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Dimitrios Karamichos, Ramin Zareian, Audrey E. Hutcheon, Jeffrey W. Ruberti, James D. Zieske; TGF-β3 Replaces Serum Action in an In Vitro Model of Corneal Matrix Development. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3646.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal fibrosis or scarring has left over 10 million people blind. Currently, therapeutic treatments are almost non-existent. Numerous researchers have attempted to develop an artificial cornea-like tissue in vitro; however, the attempts have proven problematic due to the need for the non-physiological presence of serum. In this study, we used our 3-dimensional (3D) stroma-like in vitro model (construct) to test whether transforming growth factor-beta 3 (T3) could replace serum.
Human corneal fibroblasts (HCF) were isolated and cultured in 1 or 10% serum. Upon reaching confluence, the cells were grown in either (1) 1%S: 1% serum only, (2) 1%S+T3: 1% serum + T3, (3) 10%S: 10% serum only, or (4) 10%S+T3: 10% serum ± T3, and stimulated with a stable Vitamin C (VitC) derivative for 4 weeks, allowing them to secrete a self-assembled matrix. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence microscopy (IF: whole mount and frozen) and transmission electron microscopy (TEM).
Our data shows that by 4 weeks, the HCF in 1%S+T3 stratified to a mean thickness of ~30 microns, similar to those stimulated with 10%S. 10%S+T3 reached the highest thickness of ~60 microns. In contrast, HCF with 1%S failed to stratify, resulting in a monolayer. TEM data revealed aligned extracellular matrix with long fibrils for all conditions except 1%S, and T3-stimulated constructs appeared to be more organized. With IF, we found high levels of type I and V collagen with the T3-stimulated constructs, but very little type III.
Overall, the data suggests that it is possible to stimulate matrix secretion and assembly in vitro without non-physiological levels of serum by replacement with a single growth factor, T3. T3 and serum both stimulate matrix production; however, T3 appeared to stimulate a more aligned, non-fibrotic matrix than serum. These data also suggest that T3 may play a role in stimulating assembly of the stromal matrix during development.
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