Name: Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model (Video) | JoVE Journal
Uploaded: 2011-11-05T14:00:00.000+00:00
Duration: 9 min 15 s
Description: Learn stem cell transplantation using an in vitro ischemia/reperfusion model with cardiac cells and mesenchymal stem cells. This method enables assessment of cell viability and therapeutic mechanisms.

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5 minutes

This work was supported by OTKA (Hungarian Scientific Research Fund) D45933, T049621, T&Eacute;T (Hungarian Science and Technology Foundation) A4/04, Arg-17/2006 and SIN, Bolyai, &Ouml;veges Fellowships and T&Aacute;MOP 4.2.2-08/1/KMR-2008-0004 and 4.2.1/B 09/1/KMR-2010-0001. OTKA 83803. We would like to thank William Gesztes for providing the voice-over.

1. Preparing H9c2 cardiomyoblast cells
H9c2 rat cardiomyoblasts were obtained from ATCC (Wesel, Germany) and expanded in high glucose (4.5 g/L) DMEM containing 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The H9c2 myoblast cell line is derived from embryonic rat heart, it is used as an in vitro model for both skeletal and cardiac muscle 8,9.
Prepare 12 well plate of H9c2 cells:
<ol>
	<li>Remove culture medium from Petri dish of H9c2 cells.</li>
	<li>Wash cells twice with 5 ml PBS and digest with 2-3 ml 0.05% trypsin/EDTA.</li>
	<li>Place Petri dish for 5 minutes in the 37°C incubator.</li>
	<li>Inhibit trypsin with 10 ml DMEM culture medium.</li>
	<li>Spin down the cells at 1200 RPM for 8 minutes.</li>
	<li>After removing the supernatant, resuspend cells in 1 ml DMEM culture medium and count cells with hemocytometer using Trypan Blue staining.</li>
	<li>Seed 30,000 H9c2 cells in 2 ml DMEM per well in a 12 well plate.</li>
	<li>Place the plate into the 37°C CO2 incubator for 24 hours.</li>
</ol>
2. Simulated ischemia/reperfusion
Ischemia/reperfusion was simulated in vitro by performing oxygen glucose deprivation (OGD) on H9c2 cell cultures. This procedure was performed on the stage of the Zeiss confocal microscope with the PeCon cell incubation system (Erbach, Germany) allowing the observation of the cells during OGD. 
<ol>
	<li>Remove medium by aspiration from 12 well plate of H9c2 cells.</li>
	<li>Wash cells twice with PBS.</li>
	<li>Add 3 ml glucose free medium per well.</li>
	<li>Place the plate into the cell incubation system.</li>
	<li>Start nitrogen gas to purge the oxygen out of the incubation system.</li>
	<li>Wait until the level of oxygen reaches 0.5% then start the timer. 0.5% oxygen means approximately 3 mmHg partial tension. Subject cells to 150 minutes of simulated ischemia.</li>
	<li>At the end of simulated ischemia, remove the glucose free medium from the cells.</li>
	<li>Pipette 2 ml normal culture medium to the wells.</li>
	<li>Place the 12 well plate for 30 minutes in the 37°C CO2 incubator; this is the "reperfusion period".</li>
</ol>
3. Preparing rescue mesenchymal stem cells
Bone marrow of human origin was taken into T75 flasks and diluted with Dulbecco's Modified Eagle's Medium (DMEM) culture medium containing 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin, 4 mM L-glutamine and 1 g/l glucose. The flasks were incubated at 37°C in a fully humidified atmosphere of 5% CO2 for 3 days. After the incubation period the BMSCs adhered to the surface of the flasks and the remaining components of bone marrow were eliminated by washing with PBS. The used BMSCs were between 1 and 5 passages in the experiments. The identity of cells was confirmed by the presence of lineage-specific cell surface markers with flow cytometry. Hematopoietic linage-specific surface markers (CD34, CD45) and mesenchymal surface markers (CD73, CD90, CD105 and CD166) were investigated.
During simulated ischemia, prepare rescue mesenchymal stem cells for transplantation.
<ol>
	<li>Dilute the fluorescent dye Vybrant DiD in 1:200 ratio in DMEM culture medium.</li>
	<li>Aspirate medium from the rescue cells and add 300 μl DMEM with Vybrant DiD.</li>
	<li>Place Petri dish for 30 minutes in the 37°C CO2 incubator.</li>
	<li>Remove culture medium from Petri dish of mesenchymal stem cells.</li>
	<li>Wash cells twice with 5 ml PBS and digest with 2-3 ml 0.05% trypsin/EDTA.</li>
	<li>Place Petri dish for 5 minutes in the 37°C incubator.</li>
	<li>Inhibit trypsin with 10 ml DMEM culture medium.</li>
	<li>Spin down the cells at 1200 RPM for 8 minutes.</li>
	<li>After removing the supernatant, resuspend cells in 1 ml DMEM culture medium and count cells with hemocytometer using Trypan Blue staining.</li>
	<li>After 30 minutes at the end of the reperfusion add 20,000 mesenchymal stem cells per well to the postischemic H9c2 cardiac myoblasts.</li>
	<li>Place co-culture of cells into the 37°C CO2 incubator for 24 hours.</li>
</ol>
4. Analysis with confocal microscopy
After the simulated ischemia/reperfusion injury, cells are incubated in normal culture medium for 24 hours.
<ol>
	<li>After 24 hours wash cells twice with PBS.</li>
	<li>Stain the cells in 500 μl live/dead dye solution for 30 minutes (5 nM calcein and 400 nM Ethidium-homodimer-2 in PBS) (5x104 cell/ml).</li>
