Mouse L929 fibroblasts and normal human dermal fibroblasts (NHDFs) were constructed into three-dimensional (3-D) multilayered tissues by directly coating them with nano-films consisting of fibronectin (FN) and gelatin (G) onto the surface of the cell layer using layer-by-layer assembly. The one-, two- and five-layered (1L, 2L and 5L) tissues were cultured for 1month in order to evaluate their long-term survival and structural changes. L929 cells in the 3-D tissues showed excessive proliferation throughout the culture period, regardless of the starting layer number. The cross-sectional images stained with hematoxylin and eosin revealed heterogeneous and random increases in the thickness of their layered structures, probably due to the immortalized property of L929 fibroblasts. On the other hand, NHDFs, which are primary cells, showed high stability in their amount of DNA in the multilayered structures, and their thicknesses were completely maintained even after 1month of incubation. To evaluate the living cell density in each layer of 5L tissues during the culture period, 5L NHDFs were fluorescently labeled with LIVE/DEAD reagent and analyzed by confocal laser scanning microscopy. Although the upper layers did not show any dead cells, the bottom layers showed pieces of dead cell nuclei depending on culture time. However, the living cell densities in all layers were almost the same, even after 1month of culture, suggesting that the 5L structures were completely filled with living cells. These findings from the multilayered tissue constructs will provide important information not only for the construction of 3-D engineered tissues in tissue engineering but also on 3-D cell culture in biological science generally.