Facile and Green Fabrication of Porous Hydrogels Based on Gelatin Microsphere Porogens for 3D Immune Cell Culture

Porous hydrogels are critical for tissue engineering and regenerative medicine, as they mimic the native extracellular matrix to support cell infiltration and mass transport. A common strategy for engineering pore structures involves the incorporation and subsequent removal of sacrificial porogen templates (e.g., crystals or microspheres). Although this approach offers excellent control over pore architecture, it often suffers from complex procedures and biosafety concerns arising from incomplete template removal. In this work, we present a simple, biocompatible, and versatile templating approach. By systematically investigating the coacervation parameters, we produced gelatin microspheres (GSs) with tunable diameters from 7 µm to 300 µm via a green, instrument-free, and scalable process. Using GSs of 20-160 µm as porogens, we obtained alginate hydrogels with adjustable viscoelasticity, stiffness, and pore sizes. We then validated two cell-loading strategies for bulk porous alginate hydrogels using immortalized human T (Jurkat) cells: (i) post-seeding into pre-formed pores supported high-density, long-term, and organized cell aggregates with >90% viability; (ii) in situ encapsulation (prior to pore formation) yielded >80% viability and preserved the cluster-forming growth characteristics of Jurkat cells. Moreover, composites of smaller GSs (7-20 µm) with alginate could be syringe-extruded into stable, sub-millimeter porous filaments, demonstrating the potential for 3D printing. Collectively, this work provides a promising platform for three-dimensional culture of immune cells.