fibrillation.">Enhanced Cardiomyocyte NLRP3 Inflammasome Signaling Promotes Atrial Fibrillation.</a> Circulation. 138 (20), 2227-2242 (2018).</li><li>Cha, Y., Redfield, M. M., Shen, W., Gersh, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+Fibrillation+and+Ventricular+Dysfunction:+A+Vicious+Electromechanical+Cycle.">Atrial Fibrillation and Ventricular Dysfunction: A Vicious Electromechanical Cycle.</a> Circulation. 109 (23), 2839-2843 (2004).</li><li>Issa, 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=Left+atrial+size+and+heart+failure+hospitalization+in+patients+with+diastolic+dysfunction+and+preserved+ejection+fraction.">Left atrial size and heart failure hospitalization in patients with diastolic dysfunction and preserved ejection fraction.</a> Journal of Cardiovascular Echography. 27 (1), 1-6 (2017).</li><li>de Bold, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+natriuretic+factor:+a+hormone+produced+by+the+heart.">Atrial natriuretic factor: a hormone produced by the heart.</a> Science. 230 (4727), 767-770 (1985).</li><li>McGrath, M. F., de Bold, M. L., de Bold, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endocrine+function+of+the+heart.">The endocrine function of the heart.</a> Trends in Endocrinology and Metabolism. 16 (10), 459-477 (2005).</li><li>Doevendans, P. A., Daemen, M. J., de Muinck, E. D., Smits, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+phenotyping+in+mice.">Cardiovascular phenotyping in mice.</a> Cardiovascular Research. 39 (1), 34-49 (1998).</li><li>Banerjee, I., Fuseler, J. W., Price, R. L., Borg, T. K., 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=Determination+of+cell+types+and+numbers+during+cardiac+development+in+the+neonatal+and+adult+rat+and+mouse.">Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse.</a> American Journal of Physiology. 293, 1883-1891 (2007).</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, 1395-1403 (2003).</li><li>Omatsu-Kanbe, 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=Identification+of+cardiac+progenitors+that+survive+in+the+ischemic+human+heart+after+ventricular+myocyte+death.">Identification of cardiac progenitors that survive in the ischemic human heart after ventricular myocyte death.</a> Scientific Reports. , 7 (2017).</li><li>Song, 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=Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors.">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485, 599-604 (2012).</li><li>Limana, F., 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=bcl-2+overexpression+promotes+myocyte+proliferation.">bcl-2 overexpression promotes myocyte proliferation.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 6257-6262 (2002).</li><li>Pinto, A. R., 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=Revisiting+cardiac+cellular+composition.">Revisiting cardiac cellular composition.</a> Circulation Research. 118 (3), 400-409 (2015).</li><li>Chen, W., Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+inflammatory+and+fibrogenic+pathways+in+heart+failure+associated+with+aging.">The role of inflammatory and fibrogenic pathways in heart failure associated with aging.</a> Heart Failure Reviews. 15, 415-422 (2010).</li><li>Daly, M. J., Elz, J. S., Nayler, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contracture+and+the+calcium+paradox+in+the+rat+heart.">Contracture and the calcium paradox in the rat heart.</a> Circulation Research. 61 (4), 560-569 (1987).</li><li>Li, Z., Sharma, R. V., Duan, D., Davisson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+to+adult+mouse+cardiomyocytes+is+selectively+influenced+by+culture+medium.">Adenovirus-mediated gene transfer to adult mouse cardiomyocytes is selectively influenced by culture medium.</a> Journal of Gene Medicine. 5 (9), 765-772 (2003).</li></ol>

