Myoblast cell lines are grown and differentiated readily in cell culture. Two cell lines typically used for investigating the growth and differentiation of muscle are the mouse cell line C2C12 and the rat cell line L6. The differentiation of these cells in vitro requires a switch from a serum-rich medium to a less rich medium after the cells have reached confluence. Since the components present in serum are not well characterized, the use of a better defined medium for these studies was investigated. C2C12 and L6 myoblasts were differentiated in both serum-containing and serum-free media. The differentiation state of these cultures was then tested both microscopically and biochemically. Cultures were checked for myotube formation, the activity of creatine phosphokinase and the presence of sarcomeric actin. In C2C12 cells, the extent of differentiation was greater in the serum-free than in the serum-containing system. In both media types, the C2C12 cells produced sarcomeric actin, showing the presence of sarcomere structure in the myotubes. In L6 cells, however, myotubes were readily formed in medium containing 2% horse serum, but not in the serum-free system. In addition, the ability of C2C12 cells to differentiate on substrates coated with extracellular matrix proteins was shown to be media-dependent. The presence of extracellular matrix proteins did not enable L6 cells to form myotubes when cultured in serum-free media. Primary cultures of chick myoblasts were able to differentiate in both media tested, with Dulbecco’s modified Eagle medium containing horse serum being a more efficient medium for cell fusion. This study shows a divergence in muscle cell line responses in three cell lines, two of which are typically used as ‘model systems’ for understanding muscle growth and development.

Keywords:
Myogenesis, Differentiation, Myoblast, Serum-free culture
1.
Causey, A.L., R.M. Wooten, L.W. Clem, J.E. Bly (1994) A serum-free medium for human primary T lymphocyte culture. J Immunol Methods 175: 115–121.
2.
Dollenmeier, P., D.C. Turner, H.M. Eppenberger (1981) Proliferation and differentiation of chick skeletal muscle cells cultured in a chemically defined medium. Exp Cell Res 135: 47–61.
3.
Dourdin, N., J.J. Brustis, D. Balcerzak, N. Elamrani, S. Poussard, P. Cottin, A. Ducastaing (1997) Myoblast fusion requires fibronectin degradation by exteriorized m-calpain. Exp Cell Res 235: 385–394.
4.
Edmonson, D.G., E.N. Olsen (1993) Helix-loop-helix proteins as regulators of muscle-specific transcription. J Biol Chem 268: 755–758.
5.
Ellis, D.G., Q. Cheng, D.A. Lee (1996) The effects of growth factors on Tenon’s capsule fibroblasts in serum-free culture. Curr Eye Res 15: 27–35.
6.
Engvall, E. (1993) Laminin variants: Why, where and when? Kidney Int 43: 2–6.
7.
Florini, J.R., S.B. Roberts (1979) A serum free medium for the growth of muscle cells in culture. In Vitro 15 983–992.
8.
Foster, R.F., J.M. Thompson, S.J. Kaufman (1987) A laminin substrate promotes myogenesis in rat skeletal muscle cultures. Analysis of replication and development using antidesmin and anti-BrdUrd monoclonal antibodies. Dev Biol 122: 11–20.
9.
Gilpin, B.J., F. Loechel, M.G. Mattei, E. Engvall, R. Albrechtsen, U.M. Wewer (1998) A novel, secreted form of human ADAM 12 (meltrin alpha) provokes myogenesis in vivo. J Biol Chem 273: 157–166.
10.
Gogos, J.A., R. Thompson, W. Lowry, B.F. Sloane, H. Weintraub, M. Horwitz (1996) Gene trapping in differentiating cell lines: Regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion. J Cell Biol 134: 837–847.
11.
Goto, S., K. Muyazaki, T. Funabiki, H. Yasumitsu (1999) Serum free culture conditions for analysis of secretory proteinases during myogenic differentiation of mouse C2C12 myoblasts. Anal Biochem 272: 135–142.
12.
Guerin, C.W., P.C. Holland (1995) Synthesis and secretion of matrix-degrading metalloproteases by human skeletal muscle satellite cells. Dev Dyn 202: 91–99.
13.
Harper, J.M.M., J.B. Soar, P.J. Buttery (1986) Changes in protein metabolism of ovine primary muscle cultures on treatment with growth hormone, insulin, insulin-like growth factor or epidermal growth factor. J Endocrinol 112: 87–96.
14.
