作者感谢 Woitasky 和彼得沃尔克的技术援助。此外, 作者还感谢 Mrs. (医学作家 ACCEDIS) 在准备手稿方面的帮助。此手稿由 DFG (Schlu 324/7-1) 资助。

这项调查是根据美国国立卫生研究院 (NIH 出版物 No. 85-23, 修订 1996) 所出版的实验室动物的护理和使用指南进行的。通常, 雄性大鼠年龄在3到4月, 平均重量为 250-350 克, 用于本协议。一只老鼠的心脏是足够为20文化菜 (1 毫升每盘; 内径:35 毫米) 与近似细胞密度 1.5 x 10 4 cells/1000 mm 2 . 
 1. 培养基和试剂的制备 <ol> <li> 肌酸-肉碱-牛磺酸培养基 (cct 培养基) 注: cct 培养基是一种以培养基199为基础的复合肌酸、肉碱和牛磺酸的培养基。
	<ol> <li> 准备 1 L 中 199: 添加3.6 克 Hepes 和混合为 1 h。然后添加655.5 毫克肌酸 (5 毫米), 395.4 毫克肉碱 (2 毫米), 和625.5 毫克牛磺酸 (5 毫米)。肉碱和牛磺酸改变 pH 值和 #60; 7。为了抑制任何污染细胞的生长, 例如 内皮细胞或成纤维细胞, 增加10和 #181; M 胞嘧啶和 #946; d-arabinofuranoside 到培养基。调整 pH 值与氢氧化钠 (2 毫米) 到7.4 和无菌过滤器媒介。将 CCT 介质存储在4和 #176; C. </li>
	</ol> </li> <li> 鲍威尔介质 <ol> <li> 为 1 L 鲍威尔介质, 溶解 6.43 g 氯化钠 (110 毫米) 与 0.19 g 氯化钾 (2.5 毫米), 0.16 克 2 PO 4 (1.2 mm), 0.3 g MgSO 4 7H 2 O (1.2 mm), 5.96 g Hepes (25 mm), 和 1.98 g D (+)-水中无菌的葡萄糖一水合物 (10 毫米)。用氢氧化钠 (2 米) 调节 pH 值 7.4, 无菌过滤介质。在4和 #176 存储鲍威尔介质; C. </li>
	</ol> </li> <li> 氯化钙 (CaCl 2 ) <ol> <li> 准备 100 mM CaCl 2 解决方案 (50 mL), 并准备包含500和 #181 的等分; L CaCl 2 。在-20 和 #176 冻结等分; C. </li>
	</ol> </li> <li> 准备培养基 <ol> <li> 准备三细胞培养基: 镀培养基、电镀介质和洗涤介质。使用 CCT 介质作为所有三介质的基础 ( 表 1 )。计算每个培养皿1毫升的 CCT 介质。因此, 为20培养皿准备20毫升 CCT 培养基 (内径:35 mm). </li>
		<li> 细胞培养板: 将每个细胞培养板 (内径:35 毫米) 与1毫升镀培养基涂层。将涂覆的盘子储存在37和 #176; C 隔夜或至少2小时前使用. </li>
	</ol> </li> </ol> 2。成人心肌细胞的分离 <ol> <li> 共生灌注系统的制备 <ol> 热镀介质和洗涤介质到37和 #176; 解冻500和 #181 的管子; CaCl <li> 2 , 重25毫克胶原酶. 
		<li> 冲洗共生灌注系统与 aqua 无菌, 事后让鲍威尔介质循环系统5分钟. </li>
		<li> 用80毫升鲍威尔培养基填充共生灌注系统, 不带任何气泡, 用95% 氧气将介质充气. </li>
		<li> 用40毫升的鲍威尔培养基制备试管 (50 毫升), 将其加热到37和 #176; C, 用95% 的氧气将其充气. </li>
		<li> 准备一个长度约25厘米的螺纹, 用于将卸下的心脏连接到套管. </li>
		<li> 脱脂的刀片与酒精 (70% 按体积) 和固定它的菜刀。将塑料盘夹入菜刀. </li>
	</ol> </li> <li> 提取心脏 <ol> <li> 麻醉4% 到5% 异氟醚的雄性大鼠, 并将其与颈椎脱位一起牺牲。用腹式剪开腹部后侧的肋弓, 用同样的剪刀, 切开隔膜打开胸腔. </li>
		<li> 取出心脏, 连同肺和胸腺, 通过在胸腔上方切除胸腺高颅。立即将物料转移到 ice-cold 盐水溶液中. </li>
		<li> 从心脏取出肺和胸腺, 用解剖剪刀 (大) 和用胶囊钳固定材料, 将后者转移到新的盐水溶液中. </li>
	</ol> </li> <li> 隔离 <ol> <li> 从心脏取出多余的组织, 如胸腺、气管、脂肪和结缔组织的残留物, 使用胶囊钳和解剖剪刀 (大或小)。发现主动脉和切断它与解剖剪刀 (大或小) 之间的第一和第二鳃弓. </li>
