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Cell-membrane permeability to water (Lp) and cryoprotective agents (Ps) of a cell type is a crucial cellular information for achieving optimal cryopreservation in the biobanking industry. In this work, a new micro-vortex system was developed to virtually trap cells of interest in a flow-driven hydrodynamic circulation passively formed at the expansion region in a microfluidic channel. Trapped cells remain in suspension and flow along with the streamline of the localized vortex, thus, featuring no physical contact with the device structure, and furthermore supporting a pragmatic assumption of 100% sphericity and active surface area of cell membrane for estimating actual cell volume from two-dimensional images. Cell membrane permeability was therefore able to be accurately determined by directly visualizing, with automatic cell recognition from high-speed videos, the transient profile of cell-volume change in response to a sudden osmotic gradient instantaneously (within 2 seconds) applied between the intracellular and extracellular environments. An acute T-cell lymphoma cell line (Jurkat) was used as a model to examine the newly-adopted micro-vortex technology in this research, and our results indicate a moderately higher values (Lp = 0.34 μm min-1 atm-1 for the binary system, Lp = 0.16 μm min-1 atm-1 and Ps = 0.55 × 10-3 cm min-1 for the ternary system) than those in prior arts utilizing contact-based cell-trapping techniques, manifesting an significant influence of active surface area on the determination of cell-membrane permeability.