Name: simultaneous isolation of high quality cardiomyocytes, endothelial cells, and fibroblasts from an adult rat heart
Uploaded: 2017-05-19T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart at JoVE.com

L. Rinaldi、S.Schäffer、D. Reitz、H. Thomas、A. Weberの技術サポートは感謝しています。著者はまた、広範囲にわたる校正と原稿の言語編集に感謝したいと思います。この研究はGiessen Anschubsfinanzierungの助成によりM. AslamとD.Gündüzに付与されました。

この調査は、米国国立衛生研究所（NIH Publication No. 85-23 1985）に掲載された実験動物の管理と使用のガイドラインに準拠し、Giessen大学の地域倫理委員会によって承認された。この研究では体重200〜250gの成体雄Wistar系ラットを用いた。 1.オートクレーブ<ol><li>処置を行う前の少なくとも1日は、すべての手術器具、ピペットチップ、パスツールピペットを121℃で30分間、オ?...

<ol>
	<li>Zaragoza, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+cardiovascular+diseases.">Animal models of cardiovascular diseases.</a> J Biomed Biotechnol. 2011, 497841 (2011).</li><li>Claycomb, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HL-1+cells:+a+cardiac+muscle+cell+line+that+contracts+and+retains+phenotypic+characteristics+of+the+adult+cardiomyocyte.">HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.</a> Proc Natl Acad Sci U S A. 95 (6), 2979-2984 (1998).</li><li>Barbieri, S. S., Weksler, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke+cooperates+with+interleukin-1beta+to+alter+beta-catenin+trafficking+in+vascular+endothelium+resulting+in+increased+permeability+and+induction+of+cyclooxygenase-2+expression+in+vitro+and+in+vivo.">Tobacco smoke cooperates with interleukin-1beta to alter beta-catenin trafficking in vascular endothelium resulting in increased permeability and induction of cyclooxygenase-2 expression in vitro and in vivo.</a> FASEB J. 21 (8), 1831-1843 (2007).</li><li>Piper, H. M., Probst, I., Schwartz, P., Hutter, F. J., Spieckermann, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+of+calcium+stable+adult+cardiac+myocytes.">Culturing of calcium stable adult cardiac myocytes.</a> J Mol Cell Cardiol. 14 (7), 397-412 (1982).</li><li>Xu, X., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+adult+rat+heart+myocytes.">Primary culture of adult rat heart myocytes.</a> J Vis Exp. (28), (2009).</li><li>G&#252;nd&#252;z, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+extracellular+ATP+protects+against+acute+reperfusion+injury+in+rat+heart+endothelial+cells.">Accumulation of extracellular ATP protects against acute reperfusion injury in rat heart endothelial cells.</a> Cardiovasc.Res. 71 (4), 764-773 (2006).</li><li>Brilla, C. G., Zhou, G., Matsubara, L., Weber, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+metabolism+in+cultured+adult+rat+cardiac+fibroblasts:+response+to+angiotensin+II+and+aldosterone.">Collagen metabolism in cultured adult rat cardiac fibroblasts: response to angiotensin II and aldosterone.</a> J Mol Cell Cardiol. 26 (7), 809-820 (1994).</li><li>Shahzad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+involved+in+postconditioning+protection+of+cardiomyocytes+against+acute+reperfusion+injury.">Mechanisms involved in postconditioning protection of cardiomyocytes against acute reperfusion injury.</a> J Mol Cell Cardiol. 58, 209-216 (2013).</li><li>G&#252;nd&#252;z, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+Stabilizes+Microvascular+Endothelial+Barrier+Function+via+Phosphatidylinositol+3-Kinase/Akt-Mediated+Rac1+Activation.">Insulin Stabilizes Microvascular Endothelial Barrier Function via Phosphatidylinositol 3-Kinase/Akt-Mediated Rac1 Activation.</a> Arterioscler.Thromb.Vasc.Biol. 30, 1237-1245 (2010).</li><li>Lipps, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-terminal+fragment+of+cardiac+myosin+binding+protein-C+triggers+pro-inflammatory+responses+in+vitro.">N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.</a> J Mol Cell Cardiol. 99, 47-56 (2016).</li><li>Abdallah, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+Ca2++cycling+and+mitochondrial+permeability+transition+pores+promotes+reperfusion-induced+injury+of+cardiac+myocytes.">Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes.</a> J Cell Mol Med. 15 (11), 2478-2485 (2011).</li><li>G&#252;nd&#252;z, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ticagrelor+on+endothelial+calcium+signalling+and+barrier+function.">Effect of ticagrelor on endothelial calcium signalling and barrier function.</a> Thromb Haemost. , (2016).</li><li>G&#252;nd&#252;z, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+effects+of+ATP+and+adenosine+on+barrier+function+of+rat+coronary+microvasculature.">Opposing effects of ATP and adenosine on barrier function of rat coronary microvasculature.</a> J Mol.Cell Cardiol. , (2012).</li></ol>

