Integrated Microfluidic Platform for Inertial Separation and Encapsulation of Single Cells in Droplets

Abstract

The ability to sort and separate large cellular subpopulations based on their intrinsic properties underpins a wide range of biological, diagnostic, and therapeutic applications. For many of these applications, maintaining cellular homogeneity within the confinement of a droplet is critical for accurate quantitative analysis and subsequent processing. Although microfluidic platforms have successfully enabled the separation of cellular subpopulations from heterogeneous samples, the lack of droplet-based encapsulation post separation remains a major bottle neck for achieving high-throughput single-cell analysis.Here, we address this limitation by developing an integrated microfluidic device that enables size-based cell separation and simultaneously encapsulating single cells into picolitre droplets. The device overcomes unstable encapsulation of cells by uniformly spacing cells prior to the encapsulation process. Proof-of-concept experiments achieved a single-particle encapsulation efficiency of 60% for 15 µm polystyrene beads, exceeding the Poisson limit of ~35% single occupancy. Size-based separation of 15-µm particles from 10-µm particles yielded a separation efficiency of 94.39%, with nearly 60% of the separated particles successfully encapsulated as single particles in droplets. Validation experiments using MDA-MB-231 cancer cells dispersed in white blood cells (WBCs) demonstrated a 92.74% separation efficiency, with approximately 28% of cancer cells encapsulated as single cells within droplets.