Magnetic resonance imaging (MRI) using magnetic nanoparticles has been used to diagnose vascular diseases as well as to monitor transplanted cells and tissues. In this study, we synthesized magnetic iron oxide nanoparticles (TMADM-03), electrically charged by the presence of a cationic end-group substitution of dextran, and observed these nanoparticles inside three-dimensional models of HepG2 spheroids, which mimic tissues. Patterned cell array glass disks were prepared to visualize the presence of TMADM-03 uptaken by HepG2 spheroids using transmission electron microscopy (TEM). The HepG2 cells (2 × 10(5) cells) were inoculated onto Cell-able™ 12-well plates. After 48 h of culture, the cells were incubated with 75 µg Fe/ml TMADM-03 in culture medium for 24 h. To investigate the cellular function of the HepG2 spheroids, the albumin secretion was evaluated by an ELISA. The albumin secretion after incubation for 24 h was reduced compared with the secretion prior to the addition of TMADM-03. TEM image samples were prepared in a planar direction or a vertical direction to the HepG2 spheroids on patterned cell array glass disks. The incorporation of TMADM-03 inside the HepG2 spheroids was confirmed. In addition, TMADM-03 could be observed in the deeper layers of the spheroids, and this was localized in the lysosomes. These data suggest that the novel magnetic iron oxide nanoparticles invade three-dimensional HepG2 spheroids.

Establishing a rapid in vitro evaluation system for drug screening is essential for the development of new drugs. To reproduce tissues/organs with functions closer to living organisms, in vitro three-dimensional (3D) culture evaluation using microfabrication technology has been reported in recent years. Culture on patterned substrates with controlled hydrophilic and hydrophobic regions (Cell-ableTM) can create 3D liver models (miniature livers) with liver-specific Disse luminal structures and functions. MRI contrast agents are widely used as safe and minimally invasive diagnostic methods. We focused on anionic polysaccharide magnetic iron oxide nanoparticles (Resovist®) and synthesized the four types of nanoparticle derivatives with different properties. Cationic nanoparticles (TMADM) can be used to label target cells in a short time and have been successfully visualized in vivo. In this study, we examined the morphology of various nanoparticles. The morphology of various nanoparticles showed relatively smooth-edged spherical shapes. As 3D liver models, we prepared primary hepatocyte–endothelial cell heterospheroids. The toxicity, CYP3A, and albumin secretory capacity were evaluated in the heterospheroids labeled with various nanoparticles. As the culture period progressed, the heterospheroids labeled with anionic and cationic nanoparticles showed lower liver function than non-labeled heterospheroids. In the future, there is a need to improve the method of creation of artificial 3D liver or to design a low-invasive MRI contrast agent to label the artificial 3D liver.