Microfluidic generation of alginate microbeads containing in situ-formed apical papilla stem cell spheroids and NecroX-5-loaded nanoparticles

Abstract

Mesenchymal stem/stromal cells (MSC) are key players in regenerative medicine due to their immunomodulatory properties and ability to promote tissue repair. However, their therapeutic efficacy is often limited by rapid clearance following transplantation. MSC spheroids have shown enhanced functional properties, and we hypothesize that encapsulating them within hydrogel microbeads could offer additional protection and improve their viability. We developed a continuous, single-emulsion droplet-based microfluidic protocol that forms MSC spheroids in situ within alginate droplets overnight (In-Bead), before cross-linking and bead retrieval. Unlike double-emulsion approaches, the single-emulsion format yields homogeneously distributed nanomedicines, and the short, cell-permissive cross-linking produces ultra-soft, tissue-mimetic (~150 Pa) beads that remain retrievable. SCAP were successfully encapsulated within 275 um alginate microbeads, forming spheroids of approximately 80 um in diameter. Encapsulated SCAP spheroids retained their immunomodulatory properties. The process was further optimized by incorporating nanomedicines into the alginate solution before the formation of droplets, thus forming hybrid beads (Sph.Beads/NP). Nanomedicines were loaded with NecroX-5, a necrosis inhibitor, to improve SCAP viability further. Live/Dead assays indicated a protective effect of the nanomedicines, supporting the potential of this system for advanced cell delivery in regenerative applications.