Advancing technical understanding of the double-seeded gill cell culture system for drug uptake studies in fish

Environmental risk assessment (ERA) is required for new drug approvals in Europe and the United States, driving the demand for reliable bioassays to model active pharmaceutical ingredient (API) uptake in fish. The rainbow trout (Oncorhynchus mykiss) primary gill cell culture system, developed on double-seeded inserts (DSIs), mimic key properties of intact gill tissue, which includes high transepithelial electrical resistance (TEER) and shows promise for assessing API uptake in freshwater fish. Although previous studies have demonstrated its potential, broader application has been limited by variability in achieving high TEER values and tolerance to apical water exposure, both critical for replicating functional gill epithelium. In this study, we reassessed the DSI methodology in detail and explored key factors influencing assay success. These included the donor fish's body size/developmental stage, potential seasonal influences, and conditions of cell washing and insert-membrane precoating. In most DSI cultures, we observed a sharp TEER decline-near-baseline within 24 hr-after apical freshwater application. This prompted us to implement a practical adjustment using serum-free L-15 medium for apical exposure, which effectively maintained TEER and enabled reproducible API transfer data across different classes and doses of APIs. Expanding on previous findings, this work provides critical insights into the technical variables affecting DSI culture success and offers practical refinement to enhance its reproducibility, supporting standardization and future application in regulatory ERA frameworks. Nonetheless, further optimization of the DSI system, particularly to improve apical freshwater tolerance, is needed. A valuable next step may be to investigate the composition of isolated gill cell populations and gain a deeper understanding in their roles in the reconstitution of a functional gill epithelium in vitro.