3D tissue formation by stacking detachable cell sheets formed on nanofiber mesh

We present a novel approach for assembling 3D tissue by layer-by-layer stacking of cell sheets formed on aligned nanofiber mesh. A rigid frame was used to repeatedly collect aligned electrospun PCL (polycaprolactone) nanofiber to form a mesh structure with average distance between fibers 6.4um. When human umbilical vein endothelial cells (HUVECs), human foreskin dermal fibroblasts, and skeletal muscle cells (C2C12) were cultured on the nanofiber mesh, they formed confluent monolayers and could be handled as continuous cell sheets with areas 3x3cm² or larger. Thicker 3D tissues have been formed by stacking multiple cell sheets collected on frames that can be nested (i.e. Matryoshka dolls) without any special tools. When cultured on the nanofiber mesh, skeletal muscle, C2C12 cells oriented along the direction of the nanofibers and differentiated into uniaxially aligned multinucleated myotube. Myotube cell sheets were stacked (upto 3 layers) in alternating or aligned directions to form thicker tissue with ~50 um thickness. Sandwiching HUVEC cell sheets with two dermal fibroblast cell sheets resulted in vascularized 3D tissue. HUVECs formed extensive networks and expressed CD31, a marker of endothelial cells. Cell sheets formed on nanofiber mesh have a number of advantages, including manipulation and stacking of multiple cell sheets for constructing 3D tissue and may find applications in a variety of tissue engineering applications. Cell sheet engineering has attracted interest as a clinical study to replacing diseased tissue with confluently cultured cell sheets in vitro. Recent advances in cell sheet engineering have resulted in highly viable, cell-dense constructs based on studies with thermo-responsive polymer substrates. Although these advances have provided a powerful tool for cell sheet manipulation, we consider a different aspect of approach for easy and labor-independent method to obtain multiple number of cell sheets with large surface area. In order to achieve these difficulties, we used cell sheets formed on aligned electrospun PCL nanofiber membranes to assemble thick 3D tissues comprising multiple layers of cell sheets. Tissue engineering aims to replace diseased organs or tissues by combination of cells, scaffold materials and biochemical factors. Among various approaches, many groups have researched to fabricate 2D cellular sheets as an assembling unit which can be stacked with arbitrarily combined thick tissue in vitro. In this study, we cultured various types of cell on electrospun biocompatible nanofibrous mat to form cellular sheet and combined to multiple sheets by layer-by-layer stacking with laser-cut acrylic frames or inserting to 3D printer fabricated support.