Flat Cell Culturing Surface May Cause Misinterpretation of Cellular Uptake of Nanoparticles

In vitro cellular uptake of nanoparticles (NPs) is typically evaluated using a monolayer of cells seeded on a 2D culture plate, with the assumption of reliable and reproducible outcomes. However, recent developments reveal that 2D culture may produce errors in the measurement of cellular uptake of NPs due to issues including sedimentation and diffusion of NPs in cell-culture media. To shed more light on the effect of culture methods on the uptake of NPs, the same number of prostate cancer cells is cultured in 2D and 3D substrates and their uptake of quantum dots (QDs, as a model NP) and entrance mechanisms are assessed. Significantly fewer QDs are taken up, but they are more evenly distributed among the cells, in the 3D compared to the 2D culture method; in addition, QDs enter the cells via different mechanisms of endocytosis in 2D than they do in 3D approaches. Findings regarding cell cycle phase distribution also vary between 3D and 2D samples, which results in a significantly lower percentage of QDs being taken up in 3D compared to 2D culture. These findings indicate that the culture environment drastically influences NP–cell readouts, which may lead to misinterpretation of in vitro outcomes.