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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

本稿では、マウスの心臓から大人のCMの成功単離および培養のために必要な技術を説明しています。我々の技術は、上記の方法を用いてCM機能および興奮性のその後の研究を可能にする。大人のCMの機能性を研究するための重要なパラメータは、孤立したCMの健康と品質です。上述したように、我々の技術は、アデノウイルス/レンチウイルスの培養における感染、および細胞興奮性と収縮機能?...

心血管疾患のマウスモデルは、潜在的な治療標的に1,3を識別するための基本的な病気のメカニズムの1,2の解明だけでなく、のための効果的なツールとして役立ってきた。具体的には、（例えば、圧力過負荷など）後天性心疾患の両方のマウスモデル4,5およびトランスジェニックマウスモデルの使用は、心臓病6-8の我々の理解を進めてきた。単一細胞のレベルで心臓11-13に細胞の興奮と興奮収縮連関の根底にある個々のタンパク質におけるシグナル伝達カスケード3,9,10及び変更を研究するための細胞培養技術の使用は、 インビボのマウスモデルを補完している。しかしながら、成人のCMの構造と機能を反映十分な細胞株の欠如は、重大な制限となっている。研究者らは、異種発現系において、イオンチャネルなどの個々のタンパク質を研究することによってこの問題を克服しようとしてきた<suP&gt; 14、およびこれらの研究は、イオンチャネルの生物物理学やタンパク質輸送の面で有益な情報を提供してくれたが、CMのネイティブ微小環境の不十分な表現は重要な制限である。第二に、これらの異種細胞のほとんどが成熟した収縮装置を有していないので、収縮機能および細胞興奮性と収縮との間の複雑な相互作用を研究することは不可能であった。この理由のために、研究者は、それらのインビトロ機能的研究の多くで主要な心臓細胞培養物になっている。最後に、単離された心筋細胞の研究は、瘢痕または線維症と繊維配向の影響を含む多細胞調製物の交絡因子なしで収縮機能の評価を可能にする。 原発新生児ラット心室心筋細胞（NRVMs）が文化に比較的容易に、遺伝子発現15を操作するためにアデノウイルスおよびレンチウイルスに感染することができているNDしたがって首尾よく1使用されますが、自分自身の限界を持っているされています。これらは生理的微小環境1を提供し、シグナリング·フィールドの主力してきたが、形態およびNRVMsと大人のcardiomyoctyesの細胞内組織との間の実質的な違いは、彼らに大人の心の中のイオンフラックスと興奮収縮連関の研究には不十分モデルを作成。最も顕著なのはNRVMsは決定的なT-管状サブシステム4を欠いている。 Ca 2 +のフラックスとダイナミクスが成熟T-管状と筋小胞体（SR）構造6、Ca 2 +の動態とNRVMsにおける心収縮性の機能研究に決定的に依存しているので、成人の心筋細胞でこれらの重要なプロセスを正確に反映していない。さらに、シグナル伝達経路の一部のコンポーネントは、それによって病気のプロセスとそのIを研究するための別の制限を提供し、新生仔および成体マウス9の間で異なるNRVMs中の細胞の興奮と収縮性に対するMPACT。最後に、収縮機構の分布は、収縮測定の精度を制限する多方向不均一な細胞短縮につながる。 孤立した大人の心筋細胞の使用は、より正確なin vitroのモデル化システムを提供しています。マウスの遺伝子操作によって可能になった知識の驚くべき成長は、マウスから機能的な孤立心筋細胞を得ることの重要性を強調している。実際には、マウスモデルから分離され、成人のCMの特徴付けは、多くの生物学的および病理学的事象を解明しました。トランスジェニックマウスモデルから分離されたCMは、それによって中から得られた情報を補完し、そのような虚血/再灌流17,18として疾患モデルでのゲインまたはタンパク質の機能喪失単セル2,16の収縮特性上、及び生存率の研究を可能にした上のin vivo試験SEマウス。 （生理肥大をモデル化するための）や運動5,21検査のために可能にする（模倣高血圧や大動脈弁狭窄症は、そのような横方向の大動脈狭窄誘発の圧負荷など、）後天性心疾患3,19,20のマウスモデルから単離した成人のCMの使用単セルのレベルでの細胞の興奮と興奮収縮連関と、これらのプロセスに関与するシグナル伝達カスケードの相互作用の。さらに、成人のCMでアデノウイルス駆動性遺伝子発現を用いて遺伝子発現を操作する能力は、私たちの複雑なシグナル伝達経路の成分を分析する機会を与える。 電気生理学の観点から、全セル電圧および単離された成体のCMに電流クランプ実験は、ベースラインでのイオンフラックスの性質を解明することで、種々の疾患状態において重要であった。なぜなら、細胞膜の複雑な構造と差動proteの成人のCMとNRVMsまたは異種の細胞株の間に足場構造で、成人の細胞にパッチを適用する機能は、特定の膜タンパク質、構造タンパク質、および成人の心臓の電気生理学的コンポーネントのイオンチャネル相互作用パートナーの影響のより良い表現を提供します。 大人のマウスの心筋細胞を研究する上で、このような顕著な利点にもかかわらず、成体マウスの心筋細胞を分離して培養したウイルスを用いてさらに遺伝子操作を可能にするために実行可能なマウスの心筋細胞を分離し、培養液中で、それらを維持するための方法論の体系的かつ正確な説明の必要性を促し、挑戦しているベクトル。これまでの研究では、急性単離したマウスの成人CMSまたは培養したラットの成人のCMのいずれかを使用している。後者は、成体マウスのCMよりも文化に容易であり、in vitroでの遺伝子発現を操作するほとんどの実験は、ラット大人のCMを使用している。いくつかの研究が正常に変更し、functiをを調査した実験の範囲に大きな制約を提示し、マウスの大人のCMSのonal遺伝子発現。そこで、ここでは詳細に隔離7,8,22、文化3,10,15,23、アデノウイルス感染11-13,15、および成体マウスの心室cardiomyoctyesの機能解析のための以前の研究から変更などの方法を、紹介します。この分離プロトコル我々は、最大72時間のために正常に培養し、一過性にアデノウイルスでトランスフェクションしていたCa 2 +トレラント、興奮性心筋細胞における結果。これらの単離された細胞の機能は、説明するMMSYS撮像システム14,24およびパッチクランプを用いて評価することができる。

