Long-term culture of muscle explants from Sparus aurata

Funkenstein, B., Balas, V., Skopal, T., Radaelli, G., Rowlerson, A.
Tissue and Cell 23 (2006) 399-415


Summary:

This paper is about the development of a fish myoblast cell line from Sparus aurata fry muscle explants. The mechanisms of somatic growth and development, regulated by hormones, growth factors, and cytokines, are relatively well conserved among vertebrates. Fish skeletal muscle, like all vertebrate muscle, develops from myoblasts in the somatic mesoderm, and they can continue to grow significantly into juvenile life through continuous hyperplasia and hypertrophy. Hyperplasia is the proliferation of cells within an organ or tissue beyond that which is normally seen, and can result in the gross enlargement of an organ/tissue. Hyperplasmic growth of muscle refers to an increase in muscle due to the formation of new fibers from myogenic precursor cells. Hypertrophy is the increase in the size of an organ or area of tissue due to an increase in the size of cells, not a change in the number of cells. Hypertropic growth requires existing fibers to acquire additional nuclei, which they can do by fusing with mononucleate myoblasts. It has not yet been fully established if the same or different myogenic precursors underlie these two growth mechanisms. Many labs have previously attempted to develop primary cultures of fish myoblasts to further investigate this, however, although the myoblasts can be isolated and grown in culture, they do not proliferate. The authors of this paper were successful in developing a culture system of proliferative fish muscle cells.
In order to create the cell cultures, they took white muscle explants from Sparus aurata fry. The explants were cultured in both sterile cell culture flasks and on glass cover slips. After various culture periods the explants were either dehydrated, embedded in paraffin and sliced using a microtome, fixed to the cover slips and stained using immunohistochemistry, or solublized in order to study proteins. Anti-PCNA was used to assess cellular proliferation, and the TUNEL assay was used to follow apoptosis in the original muscle fibers. Early differentiation was tracked by staining with anti-Myf5, and terminal differentiation by staining for desmin, myosin and parvalbumin. RT-PCR was also performed on some muscle explants to study gene expression.
The results of this study show that muscle explants from Sparus aurata can be grown in long-term culture. New fibers will appear as the original ones degenerate. The in vitro system mimics parts of the in vivo situation of damaged muscle and subsequent regeneration. The system should be useful for studying the interaction between growth inhibitors, like MSTN, and growth factors, like IGFs, as well as for studying fish muscle cell precursors.

Significance:
This article is important because it is declaring the development of a new cell line from primary tissue. Use of this new cell line in future studies could provide further insight into muscle regeneration and myogenic precursor proliferation. This may eventually lead to promising future research in human skeletal muscle/muscle regeneration. The development of new cell lines is important in and of itself because it could positively impact research involving in vitro cultured cells. New cell lines could be used to help validate previous experiments by providing new environments in which researchers can test their hypothesis. They can also enable studies that were previously difficult to conduct, for example the effect of IGFs on fish skeletal muscle cells should now be easier to follow.
Additionally, many of the techniques used in this study were covered in class. This is a great application of the tools we have been learning to use in lab! Yay!