Emerging collective behaviors in biological tissues are largely controlled by the structure of the extracellular matrix (ECM). Notably, cells can deposit aligned ECM fibers guiding neighboring cells and leading to the formation of large domains of common cell orientation. In these active cell sheets, multilayers form at conflicts of orientation such as defects. At an early stage, bilayering corresponds to a 2D-to-3D transition that is critical in several biological processes in embryogenesis or cancer development but whose physical mechanism is still unclear. We have recently shown that aligned ECM on a surface can be mimicked by synthetic micro-patterned grooves. We then make mosaic surfaces with juxtaposed domains of uniform orientation recapitulating in a controlled way defect-like conflicts of orientation. The angle between the two orientations is one of our control parameters. Culturing a cell monolayer on such substrates can result in “crisscross” bilayers where the two layers are perpendicularly oriented. By monitoring quantitatively the formation of these bilayers in space and time, the fields of orientation or velocity can be quantitatively compared to the predictions of active matter theories developed in parallel by our collaborators in the laboratory. Other geometries can provide complementary information contributing to unravel the mechanism of formation of bilayers from a 2D monolayer as a first step in our understanding of the formation of 3D tissues.