Space-efficient method for high-throughput generation of uniform cell-laden hollow agarose microcapsules

Hollow hydrogel microcapsules isolate cells while allowing diffusion of nutrients and factors, supporting applications from single-cell analysis and clonal expansion to spheroid formation inside them. Conventional encapsulation relies on bulky instruments such as pumps and pressure controllers, hindering use in confined environments such as anaerobic chambers. Here we present a space-efficient method combining pump-free, −20 kPa negative-pressure droplet generation with particle-templated emulsification to produce millions of ∼70 µm hollow agarose microcapsules using only a sealed bottle and a vortex mixer within a 30 cm × 30 cm footprint. The system maintains ∼491 ± 2 drops/s and yields (2.26 ± 0.09) × 10 ⁶ capsules per batch with CV < 4% (N = 3). Operating entirely inside an anaerobic chamber, we encapsulated mouse gut bacteria and quantified population-level proliferation over four days. This space-efficient workflow enables high-throughput encapsulation under spatial constraints such as biosafety cabinets or anaerobic chambers, providing a practical foundation for downstream single-cell and clonal analyses.