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 &mu;g/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:
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	<li>Remove cu...

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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...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>calcein-AM</td>
			<td>Molecular Probes</td>
			<td>L3224, C3099</td>
			<td><a href="http://www.invitrogen.com">http://www.invitrogen.com</a></td>
		</tr>
		<tr>
			<td>ethidium homodimer-2</td>
			<td>Molecular Probes</td>
			<td>L3224, E3599</td>
			<td><a href="http://www.invitrogen.com">http://www.invitrogen.com</a></td>
		</tr>
		<tr>
			<td>Vybrant DiD</td>
			<td>Molecular Probes</td>
			<td>V22887</td>
			<td><a href="http://www.invitrogen.com">http://www.invitrogen.com</a></td>
		</tr>
	</tbody>
</table>
Table 1. Reagents.
<table border="1">
	<tbody>
		<tr>
			<td>Name of the equipment</td>
			<td>Company</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>PeCon cell incubation system for Zeiss microscopes</td>
			<td>PeCon GmbH</td>
			<td><a href="http://www.pecon.biz/">www.pecon.biz/</a></td>
		</tr>
	</tbody>
</table>
Table 2. Equipment.

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.

Watch this Scientific Journal Video about Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model at JoVE.com

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 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 ...

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14.0K Views.  Semmelweis University. Dimostriamo come impostare uno In vitro Ischemia / riperfusione modello e come valutare l'effetto della terapia con cellule staminali su postischemica cellule cardiache.