Serum-free cultivation of bovine stromal fibroblasts

Abstract

The purpose of the present study was to conduct a comparative evaluation of the effect of several serum-free culture conditions on adhesion, population doubling, cryopreservation and PDGF-induced effects on cell proliferation of bovine stromal fibroblasts (BSF). Additionally, these effects were compared to serum-containing cultures. Only second-passage BSF were used. Cells were cultured using four different culture media (WM/F12, WM/F12 + FCS 1%, LR-1, DMEM). After 24 h, plating efficiency was determined using a cell-counter system. Subsequently, the cells were seeded at a density of 100 cells/mm2 and cultured for 10 days using the different culture media. Cell number was determined at day 2, 4, 7 and 10 after seeding. Furthermore, the effect of 50 ng/ml PDGF-BB on the proliferation of BSF was tested for these conditions. Cell vitality was determined after cryopreservation of two weeks for each culture medium. The plating efficiency of BSF ranged from 50.2 to 55.5% for the serum-free culture media in contrast to serum-containing conditions, where plating efficiency was 94.8%. With WM/F12 + FCS 1%, a population doubling of 1.27 was observed after an incubation period of 10 days. In contrast, cultivation under serum-free conditions caused neither significant cell proliferation nor cell loss. The stimulation of cell proliferation with PDGF-BB was shown to be 28% (LR1), 40% (WM/F12 + FCS 1%) 76% (WM/F12) and 95% (DMEM) compared to the control. While cell vitality after cryo-preservation was found to be 62.7% using WM/F12 + FCS 1%, vitality using serum-free media was 12.6-22.8%. The results of the present study demonstrate that with respect to optimal cell adhesion and cell vitality after cryo-preservation, serum-containing media should be used. BSF cultured under the serum-free conditions used in the present study can be maintained quiescent and vital for at least 10 days. Therefore, these serum-free media are useful for cell-culture studies (e.g., determination of proliferation and cytotoxicity).