Morphological and Functional Changes of Rat Hepatocytes by Vertical Cell-Cell Adhesion in Three-Dimensional Stacked-Up Culture

Vertical cell-cell contact is an important difference between 2D- and 3D-culture models. We hypothesized that this difference in cell-cell contacts is critical in maintaining hepatocyte functions. Here we developed a simple culture technique to investigate the effect of the vertical cell-cell contacts on morphogenesis, proliferation, and differentiation of hepatocytes. Two separate monolayers of primary rat hepatocytes were first cultured on a microporous membrane and a culture dish and one was then inverted and placed on top of the other to create a 3D-configuration. Imaging techniques revealed that hepatocytes recovered cell polarity and formed bile canaliculi in response to the addition of the vertical cell-cell contacts. Quantitative analysis revealed that the cells exhibited cuboidal cell shape with vertical cell-cell contacts and maintained differentiated functions while their proliferation activity was inhibited. In contrast, hepatocytes in a monolayer gradually flattened due to the lack of vertical cell-cell contacts, resulting in cells losing differentiated functions which correlated to the deformation of cell shape. Controlling 2D- and 3D-configurations is important in switching growth and differentiation of hepatocytes. These results implicate how we apply 2D- and 3D-cultures for tissue engineering of the liver.