Rapid Construction of Three‐Dimensional Multilayered Tissues with Endothelial Tube Networks by the Cell‐Accumulation Technique

The in vitro construction of living tissue or organ models, which are highly organized with various types of cells and extracellular matrices (ECM) and allow for the evaluation of tissue functions, have attracted increasing attention in the fi elds of tissue engineering and drug assessment. [ 1–3 ] In particular, the fabrication of vascularized thick and complex tissues is a key challenge for transplantable constructs or angiogenesis models. [ 4 ] Although various functional scaffolds possessing specifi c cell-adhesive and degradability-controllable properties have achieved notable advances in tissue regeneration, [ 5–8 ] three-dimensional (3D) tissue models which precisely control the cell type, cell alignment, and cell-cell interactions in all three dimensions have not been developed yet. A conventional approach using biodegradable scaffolds has several limitations in developing 3D tissue constructs which satisfy the above requirements. Recently, several bottom-up approaches such as a cell sheet, [ 9–11 ] magnetic liposomes, [ 12 , 13 ] and cell-containing gel layers [ 14 ] have been reported for the construction of multilayered tissues. These methods are intriguing examples of a bottom-up approach, but have limitations due to the complicated manipulation of fragile cell sheets or the remains of magnetic particles in the cells. We have developed a simple bottom-up approach by preparing nanometer-sized ECM fi lms on cell surfaces. [ 15–17 ] Less than 10 nm-thick fi bronectin-gelatin (FN-G) fi lms prepared by layer-by-layer (LbL) assembly [ 18–20 ] promoted cell-cell interactions like a natural ECM, and thus we successfully fabricated over fi ve layered (5L) tissue models such as blood vessels, skeletal muscle, and connective tissue. Although this technique is simple and versatile enough to develop the multilayered constructs while controlling the cellular type and location, the fabrication of 2L tissues is limited due to the time required for stable cell adhesion. Therefore, a simple and rapid approach is strongly desired for the in vitro construction of thick multilayered tissue models. In this study, we developed a simple and rapid bottom-up approach, called the cell-accumulation technique, by a single