Toxicity evaluation methods for nanomaterials often involve animal experiments and in vitro studies using cells and bacteria. Various metal nanoparticles, including platinum, silver, and zinc, are used in the medical field. Platinum nanoparticles can decompose superoxide radicals (O₂⁻) within cells, showing superoxide dismutase (SOD)-like activity. This study examined the effects of equal-volume mixtures of platinum and silver nanoparticles, and platinum and zinc nanoparticles, on SOD-like activity, oxidative stress, and cell viability compared to individual silver and zinc nanoparticles. Results indicated that the SOD-like activity was highest (97.61%) with the platinum-silver mixture, while it was lower with the platinum-zinc mixture. Oxidative stress results showed no significant differences, and cell viability under mixed conditions was intermediate between the single metals with no specific increase or decrease. The study suggests that a platinum-silver mixture exhibits higher SOD-like activity and slightly suppresses intracellular ROS generation compared to individual metals. Further research is needed to determine the relationship between ROS, aging, and carcinogenesis using alternative methods in animal experiments. Additionally, elucidating the functions and applications of metal nanomaterials under various mixing conditions is important for future applications.

Cytoskeletal tension changes play a key role in cellular responses to physical and mechanical cues. The FRET tension sensor Actinin-sst-FRET GR is a powerful tool for studying intracellular tension changes, offering the advantage of measuring intracellular mechanics in situ under culture conditions. However, its application faces challenges of low transfection efficiency and poor protein expression levels, particularly in primary cells. In this study, we developed an adenovirus vector for Actinin-sst-FRET GR and validated its expression in rat bone marrow mesenchymal stem cells (rMSCs). The adenoviral transduction approach significantly improved the efficiency of gene delivery and protein expression levels compared to chemical transfection methods. The sensor introduced via adenoviral infection exhibited FRET activity and normally responded to changes in actin cytoskeletal tension. Our findings demonstrate that adenoviral transduction is a highly effective gene delivery method for intramolecular FRET biosensors in stem cells, addressing key limitations of traditional methods. This approach provides a promising platform for in vitro investigations of intracellular mechanics across diverse cell types, including primary and stem cells, and serves as an alternative to animal experiments.

Co-culture systems with culture inserts are commonly used to study cell-cell interactions mediated by soluble factors. However, the inclusion of culture inserts reduces the air-medium interface for cells cultured in the bottom wells, potentially leading to insufficient oxygen supply. This study investigated the impact of culture conditions on human hepatocytes when cultured with culture inserts. The results showed that using culture inserts increased cell death and decreased mitochondrial activity in hepatocytes grown at the bottom wells of standard multi-well plates. Additionally, bile canaliculi formation was impaired in hepatocytes derived from a chimeric mouse with a humanized liver. To address these limitations, we replaced the standard culture plates with InnoCell^TM T-plates, which facilitate oxygen supply from the bottom of the wells, and assessed cellular functions. Using the InnoCell^TM T-plate significantly reduced cell death, increased the presence of mitochondria with high membrane potential, and restored bile canaliculi formation. Furthermore, CYP3A4 activity, one of the key drug metabolizing enzymes of hepatocytes, was notably enhanced. These findings demonstrate that the reduced functionality of hepatocytes in co-culture systems with culture inserts can be mitigated by improving oxygen delivery through the use of InnoCell^TM T-plates. This approach supports improved cell survival and enables more accurate observation of cell-cell interactions. Co-culture systems with culture inserts are increasingly recognized as part of Microphysiological Systems (MPS). The above approach will lead to the specification of the alternatives to animal experiments and the consideration points to be made in MPS.