Name: development of a cell co-culture model to mimic cardiac ischemia/reperfusion in vitro
Uploaded: 2021-10-13T14:00:00
Description: Watch this Scientific Journal Video about Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro at JoVE.com

这项工作部分得到了美国退伍军人事务部生物医学实验室研发服务（I01 BX003482）和M.L.R.机构资金的支持。

1. 实验准备/电镀
<ol><li>根据制造商的说明维护CM和EC。<ol>
<li>当两个细胞系从供应商处到达时，解冻它们。用新鲜培养基洗涤后，将板放入T25烧瓶中。建议从购买细胞的同一供应商处购买每种细胞培养基。第二天，用培养基刷新细胞，并在汇合时使用。</li>
		<li>将细胞培养箱保持在37°C，21%O2，5%CO2，74%N2 并保持加湿。 注意：从C57BL / 6小鼠的冠状动脉中分离...

<ol>
	<li>Hausenloy, D. J., 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=Ischaemic+conditioning+and+targeting+reperfusion+injury:+a+30+year+voyage+of+discovery.">Ischaemic conditioning and targeting reperfusion injury: a 30 year voyage of discovery.</a> Basic Research in Cardiology. 111 (6), 70 (2016).</li><li>Hausenloy, D. J., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+ischemia-reperfusion+injury:+a+neglected+therapeutic+target.">Myocardial ischemia-reperfusion injury: a neglected therapeutic target.</a> The Journal of Clinical Investigation. 123 (1), 92-100 (2013).</li><li>Gottlieb, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+pathways+in+acute+ischemia/reperfusion+injury.">Cell death pathways in acute ischemia/reperfusion injury.</a> Journal of Cardiovascular Pharmacology and Therapeutics. 16 (3-4), 233-238 (2011).</li><li>Colliva, A., Braga, L., Giacca, M., Zacchigna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell-cardiomyocyte+crosstalk+in+heart+development+and+disease.">Endothelial cell-cardiomyocyte crosstalk in heart development and disease.</a> The Journal of Physiology. 598 (14), 2923-2939 (2020).</li><li>Bogdanowicz, D. R., Lu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+cell-cell+communication+in+co-culture.">Studying cell-cell communication in co-culture.</a> Biotechnology Journal. 8 (4), 395-396 (2013).</li><li>Salzman, M. M., Bartos, J. A., Yannopoulos, D., Riess, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poloxamer+188+protects+isolated+adult+mouse+cardiomyocytes+from+reoxygenation+injury.">Poloxamer 188 protects isolated adult mouse cardiomyocytes from reoxygenation injury.</a> Pharmacology Research &amp; Perspectives. 8 (6), 00639 (2020).</li><li>Kalogeris, T., Baines, C. P., Krenz, M., Korthuis, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+ischemia/reperfusion+injury.">Cell biology of ischemia/reperfusion injury.</a> International Review of Cell and Molecular Biology. 298, 229-317 (2012).</li><li>Martindale, J. J., Metzger, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+of+increased+cellular+oxidative+stress+and+myocardial+ischemia+reperfusion+injury+by+directed+sarcolemma+stabilization.">Uncoupling of increased cellular oxidative stress and myocardial ischemia reperfusion injury by directed sarcolemma stabilization.</a> Journal of Molecular and Cellular Cardiology. 67, 26-37 (2014).</li><li>vander Meer, A. D., Orlova, V. V., ten Dijke, P., vanden Berg, A., Mummery, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+co-cultures+of+human+endothelial+cells+and+embryonic+stem+cell-derived+pericytes+inside+a+microfluidic+device.">Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device.</a> Lab on a Chip. 13, 3562-3568 (2013).</li><li>Bidarra, S. J., 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=Phenotypic+and+proliferative+modulation+of+human+mesenchymal+stem+cells+via+crosstalk+with+endothelial+cells.">Phenotypic and proliferative modulation of human mesenchymal stem cells via crosstalk with endothelial cells.</a> Stem Cell Research. 7, 186-197 (2011).</li><li>Campbell, J. J., Davidenko, N., Caffarel, M. M., Cameron, R. E., Watson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifunctional+3D+co-culture+system+for+studies+of+mammary+tissue+morphogenesis+and+stem+cell+biology.">A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.</a> PloS One. 6, 25661 (2011).</li><li>Mehes, E., Mones, E., Nemeth, V., Vicsek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+motion+of+cells+mediates+segregation+and+pattern+formation+in+co-cultures.">Collective motion of cells mediates segregation and pattern formation in co-cultures.</a> PloS One. 7, 31711 (2012).</li><li>Miki, 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=The+advantages+of+co-culture+over+mono+cell+culture+in+simulating+in+vivo+environment.">The advantages of co-culture over mono cell culture in simulating in vivo environment.</a> The Journal of Steroid Biochemistry and Molecular Biology. 131 (3-5), 68-75 (2012).</li><li>Moraes, C., Mehta, G., Lesher-Perez, S. C., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organs-on-a-chip:+a+focus+on+compartmentalized+microdevices.">Organs-on-a-chip: a focus on compartmentalized microdevices.</a> Annals of Biomedical Engineering. 40 (6), 1211-1227 (2012).</li><li>Campbell, J., Davidenko, N., Caffarel, M., Cameron, R., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifunctional+3D+co-culture+system+for+studies+of+mammary+tissue+morphogenesis+and+stem+cell+biology.">A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.</a> PLoS One. 6, 25661 (2011).</li><li>Wu, M. H., Huang, S. B., Lee, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+cell+culture+systems+for+drug+research.">Microfluidic cell culture systems for drug research.</a> Lab on a Chip. 10, 939-956 (2010).</li></ol>

