Name: isolation and cryopreservation of neonatal rat cardiomyocytes
Uploaded: 2015-04-09T14:00:00.000+00:00
Duration: 7 min 5 s
Description: Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor&#8217;s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

下面的协议是专为心肌细胞的新生幼鼠（10-14幼仔）一窝隔离。如果产仔显著不同，该过程可能需要进行调整以补偿。小狗应该在48±6小时岁。所有的程序在此已批准北卡罗莱纳州立大学的机构动物护理和使用委员会（IACUC）。 1.细胞分离制备注:隔离之前执行以下步骤的一天。 <ol><li>高温高压灭菌以下:大剪刀，小剪刀，小尖镊子，镊子大。使用重力周期为60分钟，在最小的121℃，30分钟的干燥时间。 </li><li>将加入40ml 0.1％胰蛋​​白酶溶液在冰箱中解冻O / N。冷藏汉克的平衡盐溶液（HBSS）如果尚未感冒。制备一个20％的胎牛血清（FBS）的股票介质溶液。 <ol><li> 400毫升的短路电流OVE氏改良的Dulbecco氏培养基（IMDM）加入100 ml的FBS，加入5ml L-谷氨酰胺（200毫米），2.5 ml庆大霉素（10毫克毫升-1），和0.9毫升2-巯基乙醇。使用消毒真空过滤。商店媒体在4℃长达3周，这是可伸缩的较小或较大的体积。稀释20％FBS的库存媒体1:1的IMDM，以产生10％FBS的NRCM介质。在小批量只会使防止反复加热和冷却介质。 </li></ol></li></ol> 2.细胞分离第1天<ol><li>在培养罩执行以下工作，以防止样品污染。为了获得最佳的时机启动这项工作在下午，因为有一个O / N孵化。 </li><li>无菌培养罩内做好准备工作区。将板凳垫在培养罩。填充两个250毫升烧杯，用70％的乙醇，并放置手术工具在其中。将小桶冰块的引擎盖。确保它是大到足以容纳数管和150毫米的菜。紫外线消毒罩FOR 10分钟。置于冰上40毫升0.1％胰蛋​​白酶。 </li><li>检索从母亲的乳鼠幼仔。为了使动物保暖，直到使用，将它们放置在一个绝缘容器，如一个冰桶包装在板凳垫，让他们温暖。 </li><li>填写150mm的培养皿中，用25ml HBSS并放置冰上。展开2-3纱布垫和放置在工作区域。通过在脖子后面的皮肤保持小狗，用70％的乙醇，并装在罩喷洒它。注:为了更好地保持无菌，这是可以由另一个人谁再放置清洗小狗在引擎盖完成。 </li><li>通过在脖子后面的皮肤保持小狗。然后使用大型剪刀，在一个快速，强大的切割断头安乐死。吸干血的纱布。注:由于在IACUC要求差异，这可能是更合适的清洗用乙醇小狗擦拭而不是喷洒它。一定要检查与大学IACUC为自己的这一步的指引。 </li><li>继续削减声道的前侧通过胸腔共青。挤在脖子上的皮肤，帮助打开胸腔，直至心脏是可见的。用小剪刀，取出心脏和地方冰上HBSS培养皿。重复其他幼仔。 </li><li>取下样品任何非心脏组织，摇动HBSS轻轻洗心。转移的心脏组织，以含有新鲜HBSS另一个培养皿中，并进一步清洗组织。切割组织进一步直到作品是1-3毫米3，然后转移到胰蛋白酶溶液。广场上的摇杆在4℃冰箱中O / N胰蛋白酶溶液。 </li></ol>细胞分离第2天注意:当吸在下面的步骤中，使用移液管，而不是一个真空管线。组织容易移动，并且真空管线将堵塞或仍然去除组织含有细胞。最好是用吸管吸出用于代替。如果组织进入吸管，吸管背出将其删除。 <ol><li>使吨10％NRCM媒体的重稀土等分:一是用25毫升和两个15毫升。将一份25毫升管媒体在37℃水浴。添加4毫升HBSS 5个不同的15毫升锥形管，并把所有的冰。标签每次15毫升管＃1-5。 </li><li>加40毫升的HBSS到50ml锥形管中。加入10 mL HBSS的胶原酶，吸管暂停，并结合解决方案，以创造50毫升1毫克毫升-1胶原酶溶液。消毒用0.22微米真空过滤器，并储存在室温。 </li><li>检索心脏组织/胰蛋白酶管。保证胰蛋白酶清晰，组织显得蓬松。