Background: Mesenchymal stem/stromal cells (MSCs) with two intrinsically comprehensive characteristics, low immuno-genicity and smart multi behavior (pro- and anti-inflammatory) in response to signals, have been broadly manufactured and utilized in numerous clinical trials. Due to the heterogeneous nature of MSCs and upcoming senescence, allogeneic therapies have not been used as widely as autologous ones. Mainly, au-tologous therapy raises costs and time of remedies. Moreover, quality, scalability, and sustainability as other key elements of successful manufacturing, have been missed largely with au-tologous strategy.Materials and Methods: In this study, we developed a method based on the subfractionation culturing method (SCM) to sepa-rate non-adherent mesenchymal progenitors (NAMPs) as more potent and proliferative MSCs in good manufacturing practice (GMP) environment to overcome the aforementioned hurdles. Notably, to reduce the overall costs, four banking steps were designed including seed stock, initial, master and working cell banks (ICB, MCB, and WCB). After picking up colonies in pri-mary culture of bone marrow and subculture until passage 3, cells were frozen in 2-3 vials as seed stock. A small amount of each colony was passaged to reveal passageable ones in 11-12 more serial passages. Passageable colonies from seed stock were thawed to establish ICB, MCB, and WCB at P5 ± 1, P10 ± 1, and P15 ± 1, respectively. Each step has passed it necessitate assays, however, in MCB in addition to biological properties (morphology, immunophenotype, differentiation, senescence, growth curve and doubling time), functional potentials (cy-tokine secretion, immunosuppressive effect and migration as-says) were also considered as determinants for screen colonies to fit specific diseases. Moreover, in WCB biosafety concerns (immunogenicity, genetic stability, and tumorigenicity) were considered as release tests to establish the end of production cell bank (EoPCB).Results: In the end, we reached to three colonies that meet all characteristics and potentially could cure more than 5,000 pa-tients with average cell demand of 1.5 ×108 cells.Conclusion: Therefore, our GMP-compliant cell bank could potentially support many clinical trials with low cost, high qual-ity and sustainable sources from a small amount of aspirated BM.