Continuous fish muscle cell line with capacity for myogenic and adipogenic-like phenotypes

Cell-cultivated fish offers the potential for a more ethical, sustainable, and safe seafood system. However, fish cell culture is relatively understudied in comparison to mammalian cells. Here, we established and characterized a continuous Atlantic mackerel (Scomber scombrus) skeletal muscle cell line ("Mack" cells). The cells were isolated from muscle biopsies of fresh-caught fish, with separate isolations performed from two distinct fish. Mack1 cells (cells from the first isolation) were cultured for over a year and subcultured over 130 times. The cells proliferated at initial doubling times of 63.9 h (± 19.1 [SD]{.caps}). After a spontaneous immortalization crisis from passages 37--43, the cells proliferated at doubling times of 24.3 h (± 4.91 [SD]{.caps}). A muscle phenotype was confirmed through characterization of muscle stemness and differentiation via paired-box protein 7 and myosin heavy chain immunostaining, respectively. An adipocyte-like phenotype was also demonstrated for the cells through lipid accumulation, confirmed via Oil Red O staining and quantification of neutral lipids. New qPCR primers ([HPRT]{.caps}, [PAX3B]{.caps}, [MYOD1]{.caps}, [MYOG]{.caps}, [TNNT3A]{.caps}, and [PPARG]{.caps}) were tailored to the mackerel genome and used to characterize mackerel cell genotypes. This work provides the first spontaneously immortalized fish muscle cell line for research, ideally serving as a reference for subsequent investigation.