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

Abstract

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.

Microfluidic Platform to Transduce Cell Viability to Distinct Flow Pathways for High-Accuracy Sensing

“…Transient adhesion between cells and microchannel surfaces can affect the cell trajectory . The magnitude of the adhesion can be modified using surface treatments.…”

“…Transient adhesion between cells and microchannel surfaces can affect the cell trajectory. 39 The magnitude of the adhesion can be modified using surface treatments. In occasional events, we observed adhesion of nonviable cells to the microfluidic channel surface that caused an increased ridge interaction time and a pile up of the cells near the ridges.…”

Magnetic Ranking Cytometry: Profiling Rare Cells at the Single-Cell Level

“…This approach enables sorting cells according to their biochemical and functional properties. However, several microfluidic approaches have exploited physical parameters, such as size, viscoelasticity, stiffness, and adhesiveness to enable cell sorting into distinct capture zones.…”

Recent Advances in Microfluidic Techniques for Systems Biology

“…Functionalized microchannel walls with different antibodies were also used to profile surface markers of single cells using the same measurement scheme . Other than high-throughput characterizations, the continuous-flow based strategy has also been used for mechanical property-based cell sorting in various medical applications, such as capturing circulating tumor cell clusters, examining heterogenic cancer cell chemotherapy responses, and screening drug compounds by cell viability. − …”