分离培养和成年小鼠心肌细胞转导
Summary
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.
Abstract
培养的心肌细胞可用于研究是否可使用那些对体内系统补充技术心肌生物学。例如， 在体外培养的纯度和可访问性使得在生化分析，实时成像，和电精细控制。心肌细胞长期培养提供了访问，不能在短期内培养进行额外的实验方法。例如，去分化，细胞周期再入，和细胞分裂的体外调查迄今基本上被限制在大鼠心肌细胞，这似乎是在长期培养更加健壮。然而，转基因小鼠线和发达的疾病模型的丰富的工具集的可用性使鼠标系统的心脏研究的吸引力。尽管一些报道成年小鼠心肌细胞分离存在的，一些研究展示自己的长期培养。这里介绍的，是为一步一步的方法成年小鼠心肌细胞的分离和长期培养。首先，逆行的Langendorff灌注被用来有效地消化用蛋白酶的心脏，接着通过重力沉降纯化。一段去分化下述分离后，将细胞逐渐附着到培养，并且可以是数周培养。腺病毒的细胞裂解物，用于有效地转导的分离的心肌细胞。这些方法提供了一种简单，但功能强大的模型系统来研究生物学的心脏。
Introduction
培养的心肌细胞经常用于监测在体外良好控制的环境中细胞的行为。例如，形态，电气，生化或机械细胞性质可在工程衬底进行研究，在确定的培养基1,2，以及响应于小分子药物，肽，基因调控，3或电刺激。4蜂窝含量可以还使用定义共培养物来控制。5 这些体外实验是在大的药物或遗传筛选有用和体内方法各种类型涉及心肌生物学调查补充。
长期的文化使那些需要长的时间内，实现表型改变的实验途径。适时地例子是成年哺乳动物心肌细胞增殖，其中去分化，细胞周期再入，和细胞分裂典型地研究过本身的veral天至数周，6,7这里，延长培养时间有利于遗传操作，7,8分化的功能（ 例如，肌节拆卸）9和潜在的转录分化。6随后细胞周期重新进入和细胞分裂，需要更长的时间培养观察，特别是如果多轮师都是实验性的目标。心肌细胞周期的重要性是中央在心脏再生，其中，预先存在的心肌细胞的分化和增殖已显示负责在斑马鱼和新生小鼠心脏再生几个最近的关键科学著作。10-12。因此，有可能刺激分化和细胞周期重新进入哺乳动物成年心肌留在人的心脏再生13-15的一个关键问题
I N体外实验研究哺乳动物心脏的细胞周期肌细胞已主要使用鼠源，由于其相对容易长期培养的相比小鼠模型16然而，鼠系统提供的充分表征的基因的工具和疾病模型是在体外 和体内有用资源丰富协议。例如，基于Cre的谱系追踪，使预先存在的心肌细胞的鉴定如在体内的新生小鼠心脏再生心肌的一个来源。12 体外谱系追踪新生小鼠的心肌细胞的研究已启用与基质相互作用的检查细胞通过共培养成纤维细胞。5然而，由于它的挑战，17报道很少成年小鼠心肌细胞的分离和长期培养的存在。18,19
单独短期培养存活成年小鼠心肌细胞的分离被称为是一个具有挑战性的任务。这protocOL提供了有关如何实现从可用于短期和长期的调查成年小鼠心肌细胞存活一步一步的指导。使用此协议分离的心肌细胞可以与腺病毒载体20,21和培养周有效地转。这些方法提供了强大的系统来研究生物学心肌体外 。
本文所描述的方法是基于从使用的Langendorff逆行灌注的变化之前的作品几个元素。18,22虽然几个协议已发表 关于短期培养和研究中，23-25 的优点的成年小鼠心肌细胞的隔离该协议是能够培养分离的心肌细胞长期的。这将是在涉及异位基因表达并且需要延长的时间段，例如在细胞的初始化策略细胞过程的研究是有用的。
Protocol
这里列出的所有程序已获得批准的机构动物使用及护理委员会在加州大学旧金山分校。 注：简单地说，来自小鼠胸腔中提取的心脏后，冠状动脉逆行灌注被用来有效地消化用胶原酶和蛋白酶XIV细胞外基质。然后心室被隔离，机械分离并过滤成一个单一的细胞悬浮液。重力沉降进行分离的心肌细胞，这是从基质细胞样的成纤维细胞和内皮细胞可以通过高密度分离?…
Representative Results
野生型成人ICR（CD1）小鼠心脏通常产生从一个成功的隔离500,000到1,000,000心肌细胞。分离后立即将细胞保持大多棒状外观（ 图3A）与完整肌节和可用于涉及心肌收缩功能研究。棒状心肌（90％以上）的高百分比是有效灌注和消化的指示。可行的心肌细胞将是大的（〜100 – 200微米长）和出现有在光照下（ 图3A）的急剧外膜。免疫染色特定心肌肌节…
Discussion
分离出心肌细胞的整体健康取决于该协议的几个重要方面。首先，从心脏提取到灌注的时间是非常关键的，应在5分钟以下进行。钙的除去有助于解离细胞-细胞相互作用，但能细胞健康长期负面影响。29-32。因此，我们发现它足以通过EGTA灌注的最初几分钟期间去除钙（乙二醇四乙酸）螯合， 22但很快消化和随后的步骤中恢复钙。