</ol>
The evaluation of confocal images for live and dead cells selected by morphology and fluorescence can be performed with the ImageJ software (National Institutes of Health, USA). In case of co-cultures, MSCs can be distinguished from H9c2 cells due to their Vybrant DiD cell labeling. The ratio of dead cells can be evaluated in 4 independent fields of view (objective 10x) for each culture in a blind fashion10.
H9c2 cardiomyoblasts cultured on 42 mm glass coverslips can also be investigated with time lapse video microscopy during and after ischemia/reperfusion injury using the cell incubation system of the confocal microscope. Cell-to-cell interactions such as cell fusion and formation of intercellular nanotubes can be studied over time.
5. Analysis with flow cytometry
After the simulated ischemia/reperfusion injury, cells are incubated in normal culture medium for 24 hours.
<ol>
	<li>After 24 hours, harvest the culture media. It is very important to collect the culture medium also because some dead cells could have detached from the culture dish surface during the 24 hour incubation.</li>
	<li>Wash cells twice with PBS and digest with 0.05% trypsin-EDTA.</li>
	<li>Inhibit trypsin with DMEM culture medium.</li>
	<li>Spin down the cells and the harvested culture medium at 1200 RPM (approximately 150-200g) for 8 minutes.</li>
	<li>Discard the supernatant.</li>
	<li>Suspend the cells in 500 μL live/dead dye solution for 30 minutes (5 nM calcein and 400 nM Ethidium-homodimer-2 in PBS) (5x104 cell/ml).</li>
	<li>Transfer cells to flow cytometry tubes.</li>
	<li>Start the CellQuest Pro software.</li>
	<li>To set up the flow cytometer for measurement, prepare control samples of live and dead cells.
		<ol class="lalpha">
			<li>Plate H9c2 cells at a density of 3x104 in 12-well plates as described in point 1.</li>
			<li>Prepare live cell controls with single trypsinisation as described in point 1.</li>
			<li>Prepare dead cell controls by treatment with 10 μM H2O2 for 1 hour. </li>
			<li>After trypsinisation, resuspend live and dead cell controls in 500 μl live/dead dye solution (5 nM calcein-AM and 400 nM Ethidium-homodimer-2 in PBS).</li>
		</ol>
	</li>
	<li>The instrument settings should be adjusted so the living cells are calcein positive and ethidium homodimer negative, while the dead cells are calcein negative and ethidium homodimer positive.</li>
	<li>The percentage of these populations can be expressed as bars on a graph and statistical analysis can be performed on these values.</li>
	<li>Measure the different experimental groups.</li>
</ol>
6. Representative results:
Our results showed that added mesenchymal stem cells could improve the survival of postischemic cardiac cells. Confocal microscopy images revealed direct cell-to-cell interactions, such as partial membrane connection or intercellular nanotubes, which probably play an important role in the observed protection10. These nanotubes span over distances of several cell diameters, and their diameters were between 200 and 500 nm (white arrows).
<img src="/files/ftp_upload/3575/3575fig1.jpg" alt="Fluorescence microscopy image; cell labeling with green and red markers, scale bar 50 µm." data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Nanotube formation between cells. Nanotube formation (white arrow) was observed between Vybrant DiO labeled cardiomyoblasts and Vybrant DiD labeled mesenchymal stem cells.
Flow cytometry analysis showed the populations of surviving and necrotic cardiomyoblasts and mesenchymal stem cells. Interestingly, we also observed a third, ‘injured’ cardiomyoblast cell population which contained calcein but also contained the indicator of the dead cells, ethidium homodimer (Figure 2). 
<img src="/files/ftp_upload/3575/3575fig2.jpg" alt="Flow cytometry dot plot, cell viability analysis: live, injured, dead, and debris cell populations." data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Representative dot-plot of the different cell populations after simulated ischemia.
The evaluation of several experiments showed that addition of mesenchymal stem cell to postischemic H9c2 cardiomyoblast increased significantly the percentage of living cells compared to the cardiac myoblasts cultured alone after ischemia (Figure 3).
<img src="/files/ftp_upload/3575/3575fig3.jpg" alt="Bar chart showing percentage of live cells in control, I-R model, and I-R+BMSC conditions, analysis." data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Effect of stem cell treatment on postischemic cardiomyoblasts. Percentage of living cells was significantly reduced after simulated ischemia compared to control (control vs. I-R model: 90.36±2.60 vs. 10.52±0.48, *** p&gt;0.001), which increased after addition of mesenchymal stem cells (I-R model vs. I-R + BMSC: 10.52±0.48 vs. 25.21±2.13, *** p&gt;0.001). Data represent mean±SEM. Statistical analysis of data was carried out using one-way analysis of variance with Tukey's multiple comparison post hoc test.

