2009, 2(4):331-337.
Abstract:
AIM: To optimize the conditions for in vitro culture of retinal pigment epithelium (RPE) cells, we characterized expressions of various growth factors in RPE cells, including tumor necrosis factor (TNF-α), vascular endothelial growth factor (VEGF), β fibroblast growth factor (βFGF), transforming growth factor β2 (TGFβ2), and interferon-γ (IFN-γ). We also studied expressions of caspase-3 under different concentrations of fetal bovine serum (FBS) with insulin- transferrin-sodium selenite (ITS) supplement.
METHODS: First, we investigated if the expressions of TNF-α, VEGF, βFGF, TGFβ2, IFN-γ, and caspase-3 in FBS and ITS with of concentration. Second, we cultured primary RPE cells from eyes of forty C57 BL/6 mice in standard dulbecco's modified eagle's medium (DMEM) containing 20, 40, 100mL/L FBS and 20, 40, 100mL/L FBS together with 10g/L ITS. Immunohistochemical staining and cell counting were performed to verify the existence and growth condition of RPE cells. The expressions of TNF-α, VEGF, βFGF, TGFβ2 and IFN-γ were determined using cells and supernatant from passage-3 to -4 primary RPE cell after 48 hours of culture with RT-PCR and enzyme-linked immunosorbent assays (ELISA). The expression of casepase-3 was determined via Western blotting. The major outcome measurement was the expression level of growth factors in cultured RPE cells and the experiment design was to expose the RPE cells to different culture medium.
RESULTS: TNF-α, VEGF, βFGF, TGFβ2, but not IFN-γ, were expressed and the expressions increased with concentration. No expression of the aforementioned genes was detected in presence of ITS. The primary cultures of RPE cells were successfully established. TNF-α, VEGF, βFGF, TGFβ2 (but no IFN-γ) and the active caspase-3 were detected in 20, 40, 100mL/L FBS or 20, 40, 100mL/L FBS combined with 10g/L ITS; the expressions were upregulated with increasing concentration of FBS. There was no significant difference in the expression of growth factors between these groups. However, significant differences were shown among different concentration of FBS (P＜0.01). The lowest expression was observed in 20mL/L FBS or 20mL/L FBS combined with 10g/L ITS medium with RPE cells. But RPE cells were shown in better growth condition in 20mL/L FBS combined with 10g/L ITS.
CONCLUSION: TNF-α, VEGF, βFGF, TGFβ2 and caspase-3 are expressed in RPE cells and supernatants. The production of above 20mL/L FBS combined with 10g/L ITS in DMEM may be the ideal cell culture medium that supports the normal growth of RPE cells.
