Conditions for the culture of bovine embryonic myogenic cells.
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.
Woods
T L
TL
Department of Animal Science, University of Nebraska, Lincoln 68583-0908, USA.
Smith
C W
CW
Zeece
M G
MG
Jones
S J
SJ
D000818:Animals; D002417:Cattle; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D003402:Creatine Kinase / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D005333:Fetus / Q000166:cytology*; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000201:enzymology; D018482:Muscle, Skeletal / Q000166:cytology / Q000196:embryology / Q000201:enzymology
D003470:Culture Media; D003402:Creatine Kinase