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Content Provider | World Health Organization (WHO)-Global Index Medicus |
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Author | Cselenyák, Attila Benko, Zsolt Szepes, Mónika Kiss, Levente Lacza, Zsombor |
Description | Author Affiliation: Cselenyák A ( Institute of Human Physiology and Clinical Experimental Research, Semmelweis University. attila.cselenyak@eok.sote.hu) |
Abstract | 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 the mechanisms involved in the treatment process. 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. 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. |
File Format | HTM / HTML |
e-ISSN | 1940087X |
DOI | 10.3791/3575 |
Journal | Journal of Visualized Experiments |
Issue Number | 57 |
Language | English |
Publisher | MyJove Corp. |
Publisher Date | 2011-11-05 |
Publisher Place | United States |
Access Restriction | Open |
Subject Keyword | Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Mesenchymal Stem Cell Transplantation Reperfusion Injury Surgery Flow Cytometry Mesenchymal Stromal Cells Cytology Microscopy, Confocal Research Support, Non-u.s. Gov't Video-audio Media |
Content Type | Text |
Resource Type | Article |
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