Submitted to: Cloning and Stem Cells
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/16/2003
Publication Date: 6/1/2004
Citation: Talbot, N.C., Powell, A.M., Caperna, T.J. 2004. Colony-formation efficiency of bovine fetal fibroblast cell lines cultured with low oxygen, hydrocortisone, L-carnosine, or BFGF. Cloning and Stem Cells. 6(1):35-45. Interpretive Summary: Somatic cell nuclear transfer (NT), or nuclear cloning, depends upon replacing the nucleus of the cow egg with the nucleus of a somatic cell, or body cell. The most commonly used body cell for NT is the fibroblast, a connective tissue cell found throughout the body that is easy to isolate and grow in culture. The present study compared six independently derived bovine fetal fibroblast cell lines under various conditions of culture to determine if difference existed between the cell lines. The comparison was made on the basis of the cells of a cell line to grow out to a colony of 16 or more cells from a single cell attaching to the culture dish. Culture conditions tested include low oxygen (5% vs. 20%), and the addition of either hydrocortisone, L-carnosine, basic fibroblast growth factor (bFGF), or different levels of fetal bovine serum (FBS) to the medium. The results showed that there were statistically significant difference between cell lines in their growth responses to the various culture conditions. However, with the exception of bFGF and higher levels of FBS, none of the culture conditions caused a beneficial response in the cells lines, i.e., an increase in the number and size of cell colonies. The study shows that culture treatments of putative benefit to colony-forming efficiency in rodent or human fibroblasts are not necessarily of benefit to cultures of bovine fibroblasts.
Technical Abstract: A comparison of colony-formation efficiency (CFE) was made between six independent bovine fetal fibroblast (BFF) cell lines used in somatic cell nuclear transfer. Variation in CFE was assessed under different culture conditions. The conditions examined were ambient atmosphere culture vs. 5% oxygen culture, three levels of fetal bovine serum (FBS) in the medium (5%, 10% or 20%), and the amendment of 10% FBS medium with basic fibroblast growth factor (1 ng/ml), carnosine (20 mM), or hydrocortizone (1 ¿M). The six BFF cell lines showed significant differences from one another in CFE. No significant difference in CFE was found with reduced oxygen culture. Carnosine also had no significant effect on CFE. A FBS concentration of 10% was found to produce the best overall CFE. Hydrocortisone treatment reduced the growth (size) of colonies although the number of colonies formed, i.e., surviving was not affected. Basic FGF increased the size of colonies but the number of colonies formed was not affected. The results showed that different BFF cell lines varied significantly in their CFE. Also, medium amendments (with the exception of bFGF) or culture conditions that have shown positive effects on the growth and survival on fibroblast of other species failed to show significant positive CFE effects in the BFF cell lines tested.