Helsinki, E.M., K.L. Bielat, G.M. Ovak, J.L. Pauley (1988) Long-term cultivation of functional human macrophages in Teflon dishes with serum-free media. J Leukoc Biol 44: 111–121.
15.
Kroll, T.G., B.P. Peters, C.M. Hustad, P.A. Jones, P.D. Killen, R.W. Ruddon (1994) Expression of laminin chains during myogenic differentiation. J Biol Chem 269: 9270–9277.
16.
Kuhl, U., R. Timpl, K. von der Mark (1982) Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes. Dev Biol 93: 344–354.
17.
Kwak, K.B., J. Kambayashi, M.S. Kang, D.B. Ha, C.H. Chung (1993) Cell-penetrating inhibitors of calpain block both membrane fusion and filamin cleavage in chick embryonic myoblasts. FEBS Lett 323: 151–154.
18.
Lasser, A.B., S.X. Skapek, B. Novich (1994) Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr Opin Cell Biol 6: 788–794.
19.
Mayne, R., R.D. Sanderson (1985) The extracellular matrix of skeletal muscle. Coll Relat Res 5: 449–468.
20.
Minotto, S., B.M. Scicchitano, C. Nervi, S. Scarpa, M. Lucarelli, M. Molinaro, S. Adamo (1998) Vasopressin and insulin-like growth factors synergistically induce myogenesis in serum-free medium. Cell Growth Differ 9: 155–163.
21.
Miralles, F., D. Ron, M. Baiget, J. Felez, P. Munoz-Canoves (1998) Differential regulation of urokinase-type plasminogen activator expression by basic fibroblast growth factor and serum in myogenesis. Requirement of a common mitogen-activated protein kinase pathway. J Biol Chem 272: 2052–2058.
22.
Podleski, T.R., I. Greenberg, J. Schlessinger, K.M. Yamada (1979) Fibronectin delays the fusion of L6 myoblasts. Exp Cell Res 122: 317–326.
23.
Slunt, J.B., E.A. Taketomi, T.A. Platts-Mills (1997) Human T-cell responses to Trichophyton tonsurans: Inhibition using the serum free medium Aim V. Clin Exp Allergy 27: 1184–1192.
24.
Thyberg, J., A. Hultgardh-Nilsson (1994) Fibronectin and the basement membrane components laminin and collagen type IV influence the phenotypic properties of subcultured rat aortic smooth muscle cells differently. Cell Tissue Res 276: 263–271.
25.
Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, E. Engvall (1996) Merosin and laminin in myogenesis; specific requirement for merosin in myotube stability and survival. J Cell Biol 134: 1483–1497.
26.
Von der Mark, H., M. Ocalon (1989) The differentiation and redifferentiation of myoblasts is triggered by fibronectin and laminin. Differentiation 40: 150–157.
27.
Weintraub, H., R. Davis, S. Tapscott, M. Thayer, M. Krause, R. Benezra, T.K. Blackwell, D. Turner, R. Rupp, S. Hollenberg, Y. Zhuang, A. Lasser (1991) The myoD gene family: Nodal point during specification of the muscle cell lineage. Science 251: 761–766.
28.
Yablonka-Reuveni, Z., A.J. Rivera (1994) Temporal expression of regulatory and structural muscle proteins during myogenesis of satellite cells on adult rat fibers. Dev Biol 164: 588–603.
29.
Yagami-Hiromasa, T., T. Sato, T. Kurisaki, K. Kamijo, Y. Nabeshima, A. Fujisawa-Sehara (1995) A metalloprotease-disintegrin participating in myoblast fusion. Nature 377: 652–656.
30.
Yeoh, G., H. Holtzer (1977) The effect of cell density, conditioned medium and cytosine arabinoside on myogenesis in primary and secondary cultures. Exp Cell Res 104: 63–78.
31.
Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, Y. Nabeshima (1998) Cell heterogeneity upon myogenic differentiation: Down-regulation of Myo-D and Myf-5 generates ‘reserve cells’. J Cell Sci 111: 769–779.
32.
Yoshiko, Y., K. Hirao, K. Sakabe, J. Takezawa, N. Maeda (1996) Autonomous control of expression of genes for insulin-like growth factors during the proliferation and differentiation of C2C12 mouse myoblasts in serum-free culture. Life Sci 59: 1961–1968.
© 2000 S. Karger AG, Basel
2000
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.