作者没有什么可透露的。

使用此处描述的程序分离的细胞的质量取决于许多可控因素，这些过程由培养细胞的细胞产量和整体健康决定。从小鼠本身开始，据记载，强加于动物的压力会对培养物中的细胞产量和生存能力产生负面影响，这大概是由于系统性皮质醇、儿茶酚胺和心脏组织2、5、7的超合同状态。出于这些原因，应采取措施避免在牺牲前惊动动物。这些措施包括覆盖动物的笼子，在牺牲前限制在动物笼子外的时间。肝素和许多巴比妥酸盐通常用于安乐死，可以影响信号通路:因此，应相应地定制最佳安乐死方法。动物的年龄对分离细胞的质量和生存能力有相当大的影响，很可能是由于间质纤维化的逐渐积累与衰老过程同时发生，这可能会影响组织消化25。在此处没有提供的数据中，虽然上述方法适用于78周大的小鼠，但这些老动物的细胞质量较低。
在描述的隔离过程中，最关键的一步，以及其他具有逆行香水的Langendorff装置的协议，是心脏的精制和初始注入。为了获得最佳结果，从心脏切除到上升主动脉的坎化和注入的启动，时间不应超过90秒。除了时间，另外两个重要因素是管子的深度和从输液装置中引入空气浮雕的可能性。因此，管子应推进到上升主动脉，以免进入主动脉根和阻碍主动脉瓣，这将损害冠状动脉的注入。
在消化过程中，定期测试心脏的刚度，避免长时间接触消化酶拼贴酶，降低心脏肌细胞钙耐受性。上述关于钙重新引入孤立的心室肌细胞培养物的协议旨在通过商店操作的钙通道不当钙流入来限制心脏肌细胞死亡。应当指出，逐步钙重新引入不应为孤立的脑膜肌细胞培养进行，因为这将促进细胞死亡在短期和长期文化12。为进一步预防，这里使用的香水和消化缓冲包括心脏肌肉收缩抑制剂丁尼迪翁单氧体（BDM），以避免孤立的肌细胞的过度竞争，以及钙悖论，两者都影响肌细胞的生存能力26。然而，应该注意从BDM到布比斯塔汀的转换，因为它是维护分离心脏肌细胞介质的首选抗收缩剂。在未显示的数据中，布比斯塔汀为孤立的心脏肌细胞的长期培养赋予了更大的生存能力。
隔离后立即考虑心脏细胞，特别是肌细胞的长期培养的影响是很重要的。此处描述的心脏非肌细胞分离和培养方案基于利用不同心脏细胞的不同密度和粘合特性的常见方法。非肌细胞的好处是它们在文化上具有很高的扩张潜力:因此，与心脏肌细胞不同，它们适合通过永久存在。然而，众所周知，培养条件，包括中等补充FBS，可以影响心脏肌细胞功能27。这里描述的文化媒体旨在优化生存能力和限制功能疯狂，特别是对于孤立的体外肌细胞。虽然在缺乏卵泡补充的情况下，在培养后分离的心脏肌细胞中未观察到明显受损的收缩能力，但专注于电生理学、收缩性和其他基于体内的单细胞分子信号的研究应在隔离后不久进行，此时肉体结构和分子特征仍与完整的心脏相似。
室外肌细胞的一个标志性特征是它作为具有巨大分泌能力的内分泌细胞的月光能力，以及它们的收缩功能。在生理条件下，心律间肌细胞产生大量的ANP，储存在内质性视网膜和大型密集核心分泌颗粒中，在接受刺激16、17后，这些颗粒准备进行调节性外分泌。虽然许多孤立的心房肌细胞研究侧重于其独特的电生理特性，但这是首次设计培养基的研究。这允许长期生存，以及促进内分泌的维持功能和收缩功能的房内分泌肌细胞。这种新的培养方法，以及同时将所有细胞类型与心房和心室从单个小鼠心脏中分离出来，对于研究心房和心室肌细胞的生理和病理生理特性将非常有用和有效。

