Introduction:The adult stem cells used in clinical practice are: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). These cells, present in the bone marrow, play a key role in the physiological renewal and repair of injured tissues. MSCs can differentiate into a variety of cell types including bone cells, cartilage cells, connective cells etc., and they play an important role in the repair of tissue injured by trauma or disease. In view of all this, all the technologies for the isolation and the re-implantation, after minimal manipulation, of autologous stem cells are very interesting, also to make the use of stem cell therapy more widespread in the orthopaedic field.The aim of this study was to verify the RegenKit method of selection and enrichment of autologous mesenchymal and hematopoietic stem cell fractions from bone marrow, used intraoperatively, in order to define a standard therapeutic application.Materials and methods:Separation of MSCs using RegenKit: 32 ml of bone marrow aspirate is collected and divided into 4 Regen THT tubes, and then centrifuged directly in the operating room. The erythrocytes are pelletted at the bottom of the tube, while the mononuclear cells collect in the layer between the gel selector and plasma. The tubes are agitated to suspend the mononuclear cells in the plasma. The content of one of these tubes is used for a cellular efficiency and yield analysis. The sample is stratified on a lymphocyte separation medium gradient (density 1.077 g/ml, Lonza) and centrifuged at 1000xg for 30 min. The mononuclear cells are collected, washed in DPBS without Ca and Mg and seeded on a 100 mm culture plate, in growth culture medium (GM) Ham?s F12, modified according to Coon, with the addition of FBS 10%, penicillin 100 IU/ml, streptomycin 100 g/ml. Separation of control MSCs: A bone marrow aspirate sample not centrifuged and submitted to the same laboratory procedures is used as control. Cellular yield: 48 hours after seeding, the adherent cells (MSCs) are counted directly on the growth medium. The cells present in 30 ocular fields are counted using an appropriate standard grid; the number obtained is normalised using an appropriate conversion coefficient.Results:10 samples from patients were prepared using the RegenKit method and 10 cell lines were obtained. The results showed that:Conclusions: This study describes how, with the RegenKit device, it is possible to separate and concentrate the mono-nuclear cells derived from bone marrow aspirate, obtaining a high cellular yield. The future prospects are to verify the osteogenic ability of these cells, confirming clinical results.