By the use of a MatriGrid (R) we have established a three-dimensional high density cell culture. The MatriGrid (R) is a culture medium permeable, polymeric scaffold with 187 microcavities. In these cavities (300m diameter and 207m deep) the cells can growth three-dimensionally. For these experiments we measured the oxygen consumption of HepG2 cell cultures in order to optimize cultivation conditions. We measured and compared the oxygen consumption, growth rate and vitality under three different cultivation conditions: monolayer, three-dimensional static and three-dimensional actively perfused. The results show that the cells in a three-dimensional cell culture consume less oxygen as in a monolayer cell culture and that the actively perfused three-dimensional cell culture in the MatriGrid (R) has a similar growth rate and vitality as the monolayer culture. Biotechnol. Bioeng. 2013; 110:2504-2512. (c) 2013 Wiley Periodicals, Inc.