The effect of cell spot sizes (micropattern sizes) on the morphological and functional behaviors of micropatterned hepatocytes was investigated. Three similar cell chips with collagen spot sizes of 200 mu m (chip 200), 500 mu m (chip 500), and 800 mu m (chip 800) were prepared by the microcontact printing method: the region outside the collagen spots was modified with polyethylene glycol to create the non-adhesion area. The total collagen area of these chips was kept similar. Although the morphology of rat hepatocytes on chips 500 and 800 showed an almost single-cell layer on the collagen spots similar to the random monolayer culture, hepatocytes on chip 200 gathered to the center of the collagen spot and formed multi-cell layers exhibiting a semi-spheroid form. The expression levels of cell-cell interaction molecules (cadherin and connexin-32) and cell function activities (albumin secretion and ammonia removal) were higher in the hepatocytes on chip 200 compared to those in other chip conditions and the random monolayer. These effects may relate to the boundary length obtained from spot size and spot number of collagen, and the cell chip with large boundary region may be superior in maintaining the functional expression of micropatterned hepatocytes. (C) 2010 Elsevier B.V. All rights reserved.