Abstract
Skeletal muscle myoblastic cell lines can provide a valuable new in vitro model for the exploration of the mechanisms that control skeletal muscle development and its associated molecular regulation. In this study, the skeletal muscle tissues of grass carp were digested with trypsin and collagenase I to obtain the primary myoblast cell culture. Myoblast cells were obtained by differential adherence purification and further analyzed by cryopreservation and resuscitation, chromosome analysis, immunohistochemistry, and immunofluorescence. A continuous grass carp myoblast cell line (named CIM) was established from grass carp (Ctenopharyngodon idellus) muscle and has been subcultured > 100 passages in a year and more. The CIM cells revived at 79.78–95.06% viability after 1–6 months of cryopreservation, and shared a population doubling time of 27.24 h. The number of modal chromosomes of CIM cells was 48, and the mitochondrial 12S rRNA sequence of the CIM cell line shared 99% identity with those of grass carp registered in GenBank. No microorganisms (bacteria, fungi, or mycoplasma) were detected during the whole study. The cell type of CIM cells was proven to be myoblast by immunohistochemistry of specific myogenic protein markers, including CD34, desmin, MyoD, and MyHC, as well as relative expression of key genes. And the myogenic rate and fusion index of this cell line after 10 days of induced differentiation were 8.96 ~ 9.42% and 3–24%, respectively. The telomerase activity and transfection efficiency of CIM cell line were 0.027 IU/mgprot and 23 ~ 24%, respectively. These results suggest that a myoblast cell line named CIM with normal biological function has been successfully established, which may provide a valuable tool for related in vitro studies.
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Acknowledgements
The authors thank the College of Fisheries of Huazhong Agricultural University for the infrastructure provided. Finally, thanks to everyone who contributed to this experiment.
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This work was supported by the National Natural Science Foundation of China (Grant No.: 32072950).
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Conceptualization: Xianmei Long, Wangwang Chen, Qingsong Tan; formal analysis: Xianmei Long, Guoqing Liu, Qingsong Tan; investigation: Xianmei Long, Wangwang Chen, Wenguang Hu; methodology: Xianmei Long, Qingsong Tan; data curation: Xianmei Long, Wangwang Chen; writing — original draft: Xianmei Long; Resources: Guoqing Liu; project administration: Qingsong Tan; writing — review and editing: Xianmei Long, Qingsong Tan. All authors read and approved the final manuscript.
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All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the UK Animals (Scientific Procedures). What is more, the animal sample collection and experimental protocols were approved by the Institutional Ethics Committee of Huazhong Agriculture University (NO. FISH-2020–04-03). All animal handling and methods were performed according to the relevant guidelines.
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Long, X., Chen, W., Liu, G. et al. Establishment and characterization of a skeletal myoblast cell line of grass carp (Ctenopharyngodon idellus). Fish Physiol Biochem 49, 1043–1061 (2023). https://doi.org/10.1007/s10695-023-01246-w
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DOI: https://doi.org/10.1007/s10695-023-01246-w