Stiffness based enrichment of leukemia cells using microfluidics.

To improve the survival rate of cancer patients, new diagnosis strategies are necessary to detect lower levels of cancer cells before and after treatment regimens. The scarcity of diseased cells, particularly in residual disease after treatment, demands highly sensitive detection approaches or the ability to enrich the diseased cells in relation to normal cells. We report a label-free microfluidic approach to enrich leukemia cells from healthy cells using inherent differences in cell biophysical properties. The microfluidic device consists of a channel with an array of diagonal ridges that recurrently compress and translate flowing cells in proportion to cell stiffness. Using devices optimized for acute T cell leukemia model Jurkat, the stiffer white blood cells were translated orthogonally to the channel length, while softer leukemia cells followed hydrodynamic flow. The device enriched Jurkat leukemia cells from white blood cells with an enrichment factor of over 760. The sensitivity, specificity, and accuracy of the device were found to be > 0.8 . The values of sensitivity and specificity could be adjusted by selecting one or multiple outlets for analysis. We demonstrate that low levels of Jurkat leukemia cells (1 in 10 4 white blood cells) could be more quickly detected using flow cytometry by using the stiffness sorting pre-enrichment. In a second mode of operation, the device was implemented to sort resistive leukemia cells from both drug-sensitive leukemia cells and normal white blood cells. Therefore, microfluidic biomechanical sorting can be a useful tool to enrich leukemia cells that may improve downstream analyses.

Microfluidic cell sorting by stiffness to examine heterogenic responses of cancer cells to chemotherapy.

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.

Continuous Sorting of Cells Based on Differential P Selectin Glycoprotein Ligand Expression Using Molecular Adhesion.

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.

Normal saline is associated with increased sickle red cell stiffness and prolonged transit times in a microfluidic model of the capillary system.

OBJECTIVE: Vaso-occlusive crisis (VOC) is a complex process that occurs in patients with sickle cell disease (SCD) and is often associated with pain and urgent hospitalization. A major instigator of VOC is microvascular obstruction by pathologically stiffened sickle red blood cells (RBCs), and thus, therapy relies heavily on optimizing intravenous fluid (IVF) hydration to increase RBC deformability. However, no evidence-based guidelines regarding the choice of IVF currently exist. We therefore analyzed alterations in biomechanical properties of sickle RBCs isolated from patients with homozygous SCD (hemoglobin SS) after exposure to different osmolarities of clinical IVF formulations. METHODS: Atomic force microscopy (AFM) was used to assess stiffness of RBCs after exposure to different IVFs. A microfluidic model of the human capillary system was used to assess transit time (TT) and propensity to occlusion after exposure to the different IVF formulations. RESULTS: Sickle RBCs exposed to normal saline (NS) had increased stiffness, TTs, and propensity to microchannel occlusion compared to other osmolarities. CONCLUSION: NS, an IVF formulation often used to treat patients with SCD during VOC, may induce localized microvascular obstruction due to alterations of sickle RBC biomechanical properties.