Influence of storing muscle tissue in the cold on in vitro myogenic cell line development from the black sea bream (Acanthopagrus schlegelii).

With advances in biotechnology and tissue engineering, the development of fish muscle cell lines from aquatic resources has gained increasing attention. However, most studies rely on immediate tissue processing after sampling, and few studies have investigated the potential of establishing cell lines from stored samples. In this study, we evaluated the potential for establishing skeletal muscle cell lines from black sea bream (Acanthopagrus schlegelii) tissue stored at 4 ℃ for 0, 2, 5, 10, 20, and 30 days. Primary culture was conducted using two methods: explant culture and dissociated culture. Cell line development was assessed in relation to cold storage duration, and established cell lines were characterized for their growth and differentiation capacities at early (passage 5) and late (passage 20) stages. To further investigate the effects of cold storage, we examined cell yield and viability after tissue dissociation, as well as histological and ultrastructural changes in the tissue. Results demonstrated that explant culture allowed cell line development from tissues stored up to 10 days, whereas dissociated culture was effective only up to 5 days. All cell lines established via dissociated culture retained the ability to differentiate into myotubes. Tissue-level analysis indicated that cell viability remained above 80% for up to 5 days, although the number of isolated cells decreased with prolonged storage. Histological observations revealed gradual expansion of interstitial spaces and increased tissue disorganization, particularly after 5-10 days. Transmission electron microscopy further showed progressive ultrastructural damage, with an increasing number of vacuolated structures over time. These findings suggest that short-term cold storage less than 5 days preserves the potential for muscle cell line establishment, while tissue quality deteriorates with longer storage durations. The results provide practical insights into the storage and transport of fish cell resources, highlighting the need for optimized preservation conditions for extended storage periods.