利用蛋白酶十四大大提高比单?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该项目由加州大学旧金山分校计划突破生物医学研究（由桑德勒基金会提供部分资助），美国国立卫生研究院途径来独立奖（R00HL114738）和爱德华马林克罗特小基金会。 JJ是由来自美国国立卫生研究院（T32HL007731）的博士后奖学金支持。作者在此工作，并不一定代表美国国立卫生研究院的官方意见的内容负责。
Materials
| Equipment | |||
| Heated water jacket | Radnoti | 158831 | |
| Circulating heated water bath, Isotemp | Fisher Scientific | 3013 | |
| Laboratory pump | Watson-Marlow | 323 | |
| Hemostats | Exelta | 63042-090 | |
| Tissue forceps | VWR | 470128034 | |
| Dumont #7 curved forceps | FST | 91197-00 | |
| Dumont #5 fine forceps | FST | 11251-20 | |
| Small dissection scissors | VWR | 470128034 | |
| Extra fine bonn scissors | FST | 14084-08 | |
| Fine spring scissors | FST | 91500-09 | |
| Name | Company | Catalog Number | Comments |
| Materials | |||
| NaCl | Sigma | S9888 | |
| KCl | Sigma | P9541 | |
| Na2HPO4-7H2O | Fisher | S25837 | |
| MgSO4-7H2O | Fisher | S25414 | |
| Taurine | Sigma | 86329 | |
| Butane dione monoxime (BDM) | Sigma | B0753 | |
| HEPES | Fisher | BP310100 | |
| Glucose | Sigma | G-7021 | |
| Insulin | Novo Nordisk | 393153 | |
| EGTA | Amresco | 0732-288 | |
| Protease, type XIV | Sigma | P5147 | |
| Collagenase II | Worthington | LS004176 | |
| MEM | Corning | 15-010-CV | |
| FBS, heat inactivated | JRS | 43613 | |
| Primocin | Invitrogen | NC9141851 | |
| Ethyl Carbamate | Alfa Aesar | AAA44804-18 | |
| 215 micron mesh | Component supply | U-CMN-215-A | |
| 20 G blunt ended needle | Becton Dickinson | 305183 | |
| 20 G beveled needle | Becton Dickinson | 305176 | |
| Lab tape | VWR | 89097-990 | |
| Surgical tape | 3M | 1527-0 | |
| Silk suture, 7-0 | Teleflex | 15B051000 | |
| Mouse anti-alpha-actinin antibody | Sigma | A7811 | |
| Alexa Fluor 488 goat anti-mouse IgG1 antibody | Thermo Fisher | A21121 |
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Cite This Article
Judd, J., Lovas, J., Huang, G. N. Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes. J. Vis. Exp. (114), e54012, doi:10.3791/54012 (2016).