原细胞培养是一种不可或缺的资源，为心脏肌细胞功能的详细机械研究提供了一个受控的环境。由于其更持久的性质和容易隔离，新生儿大鼠心室和心室肌细胞一直是这种细胞培养物的共同来源1。然而，成人小鼠心房和心室肌细胞（AMAM和AMVM）是非常可取的体外研究，因为他们的分子和功能特征更好地模仿成人心脏细胞。因此，他们已成为相关的研究与心脏病理学，其中大多数发展在成人2。
此外，转基因和疾病小鼠模型的可用性和使用扩展了孤立的成人心脏肌细胞的效用。鼠标AMVM的短期和长期研究的隔离和文化协议已在许多以前的出版物2，3，4，5，6，7，8，9，10，11中进行了描述。相比之下，对于 AMAM 的隔离，很少描述协议。此外，那些描述主要是优化为急性研究新鲜分离的细胞，没有长期的培养协议描述至今11，12，13。因此，AMAM 隔离协议的设计并不旨在为 AMM 的隔离和文化提供已发布的协议的实用性和多功能性。此外，虽然对AMAM和AMVM隔离的开创性研究已被证明是足智多谋的，但目前还没有关于AMAM和AMVM最佳并发隔离和文化的协议，这导致每次准备都能够有效地使用整个心脏。
到目前为止，已发布的AMAM和AMVM隔离协议并不是为同时隔离这两种细胞类型而设计的，因为大多数关于心房和心室功能的研究都有一个特定于腔室的焦点。例如，AMAM主要用于研究心房肌细胞电生理学，部分原因是对心房颤动（AF）的兴趣，心房颤动是美国最常见的心律失常。然而，AF不是一种孤立地影响腹膜的疾病，它被牵连为在轻度到重度左心室功能障碍14中起因果作用。此外，心电图从心力衰竭患者与保留的弹射分数（HFPEF）已经表明，左心房大小是最有力的预测之一，易感性心力衰竭15。
中庭除了在电生理学和收缩性方面的作用外，还是一种内分泌器官，分泌心电因子（即心房内科肽[ANP]），以平衡调节血压和体积16，17。此外，ANP（大概来自心室肌细胞）在心室肌细胞16，17中具有突出的保护和抗肥大作用。虽然不同疾病状态的腹膜和心室之间有很强的神经荷尔蒙交流的含义，但这种交流背后的机制尚未得到充分探讨。这一点进一步体现了研究的激增，重点是1）非肌细胞（特别是心脏成纤维细胞和免疫细胞）在患病心脏中的作用，2）心脏重塑作为疾病的功能直接影响心脏肌细胞的生存能力和全球心脏功能18，19，20，21，22。因此，从心室和心室研究心脏细胞是获得更全面地了解它们在心脏病理生理学中的作用的必要方法。
以下协议描述了在生理和病理生理条件下，心室和心室肌细胞和非肌细胞与单个小鼠心脏的同步分离。此外，该方法是首次描述维持心室心肌细胞培养所必需的最佳条件，因为维持心室肌细胞培养的条件已经公布。

