Maintenance of highly contractile tissue-cultured avian skeletal myotubes in collagen gel
- 163 Downloads
Highly contractile skeletal myotubes differentiated in tissue culture are normally difficult to maintain on collagen-coated tissue culture dishes for extended periods because of their propensity to detach as a sheet of cells from their substratum. This detachment results in the release of mechanical tension in the growing cell “sheet” and, consequently, loss of cellular protein. We developed a simple method of culturing high density contractile primary avian myotubes embedded in a collagen gel matrix (collagel) attached to either a stainless steel mesh or nylon support structure. With this system the cells are maintained in a highly contractile state for extended periods in vitro under tension. Structural integrity of the myotubes can be maintained for up to 10 d in basal medium without serum or embryo extract. Total cellular protein and myosin heavy chain accumulation in the cells can be maintained for weeks at levels which are two to three times those found in timematched controls that are under little tension. Morphologically, the myotubes are well differentiated with structural characteristics of neonatal myofibers. This new collagel culture system should prove useful in the analysis of in vitro gene expression during myotube to myofiber differentiation and its regulation by various environmental factors such as medium growth factors, innervation, and mechanical activity.
Key wordsskeletal muscle differentiation collagen myosin external lamina
Unable to display preview. Download preview PDF.
- 1.Ashmore, C. R.; Summers, P. J. Stretch-induced growth in chicken wing muscles: Myofibrillar proliferation. Am. J. Physiol. 51:C93-C97; 1981.Google Scholar
- 3.Buckingham, M. Muscle cells in tissue culture. Int. Rev. Biochem. 15:315–322; 1977.Google Scholar
- 10.Fambrough, D. M.; Bayne, E. K.; Gardner, J. M., et al. Monoclonal antibodies to skeletal muscle cell surface. In: Brockes, J., ed.Neuroimmunology, New York: Plenum Press; 1982: 49–89.Google Scholar
- 11.Fischman, D. A. Myofibrillar assembly in skeletal muscle. In: Bourne, G. H., ed. The structure and function of muscle. vol. 1. New York: Academic Press; 1973:75–148.Google Scholar
- 14.Hay, E. D. Cell biology of the extracellular matrix. New York: Plenum Press; 1982.Google Scholar
- 22.Lowry, O. H.; Rosebrough, N. J.; Farr, A. L., et al. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1954.Google Scholar
- 24.Murray, M. R. Skeletal muscle in culture. In: Bourne, G. H., ed. Structure and function of muscle, vol. I, part I. New York: Academic Press; 1972: 237–299.Google Scholar
- 26.Sheehan, D. C.; Hrapchak, B. B. Theory and practice of histochemistry, 2nd ed. St. Louis: C. V. Mosby Co.; 1980: 143.Google Scholar
- 33.Waterlow, J. C.; Garlick, P. J.; Millward, D. J., Editors. Protein turnover in mammalian tissues and in the whole body. Amsterdam, Netherlands: North-Holland; 1978:529–594.Google Scholar