Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (51): 8206-8211.doi: 10.3969/j.issn.2095-4344.2014.51.002
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Liu Tao1, Liu Gui-xiang1, Yin Ya-nan1, Zhou Juan2
Online:2014-12-10
Published:2014-12-10
Contact:
Liu Gui-xiang, M.D., Professor, Department of Ophthalmology, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
About author:Liu Tao, Studying for master’s degree, Department of Ophthalmology, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
Supported by:the Natural Science Foundation of Shandong Province, No. ZR2013HM014
CLC Number:
Liu Tao, Liu Gui-xiang, Yin Ya-nan, Zhou Juan. Primary culture and identification of kitten extraocular muscle myoblasts[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(51): 8206-8211.
Separation and culture in vitro of myoblasts Myoblasts from the extraocular muscle were treated by two-step combined differential attachment (pancreatic enzyme and type II collagenase), and the cell survival rate was (91.0±2.1)% via trypan blue staining. After observation, cells cultured separately were greatly different from the myoblasts in the late phase, and would not merge into a myotube. The isolated original generation of myoblasts was in spherical shape under inverted microscope with strong refraction (Figure 1A). The cells began to be adhered to the wall after 12 hours of culture (Figure 1B), became fusiform or spindle shape gradually after 24 hours, and completely adhered after 72 hours (Figure 1C). The myoblasts were arranged parallel to the long axis. When myoblasts completely adhered and grew for a period of time, many myoblasts which were close with each other began to fuse. If cell fusion rate was more than 80% still without digestion and generation of the cells, myoblasts would appear to have widespread and irreversible fusion. On the 72nd hour of differentiation culture, a great mass of myotube cell formed (Figure 1D). After 30 minutes of subculture, the generated cells began to adhere to the wall and fully attached after 12 hours (Figures 1E, F)."
Identification and purity results of myoblasts Desmin filaments of myoblast antibody were stained. The cytoplasm was dyed brown or brownish red with one or two light-dyed cell nuclei. If more than 90% of the cells presented positive reaction, the cultured cells were extraocular muscle cells, as shown in Figure 2. After 8 days of primary culture, myoblasts were given immunochemical staining and then positive cells were counted. The cell purification rate was (95.0±3.8)%."
Proliferation ability of myoblasts Myoblasts seeded onto 24-well plates were adhered to the wall quickly, and completely adhered after 12 hours with a larger growth rate. The cells entered the logarithmic phase on the 2nd day, while the growth slowed significantly on the 6th day due to contact inhibition. The cell growth curve is shown in Figure 3."
Differentiation capability of myoblasts The myoblasts were added with differentiation medium and fused in large amount. The fusion rate was increased significantly at 24 and 48 hours and reached the peak at 72 hours. After that, the variation tended to be stable, as shown in Figure 4. The myoblasts were arranged parallelly and mutually fused into myotubes. Multiple nuclei were arranged in the middle of the myotube (Figure 1D). Myotube formation is a sign of skeletal satellite cell differentiation[7]."
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