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% CO2, 74% N2 and keep it humidified. 
		NOTE: The Mouse Primary Coronary Artery Endothelial Cells used in this protocol are isolated from coronary arteries of C57BL/6 mice, and Adult Mouse Cardiomyocytes are isolated from adult C57BL/6J mouse hearts and commercially obtained (see Table of Materials).</li>
	</ol>
	</li>
	<li>Plate CMs under sterile conditions onto the bottom of 24-well plates (inferior layer). 24 h later, plate ECs into the insert (or superior portion) of the co-culture well. 24 h after EC plating, place EC inserts onto CM plate bases, starting the co-culture period. Allow the cells to co-culture for at least 12-24 h before use. These steps are described in detail below.
	<ol>
		<li>Estimate CM cell line confluency under a light microscope. When cells appear to be 90%-100% confluent in culture flasks, proceed with experimental plating.</li>
		<li>Remove the media from the flasks containing confluent cell cultures and trypsinize with 3-5 mL of Trypsin/Ethylenediaminetetraacetic acid (EDTA) for T25 flasks. Agitate the flask gently, incubate at 37 &#176;C for 2-5 min, and then assess the enzymatic progress under a light microscope. Once the cells start to round up, use a cell scraper to detach the cells from the surface.</li>
		<li>After detachment, inactivate the trypsin solution by adding the trypsin/cell solution to a 50 mL tube containing 10 mL of media for T25 flasks. Centrifuge the cell suspension at 120 x g for 2 min to obtain a soft cell pellet. Remove the supernatant and resuspend the pellet in 5 mL of media.</li>
		<li>To perform cell counting and confirm cell viability, mix an aliquot of 10&#160;&#181;L of resuspended cells and 10&#160;&#181;L of trypan blue dye. Count the living cells using a cell counter. Dilute the cells with fresh regular media to achieve the desired seeding densities. Volumes are calculated depending on the desired seed density and cell concentration of stock solutions. All plating should be performed under sterile conditions.</li>
		<li>Plate CMs at seeding densities of 300,000 per well onto the bottom of a 24-well plate pre-coated with extracellular matrix. Maintain the cells at 37 &#176;C with 21% O2 and 5% CO2 overnight.</li>
		<li>After 24 h, plate ECs into inserts at an optimal plating density of 100,000 per insert (culture area is 33.6 mm2), (Figure 1). After 24 h of EC plating, place EC inserts inside the CM wells, initiating co-culture. Allow cells to co-culture for 12-24 h before performing the experiments.</li>
		<li>Ensure that, by the time experiments are performed, both CMs and ECs have reached 80%-90% confluency.</li>
	</ol>
	</li>
</ol>
2. Hypoxia/reoxygenation to simulate ischemia/reperfusion injury&#160;&#160;In Vitro
NOTE: The following steps need to be performed as described, do not pause in-between.
<ol>
	<li>Prepare the hypoxic media by pouring ~25 mL of media in a 50 mL conical tube. Air-seal the top with a silicone membrane and use a sterilized pipette to punch a hole. Create another hole, this time leaving the pipette submerged approximately 2/3 into the media.</li>
	<li>Flush the media with hypoxic gas (0.0125% O2, 5% CO2, 94.99% N2) for 5 min at a flow rate of 30 L/min. This minimizes O2 dissolved in the media when hypoxia starts (step 2.5).</li>
	<li>Discard the media of the 24-well plates or culture inserts containing attached cells and wash with 100 &#181;L/well of 10% Phosphate Buffered Saline (PBS) very gently. Afterwards, add 500 &#181;L of the freshly prepared hypoxic media to each well of the plates or inserts. 
	NOTE: Hypoxia in the media is interrupted as briefly as possible during this step before true hypoxia for cells and media starts in step 2.5.
	<ol>
		<li>In the normoxic control group, replace the original media with fresh normal media containing glucose and serum.</li>
	</ol>
	</li>
	<li>Humidify the hypoxia chamber by placing a Petri-dish filled with sterile water within the chamber. Next, place the plates comprising the hypoxic groups into the chamber.</li>
	<li>Flush the hypoxia chamber with hypoxic gas (0.0125% O2, 5% CO2, 94.99% N2) for 5 min at a flow of 30 L/min. Place the chamber in the 37 &#176;C incubator for 24 h.</li>
	<li>After hypoxia, cultivate CMs and ECs under normal conditions. To do so, discard the old media of the plates/inserts, replace it with normal media (containing glucose and serum; 500 &#181;L of each well/insert) and store it in the incubator under normal culture conditions (21% O2, 5% CO2, 74% N2, 37 &#176;C) for 2 h to mimic reperfusion.</li>
	<li>To control for any effects of media replacement, change the media of normoxic cells at the same time as the hypoxic media is changed. 
	NOTE: Figure 2 provides a schematic overview of this protocol.</li>
</ol>
3. Endpoint assessment
<ol>
	<li>Transfer the cell culture media from the 24-well plate into a 96-well plate. Use 200 &#181;L of media from each well of the 24-well plate and equally distribute into four wells of the 96-well plate, accordingly. Use one plate each for normoxia versus hypoxia versus HR.</li>
	<li>Determine the degree of cellular injury, e.g., by measuring absorbance for LDH using a cytotoxicity assay kit following the manufacturer&#39;s instructions. Perform the assay in a 96-well plate as instructed in the assay protocol.</li>
</ol>
4. Statistics
<ol>
	<li>Display the parametric data as mean/standard deviation and non-parametric data as box plots with median/interquartile range. Adequate parametric versus non-parametric multiple comparisons tests with acceptable post-hoc tests to decrease the chance for a type-1 error should be performed. Significance is typically set at an alpha of 0.05 (two-tailed).</li>
	<li>For all the experiments carried out in the current study, perform at least three well replicates within one experiment. There were three to six repetitions of each experiment used for statistical analysis.</li>
</ol>

