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The role of astrocytes in brain function has received increased attention lately due to their critical role in brain development and function under physiological and pathophysiological conditions. However, the biological evaluation of soft material nanoparticles in astrocytes remains unexplored. Here, the interaction of crosslinked hybrid vesicles (HVs) and either C8-D1A astrocytes or primary astrocytes cultured in polystyrene tissue culture or floatable paper-based chips is investigated. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) (P1) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine lipids are used for the assembly of HVs with crosslinked membranes. The assemblies show no short-term toxicity towards the C8-D1A astrocytes and the primary astrocytes, and both cell types internalize the HVs when cultured in 2D cell culture. Further, it is demonstrated that both the C8-D1A astrocytes and the primary astrocytes could mature in paper-based chips with preserved calcium signaling and glial fibrillary acidic protein expression. Last, it is confirmed that both types of astrocytes could internalize the HVs when cultured in paper-based chips. These findings lay out a fundamental understanding of the interaction between soft material nanoparticles and astrocytes, even when primary astrocytes are cultured in paper-based chips offering a 3D environment., (© 2022 The Authors. Advanced Biology published by Wiley-VCH GmbH.)