协议中的关键步骤 细胞共培养模型已被用于研究心脏保护的细胞机制。因此，如何创建两个独立的层，它们之间有有意义的距离，对于开发合适的共培养模型至关重要。研究模拟IR（即HR损伤）的一个挑战是，不仅缺血（缺氧）本身，而且再灌注（再氧合）也会加重细胞功能障碍。因此，一个现实的模型需要反映这些特征，例如，证明缺氧后再氧合的损伤充分增加，而不是单独缺氧...

缺血性心脏病是全世界死亡和残疾的主要原因1，2。然而，再灌注的治疗过程本身可引起心肌细胞死亡，称为心肌缺血/再灌注（IR）损伤，对此仍然没有有效的补救措施3。内皮细胞（EC）已被建议通过分泌旁分泌以及细胞间相互作用来保护心肌细胞（CMs） 4。
细胞共培养模型已被广泛用于研究自分泌和/或旁分泌细胞 - 细胞相互作用对细胞功能和分化的作用。在共培养模型中，混合共培养是最简单的，其中两种不同类型的细胞以所需的细胞比5在单个培养室内直接接触。然而，鉴于混合群体，细胞类型之间的单独处理和单细胞类型的下游分析并不容易实现。
先前的研究表明，缺氧和缺血性损伤对细胞膜的完整性造成显着损害，通过乳酸脱氢酶（LDH）的释放来测量。这种损伤在复氧时加重，类似于再灌注损伤6，7，8。当前方案的目标是测试假设，即EC的存在可以剂量依赖性地衰减由缺氧和再氧合（HR）引起的CM的细胞膜泄漏，并且可以通过改变两个细胞系之间的接触距离来优化EC的保护作用。因此，我们采用了三种类型的细胞培养插入物和小鼠原发性冠状动脉内皮细胞和成年小鼠心肌细胞。这些由康宁、默克密理博和格瑞纳Bio-One品牌设计的插入物使我们能够创建三种不同的细胞培养串扰条件，细胞间距离分别为0.5、1.0和2.0 mm。在每种情况下，每个刀片镀100，000个EC。
此外，为了确定共培养中ECs的密度是否有助于该模型中的HR损伤衰减，我们研究了EC浓度与CM释放LDH之间的剂量 - 反应关系。
本报告为调查人员提供了一种循序渐进的方法，使他们能够利用这一重要模型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Adult Mouse Cardiomyocytes (CMs)</td>
			<td >Celprogen Inc</td>
			<td >11041-14</td>
			<td >Isolated from adult C57BL/6J mouse cardiac tissue</td>
		</tr>
		<tr >
			<td >Automated Cell Counter Countess II</td>
			<td>Invitrogen</td>
			<td>A27977</td>
			<td>Cell counting for calculating cell numbers</td>
		</tr>
		<tr >
			<td >Bio-Safety Cabinet</td>
			<td>Nuaire</td>
			<td>NU425400</td>
			<td>Cell culture sterile hood</td>
		</tr>
		<tr >
			<td >Cell Culture Freezing Medium</td>
			<td>Cell Biologics Inc</td>
			<td>6916</td>
			<td>Used for cell freezing for long term cell line storage</td>
		</tr>
		<tr >
			<td >Cell Culture Incubator</td>
			<td>Nuaire</td>
			<td>Nu-5500</td>
			<td>To provide normal cell living condition (21%O2, 5%CO2, 74%N2, 37&#176;C, humidified)</td>
		</tr>
		<tr >
			<td >Cell Culture Incubator Gas Tank</td>
			<td>A-L Compressed Gases</td>
			<td>UN1013</td>
			<td>Gas needed for cell culture incubator&#160;</td>
		</tr>
		<tr >
			<td >Cell Culture Inserts A (0.5 mm)</td>
			<td>Corning Inc</td>
			<td>353095</td>
			<td>Used for EC-CM co-culture</td>
		</tr>
		<tr >
			<td >Cell Culture Inserts B (1.0 mm)</td>
			<td>Millicell Millipore</td>
			<td>PIHP01250</td>
			<td>Used for EC-CM co-culture</td>
		</tr>
		<tr >
			<td >Cell Culture Inserts C (2.0 mm)</td>
			<td>Greiner Bio-One</td>
			<td>662640</td>
