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

Summary

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.

Abstract

Stem cell transplantation protocols are finding their way into clinical practice^1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research^4,5. 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 plates^7,8. 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.

Protocol

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: Remove cu…

Discussion

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…

Materials

Name of the reagent	Company	Catalogue number	Comments
calcein-AM	Molecular Probes	L3224, C3099	http://www.invitrogen.com
ethidium homodimer-2	Molecular Probes	L3224, E3599	http://www.invitrogen.com
Vybrant DiD	Molecular Probes	V22887	http://www.invitrogen.com

Table 1. Reagents.

Name of the equipment	Company	Comments (optional)
PeCon cell incubation system for Zeiss microscopes	PeCon GmbH	www.pecon.biz/

Table 2. Equipment.