<ol><li>Hausenloy, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischaemic+conditioning+and+targeting+reperfusion+injury%3A+a+30+year+voyage+of+discovery%5BTitle%5D)+AND+%22Basic+Research+in+Cardiology%22%5BJournal%5D)">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/pubmed?term=((Myocardial+ischemia-reperfusion+injury%3A+a+neglected+therapeutic+target%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">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/pubmed?term=((Cell+death+pathways+in+acute+ischemia%2Freperfusion+injury%5BTitle%5D)+AND+%22Journal+of+Cardiovascular+Pharmacology+and+Therapeutics%22%5BJournal%5D)">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/pubmed?term=((Endothelial+cell-cardiomyocyte+crosstalk+in+heart+development+and+disease%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">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/pubmed?term=((Studying+cell-cell+communication+in+co-culture%5BTitle%5D)+AND+%22Biotechnology+Journal%22%5BJournal%5D)">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/pubmed?term=((Poloxamer+188+protects+isolated+adult+mouse+cardiomyocytes+from+reoxygenation+injury%5BTitle%5D)+AND+%22Pharmacology+Research+%26+Perspectives%22%5BJournal%5D)">Poloxamer 188 protects isolated adult mouse cardiomyocytes from reoxygenation injury.</a> Pharmacology Research & 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/pubmed?term=((Cell+biology+of+ischemia%2Freperfusion+injury%5BTitle%5D)+AND+%22International+Review+of+Cell+and+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Uncoupling+of+increased+cellular+oxidative+stress+and+myocardial+ischemia+reperfusion+injury+by+directed+sarcolemma+stabilization%5BTitle%5D)+AND+%22Journal+of+Molecular+and+Cellular+Cardiology%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+co-cultures+of+human+endothelial+cells+and+embryonic+stem+cell-derived+pericytes+inside+a+microfluidic+device%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((Phenotypic+and+proliferative+modulation+of+human+mesenchymal+stem+cells+via+crosstalk+with+endothelial+cells%5BTitle%5D)+AND+%22Stem+Cell+Research%22%5BJournal%5D)">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/pubmed?term=((A+multifunctional+3D+co-culture+system+for+studies+of+mammary+tissue+morphogenesis+and+stem+cell+biology%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">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/pubmed?term=((Collective+motion+of+cells+mediates+segregation+and+pattern+formation+in+co-cultures%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">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/pubmed?term=((The+advantages+of+co-culture+over+mono+cell+culture+in+simulating+in+vivo+environment%5BTitle%5D)+AND+%22The+Journal+of+Steroid+Biochemistry+and+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Organs-on-a-chip%3A+a+focus+on+compartmentalized+microdevices%5BTitle%5D)+AND+%22Annals+of+Biomedical+Engineering%22%5BJournal%5D)">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/pubmed?term=((A+multifunctional+3D+co-culture+system+for+studies+of+mammary+tissue+morphogenesis+and+stem+cell+biology%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Microfluidic+cell+culture+systems+for+drug+research%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">Microfluidic cell culture systems for drug research.</a> Lab on a Chip. 10, 939-956 (2010).</li>
</ol>

The authors declare no conflicts of interest.

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><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%O&lt;sub&gt;2&lt;/sub&gt;, 5%CO&lt;sub&gt;2&lt;/sub&gt;, 74%N&lt;sub&gt;2&lt;/sub&gt;, 37&amp;deg;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&amp;nbsp;</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&amp;nbsp;</td><td>C20301</td><td>LDH measurement kit</td></tr><tr><td>ECs Cell Culture Flasks T25</td><td>Fisher Scientific&amp;nbsp;</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%O&lt;sub&gt;2&lt;/sub&gt; 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%O&lt;sub&gt;2&lt;/sub&gt; Cylinder)</td><td>A-L Compressed Gases</td><td>UN1956</td><td>Used to flush hypoxic medium and chamber (0.01%O&lt;sub&gt;2&lt;/sub&gt;/5%CO&lt;sub&gt;2&lt;/sub&gt;/94.99N&lt;sub&gt;2&lt;/sub&gt;)</td></tr><tr><td>Microscope&amp;nbsp;</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...

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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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3.6K Views.  Vanderbilt University Medical Center. 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.

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

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Medicine

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Bioengineering

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Neuroscience

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

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

Summary

Explore More Videos

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Slicing and Culturing Pig Hearts under Physiological Conditions

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model