Image Analysis of Proliferation and Fusion in Cultured Satellite Cells
Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14 after plating and the histological characteristics of the cultures were defined. The cell cycle phases determined by examining Feulgen-stained cultures with a cell image processor SAMBA 200. Nuclei were automatically analysed with two different methods which made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes. After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of fetal calf serum, this percentage increased until day 3 after plating. Histogram analysis showed that, at that time, the DNA content of 2 8, 2% of the cell population was higher than 3C, and image analysis showed that 42% of the S and G2 phase cells were involved in DNA synthesis. From day 4, the proliferation rate gradually slowed down until day 8, when less than 5% cells had a DNA content higher than 3C. After day 8, when numerous myotubes differentiated, the image analysis method revealed that the percentage of S and G2 phase cells involved in DNA synthesis had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed for the first time in satellite cells before cell fusion. These methods of analysis gave the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscular tissue. This now makes it possible to study the effects of growth-promoting substances in vitro.
KeywordsTrypsin Creatine Collagenase Laminin
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