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

Abstract

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). After a spontaneous immortalization crisis from passages 37–43, the cells proliferated at doubling times of 24.3 h (± 4.91 SD). 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, PAX3B, MYOD1, MYOG, TNNT3A, and PPARG) 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.

Investigating the Impact of Microplastics on Fish Muscle Cell Proliferation and Differentiation: Enhancing Food Safety in Cultivated Meat Production

“…RT-qPCR was performed in a CFX Opus 96 thermocycler (Bio-Rad, Hercules, California, USA) using the TB Green Advantage qPCR premix (Takara Bio, Kutatsu, Japan) and the oligonucleotides specified in Table 1. Primers were designed using a reference genome for southern bluefin tuna (Thunnus maccoyii; NBCI RefSeq GCF_910596095.1) by Saad et al (2023). Three technical replicates were performed per sample and gene, as well as for the - RT and no template controls (NTC).…”

“…The mackerel cell lines (MACK2) used in this study were obtained from Dr. David L. Kaplan Lab at Tufts University. The cell preparation followed the protocol described in Saad et al (2023). Briefly, frozen cells at passage 81 were thawed using 9 mL of complete growth medium, which comprised Leivovitz's L-15 medium (Gibco™, Billings, MT, US) and supplemented with 20% fetal bovine serum (FBS, Gibco™, Billings, MT, US), 1 ng/mL FGF2-G3 (human) growth factor (Defined Bioscience, San Diego, CA, US), 20 mM HEPES (Gibco™, Billings, MT, US), and 1% Antibiotic-Antimycotic (Gibco™, Billings, MT, US).…”

“…To observe the influence of microplastics on cell differentiation, immunostaining for myosin heavy chain was conducted following the protocol outlined in Saad et al (2023).…”

“…While the presence and potential hazards of microplastics are increasingly acknowledged, understanding the exact mechanisms by which they influence cellular functions remains a critical research frontier (O’Neill & Lawler, 2021; Thornton Hampton et al, 2022). To understand the cellular impacts of microplastic exposure more comprehensively, we utilized Atlantic mackerel ( Scomber scombrus ) skeletal muscle cell lines, previously established and characterized by Saad et al (2023), given their relevance to cultivated meat processing. This study employed fluorescent polyethylene microspheres (10-45 µm) as representative microplastics, a size range previously documented in fish (Makhdoumi et al, 2023; Thiele et al, 2021).…”

Immortalized Bovine Satellite Cells for Cultured Meat Applications

“…6−9 To date, numerous strategies have been explored to immortalize myogenic precursors. These include spontaneous immortalization through random mutations, 10 infection with simian virus 40 (SV40) or expression of the SV40 Large T antigen, 11,12 and ectopic expression of telomerase reverse transcriptase (TERT) and Cyclin dependent kinase 4 (CDK4). 13,14 In the latter case, TERT acts to elongate telomeres to counteract doubling-induced telomere shortening, while CDK4 promotes progression through the G1/S transition, thereby providing an "engine" for cell division.…”