Intracellular crowding links dimensionality to cell fate through a mechano-metabolic signalling axis

Three-dimensional (3D) cell culture systems exhibit more native-like cell behaviour compared to conventional two-dimensional (2D) cell culture systems. However, it remains largely unknown how dimensionality alters cell behaviour. Here, we identify intracellular crowding as a key biophysical parameter altered by cell culture dimensionality, which directs cell fate over a mechano-metabolic axis. Specifically, culture dimensionality controlled intracellular crowding by altering cell volume, which was confirmed across multiple cell types and cell culture platforms. Using chondrogenesis as a model system, we demonstrated that dimensionality-induced intracellular changes lead to improved chondrogenic stem cell differentiation in microtissue culture via a FOXO1 signalling axis. Our findings highlight intracellular crowding as an important parameter of cell culture systems, and present a novel strategy for engineering biomimetic cell culture systems, which has implications for a multitude of applications including disease modelling, in vitro drug screening models, developmental biology, and cell-based therapies.