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Cytotechnology

, Volume 13, Issue 1, pp 55–60 | Cite as

A rapid preparation of primary cultures of mouse skeletal muscle cells

  • Laurent Metzinger
  • Philippe Poindron
  • Anne-Catherine Passaquin
Technical Report

Abstract

We describe a rapid and reproducible technique for establishing primary cultures of skeletal muscle cells from mouse origin. This method was aimed at avoiding extensive enzymatic proteolysis which is commonly used for preparation of primary skeletal muscle cultures. It relies on a Stomacher® blender that allows a rapid and regular mechanical dissociation of muscle samples by repeated shocks. Cultures have been compared to those obtained by a modification of the method of Yaffé (1993) based on tryptic dissociation of rat muscle thighs. The time of preparation was reduced to 1 h and 15 min as compared to 4 h with the technique of Yaffé. Both cultures displayed similar morphologies and exhibited comparable myogenesis processes. Cellular yield, rate of myotube formation and myotube numbers were similar. The expression of myogenesis markers were identical as assessed by determination of acetylcholine receptor number, creatine kinase activity and level of myosin light chain.

Key words

primary muscle cell culture mouse myogenesis markers Stomacher blender trypsinization 

Abbreviations

AChR

Acetylcholine receptor

CK

creatine kinase (EC 2.7.3.2)

PBS

Phosphate buffered saline-free of Ca2+ and Mg2+

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References

  1. André P, Braun S, Passaquin A-C, Coupin G, Bartholeys J, Warter J-M and Poindron P (1988) Rat interferon enhances the expression of acetylcholine receptors in rat myotubes in culture. J. Neurosci. Res. 19: 297–302.Google Scholar
  2. Askanas V, Cave S, Martinuzzi A and Engel WK (1986) Glucocorticoids increase number of acetylcholine receptors (AChRs) and AChRs aggregates in human muscle cultured in serum-free hormonally/chemically defined medium. Neurology 36: 241.Google Scholar
  3. Blau HM and Webster C (1981) Isolation and characterization of human muscle cells. Proc. Natl. Acad. Sci. USA 78: 5623–5627.Google Scholar
  4. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72: 248–254.Google Scholar
  5. Braun S, Tranchant C, Vilquin J-T, Labouret P, Warter J-M and Poindron P (1989) Stimulating effect of prednisolone on acetylcholine receptor expression and myogenesis in primary cultures of newborn rat muscle cells. J. Neurol. Sci. 92: 119–131.Google Scholar
  6. Cole R and de Vellis J (1989)_Preparation of astrocyte and oligodendrocyte cultures from primary rat glial cultures. In: Shahar A, de Vellis J, Vernadakis A and Haber B (eds) A Dissection and Tissue Culture Manual of the Nervous System. Alan R. Liss, Inc., 121–133.Google Scholar
  7. Fong P, Turner PR, Denetclaw WF and Steinhardt RA (1990) Increased activity of calcium leak channels in myotubes of Duchenne human andmdx mouse origin. Science 250: 673–676.Google Scholar
  8. Guerriero V and Florini JR (1980) Dexamethasone effects on myoblast proliferation and differentiation. Endocrinology 106: 1198–1202.Google Scholar
  9. Konigsberg IR (1979) Skeletal myoblasts in culture. Methods Enzymol. 43: 511–527.Google Scholar
  10. Kühl U, Timpl R and von der Mark K (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.Google Scholar
  11. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the bacteriophageT4. Nature 227: 680–688.Google Scholar
  12. Metzinger L, Passaquin A-C, Warter J-M and Poindron P (1993) α-methylprednisolone promotes skeletal myogenesis in dystrophin-deficient and control mice cultures. Neurosci. Lett. 155: 171–174.Google Scholar
  13. Partridge TA (1991) Muscle transfection made easy. Nature 352: 757–758.Google Scholar
  14. Passaquin A-C, Metzinger L, Léger JJ, Warter J-M and Poindron P (1993) Prednisolone enhances myogenesis and dystrophin-related protein in skeletal muscle cell cultures frommdx mouse. J. Neurosci. Res. 35: 363–372.Google Scholar
  15. Richler C and Yaffe D (1970) Thein vitro cultivation and differentiation capacities of myogenic cell lines. Dev. Biol. 23: 1–22.Google Scholar
  16. Walsh FS and Ritter MA (1981) Surface antigen differentiation during myogenesis in culture. Nature 289: 60–64.Google Scholar
  17. Watt DJ, Lambert K, Morgan JE, Partridge TA and Sloper JC (1982) Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse. J. Neurol. Sci. 57: 319–331.Google Scholar
  18. Yaffé D (1973) Rat skeletal muscle cells. In: Kruse P and Patterson MK (eds) Tissue Culture: Methods and Applications, New York, San Francisco, London: Academic Press, 106–114.Google Scholar
  19. Yasin R, Van Beers G, Nurse KCE, Al-ani S, Landon DN and Thompson EJ (1977) A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells. J. Neurol. Sci. 32: 347–360.Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Laurent Metzinger
    • 1
  • Philippe Poindron
    • 1
  • Anne-Catherine Passaquin
    • 1
  1. 1.Département d'Immunologie, d'Immunopharmacologie et de Pathologie, Equipe de Biologie de la cellule musculaireUniversité Louis Pasteur (ULP)Illkirch CedexFrance

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