Abstract Background Cardiovascular cell therapy represents a promising field, with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study (“Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease”) consists of fresh mononuclear cells (MNC) isolated from autologous bone marrow (BM) through density gradient centrifugation on standard Ficoll-Paque. Cells are tested for safety (sterility, endotoxin), identity/potency (cell count, CD45/CD34/CD133, viability) and purity (contaminant granulocytes and platelets). The aims of the present work were (1) to optimize the cell manufacturing process in order to reduce contaminants and (2) to implement additional assays in order to improve product characterization and evaluate product stability. Methods BM-MNC were isolated by density gradient centrifugation on Ficoll-Paque. The following process parameters were optimized throughout the study: gradient medium density; gradient centrifugation speed and duration; washing conditions. Differential cell count was performed by an automated hematology cell analyzer. Immunophenotype and cell viability were determined by flow cytometry. Functional hematopoietic and mesenchymal precursors and cells with angiogenic potential were assessed by colony-forming assays, cell invasion capacity by a fluorimetric assay. Sterility was tested using an automated microbial detection system, endotoxin by a kinetic chromogenic Limulus amebocyte lysate test. T-test was used for statistical analysis. Results A new manufacturing method was set up, based on gradient centrifugation on low density Ficoll-Paque, followed by 2 washing steps, of which the second one at low speed. It led to significantly higher removal of contaminant granulocytes and platelets, improving product purity; the frequencies of CD34 + cells, CD133 + cells and functional hematopoietic and mesenchymal precursors were significantly increased. The process was successfully validated according to Good Manufacturing Practices. The resulting ATMP mainly consisted of viable MNC including CD34 + and CD133 + cell subsets (2.98% ± 1.90% and 0.83% ± 1.32%, respectively), CD184/CXCR4 + cells (34% ± 15%), .