このプロジェクトは、（サンドラー財団によって部分的に資金提供）画期的な生物医学研究のためのUCSFプログラム、独立賞にNIH経路（R00HL114738）、およびエドワード・マリンクロット・ジュニア財団によって資金を供給されました。 JJは、NIH（T32HL007731）からポスドクフェローシップによってサポートされていました。著者は、必ずしもNIHの公式見解を示すものではありません。この作品の内容について、単独で責任を負うものとします。

The authors have nothing to disclose.

孤立した心筋細胞の全体的な健康状態は、このプロトコルのいくつかの重要な側面に依存します。まず、灌流に対する心臓の抽出から時間が重要であり、5分以内に行われるべきです。カルシウムの除去は、細胞間相互作用を解離するのに役立ちますが、負の細胞の健康、長期的に影響を与える可能性がある。29-32したがって、我々はそれが十分なEGTA（エチレングリコール四酢酸）キ?...

培養心筋細胞は、しばしば、インビトロで十分に制御された環境における細胞の挙動を監視するために使用されます。例えば、形態学的、電気的、生化学的、または機械的な電池特性は、定義された培地中で1,2、および小分子薬剤、ペプチド、遺伝子調節、3または電気的刺激に応答して操作された基板上に研究することができる。4細胞の含有量ができますまた、定義された共培養を使用して制御される。5これらのインビトロ実験は、大きな薬物または遺伝子スクリーニングにおいて有用であり、心筋細胞の生物学に関わる研究の様々なタイプのためのインビボの方法で補います。 長期培養は、表現型の変化を達成するために長時間を必要とする実験的な道を可能にします。タイムリーな例は、脱分化、細胞周期に再進入し、細胞分裂は、典型的には、それ自体の上に検討されている成体哺乳類の心筋細胞の増殖、のものです週にveral日。ここで6,7、延長培養時間は、遺伝子操作、7,8-機能脱分化（ 例えば、筋節解体）9と、潜在的に転写脱分化を促進する。6その後の細胞周期の再入国と細胞分裂がさらに長い培養期間を必要とします分裂複数回の実験の目標である場合は特に、観察しました。心筋細胞周期の重要性は、既存の心筋細胞の脱分化と増殖は、ゼブラフィッシュおよび新生児マウスにおける心臓の再生を担うことが示されている心臓再生における最近のいくつかの重要な科学的な作品、の中心である。10月12日このように、可能性哺乳類の成体の心筋細胞に脱分化、細胞周期の再入国を刺激することは、人間の心臓の再生13-15で重要な問題のまま I nは 、哺乳動物の心臓の細胞周期を研究するインビトロ実験筋細胞は、主に起因するマウスモデルと比較して、長期培養のが比較的容易に、ラットのソースを使用していた。16しかし 、マウス系は両方のin vitroおよびin vivoで有用である十分に特徴付けられたトランスジェニックツールや疾患モデルの豊富なリソースを提供していますプロトコル。例えば、Creをベース系統のトレースは、in vivoでの新生児マウスの心臓に心筋を再生するソースとして既存の心筋細胞の同定を可能にした。系譜トレース新生児マウス心筋細胞のin vitro試験において 、12は間質との相互作用の検討を有効にしています線維芽細胞との共培養による細胞。5しかし、その課題に、17ほとんど報告が成体マウスの心筋細胞の単離および長期的な文化の存在。18,19 単独での短期培養のための実行可能な成体マウス心筋細胞の単離は困難な作業であることが知られています。このprotocオールは、短期および長期の調査の両方に使用することができる成体マウスから実行可能な心筋細胞を達成する方法について順を追って説明します。このプロトコルを使用して単離された心筋細胞を効率的にアデノウイルスベクター週間20,21培養で形質導入することができます。これらの方法には、in vitroで心筋細胞生物学を研究するための強力なシステムを提供します。 本明細書に記載の方法は、ランゲンドルフ逆行性灌流のバリエーションを使用して、以前の作品からいくつかの要素に基づいています。18,22、いくつかのプロトコルは、短期培養および研究のための成体マウスの心筋細胞の単離に公開されているが、23-25 ​​利点のこのプロトコルは、培養する能力孤立した心筋細胞の長期的です。これは、異所性遺伝子発現に関与すると、携帯リプログラミング戦略のように長期間を要する細胞プロセスの研究に有用であろう。

