Quantitating distance-dependent, indirect cell–cell interactions with a multilayered phospholipid polymer hydrogel

Abstract

Multilayered polymer hydrogels containing living cells were assembled for assessing the distance-dependent effects of soluble factors secreted by stroma cells on tumor cell cycle progression in vitro. A layer of tumor cells and a layer of stroma cells were separated with finely controlled spacing in a multilayered sandwich composed of a 2-methacryloyloxyethyl phosphorylcholine polymer and poly(vinyl alcohol) hydrogel. We demonstrated the utility of this tool for investigating intercellular communication between human cervical cancer HeLa cells and supportive stromal L929 fibroblast cells in co-culture. Time-lapse microscopic analyses of HeLa cells showed short distances (15 μm) between tumor cells and stroma cells induced a continuous increase in the percentage of HeLa cells in the S/G2/M phases of the cell cycle, while longer distances (70 μm) between the cells caused a slower increase followed by a sharp increase in the percentage of cells in S/G2/M phases. One possible explanation is gradient formation in the diffusion-dominant multilayer hydrogels by water-soluble factors such as those inducing growth, differentiation, and proliferation. This study provides insights into the potential effects of diffusion of soluble factors and related distance-dependent effects on cell behavior, which may contribute to the design of future co-culture systems.