Construction of Multi-layered Cell Sheet Using Magnetite Nanoparticles and Magnetic Force

Abstract

Novel technologies to establish 3D tissue-like constructs are desired for tissue engineering. In the present study, magnetic force and magnetite nanoparticles were used to construct a layered mesenchymal stem cell (MSC) sheet, a layered cardiomyocyte sheet, and a layered fibroblast sheet involving capillaries. Magnetite cationic liposomes (MCLs), which have a positive surface charge, were taken up by the target cells. When a magnet was set under a tissue culture dish, magnetically labeled target cells were attracted and then adhered to form a layered cell sheet. For MSC sheets, MSCs within the sheets maintained an in vitro multi-differentiation ability into osteoblasts, adipocytes or chondrocytes after a 21-day culture period using each induction medium. By using an electromagnet, MSC sheets were harvested and transplanted into the bone defect in cranium of nude rats. Histological observation revealed that new bones surrounded by osteoblast-like cells were formed in the defect area of rats on 14 days after transplantation with MSC sheets. For cardiomyocyte sheets, the immuno-fluorescence staining of connexin43 revealed the existence of gap junctions within the cardiomyocyte sheets. Moreover, electrical connection within the cardiomyocyte sheets was confirmed by using extracellular potential mapping. For fibroblast sheets, normal human dermal fibroblasts (NHDFs) sheets contained the major dermal extracellular matrix (ECM) components (fibronectin and type I collagen). Human umbilical vein endothelial cells (HUVECs) were co-cultured with NHDF sheets, resulted in tube-like formation of HUVECs, resembling early capillaries, within NHDF sheets after short-term 3D co-culture. These results suggest that this novel use of magnetite nanoparticles and magnetic force, which we refer to as “magnetic force-based tissue engineering (Mag-TE)”, offers a major advancement in tissue engineering.