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<li>Sardao, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vital+imaging+of+H9c2+myoblasts+exposed+to+tert-butylhydroperoxide--characterization+of+morphological+features+of+cell+death%5BTitle%5D)+AND+%22BMC+Cell+Biol%22%5BJournal%5D)">Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide--characterization of morphological features of cell death.</a> BMC Cell Biol. 8, 11-11 (2007).</li>
<li>Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological%2C+biochemical%2C+and+electrophysiological+characterization+of+a+clonal+cell+%28H9c2%29+line+from+rat+heart%5BTitle%5D)+AND+%22Circulation+research%22%5BJournal%5D)">Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart.</a> Circulation research. 69, 1476-1476 (1991).</li>
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No conflicts of interest declared.

The demonstrated protocol is an in vitro approach to the much more complex issue of stem cell therapy in myocardial infarct, with all the advantages and disadvantages of such a model. Obviously it cannot reflect the complex (e.g. immunological) events taking place during and after myocardial infarct but can focus on the direct effects of the added cells on the postischemic cells. The effects of simulated ischemia on H9c2 cardiomyoblasts highly depend on the time of OGD, on the passage number of the used cells an...

stem cell transplantation in an in vitro simulated ischemia/reperfusion model

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.

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 ...

Cardiac derived cells are placed onto cell culture plates for simulated ischemia. Reperfusion model ischemia is achieved with 150 minutes in a glucose free medium, while the oxygen level is below 0.5%This ischemic period leads to the shrinking and blabbing of most cells. Then normal media and oxygen levels are reintroduced to simulate reperfusion.Mesenchymal stem cells are added and cultivated together with the damaged cells. After 24 hours of co cultivation cells are stained with calcium and Ethereum homodimer To differentiate between live and dead cells, confocal microscope, it clearly shows that two cell populations and their interactions flow cytometry analysis reveals three clusters of damaged cells, which can be plotted on a graph and analyzed statistically Stem cell transplantation. Protocols are finding their way into clinical practice, getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research.Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate mechanisms involved in the treatment process. We designed an experimental protocol in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia, perfusion injury and after stem cell transplantation. Our design criteria, our fully in vitro system, but only the host and the donor cells are present.So it is isolated from the surrounding tissue. Continuous measurement of cell viability before, during, and after the injury for up to 24 hours. And clear identification of both cell populations so the fate of the grafted cells can be traced.In order to reach this goal, we set up a protocol of oxygen, glucose deprivation, which is commonly abbreviated as OGD, followed by a reoxygenation period. Our choice of host cells was age nine C two cells via known red cardio myoblast cell line. After OGD, the damaged cells were supported by transplantation of bone marrow derived mesenchymal stem cells by adding them to the culture.This setup allowed us to investigate the intercellular connections using confocal forest and microscopy and to quantify the survival rate of damaged cells by flow cytometry. Let's get to work and see how we do it. Remove culture medium from H nine C two cells.Wash cells twice with PBS and digest with 0.05%trypsin EDTA place Petri dishes for five minutes. In a 37 degree incubator, inhibit trypsin with DMEM culture medium. Spin down the cells at 1200 RPM for eight minutes.After removing the super natin resuspend cells in one millimeter DM EM culture, medium count cells with he cytometer using triam blue staining C 30, 000 H nine C two cells per well. In a 12 well plate, place the plate into the 37 degree carbon dioxide incubator for 24 hours. Remove medium by aspiration wash cells twice with PBS.Add three millimeters glucose free medium. Place the plate into the cell incubation system. Start nitrogen gas to purge the oxygen out of the incubation system.Wait until the level of oxygen reaches 0.5%Then start the timer, 0.5%Oxygen means approximately three millimeters of mercury partial tension subject cells to 150 minutes of simulated ischemia. During