Expansion of Functional Human Salivary Acinar Cell Spheroids with Reversible Thermo-Ionically Crosslinked 3D Hydrogels

This study reports a new class of reversible thermo-ionically crosslinked composite hydrogels—alginate–gelatin (AG), AG–collagen (AGC), and AG–hyaluronic acid (AGHA)—designed to support the expansion and differentiation of human salivary acinar (NS-SV-AC) and primary salivary functional unit (SFU) spheroids. Mechanical properties (≈11 kPa) mimicked native salivary gland tissue. Among the tested hydrogels, AGHA yielded large (>100-cell) spheroids with >93% viability, sustained metabolic activity, and robust expression of acinar markers (AQP5, ZO-1, NKCC1, α-amylase). Upon β-adrenergic stimulation with isoprenaline, AGHA-cultured spheroids showed increased α-amylase granule formation and secretion activity, confirming preserved acinar functionality. This low-cost, reversible hydrogel system allows recovery of intact spheroids via ion chelation at 37 °C and demonstrates high translational potential for regenerative salivary gland bioengineering.