Recent scientific explorations in search of novel sources for autologous transplantation transpired an alternative source of MSCs (mesenchymal stem cells) derived from omentum fat. The scarcity of experimental evidences probing into the biosafety concerns of omentum fat-derived MSC under prolonged culture conditions limits its applicability as an efficient tool in regenerative medicine. This study, thus, aims to optimize human omentum fat-derived MSC in four different media [DMEM (Dulbecco's modified Eagle's medium) LG (low glucose), DMEM KO (knock out), α-MEM (α-minimal essential media) and DMEM F12] in the facets of phenotypic characterization, growth kinetics, differentiation and karyotyping under prolonged culture. The cells exhibited a similarity in expression profile for the majority of markers with evidential variations in certain markers. The relevance of omentum fat-derived MSCs became evident from its triumphant differentiation potential and karyotypic stability substantiated even at later passage. The results obtained from growth curve and PDT (population doubling time) lead to optimization of appropriate media for omentum fat-derived stem cell research, thereby bringing omentum fat into the forefront of regenerative medicine.