SERUM-FREE CULTIVATION OF BOVINE STROMAL FIBROBLASTS

The purpose of the present study was to conduct a comparative evaluati on of the effect of several serum-free culture conditions on adhesion, population doubling, cryopreservation and PDGF-induced effects on cel l proliferation of bovine stromal fibroblasts (BSF). Additionally, the se effects were compared to serum-containing cultures. Methods: Only s econd-passage BSF were used. Cells were cultured using four different culture media (WM/F12, WM/F12 + FCS 1%, LR-1, DMEM). After 24 h, plati ng efficiency was determined using a cell-counter system. Subsequently , the cells were seeded at a density of 100 cells/mm(2) and cultured f or 10 days using the different culture media. Cell number was determin ed 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 conditi ons. Cell vitality was determined after cryopreservation of two weeks for each culture medium. Results: The plating efficiency of BSF ranged from 50.2 to 55.5% for the serum-free culture media in contrast to se rum-containing conditions, where plating efficiency was 94.8%. With WM /F12 + FCS 1%, a population doubling of 1.27 was observed after an inc ubation period of 10 days. In contrast, cultivation under serum-free c onditions 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 t o the control. While cell vitality after cryopreservation was found to be 62.7% using WM/F12 + FCS 1%, cell vitality using serum-free media was 12.6-22.8%. Conclusions: The results of the present study demonstr ate that with respect to optimal cell adhesion and cell vitality after cryopreservation, serum-containing media should be used. BSF cultured under the serum-free conditions used in the present study can be main tained quiescent and vital for at least 10 days. Therefore, these seru m-free media are useful for cell-culture studies (e. g., determination of proliferation and cytotoxicity).