		<li> 启动共生灌注系统的滴水。将心脏放在共生灌注系统的套管上, 然后先用鳄鱼钳夹住它, 之后再用准备好的螺纹。冲洗心脏, 直到它没有血液. </li>
		<li> 溶解25毫克胶原酶在 5-6 毫升温暖鲍威尔培养基和添加12.5 和 #181; L CaCl 2 (30 和 #181; M). </li>
		<li> 关闭循环通过移动一个玻璃漏斗, 这是连接与共生灌注系统, 在滴水的心脏和添加解决胶原酶的灌注系统。开始灌注25分钟, 滴速为每秒1滴. 
		注: 在灌注过程中, 心脏会肿胀并出现蜡状. </li>
		<li> 停止灌注25分钟后, 从共生灌注系统中取出心脏。从心脏中取出主动脉、心房和结缔组织, 并打开右心室和左侧脑室. </li>
		<li> 在90和 #176 的角度劈两次心; (切割宽度: 0.7 毫米; 速度: 0.15 厘米/秒)。手动重复此过程, 两个手术刀为十年代的每边. </li>
		<li> 将12毫升的灌注介质转换为新管 (50 毫升)。将细胞浆倒入这种培养基中, 再在37和 #176 消化细胞五分钟; c. 每分钟混合解决方案. </li>
		<li> 通过尼龙网 (200 和 #181; m) 将溶解的心脏过滤到一个新的管子 (50 毫升). </li>
		<li> 离心过滤解决方案在 29 x g, 3 分钟. 丢弃上清, 添加6毫升温暖的鲍威尔培养基, 包括12.5 和 #181; CaCl 2 (250 和 #181; M) 到细胞颗粒。通过平滑的震动运动重颗粒。再次离心 29 x g, 2 分钟丢弃上清, 加入6毫升暖鲍威尔培养基取代25和 #181; L CaCl 2 (500 和 #181; M)。通过轻柔的摇动动作溶解细胞颗粒, 加入12毫升温暖的鲍威尔培养基, 包括120和 #181; L CaCl 2 (1 mM)。离心第三次在 16 x g 为1分钟。再次, 清除上清. </li>
		<li> 将电池颗粒与5ml 电镀介质混合. </li>
		<li> 从区域性板中删除镀介质。转移1毫升电镀介质, 包括分离的心肌细胞, 到每个培养板。在37和 #176 孵育新鲜分离的心肌细胞; C 为 1 h. </li>
		<li> 从培养皿中取出电镀介质。在每种培养基上加入1毫升洗涤培养基, 并在37和 #176 中储存板材; C 最多六天, 不改变培养基. </li>
		<li> 用于调查不同化学品和处理对 ARVC 的影响, 首先通过洗涤介质来刷新电镀介质, 然后添加不同的化学物质. 
		注: 用光镜评价: 每个细胞准备, 150 至300心肌细胞应监测每天通过光镜。根据它们的外观将所有计数的心肌细胞细分为组 ( 例如, 和 #34; 杆形和 #34; #34; #34; #34; #34; 不寻常的外表和 #34 #34。类别和 #34; 传播和 #34; 包括所有具有伪足样结构的心肌细胞。#34; 不寻常的外表和 #34; 包括所有 ARVC, 表面不规则, 没有可检测的完整细胞膜. </li>
	</ol> </li> </ol> 3。例如实验 <ol> 成人心肌细胞的荧光染色/免疫荧光着色 <ol> 在共聚焦激光显微镜下对 ARVC 的形态学和结构转换进行了分析。利用笔 TRITC 研究 #34; 杆形和 #34;; #34; #34;; #34; 传播和 #34; ARVC。根据制造商和 #39 的协议执行染色。在参考 Nippert et al. 中给出了笔-TRITC 染色的一个例子 11 . 利用荧光/免疫荧光染色, 培养成人心肌细胞的 de-and re-differentiation 在实验治疗之间的差异 ( 例如, Swiprosin-1, ionomycin) 可以被调查. </li>
	</ol> </li> <li> 实时定量 rt-pcr (qRT pcr) <ol> <li> 执行 qRT pcr 研究不同基因 ( OSM、Swiprosin-1、#946、MHC) 在 ARVC 培养过程中 mRNA 表达的变化。为充分的样品大小, 使用更大的文化盘子 (内径:60 毫米) 与2毫升容量。ARVC 的五种文化菜肴有一个样品。根据制造商和 #39 的协议, 执行 mRNA 的分离和 cDNA 的转化. </li>
	</ol> </li> <li> 免疫技术 <ol> <li> 执行西墨, 以调查在 ARVC 的培养过程中蛋白质表达的变化 (如 Swiprosin-1)。每个样品使用一个培养皿 (1 毫升). </li>
	</ol>
	</li>
</ol>

