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Wenjing Wu, Shrestha Priyadarsini, James D Zieske, Dimitrios Karamichos; Comparison of human corneal fibroblasts cultured in human or fetal bovine serum in an in vitro 3D model. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2358.
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© ARVO (1962-2015); The Authors (2016-present)
TGF-β isoforms are known for their effects on corneal fibrosis with TGF-β1 and -β2 being pro-fibrotic and TGF-β3 being anti-fibrotic. The purpose of this study was to investigate the differences between human serum (HS) and fetal bovine serum (FBS) on human corneal fibroblasts (HCF) in an in vitro 3D culture model, as well as, examine HS’s influence on corneal stromal fibrosis.
HCF were cultured for 2 weeks in EMEM + 0.5mM Vitamin C (2-O-α-D-glucopyranosyl-L-ascorbic acid) with either HS or FBS ± TGF-β1, -β2, or -β3. After 2 weeks, samples were collected, processed for qRT-PCR and/or western blot, and examined for alterations in fibrotic markers—α-smooth muscle actin (α-SMA) and collagen types III (Col III) and V (Col V)—as well as, focal adhesion kinase (FAK) and matrix metalloproteinases 1 (MMP1) and 3 (MMP3).
In HCF controls, Col III was significantly downregulated (p<0.05) and MMPs 1 and 3 (p<0.0001 for both) were upregulated in HS compared to FBS; however, there was no difference in α-SMA or Col V expression. For all TGFβ isoforms, α-SMA showed no significant difference between serums; however, Col III was significantly decreased (p<0.004) and Col V was significantly increased (p<0.0005) in HS as compared with FBS samples. When compared with their respective serum controls, both MMP1 and 3 in HS were significantly downregulated by the TGFβ isoforms, especially TGF-β3 (p<0.05); however, in the FBS samples, MMPs 1 and 3 were both upregulated with TGFβ stimulation. Interestingly, TGF-β1 stimulated FAK (p<0.05), while TGF-β3 appeared to have no effect.
Our results show that HS could potentially replace FBS in our in vitro culture model, and offer a more translational in vitro corneal stroma equivalent. The use of HS may also provide insights into the development of a fibrotic phenotype. For example, HCF cultured with HS and TGF-β3 appeared to significantly inhibit degradation enzymes (ie. MMPs 1 and 3) and promote a non-fibrotic phenotype.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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