The microfluidic ratchet mechanism filters cells through a funnel-shaped micro-pillar array with minimum gap widths (or pore sizes) that gradually decrease from the bottom row to the top row of the array (Fig. 1A). The sample cells are infused at the bottom-left corner of the matrix, and transported through the array using a vertical oscillatory flow, at the same time as a constant horizontal cross flow. These flows combine to propel the cells in a zigzag diagonal path through the constriction matrix with each oscillation cycle, until they reach a limiting pore size that prevents further upward transport (Fig. 1A). Upon reaching this point, the cells oscillate between two funnel rows, allowing the horizontal flow to divert them toward a specific outlet.Figure 1Principle and design of the microfluidic ratchet cell sorting device. (A) Schematic illustration of the cell sorting region consisting of a 2D array of funnel microstructures with progressively smaller pore sizes from the bottom to the top row. Unprocessed whole blood is introduced at the bottom-left corner of the array while cells flow along a diagonal path under a combined biased oscillatory flow and cross flow. Smaller and softer RBCs flow into the outlets lining the top of the sorting region while the flow of leukocytes is restricted based on their size and deformability, which divert each cell into specific outlets. Colored arrows indicate the ratchet motions of erythrocytes and different subsets of leukocytes. (B,C) The underlying principle of the microfluidic ratchet involves selectively transporting cells using a ratcheting effect. Smaller and more deformable cells flow across tapered constrictions during forward flow and are prevented from returning during reverse flow. Larger and less deformable cells are trapped by the constriction and released with each reverse flow. (D) Image of the microfluidic device infused with different food color dyes illustrating components of the microfluidic device including the sorting region, sample inlet (SI), cross-flow inlet (CFI), oscillatory flow inlets (OSC1 and OSC2), and nine outlets (O1–O9).