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 attrib-uted 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 chal-lenge for reproducibility and cross-study comparisons. We therefore developed a novel tool (Automated Layer Analysis, "ALAn") to characterize architecture in an unbiased manner. Us-ing 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 de-velopment 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 three-dimensional 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 epi-thelial cell culture.
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