Abstract:

Objective　To compare morphologic and functional features of C2C12 cells cultured on monolayer or Sylgard 184.   Methods    C2C12 cells were seeded on the culture dishes or the Sylgard 184 slots modified by type I collagen and Matrigel Matrix respectively. 2% horse serum was added into DMEM for priming the cell differentiation after the cell proliferating and confluencing about 80%. The morphologic and functional features were explored, adopting the inverted microscope, RT-PCR, and immunoflurecence. Results: After 5 day's induction, C2C12 on Sylgard 184 slots fused into polynuclear and polar myotubes. At the culturing time of 17d, the myotubes became matured, with extended diameter, paralleled distribution and the further confluence. This constructed muscle tissue showed automatic contractility, with the thickness of about 0.15 mm. Under the scanning electron microscopy, myotubes closely arranged and overlapped with each other. Compared with that of spatial culture, the differentiated myotubes of monolayer culture was tiny, arranged more randomly. The results of RT-PCR proved that, spacial culture prompted to the expression of MyoD and Myogenin mRNAs, compared to monolayer cultures, as well the protein levels of Desmin, F-actin, MHC, and nAChR. Conclusions   Compared with the traditional monolayer culture, three-dimensional culture is preferential to the differentiation, paralleled arrangement and the expression of functional molecules of myotubes in vitro, which is a favorable model to explore the development and stress loading of muscle tissue, as well the principals of skeletal muscle diseases.

Key words: C2C12, Sylgard 184, Three-dimensional culture, Monolayer culture