Intracellular transport of macromolecules is crucial for the proper functioning of most cellular processes. Although intracellular crowding is known to strongly alter macromolecule mobility, how cytoplasmic structures physically modulate diffusion remains largely unexplored. Here, we investigated the mechanisms by which cytoplasmic crowding controls diffusivity using live-cell experiments and porous media modeling approaches. Confocal microscopy combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy measurements revealed an anticorrelation between free green fluorescent protein diffusivity and the heterogeneous cytoplasmic structure abundance in live mammalian cells. This motivated the development of a multiscale model, where the cytoplasm is treated as a hierarchical porous medium with nanometric and micrometric obstacles. Numerically solving the model allowed us to predict the effective cytoplasmic diffusion coefficient for various obstacle volume fractions and to identify tortuous and porous hydrodynamic hindrances as key diffusion reduction mechanisms. Comparison with our experimental results highlighted the importance of hydrodynamic interactions between diffusing molecules and nanometric obstacles. Importantly, we found that the effective cytoplasmic diffusivity was not dependent on specific intracellular regions but rather on the local intracellular obstacle volume fraction. Finally, the model was extended to predict the diffusivity of larger macromolecules, showing excellent agreement with literature data for several macromolecules and cell lines. This study provides insights into the physical mechanisms impeding intracellular diffusion, demonstrating the potential of porous media modeling approaches to predict transport mechanisms in dynamic or heterogeneous intracellular structures, as in cell motility, blebbing, and apoptosis.

Cells within biological tissue are constantly subjected to dynamic mechanical forces. Measuring the internal stress of tissues has proven crucial for our understanding of the role of mechanical forces in fundamental biological processes like morphogenesis, collective migration, cell division, or cell elimination and death. Previously, we have introduced Bayesian inversion stress microscopy (BISM), which relies on measuring cell-generated traction forces in vitro and has proven particularly useful to measure absolute stresses in confined cell monolayers. We further demonstrate the applicability and robustness of BISM across various experimental settings with different boundary conditions, ranging from confined tissues of arbitrary shape to monolayers composed of different cell types. Importantly, BISM does not require assumptions on cell rheology. Therefore, it can be applied to complex heterogeneous tissues consisting of different cell types, as long as they can be grown on a flat substrate. Finally, we compare BISM to other common stress measurement techniques using a coherent experimental setup, followed by a discussion on its limitations and further perspectives.