<table border="0" cellpadding="0" cellspacing="0" style="width:668px;" width="669">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">(-)-Blebbistatin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">B0560</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">1 Liter Water Jacketed Reservoir</td>
			<td style="width:175px;">Radnoti</td>
			<td style="width:116px;">120142-1</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">2,3-Butanedione monoxime</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">B0753</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">5-0 Silk Suture Thread</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:116px;">18020-50</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Adenosine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">A9251</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Bovine Serum Albumin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">A6003</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bubble Trap Compliance Chamber</td>
			<td style="width:175px;">Radnoti</td>
			<td style="width:116px;">130149</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Calcium Chloride Anhydrous</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:116px;">C614-500</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Carnitine hydrochloride</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">C9500</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Collagenase type 2</td>
			<td style="width:175px;">Worthington Biochemical Corporation</td>
			<td style="width:116px;">LS004176</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Creatine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">C0780</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dexamethasone</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">D2915</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DMEM/F12 (1:1; 1X)</td>
			<td style="width:175px;">Gibco</td>
			<td style="width:116px;">11330-032</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dumont #7 - Fine Forceps</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:116px;">11274-20</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Epifluorescent micropscpe</td>
			<td style="width:175px;">Olympus X70</td>
			<td style="width:116px;">IX70</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Fetal Bovine Serum (Heat Inactivated)</td>
			<td style="width:175px;">Omega Scientific</td>
			<td style="width:116px;">FB-12</td>
			<td style="width:163px;">Lot# 206018</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fine Scissors - Sharp</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:116px;">14060-09</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Graefe Forceps</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:116px;">11051-10</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Headband Magnifiers</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:116px;">28030-04</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hemacytometer (Bright-Line)</td>
			<td style="width:175px;">Hausser Scientific</td>
			<td style="width:116px;">1475</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HEPES (1M)</td>
			<td style="width:175px;">Gibco</td>
			<td style="width:116px;">15630-080</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Inosine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">I4125</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Insulin-Transferrin-Selenium-X</td>
			<td style="width:175px;">Gibco</td>
			<td style="width:116px;">51500-056</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isotemp 105 Water Bath</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:116px;">NC0858659</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isotemp 3006</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:116px;">13-874-182</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Joklik Modified Minimum Essential Media</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">M-0518</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Laminin (Natural, Mouse)</td>
			<td style="width:175px;">Gibco</td>
			<td style="width:116px;">1795024</td>
			<td style="width:163px;">Lot# 1735572</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">L-Glutamine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">G8540</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Masterflex C/L Single-Channel Variable-Speed Compact Pump</td>
			<td style="width:175px;">Cole-Palmer</td>
			<td style="width:116px;">EW-77122-24</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Minimum Essential Medium (MEM 1X)</td>
			<td style="width:175px;">Gibco</td>
			<td style="width:116px;">12350-039</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Molecular Biology Grade Water</td>
			<td style="width:175px;">Corning</td>
			<td style="width:116px;">46-000-CM</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Pen Strep Glutamine (100X)</td>
			<td style="width:175px;">Gibco</td>
			<td style="width:116px;">10378-016</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Spring Scissors - 6mm Cutting Edge</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:116px;">15020-15</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Taurine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:116px;">T-8691</td>
			<td style="width:163px;"></td>
		</tr>
	</tbody>
</table>

simultaneous isolation and culture of atrial myocytes, ventricular myocytes, and non-myocytes from an adult mouse heart

从小鼠身上分离和培养心脏肌细胞对于进一步理解心脏生理学和病理生理学至关重要。虽然从新生儿小鼠心脏中分离肌细胞相对简单，但成人小鼠心脏的肌细胞是首选。这是因为与新生儿细胞相比，成人肌细胞更准确地重述细胞功能，因为它发生在成人心脏的体内。 然而，从技术上讲，很难在必要的数量和可行性中分离成年小鼠心脏肌细胞，这导致了实验僵局。此外，公布的程序是专门隔离心室或心室肌细胞，牺牲心室和心室非肌细胞。这里描述的是一个详细的方法，隔离心室和心室心脏肌细胞，以及心室和心室非肌细胞，同时从一个单一的老鼠心脏。此外，还提供了最佳细胞特异性培养方法的详细信息，可增强细胞的生存能力和功能。该协议不仅旨在加快成人穆林心脏细胞分离的过程，而且旨在提高细胞的产量和存活率，用于研究心室和心室心脏细胞。

