Name: isolation and cryopreservation of neonatal rat cardiomyocytes
Uploaded: 2015-04-09T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor&#8217;s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

以下のプロトコルは、新生児仔ラット（10〜14匹）の1リットルから心筋細胞を分離するために設計されています。産子数が大幅に異なる場合は、手順は、補償するためにスケーリングされなければならないことがある。子犬は約48±6時間古いすべきである。すべての手順は、ここにノースカロライナ州立大学施設内動物管理および使用委員会（IACUC）によって承認されている。 <p class="jove_ti...

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このプロトコルは、凍結保存され、単離されるべきNRCMsを可能にし、解凍した。細胞を解凍すると、手順の重要な部分である。 DMSOの希釈系列をゆっくりとセル14からDMSOを除去するために使用される。これは、細胞が、解凍後すぐに死ぬに特に敏感であるようにDMSOの抽出を迅速に行うことが重要である。多かれ少なかれ細胞は解凍のために必要とされる場合は、必要に応じて、DMSO溶?...

心筋細胞の細胞培養は、現代の心臓の研究における重要なツールである。単離および培養成体ラット心筋細胞1よりも容易であるので、新生児ラット心筋細胞（NRCMs）が一般的に使用される。 NRCM方法はまだ長い分離手順とディッシュ内の限られた細胞増殖を含む、いくつかの制限を有する。最も一般的な作業2-6の4-48時間を必要とNRCMsの単離のための多数のプロトコルがあります。さらに、細胞はしばしば2,4-7 1~2日齢のラットの仔から単離される。出産のタイミングは予測できないと研究室で他の作業と競合することができます。細胞のわずかな量は、実験に必要とされる場合に単離が非効率的で無駄にすることができる。分離時間を短縮することにワークフローのフォーカスを改善する上でほとんどの努力は、まだこれは子犬の出産のタイミングの問題を解決しない。 代替案として、多くのラボでは、利用胚性幹細胞（ESC）または人工多能性幹細胞（iPS細胞）に由来する心筋細胞。しかし、再プログラミングおよび/または分化のプロセスも非常に時間がかかり、コストがかかる可能性がある。 インビトロ筋細胞モデルとして、これらの細胞を使用した場合、他の問題が存在し得る。両方ESC-とiPSC-由来の心筋細胞を初代心筋8-10からの電気の違いを示すことが示されている。 解離NRCMs冷蔵11を用いて、数日間保存することが可能であり、まだこれは、長期保存を可能にしない。液体窒素は、典型的には、時間の大きな期間、細胞を保存するために使用されるが、ジメチルスルホキシド（DMSO）などの凍結防止剤を必要としている。これまでの研究では、凍結メディア5-10％DMSOの間の理想的な濃度はNRCMsの凍結保存を可能にすることが示されている、まだその時でさえ生存率は低い12のまま。 DMSOは、無料の間に細胞を保護するのに役立ちますが、熱意は、それは1.5％13以上の濃度で細胞に対して毒性であり得る。以前の研究は、ゆっくり細胞からDMSOを除去し、細胞生存率14を改善することができることを示している。 我々は、単離後に凍結保存細胞によるNRCM細胞ベースのアッセイの効率を改善しようとした。これは、細胞を解凍し、必要に応じて、単離および動物の消費の頻度を低減する際に使用することを可能にする。この方法を使用して、我々はそれがNRCMsを凍結保存し、後で使用するために解凍することが可能であることを示す。解凍後、細胞は、許容可能な生存能力を維持し、自然発生的にα-筋節アクチニン（α-SA）との契約について陽性であるNRCM文化を作り出す。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IMDM (+25mM HEPES +L-glutamine)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>With added 25mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (+Ca +Mg)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td>With added calcium and magnesium, pH 7.1-7.4</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Gibco</td>
			<td>25300-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS5</td>
			<td>BioLife Solutions</td>
			<td>205102</td>
			<td>Freezing media</td>
		</tr>
		<tr>
			<td>Cryogenic Vial</td>
			<td>Corning</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C1889-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m Cell Strainer</td>
			<td>Greiner Bio-One</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizing Vacuum Filter (0.22&#956;m)</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical</td>
			<td>Corning</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>Cellgro</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr Frosty Freezing Container</td>
			<td>ThermoScientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell EZ&#160;SLIDES</td>
			<td>Millipore</td>
			<td>PEZGS0416</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-sarcomeric actinin antibody</td>
			<td>Sigma</td>
			<td>A7811</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine</td>
			<td>BD Biosciences</td>
			<td>51-7581KZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cryopreservation of neonatal rat cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

新生児ラット心筋細胞の単離後、細胞を液体窒素中に凍結しており、少なくとも数ヶ月間保存することができる。典型的に解凍すると、トリパンブルー分析により決定生存率は40〜60％の間であろう。にもかかわらず、これは適切な播種し、培養して、細胞が（ 図1）を増殖する、他の細胞型より低い。収縮性を確認するために、細胞を、位相差顕微鏡を使用して画像化すること?...

Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

新生児ラット心筋細胞の単離と凍結保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

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