			<td>Used for EC-CM co-culture</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td>Anstel Enterprises Inc</td>
			<td>4235</td>
			<td>For cell culture plating and passaging</td>
		</tr>
		<tr >
			<td >CMs Cell Culture Flasks T25</td>
			<td>Celprogen Inc</td>
			<td>E11041-14</td>
			<td>Used for CMs regular culture, coated by manufacturer</td>
		</tr>
		<tr >
			<td >CMs Cell Culture Medium Complete</td>
			<td>Celprogen Inc</td>
			<td>M11041-14S</td>
			<td>CMs culture complete medium</td>
		</tr>
		<tr >
			<td >CMs Cell Culture Medium Complete Phenol free</td>
			<td>Celprogen Inc</td>
			<td>M11041-14PN</td>
			<td>CMs culture medium without phenol red used during LDH measurement</td>
		</tr>
		<tr >
			<td >CMs Cell Culture Plates 96 well</td>
			<td>Celprogen Inc</td>
			<td>E11041-14-96well</td>
			<td>Used for experiments of LDH measurement, coated by manufacturer</td>
		</tr>
		<tr >
			<td >CMs Hypoxia Cell Culture Medium</td>
			<td>Celprogen Inc</td>
			<td>M11041-14GFPN</td>
			<td>CMs cell culture under hypoxic condition (glucose- and serum-free)</td>
		</tr>
		<tr >
			<td >Countess cell counting chamber slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td>Counting slides used for cell counter</td>
		</tr>
		<tr >
			<td >Cyquant LDH Cytotoxicity Kit</td>
			<td>Thermo Scientific&#160;</td>
			<td>C20301</td>
			<td>LDH measurement kit</td>
		</tr>
		<tr >
			<td >ECs Cell Culture Flasks T25</td>
			<td>Fisher Scientific&#160;</td>
			<td>FB012935</td>
			<td>Used for ECs regular culture</td>
		</tr>
		<tr >
			<td >ECs Cell Culture Medium Complete</td>
			<td>Cell Biologics Inc</td>
			<td>M1168</td>
			<td>ECs culture complete medium</td>
		</tr>
		<tr >
			<td >ECs Cell Culture Medium Complete Phenol free</td>
			<td>Cell Biologics Inc</td>
			<td>M1168PF</td>
			<td>ECs culture medium without phenol red used during LDH measurement</td>
		</tr>
		<tr >
			<td >ECs Cell Culture Plates 96 well</td>
			<td>Fisher Scientific (Costar)</td>
			<td>3370</td>
			<td>Used for experiments of LDH measurement</td>
		</tr>
		<tr >
			<td >ECs Culture Gelatin-Based Coating Solution</td>
			<td>Cell Biologics Inc</td>
			<td>6950</td>
			<td>Used for coating flasks and plates for ECs</td>
		</tr>
		<tr >
			<td >ECs Hypoxia Cell Culture Medium</td>
