A novel tool for the unbiased characterization of epithelial monolayer development in culture

Abstract

The function of an epithelial tissue is intertwined with its architecture. Epithelial tissues are often described as pseudo-two-dimensional, but this view may be partly attributed to experimental bias: many model epithelia, including cultured cell lines, are easiest to image from the “top-down.” We measured the three-dimensional architecture of epithelial cells in culture and found that it varies dramatically across cultured regions, presenting a challenge for reproducibility and cross-study comparisons. We therefore developed a novel tool (Automated Layer Analysis, “ALAn”) to characterize architecture in an unbiased manner. Using ALAn, we find that cultured epithelial cells can organize into four distinct architectures and that architecture correlates with cell density. Cells exhibit distinct biological properties in each architecture. Organization in the apical-basal axis is determined early in monolayer development by substrate availability, while disorganization in the apical-basal axis arises from an inability to form substrate connections. Our work highlights the need to carefully control for 3D architecture when using cell culture as a model system for epithelial cell biology and introduces a novel tool, built on a set of rules that can be widely applied to epithelial cell culture. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

The mechanical influence of densification on epithelial architecture

“…Cultured monolayer architecture changes as the layer densifies ( S1 Fig ) [ 8 ], suggesting the possibility that epithelial architecture arises from cell crowding. We used our computational model to test this possibility, varying cell density by limiting the amount of available basal substrate.…”

“…The ratio between the maximum slopes on either side of the shoulder, defined here as the Lateral-to-Apical Shape Index, is therefore an indicator of architecture development. Our image analysis pipeline ALAn defines Mature architectures as those in which the Lateral-to-Apical Shape Index is greater than or equal to 1 [ 8 ].…”

“…ALAN does not use the Lateral-to-Apical Shape Index to distinguish Intermediate architectures from Immature architectures. Rather, these are distinguished by A) an apically-biased asymmetry in the actin intensity plot and B) a change in the relationship between peak actin intensity and peak nuclear distribution across the apical-basal axis, such that the former is apical to the latter (meaning that an apical surface has started to develop in the cultured tissue) [ 8 ]. We examined how this Index evolves in early architectures, and whether it could be used as a means of assessing architecture development within the Intermediate group.…”

“…Because cell shapes and cell packing are intertwined, cell shape regularity (as measured by circularity) can be used to reflect packing efficiency. We previously described that the apical cell shapes of confluent cultured epithelial cells become more regular as they proliferate and the cultured tissue densifies [ 8 ] ( S1 Fig ). Cell shapes in the plane of the tissue are widely studied in the context of understanding and predicting the rheological properties of epithelial tissues.…”

“…An unbiased image analysis pipeline called ALAn (Automated Layer Analysis) was recently developed to measure apical-basal architecture in Madin-Darby Canine Kidney (MDCK) cultured epithelial cell layers [ 8 ]. ALAn recognizes a developmental series of three organized architectures (Immature, Intermediate, and Mature), and a fourth category of exclusion (Disorganized) based on the average shape and nuclear position of component cells in the apical-basal plane of the tissue [ 8 ].…”