这项工作是由国家心脏，肺和血液研究所资助的HL-36573的支持。我们感谢大卫·索瓦先生编辑这篇稿子。

对小鼠的研究都按照程序，美国国立卫生研究院的指导方针进行，而协议已获托莱多大学的机构动物护理和使用委员会，医学与生命科学学院。 1，灌注系统准备<ol><li>隔离的前一天，填充整个灌注系统（包括水库）用快速碱性无残留洗涤剂的溶液。让溶液浸泡到系统中的几个小时或过夜。 </li><li>第二天早上，调节热循环温度至37℃，并让溶液升温。除去洗涤剂溶液并彻底用灭菌蒸馏水冲洗系统。不要忘了所有的偏科。 </li><li>通过恒蠕动泵填补了系统灌注缓冲液，绘制气泡从主动脉腔（气泡陷阱）与侧置式注射器保护在心脏冠状动脉闭塞。蠕动泵的流量应定期检查。 </li></ol> 2，缓冲溶液之制备，文化传媒，和菜<ol><li>灌注缓冲液:请在使用前500毫升的1X灌注缓冲。此无钙缓冲液含有113 mM氯化钠，4.7 mM的氯化钾，0.6mM的KH 2 PO 4，0.6毫米的Na 2 HPO 4，1.2 mM的MgSO 4干燥，10毫钠HEPES，12mM的碳酸氢钠 ，10mM的KHCO 3，0.032毫酚红，30 mM的牛磺酸，10毫米的BDM，和5.5毫米的葡萄糖。将pH调节至7.0，用2M HCl和通过0.2微米的过滤器，过滤存储在4℃下 注意:a）本灌注溶液应准备在同一天使用时。在某些情况下，它是可以接受的，以存储少于3天的溶液在4℃下B）这个缓冲区具有较高的钾浓度达15.3毫米，这可以保持心肌细胞静止期，减少ATP的Ç【消耗和增加的Ca 2 +的容错能力。 </li><li>消化缓冲液（300单位/毫升，25〜50毫升/心脏）:准备之前，为了灌注。称取II型胶原酶的总活性为15,000 U，溶解在培养罩50毫升灌注缓冲。加入25微升100毫米氯化钙2（原液，过滤消毒）做最后的钙浓度为50微米。暖缓冲到37℃时准备好隔离。不像胰蛋白酶，胶原酶工作在50-100微米的Ca 2 +的存在下最好。 </li><li>终止缓冲液（50毫升/心脏）:操作中培养罩。混合45毫升的灌注缓冲液用5ml无菌胎牛血清。 <ol><li> Ca 2 +的溶液I（12.5微米的Ca 2 +）:加入2.5微升100mM氯化钙2〜20的停止缓冲液，混匀。 </li><li> Ca 2 +的溶液II（100μM的Ca 2 +）:添加10微升100mM的CaCl 2至10ml的终止缓冲液，混匀。 </li><li> Ca 2 +的溶液III（400μMCa 2 +的）:加入40μl100毫米氯化钙2〜10毫升挡车器，混匀。 </li><li> Ca 2 +的溶液IV（900μM的Ca 2 +）:添加90微升100mM的CaCl 2至10ml的终止缓冲液，混匀。 </li></ol></li><li>电镀液（50毫升/心脏）:传输43毫升MEM中含有2mM L-谷氨酰胺和1.26毫米氯化钙2到无菌的50毫升锥形管中培养罩和加入5 ml的FBS，1毫升的BDM原液（ 500 mM时，过滤器灭菌）和0.5 ml青霉素/链霉素（10,000 U /毫升）。混合和2％的CO 2，37℃培养箱松瓶盖平衡2-3小时前，心肌细胞隔离。右肌细胞电镀之前，加入0.5毫升200毫摩尔ATP（pH值7.2，料液，过滤灭菌）到培养基中。 </li><li>培养基（50毫升/心脏）:传输48毫升含有2mM L-谷氨酰胺和1.26毫米氯化钙2到无菌的50毫升锥形管中培养罩MEM中并加入1毫升BDM储备溶液（500 mM时，过滤接ř消毒），和0.5 ml青霉素/链霉素（10,000单位/毫升）。混合和平衡在2％的CO 2，37℃培养箱前肌细胞隔离。在使用之前，加入0.5毫升100毫克/毫升BSA（过滤灭菌的）到培养基中。 注意 :使用2％CO 2培养箱中有利于维持培养基的pH在6.9-7.0，这有助于myoctyes保持它们的棒状形态长达24小时。 </li><li>层粘连蛋白（1-2微克/厘米2） -包被的培养皿或板:传输200微升的1毫克/毫升的天然小鼠层粘连蛋白20的灭菌PBS（W / O型钙和镁）的溶液并混合。加入1 ml至35毫米的菜肴，2.5毫升到60毫米的菜肴，或5毫升到100毫米的菜，摇到均匀覆盖面，并将其放置在2％的CO 2，37℃培养箱中培养，直到肌细胞电镀。 注意:如果菜肴或板不是在天涂覆使用，它们可以被用石蜡膜密封并储存于4℃过夜（慢涂层）。密封是热曲IRED以防止蒸发和污染。涂层板可保持在4℃下长达1周。 </li></ol> 3，小鼠心脏拆卸和插管<ol><li>浸泡剪刀和镊子在70％乙醇浸泡30分钟，让他们干。 </li><li>权衡鼠标到0.1克。记录其体重，品系，性别和出生日期。 </li><li>注入200μL肝素（100 IU /鼠标）IP 10分钟前麻醉，防止血液凝固的冠状动脉。麻醉小鼠200毫克氯胺酮/ kg体重和10毫克/公斤体重甲苯噻嗪腹腔注射，等待5-10分钟，直至鼠标停止响应的尾巴/脚趾捏。 </li><li>轻轻固定的前爪和后爪的pinnable工作表面（ 即发泡胶）在动物手术托盘或桌子板凳附近灌注系统确保鼠标在仰卧位。 </li><li>擦拭胸部和腹部，用70％的乙醇。做中线的皮肤我ncision从中间腹部有手术剪刀隔膜。 </li><li>切割膜片与另一手术剪刀按住胸骨弧形锯齿钳。削减双边和retroflect胸廓暴露心脏。 </li><li>提起心脏稍微用弯曲细小锯齿和无创伤钳和解剖的心脏出了胸腔尽可能靠近背侧胸壁。 </li><li>在心脏转移至有冷灌注缓冲液100毫米的菜。精心修剪掉的结缔组织，如肺，胸腺和支气管。 </li><li>识别主动脉及其分支颅，通常隐藏在胸腺和脂肪垫。切开主动脉低于其首支13，抓住主动脉壁，解除心脏，稍微滑了到灌注液填充主动脉插管（1.0毫米外径不锈钢套管用钝/平头和凹口1到2毫米以上使用两个超精细尖F或a 22政可重复使用的喂食针）;尖orceps。 注意:a）本灌注流体应当心脏插管过程中滴落，以防止气泡进入冠状动脉; b）不断插管的尖端正上方的主动脉瓣。 </li><li>夹住主动脉插管并结扎主动脉插管用6-0手术丝。整个插管时间不超过120秒。 </li></ol> 4，心脏灌注和消化<ol><li>用无钙灌注缓冲液灌注心脏在4毫升的流速/分钟约4-5分钟，直到流出物变得清晰，然后切换到含有50微米氯化钙消化缓冲液和灌注的3.5-20分钟根据小鼠品系，灌注压和胶原酶活性。如果适用，当消化增加的主动脉压力70-80毫米汞柱。必要时，酶灌流液可以循环进行消化。通常情况下，300 U / ml的酶缓冲液将在4毫升/分钟的流量在10〜12分钟消化心脏;如果施加负荷压力在70毫米汞柱，300 U / ml的将仅3.5-5.5分钟以4毫升/分钟的流速。 </li><li>终止消化时，心脏变得略显苍白和松弛。它应该是海绵状时，轻轻挤压。 </li></ol> 5，细胞分离和重新引入钙<ol><li>从插管用70％乙醇浸泡的钳子拉主动脉，并把心脏成含有2.5毫升消化缓冲液中的无菌的60毫米培养皿中。搬入培养罩采用无菌物品供应，保持留意无菌技术，这和后续步骤。 </li><li>移除主动脉，心房和大血管细手术剪。轻轻挑逗心室成10-12小块，用两个细尖镊子。 </li><li>轻轻吸取的心脏碎片和细胞上下〜10倍与10毫升的移液管，然后转移到一个15毫升的聚丙烯锥形管。涮菜用口含12.5微米氯化钙和转让7.5毫升钙溶液这个进入管，从而导致10毫升的最终体积。 </li><li>轻轻吸取细胞悬液上下用细尖移液管驱散大块的心脏组织。 </li><li>然后离心3分钟，以20×g离心以分离出小的非心肌细胞的细胞，如内皮细胞和成纤维细胞。 </li><li>吸上清，重悬肌细胞沉淀在10毫升钙的解决方案，我用100微升200毫摩尔ATP（pH值7.2，储备液，过滤除菌）的补充。离开该管在通风橱中3-5分钟。 </li><li>传输重复10微升等分至血细胞计数器计数杆状和圆细胞。计算细胞总数，并计算棒状细胞的百分比。 </li><li>离心细胞为3分钟，在20×g下。除去上清，悬浮颗粒在10毫升钙溶液II含有100微米氯化钙 。吹打分散的细尖移液管的细胞向上和向下。 </li><li>分别用钙溶液III（400微米氯化钙 ）和IV（900微米氯化钙 ），重复上述步骤（5.8）。 </li><li>对于小鼠心肌细胞原代培养，悬浮最终心肌细胞沉淀在5毫升电镀液中。计数细胞总数和计算的杆状细​​胞的百分比。 </li></ol> 6。细胞培养<ol><li>调整的杆状细胞浓度至25000杆状细胞/ 50-ml管毫升。使用10毫升吸管暂停他们也轻轻吸取的细胞。避免在移液管和管细胞沉淀，以确保平等的细胞密度为每道菜或板。 </li><li>吸了层粘连蛋白涂层溶液后，将镀成肌细胞的菜肴或板。轻轻滑动培养罩的表面上的碗碟或板向前和向后和侧到侧的十字图案的三到四倍。 </li><li>放置碗碟或板在2％二氧化碳2培养箱中在37℃下孵育1-3小时，以允许肌细胞附着在约80％的速度。 </li><li>附件后，轻轻吸了含有独立的细胞和细胞碎片的媒介。轻轻培养基加到菜肴或板的两侧。由一个执行这种媒介的变化之一。立即返回菜肴或板的孵化器。 </li><li> O / N培养后，细胞可改为短期细胞信号转导的研究之前，在相同的培养基无BDM。保持在BDM长期培养的肥大或凋亡检测。 注 :BDM（2,3 -丁二酮肟）能抑制肌球蛋白ATP酶活性，防止收缩。其抑制肌球蛋白为基础的收缩是可逆的。据报道，BDM可能有一些潜在的副作用，如作为一种非特异性磷酸酶，从SR增加钙释放，并改变游离钙离子浓度和蜂窝电PROPE的rties，所以治疗前，BDM应该被删除。此外，在某些情况下，一个特定的肌球蛋白II抑制剂II型肌球蛋白可以是一种替代药物，抑制心肌收缩8,9。然而，在我们的情况下，BDM比II型肌球蛋白更好地为求心肌细胞的质量和数量。 </li></ol>

