Drug-induced liver injury (DILI) is a major cause of drug attrition and market withdrawal. Compounds associated with DILI typically exhibit some degree of mitochondrial liability, as empirically observed in multiple previous studies. Among the various mitochondrial toxicities, mitochondrial permeability transition (MPT) has been implicated in severe DILI. Conventional MPT assessments rely on swelling assays using mitochondria isolated from fresh animal tissues, which limits their applicability to human-based systems and high-throughput screening. To address this, we developed and validated a platelet-based approach to detect MPT by leveraging their known responsiveness to CypD-dependent mitochondrial perturbation. We evaluated 22 compounds using both a conventional mitochondrial swelling assay with mouse liver mitochondria and platelet-based activation assay. A correlation was observed between the two methods (r = 0.848), suggesting general concordance. Furthermore, we applied this assay to human platelets obtained from transfusion-ineligible platelet products and observed MPT-dependent activation by compounds such as diclofenac. However, lot-to-lot variability highlighted the challenges in terms of reproducibility. These findings suggest that platelets can serve as mechanistically relevant and accessible tools for detecting MPT induction and offer a potential alternative to animal-derived mitochondrial assays. This approach may contribute to the early identification of mitochondrial liabilities during drug development.

Functional cells derived from human iPS cells (hiPSC) show potential for use in the evaluation of drug toxicity and kinetics. Recently, there has been an increasing demand for alternative in vitro methods to animal testing, and hiPSC-derived cells such the nerve, cardiomyocyte and airway epithelium and so on are being used for the evaluation of drug toxicity and kinetics.

hiPSC cardiomyocytes (CMs) have ion channels expressed in human heart tissue, such as hERG channels, and are therefore expected to be a tool for predicting the risk of torsade de pointes (TdP) caused by pharmaceuticals. In the myocardium, the majority of ATP is synthesized in mitochondria. Therefore, oxygen supply to hiPSC-CMs is important for investigating their physiological properties. In the present study, we evaluated the effect of oxygen-permeable plate on hiPSC-CM culture. Additionally, we examined its effects on drug responsiveness. Unlike previous reports using other culture systems, hiPSC-CMs cultured on oxygen-permeable plates showed the possibility of evaluating cardiac toxicity.

Microphysiological systems (MPS) provide a basis for culturing human hepatocytes and mimicking their function in viro. However, human hepatocytes do not proliferate and must be seeded at a high density. In a closed system device, such as the MPS, the recommended seeding density does not allow for good adhesion. In this study, we demonstrated that the perfusion of lower channel immediately after seeding in a double-layered MPS, Fluid3D-X^®, which has two-flow channels, improved cell adhesion and mitochondrial activity and enabled the healthy culture of hepatocytes, even when seeded at the recommended seeding density. Furthermore, in Fluid3D-X^®, which has a long flow channel, increasing the medium flow rate in the upper and lower two channels maintains cell viability throughout the entire channel, suggesting that even human hepatocytes with high oxygen demand can be cultured in a healthy manner. The approach developed in this study is expected to guide the broader application of MPS devices in studies of human hepatocytes.

Hydrocortisone is a key regulator of energy metabolism, including the metabolic pathways of glucose and lipids. It is widely used to promote hepatic function in cultured hepatocytes. However, the relationship between hepatocyte morphology and hydrocortisone stimulation has not been thoroughly investigated. In this study, we evaluated the differential responsiveness of primary rat hepatocytes to hydrocortisone in monolayer and spheroid culture systems. Regardless of the presence or absence of hydrocortisone, the cells exhibited a flattened morphology in monolayer cultures, but formed organized three-dimensional structures with smooth surfaces in spheroid cultures. Hydrocortisone upregulated integrin expression in monolayers and cadherin expression in spheroids. In terms of hepatic metabolism, hydrocortisone upregulated the expression of genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase, Pepck), amino acid metabolism (tryptophan 2,3-dioxygenase, To), and the urea cycle (arginase 1, Arg1), particularly in spheroid culture. Albumin secretion and drug-metabolizing activity increased in a concentration-dependent manner in both culture systems, with drug metabolism notably enhanced in spheroids. These findings highlight that hydrocortisone is a critical factor in maintaining hepatocyte function, with the responsiveness of spheroids being superior to that of monolayers.

The o06 strain of Bombyx mori silkworms provided by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) NBRP possesses uric acid production capability but lacks the ability to transport/accumulate uric acid in dermal cells . We subjected these silkworms to glucose loading to create a pseudo-pathological model of hyperglycemia originating from hyperuricemia. Compared to mice and rats, this model possesses a uric acid metabolism system closer to that of humans, enabling evaluation of the efficacy of XO (Xanthine Oxidase) inhibition and HGPRT (Hypoxanthine Guanine Phosphoribosyl Transferase) activation in lowering blood uric acid levels. Furthermore, this model demonstrated blood sugar reduction and hyperglycemia improvement with human insulin and oral antidiabetic drugs. Moreover, this model suggested significant effects with various natural functional food components known to improve hemolymph uric acid and blood sugar levels. In this study, we used the o06 strain of silkworms, optimized their rearing and feeding conditions, along with the hemolymph sugar levels analysis method for this strain, to explore the potential of this novel evaluation model.