Name: isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes
Uploaded: 2020-10-10T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes at JoVE.com

这项工作得到了辛辛那提大学医学院病理学和实验室医学系对奥努尔·卡尼西卡克博士的资助;国家卫生研究院（R01HL148598）向奥努尔·卡尼西卡克博士提供赠款。奥努尔·卡尼西卡克博士获得美国心脏协会职业发展奖（18CDA34110117）的支持。Perwez Alam博士得到美国心脏协会博士后资助（AHA_20POST35200267）。Malina J. Ivey 博士得到 NIH T32 赠款 （HL 125204-06A1） 的支持。

所有实验都应按照美国国家卫生研究院（NIH）发布的《实验室动物护理和使用指南》的指南进行。视频中显示的所有协议都经过了辛辛那提大学医学院动物护理和使用委员会 （IACUC） 的批准。
1. 从成年小鼠（和大鼠）中提取心脏前的准备
<ol><li>根据表 1 和表2中给出的小鼠和小鼠隔离 的配方，准备相应的灌注、酶和停止溶液。通过 0.22 μm 过滤器过?...

<ol>
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迫切需要建立成人心肌细胞分离和长期培养的规程，以进行细胞特定的机械研究。只有少数报告讨论成人CM隔离协议，甚至更少用于成人小鼠CM15，16，17,16,的长期培养。研究表明，成年大鼠CM对体外培养的耐受性比成年大鼠CM10、11、12,11,高。在这份报告中，?...

心脏病是全世界主要死亡原因之一。几乎所有类型的心脏损伤都会导致成人心肌细胞 （CMs） 的显著损失。由于成人CM1的衰老性质，成年哺乳动物的心脏无法修复其心脏损伤。因此，任何对成年哺乳动物心脏的侮辱都会导致CMs的永久丧失，导致心脏功能下降和心力衰竭。与成年哺乳动物不同，斑马鱼和纽特心脏等小动物可以通过现有的CM增殖2、3、43再生心脏损伤。2正在全球范围内努力通过增殖和非增殖方法寻找一种新的治疗性治疗性治疗性治疗性治疗手段。在过去的几十年里，已经开发出各种基因小鼠模型来研究心脏损伤和修复。然而，在体内使用动物模型仍然是一种昂贵的方法，从二次效应中破译细胞自主机制的复杂性更高。此外，体内系统很难分析从CM诱导心脏保护信号的药理干预的CM特异性效果。
此外，成人CM的长期文化对于进行CM增殖分析是必要的。CM增殖检测需要至少4-5天细胞被诱导到细胞周期，并在此之后获得准确的数据。此外，利用分离的CMs进行电生理学研究、药物筛选、毒性研究和Ca+平衡研究的研究都需要改进培养系统5、6、7。,6,7此外，最近的研究表明，从CMs（心因子）8，9中分泌的细胞因子具有心脏保护意义。为了研究这些心因子在心脏修复和再生过程中的治疗作用和分子机制，需要长期培养。
成年大鼠CMs在体外系统10、11、12中，足够坚固，可以进行单细胞分离和长期培养。然而，成人小鼠CM是极大的兴趣的体外测定，由于各种转基因小鼠模型的可用性，这允许设计和执行各种创新分析，这是不可能与大鼠CM13。与成年大鼠CM分离相比，从成年小鼠心脏获得单细胞悬浮是相当具有挑战性的，成人小鼠CM在培养中的长期培养更具挑战性。
成人CM隔离从老鼠和老鼠的心脏使用兰根多夫系统已经建立，以研究CM功能5,5，14，15。,15在这里，我们详细介绍了成人CM从大鼠和小鼠分离的协议，以及经过修改的长期培养，转染和CM增殖的分离细胞。

