We have developed a label-free and passive method for the early isolation of activated T-cells and the first technique that enables the isolation prior to the display of cellular surface markers. T-cells are white blood cells whose activation is a critical step in adaptive immune response. The isolation of activated T-cells from naive cells is important for their study and downstream use.

The conventional methods of isolating activated T-cells rely on the use of antibodies specific to surface markers. However, it can take many hours and as much as a full day after activation for the markers to be displayed; greatly limiting fast detection and selection. In contrast, changes in cell metabolism occur within minutes after activation.

The presented work is based on our recently developed sorting platform dubbed "Sorting by Interfacial Tension" (SIFT) that sorts droplets based on pH. After bead activation (CD3/CD28) and a brief incubation on chip, droplets containing activated T-cells display a lower droplet pH due to proton secretion associated with increased glycolysis. Under specific surfactant conditions, a change in pH can lead to a concurrent increase in droplet interfacial tension. The sorting of activated T-cells on chip is hence achieved as flattened droplets are displaced as they encounter a micro-fabricated trench oriented diagonally with respect to the direction of flow.

The pH of droplets containing cells was studied as a function of time of activation. Longer activation times led to a lower droplet pH with a change in droplet pH observed in as little as 15 minutes of activation. Droplets containing activated cells (compared to naive cells or an empty droplet) have a lower pH due to the enhanced metabolism, and hence higher interfacial tension, leaving the trench at a different lateral position. This leads to an enrichment of activated T-cells from an initial mixed population of activated (15 minutes) and naive T-cells. Preliminary results led to an enrichment from 28% to 75% of activated T-cells. Moreover, since the pH change is correlated to the level of activation, the technique allows the isolation of T-cells with the highest activation, an important subset of cells for study and potentially immunotherapy.

This novel label-free technique for the early detection, enrichment and isolation of activated T-cells in minutes rather than hours can have broad usage as a biotechnology tool and for the study and selection of T-cells for immunology and immunotherapy.