<table border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
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			<td height="21" style="height:21px;width:295px;">Equipment</td>
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			<td style="width:99px;"></td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated water jacket</td>
			<td>Radnoti</td>
			<td>158831</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Circulating heated water bath, Isotemp</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:99px;">3013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Laboratory pump</td>
			<td style="width:148px;">Watson-Marlow</td>
			<td style="width:99px;">323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hemostats</td>
			<td style="width:148px;">Exelta</td>
			<td style="width:99px;">63042-090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tissue forceps</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">470128034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #7 curved forceps</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">91197-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #5 fine forceps</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small dissection scissors</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">470128034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Extra fine bonn scissors</td>
			<td>FST</td>
			<td style="width:99px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fine spring scissors</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">91500-09</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Name</td>
			<td style="width:148px;">Company</td>
			<td style="width:99px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Materials</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">NaCl</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">KCl</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4-7H2O</td>
			<td style="width:148px;">Fisher</td>
			<td style="width:99px;">S25837</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4-7H2O</td>
			<td style="width:148px;">Fisher</td>
			<td style="width:99px;">S25414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">86329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butane dione monoxime (BDM)</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">B0753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:148px;">Fisher&#160;</td>
			<td style="width:99px;">BP310100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">G-7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Insulin</td>
			<td style="width:148px;">Novo Nordisk</td>
			<td style="width:99px;">393153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EGTA</td>
			<td style="width:148px;">Amresco</td>
			<td style="width:99px;">0732-288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Protease, type XIV</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Collagenase II</td>
			<td style="width:148px;">Worthington</td>
			<td style="width:99px;">LS004176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MEM</td>
			<td style="width:148px;">Corning</td>
			<td style="width:99px;">15-010-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">FBS, heat inactivated</td>
			<td style="width:148px;">JRS</td>
			<td style="width:99px;">43613</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Primocin</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:99px;">NC9141851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Ethyl Carbamate</td>
			<td style="width:148px;">Alfa Aesar</td>
			<td style="width:99px;">AAA44804-18</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:295px;">215 micron mesh</td>
			<td style="width:148px;">Component supply</td>
			<td style="width:99px;">U-CMN-215-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">20 G blunt ended needle</td>
			<td style="width:148px;">Becton Dickinson</td>
			<td style="width:99px;">305183</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">20 G beveled needle</td>
			<td style="width:148px;">Becton Dickinson</td>
			<td style="width:99px;">305176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Lab tape</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">89097-990</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:295px;">Surgical tape</td>
			<td style="width:148px;">3M</td>
			<td style="width:99px;">1527-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Silk suture, 7-0</td>
			<td style="width:148px;">Teleflex</td>
			<td style="width:99px;">15B051000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Mouse anti-alpha-actinin antibody</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">A7811</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Alexa Fluor 488 goat anti-mouse IgG1 antibody</td>
			<td style="width:148px;">Thermo Fisher</td>
			<td style="width:99px;">A21121</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, culture and transduction of adult mouse cardiomyocytes

培養心筋細胞がインビボ系に相補的な技術を使用して心筋細胞生物学を研究するために使用することができます。例えば、 インビトロ培養の純度とアクセシビリティは、生化学分析、ライブイメージング、および電気生理学を細かく制御を可能にします。心筋細胞の長期培養は、短期培養で完了することができない追加の実験的アプローチへのアクセスを提供しています。例えば、脱分化、細胞周期の再進入、および細胞分裂のインビトロ研究は、これまで主として長期培養においてより堅牢であると思われるラット心筋細胞に限定されています。しかし、トランスジェニックマウス系統とよく発達した疾患モデルの豊富なツールセットの利用可能性は、心臓研究のためのマウスのシステムは魅力的です。いくつかの報告は、成体マウスの心筋細胞の単離の存在するが、いくつかの研究は、それらの長期培養を実証します。ここで紹介する、ステップ・バイ・ステップ方式であります単離および成体マウスの心筋細胞の長期培養。まず、逆行ランゲンドルフ灌流を効率的に重力沈降精製を行っプロテアーゼ、と心を消化するために使用されます。単離後脱分化の期間の後、細胞は徐々に培養に付着し週間培養することができます。アデノウイルスの細胞溶解物を効率的に単離された心筋細胞を形質導入するために使用されます。これらの方法は、心臓の生物学を研究するために、シンプルでありながら強力なモデルシステムを提供します。

野生型大人のICR（CD1）マウスの心臓は、一般的に成功したアイソレーション〜500,000百万〜1心筋細胞が得られます。すぐに分離した後、細胞は無傷のサルコメアとほとんど棒状の外観（ 図3A）を維持し、心筋収縮を伴う機能的研究のために使用することができます。 （90％以上）の棒状の心筋細胞の高い割合は、効果的な灌流および消化の指標です。生存...

Watch this Scientific Journal Video about Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes at JoVE.com

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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.

成体マウスの心筋細胞の単離、培養および形質導入

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

isolation-culture-and-transduction-of-adult-mouse-cardiomyocytes

Research

JoVE Journal

Biology

26.4K ビュー.  University of California, San Francisco. 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. 

ビデオ: Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成体マウスの心筋細胞の単一細胞転写プロファイリング

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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.

新生仔マウス心筋細胞の単離および培養

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.

アイソレーション、文化、および成体マウスCardiomyoctyesの機能解析

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

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

マウス心筋細胞の単離と生理解析

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

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

成体マウス切歯から歯科上皮幹細胞の単離および培養

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

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.

細胞シグナリングのための成体マウスの心筋細胞の単離と文化<em&gt; in vitroで</em&gt;心肥大

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...