Determining Influence of Culture Media and Dose-Dependent Supplementation with Basic Fibroblast Growth Factor on the Ex Vivo Proliferative Activity of Domestic Cat Dermal Fibroblasts in Terms of Their Suitability for Cell Banking and Somatic Cell Cloning of Felids

Abstract

Abstract Dermal fibroblasts are commonly used as donors of genetic material for somatic cell nuclear transfer in mammals. Basic fibroblast growth factor (bFGF) is a cytokine that regulates proliferation and differentiation of different cell types. The study was aimed at optimizing the cell culture protocol for cat dermal fibroblasts by assessing the influence of culture media and different doses of bFGF on proliferation of fibroblasts and their viability in terms of cell banking and somatic cloning of felids. In Experiment I, skin biopsies of domestic cats were cultured in DMEM (D) and/or DMEM/F12 (F), both supplemented with 5 ng bFGF/ml (D-5, F-5, respectively). After the primary culture reached ~80% of confluency, the cells were passaged (3–4 times) and cultured in media with (D-5, F-5) or without (D-0, F-0) bFGF. To determine the optimal doses of bFGF, in Experiment II, secondary fibroblasts were cultured in DMEM with 0 (D-0), 2.5 (D-2.5), 5 (D-5) or 10 (D-10) ng bFGF/ml. The results showed that in D-5 the cells proliferated faster than in D-0, F-5 and F-0. Due to their poor proliferation, passages IV were not performed for cells cultured in F-0. In experiment II, a dose-dependent effect of bFGF on proliferation of cat dermal fibroblasts was found. In D-5 and D-10, the cells exhibited higher (P<0.05) proliferation compared with D-0. In D-2.5 the cells showed a tendency to proliferate slower than in D-5 and D-10 and at the same faster than in D-0. In conclusion. DMEM supplemented with bFGF provides better proliferation of domestic cat dermal fibroblasts culture than DMEM/F12. Supplementation of culture medium with bFGF has a beneficial effect on cat dermal fibroblast proliferation and could be recommended for addition to culture media.