Applying high‐throughput analysis to understand cell proliferation under different adhesion conditions

Abstract

Mammalian cell culture is a staple of biomedical research, and yet it has long been recognized that the conditions in which cells are cultured do not reflect the cellular environment in vivo. Adherent cells are cultured on a plastic surface with an elastic modulus over 10,000‐fold stiffer than what is experienced by neuronal, epithelial and muscle cells in living tissues. These altered conditions have a variety of effects on cellular behaviors, especially in regard to cell shape, motility and division. Previous work in the lab suggest that cell adhesion and motility play a role in the mechanics of cytokinesis in adherent cells. To test this hypothesis, we are conducting a study of the cell cycle and cell division dynamics of human Retinal Pigmented Epithelial (RPE1) cells cultured on substrates of differing mechanical properties. We have genetically engineered lines of RPE1 cells that express either a nuclear Red Fluorescent Protein (RFP) marker or a two‐color fluorescent cell cycle biosensor that respectively quantify cell number and cell cycle timing using a high‐throughput imaging system. The doubling time as well as the lengths of each phase of the cell cycle will be measured in cells grown in matrices with decreasing stiffness, and these experiments will also allow us to define a minimal matrix stiffness that will still afford successful cytokinesis. These data will then form the foundation for future studies where we perturb different components of the cytokinetic machinery and test the ability of cells to compensate through increased cell adhesion and motility.