允许组织沉降到管和抽吸的角落尽可能多的液体尽可能。 </li><li>加25毫升温10％FBS的NRCM媒体到管和旋流的手。帽和旋转在37℃水浴中2分钟。吸液了。组织应该浮动在这个步骤;如果是这样，从该管的底部吸出。 </li><li>加入10 mL胶原酶。旋转水浴2分钟，或直至溶液浑浊。曲ickly吸出液体。加入10 mL新鲜胶原酶含有该组织管和旋转水浴2分钟。移液器混合，转移溶液到管＃1中，并放置在冰上。它可能无法除去所有的液体。重复步骤3.2-3.6为其他三个的15毫升锥形管中。传送所有其余的液体到管＃5。 </li><li>离心15ml试管在410 RCF 8分钟。而离心，放置15毫升10％FBS NRCM媒体管水浴。放置在50ml锥形管中的顶部40微米的细胞过滤，并用2毫升冷HBSS中湿。 </li><li>吸取上清液降速细胞。加入6毫升冷HBSS中，以每管重悬。通过过滤到50ml锥形移液器细胞悬液，以消除任何组织。冲洗各15毫升管的两侧用3ml冷HBSS中，并加入到过滤器。离心细胞，在410 RCF离心10分钟。吸上清。重悬细胞沉淀在10ml 10％FBS的NRCM媒体和传输至T-75烧瓶中。冲洗锥形用5ml媒体，并加入到FL问。 </li><li>孵育在37℃下1小时和5％的CO 2。将过去的15毫升10％FBS NRCM媒体管进入水浴。转让T-75到一个新的T-175瓶内容物，冲洗T-75用15毫升的媒体，并加入到T-175。孵育1小时。 </li></ol> 4.冷冻保存<ol><li>广场上冰冻结介质。从T-175到50毫升锥形管转移细胞媒体，并收集10微升细胞悬液用台盼蓝细胞计数。离心在410 RCF 5分钟。而离心，计数使用细胞计数器细胞。 </li><li>吸文化传媒，并在每毫升2-4万个细胞浓度冷冻介质悬浮细胞。将1毫升细胞悬液在低温小瓶，然后把试管放入一个控制冷却速度冷冻集装箱，并将其放置在-80℃冷冻O / N。在1-2天内，在液氮中传送小瓶。 </li></ol> 5.细胞解冻<ol><li>大衣盘中纤维连接蛋白溶解250微升纤维连接蛋白（股票浓度为1毫克毫升-1）到9.75毫升水（终浓度为0.025毫克毫升-1）的; 60。加入足够的溶液以覆盖培养板，孵育在37℃下至少30分钟。 <ol><li>四的15毫升锥形管，使DMSO /媒体混合如下加入DMSO至20％媒体。添加（5％）9.5毫升介质用500μlDMSO中。加（2.5％）9.75毫升介质用250μlDMSO中。加入10 mL媒体（你两个人这些）。 </li></ol></li><li>加热在37℃水浴中上述介质的混合物。从液氮中取出细胞，并放在干冰。当媒体的混合物是温暖的，解冻的细胞在37℃水浴，直到刚刚解冻。不要让细胞在水浴中太久，因为这可能会损坏电池。 </li><li>传送低温小瓶的内容对5％的DMSO /介质管。离心在410 RCF 8分钟。吸上清留下细胞沉淀。加2.5％的DMSO /介质管内容物至细胞沉淀且resuspend。离心在410 RCF 8分钟。吸上清留下细胞沉淀。 </li><li>添加一个0％DMSO /媒体管内容细胞沉淀重悬。离心在410 RCF 8分钟。吸上清留下细胞沉淀。 </li><li>添加第二个0％DMSO /媒体的内容对细胞沉淀重悬。就拿细胞计数小样本。离心在410 RCF 8分钟。而离心细胞计数在血球使用台盼蓝标记死细胞。记录细胞量，以及生存力。 </li><li>吸上清留下细胞沉淀。悬浮在10％NRCM媒体（可选:加100μM溴脱氧尿苷（尿嘧啶））和板细胞纤维连接蛋白涂层的菜肴。 </li></ol> 6.细胞培养<ol><li>板细胞在每平方厘米10万左右的细胞。调整的基础上的预定用途培养物的接种密度。通过在1:10的比例稀释的20％中的介质的IMDM制备2％NRCM介质。下面是一个典型的培养物的轮廓。&lt;/ li&gt; <li>第1天，冲洗死细胞客场以热烈的IMDM，加入10％NRCM（可选100μM的BrdU）。第2天，冲洗死细胞客场以热烈的IMDM，加入10％NRCM。第3天，加2％NRCM。第4天加2％NRCM。在第5天保证在细胞融合和跳动。保持培养中的细胞对周围一周。在这一点上的成纤维细胞可能开始显著长大了新农合。 </li></ol>

