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Cardiomyocyte progenitor cells isolated from cardiac tissue surgical waste or from fetal heart tissue. The cells are spindle-shaped with a high nucleus-to-cytoplasm ratio.
For preparation of single-cell suspensions, the tissue is cut into small clumps in M-buffer containing PBS supplemented with EDTA (2 mM) and FBS (1%), than washed with cold PBS and digested by treatment with collagenase A. The tissue is ground on a cell strainer (40-mm), washed with M-buffer and resuspended in DMEM supplemented with FBS (10%) and penicillin/streptomycin (1X). CMPCs are isolated using clonogenic isolation or magnetic cell sorting (MACS). When using the method of clonogenic isolation, the cells (5 cells/ml) are plated on gelatin (0.1%)-coated 96-well plates, in growth medium comprised of M199 and EGM-2 (3:1) supplemented with FBS (10%), penicillin/streptomycin (1X) and MEM nonessential amino acids (1X). When using the method of MACS isolation, cells are isolated using anti-Sca-1 microbeads and then plated on gelatin (0.1%)-coated plates in the growth medium described above. After 3 days, the isolated CMPCs are cultured in growth medium supplemented with bFGF (10ng/ml).