Tissues are composed of multiple cells with well-organized three-dimensional (3-D) structure. Tissue-engineered constructs require well-defined 3-D chemical and spatial control over cells to assemble them into a functional structure that more faithfully mimics the tissue in vivo. Pattern formation is a hallmark of the precise control of cell behavior to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis. However, most of patterning is two-dimensional (2-D). It is challenge to assemble the 2-D patterned surfaces with cells into 3-D structure. Here, using low-pressure carbon dioxide, we demonstrated a biologically permissive fusion approach to assembling polymeric well-defined microstructure with embryonic stem (ES) cells grown on it into a multilayer cell-scaffold constructs. The mouse ES cells in the assembled constructs are viable, maintain the ES cell-specific gene expression of Oct-4, a canonical marker of pluripotency, and can further form embryonic bodies. In addition to tissue engineering, this CO2-assisted bio-assembly method can have wide applications in polymer-based MEMS/NEMS, such as biochips and drug/gene delivery devices.