Recloning and Characterization of C2C12 Myoblast and Its Clonal Derivatives

Pekik Wiji Prasetyaningrum, Endah Puji Septisetyani, Ahmad Suyoko, Adi Santoso

Abstract


The C2C12 myoblasts are adult murine muscle stem cells which isolated after injury to induce muscle regeneration. The cells are widely used in pharmaceutical and biological researches to represent skeletal muscle cells. In our laboratory, we utilize the cells for glucose uptake assay after insulin treatment and studying the muscle regeneration. In this study we conducted recloning of C2C12 cells by limiting dilution cloning (LDC) and investigated the biological properties incuding cell proliferation, adhesion and differentiation of the clonal cells in comparison to the parental cells. Cell proliferation rate had been determined by WST assay, cell adhesion had been observed after cell detachment by EDTA and cell differentiation into multinucleated myotube had been investigated after induction and incubation with horse serum. As results, two clonal derivatives of C2C12 myoblast cells had been retrieved by LDC and used for cell assays. Moreover, the results indicated that parental cells showed faster proliferation rate and better differentiation ability than that of clonal cells. In the contrary the parental cells exhibited weaker adhesion rate than clonal cells. To conclude, C2C12 parental cells are better for performing the glucose uptake or muscle regeneration assays since they showed better differentiation capability.


Keywords: C2C12 cells, cells differentiation, myoblast, myotube, recloning.


Full Text:

PDF

References


Acharya, B.R. and Yap, A.S., 2016, Cell–Cell Adhesion and the Cytoskeleton, Encyclopedia of Cell Biology, 2, 704–712. CrossRef

Černochová, P., Blahová, L., Medalová, J.,Necas, D., Michlicek, M., Kaushik, P., et al., 2020, Cell type specific adhesion to surfaces functionalised by amine plasma polymers, Sci. Rep., 10, 9357. CrossRef

Cheng, C.S., El-Abd, Y., Bui, K., Hyun, Y-E., Hughes, R.H., Kraus, W.E., et al., 2014, Conditions that promote primary human skeletal myoblast culture and muscle differentiation in vitro, Am J Physiol Cell Physiol, 306(4), C385-C395. CrossRef

Cornelison, D., 2008, Context matters: In vivo and in vitro influences on muscle satellite cell activity, J. Cell. Biochem., 105(3), 663-669. CrossRef

Freshney, R.I., 2015, Culture of animal cells: a manual of basic technique and specialized applications, 7th ed, John Wiley & Sons.

Gurdon, J.B., Lemaire, P. and Kato, K., 1993, Community Effects and Related Phenomena in Development, Cell, 75(5), 831–834. CrossRef

Huang, H.L., Hsing, H.W., Lai, T.C., Chen, Y.W., Lee. T.R., Chan, H.T., et al., 2010, Trypsin-induced proteome alteration during cell subculture in mammalian cells, J. Biomed. Sci., 17(1), 1-10. CrossRef

Mangnall, D., Bruce, C. and Fraser, R.B., 1993, Insulin-stimulated glucose uptake in C2C12 myoblasts, Biochem Soc Trans, 21(4), 438S. CrossRef

Martín-Pardilos, A., Chiva, Á.V., Vargas, G.B., Blanco, P.H., Cid, R.P., et al., 2019, The role of clonal communication and heterogeneity in breast cancer, BMC Cancer, 19, 666. CrossRef

Murphy, S.M., Kiely, M., Jakeman, P.M., Kiely, P.A. and Carson, B.P., 2016, Optimization of an in vitro bioassay to monitor growth and formation of myotubes in real time, Biosci Rep., 36(3), e00330. CrossRef

Priola, J.J., Calzadilla, N., Baumann, M., Borth, N., Tate, C.G. and Betenbaugh, M.J., 2016, High‐throughput screening and selection of mammalian cells for enhanced protein production, Biotechnology Journal, 11(7), 853-865. CrossRef

Rodgers, B.D., Wiedeback, B.D., Hoversten, K.E., Jackson, M.F., Walker, R.G. and Thompson, T. B., 2014, Myostatin stimulates, not inihibits, C2C12 myoblast proliferation, Endocrinology, 155(3), 670-675. CrossRef

Scharner, J. and Zammit, P. S., 2011, The muscle satellite cell at 50: the formative years, Skeletal Muscle, 1(1), 28. CrossRef

Schubert, R., Strohmeyer, N., Bharadwaj, M., Ramanathan, S.P., et al., 2014, Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy, FEBS letters, 588(19), 3639-3648. CrossRef

Septisetyani, E.P., Prasetyaningrun, P.W. and Santoso A., 2021, Naringin May Alleviate Doxorubicin Cytotoxic Effects In C2C12 Myoblastcells, IOP Conference Series: Earth and Environmental Science, 762(1), 012027. CrossRef

Tachibana, I. and Hemler, M.E., 1999, Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance, J Cell Biol, 146(4), 893-904. CrossRef

Tanaka, K., Sato, K., Yoshida, T., Fukuda, T., Hanamura K., et al., 2011, Evidence for cell density affecting C2C12 myogenesis: possible regulation of myogenesis by cell-cell communication, Muscle Nerve, 44(6), 968-77. CrossRef

Wong, C.Y., Al‐Salami, H. and Dass, C.R., 2020, C2C12 cell model: its role in understanding of insulin resistance at the molecular level and pharmaceutical development at the preclinical stage, Journal of Pharmacy and Pharmacology, 72(12), 1667-1693. CrossRef

Yaffe, D. and Saxel, O., 1997, Serial Passaging and Differentiation of Myogenic Cells Isolated from Dystrophic Mouse Muscle, Nature, 270, 725–727. CrossRef

Yang, M., Wei, D., Mo, C., Zhang, J., Wang, X., Han, X., et al., 2013, Saturated fatty acid palmitate-induced insulin resistance is accompanied with myotube loss and the impaired expression of health benefit myokine genes in C2C12 myotubes, Lipids in Health and Disease., 12, 104. CrossRef

Zhou, H., Weir, M.D. and Xu, H.H., 2011, Effect of cell seeding density on proliferation and osteodifferentiation of umbilical cord stem cells on calcium phosphate cement-fiber scaffold, Tissue Engineering Part A, 17(21-22), 2603-2613. CrossRef




DOI: http://dx.doi.org/10.14499/indonesianjcanchemoprev12iss2pp99-105

Copyright (c) 2021 Pekik Wiji Prasetyaningrum, Endah Puji Septisetyani, Ahmad Suyoko, Adi Santoso

Indexed by:

               

               

      

 

Indonesian Society for Cancer Chemoprevention