<ol><li>Louch, W. E., Sheehan, K. A., Wolska, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+in+cardiomyocyte+isolation%2C+culture%2C+and+gene+transfer%5BTitle%5D)+AND+%22J.+Mol.+Cell%22%5BJournal%5D)">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> J. Mol. Cell. 51, 288-298 (2011).</li>
<li>Gross, D. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGross%2C+DR+%22Isolated+heart+preparations%2C+problems%2C+and+pitfalls.+In%3A+Animal+Models+in+Cardiovascular+Research%22">Isolated heart preparations, problems, and pitfalls. In: Animal Models in Cardiovascular Research</a>. Gross, D. R. , 3rd edition, Springer. 109-130 (2009).</li>
<li>Schlüter, K. D., Piper, H. M. Isolation and culture of adult ventricular cardiomyocytes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSchl%C3%BCter%2C+KD+%22Practical+Methods+in+Cardiovascular+Research%22">Practical Methods in Cardiovascular Research</a>. Dheim, S., Mohr, F. W., Delmar, M. , Springer. 557-567 (2005).</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/pubmed?term=((Isolation+and+culture+of+adult+cardiac+myocytes+for+signaling+studies%5BTitle%5D)+AND+%22AfCS+research+reports%22%5BJournal%5D)">Isolation and culture of adult cardiac myocytes for signaling studies.</a> AfCS research reports. 1 (5), 1-9 (2003).</li>
<li>O'Connell, T. D., Rodrigo, M. C., Simpon, P. C. Isolation and culture of adult mouse cardiac myocytes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aO%27Connell%2C+TD+%22Methods+in+Molecular+Biology%22">Methods in Molecular Biology</a>. Vivanco, F. 35, Humana Press. 271-296 (2007).</li>
<li>Nilolskaya, A., Sharma, V. Cell culture models and methods. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNilolskaya%2C+A+%22Cardiac+Electrophysiology+Methods+and+Models%22">Cardiac Electrophysiology Methods and Models</a>. Sigg, D. C., Laizzo, P. S., Xiao, Y. F. , Springer. 213-235 (2010).</li>
<li>Merx, M. W., Schrader, M. The working heart. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMerx%2C+MW+%22Practical+Methods+in+Cardiovascular+Research%22">Practical Methods in Cardiovascular Research</a>. Dhein, S., Mohr, F. W., Delmar, M. , Springer-Verlag. Berlin Heidelberg, Germany. 173-189 (2005).</li>
<li>Kabaeva, Z., Zhao, M., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blebbistatin+extends+culture+life+of+adult+mouse+cardiac+myocytes+and+allows+efficient+and+stable+transgene+expression%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 1667-1674 (2008).</li>
<li>Lou, Q., Li, W., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+dynamic+instability+and+wavelength+in+arrhythmia+maintenance+as+revealed+by+panoramic+imaging+with+blebbistatin+vs.+2%2C3-butanedione+monoxime%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">The role of dynamic instability and wavelength in arrhythmia maintenance as revealed by panoramic imaging with blebbistatin vs. 2,3-butanedione monoxime.</a> Am. J. Physiol. Heart Circ. Physiol. 302, 262-269 (2012).</li>
<li>Liu, C. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduction+of+Na%2FK-ATPase+potentiates+Marinobufagenin-induced+cardiac+dysfunction+and+myocyte+apoptosis%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Reduction of Na/K-ATPase potentiates Marinobufagenin-induced cardiac dysfunction and myocyte apoptosis.</a> J. Biol. Chem. 287 (20), 16390-16398 (2012).</li>
<li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Different+roles+of+the+cardiac+Na%2B%2FCa2%2B-exchanger+in+ouabain-induced+inotropy%2C+cell+signaling%2C+and+hypertrophy%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">Different roles of the cardiac Na+/Ca2+-exchanger in ouabain-induced inotropy, cell signaling, and hypertrophy.</a> Am. J. Physiol. Heart Circ. Physiol. 304, 427-435 (2013).</li>
<li>Liu, L. J., Zhao, X. C., Pierre, S. V., Askari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+PI3K-AKT+signaling+pathway+with+digitalis-induced+hypertrophy+of+cardiac+myocytes%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Cell+Physiol%22%5BJournal%5D)">Association of PI3K-AKT signaling pathway with digitalis-induced hypertrophy of cardiac myocytes.</a> Am. J. Physiol. Cell Physiol. 293, 1489-1497 (2007).</li>
<li>Flynn, J. M., Santana, L. F., Melov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+Cell+Transcriptional+Profiling+of+Adult+Mouse+Cardiomyocytes%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes.</a> J. Vis. Exp. (58), (2011).</li>
</ol>

