BACKGROUND: Adipose stem/stromal cells (ASCs) are a type of adult/tissue mesenchymal stem cell that may be isolated in abundance from adipose tissue, but, have a limited lifespan in vitro. Typical culture conditions involve the use of serum to enhance cell attachment and as a source of growth factors. These cells or their secretome have potential therapeutic effects, making it crucial to develop stable ASC lines and to eliminate serum supplements from the culture media to improve culture stability, secretory factor scalability and to improve safety. OBJECTIVES: To develop immortalized ASC lines from primary cultures and formulate a superior, completely defined Serum-Free and Xeno-Free media (SFM) that supports the growth of these cells in vitro and may be utilized for clinical applications. METHODS: ASCs were isolated with enzymatic treatment. Immortalization was performed by transduction with SV40 and hTERT genes. Primary and immortalized cells were cultured in vitro with our SFM and characterized. RESULTS: Co-transductions successfully immortalized ASCs resulting in lines with characteristic ASC morphologies, marker expression, stemness and similar differentiation potentials to mother lines and population doubling levels of greater than 70 thus far. Our complete SFM also supported the growth and proliferation of non-immortalized ASCs up to passage 14 in vitro while maintaining their mesenchymal stem cell characteristics. All cells were successfully cultured in monolayers and three-dimensional scaffolds, were able to form structures like embryoid bodies, and, differentiated to adipocytes, chondrocytes and osteocytes. CONCLUSIONS: Human ACS were successfully immortalized. Our SFM is suitable for culturing immortalized and primary human ASCs which maintain their stemness and secrete fewer pro-inflammatory chemokines as compared to ASCs grown in traditional serum supplemented media. These ASCs or their secretome may be used for downstream regenerative medicine applications. [Am J Transl Med 2021. 5 (2): 76-102].