Portiér G L, Benders A G, Oosterhof A, Veerkamp J H, van Kuppevelt T H
Department of Biochemistry, University of Nijmegen, The Netherlands.
In Vitro Cell Dev Biol Anim. 1999 Apr;35(4):219-27. doi: 10.1007/s11626-999-0030-8.
The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C2C12 and rat L6, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C2C12 myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca2+ concentration. L6 myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the epsilon-subunit of AChR.C2C12 cells show spontaneous contraction and response of cytosolic Ca2+ to various stimulants in contrast to L6 cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.
培养细胞的分化程度取决于培养基的成分。两种常用的成肌细胞系，小鼠C2C12和大鼠L6，通常在低浓度马血清中分化。在本研究中，我们比较了马血清与含有低百分比Ultroser G和大鼠脑提取物的培养基的效果。根据各种生化、（免疫）组织化学和细胞生理参数评估成熟程度。在分化阶段用Ultroser G和大鼠脑提取物替代马血清，基于肌酸激酶活性和肌管直径，两种细胞系的肌管成熟程度更高。此外，C2C12肌管显示出横纹，含有更高百分比的肌酸激酶肌肉特异性同工酶MM，乙酰胆碱受体（AChR）簇增加九倍，形成连续的基底膜，并且静息细胞质Ca2+浓度较低。L6肌管的AChR簇增加五倍，AChRε亚基的mRNA表达增加两倍。与不显示自发收缩的L6细胞相比，C2C12细胞显示出自发收缩以及细胞质Ca2+对各种刺激的反应。这些研究表明，Ultroser G/脑提取物培养基可使两种细胞系的分化程度更高，但适合用作分化标志物的参数在两种细胞系之间似乎有所不同。
