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 
			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 &#956;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

15.4K 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 Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

科研

医学

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 ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

使用左前部降冠动脉的永久结扎的月心肌梗死模型

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

超声波增强原成人成纤维细胞隔离

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

使用皮肤心肌细胞进行心脏功能的体外评估

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

大鼠骨髓骨髓的隔离和培养

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

使用荧光标记肉瘤蛋白缩短多能干细胞衍生心肌细胞的肉瘤。

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

朗根多夫方法的修改，用于从成年小鼠中同时分离心房和心室肌细胞

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...