この記事では、心筋細胞、内皮細胞、および線維芽細胞の単離および培養のための再現可能なプロトコールが記載されている。このプロトコルは、ただ1つの細胞型ではなく、心臓組織、 すなわち心筋細胞、内皮細胞および線維芽細胞の主要な細胞型の同時かつ高品質の単離を記載する。単離手順の重要なステップは、心臓組織の適切な消化である。消化が不完全であれば、多数の筋?...

げっ歯類のモデルは、健康や病気の心血管生理学の理解を広げるためのツールとして長い間使われてきました。これらの動物モデルは、臓器レベルで疾患の病態生理を理解し、心血管疾患の治療に使用される様々な薬理学的薬剤の薬物動態および薬力学を分析することを可能にするが、心血管疾患発症の分子メカニズムの理解および細胞型は、 インビトロ細胞培養モデルの使用を必要とする。この目的のために、心臓血管系からの異なる不死化細胞株が開発された。しかしながら、新たに単離された初代細胞は、生理学的および機能的に生きた組織および生物に関連している。 心臓は、心臓血管のあらゆる主要なタイプの細胞を含む汎用性のある臓器であるラット心臓は依然として心臓血管生理学の理解のために一般的に使用されているモデルである。ここ数十年の間に、個々の細胞型を心臓組織から単離するための異なる方法が記載されている。しかし、これらの方法は、ある特定の細胞型の単離にのみ焦点を当て、細胞分析のためにもはや使用することができない他の型の細胞を喪失させる。ここでは、主要な細胞タイプの心臓組織、すなわち心筋細胞、内皮細胞および線維芽細胞の同時かつ高品質の単離を可能にする最適化されたプロトコールが記載されている。これらの細胞型はすべて、異なる実験設定8,9,10および同じ動物の細胞 - 細胞相互作用の分析に使用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="717">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">anti-vWF</td>
			<td style="width:133px;">Santa Cruz Biotech.</td>
			<td style="width:119px;">SC-14014</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">102378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carnitin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Collagenase type II</td>
			<td style="width:133px;">Worthington</td>
			<td style="width:119px;">LS004176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Creatin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">D-Glucose</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">108342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dil-Ac-LDL</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">L3484</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">EDTA Solution (0.2 M)</td>
			<td style="width:133px;">Biochrome AG</td>
			<td style="width:119px;">L2113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Embeding solution</td>
			<td style="width:133px;">Citiflour</td>
			<td style="width:119px;">AF1-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Endothelial cell medium MV2</td>
			<td style="width:133px;">PromoCell</td>
			<td style="width:119px;">C-22022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Foetal calf serum (FCS)</td>
			<td style="width:133px;">Biochrome AG</td>
			<td style="width:119px;">S0615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Gentamicin</td>
			<td style="width:133px;">Serva Chemicals</td>
			<td style="width:119px;">47991</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">HEPES</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Isoflurane</td>
			<td style="width:133px;">Abbott</td>
			<td style="width:119px;">TU 061219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Laminin&#160;</td>
			<td style="width:133px;">Roche/Sigma</td>
			<td style="width:119px;">11243217001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">M199 medium</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">11150059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">M199 medium (Powder)</td>
			<td style="width:133px;">Biochrome AG</td>
			<td style="width:119px;">T061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Magnesium sulphate</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">63138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mouse anti-rat CD31 antibody (TLD-3A12)</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">MA1-81051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">NaCl solution (0.9%), Sterile</td>
			<td style="width:133px;">B. Braun</td>
			<td style="width:119px;">30820080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Pan mouse IgG beads (Dynabeads)</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">11041</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Paraformaldehyde (PFA) 4% Solution</td>
			<td style="width:133px;">Santa Cruz Biotech.</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-Streptomycin</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Phosphate buffer saline (PBS) 1x&#160;</td>
			<td style="width:133px;">PAN-Biotech</td>
			<td style="width:119px;">P04-36500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plastic consumables</td>
			<td style="width:133px;">Greiner Bio-One</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Potassium Chloride</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">4933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Potassium dihydrogen phosphate</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">7873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium Chloride</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">6404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium Hydroxide Solution (2 N)</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">109136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sterile filtration system</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">5660020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Taurine</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">TO-PRO</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">T3605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypsin-EDTA Solution (10X)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Water, Sterile</td>
			<td style="width:133px;">B. Braun</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous isolation of high quality cardiomyocytes, endothelial cells, and fibroblasts from an adult rat heart

