Advances in cell culture technology have led to fish cell lines being used as cost-effective, reproducible, and ethically favourable instruments in ecotoxicology. The development of new lines contributes to reducing animal experiments and improves model diversity. The brown trout (Salmo trutta), an important bioindicator due to its sensitivity to pollutants, is still underrepresented in cell culture systems. In this study, two novel larvae-derived cell lines, STRlar1 and STRlar2, were established and maintained for over 40 passages. Although derived from sibling larvae of the same parental strain, the two lines showed marked differences in growth dynamics and toxicological responses to ethanol, dimethyl sulfoxide, isopropanol, and acetone. STRlar2 showed greater sensitivity to all chemicals tested, while STRlar1 exhibited longer proliferation and higher impedance, suggesting stronger substrate adhesion. These differences emphasise the importance of cellular heterogeneity, even in closely related lines. Our results underline the need to carefully validate new in vitro models and caution against relying on single cell lines. To improve the robustness and reliability of ecotoxicological assessments, we recommend the use of multiple independently derived lines that better reflect biological variability and reflect in vivo complexity.

Sturgeons, once resilient enough to outlive dinosaurs, are now critically endangered. All 26 species of Acipenseriformes face extinction due to anthropogenic causes. Despite their ecological and economic significance, sturgeon research lacks essential tools such as larval cell lines; the Cellosaurus database lists only one larval cell line (AOXlar7y from Atlantic sturgeon). Larval stages are key to understand fish development, representing a transitional phase between embryonic and adult life that is highly sensitive to temperature shifts, oxygen depletion and pollution. Larval cell lines therefore provide potential in vitro models for studying development and stress responses in endangered species. This study focused on establishing and initially characterizing five novel larval cell lines from siblings of the Siberian sturgeon (Acipenser baerii). The lines proved viable for long-term culture, bio-banking and transfer, displaying different morphologies ranging from epithelial-like to fibroblast-like. Functional assays showed variable mitochondrial activity and extracellular acidification rates. A preliminary targeted gene expression analysis revealed similarity to whole larvae within early passages and in vitro adaptations for certain genes (gapdh, vim, col1a1, pcna). These sibling-derived cell lines hold potential as in vitro tools to deeper explore the biology of Siberian sturgeon larvae and support conservation-focused research.

Caviar extract is widely used in luxury cosmetics and is generally defined as a homogeneous ingredient derived exclusively from sturgeon eggs. However, its molecular composition remains inadequately characterised. In this study, proteomic analyses were performed on 14 commercial caviar samples from different sturgeon species and geographical origins, examined independently in two laboratories. Across three single-origin samples (Germany, Poland, China), 1437 protein groups and 3452 unique peptides were identified, with consistent overlaps with sturgeon ovarian fluid (≥25–38 proteins). Extending this analysis across multiple species confirmed the presence of ovarian fluid-associated proteins in all examined groups, including 69 in Acipenser baerii, 61 in A. gueldenstaedtii, 55 in A. schrenckii, and 49 in H. huso. The results showed that ovarian fluid is consistently co-extracted during standard roe harvesting processes due to its strong egg surface adhesion, making this co-extraction intrinsic rather than incidental. Proteomic profiling revealed a complex mixture of egg- and ovarian fluid-derived proteins, including zona pellucida glycoproteins, immunoglobulins, complement components, proteases, coagulation factors, and antioxidant enzymes. Many of these proteins influence skin biology, suggesting that the functional effects attributed to caviar extract may partially originate from ovarian fluid constituents rather than yolk-derived nutrients alone. At the same time, the presence of immune-active and enzymatic proteins raises important questions regarding safety assessment and regulatory oversight. Species-specific proteomic clustering also indicates considerable composition heterogeneity, challenging assumptions of ingredient standardisation. Together, these results highlight a discrepancy between current definitions and the molecular reality and underscore the need for improved molecular characterisation, updated regulatory definitions, and the consideration of alternative, cell-based production strategies for cosmetic applications.

Reliable fish cell models are essential for studying cellular processes under controlled in vitro conditions and thereby reducing the need for animal experiments. In this study, we established and characterized OMYlar5, a novel larval cell line derived from rainbow trout (Oncorhynchus mykiss). OMYlar5 demonstrated sustained proliferative capacity over at least 60 passages, indicating spontaneous immortalization and its suitability for long-term applications in fish biology and aquaculture research. To investigate temperature-dependent cellular responses, cells were cultured at 18, 20, and 22 °C, and changes in growth, morphology, cytoskeletal organization, stress responses, and gene expression were analyzed. Increased temperature enhanced cell proliferation, whereas the expression of the proliferation-associated genes pcna and c-myc remained unchanged. Temperature-dependent alterations in cell morphology and F-actin organization were observed, with enhanced actin bundle formation at 22 °C. However, no significant induction of Hsp70 or Hsp90 was detected at the transcript or protein level, suggesting that the tested temperature range was not associated with detectable induction of these heat shock proteins. Furthermore, OMYlar5 cells expressed nanog, nes, pax3, and sox9, whereas pou5f1 and sox2 were expressed at very low levels. These expression patterns provide information on the molecular characteristics of the cell line. Overall, OMYlar5 may provide a useful in vitro model for investigating temperature responses, cellular physiology, and developmental processes in rainbow trout.