Millipore Technical Publications |
 | PO- ps1235en00 | |
Cardiac Stem Cell Isolation | ||
| Lit No: | ps1235en00 | |
| Year: | 2008 | |
| The striated muscle cells of the heart represent the functional contractile unit permitting blood flow throughout the circulatory system. Adult murine cardiomyocytes provide an excellent model system for the study of cardiovascular diseases. Methods to isolate cardiomyocytes have traditionally relied upon mechanical mincing and enzymatic treatment of ventricular tissues followed by the use of cell strainers, which risks introducing contamination to the sample. These methods often yield mixed populations of cells. Cell heterogeneity makes analyses of discrete cell populations difficult. Here we report a novel method for the isolation and purification of cardiomyocytes from endogenous cardiac stem cells (CSCs). Ventricular heart tissues were isolated using sterile tissue dissection techniques from C57/BL6 mice and transferred to CSC Isolation Buffer. These tissues were subsequently minced and transferred to a Cardiac Tissue Dissociation Buffer. Following incubation and tituration, the cell suspension was filtered through a Steriflip unit, a self-contained, vacuum-assisted apparatus with a modified 100 μm nylon mesh membrane. The cell mixture was further p gradient centrifugation. Freshly isolated CSCs can either be further expanded for up to 2 weeks in culture in CSC maintenance Medium or differentiated into cardiomyocytes by culture in Cadiomyocyte Differentiation Medium. Characterization of the differentiated cardiomyocytes was accomplished using a panel of antibodies which include, tropomyosin, desmin, actinin, troponin I. Collectively our results demonstrate a reliable and reproducible method for the isolation and culture of ventricular cardiomyocytes and endothelial cells and should facilitate the use of these purified cells for a wide array of biological applications. | |
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