ラットは、心臓血管研究で使用される重要な動物モデルであり、心臓肥大、線維症、およびアテローム性動脈硬化症のような心臓血管疾患進行の分子メカニズムのインビトロ分析のためにラットの心臓細胞が日常的に使用される。これらの細胞機構を理解するために、循環系から不死化細胞株を開発するために様々な試みがなされてきたが、初代細胞は、そのような研究のためにより自然かつ近似したin vivo環境を提供する。したがって、特定の細胞型に取り組んでいる異なる研究室では、目的のラット心臓細胞の個々の型を単離するためのプロトコールが開発されている。しかし、複数の細胞タイプの分離を可能にするプロトコルは欠けている。ここでは、単一の調製物から高品質の主要な心臓細胞型（心筋細胞、内皮細胞および線維芽細胞）の単離を可能にする最適化されたプロトコールが記載されており、細胞分析に使用する。これにより、利用可能なリソースを最も効率的に使用することができ、時間を節約し、研究費を削減することができます。

<html>単離手順は、計画された実験のために使用することができる70〜80％の生存可能な棒状の線条体心筋細胞（ 図2Aおよび2C ）の収率をもたらす。私たちの研究室では、心筋細胞はCa 2+シグナル伝達の解析に日常的に使用されています。 図5Aは、Fura-AM（5μM）を負荷した心筋細胞における虚血/再灌流に応答する細胞内カル?...

Watch this Scientific Journal Video about Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart at JoVE.com

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

ラット心臓から様々な心臓細胞型を単離するためのいくつかのプロトコールが開発され、記載されている。ここでは、単一の調製物から高品質の主要な心臓細胞型（心筋細胞、内皮細胞および線維芽細胞）の単離を可能にし、実験コストを削減する最適化プロトコルが記載されている。