<ol>
	<li>Louch, W. E., Sheehan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+cardiomyocyte+isolation,+culture,+and+gene+transfer.">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> Journal of Molecular and Cellular Cardiology. 51 (3), 288-298 (2011).</li><li>Miragoli, M., Salvarani, N., Rohr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblasts+Induce+Ectopic+Activity+in+Cardiac+Tissue.">Myofibroblasts Induce Ectopic Activity in Cardiac Tissue.</a> Circulation Research. 101, 755-758 (2007).</li><li>Simpson, P., Savion, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+rat+myocytes+in+single+cell+cultures+with+and+without+proliferating+nonmyocardial+cells.+Cross-striations,+ultrastructure,+and+chronotropic+response+to+isoproterenol.">Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure, and chronotropic response to isoproterenol.</a> Circulation Research. 50 (1), 101-116 (1982).</li><li>Simpson, P., McGrath, a., Savion, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocyte+hypertrophy+in+neonatal+rat+heart+cultures+and+its+regulation+by+serum+and+by+catecholamines.">Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines.</a> Circulation Research. 51 (6), 787-801 (1982).</li><li>Rohr, S., Fl&#252;ckiger-Labrada, R., Kucera, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolithographically+defined+deposition+of+attachment+factors+as+a+versatile+method+for+patterning+the+growth+of+different+cell+types+in+culture.">Photolithographically defined deposition of attachment factors as a versatile method for patterning the growth of different cell types in culture.</a> Pfl&#252;gers Archive : European Journal of Physiology. 446 (1), 125-132 (2003).</li><li>Zhang, B., Green, J. V., Murthy, S. K., Radisic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+enrichment+of+functional+cardiomyocytes+using+microfluidic+deterministic+lateral+flow+displacement.">Label-free enrichment of functional cardiomyocytes using microfluidic deterministic lateral flow displacement.</a> PloS One. 7 (5), e37619 (2012).</li><li>Boateng, S. Y., Hartman, T. J., Ahluwalia, N., Vidula, H., Desai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+fibroblast+proliferation+in+cardiac+myocyte+cultures+by+surface+microtopography.">Inhibition of fibroblast proliferation in cardiac myocyte cultures by surface microtopography.</a> American Journal of Physiology. Cell Physiology. 285 (1), C171-C182 (2003).</li><li>Fu, J. -. D., Li, 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=Crucial+role+of+the+sarcoplasmic+reticulum+in+the+developmental+regulation+of+Ca2++transients+and+contraction+in+cardiomyocytes+derived+from+embryonic+stem+cells.">Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca2+ transients and contraction in cardiomyocytes derived from embryonic stem cells.</a> FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology. 20 (1), 181-183 (2006).</li><li>Liu, J., Fu, J. D., Siu, C. W., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Functional+sarcoplasmic+reticulum+for+calcium+handling+of+human+embryonic+stem+cell-derived+cardiomyocytes:+insights+for+driven+maturation.">a Functional sarcoplasmic reticulum for calcium handling of human embryonic stem cell-derived cardiomyocytes: insights for driven maturation.</a> Stem Cells. 25 (12), 3038-3044 (2007).</li><li>Knollmann, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell-derived+cardiomyocytes:+boutique+science+or+valuable+arrhythmia+model.">Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model.</a> Circulation Research. 112 (6), 969-976 (2013).</li><li>Uchida, T., Nagayama, M., Taira, T., Shimizu, K., Sakai, M., Gohara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+temperature+range+for+low-temperature+preservation+of+dissociated+neonatal+rat+cardiomyocytes.">Optimal temperature range for low-temperature preservation of dissociated neonatal rat cardiomyocytes.</a> Cryobiology. 63 (3), 279-284 (2011).</li><li>Miyamura, K., Nagayama, 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=Evaluation+of+viability+of+cryopreserved+rat+cardiac+myocytes+and+effects+of+dimethyl+sulfoxide+concentration+on+cryopreservation.">Evaluation of viability of cryopreserved rat cardiac myocytes and effects of dimethyl sulfoxide concentration on cryopreservation.</a> Cryobiology. 59 (3), 375 (2010).</li><li>Rana, P., Anson, B., Engle, S., Will, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human-induced+pluripotent+stem+cell-derived+cardiomyocytes:+bioenergetics+and+utilization+in+safety+screening.">Characterization of human-induced pluripotent stem cell-derived cardiomyocytes: bioenergetics and utilization in safety screening.</a> Toxicological Sciences: An Official Journal of the Society of Toxicology. 130 (1), 117-131 (2012).</li><li>Yokomuro, H., Mickle, D. a. G., Weisel, R. D., Li, R. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+conditions+for+heart+cell+cryopreservation+for+transplantation.">Optimal conditions for heart cell cryopreservation for transplantation.</a> Molecular and Cellular Biochemistry. 242 (1-2), 109-114 (2003).</li><li>Simpson, P., McGrath, a., Savion, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocyte+hypertrophy+in+neonatal+rat+heart+cultures+and+its+regulation+by+serum+and+by+catecholamines.">Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines.</a> Circulation Research. 51 (6), 787-801 (1982).</li><li>Evans, H. J., Western Goodwin, 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=array+analysis+of+cell+cycle+protein+changes+during+the+hyperplastic+to+hypertrophic+transition+in+heart+development.">array analysis of cell cycle protein changes during the hyperplastic to hypertrophic transition in heart development.</a> Molecular and Cellular Biochemistry. 303 (1-2), 189-199 (2007).</li><li>Golden, H. B., Gollapudi, D., 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=Methods+in+Molecular+Biology.">Methods in Molecular Biology.</a> , 205-214 (2012).</li><li>Zlochiver, S., Mu&#241;oz, V., Vikstrom, K. L., Taffet, S. M., Berenfeld, O., Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotonic+myofibroblast-to-myocyte+coupling+increases+propensity+to+reentrant+arrhythmias+in+two-dimensional+cardiac+monolayers.">Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers.</a> Biophysical Journal. 95 (9), 4469-4480 (2008).</li><li>Chang, M. G., Zhang, Y., 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=Spiral+waves+and+reentry+dynamics+in+an+in+vitro+model+of+the+healed+infarct+border+zone.">Spiral waves and reentry dynamics in an in vitro model of the healed infarct border zone.</a> Circulation Research. 105 (11), 1062-1071 (2009).</li></ol>

