Thin film hydration methods are widely used to assemble giant unilamellar vesicles (GUVs), but their efficiency drops sharply in solutions of physiological ionic strength due to enhanced adhesion between stacked lipid bilayers, which suppresses bud and foam-like mesophase formation. Here, polymer-coated nanocellulose paper (PCP) is introduced as a nanostructured substrate that overcomes this limitation. Hydration of lipid films on PCP, termed PCP-assisted hydration, achieves high GUV yields across a broad temperature range (4 °C-45 °C) using diverse soluble polymers, including ultralow-gelling agarose, hyaluronic acid, dextran, carrageenan, and polylysine. The nanoscale curvature of the cellulose fibers and the osmotic pressure generated by polymer dissolution act synergistically to promote membrane budding even under physiological salt conditions. The approach is scalable-supporting GUV production from millimeter-sized pieces to large-format sheets-and biocompatible, enabling encapsulation of complex biochemical systems such as cell-free expression mixtures and actin-fascin assemblies. PCP-assisted hydration thus provides a robust, versatile, and high-yielding platform for generating functional GUVs under physiological conditions.
Keywords: actin; artificial tissues; cell‐free expression; giant unilamellar vesicles; synthetic cells.
© 2025 The Author(s). Small Methods published by Wiley‐VCH GmbH.