Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering.

Reconstructing a vascular network is a common task for three-dimensional (3-D) tissue engineering. Three-dimensional stratified tissues were created by stacking cell sheets, and the co-culture with endothelial cells (ECs) in the tissues was found to lead to in vitro pre-vascular network formation and promoted in vivo neovascularization after their transplantation. In this study, to clarify the effect of tissue fabrication process on a pre-vascular network formation, human origin ECs were introduced into the stratified tissue in several different ways, and the behavior of ECs in various positions of the 3-D tissue were compared each other. Human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), and their mixture were harvested as an intact cell sheet from temperature-responsive culture dish at low-temperature (<20 degrees C). Single mono-culture EC sheet was stacked with two NHDF-sheets in different orders, and 3 co-cultured cell sheets were layered by a cell sheet collecting device. Morphological analyses revealed that pre-vascular networks composing of HUVECs were formed in all the triple layer constructs. Confocal microscope observation showed that the pre-vascular networks formed tube structures like a native microvasculature. These data indicate that the primary EC positioning in 3-D tissues may be critical for vascular formation.