Serum-free cultivation of human Tenon's capsule fibroblasts

Purpose. In the present study, the effect of three different serum-free and one serum-containing control medium on adhesion, proliferation, cryopreservation and PDGF-induced effects on cell proliferation of human Tenon's capsule fibroblasts (HTF) was compared. Materials and methods. Third passage HTF were suspended in four different culture media (WM/F12, WM/F12/FCS 1%, LR-I, DMEM) and plating efficiency was determined after 24h using a cell counter system. Subsequently, cells were seeded at a density of 50/mm(2) and cultured for ten days using the different culture media. Cell number was determined at day 2, 4, 7 and 10 after seeding. Furthermore, HTF cultured under the different conditions were stimulated by PDGF-BB [50 ng/ml]. Additionally, cell vitality after two weeks cryopreservation in five different culture media (WM/F12, WM/F12/FCS 1%, WM/F12/FCS 20%, LR-1, DMEM) was determined. Results. The plating efficiency of HTF when seeded in serum-free medium ranged from 55.3% to 59.6%. Using serum containing WM/F12/FCS 1% a slightly higher plating efficiency of 74.8% was obtained. Proliferation assays revealed population doublings of 0.77 with WM/F12/FCS 1% after an incubation period of 10 days. Cultivation of HTF using serum-free conditions did not cause significant cell proliferation but a slight cell loss which ranged from 23.1% to 34%. Addition of PDGF-BB resulted in a significant increase in cell proliferation with WM/F12/FCS 1%, WM/F12 and DMEM. After two weeks of cryopreservation in WM/F1Z, LR-1, DMEM, WM/F12/FCS 1% and WM/F12/FCS 20%, only the application of high serum concentrations led to sufficient preservation of cell vitality with a plating efficiency of 82.9%. Discussion. The results of the present study demonstrate that the use of serum-containing media is mandatory for cryopreservation of HTF. Seeding of cells can be performed either with serum or without serum. HTF cultured under serum-free conditions can be maintained quiescent with a sufficient number of cells remaining vital. The serum-free media used in this study can be applied for the investigation of cytokine effects on HTF.