<table border="1">
<tbody>
 <tr>
 <td>Name of 			Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Sigma</td>
 <td>S7653</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>Sigma</td>
 <td>P9333</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium Chloride</td>
 <td>Sigma</td>
 <td>M8266</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H3375</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Phosphate 			Monobasic</td>
 <td>Sigma</td>
 <td>S8282</td>
 <td></td>
 </tr>
 <tr>
 <td>D-glucose minimum</td>
 <td>Sigma</td>
 <td>G8270</td>
 <td></td>
 </tr>
 <tr>
 <td>Taurine</td>
 <td>Sigma</td>
 <td>T0625</td>
 <td></td>
 </tr>
 <tr>
 <td>2,3-Butanedione 			monoxime</td>
 <td>Sigma</td>
 <td>B0752</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase B</td>
 <td>Roche Applied 			Science</td>
 <td>11088807001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche Applied 			Science</td>
 <td>11088858001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Protease 			XIV from Streptomyces griseus</td>
 <td>Sigma</td>
 <td>P5147</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin from Bovine 			Serum</td>
 <td>Sigma</td>
 <td>A2153</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride</td>
 <td>Sigma</td>
 <td>C8106</td>
 <td></td>
 </tr>
 <tr>
 <td>Minimum Essential 			Media</td>
 <td>Sigma</td>
 <td>51411C</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin solution 			from bovine serum</td>
 <td>Sigma</td>
 <td>A8412</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Sigma</td>
 <td>G3126</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Sigma</td>
 <td>P4333</td>
 <td></td>
 </tr>
 <tr>
 <td>Insulin-transferrin-sodium 			selenite media supplement</td>
 <td>Sigma</td>
 <td>I1884</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Chloride</td>
 <td>Sigma</td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sial/289329?lang=en&amp;region=US" target="_blank">289329</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Sigma</td>
 <td>G3291</td>
 <td></td>
 </tr>
 <tr>
 <td>Adenosine 			5'-triphosphate magnesium salt</td>
 <td>Sigma</td>
 <td>A9187</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylene 			glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic 			acid</td>
 <td>Sigma</td>
 <td>E3889</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Hydroxide 			Solution</td>
 <td>Sigma</td>
 <td>232041</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetraethylammonium 			hydroxide solution</td>
 <td>Sigma</td>
 <td>86643</td>
 <td></td>
 </tr>
 <tr>
 <td>OptiVisor optical 			glass binocular 			visor</td>
 <td>Dohegan Optical 			Company Inc.</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue 			forceps, 5.5", 1x2 teeth</td>
 <td>Roboz Scientific</td>
 <td>RS-8164</td>
 <td></td>
 </tr>
 <tr>
 <td>Moloney 			forceps - 4.5" (11.5 cm) long slight curve, serrated </td>
 <td>Roboz Scientific</td>
 <td>RS-8254</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont 			#3 Forceps, Dumostar, tip size 0.17 x 0.10mm</td>
 <td>Roboz Scientific</td>
 <td>RS-4966</td>
 <td></td>
 </tr>
 <tr>
 <td>Packer 			Mosquito Forceps 5" Straight Flat</td>
 <td>Roboz Scientific</td>
 <td>RS-7114</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro 			Dissecting Scissors 4.5" Curved Sharp/Sharp </td>
 <td>Roboz Scientific</td>
 <td>RS-5917</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Dissecting Scissors 3.5" Straight Sharp/Sharp20mm</td>
 <td>Roboz Scientific</td>
 <td>RS-5907</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation, culture, and functional characterization of adult mouse cardiomyoctyes

