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Purpose: To investigate stimulatory effects of PDGF-AA, PDGF-AB, PDGF-BB, bFGF, IL-1beta, TGF-beta1 and TGF-beta2 on the proliferation and myofibroblast transformation of cultured human Tenon's capsule fibroblasts and to characterize expression of PDGF- and TGF-beta-receptors in these cells., Methods: To determine cell proliferation, cell number of 2nd passage cultured human Tenon's capsule fibroblasts was measured before and after addition of growth factors using a computer-based cell counter system. Immunoblotting was used to detect and quantitate alpha-smooth-muscle actin (alpha-SMA) expression. Expression of PDGF- and TGF-beta-receptor mRNA was detected by RT-PCR, expression of the corresponding protein was demonstrated using Western blot., Results: A significant increase in proliferation (p < or = 0.05) was detected after exogenous stimulation with PDGF-AA (10 ng/ml and 100 ng/ml), PDGF-AB (10 ng/ml and 100 ng/ml), PDGF-BB (10 ng/ml and 100 ng/ml), bFGF (100 ng/ml), IL-1beta (1 ng/ml and 10 ng/ml), TGF-beta1 (0.5 ng/ml) and TGF-beta2 (0.5 ng/ml). Both TGF-beta1 and TGF-beta2 stimulated expression of alpha-SMA in a dose dependent manner with peak activity at a concentration of 50 ng/ml (TGF-beta1) and 500 ng/ml (TGF-beta2). Protein and mRNA of PDGF-receptor type alpha and type beta and TGF-beta-receptors type I, II and III are expressed in cultured human Tenon's capsule fibroblasts., Conclusions: The present investigation strongly supports the hypothesis that PDGF-isoforms are major stimulators of proliferation of Tenon's capsule fibroblasts after glaucoma filtering surgery while TGF-beta-isoforms are essential for the transformation of Tenon's capsule fibroblasts into myofibroblasts.

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-1, DMEM) and plating efficiency was determined after 24h using a cell counter system. Subsequently, cells were seeded at a density of 50/mm2 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/F12, 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.