Tuning extracellular fluid viscosity to enhance transfection efficiency

Gene therapies and cellular programming rely on effective cell transfection. Despite continuous advancements in carrier development and transfection techniques to enhance efficiency, the biophysical parameter of extracellular fluid viscosity has been largely overlooked. Here we report a substantial impact of culture media viscosity on transfection efficiency of several delivery vehicles, including lipid nanoparticles, polyplexes, adeno-associated vectors and lentiviral vectors across a range of cell types. We observed substantially increased transfection efficiencies for lipid nanoparticles and polyplexes when the media viscosity matched that of biological fluids (2.0–4.0 centipoise (cP)). This enhancement correlates with higher levels of cellular uptake and improved endosomal escape. Moreover, cells cultured in optimized viscosity conditions exhibit a different profile of uptake pathways compared with those cultured at the standard viscosity of 0.8 cP. This discovery highlights the critical role of media viscosity in the transfection process and provides an additional method to optimize gene delivery and cell programming processes, potentially reducing production costs and increasing the accessibility of gene and cell therapies. Gene therapies and cellular programming rely on effective cell transfection. Here it is shown that optimizing the viscosity of cell culture media to match that of biological fluids substantially enhances the transfection efficiency for various gene delivery vehicles across different cell types.