Supercharged Protein Nanosheets for Cell Expansion on Bioemulsions

Cell culture at liquid-liquid interfaces, for example, at the surface of oil microdroplets, is an attractive strategy to scale up adherent cell manufacturing while replacing the use of microplastics. Such a process requires the adhesion of cells at interfaces stabilized and reinforced by protein nanosheets displaying not only high elasticity but also presenting cell adhesive ligands able to bind integrin receptors. In this report, supercharged albumins are found to form strong elastic protein nanosheets when co-assembling with the co-surfactant pentafluorobenzoyl chloride (PFBC) and mediate extracellular matrix (ECM) protein adsorption and cell adhesion. The interfacial mechanical properties and elasticity of supercharged nanosheets are characterized by interfacial rheology, and behaviors are compared to those of native bovine serum albumin, human serum albumin, and α-lactalbumin. The impact of PFBC on such assembly is investigated. ECM protein adsorption to resulting supercharged nanosheets is then quantified via surface plasmon resonance and fluorescence microscopy, demonstrating that the dual role supercharged albumins are proposed to play as scaffold protein structuring liquid-liquid interfaces and substrates for the capture of ECM molecules. Finally, the adhesion and proliferation of primary human epidermal stem cells are investigated, at pinned droplets, as well as on bioemulsions stabilized by corresponding supercharged nanosheets. This study demonstrates the potential of supercharged proteins for the engineering of biointerfaces for stem cell manufacturing and draws structure-property relationships that will guide further engineering of associated systems.