成体ラット心臓からの高品質心筋細胞、内皮細胞および線維芽細胞の同時単離

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

The overall goal of this procedure is to simultaneously isolate viable cardiomyocytes, endothelial cells, and fibroblasts from the rat heart for their individual in vitro analyses. This method can help answer key questions in the cardiovascular field about the signaling mechanisms involved in cardiac hypertrophy, ischemia reperfusion injury, endothelial function, and cardiac fibrosis. The main advantage of this technique is that all of the major cardiac cells can be isolated simultaneously, potentially reducing the associated research costs and experimental animal numbers.After one distilled water and one 50 milliliter Powell medium flush, replace the medium with 80 milliliters of fresh Powell medium, and use a glass Pasteur pipette from the glass cylinder to continuously perfuse the medium with carbogen. Next, use two pairs of thin curved forceps to mount a freshly harvested rat heart via the aorta onto the Langendorff perfusion system, and place the end of the perfusion system cannula between the first aortic branch and the aortic valve. Fix the aorta with a crocodile clamp.Open the valve of the cannula, and then fix the heart with a surgical suture. Allow 35 milliliters of the perfusion medium to pass through the heart drop wise, removing any residual blood. Place the collecting funnel under the heart, and start the peristaltic pump to recirculate the perfusion medium from the collecting funnel to the reservoir.Add collagenase solution to the perfusion medium, and perfuse the heart with the recirculating collagenase solution for 30 minutes. At the end of the digestion, use scissors to remove the heart from the Langendorff system and place it in a glass Petri dish. Now remove both atria and any leftover fatty tissue from the heart.To avoid epithelial cell contamination, use two forceps to carefully peel away the pericardium. Cut the heart down the middle, and place the halves into the tissue chopper. Mince the heart two to three times, and transfer the tissue pieces into a 15 milliliter conical tube containing 12 milliliters of digestion buffer from the Langendorff perfusion.Digest the tissue fragments in a water bath for five to 10 minutes, occasionally mixing with a five milliliter disposable plastic pipette. Then filter the cell suspension through a 100 micrometer sieve into a 50 milliliter conical tube. Flush the perfusion system with a minimum of one liter of distilled water followed by 100 milliliters of 0.1 normal sodium hydroxide for 30 minutes, then flush the system with another two to three liters of distilled water, and dry the apparatus with flushed air.Collect the filtered cells by centrifugation, and use a disposable pipette to carefully transfer the endothelial cell and fibroblast containing supernatant into a new 50 milliliter tube. Resuspend the pellet in six milliliters of calcium solution one. After one minute, collect the cells with a second centrifugation, and resuspend the pellet in six milliliters of calcium solution two.After one minute, dilute the cell suspension with the addition of 12 milliliters of calcium solution three, and mix well by gentle tilting. After another centrifugation, resuspend the pellet in 20 milliliters of pre-warmed CCT medium. Then aliquot one milliliter of cells to each of 20 sterile 35 millimeter cell culture dishes pre-coated with laminin, and place the cells in a carbon dioxide free cell culture incubator, replacing the medium with two milliliters of fresh CCT medium to remove any dead cells after two hours.Now centrifuge the endothelial cell and fibroblast containing supernatant, and resuspend the pellet in 1.5 milliliters of endothelial cell isolation buffer. Transfer the cells to a two milliliter sample tube, and add mouse anti-rat CD31 antibody to the cells for a 30 minute incubation at four degrees Celsius with end to end rotation. At the end of the incubation, add Pan anti-mouse IGG beads for a 20 in incubation at four degrees Celsius, with end to end rotation.At the end of the bead incubation, place the cells in a magnetic rack for two minutes, and use a one milliliters pipette to carefully remove the mostly fibroblast containing supernatant. See the fibroblasts on a 10 centimeter culture dish containing 10 milliliters of fibroblast cell culture medium, and place the cells in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide for one hour. Next, add one milliliter of wash buffer to the tube of endothelial cells and cap the tube.Gently shake the cells four to five times to resuspend the beads. Then return the tube to the magnet, and remove the wash buffer after one minute. After the last wash, replace the wash buffer with one milliliter of MV2 endothelial cell culture medium, and seed the endothelial cells into a single well of a 12 well plate, for their overnight culture in the cell culture incubator.At the end of the fibroblast incubation, wash the attached cells with an appropriate volume of pre-warmed PBS two to three times. Then, add pre-warmed fresh fibroblast cell culture medium to the cells, and return the fibroblasts to the cell culture incubator. The next morning, replace the supernatant on the endothelial cell culture with fresh endothelial cell culture medium, and return the cells to the incubator, replacing the medium every two to three days, until semi-confluency.Then wash the culture of attached fibroblast cells with fresh pre-warmed PBS as just demonstrated, and feed the cells with fresh pre-warmed medium. The cardiomyocyte isolation procedure yields a 70 to 80%pure, viable, rod-shaped, striated cardiac cell population. Intracellular calcium oscillation analysis of isolated cardiomyocytes loaded with fura AM, in response to ischemia repurfusion in cardiomyocytes can then be performed, as well as analysis of the changes in calcium signaling in magnetic bead isolated endothelial cells and fibroblasts, after the addition of ATP.Once mastered, this technique can be completed in three hours if it performed properly. While attempting this procedure, it is important to remember to set up this perfusion system properly, and to promptly fix the heart in the system as soon as it is ready. After it's development, this technique paved the way for researchers in the field of cardiovascular research to explore the signaling involved in different cardiovascular pathophysiologies.After watching this video, you should have a good understanding of the basic principals of cardiac cell isolation and culture. Don't forget that working with surgical instruments and cell culture reagents can be extremely hazardous, and that precautions, such as careful usage of the instruments and the use of gloves should always be taken while performing this procedure.

Research

JoVE Journal

Biology

Primary Culture of Adult Rat Heart Myocytes

成体ラットの心筋細胞の初代培養

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

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

大人の線維芽細胞から再プログラムヒト人工多能性幹細胞のコロニーを選択し、分離

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation of Embryonic Ventricular Endothelial Cells

胚心室内皮細胞の単離

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Murine Valve Endothelial Cells

ネズミ弁内皮細胞の単離

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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

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

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 ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

免疫細胞化学とプロイド分析のための固定心からの心筋細胞の分離

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...