Woods T L, Smith C W, Zeece M G, Jones S J
Department of Animal Science, University of Nebraska, Lincoln 68583-0908, USA.
Tissue Cell. 1997 Apr;29(2):207-15. doi: 10.1016/s0040-8166(97)80020-1.
The objective of this experiment was to determine the growth characteristics of bovine embryonic muscle cells and to optimize the growth conditions for these cells using commercially-prepared media and sera. In the first study, the growth of muscle cells isolated from the hindlimb was determined by measuring DNA content. The DNA concentration was lowest (P < 0.001) at 24 h post-plating and increased to a maximum at approximately 60 h. The slopes of creatine kinase activity and fusion index curves were similar to the DNA; however, the creatine kinase activity achieved a maximum at 140 h post-plating, while the fusion index reached maximum at 120 h. In the second study, cells were cultured on different substrata, either plastic, gelatin, or collagen. There were no differences (P > 0.05) in the cell growth rates for any of the three substrata. In the third study, cells were grown in 10% fetal bovine serum (FBS) and either a balanced salt solution (BSS; 30 mM Hepes, 10 mM glucose, 120 mM NaCl, 2.5 mM Na2HPO4, and 3 mM KCl), McCoy's 5A, Dulbecco's Minimal Essential Medium/Ham's F12 (DMEM/F12), or 70% DMEM/20% M-199. Cell numbers adhering to the plate at 26 h post-plating were different (P > 0.001) between each medium (DMEM/M-199 > McCoy's 5A > DMEM/F12 > BSS). Cell proliferation rates for each treatment medium were greatest for DMEM/M-199, followed by McCoy's 5A, DMEM/F12, and BSS. Cell differentiation was highest (P < 0.05) in the DMEM/F12, followed by McCoy's 5A, DMEM/M-199, and BSS. In the final study, the cells were treated with different sources of serum added at 10% to DMEM/M-199. The sera consisted of FBS, newborn calf serum (NCS), horse serum (HS) and iron-supplemented calf serum (Fe(2+)-CS). The cells were added to each well at 10(4) cells. At 24 h post-plating, the serum-free, NCS, and FBS-treated cell numbers were greater (P < 0.05) than the cells treated with HS or Fe(2+)-CS, which may reflect the efficient adherence to the surface or faster adaptation to the serum by the cells. The proliferation rate was greatest (P < 0.001) for the cells treated with Fe(2+)-CS, followed by FBS = NCS, HS, and no serum. Therefore, the muscle cells obtained from bovine embryos grow and differentiate similar to muscle cells from other species. The optimal growth medium for growing these cells in vitro is DMEM/M-199 plus 10% Fe(2+)-CS, while the optimal differentiation medium is McCoy's 5A.
本实验的目的是确定牛胚胎肌肉细胞的生长特性，并使用市售培养基和血清优化这些细胞的生长条件。在第一项研究中，通过测量DNA含量来确定从后肢分离的肌肉细胞的生长情况。接种后24小时DNA浓度最低（P<0.001），并在约60小时时增加到最大值。肌酸激酶活性和融合指数曲线的斜率与DNA相似；然而，肌酸激酶活性在接种后140小时达到最大值，而融合指数在120小时达到最大值。在第二项研究中，细胞在不同的基质上培养，即塑料、明胶或胶原蛋白。三种基质中任何一种的细胞生长速率均无差异（P>0.05）。在第三项研究中，细胞在10%胎牛血清（FBS）和平衡盐溶液（BSS；30 mM Hepes、10 mM葡萄糖、120 mM NaCl、2.5 mM Na2HPO4和3 mM KCl）、McCoy's 5A、Dulbecco's Minimal Essential Medium/Ham's F12（DMEM/F12）或70% DMEM/20% M-199中培养。接种后26小时附着在平板上的细胞数量在每种培养基之间有所不同（P>0.001）（DMEM/M-199>McCoy's 5A>DMEM/F12>BSS）。每种处理培养基的细胞增殖速率以DMEM/M-199最大，其次是McCoy's 5A、DMEM/F12和BSS。细胞分化在DMEM/F12中最高（P<0.05），其次是McCoy's 5A、DMEM/M-199和BSS。在最后一项研究中，细胞用添加到DMEM/M-199中的10%不同来源血清处理。血清包括FBS、新生牛血清（NCS）、马血清（HS）和补铁牛血清（Fe(2+)-CS）。以10(4)个细胞接种到每个孔中。接种后24小时，无血清、NCS和FBS处理的细胞数量比HS或Fe(2+)-CS处理的细胞多（P<0.05），这可能反映了细胞对表面的有效附着或对血清的更快适应。Fe(2+)-CS处理的细胞增殖速率最大（P<0.001），其次是FBS=NCS、HS和无血清。因此，从牛胚胎获得的肌肉细胞的生长和分化与其他物种的肌肉细胞相似。体外培养这些细胞的最佳生长培养基是DMEM/M-199加10% Fe(2+)-CS，而最佳分化培养基是McCoy's 5A。
