Characterizing the membrane physiochemical properties of cells is important for understanding the physiology and fate of cells; separating cells based on their membrane signature is instrumental in cell manufacturing and regenerative medicines. To achieve rapid membrane-based cell separation in a biologically compatible environment, we develop a centrifugation-powered 3D multiphase microfluidic device. By characterizing the translocation of single cells through the interface of an aqueous two-phase systems (ATPS) under centrifugal forces, we extract the membrane physiological properties of cells, such as hydrophobicity and surface heterogeneity, and separate them based on these properties. Currently, we apply this method to purify osteoarthritis patient-derived chondrocytes for cartilage tissue regeneration and characterize muscle progenitor cells for in vivo skeletal muscle reconstruction.