The specific properties responsible for the stability and function induced by three-dimensional (3D) cellular constructs were evaluated and compared to a monolayer structure. 3D-cellular multilayers composed of human fibroblast cells (FCs) and human umbilical vascular endothelial cells (ECs) were fabricated by a hierarchical cell manipulation technique. Interestingly, the ECs adhered homogeneously onto four-layered (4L) FCs, and tight-junction formation was widely observed at the centimeter scale, while heterogeneous EC domain structures were observed on the monolayered (1L) FCs. The production of heat shock protein70 (Hsp70) and interleukin-6 (IL-6) from the cellular structures were investigated to elucidate any 3D-structural effect on cellular function. The Hsp70 expression of the ECs decreased after adhesion onto the 4L-FC structure as compared with the EC monolayer. Surprisingly, the Hsp70 production response to heat shock increased drastically by approximately 10-fold as compared with a non-heat shock by 3D structure formation, whereas the monolayer structures showed no change. Moreover, the production of the inflammatory cytokine IL-6 decreased significantly depending on the layer number of FCs. To the best of our knowledge, this is the first report on a basic, 3D-structural effect on cellular stability and function. These findings could be important for not only tissue engineering, but also for basic cell biology. (C) 2010 Elsevier Inc. All rights reserved.