ischemia, cells are getting detached from the surface and their membrane starts blabbing. We found that each population needs slightly different lengths of time, anti reaching a 60 to 80%of cell image.That's why we suggest to begin with the two R incubation. And if it's not enough, increase the time of OGD. In our case, age nine C two cells typically need 150 to 70 minutes of OGD At the end of simulated ischemia.Remove the glucose free medium from the cells pipette normal culture medium to the wells. Place a 12 well plate for 30 minutes in the 37 degree carbon dioxide incubator. This is the reperfusion period.During simulated ischemia, prepare rescue cells for transplantation. Dilute the fluorescent diet, vibrant DID in one to 200 ratio in DMEM culture. Medium aspirate medium from the rescue cells and add 300 microliters DMEM from vibrant Brent Place Petri dishes for 30 minutes in the 37 degree carbon dioxide incubator.After 30 minutes at the end of the reperfusion at thousand mesenchymal stem cells per well to the post ischemic H nine C two cardiac myoblasts. The initial steps are the same as H nine C two seeding washing two times with PBS trypsin and counting. Place co culture of cells into the 37 degree carbon dioxide incubator for 24 hours.After 24 hours. Harvest a culture media and the adherence cells from wells with ization. It is very important to collect a culture medium also because some dead cells could have a detached from the cultured dish surface during the 24 hour incubation.Suspend the cells in 500 microliter live, dead, die solution, five NMO calcium, and 400 NMO Ethereum homodimer in PBS five times 10 to the power of four cell per milliliters. In case of co cultures, mesenchymal stem cells are distinguished from H nine C two cells by the respective fluorescent cell labeling. This way it is possible to detect cell fusion and cell to cell contact sites.The ratio of dead cells can be evaluated in four independent fields of view, using a 10 times objective for each culture in a blind fashion, which gives a reproducible cell count result. Transfer cells to flow cytometric tubes. Start the cell Quest Pro software to set up the flow cytometer for measurement.Prepare control samples of live and dead cells. Living cells are cultured at the same density in normal conditions and stain with calcium. Dead cells are prepared with one hour treatment with 10 micromole hydrogen peroxide stained with Ethereum, homodimer, and harvested in the same manner the instrument settings should be adjusted so the living cells are calcium positive and Ethereum homodimer negative.While the dead cells are calcium negative and Ethereum homodimer positive, the percentage of these populations can be expressed as bars on a graph and statistical analysis can be performed On these values. We found that the addition of healthy stem cells increase the ratio of surviving cells in the population. Interestingly, we also observed that the cells which were saved after the injury can be gated out from those which were dead, but also from those that remained alive.We believe that this population warrants further investigations. After watching this video, you should have a good understanding of how to conduct simulated ischemia experiments with in vitro stem cell transplantation in a standardized way. So that's it.Thanks for watching and good luck with your experiments. In case you have any technical questions, do not hesitate to contact us. We'll be there to help out.

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Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

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This paper describes ischemia-reperfusion (I/R) modeling in a 3-day chick embryo using a spinal needle customized hook to better understand I/R development and treatment. This model is simple, quick, and inexpensive.

Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

Here, we describe an in vitro protocol to model hypoxia/reoxygenation damage in the highly metabolic proximal tubular epithelial cells. This model is designed to induce metabolic stress-induced damage, which can be measured through the expression of proximal tubule damage markers and assessment of mitochondrial respiratory function.

Modeling Hypoxia/Reoxygenation Injury in Proximal Tubular Epithelial Cells

Learn stem cell transplantation using an in vitro ischemia/reperfusion model with cardiac cells and mesenchymal stem cells. This method enables assessment of cell viability and therapeutic mechanisms.

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

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