Advancing Mytilus galloprovincialis Primary Cell Cultures as Models for Bioactive Compound Screening and...

Marine bivalves represent promising experimental models for the development of in vitro approaches in environmental biology, ecotoxicology, and bioactive compound screening. However, their application remains limited compared with that of more established vertebrate and mammalian cell systems, mainly due to the lack of standardized and reproducible protocols for maintaining primary cell cultures. This study aimed to establish and optimize a short-term primary cell culture protocol using gill and digestive gland tissues from the marine mussel Mytilus galloprovincialis. Primary cultures were prepared at four initial cell densities, ranging from 2.5 to 10 × 10⁶ cells mL^-1, and monitored over 72 h through cell density evaluation and metabolic activity assessment. Cells from both tissues were successfully maintained in short-term culture, predominantly showed rounded to spheroidal morphologies. Nevertheless, tissue-specific responses were observed. Gill-derived cultures displayed greater morphological homogeneity and stability across the tested densities, whereas digestive gland cultures showed greater cellular heterogeneity, aggregation, and stronger density-dependent responses. Metabolic activity remained stable during the first 24 h, while more evident differences among cell densities emerged after 48 and 72 h. Overall, intermediate cell densities, particularly 5 × 10⁶ and 7.5 × 10⁶ cells mL^-1, provided the most suitable balance between cell maintenance and metabolic performance. This optimized methodological framework supports the further development of mussel primary cell cultures as reproducible and ecologically relevant in vitro models for studying early cellular responses and for preliminary environmental safety assessment.