<ol>
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所显示的结果是 Franziska Nippert 博士论文的一部分。

成年心肌细胞的行为在体内受许多与其他细胞的相互作用 (如: 如, 神经元, 内皮细胞, 成纤维细胞, 炎症细胞) 和电合, 他们形成1。因此, 研究成年心肌细胞的应激适应需要 ARVC 的分离和培养。分离和培养 ARVC 的主要作用是: 1) 从细胞外基质和细胞间接触;2) 将它们与收缩刺激断开;3) 迫使他们从三维组织适应 two-dimensional 环境。在这些条件下, ARVC 开始 de-and re-differentiation...

成年心肌细胞在体内的工作, 作为一个电子合的基础上细胞细胞之间的联系。此外, 它们受邻近细胞的影响, 如心脏成纤维细胞、内皮细胞、神经元和炎症细胞1。为了研究心肌细胞适应其细胞内组织改变负荷状况的能力, 心肌肥厚是导致心力衰竭的最初步骤, 成年心室大鼠的分离和培养心肌细胞 (ARVC) 是必需的2,3,4。从历史上看, 心肌细胞首先从胚胎小鸡心脏中分离出来5,6。几年后, 首次分离的终末分化心肌细胞使用钙消耗7描述。然而, 这些成年心肌细胞不能耐受钙, 因此不能用于功能性化验。最后, 在 1976年, 一项新的协议使鲍威尔和扭转在生理情况下调查成年心室心肌细胞8。作为第一步, 他们在低钙浓度下分离成年心肌细胞, 然后在逐步的过程中增加钙对生理浓度。今天, 大多数用于分离和培养成年心肌细胞的协议与此钙协议一起使用, 胶原酶用于酶消化致密细胞-细胞接触1。
为成功的耕种, 胎儿小牛血清 (FCS) 或 oncostatin M (OSM) 是需要的。ARVC 执行 de-and re-differentiation 与广泛的结构变化, 包括肌拆卸和改革9,10,11,12。这一过程伴随着重新的胎儿型基因, 如β-肌球蛋白重链 (β-MHC), 如已知的肥大, 和形成的伪足样结构, 也称为传播4,11,13. 此外, swiprosin-1 (EFHD2), 一个新发现的蛋白质, 发挥了重要作用的过程中 re-differentiation 栽培 ARVC11。结果, ARVC 在文化中转化为广泛的多态细胞, 在培养的两个到三周后自发地出现收缩2,4,14。
最近的发现表明, 心脏 re-and de-differentiation, 因为它发生在文化条件下模仿功能在心脏重塑10,15期间看到的在体内。心脏重塑是心脏疾病的一个关键过程16。由于心脏疾病仍然是工业化社会的主要死亡原因, 对成人心肌细胞生物学的更好的了解是重要的 (卫生组织; 2015)。ARVC 的分离和培养有助于制定新的治疗心脏疾病的策略和药物。通过这份手稿, 为 ARVC 的分离和培养提供了一个协议。此外, 该方法的某些关键部分在讨论部分中进行了突出显示。