野生类型 10 周大的 C57b6/j 小鼠心脏通常导致 75，000~150，000 心室肌细胞和 1.0×1.5 x 106心室肌细胞， 相当于心室和心室肌细胞18，19的约30%-50%的收益率。在隔离期间和隔离后立即，可行的心肌细胞应出现杆状和非收缩。大多数分离的心脏肌细胞应该适应这种形态，这是有效注入的迹象。棒形形态也可以是生存能力的预测器。该协议旨在提高从患病小鼠心脏中分离出的肌细胞和非肌细胞的产量和存活率。此外，它已在压力超载引起的心力衰竭模型中进行了测试（未显示数据）。
为了确认心肌细胞和非肌细胞与心室组织充分和可复制的分离，在培养的不同日子观察和拍摄细胞（图2）。此外，还进行了定量反向转录聚合酶链反应 （qRT-PCR） 来测量特定于细胞类型的成绩单水平。心脏肌肉肌肽T（Tnnt2）是心脏肌细胞的标记，在心室和心室心脏肌细胞培养物（图3A）中都表现强烈。相比之下，心室心律肽（Nppa，在生理条件下通常只在成人心室心肌细胞中表达）和肌素光链2（Myl2，这是一个心室肌细胞特异性基因）分别在心室和心室心脏肌细胞培养物中强烈而具体地表达（图3B，C）。
纤维细胞标记、转录因子21（Tcf21）、血小板衍生生长因子受体A（Pdgfra）和单细胞衍生细胞标记群的分化68（Cd68）完全表现在从心房和心室分离出来的非肌细胞培养物中（图3D+F）。据估计，非肌细胞危害约65%的所有心脏细胞，其中大部分来自成纤维细胞或单细胞衍生的血统18，19，23，24。因此，这两个血统的标记被选为具有代表性的，因为这些细胞群对研究各种模型和心脏病理学的词源感兴趣。
AMAM 和 AMM 的免疫素用于 t-tubule 标记二氢丙烯（DHPR，这是一种电压依赖型 （L） 型钙通道）以及 Ryanodine 受体 （RYR2）， 在整个隔离和长期培养过程中展示了完整的 t 型管（图 4A，B）。DHPR的丰富性和局部性是心室和心室肌细胞所特有的，表明t-tubules的存在。此外，DHPR与RYR2免疫污染的同位化是完整直径结构的指标。心室和心室心肌细胞中肉瘤蛋白α-actinin的免疫素的免疫素导致预期的肉瘤斯特里特模式。肉体条纹模式用于评估孤立的心脏肌细胞的纯度和可行性与棒状形态形状和核染色与 TOPRO-3 （图 4C，D;紫色和红色）。不出所料，心室心脏肌细胞很大，平均长度为150毫米，心室心肌细胞平均长度为75毫米。此外，在免疫污染分析中，心室心脏肌细胞（但不是心室心肌细胞）表现出心室尿毒症肽（ANP）的强健表达，其染色模式是内质性晶体和分泌颗粒的本地化特征（图4C，D:绿色）。
心室心脏肌细胞特有的一个特点是，除了收缩细胞之外，它还被归类为内分泌细胞。当心外肌细胞在基底条件下分泌ANP时，分泌物会随着分泌物（即α-肾上腺炎激动剂、苯肾上腺素[PE]）而增加。此外，心室心脏肌细胞分泌ANP和共同分泌处理激素的一部分从其前体状态（亲ANP，15kD）到产品肽（ANP 3kD）16，17。这种分泌能力可以通过免疫细胞检测ANP在分离心室心脏肌细胞的介质中，以回应急性PE治疗（图4E）量化。这种心室心脏肌细胞的分泌和处理能力被发现对培养条件很敏感。因此，必须辅以二甲基肌细胞电镀介质，辅以德沙美松、胰岛素、转移素和氦。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61224/61224fig1.jpg"> 图1:逆行心脏输液、消化和细胞分离的示意图概述。 显示的是细胞与心房和心室同时从单个鼠心脏分离的主要步骤。（A） 单只老鼠的心脏通过上升的主动脉迅速被凝结，并以逆行的方式注入。（B） 心脏被分离成心室和心室组织，以进一步消化和物理分离。（C） 充分消化后，透过重力过滤将细胞分离成四个细胞部分，供日后实验使用。 <a href="https://www.jove.com/files/ftp_upload/61224/61224fig1large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61224/61224fig2.jpg"> 图2:对培养中分离的心室和心室心肌细胞和非肌细胞的形态学分析。（A） 孤立的成人小鼠心房肌细胞 （AMAM）， （B） 成人小鼠心房非肌细胞 （AMANM）， （C） 成人小鼠心室肌细胞 （AMVM）， 或 （D ）） 成人小鼠心室非肌细胞 （AMVNM） 在四室（1.7 厘米2）玻璃滑梯上镀在 5 x 105细胞/室中。相位图像是在文化中使用10倍目标在表观荧光显微镜下获得的。