			<td>Cell Biologics Inc</td>
			<td>GPF1168</td>
			<td>ECs cell culture under hypoxic condition (glucose- and serum-free)</td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>MT35011CV</td>
			<td>FBS-HI USDA-approved for cell culture and maintenance</td>
		</tr>
		<tr >
			<td >Hypoxia Chamber</td>
			<td>StemCell Technologies</td>
			<td>27310</td>
			<td>To create a hypoxic condition with 0.01%O2 environment</td>
		</tr>
		<tr >
			<td >Hypoxia Chamber Flow Meter</td>
			<td>StemCell Technologies</td>
			<td>27311</td>
			<td>To connect with hypoxic gas tank for a consistent gas flow speed</td>
		</tr>
		<tr >
			<td >Hypoxic Gas Tank (0.01%O2 Cylinder)</td>
			<td>A-L Compressed Gases</td>
			<td>UN1956</td>
			<td>Used to flush hypoxic medium and chamber (0.01%O2/5%CO2/94.99N2)</td>
		</tr>
		<tr >
			<td >Microscope&#160;</td>
			<td>Nikon</td>
			<td>TMS</td>
			<td>To observe cell condition</td>
		</tr>
		<tr >
			<td >Mouse Primary Coronary Artery Endothelial Cells (ECs)</td>
			<td>Cell Biologics Inc</td>
			<td>C57-6093</td>
			<td>Isolated from coronary artery of C57BL/6 mice</td>
		</tr>
		<tr >
			<td >NUNC 15ML CONICL Tubes</td>
			<td>Fisher Scientific</td>
			<td>12565269</td>
			<td>For cell culture process, experiments, solution preparation etc.</td>
		</tr>
		<tr >
			<td >NUNC 50ML CONICL Tubes</td>
			<td>Fisher Scientific</td>
			<td>12565271</td>
			<td>For cell culture process, experiments, solution preparation etc.</td>
		</tr>
		<tr >
			<td >Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td>Used for cell washing during culture or experiments</td>
		</tr>
		<tr >
			<td >Plate Reader</td>
			<td>BioTek Instrument</td>
			<td>11120533</td>
			<td>Colorimetric or fluorometric plate reading</td>
		</tr>
		<tr >
			<td >Reaction 96 Well Palte (clear no lid)</td>
			<td>Fisher Scientific</td>
			<td>12565226</td>
			<td>Used for LDH measurement plate reading</td>
		</tr>
		<tr >
			<td >Trypsin/EDTA for CMs</td>
			<td>Celprogen Inc</td>
			<td>T1509-014</td>
			<td>1 x sterile filtered and tissue culture tested</td>
		</tr>
		<tr >
			<td >Trypsin/EDTA for ECs</td>
			<td>Cell Biologics Inc</td>
			<td>6914/0619</td>
			<td>0.25%, cell cuture-tested</td>
		</tr>
	</tbody>
</table>

