Deterministic Single‐Cell Encapsulation in PEG Norbornene Microgels for Promoting Anti‐Inflammatory Response and Therapeutic Delivery of Mesenchymal Stromal Cells

Abstract Tissue engineering at single‐cell resolution has enhanced therapeutic efficacy. Droplet microfluidics offers a powerful platform that allows deterministic single‐cell encapsulation into aqueous droplets, yet the direct encapsulation of cells into microgels remains challenging. Here, the design of a microfluidic device that is capable of single‐cell encapsulation within polyethylene glycol norbornene (PEGNB) hydrogels on‐chip is reported. Cells are first ordered in media within a straight microchannel via inertial focusing, followed by the introduction of PEGNB solution from two separate, converging channels. Droplets are thoroughly mixed by passage through a serpentine channel, and microgels are formed by photo‐photopolymerization. This platform uniquely enables both single‐cell encapsulation and excellent cell viability post‐photo‐polymerization. More than 90% of singly encapsulated mesenchymal stromal cells (MSCs) remain alive for 7 days. Notably, singly encapsulated MSCs have elevated expression levels in genes that code anti‐inflammatory cytokines, for example, IL‐10 and TGF‐β, thus enhancing the secretion of proteins of interest. Following injection into a mouse model with induced inflammation, singly encapsulated MSCs show a strong retention rate in vivo, reduce overall inflammation, and mitigate liver damage. These translational results indicate that deterministic single‐cell encapsulation could find use in a broad spectrum of tissue engineering applications.