Morphological switch is associated with increase in cell–cell contacts, ALP, and confluence above a minimum island area to perimeter ratio

During osteogenic differentiation in vitro, stem-like cells seeded at a low-density spread and are isolated. As the cells proliferate and mature, they become more cuboidal in shape with more cell–cell contacts. However, the coordination of this switch in cell morphology from elongated to cuboidal, cell–cell contacts, and differentiation is not known. In this work, we present results from experiments and a simulation of cell proliferation on protein-micropatterned islands that, independent of island size (25–1,000 μm) or shape (circles, squares, and hollow squares), shows a distinct morphological switch that is better described as a function of island confluence than time in culture, the standard measure in cell culture experiments. The simulation and experiments show cell morphology and island cell density versus confluence collapse to a single curve for all islands if the island area to perimeter ratio is ≥25 μm. Cell–cell contacts in the simulation and alkaline phosphatase (ALP) expression in experiments, a common marker for osteogenic differentiation, show exponential growth with confluence, rapidly increasing after the switch at ≈0.5 confluence. Furthermore, cell morphology, density, contacts, and ALP are better predicted by confluence than time in culture. The variability with time in culture leads to challenges in not only interpreting data but also in comparing data across research laboratories. This simulation can be used to predict cell behavior on different size and shape islands and to plan and optimize experiments that explore cell behavior as a function of a wide range of island geometries.