OBJECTIVES: This study has intended to investigate longevity of subcutaneous fat-derived mesenchymal stem cells (SF-MSCs) under extensive culturing. It has also focused on optimization of culture media for them over prolonged periods in vitro. MATERIALS AND METHODS: We evaluated SF-MSCs with reference to phenotypic characterization, proliferative ability, karyotype stability and differentiation potency with early (P3) and late passage (P20) conditions, using four different media, DMEM-LG, ALPHA-MEM, DMEM-F12 and DMEM-KO. RESULTS: This study unravels retention of SF-MSC characteristics in facets of phenotypic expression profile (CD 90, CD 105, CD 73, CD 34, CD 29, CD 54, CD 49d, CD 117, HLA-DR, CD 166, CD 31, CD 44), proliferative characteristics, karyotyping and differentiation potency prolonged culturing to P25 in all media. Population doubling time (PDT) in Alpha MEM, DMEM LG, DMEM F 12, DMEM KO were identified to be (1.81, 1.84, 1.9, 2.08†days) at early passage and (2.93, 2.94, 3.12, 3.06†days) at late passage. As a corollary, Alpha MEM and DMEM LG serve as appropriate basal media for SF-MSC when proliferative potency is considered. CONCLUSIONS: In research, it is imperative that SF-MSC uphold their expansion potency in the aforesaid attributes in all media over extensive culturing, thereby transforming their colossal in vitro potency, with the aim of curing a wide horizon of diseases.