Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets |
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Authors: | Marcus Mau Niels Oksbjerg Charlotte Rehfeldt |
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Institution: | (1) Research Institute for the Biology of Farm Animals, Research Unit Muscle Biology and Growth, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany;(2) Faculty of Agricultural Sciences, Department of Food Science, University of Aarhus, 8830 Tjele, Denmark |
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Abstract: | To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive
compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 106 cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent
fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite
cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 103 cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by
2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well
plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus
10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation
medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing
this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds. |
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Keywords: | Piglets Porcine satellite cells Semimembranosus muscle Differentiation Serum-free medium |
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