文化の中での一次心筋細胞（CMS）を使用すると、心臓病の理解を進める上で、心臓病のマウスモデルへの強力な補完を提供してきました。具体的には、疾患状態および疾患を引き起こす突然変異により、イオンホメオスタシス、イオンチャネル機能、細胞の興奮と興奮収縮連関とその変化を研究する能力は、心臓疾患に有意な洞察をもたらした。さらに、成人のCMを模倣するための適切な不死化細胞株の欠如、文化の中で（成人のCMの構造的および機能的生体力学特性の多くを欠いている）新生児のCMの制限は、シグナル伝達経路間の複雑な相互作用の理解を妨げている成人の心臓のイオンチャネルおよび収縮特性は、大人の孤立心筋細胞を研究することの重要性を強化する。ここでは、アデノウイルスexpreによる遺伝子発現の単離、培養、操作するための方法を提示するssedタンパク質、および成体マウスの心筋細胞のその後の機能解析。これらの技術の使用は、細胞の興奮性を調節する経路、Ca 2 +の動態及び収縮性をシグナルに機械的な洞察力を開発し、心血管疾患の多くの生理学的に関連する特性評価を提供するのに役立ちます。

棒状、横紋および（自発的に拍動しない）、静止細胞（ 図5A）の成人cardiomyoctyes結果の単離。死んだ細胞は、丸いなりますし、何条線は存在しません。静止状態の細胞を培養し、遺伝子発現（ 図5Bおよび5C）を操作するためのアデノウイルスでトランスフェクトすることができる。培養24時間後、生きた細胞の形態が変化しないが、それでものCa 2 +·</s...

Watch this Scientific Journal Video about Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes at JoVE.com

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

ここでは、ランゲンドルフ灌流システムを使用して、成体マウスcardiomyoctyesの単離を記載している。得られた細胞は、電気的に休止状態、のCa 2 +·トレラントであり、培養およびアデノ又は遺伝子発現を操作するためのレンチウイルスでトランスフェクトすることができる。それらの機能は、MMSYSシステムおよびパッチクランプ技術を用いて分析することができる。

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-culture-functional-characterization-adult-mouse

Research

JoVE Journal

Biology

24.2K ビュー.  Beth Israel Deaconess Medical Center, Harvard Medical School. ここでは、ランゲンドルフ灌流システムを使用して、成体マウスcardiomyoctyesの単離を記載している。得られた細胞は、電気的に休止状態、のCa 2 +·トレラントであり、培養およびアデノ又は遺伝子発現を操作するためのレンチウイルスでトランスフェクトすることができる。それらの機能は、MMSYSシステムおよびパッチクランプ技術を用いて分析すること?...

ビデオ: Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

成体マウスから海馬歯状回の解剖

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

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 ...

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 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, 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.

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 ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

成体マウスから洞房結節筋細胞の単離、培養、および機能解析のための方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

分離と性質、大人マウス心臓から心臓の幹細胞の分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心臓線維芽細胞と筋線維芽細胞の分離と特徴付け

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...