Method for the isolation and expansion of cardiac stem cells from biopsy

A method to obtain stem cells able to repair damaged myocardiac tissue, comprising the following steps:

• fragmenting a cardiac tissue in vitro to obtain a plurality of tissue fragments;
• adhering said tissue fragments to a solid support;
• culturing said tissue fragments in a culture medium, wherein said culture medium comprises one or more nutrients and wherein said tissue fragments are sized to allow the diffusion of said nutrients to said tissue fragments;
• culturing said tissue fragments until one or more phase-bright cells form;
• harvesting the phase-bright cells;
• culturing the phase-bright cells on a treated substrate to generate one or more cardiospheres;
• wherein the cardiospheres form substantially spheroid structures of about 20 μm to about 150 μm in culture;
• wherein the cardiospheres are multicellular aggregates;
• wherein the cardiospheres comprise a mixed population of cells;
• wherein the mixed population comprises stem cells, cardiac cells, and endothelial cells,
• wherein the stem cells express at least one of the following three markers:
• CD34, c-kit and sca-1 within 12 hours of said cardiospheres being generated,
• wherein the endothelial cells express at least one of KDR, flk-1, and CD31 within 12 hours of said cardiospheres being generated,
• wherein the cardiospheres are weakly adherent or non-adherent to the treated substrate;
• and
• harvesting the cardiospheres, thereby obtaining stem cells for use in repairing damaged myocardiac tissue.