Cytoplasmic crowding drives intracellular viscoelastic increase during epithelial maturation

The cytoplasm is a crowded, active medium whose mechanical properties underpin essential cellular functions, yet how intracellular rheology adapts during tissue maturation remains unclear. Here, we examine the evolution of cytoplasmic viscoelasticity in Madin-Darby canine kidney epithelial monolayers as cell density increases. Using magnetic rotational spectroscopy, an active microrheology technique, we quantify zero-shear viscosity and elastic modulus throughout monolayer maturation. We uncover a substantial enhancement of intracellular viscoelasticity correlated with increasing cell density and a concomitant rise in cellular dry mass concentration. Hyperosmotic perturbations on subconfluent monolayers reproduce this effect, confirming cytoplasmic crowding as a driver of rheological changes. Notably, intracellular viscoelasticity scales linearly with dry mass concentration, suggesting a direct coupling between internal mechanics and cytoplasmic concentration during epithelial maturation. These findings establish a link between tissue-level organization and intracellular mechanics, highlighting cytoplasmic crowding as a regulator of epithelial homeostasis and providing a framework to connect rheology with tissue behavior.