Micropatterning is a promising technique for modulating culture environments. In this study, we investigated the effect of spheroid separation distance on their properties in a micropatterned chip of HepG2 spheroids. The basic chip design consisted of 37 collagen spots (300 mu m in diameter) in a hexagonal arrangement on a glass substrate; the region without collagen-spots was modified by polyethylene glycol to create the non-adhesive surface. Three similar chips were fabricated with gap distances between collagen-spots of 500, 1000, and 1500 mu m. HepG2 cells adhered on the collagen spots and then formed spheroids via cell proliferation. Although the albumin secretion activities of HepG2 spheroids were almost the same in all chips, inhibition of spheroid growth and anaerobic metabolism were intensified when the gap distance was less than 1000 mu m. Additionally, such phenomena which are induced by interference effects between spheroids, were more pronounced at the inside region of the chip than at the outside region. However, the interference effect between spheroids was nearly avoided when the gap distance was at least 1500 mu m. Furthermore, the concentration of dissolved oxygen between neighboring spheroids decreased as the gap distance decreased, indicating that the spheroids competed for oxygen and became hypoxic in a way that depended on the spheroid separation distance. These results indicate that the spheroid separation distance is an important factor that can modulate the spheroid properties. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.