The authors have nothing to disclose.

该协议允许新农合被孤立，冷冻，解冻。解冻的细胞是该过程的一个重要部分。一系列的DMSO稀释液被用来慢慢地从单元14除去DMSO中。重要的是DMSO中提取被快速地执​​行作为所述细胞是特别敏感解冻后立即死亡。如果需要更多或更少的细胞解冻，根据需要的DMSO溶液的体积可以按比例。 一个挑战NRCM隔离和文化是成纤维细胞的快速增殖。该协议使用预镀已显示减少的?...

心肌细胞培养是现代心脏研究的一个重要工具。乳鼠心肌细胞（新农合），因为隔离常用和文化是比成年大鼠心肌1更容易。该NRCM方法仍然有一些限制，包括长期分离过程和有限的细胞增殖的菜。有对新农合的大多数一般需要4-48小时工作2-6的隔离无数协议。此外，将细胞分离经常由1至2天龄的幼鼠2,4-7;诞生时间是不可预知和冲突，在实验室的其他工作。如果需要对实验只有少量细胞的隔离可能是低效和浪费的。最努力改善工作流程专注于减少隔离时间，但是这并没有解决定时幼崽诞生的问题。 作为替代，许多实验室利用从胚胎干细胞（ESC）或诱导的多能干细胞（iPS细胞）衍生的心肌细胞。然而，重新编程和/或分化的过程可能是非常耗时和昂贵的为好。使用这些细胞作为体外肌细胞模型时，可以有其他的问题。既ESC-和iPSC-衍生的心肌细胞已显示出表现从初级心肌8-10在电生理学差异。 解离合管能够被存储为使用冷冻11几天，然而这并允许长期存储。液氮通常用于保存细胞的时间较长期间，但是需要冷冻保护剂，如二甲基亚砜（DMSO）。此前有研究表明，在冻结媒体5-10％DMSO的理想浓度允许新农合的冷冻保存，但即使是这样的生存能力仍然很低12。虽然DMSO有助于释放过程中保护细胞诚，它可以是对细胞有毒的浓度高于1.5％的13。以前的研究已经表明，慢慢地从细胞中除去DMSO，可提高细胞活力14。 我们试图提高NRCM基于细胞的测定通过冷冻保存的细胞下述分离的效率。这允许了细胞被解冻，并且在必要时，减小隔离和动物消费的频率使用。使用这种方法，我们表明，它是可能的冷冻保存新农合和解冻它们用于在以后的时间。解冻后细胞维持可接受的可行性，并产生NRCM文化是积极的α-横纹肌辅肌动蛋白（α-SA）和合同油然而生。

