Adipose tissue-derived stromal cells (ASCs) are promising candidates for cellular therapy in the field of regenerative medicine. ASCs are multipotent mesenchymal stem cell-like and reside in the stromal vascular fraction (SVF) of adipose tissue with the capacity to secrete a plethora of pro-regenerative growth factors. Future applications of ASCs may be restricted through (trans)national governmental policies that do not allow for use of non-human-derived (non-autologous) enzymes to isolate ASC. Besides, enzymatic isolation procedures are also time-consuming. To overcome this issue, non-enzymatic isolation procedures to isolate ASCs or the SVF have been developed, such as the fractionation of adipose tissue procedure (FAT). This standardized procedure to isolate the stromal vascular fraction can be performed within 10–12 min. The short procedure time allows for intra-operative isolation of 1 mL of stromal vascular fraction derived from 10 mL of centrifuged adipose tissue. The stromal vascular fraction mostly contains blood vessels, extracellular matrix, and ASCs. However, based on the histological stainings, an interdonor variation exists which might result in different therapeutic effects. The existing interdonor variations can be addressed by histological stainings and flow cytometry. Furthermore, the re-usable open system has been replaced by a validated disposable semi-closed system with a one-hole fractionator.