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

This work was supported, in part, by the US Department of Veterans Affairs Biomedical Laboratory R&#38;D Service (I01 BX003482) and by institutional funds to M.L.R.

1. Experimental preparation/plating
<ol>
	<li>Maintain CMs and ECs according to the manufacturer&#39;s instructions.
	<ol>
		<li>Thaw both cell lines when they arrive from vendors. Plate in T25 flasks after being washed with fresh media. It is recommended to purchase each cell culture media from the same vendors the cells were purchased from. The next day, refresh the cells with media and use when confluent.</li>
		<li>Maintain the cell culture incubator at 37 &#176;C with 21% O2, 5% CO...

<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>

Critical steps in the protocol 
Cell co-culture models have been used to study cellular mechanisms of cardioprotection. How to create two separate layers with a meaningful distance between them is, thus, crucial for the development of a suitable co-culture model. A challenge in studying simulated IR, i.e., HR, injury is that not only ischemia (hypoxia) itself but also reperfusion (reoxygenation) aggravates cellular dysfunction. Therefore, a realistic model needs to reflect these characteristics by, ...

Ischemic heart disease is the leading cause of death and disability worldwide1,2. However, the treatment process of reperfusion can itself cause cardiomyocyte death, known as myocardial ischemia/reperfusion (IR) injury, for which there is still no effective remedy3. Endothelial cells (ECs) have been suggested to protect cardiomyocytes (CMs) through the secretion of paracrine signals, as well as cell-to-cell interactions4.
Cell co-culture models have been used extensively to investigate the role of autocrine and/or paracrine cell-cell interactions on cell function and differentiation. Among co-culture models, mixed co-culture is the simplest, where two different types of cells are in direct contact within a single culture compartment at a desired cell ratio5. However, separate treatments between cell types and downstream analysis of a single cell type are not readily feasible given the mixed population.
Previous studies indicated that hypoxic and ischemic insults cause significant damage to the integrity of cell membrane as measured by the release of lactate dehydrogenase (LDH). This injury is worsened upon reoxygenation, mimicking reperfusion injury6,7,8. The goal of the current protocol was to test the hypotheses that the presence of ECs can dose-dependently attenuate cell membrane leakage of CMs caused by hypoxia and reoxygenation (HR) and that the protective effect of ECs can be optimized by varying the contact distance between the two cell lines. Thus, we employed three types of cell culture inserts and Mouse Primary Coronary Artery Endothelial Cells and Adult Mouse Cardiomyocytes. The inserts, branded by Corning, Merck Millipore, and Greiner Bio-One allowed us to create three different cell culture crosstalk conditions with inter-cell line distances of 0.5, 1.0, and 2.0 mm, respectively. 100,000 ECs were plated per insert in each case.
In addition, in order to determine whether the density of ECs in co-culture contributes to HR injury attenuation in this model, we studied the dose-response relationship between EC concentration and LDH release by CMs. ECs were plated at 25,000, 50,000 and 100,000 per insert, respectively, in the 2.0 mm insert.
This report provides a step-by-step approach for investigators to use this important model to their advantage.

<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

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells (ECs) can protect cardiomyocytes (CMs) from reperfusion injury through cell-cell interactions. Co-cultures can help investigate the role of cell-cell interactions. A mixed co-culture is the simplest approach but is limited as isolated treatments and downstream analyses of single cell types are not feasible. To investigate whether ECs can dose-dependently attenuate CM cell damage and whether this protection can be further optimized by varying the contact distance between the two cell lines, we used Mouse Primary Coronary Artery Endothelial Cells and Adult Mouse Cardiomyocytes to test three types of cell culture inserts which varied in their inter-cell layer distance at 0.5, 1.0, and 2.0 mm, respectively. In CMs-only, cellular injury as assessed by lactate dehydrogenase (LDH) release increased significantly during hypoxia and further upon reoxygenation when the distance was 2.0 mm compared to 0.5 and 1.0 mm. When ECs and CMs were in nearly direct contact (0.5 mm), there was only a mild attenuation of the reoxygenation injury of CMs following hypoxia. This attenuation was significantly increased when the spatial distance was 1.0 mm. With 2.0 mm distance, ECs attenuated CM injury during both hypoxia and hypoxia/reoxygenation, indicating that sufficient culture distancing is necessary for ECs to crosstalk with CMs, so that secreted signal molecules can circulate and fully stimulate protective pathways. Our findings suggest, for the first time, that optimizing the EC/CM co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of ECs in CM-protection against simulated ischemia/reperfusion injury. The goal of this report is to provide a step-by-step approach for investigators to use this important model to their advantage.

All three types of inserts (A, B, C) used in this experiment have the same pore size of 0.4 &#181;m. The only difference among them is the insert-to-base height, which allows distances between the two co-cultured cell layers to be 0.5, 1.0 and 2.0 mm, respectively, (Figure 3) and that they are from different vendors (for details see Table of Materials).
To establish an in vitro co-culture model with separate layers of two cell lines under...

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

Spatial distance is a key parameter in assessing hypoxia/reoxygenation injury in a co-culture model of separate endothelial and cardiomyocyte cell layers, suggesting, for the first time, that optimizing the co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection.

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

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

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