<table>
	<tbody>
		<tr>
			<td>2,3-Butane Dione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>B-0753</td>
			<td></td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>APExBIO</td>
			<td>B1387</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>449709</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plate</td>
			<td>Corning Costar</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cel-miR-67</td>
			<td>Dharmacon</td>
			<td>CN-001000-01-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type2</td>
			<td>Worthington</td>
			<td>LS004177</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Graduated Transfer Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-711-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable polystyrene weighing dishes</td>
			<td>Sigma-Aldrich</td>
			<td>Z154881-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s medium</td>
			<td>Thermo Scientific</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU</td>
			<td>Life Technologies</td>
			<td>C10337</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Corning</td>
			<td>35-015-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Point High Precision Forceps</td>
			<td>Fisherbrand</td>
			<td>22-327379</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G-5400</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>1483</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent Pharmaceuticals</td>
			<td>PSLAB-018285-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>High Precision Straight Broad Strong Point Tweezers/Forceps</td>
			<td>Fisherbrand</td>
			<td>12-000-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I0516-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>P-8281</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>746436</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX</td>
			<td>Life Technologies</td>
			<td>13778-150</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M-2643</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Natural Mouse Laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Henry Schein</td>
			<td>24352</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease XIV</td>
			<td>Sigma-Aldrich</td>
			<td>P5147-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Selenium</td>
			<td>Sigma-Aldrich</td>
			<td>229865+5G</td>
			<td></td>
		</tr>
		<tr>
			<td>siMeis2</td>
			<td>Dharmacon</td>
			<td>s161030</td>
			<td></td>
		</tr>
		<tr>
			<td>siRb1</td>
			<td>Dharmacon</td>
			<td>s128325</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Blunt/SharpDissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>28252</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Very Fine Precision Tip Forceps</td>
			<td>Fisherbrand</td>
			<td>16-100-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T8158-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-smooth, beveled-edge finish scissor</td>
			<td>Fisherbrand</td>
			<td>22-079-747</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Fisher Scientific</td>
			<td>3006S</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes

成年哺乳动物心肌细胞（CMs）的外体培养是心脏生物学体外研究最相关的实验系统。成年哺乳动物CMs是端端分化的细胞，繁殖能力最小。成人CMs的后位位状态不仅限制了心肌细胞周期的进展，也限制了CMs的高效培养。此外，成人CM的长期培养对于许多研究是必要的，如CM增殖和基因表达分析。
老鼠和老鼠是两个最喜欢用于心肌细胞分离的实验室动物。虽然大鼠CMs的长期培养是可能的，但成年小鼠CMs很容易死亡，在正常情况下不能培养超过五天。因此，迫切需要优化成人 murine CMs 的细胞隔离和长期培养协议。通过此修改后的协议，可以成功地分离和培养成年小鼠和大鼠的 VM 超过 20 天。此外，与之前的报告相比，分离CM的siRNA转染效率显著提高。对于成年小鼠CM分离，Langendorf灌注方法采用最佳酶溶液和足够的时间进行完整的细胞外基质分离。为了获得纯心室的CMs，在进行分离和电镀之前，对两个腹膜进行解剖和丢弃。细胞分散在层压涂层板上，从而能够高效、快速地连接。在SIRNA转染之前，CMs被允许在4-6小时内安顿下来。培养基每24小时刷新20天，随后，CMs被固定和染色的心脏特异性标记，如肌龙宁和细胞周期标记，如KI67。

当前修改的协议允许在体外有效地隔离和培养大鼠和小鼠的 CMs。对于大鼠CM分离，共有3只成年（12周大）雄性菲舍尔344只大鼠被使用。图 1显示了手术过程中所需的手术器械和隔离装置;每个部件都已标记并描述在图例中。胶原酶类型2用于消化，从成功分离中产生大量高质量的CMS（图2A）。分离后24小时，这些细胞被转染细胞周期诱导特定的siRNA对Rb1</...

Watch this Scientific Journal Video about Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes at JoVE.com

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

在这里，我们提出了成人小鼠和大鼠心肌细胞的分离、转染和长期培养的协议。

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. ...

