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A. E. Hutcheon, R. Ren, X. Q. Guo, S. A. Melotti, J. W. Ruberti, J. D. Zieske, V. Trinkaus-Randall; Glycosaminoglycans Are Present in Long-Term 3-D Cultures of Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3919. doi: https://doi.org/.
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Previously, we demonstrated that primary human corneal fibroblasts (HCF) cultured for 8 weeks in Vitamin C secreted and deposited an extracellular matrix and formed a 3-D, multi-layer construct. Within this construct we found alternating organized collagen-like fibrils. In our present study, we examined these 3-D constructs for the presence of proteoglycans (PGs), which are normally found surrounding fibrils in the corneal stroma.
HCF were seeded on a disorganized collagen that was deposited on a polycarbonate membrane in 6-well transwell plates. The disorganized collagen was used to produce a scaffolding-free environment. The cells were cultured in medium containing a stabilized Vitamin C derivative (2-O-α-D-glucopyranosyl-L-ascorbic acid). The 3-D constructs were analyzed at 4, 8 and 11 weeks. Both TEM and confocal microscopy were used to examine the cellular architecture. For confocal microscopy, whole mounts of the constructs were stained with rhodamine phalloidin (a marker of actin filaments) and TOPRO3 (a marker of cell nuclei). The expression of PGs in the constructs and the presence of PGs in the media were examined using RT-PCR and western blotting, respectively. Cuprolinic blue, which stains sulfated moieties, was performed to localize the PGs.
HCF stratified and deposited an extracellular matrix by 4 weeks, and attained a thickness of 65 microns by 11 weeks. There was a 2 to 4-fold increase in Type I collagen in the constructs and a 2 to 4-fold increase in the sulfated PGs secreted into the medium during the same time. Both RT-PCR and western blotting detected the expression of lumican, mimecan, perlecan, syndecan, decorin, and biglycan; however, keratocan was not detected. Cuprolinic blue stained sections revealed that the sulfated PGs were most prominent in the mid-region of the constructs and were sparse in the basal region. The proteoglycans appeared as electron dense filaments or rods and were present either along collagen fibrils or in the extracellular matrix. There was an inverse correlation between cell density and the presence of matrix throughout the construct.
We have shown for the first time that primary human stromal fibroblasts cultured long-term can regulate the expression and deposition of sulfated proteoglycans.
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