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 및 A. Weber의 기술 지원은 감사하게 생각합니다. 저자는 광범위한 증거 교정과 원고의 언어 편집을 위해 Dr. E. Martinson에게 감사드립니다. 이 연구는 Giessen Anschubsfinanzierung 대학의 M. Aslam과 D. Gündüz 연구원의 지원을 받았다.

이 조사는 미 국립 보건원 (NIH Publication No. 85-23 1985)에 의해 출판 된 실험실 동물의 관리 및 사용 안내서에 따르며 Giessen 대학 지역 윤리위원회의 승인을 받았습니다. 본 연구에서는 200-250 g의 성인 남성 Wistar 쥐를 사용 하였다. 1. 고압 증기 멸균 <ol><li> 절차가 수행되기 적어도 하루 전에 121 ° C에서 30 분 동안 격리 절차에 사용되는 모든 수술 도구, 피펫 팁 및 파스퇴르 피펫?...

<ol>
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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>

이 기사에서는 심장 근세포, 내피 세포 및 섬유 아세포의 분리 및 배양에 대한 재현 가능한 프로토콜에 대해 설명합니다. 이 프로토콜은 단지 하나의 세포 유형에 반대 cardiomyocytes, 내피 세포, 그리고 섬유 아 세포와 같은 심장 조직의 주요 세포 유형의 동시 고품질 분리를 설명합니다. 격리 절차의 중요한 단계는 심장 조직의 적절한 소화입니다. 소화가 불완전하다면 많은 양의 근육 세포가...

설치류 모델은 오랫동안 건강과 질병에서 심혈 관계 생리학에 대한 우리의 이해를 넓히는 도구로 사용되어 왔습니다. 1 이 동물 모델은 기관 수준에서 질병의 병리 생리학을 이해하고 심혈관 질환 치료에 사용되는 다양한 약리학 적 약물의 약물 동력학 및 약력학을 분석하고 심혈관 질환 발병의 분자 메커니즘 및 특정 세포 유형은 시험 관내 세포 배양 모델의 사용을 필요로한다. 이 목적을 위해 심혈관 시스템으로부터의 상이한 불멸화 된 세포주가 개발되었다. 2 , 3 그러나 새로 분리 된 1 차 세포는 생리 학적으로 기능적으로 살아있는 조직과 유기체와 관련이 있습니다. 심장은 심장 혈관의 모든 주요 유형의 세포를 포함하는 다용도 장기입니다.ystem, 쥐 심장은 여전히 ​​심혈관 생리학의 이해를 위해 일반적으로 사용되는 모델입니다. 지난 수십 년 동안 심장 조직으로부터 개별 세포 유형을 분리하기위한 여러 가지 방법이 설명되었습니다. 4 , 5 , 6 , 7 그러나 이러한 방법은 특정 세포 유형의 분리에만 초점을 맞추어 더 이상 세포 유형을 잃어 버려 세포 분석에 더 이상 사용할 수 없습니다. 여기에는 심장 조직, 즉 심근 세포, 내피 세포 및 섬유 아세포의 주요 세포 유형을 동시에 고품질로 분리 할 수있는 최적화 된 프로토콜이 설명되어 있습니다. 이러한 모든 세포 유형은 다른 실험 설정 8 , 9 , 10 및 동일한 동물의 세포 - 세포 상호 작용 분석에 사용할 수 있습니다.

<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

쥐는 심장 혈관 연구에 사용되는 중요한 동물 모델이며, 랫트 심장 세포는 심근 비대, 섬유증 및 죽상 동맥 경화증과 같은 심혈관 질환 진행의 분자 메커니즘에 대한 생체 외 분석을 위해 일상적으로 사용됩니다. 이러한 세포 메커니즘을 이해하기 위해 심혈관 시스템에서 불후의 세포주를 개발하기 위해 다양한 성공을 거둔 여러 시도가 있었지만, 일차 세포는 이러한 연구를 위해보다 자연스럽고 가까운 생체 내 환경을 제공합니다. 따라서 특정 세포 유형에 종사하는 여러 실험실에서는 관심있는 쥐 심장 세포의 개별 유형을 분리하는 프로토콜을 개발했습니다. 그러나 하나 이상의 셀 유형을 격리 할 수있는 프로토콜이 없습니다. 여기에는 하나의 준비에서 고품질의 주요 심장 세포 유형 (cardiomyocytes, endothelial 세포 및 fibroblasts)의 격리를 허용하고 최적화 된 프로토콜을 설명합니다세포 분석에 사용. 이를 통해 사용 가능한 리소스를 가장 효율적으로 사용할 수 있으므로 시간을 절약하고 연구 비용을 줄일 수 있습니다.

<html> 분리 절차는 계획된 실험에 사용될 수있는 70-80 % 생존 가능하고 막대 모양의 줄무늬 심근 세포 ( 그림 2A 및 2C )의 수율을 초래합니다. 우리의 실험실에서 cardiomyocytes는 일상적으로 칼슘 2 + 신호 분석에 사용됩니다. 그림 5A 는 Fura - AM (5 μm의)와로드 된 cardiomyocytes의 허혈 / reperfusion에 대한 응답으로 세포 내 칼슘 [칼슘 2...

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

쥐 심장에서 다른 심장 세포 유형의 분리를 위해 몇 가지 프로토콜이 개발 및 기술되었습니다. 여기에는 실험 준비 비용을 줄이기 위해 단일 준비에서 고품질 주요 심장 세포 유형 (cardiomyocytes, endothelial cell, fibroblast)을 분리 할 수있는 최적화 된 프로토콜이 설명되어 있습니다.

성인 쥐 심장에서 고품질 Cardiomyocytes, 내피 세포 및 섬유 아세포의 동시 격리

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.

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JoVE Journal

Biology

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