Successful Preparation of Contrast Particle‐Loaded Human Mesenchymal Stem Cell Aggregates Using Adherent Cell Self‐Aggregation Technique

ABSTRACT Several studies have investigated the location of transplanted cells and tissue‐engineered cell constructs in the body by incorporating contrast nanoparticles into cells by endocytosis; however, these have yet to be applied clinically because of the complexity of assessing the safety of nanoparticles. In this study, we proposed that our developed adherent cell self‐aggregation technique (CAT) could be used to develop cell aggregates loaded with contrast particles of a size that would exclude the possibility of endocytosis, and aimed to prepare these aggregates followed by biological and computed tomography (CT) contrast evaluation under X‐rays. Once human bone marrow‐derived mesenchymal stem cells (HBMSCs) were seeded into culture dishes coated with CAT‐inducing polymer to form gapless cell monolayer sheets, tungsten carbide (WC) particles smaller than 1 μm or titanium (Ti) particles larger than 10 μm were added, and thus each particle deposited on the surface of the cell monolayer sheet. During the subsequent overnight incubation, spontaneous detachment and aggregation of the cell monolayer sheets with deposited WC and Ti particles occurred, forming single spherical cell aggregates (spheroids) and loading these particles. Histological analysis confirmed that Ti particles with a diameter of at least 10 μm were not endocytosed and remained attached to the outside of cells forming spheroids, while WC particles were endocytosed into the cells. The CT images of the Ti‐loaded spheroids were clearly visible along the spheroid shape under X‐ray irradiation. Then, we confirmed that there was no toxicity to the cells forming the spheroids by loading Ti particles, and the cells could sprout and proliferate by culturing the spheroids. We successfully prepared Ti particle‐loaded HBMSCs aggregates with long fiber shape (> 10 cm) by applying CAT to a culture dish with a ring‐fiber‐shaped culture groove and confirmed their clear visibility on CT images under X‐ray irradiation, as well as their containment and ejection into a catheter, demonstrating their applicability to catheter‐mediated regenerative therapy.