<a href="https://www.jove.com/files/ftp_upload/61224/61224fig2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61224/61224fig3.jpg"> 图3:对孤立细胞培养物的代表性qRT-PCR分析。 RNA是从新分离的心脏肌细胞和非肌细胞中提取的，细胞特异性基因标记的mRNA水平由qRT-PCR4确定。（A） Tnnt2， 心脏肌肉肌肽 T （心脏肌细胞标记）:（B） Nppa， 体外纳审肽 （脑膜肌细胞标记）:（C） Myl2， 肌素光链 2 （心室肌细胞标记）:（D） Tcf21， 转录因子 21 （成纤维细胞标记）:（E） Pdgfra， 血小板衍生生长因子受体 A （纤维细胞标记）:（F） Cd68，分化组68（单细胞衍生细胞标记）。数据表示平均± SEM（*p ≤ 0.05 不同于所有其他值，由 ANOVA 确定，随后由纽曼·库尔的事后分析确定）。 <a href="https://www.jove.com/files/ftp_upload/61224/61224fig3large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61224/61224fig4.jpg"> 图4:分离心室和心室心肌的代表性形态和功能分析。（A） A . AMAM 或 （B） AMVM 在四室（1.7 厘米 2 ） 玻璃滑梯上镀上 5 x 105个细胞/腔室 1 小时，以便粘附。随后，要么重新喂养心室肌细胞电镀介质，要么改为心室肌细胞维持介质，再补充布比斯塔汀16小时。文化随后被修复，然后免疫染色为RYR2（紫色），DHPR（绿色）和核污渍托普罗-3（红色）。（C） AMAM 或 （D） AMVM 被隔离和镀，然后免疫染色为阿替宁（紫色）、ANP（绿色）和 TOPRO-3（红色）。显示的是每个单元格类型的两个具有代表性的图像。（E） AMAM 在 12 口井培养皿上镀在 5 x10 5细胞/井上，在心室肌细胞电镀介质中镀 16 小时。随后，在收集媒体并接受ANP免疫细胞分析之前，AMAM用车辆或ANP分泌物（苯肾上腺素，50mM）治疗0.5小时。在免疫细胞分析之前，介质样本在500 x g下离心5分钟，以清除细胞碎片，并确保观测到的ANP是AMAM活性分泌的结果。<a href="https://www.jove.com/files/ftp_upload/61224/61224fig4large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

Watch this Scientific Journal Video about Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart at JoVE.com

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

一种方法被描述为同时从单个成年小鼠心脏的腹膜和心室中分离出肌细胞和非肌细胞。此协议可产生高度可行的心肌细胞和非肌细胞的一致产量，并详细说明表型和体外分析的最佳细胞特定培养条件。

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. ...

simultaneous-isolation-culture-atrial-myocytes-ventricular-myocytes

Research

JoVE Journal

Biology

7.2K 次观看.  San Diego State University. 一种方法被描述为同时从单个成年小鼠心脏的腹膜和心室中分离出肌细胞和非肌细胞。此协议可产生高度可行的心肌细胞和非肌细胞的一致产量，并详细说明表型和体外分析的最佳细胞特定培养条件。

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

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

科研

生物学

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

分离培养和成年小鼠心肌细胞转导

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

从成年大鼠心脏同时分离高质量心肌细胞，内皮细胞和成纤维细胞

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...