Alginate–CaCl2 Crosslinked Hydrogels for Gel-Assisted Giant Unilamellar Vesicle Formation

Abstract Giant unilamellar vesicles (GUVs) are widely used model systems for investigating the biophysical properties of cell membranes. Hydrogel swelling provides a versatile route for GUV synthesis, but agarose─the standard hydrogel substrate─possesses weak hydrogen bonding that is not easily tuned, while covalently crosslinked hydrogels require complex preparation. Here, we present an alternative hydrogel system based on alginate crosslinked with Ca2+ and immobilized with poly-L-lysine. This rapid, tunable crosslinking approach enables faster gelation, greater control, and simplified preparation. We evaluated swelling behavior, vesicle yield, and size distribution as functions of crosslinking ratio and buffer osmolality. Optimal conditions closely mirrored agarose benchmarks while offering greater control over hydrogel properties, establishing a robust substrate for biomimetic membrane studies and synthetic cell construction.