Tissues are composed of multiple cell types in a well-organized three-dimensional (3D) micro-environment. To faithfully mimic the tissue in vivo, tissue-engineered constructs should have well-defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two-dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well-defined microstructure were constructed into a multilayer cell-scaffold complex using low pressure carbon dioxide (CO2) and nitrogen (N-2). The mouse ES cells in the assembled constructs were viable, retained the ES cell-specific gene expression of Oct-4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO2 was replaced with N-2. The compressed gas-assisted bioassembly of stem cell-polymer constructs opens tip a new avenue for tissue engineering and cell therapy. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 535-542, 2009