development of a cell co-culture model to mimic cardiac ischemia/reperfusion in vitro

缺血性心脏病是全世界死亡和残疾的主要原因。再灌注可导致缺血以外的其他损伤。内皮细胞（EC）可以通过细胞间相互作用保护心肌细胞（CM）免受再灌注损伤。共培养可以帮助研究细胞 - 细胞相互作用的作用。混合共培养是最简单的方法，但由于分离处理和单细胞类型的下游分析不可行，因此受到限制。为了研究ECs是否可以以剂量依赖性方式衰减CM细胞损伤，以及是否可以通过改变两个细胞系之间的接触距离来进一步优化这种保护，我们使用小鼠原发冠状动脉内皮细胞和成年小鼠心肌细胞来测试三种类型的细胞培养插入物，其细胞间层距离在0.5， 分别为 1.0 毫米和 2.0 毫米。在仅CM中，通过乳酸脱氢酶（LDH）释放评估的细胞损伤在缺氧期间和再氧合后显着增加，当距离为2.0 mm时，与0.5和1.0 mm相比。当EC和CM几乎直接接触（0.5 mm）时，缺氧后CM的再氧合损伤只有轻度衰减。当空间距离为1.0 mm时，这种衰减显着增加，当距离为2.0 mm时，EC在缺氧和缺氧/再氧合期间都减轻了CM损伤，表明ECs需要足够的培养距离才能与CM串扰，以便分泌的信号分子可以循环并充分刺激保护途径。我们的研究结果首次表明，优化EC/CM共培养空间环境对于测试EC在模拟缺血/再灌注损伤CM保护中的作用提供了有利的 体外 模型。本报告的目标是为调查人员提供一种循序渐进的方法，以利用这一重要模型来发挥自己的优势。

本实验中使用的所有三种类型的插入物（A，B，C）具有相同的0.4μm孔径。它们之间的唯一区别是插入物到碱基的高度，这使得两个共培养的细胞层之间的距离分别为0.5，1.0和2.0 mm（图3），并且它们来自不同的供应商（有关详细信息，请参阅 材料表）。
为了建立一种 体外 共培养模型，其中两个细胞系的单独层经历HR以模拟IR损伤，我?...

Watch this Scientific Journal Video about Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro at JoVE.com

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

空间距离是评估分离内皮和心肌细胞层共培养模型中缺氧/再氧合损伤的关键参数，首次表明优化共培养空间环境对于为测试内皮细胞在心肌细胞保护中的作用提供有利的 体外 模型是必要的。

开发一种细胞共培养模型以模拟心脏缺血/体外再灌注

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells ...

