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Aruni K. Makuloluwa, Rosalind M. K. Stewart, Stephen B. Kaye, Rachel L. Williams, Kevin J. Hamill; Mass Spectrometry Reveals α-2-HS-Glycoprotein as a Key Early Extracellular Matrix Protein for Conjunctival Cells. Invest. Ophthalmol. Vis. Sci. 2020;61(3):44. https://doi.org/10.1167/iovs.61.3.44.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the composition of extracellular matrix (ECM) proteins secreted by a conjunctival epithelial cell line and to identify components that aid conjunctival epithelial cell culture.
Human conjunctival epithelial cell line (HCjE-Gi) cells were cultured in serum-free media and their ECM isolated using ammonium hydroxide. Growth characteristics were evaluated for fresh HCjE-Gi cells plated onto ECMs obtained from 3- to 28-day cell cultures. Mass spectrometry was used to characterize the ECM composition over 42 culture days. Cell adhesion and growth on pre-adsorbed fibronectin and α-2-HS-glycoprotein (α-2-HS-GP) were investigated.
Day 3 ECM provided the best substrate for cell growth compared to ECM obtained from 5- to 28-day cell cultures. Mass spectrometry identified a predominantly laminin 332 matrix throughout the time course, with progressive changes to matrix composition over time: proportional decreases in matrix-bound growth factors and increases in proteases. Fibronectin and α-2-HS-GP were 5- and 200-fold enriched as a proportion of the early ECM relative to the late ECM, respectively. Experiments on these proteins in isolation demonstrated that fibronectin supported rapid cell adhesion, whereas fibronectin and α-2-HS-GP both supported enhanced cell growth compared to tissue culture polystyrene.
These data reveal α-2-HS-GP as a candidate protein to enhance the growth of conjunctival epithelial cells and raise the possibility of exploiting these findings for targeted improvement to synthetic tissue engineered conjunctival substrates.
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