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Serum-free cultivation of bovine stromal fibroblasts

Research paper by Peter Oliver Denk, Kerstin Wunderlich, Marcus Knorr

Indexed on: 01 Mar '98Published on: 01 Mar '98Published in: Der Ophthalmologe : Zeitschrift der Deutschen Ophthalmologischen Gesellschaft



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.Methods: 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.Results: 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 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 demonstrate 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 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).