The cell-membrane permeabilities of a cell type toward water (Lp) and cryoprotective agents (Ps) provide crucial cellular information for achieving optimal cryopreservation in the biobanking industry. In this work, cell membrane permeability was successfully determined via directly visualizing the transient profile of the cell volume change in response to a sudden osmotic gradient instantaneously applied between the intracellular and extracellular environments. A new micro-vortex system was developed to virtually trap the cells of interest in flow-driven hydrodynamic circulation passively formed at the expansion region in a microfluidic channel, where trapped cells remain in suspension and flow with the streamline of the localized vortex, involving no physical contact between cells and the device structure; furthermore, this supports a pragmatic assumption of 100% sphericity and allows for the calculation of the active surface area of the cell membrane for estimating the actual cell volume from two-dimensional images. For an acute T-cell lymphoma cell line (Jurkat), moderately higher values (Lp = 0.34 μm min-1 atm-1 for a binary system, and Lp = 0.16 μm min-1 atm-1 and Ps = 0.55 × 10-3 cm min-1 for a ternary system) were measured than those obtained from prior methods utilizing contact-based cell-trapping techniques, manifesting the influence of physical contact on accuracy during the determination of cell membrane permeability.