Cancers consist of a heterogeneous populations of cells that may respond differently to treatment through drug-resistant sub-populations. The scarcity of these resistant sub-populations makes it challenging to understand how to counter their resistance. We report a label-free microfluidic approach to separate cancer cells treated with chemotherapy into sub-populations enriched in chemoresistant and chemosensitive cells based on the differences in cellular stiffness. The sorting approach enabled analysis of the molecular distinctions between resistant and sensitive cells. Consequently, the role of multiple mechanisms of drug resistance was identified, including decreased sensitivity to apoptosis, enhanced metabolism, and extrusion of drugs, and, for the first time, the role of estrogen receptor in drug resistance of leukemia cells. To validate these findings, several inhibitors for the identified resistance pathways were tested with chemotherapy to increase cytotoxicity sevenfold. Thus, microfluidic sorting can identify molecular mechanisms of drug resistance to examine heterogeneous responses of cancers to therapies.

Threedimensional particle tracking in microfluidic channel flow using in and out of focus diffraction, Flow Measurement and Instrumentation, http://dx.ABSTRACT Three-dimensional particle tracking is important to accurately understand the motion of particles within complex flow fields. We show that three-dimensional trajectories of particles within microfluidic flow can be extracted from two-dimensional bright field video microscopy. The method utilizes the defocusing that occurs as particles move out of the objective focal plane when viewed through a high numerical aperture objective lens. A fast and simple algorithm based on cross correlation to a set of reference images taken at prescribed amounts of defocus is used to extract out-of-plane particle position. In-plane particle position is determined through center point detection and therefore the particle position in all three dimensions can be constructed at each time point. Particle trajectories at high flow velocity of greater than 2 mm/s can be tracked by utilizing a high speed camera to obtain unblurred images. Three dimensional computational fluid simulations are used to validate the particle tracking methods.

Cell surface molecular adhesions govern many important physiological processes and are used to identify cells for analysis and purifications. But most effective cell adhesion separation technologies use labels or long-term attachments in their application. While label-free separation microsystems typically separate cells by size, stiffness, and shape, they often do not provide sufficient specificity to cell type that can be obtained from molecular expression. We demonstrate a label-free microfluidic approach capable of high throughput separation of cells based upon surface molecule adhesion. Cells are flowed through a microchannel designed with angled ridges at the top of the channel and coated with adhesive ligands specific to target cell receptors. The ridges slightly compress passing cells such that adhesive contact can be made with sufficient surface area without unduly affecting cell trajectories because of cell stiffness. Thus, sorting is sensitive to cell adhesion but not to stiffness or cell size. The enforced interactions between the cells and the ridges ensure that a high flow rate can be used without lift forces quenching adhesion. As a proof of principle of the method, we separate both Jurkat and HL60 cell lines based on their differential expression of PSGL-1 ligand by using a ridged channel coated with P selectin. We demonstrate 26-fold and 3.8-fold enrichment of PSGL-1 positive and 4.4-fold and 3.2-fold enrichment of PSGL-1 negative Jurkat and HL60 cells, respectively. Increasing the number of outlets to five allows for greater resolution in PSGL-1 selection resulting in fractionation of a single cell type into subpopulations of cells with high, moderate, and low PSGL-1 expression. The cells can flow at a rate of up to 0.2 m/s, which corresponds to 0.045 million cells per minute at the designed geometry, which is over 2 orders of magnitude higher than previous adhesive-based sorting approaches. Because of the short interaction time of the cells with the adhesive surfaces, the sorting method does not further activate the cells due to molecular binding. Such an approach may find use in label-free selection of cells for a highly expressed molecular phenotype.

High throughput size based separation and sorting of bioparticles and cells is critical to a variety of biomedical processing steps for medical diagnostics and pharmaceutical purification. Improving microfluidic size-based particle/cell sorting is a challenge to better address the need for generating more homogeneous subpopulations for study and use. We propose a novel advance to microfluidic sorting devices that uses three-dimensional focusing of the sample to optimally position particles to amplify the size-dependent differences in trajectories caused by differential secondary flows. The result is an increase in the purity of small particles by 35- fold and large particles by 8-fold in comparison to unfocused flow. Our simulated and experimental data reveal for the first time that positioning particles in three-dimensional space can be used to better leverage the differential lateral movement of particles with different sizes as they flow in microchannel with transverse secondary flows. The focusing approach may also be useful to improve positioning of particles with inertial channels with multiple equilibrium positions. This technique performs continuous-flow, high throughput size based sorting of millions of particles and cells in a minute without any pre and post-processing. We have also demonstrated improved enrichment and recovery of white blood cells from human blood.