<table><tbody><tr><td>IMDM (+25 mM HEPES +L-glutamine)</td><td>Gibco</td><td>12440-046</td><td>With added 25 mM HEPES and L-glutamine</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-064</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985-023</td><td></td></tr><tr><td>HBSS (+Ca +Mg)</td><td>Corning</td><td>21-020-CV</td><td>With added calcium and magnesium, pH 7.1-7.4</td></tr><tr><td>Trypsin 0.25%</td><td>Gibco</td><td>25300-056</td><td></td></tr><tr><td>Trypsin 0.05%</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Cryostor CS5</td><td>BioLife Solutions</td><td>205102</td><td>Freezing media</td></tr><tr><td>Cryogenic Vial</td><td>Corning</td><td>430659</td><td></td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C1889-50MG</td><td></td></tr><tr><td>40 &amp;mu;m Cell Strainer</td><td>Greiner Bio-One</td><td>542040</td><td></td></tr><tr><td>Sterilizing Vacuum Filter (0.22&amp;mu;m)</td><td>Corning</td><td>431118</td><td></td></tr><tr><td>50 ml Conical</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>15 ml Conical</td><td>Corning</td><td>430790</td><td></td></tr><tr><td>trypan blue</td><td>Cellgro</td><td>25-900-CI</td><td></td></tr><tr><td>Mr Frosty Freezing Container</td><td>ThermoScientific</td><td>5100-0001</td><td></td></tr><tr><td>Millicell EZ&amp;nbsp;SLIDES</td><td>Millipore</td><td>PEZGS0416</td><td></td></tr><tr><td>&amp;alpha;-sarcomeric actinin antibody</td><td>Sigma</td><td>A7811</td><td></td></tr><tr><td>Fibronectin</td><td>Corning</td><td>356008</td><td></td></tr><tr><td>Bromodeoxyuridine</td><td>BD Biosciences</td><td>51-7581KZ</td><td></td></tr></tbody></table>

isolation and cryopreservation of neonatal rat cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

以下的新生大鼠心肌细胞的分离，将细胞冷冻向下液氮，并且可以贮存至少几个月。通常在解冻时，用台盼蓝分析测定的生存能力将介于40-60％。虽然这是比其他类型的细胞时，用适当的接种和培养的细胞将增殖（ 图1）。为了验证收缩，细胞可以用相差显微镜进行成像。细胞应开始于约三天（ 图2）自发收缩。对于免疫细胞化学（ICC）分析，将细胞接种在4孔培养载玻片?...

Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

隔离和乳鼠心肌细胞冷冻保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

isolation-and-cryopreservation-of-neonatal-rat-cardiomyocytes

Research

JoVE Journal

Developmental Biology

12.5K 次观看.  College of Veterinary Medicine, North Carolina State University. The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

视频: Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

科研

发育生物学

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

神经科学

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 ...

生物学

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

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

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

代谢综合征心力衰竭模型大鼠心房心肌细胞的分离与保留射血分数的关系

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

一种减轻组织损伤的新型微创慢注射方法

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

作者聚焦:一种侵入性最小的新型细胞注射方法  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

一种侵袭性最小的新型细胞注射方法

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

生物工程

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

原代大鼠乳鼠心肌细胞的活细胞成像继腺病毒和慢病毒转导利用共聚焦旋转盘显微镜

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

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 ...

隔离和乳鼠心肌细胞冷冻保存

摘要

探索更多视频

此视频中的章节

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

成年大鼠心肌细胞的分离与培养

分离新生小鼠心肌文化和

代谢综合征心力衰竭模型大鼠心房心肌细胞的分离与保留射血分数的关系

一种侵袭性最小的新型细胞注射方法

一种侵袭性最小的新型细胞注射方法

一种侵袭性最小的新型细胞注射方法

原代大鼠乳鼠心肌细胞的活细胞成像继腺病毒和慢病毒转导利用共聚焦旋转盘显微镜

一种持续分离小鼠心肌细胞的简单有效的方法