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Bone marrow aspirate concentrate (BMAC) holds significant promise in regenerative medicine. However, its clinical application remains limited by the lack of standardized protocols, which directly impacts its effectiveness. This study aimed to compare two protocols for obtaining mesenchymal stem cells (MSCs) using a closed system for immediate surgical application. Additionally, it sought to contribute to the standardization of protocols, characterization of BM-MSC composition, and assessment of technique reproducibility. Methods: Ten patients undergoing surgery for osteoarticular conditions were enrolled. In both protocols, 30 mL of bone marrow was aspirated, yielding final volumes of 15.50 mL (decantation protocol) and 17.23 mL (centrifugation protocol) per donor. Results: MSCs isolated from the posterior iliac crest and proximal tibia demonstrated similar phenotypic profiles by flow cytometry, with comparable chondrogenic and osteogenic differentiation capacities. Fibroblast-like colony-forming units (CFU-Fs) were observed after 4–5 days in culture. The decantation protocol produced a significantly higher number of MSC-derived CFUs per milliliter. Moreover, the MSC growth surface area at day 10 was greater in the decantation group. Conclusion: The continued increase in use and proved efficacy of cell therapy treatments is already a reality in major centers with suitable laboratories. This study conducted research with a high degree of methodological refinement to optimizing the therapeutic potential of BMAC and supports it use with an autologous, sterile and cost-effective closed system to immediate intraoperative cell therapy for more consistent and effective therapeutic strategies.