我们的研究结果表明，优化内皮细胞心肌细胞共培养的空间环境对于提供有利的体外模型，用于测试内皮细胞在心肌细胞保护模拟缺血再繁殖损伤中的作用是必要的。细胞共培养模型已被广泛用于研究细胞 - 细胞相互作用对细胞功能和分化的作用。然而，在混合共培养中，细胞类型之间的单独处理和单细胞类型的下游分析并不容易实现。目前的研究旨在创建两个独立的细胞层，它们之间有有意义的距离，其中可以诱导缺氧再繁殖损伤，并研究结果和对单细胞类型的影响。通过解冻两个细胞系开始准备。用新鲜培养基洗涤后，将细胞接种在T25烧瓶中。第二天，用培养基刷新细胞，并在汇合时使用。将细胞培养箱保持在37摄氏度，含21%氧气，5%二氧化碳和74%氮气。保持加湿。在光学显微镜下估计心肌细胞系汇合度。从含有融合细胞培养物的烧瓶中取出培养基，并用每个烧瓶三至五毫升胰蛋白酶-EDTA胰蛋白酶消化。轻轻搅拌烧瓶，在37摄氏度下孵育两到五分钟。之后，在光学显微镜下评估酶的进展。分离后，通过将trysin或细胞溶液加入每个T25烧瓶中含有10毫升培养基的50毫升管中来灭活胰蛋白酶溶液。将细胞悬浮液以120×g离心两分钟，得到软细胞沉淀。离心后，除去上清液并将沉淀重悬于五毫升培养基中。接下来，进行细胞计数并确认细胞活力。混合并等分试样10微升重悬细胞和10微升台盼蓝染料，然后使用细胞计数器计数活细胞。用新鲜的常规培养基稀释细胞，以达到所需的接种密度。将24孔板用细胞外基质预涂覆，并将接种密度为每孔300， 000的板心肌细胞涂在板的底部。将细胞保持在37摄氏度，含5%二氧化碳过夜。24小时后，将内皮细胞以每插入物100， 000的最佳电镀密度将内皮细胞板放入插入物中。内皮细胞接种24小时后，将内皮细胞插入心肌细胞孔内并开始共培养。在进行实验之前，让细胞共培养12至24小时。通过将大约25毫升的培养基倒入50毫升锥形管中来制备缺氧培养基。用硅胶膜空气密封顶部，并使用无菌移液器打孔。之后，创建另一个孔，并将大约2/3的移液器浸没在介质中。以每分钟30升的流速用缺氧气体冲洗培养基5分钟，然后丢弃含有附着细胞的24孔板或培养插入物的培养基。用每孔100微升10%PBS非常温和地洗涤它们，向每个孔的板或插入物中加入500微升新鲜制备的缺氧培养基。用常氧对照组中含有葡萄糖和血清的新鲜正常培养基替换原始培养基。将装满无菌水的培养皿放入缺氧室中以加湿室。然后将含有缺氧基团的板放入腔室中。用缺氧气体以每分钟30升的流量冲洗缺氧室五分钟。之后，将腔室放在37摄氏度的培养箱内24小时。缺氧后，丢弃板或插入物的旧培养基，并在板的每种壁上加入500微升含有葡萄糖和血清的正常培养基，然后将板在正常培养条件下的培养箱内储存两小时以模拟再灌注。将细胞培养基从24孔板转移到96孔板中，然后从24孔板的每个孔中取出200微升培养基，并均匀分布到96孔板的四个孔中。使用96孔板中的细胞毒性测定试剂盒测量LDH的吸光度，并按照制造商的说明确定细胞损伤的程度。在这项研究中，评估了三种类型的插入物。所有三个插入物的孔径均为0.4微米。它们之间的唯一区别是插入物到碱基高度，允许两个共培养细胞层之间的距离为0.5，1.0和2.0毫米。使用该协议，在正常氧气条件下培养细胞，然后分成两组，常氧对照组和缺氧组。24小时后，用培养基刷新两组，并在正常氧气条件下再培养两小时，然后进行终点测定。本文比较了三种类型的培养插入物在正常氧、仅缺氧和缺氧再氧条件下评估乳酸脱氢酶释放的单独心肌细胞、单独内皮细胞和心肌细胞与内皮细胞共培养的分布。由于细胞层之间的距离为0.5毫米，与单独培养心肌细胞时的正常氧相比，缺氧导致LDH释放显着增加。然而，与仅缺氧组相比，缺氧复氧组的LDH仅略有增加。当内皮细胞和心肌细胞一起共培养时，仅缺氧条件下LDH释放的增加没有减弱。这表明，在仅缺氧条件下，与单独心肌细胞组相比，内皮细胞没有任何保护作用。然而，内皮细胞在HR期间对心肌细胞施加了温和但显着的保护。当细胞层之间的距离为1毫米时，仅在缺氧条件下，心肌细胞的LDH释放也显着增加。然而，与0.5毫米插入物不同，心肌细胞的LDH增加仅由HR优于仅缺氧条件增强。这种增加在两毫米插入时更加明显。将接种在两毫米插入物中的内皮细胞的浓度分别滴定为每插入物25，000，50，000和100，000个细胞。研究发现，共培养物中内皮细胞强度的增加导致HR引起的LDH释放的剂量依赖性衰减。然而，在常氧条件下没有看到这种效应。这种共培养方法不仅限于内皮细胞和心肌细胞。这些方法应有助于研究跨多个感兴趣器官的不同细胞系之间的特定细胞 - 细胞相互作用。

Research

JoVE Journal

Biology

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...