作者宣称，他们有没有竞争的财务权益。

为了获得最佳的准备，最关键的步骤是:1）及时小鼠心脏挂接到切除后的套管; 2）适当的心脏灌注。另外，还要注意水的质量，灌注温度，pH缓冲液，消毒，化学纯度和洁净，无污染管和室也是重要因素。 18.2MΩ分子生物学级H 2 O的强烈建议缓冲液配制。 200毫摩尔ATP储备溶液的pH值应调整到7.2，这是很容易错过的工序。 关键是要迅速查明主动脉和削减略低于公司第一分公司如由弗林13。?...

心肌细胞不增殖。也有一些心房心肌细胞系，如HL-1和AT-1细胞从小鼠心房肿瘤衍生的;然而，没有成年心室的心肌细胞可用于研究线。成年小鼠心肌细胞的原代细胞培养提供了强大的模型心脏研究在细胞和分子水平。迄今为止，它们已被广泛用于生化，生理和药理学研究1。此外，频繁使用转基因小鼠的必要了心肌细胞的分离有效的方法。纯培养允许无相互作用与其他器官的条件和全身循环，例如通过内源性神经激素和激素样因子2,3。然而，心肌细胞的成功分离是具有挑战性的。 我们这里介绍的协议是基于我们与成年大鼠cardiomyocyt经验ES和O&#39;Connell 等人 4,5中记载的方法。尤其是在2007年5，他们描述了从缓冲准备详细的技术，细胞培养和功能测定。自小鼠细胞极易受到挛缩相比，大鼠心肌细胞中，用于灌注，消化，Ca 2 +的耐受性，电镀和培养的小鼠协议的缓冲器或介质补充有非特异性兴奋-收缩偶联酶抑制剂，2， 3 - 丁二酮肟（BDM），以抑制其自发性收缩，因而生存能力和杆状细胞产量显著提高。在这里介绍的协议中，肌细胞是由改性的Langendorff灌注的II型胶原酶分离在从分离的心脏较高的钾缓冲液中。 Ⅱ型胶原酶有效地打破了细胞间基质，并释放细胞。该灌注溶液也保持细胞的新陈代谢处于较低水平6。在additi上，来自哈佛仪器，我们这里使用的离体心脏系统是精心设计的精确的温度和恒压控制7。这种方法提供了高度可重复的制剂和单一的细胞类型，这可用于在培养过夜，用于测量信号蛋白或2-3天培养心肌肥厚测定的均一群体。

