The separation of biological cells by filtration through microstructured constrictions is limited by unpredictable variations of the filter hydrodynamic resistance as cells accumulate in the microstructure. Applying a reverse flow to unclog the filter will undo the separation and reduce filter selectivity because of the reversibility of low-Reynolds number flow. We introduce a microfluidic structural ratchet mechanism to separate cells using oscillatory flow. Using model cells and microparticles, we confirmed the ability of this mechanism to sort and separate cells and particles based on size and deformability. We further demonstrate that the spatial distribution of cells after sorting is repeatable and that the separation process is irreversible. This mechanism can be applied generally to separate cells that differ based on size and deformability.

Circulating tumor cells (CTCs) are malignant cells shed into the bloodstream from a tumor that have the potential to establish metastases in different anatomical sites. The separation and subsequent characterization of these cells is emerging as an important tool for both biomarker discovery and the elucidation of mechanisms of metastasis. Established methods for separating CTCs rely on biochemical markers of epithelial cells that are known to be unreliable because of epithelial-to-mesenchymal transition, which reduces expression for epithelial markers. Emerging label-free separation methods based on the biophysical and biomechanical properties of CTCs have the potential to address this key shortcoming and present greater flexibility in the subsequent characterization of these cells. In this review we first present what is known about the biophysical and biomechanical properties of CTCs from historical studies and recent research. We then review biophysical label-free technologies that have been developed for CTC separation, including techniques based on filtration, hydrodynamic chromatography, and dielectrophoresis. Finally, we evaluate these separation methods and discuss requirements for subsequent characterization of CTCs.

We present a microfluidic ratchet that exploits the deformation of individual cells through microscale funnel constrictions. The threshold pressure required to transport single cells through such constrictions is greater against the direction of taper than along the direction of taper. This physical asymmetry combined with an oscillatory excitation can enable selective and irreversible transport of individual cells in low Reynolds number flow. We devised a microfluidic device to measure the pressure asymmetry across various geometries of funnel constrictions. Using a chain of funnel constrictions, we showed that oscillatory pressure enables ratcheting transport when the pressure amplitude and oscillation period exceeds the threshold required to transport single cells. These experiments demonstrate the potential of using this mechanism to selectively transport biological cells based on their internal mechanics, and the potential to separate cells based on cell morphology or disease state.

The separation of cells based on their biomechanical properties, such as size and deformability, is important in applications such as the identification of circulating tumor cells, where morphological differences can be used to distinguish target cancer cells from contaminant leukocytes. Existing filtration-based separation processes are limited in their selectivity and their ability to extract the separated cells because of clogging in the filter microstructures. We present a cell separation device consisting of a hydrodynamic concentrator and a microfluidic ratchet mechanism operating in tandem. The hydrodynamic concentrator removes the majority of the fluid and a fraction of leukocytes based on size, while the microfluidic ratchet mechanism separates cancer cells from leukocytes based on a combination of size and deformability. The irreversible ratcheting process enables highly selective separation and robust extraction of separated cells. Using cancer cells spiked into leukocyte suspensions, the complete system demonstrated a yield of 97%, while enriching the concentration of target cancer cells 3000 fold relative to the concentration of leukocytes.

Changes in red blood cell (RBC) deformability are associated with the pathology of many diseases and could potentially be used to evaluate disease status and treatment efficacy. We developed a simple, sensitive, and multiplexed RBC deformability assay based on the spatial dispersion of single cells in structured microchannels. This mechanism is analogous to gel electrophoresis, but instead of transporting molecules through nano-structured material to measure their length, RBCs are transported through micro-structured material to measure their deformability. After transport, the spatial distribution of cells provides a readout similar to intensity bands in gel electrophoresis, enabling simultaneous measurement on multiple samples. We used this approach to study the biophysical signatures of falciparum malaria, for which we demonstrate label-free and calibration-free detection of ring-stage infection, as well as in vitro assessment of antimalarial drug efficacy. We show that clinical antimalarial drugs universally reduce the deformability of RBCs infected by Plasmodium falciparum and that recently discovered PfATP4 inhibitors, known to induce host-mediated parasite clearance, display a distinct biophysical signature. Our process captures key advantages from gel electrophoresis, including image-based readout and multiplexing, to provide a functional screen for new antimalarials and adjunctive agents.