本视频的目的是演示成人心肌细胞分离、长期培养和转染的标准化协议。从小鼠或大鼠中隔离和培养心肌细胞，继续是研究健康和疾病心脏内心肌细胞自主生物学的基本方法。有几个问题，我们可以回答使用孤立的心肌细胞。如增殖潜力。有基因表达变化或损伤反应时的特定细胞因子表达。不幸的是，小鼠心肌细胞在正常培养条件下无法存活一周。因此，在这个视频中，我们描述了一种优化的方法，以隔离成年小鼠心肌细胞，转换它们，并培养它们远远超出21天，这种技术最初很难掌握。但通过实践，一个人可以达到约80-90%的存活在心肌细胞隔离后，约100%的转染效率。这些心肌细胞中大多数在正确的条件下可以存活过去20天。清洁和消毒您的手术器械，浸泡在70%酒精15分钟，然后用双蒸馏水清洗。水后，将工具留在空气中晾干，通过运行70%酒精，每次运行5分钟，清洁灌注器。通过运行双蒸馏水 10 分钟，提高流速有助于在酒精冲洗掉任何剩余的酒精后清洁油管。设置三个盘子来清洁切除后的心脏。在每个盘子中填充20毫升的菌细胞缓冲液。将两到三滴肝素加入每道菜，用巴斯德移液器混合，通过上下移液多次混合井。拿一个10毫升的注射器，用菌质缓冲液填充。从注射器中去除任何气泡，并将其放在第三盘中，放在倾斜位置，用胶带固定。用手术缝合准备一个松散的结，并放在针周围。麻醉前20分钟通过IP注射注射给小鼠注射肝素。以每分钟三毫升的流速通过灌注器循环菌质缓冲液。五分钟后，用酶溶液代替富液缓冲液，在过程中用氧气使酶溶液饱和，再次将酶溶液设置为每分钟三百分之三的流速。通过捏到手趾来确认麻醉，将鼠标放在手术平台上。用70%酒精对皮肤进行消毒。小心地打开胸膛，锻炼心脏，把心放在第一道菜里。通过轻轻挤压清除心脏的血液，然后将心脏转移到第二道菜，以进一步清洁心脏的血液，并去除任何非心脏组织。随后将心脏转移到第三道菜。找到大道，用你的钳子，以保持它佩顿更把它放在针周围。通过拉下你的预紧结紧，然后作出第二个结固定它。为了增加稳定性，请确定顺序并在夹子的帮助下放置。修剪任何多余的缝合线，最后使用注射器中的菌细胞缓冲液开始心脏大量，以清除心脏中剩余的任何血液。小心地从注射器中取出计算针，并将其钩到灌注器上。尝试防止任何气泡进入心脏。因为这可能会影响酶溶液和消化的流动。将水套向上移动，在大量时为心脏提供均匀的环境。让酶溶液以每分钟2到3mL的速度流过心脏。让酶溶液流过心脏两分钟。在两分钟内，将40微升的100微摩尔氯化钙溶液混合到酶溶液中。让酶溶液通过心脏再用10到15分钟。一旦水流变得光滑，心脏开始看起来棕色和柔软，你知道你有一个均匀的胶原酶分布，以实现心脏的适当消化。当你认为你的消化是完整的，从兰根多夫灌注系统的心脏，并把它放在一个16毫米的培养皿充满了5毫升的酶溶液，并把它到生物安全的脚。小心地去除脂肪和多余的脂肪组织。在钳子的帮助下，将心脏在定义的碎片中切碎。拿一个无菌的巴斯德移液器，并在45度切割其尖端。使用巴斯德移液器通过轻轻的移液上下打破心脏组织。最佳消化提供单细胞心肌细胞的悬浮后，添加五毫升停止溶液，以阻止酶活性，以避免过度消化，采取第一个50毫升锥形管，并放置一个无菌的100微米细胞过滤器。通过细胞过滤器通过心肌细胞悬浮液，去除任何大块的组织。最后用停止溶液清洗过滤器，收集任何附着的心肌细胞遗骸。将电池悬浮液在 20 GS 下离心三分钟，然后丢弃上一代。重新悬浮心肌细胞和10毫升止血溶液，每隔三分钟以10微升100毫摩尔氯化钙溶液四次混合。第四版离心机后，心肌细胞在20 GS下悬浮3分钟，并丢弃上经剂。将心肌细胞重新悬浮在培养基中，将细胞板入60毫米培养皿中，放入CO2培养箱2至3小时。两到三个小时后主要的污染物细胞类型，如纤维爆炸将坚持在菜的服务，允许心肌细胞的纯隔离。之后，将菜从培养箱中取出，在圆锥管中收集漂浮的心肌细胞，并在层压素涂24孔培养板中重播心肌细胞。四到六个小时足以让心肌细胞粘附在表面。因此，在电镀后六小时可以进行转染。RNA IMAX转染试剂用于移植具有西RNA的心肌细胞。培养基含有25微摩尔血，辅以10%FBS，我们发现更适合进行长期心肌细胞培养和诱导增殖分析。转染后，您可以通过显微镜查看心肌细胞，利用微培养箱了解细胞的延时成像效果。这些是低放大率和高放大率的转染杆形心肌细胞的亮场图像。在这里，你可以看到一天一天的成像，其中展示了成人小鼠心肌细胞形态的变化。我们确认，心肌细胞在早期点，以及D分化和长期培养后是健康和收缩的。下面是 Ki67 免疫荧光染色的例子，证明在转染后，成人心肌细胞在培养中诱导增殖。正如你所看到的使用这种技术的结果，一个人可以从老鼠和老鼠获得健康的成人心肌细胞，并培养它们进行长期研究。总之，一旦掌握了这项技术，我们研究心肌细胞远远超出了目前的培养方案。

Research

JoVE Journal

Biology

Primary Culture of Adult Rat Heart Myocytes

成年大鼠心脏心肌细胞的原代培养

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

科研

生物学

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

长期培养的人类乳腺癌标本，并利用光学投影层析成像分析

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

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 Primary Culture of Rat Hepatic Cells

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

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

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

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

从成年小鼠切牙牙源性上皮干细胞的分离和培养

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

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

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

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 ...