Extra cellular matrix (ECM) is a natural cell environment, possesses complicated nano- and macro- architecture. Mimicking this three-dimensional (3-D) web is a challenge in the modern tissue engineering. This study examined the application of a novel 3-D construct, produced by multilayered organization of electrospun nanofiber membranes, for human bone marrow-derived mesenchymal stem cells (hMSCs) support. The hMSCs were seeded on an electrospun scaffold composed of poly epsilon-caproloactone (PCL) and collagen (COL) (1:1), and cultured in a dynamic flow bioreactor prior to in vivo implantation. Cell viability after seeding was analyzed by AlamarBlue (TM) Assay. At the various stages of experiment, cell morphology was examined by histology, scanning electron microscopy (SEM) and confocal microscopy. Results: A porous 3-D network of randomly oriented nanofibers appeared to support cell attachment in a way similar to traditionally used tissue culture polysterene plate. The following 6 week culture process of the tested construct in the dynamic flow system led to massive cell proliferation with even distribution inside the scaffold. Subcutaneous implantation of the cultured construct into nude mice demonstrated good integration with the surrounding tissues and neovascularization. Conclusion: The combination of electrospinning technology with multilayer technique resulted in the novel 3-D nanofiber multilayered construct, able to contain efficient cell mass necessary for a successful in vivo grafting. The success of this approach with undifferentiated cells implies the possibility of its application as a platform for development of constructs with cells directed into various tissue types.