Drugs taken up by hepatocytes are excreted into the bile canaliculi via transporters such as bile salt export pump (BSEP). Here, human hepatocyte cell line PLC/PRF/5, which were reported to express BSEP, were cultured in three-dimensional (3D) culture using a scaffold called Cellbed and compared with that of two-dimensional (2D) monolayer culture. The results showed that the expression of sodium taurocholate co-transporting polypeptide (NTCP), the bile acid uptake system, did not change in both culture systems at the protein or gene level, whereas the expression of BSEP, the excretion system, was significantly enhanced in 3D culture at the protein and gene levels. The uptake and excretion activities of tauro-nor-THCA-24DBD, a model fluorescent substance for bile acids, by NTCP and BSEP were inhibited by Cyclosporin A, which is known to inhibit the activities by NTCP and BSEP in both culture systems, and the excretion activity of BSEP by 3D culture was approximately 70 times higher than that of 2D culture. The high expression of BSEP in this Cellbed 3D culture was accompanied by high expression of the hypoxia-induced transcription factor HIF-1α and HIF-1α-related genes, and the nuclear receptor for bile acids, FXR, which was reported to induce BSEP expression. These results suggest that in Cellbed cultured PLC/PRF/5 cells, the expression of BSEP is increased in accordance with the increased expression of HIF-1α, and is a promising evaluation system for drug efflux in vitro.

The renal proximal tubule plays a crucial role in drug metabolism and excretion and is an evaluation target in drug discovery. However, only a limited number of in vitro kidney models can be used as reliable alternatives to animal experiments. Traditional kidney cell lines lack physiological relevance due to low transporter expression, limited metabolic activity, and the absence of key renal-specific features, including tight junction formation and polarized transporter localization. In contrast, renal proximal tubular epithelial cells (RPTECs), derived from human renal proximal tubules, resemble native kidney epithelial cells by retaining key physiological properties such as active transport, selective permeability, and drug metabolism capabilities. As a result, RPTECs are widely used for nephrotoxicity studies. However, significant variability among commercially available RPTECs challenges their experimental reproducibility despite their potential as a promising alternative to animal-based renal studies. This study systematically compares multiple commercially available RPTECs, evaluating cell morphology, barrier function, and gene expression profiles to assess their suitability for in vitro kidney research. Our findings reveal substantial variability among RPTECs, emphasizing the need for careful cell selection. Notably, RPTEC/TERT1 exhibited stable barrier integrity, and showed maturation of transporter expression over time, making it a potentially reliable model for long-term studies. However, our results suggest that no single RPTEC model is universally optimal, and selection should be guided by specific applications, as transporter expression patterns and functional properties vary across RPTECs. Further standardizing and validating RPTEC-based models will be crucial for improving the reproducibility of renal studies and advancing drug discovery methodologies.

Cell inserts are widely used in in vitro permeability tests for convenience and functionality. However, cell maturity and functionality vary depending on culture conditions. Material of the cell insert altered the barrier integrity of the cultured cells, and affected the expression levels of the drug-metabolizing enzymes cytochrome P450 (CYP) 2C9 and CYP2C19 and the peptide transporter SLC15A1. Furthermore, differences in pore density among the inserts reduced P-glycoprotein (P-gp) transport capacity and gene expression in Caco-2 cells. These results provide valuable guidance for the selection of inserts for Caco-2 cell culture.

Oxygen deficiency is a major challenge in scaffold-based bone tissue engineering, often leading to impaired cell survival and limited tissue regeneration. To address this, we developed a three-dimensional (3D) oxygen-generating scaffold composed of polycaprolactone (PCL) and calcium peroxide (CPO) to enhance oxygen delivery under hypoxic conditions. Scaffolds with different CPO concentrations (PCL-only, CPO5, and CPO10) were fabricated using fused deposition modeling (FDM) 3D printing and characterized via scanning electron microscopy (SEM). MC3T3-E1 cells were cultured on these scaffolds under hypoxia (1% O₂) for seven days, and cell viability was assessed using the alamarBlue assay, while live/dead staining and confocal microscopy evaluated cell distribution. The results showed that the CPO10 scaffold exhibited the highest cell viability and survival rates, indicating its ability to sustain cellular activity through controlled oxygen release. This approach highlights the potential of oxygen-generating scaffolds in overcoming oxygen limitations in bone tissue engineering and provides the alternatives to animal experiments.

With the global trend towards the abolition of animal testing, there is an increasing demand for improving alternative methods for particulate matter (PM) inhalation toxicity assessment. In the present study, we developed a novel aerosol exposure system for alveolar cells using an atomizer. The humidity in the exposure chamber was maintained at 95%. When Calu-3 human lung epithelial cells were exposed to urban PM (CRM28) for 1 h and then cultured in an incubator under a CO₂ atmosphere, time-dependent decreases in the transepithelial electrical resistance and cell viability were observed. Interleukin-25 (IL-25) expression increased, whereas claudine18 (CLDN18) expression decreased in Calu-3 cells following CRM28 exposure. When mice were intranasally exposed to CRM28 or PM2.5, collected in Yokohama, Japan, for 7 days, increased IL-25 expression and decreased CLDN18 expression were observed in the lung tissues. Changes in the expression of IL-25 and CLDN18 were also observed in the lungs of mice stimulated with ovalbumin. Taken together, the exposure chamber developed in this study allowed aerosol exposure to cultured alveolar epithelial cells at humidity levels similar to those found in alveoli, and IL-25 and CLDN18 may be used as markers for respiratory damage caused by PM exposure.

AATEX 29(2) 64-75(2024); doi: https://doi.org/10.11232/aatex.29.64