<table><tbody><tr><td>Langendorff perfusion system</td><td>inhouse construction</td><td></td><td>double-walled with a water&lt;br/&gt; based heating system</td></tr><tr><td>Tissue chopper Mc Ilwain</td><td>Cavey Laboratory Engeneering Co. Ltd.</td><td></td><td></td></tr><tr><td>Aortic Cannula, OD 1,8 mm</td><td>inhouse construction</td><td></td><td></td></tr><tr><td>Abdominal shears</td><td>Aeskulap</td><td>BC772R</td><td></td></tr><tr><td>Capsule forceps</td><td>Eickemeyer</td><td>171307</td><td></td></tr><tr><td>Dissecting scissor large</td><td>Aeskulap</td><td>BC562R</td><td></td></tr><tr><td>Dissecting scissor small</td><td>Aeskulap</td><td>BC163R</td><td></td></tr><tr><td>Mash with Polyamid</td><td>Neolab</td><td>4-1413</td><td>mash size 200 &amp;mu;m</td></tr><tr><td>plastic disc</td><td>Cavey Laboratory Engeneering Co. Ltd.</td><td></td><td></td></tr><tr><td>Collagenase Typ II</td><td>Worthington</td><td>LS004177</td><td></td></tr></tbody></table>

isolation and cultivation of adult rat cardiomyocytes

在一个完整的心脏, 相邻的细胞影响成年心肌细胞。利用成人心肌细胞的分离和培养方法, 对其在特定的治疗和环境下的行为进行精确的研究是可行的。本论文提出了一种成功的成年大鼠心室心肌细胞 (ARVC) 的分离和培养方案。
大鼠在深麻醉下被宫颈脱位所牺牲。然后, 心脏被提取, 主动脉被发现。随后, 对共生灌注系统进行钙耗和胶原酶处理。随后, 心室组织得到切碎, 循环, 并筛选, 随后三离心步骤, 逐步增加 CaCl2 , 直到达到生理钙浓度。ARVC 被镀在细胞培养皿上。在对细胞培养基进行清爽后, ARVC 可在不改变含血清培养基的情况下培养六天。ARVC 的分离是一个钙敏感的过程。细胞内钙离子浓度的微小变化会导致分离电池的质量和生存能力下降。
新鲜地被隔绝的 ARVC 是杆形状。在耕种的第一天他们丢失了杆形状的形态学并且形成伪足象结构 (传播)。在这种形态学形成过程中, ARVC 最初降低其收缩元素, 然后通过肌动蛋白应力纤维和de 从头sarcomerogenesis 进行改革。经过一个星期的耕种, 大多数 ARVC 显示一个广泛的外观与一个明确的检测交叉条纹。这个过程是敏感的细胞内钙浓度, 作为治疗与 ionomycin 减弱传播。de-and re-differentiation 的关键标志是β-肌球蛋白重链 (β-MHC)、oncostatin M (OSM) 和 swiprosin-1 (EFHD2)。最近的研究表明, 在培养条件下发生的心脏 re-and de-differentiation 模仿心脏重塑过程中出现的体内的特征。因此, ARVC 的分离和培养在了解心肌细胞生物学方面起着关键的作用。

文化中的成人心肌细胞:图 1显示了最新隔离的成人心肌细胞在最后一次洗涤后2小时的概况。大约75% 的所有心肌细胞都有一个杆状形态。其余的25% 显示了一个不寻常的外观与圆形的形态学和没有可检测完整的细胞膜 (图 1)。在耕种的末端 (天 6), 15% 所有心肌细胞显示传播, 大约10% 保持在一个圆的形态学, 不用伪足象?...

Watch this Scientific Journal Video about Isolation and Cultivation of Adult Rat Cardiomyocytes at JoVE.com

Isolation and Cultivation of Adult Rat Cardiomyocytes

在这里, 我们提出了一个分离和培养成年大鼠心室心肌细胞 (ARVC) 的协议。隔离 ARVC 可用于短期和长期种植。ARVC 的分离和培养可在开发新的心脏疾病治疗方案中发挥关键作用。

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 ...

isolation-and-cultivation-of-adult-rat-cardiomyocytes

Research

JoVE Journal

Medicine

15.5K 次观看.  Justus Liebig University Giessen. 在这里, 我们提出了一个分离和培养成年大鼠心室心肌细胞 (ARVC) 的协议。隔离 ARVC 可用于短期和长期种植。ARVC 的分离和培养可在开发新的心脏疾病治疗方案中发挥关键作用。

视频: Isolation and Cultivation of Adult Rat Cardiomyocytes

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.

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 ...

科研

发育生物学

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 ...

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 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 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 ...

医学

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

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

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

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

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 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 ...

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 ...