In this work, we demonstrated the use of a new technique (Celector ®, Stem Sel Ltd., Bologna, Italy) to isolate homogeneous stem cells directly from a clinical sample, the BMC, in a label-free mode. Therefore, new isolation approaches are needed in order to homogenize the cell culture among patients and centres. The inability to isolate a pure population of MSCs has consequences in different fields, from basic research to therapeutic applications caused by differences in the proliferation rate, distinct differentiation program, and self-renewal capacity of each cell subset. The International Society for Cellular Therapy has proposed a set of criteria to define MSCs, but the list of biomarkers is not fulfilling, and other tests, such as differentiation towards different lineages and immunomodulation, need to be taken into account to prove the stemness potential. The identity of the “best” marker for MSC identification, in both human and mouse systems, remains debated. Antibody-labelled techniques necessitate a high number of cells and the expression of multiple markers, which are not always available and can be co-expressed in different cell populations. The density gradient and antibody labelling system require several hours of labour-intensive multi-step processing, consisting of centrifugation and washing steps, potentially resulting in a significant amount of target stem cell loss. Once mononuclear cells are concentrated, the gold-standard isolation technique is to exploit the plastic adherence properties of MSCs that lead to a heterogeneous population in culture, with plastic and non-plastic adherent cells, whose co-culture interactions and functional properties are unknown. Cells can be enriched by a density gradient system and centrifugal concentrator. Once BM is harvested by aspiration, different types of cells are collected simultaneously and the isolation procedures can differ in the yield, but do not alter the balance of the different cell types. To understand its regenerative activity, studies are conducted to explore the biology and characteristics of each cell component. It is important to notice that BM clot as a 3D environment supports the chondrogenesis and osteogenesis of MSCs, making this system ideal for cartilage and/or bone repair. In the orthopedic field, the use of bone marrow concentrate (BMC) in substitution of or addition to the procedure of marrow stimulation, provides interesting results because it contains not only hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) as a source for regenerating tissues but also accessory cells that support angiogenesis and vasculogenesis by producing several growth factors. This allows selective cell identification with a potential impact on regenerative medicine approaches in the orthopedic field and clinical applications.īone marrow (BM) is one of the most studied sources of MSCs, whose therapeutic potential has been explored in several diseases, proving its efficacy in clinical trials, from heart failure to grafts versus host disease and other pathologies. This new technology provides an effective method to separate MSCs from fresh BM, maintaining their native characteristics and avoiding cell manipulation. In addition, the technology depletes dead cells, removing unwanted red blood cells and non-progenitor stromal cells from the biological sample. Multipotent MSCs were found in F1: higher CFU-F activity and differentiation potential towards mesenchymal lineages compared to the other fractions. Based on cell live-imaging and the sample profile, it was possible to highlight three fractions (F1, F2, F3), and the collected cells were evaluated in terms of their morphology, phenotype, CFU-F, and differentiation potential. BM obtained from patients operated for osteochondral defects was directly concentrated in the operatory room and then analyzed using the new technology. To overcome this limitation, we investigated the use of a new separative technology to isolate MSCs from BM it label-free separates cells based solely on their physical characteristics, preserving their native physical properties, and allows real-time visualization of cells. Several methods are used for their isolation such as gradient separation or centrifugation, but these methodologies are not direct and, thus, plastic adherence outgrowth or magnetic/fluorescent-activated sorting is required.

Mesenchymal stem cells (MSC) make up less than 1% of the bone marrow (BM).

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