Three Dimensional Dynamics of Epithelial Monolayers

Abstract

Cells in epithelial monolayers are commonly quantified using a projected area and implicitly assuming constant cell volume and prism-shaped cells. These 2.5D assumptions ignore the volume and mass dynamics that may accompany density fluctuation in confluent space-filling tissues but remain inaccessible to the labelling- and intensity-based imaging techniques used to date. Here, we use time-lapse quantitative phase imaging (QPI) to obtain spatially and temporally resolved maps of height, volume, and dry mass in MDCK epithelial monolayers under physiological conditions. Three findings follow. (i) Cellular dry mass concentration is maintained to within [~] 4.5 % across the monolayer and over time, even during large-amplitude pulsations, ruling out fluid (water) transport as the dominant driver of height and area dynamics. (ii) The mean height of the monolayer increases from [~] 5.5 to [~] 9 {micro}m and the mean cell volume decreases by [~] 25 % as the cell density doubles, evidence of contact inhibition of cell size rather than constant cell volume. Cell heights vary up to 30%, oscillate with a [~] 5 h period, and follow gamma-shaped distributions. (iii) At cellular scales, both segmented-cell tracking and continuum mass-flux analysis show that projected cell volume is not conserved; mass conservation is recovered only after coarse-graining over [~] 2 cell diameters and [~] 1.6 h. Two non-exclusive mechanisms may explain this: non-prismatic cell geometry produces apparent volume fluctuations even at constant true volume, and periodic mass exchange between cells and the ECM produces genuine cyclic changes in cellular dry mass. A propagating elastic mode in the dry-mass structure factor (cs [~] 15 {micro}m/h) further indicates that the monolayer behaves as a viscoelastic active medium rather than a plug-flow fluid. Each of these results requires simultaneous, time-resolved access to height and dry mass at the cellular scale and is therefore newly visible with QPI applied to epithelial monolayers. Together, they question the common 2.5D assumptions of prism-shaped cells, constant volume, plug-flow kinematics, and argue that quantitative continuum and cell-based models of epithelial dynamics must incorporate dry-mass-density regulation and 3D cell geometry.