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。

<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>

著者は宣言することは何もない。

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 ...

simultaneous-isolation-high-quality-cardiomyocytes-endothelial-cells

Research

JoVE Journal

Biology

13.2K ビュー.  University Hospital Giessen. 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。 

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

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

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

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

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

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

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

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

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

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

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

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

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

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 ...

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 ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

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 ...