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[Serum-free cultivation of bovine stromal fibroblasts]
P O Denk1, K Wunderlich, M Knorr
1Allgemeine Augenheilkunde mit Poliklinik, Universitäts-Augenklinik Tübingen.
Unlabelled:
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 cryo-preservation was found to be 62.7% using WM/F12 + FCS 1%, 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 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).