<table><tbody><tr><td>Isolated heart system for small rodent</td><td>Harvard Apparatus</td><td>IHSR-mouse 73-4019</td><td></td></tr><tr><td>Aortic Cannula, OD 1.0 mm</td><td>Harvard Apparatus</td><td>73-2816</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Fine science tools</td><td>11294-00</td><td></td></tr><tr><td>Straight sharp serrated scissors</td><td>Fine science tools</td><td>14070-12</td><td></td></tr><tr><td>Serrated Graefe forceps</td><td>Fine science tools</td><td>11050-10</td><td></td></tr><tr><td>Curved, serrated Graefe forceps</td><td>Fine science tools</td><td>11052-10</td><td></td></tr><tr><td>Straight Mini Serrefines clamps</td><td>Fine science tools</td><td>18054-28</td><td></td></tr><tr><td>MEM</td><td>Gibco</td><td>11575-032</td><td></td></tr><tr><td>Collagenase II</td><td>Worthington</td><td>4176</td><td></td></tr><tr><td>Mucosal universal detergent</td><td>Sigma</td><td>Z637181</td><td>Mucasol is a fast alkaline residue-free detergent.</td></tr></tbody></table>

isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy

技术进步已经使转基因小鼠，包括转基因和基因敲除小鼠，在许多研究领域必不可少的工具。成人的心肌细胞被广泛接受为一个很好的模式心脏细胞生理学与病理生理学，以及医药干预。转基因小鼠排除需要复杂的心肌细胞的感染过程，以产生期望的基因型，这是低效的，由于心肌细胞"终末分化。隔离的高数量和质量心肌功能和文化将极大地有利于心血管疾病的研究，并为细胞信号转导研究和药物开发的重要工具。在这里，我们描述的，可以用很少的培训来实现成年小鼠心肌细胞分离一套行之有效的方法。小鼠心脏被切除，插管到离体心脏系统，然后用无钙及高磷灌注otassium缓冲其次是Ⅱ型胶原酶消化的Langendorff逆行灌注模式。这个协议产生一致的结果为功能性成年小鼠心肌细胞来自各种转基因小鼠的集合。

1，成功的定量分离两个标准用于定量隔离的成功:首先，心肌细胞的分离的总数，和第二，杆状耐钙圆非容错肌细胞的比率。通常这个协议需要大约75-90分钟从心脏切除到心肌细胞电镀和从一个成年小鼠心脏的产量大约100万杆状心肌（共收获细胞70-90％）。这可能与小鼠体重和应变而变化。通常情况下，0.7-1.0万元杆状细胞可以从〜25克C57BL/6J小鼠，从〜35克黑瑞鼠标1.2-1.6万元杆状...

Watch this Scientific Journal Video about Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy at JoVE.com

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

我们描述了成年小鼠心肌细胞分离的可靠方法。该协议得到了一致的结果为从各种转基因小鼠的功能成年心肌文化。

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

isolation-culture-adult-mouse-cardiomyocytes-for-cell-signaling-vitro

Research

JoVE Journal

Biology

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

47.7K 次观看.  University of Toledo College of Medicine and Life Sciences. 我们描述了成年小鼠心肌细胞分离的可靠方法。该协议得到了一致的结果为从各种转基因小鼠的功能成年心肌文化。 

视频: Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

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 ...

科研

生物学

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

成年大鼠心肌细胞的分离与培养

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

医学

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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 and Physiological Analysis of Mouse Cardiomyocytes

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

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

鼠心肌细胞的分离及生理分析

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

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 ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

一种持续分离小鼠心肌细胞的简单有效的方法

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

成年小鼠和大鼠心肌细胞的隔离、转染和长期培养

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

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

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

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

来自成人鼠心脏的心外肌细胞、心室肌细胞和非肌细胞的同步隔离和文化

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

隔离成年小鼠心肌细胞的细胞信号传导和文化及

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