The fractionation of the cell sample into the outlets after sorting is determined by the dimensions and arrangement of the funnel pores within the sorting matrix. The funnel array consists of 35 rows and 630 columns, occupying an overall area of 1875 × 9525 µm. The funnel pore size is kept constant for each set of four rows and progressively decreases with each row from 8 µm at the bottom to 2 µm at the top of the sorting region. The gap between each row of funnels is 50 µm. As a result, the 35 rows of funnel array sort the input whole blood into 9 fractions in outlet 1 to 9 (O1–O9), with O1 corresponding to the most deformable fraction and O9 corresponding to the least deformable fraction. This geometry of the funnel constrictions is designed to deform cells laterally through 8, 6.5, 5.5, 4.5, 4, 3.5, 3, 2.5 and 2 µm pore sizes, while providing stress relief in the orthogonal direction, which is kept constant at a thickness of 11–15 µm. Fluid flow in the sorting matrix is manipulated through supporting microchannel networks, including a blood sample inlet (SI), cross-flow inlet (CFI) and oscillation flow inlets (OSC1 and OSC2), as illustrated in Fig. 1D. The design of the microchannel networks follows a hydrodynamic resistance model, which determines the filtration forces applied across the funnel filter and the flow rate inside the sorting matrix. A detailed hydrodynamic model of fluid flow in this microstructure is presented in the supplemental materials. Based on this prototype design, the microfluidic ratchets are able to process whole blood at a rate of 5 µl per hour. While this throughput is low compared to other biophysical cell separation methods10–12, 14, 15, the throughput of this device could ultimately be improved by increasing the filtration area, as well as by paralleling the mechanism.