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Huifang Zhou, Qiongyu Guo, Michael Grant, Jennifer Elisseeff; Reconstruction of Conjunctival Epithelium with Goblet Cells by Collagen Vitrigel. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4681.
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The goal of this study is to evaluate the potential suitability of collagen vitrigel (CV) membrane as a substrate for engineering conjunctival epithelium with goblet cells.
Conjunctival epithelial cells were isolated from rabbit conjunctiva and were cultured in DMEM/F12 containing 10% FBS, 5 µg/mL human recombinant insulin, 0.05 mM hydrocortisone and 10 ng/mL EGF. The collagen type I vitrigel membrane was prepared in 6-well culture plates as previously described . Conjunctival cells were seeded onto the CV and were compared to identical cultures on either tissue culture plastic (TCP) or a standard collagen membrane. The viability and morphology of conjunctival cells on the different substrates were investigated. Antibodies, including MUC5AC, specific for goblet cells, CK19 for conjunctival epithelium cells, and CK3 for corneal epithelium cells, were used to characterize the cultured cells.
Conjunctival epithelial cells demonstrated a high colony-forming capacity that decreased with culture. The CV was a transparent membrane with sufficient mechanical properties with the average thickness of about 50 µm. The conjunctival epithelial cells were easy to adhere to the CV membrane, and exhibited high viability (Figure 1) and proliferated faster than control groups. The cells on the CV maintained their phenotype, as shown in Figure 2 with the presence of CK19-positive conjunctival epithelium cells and MUC5AC-positive goblet cells. No CK3-positive cornea epithelial cells were detected.
The CV membrane is a suitable substrate for in vitro culture of conjunctival epithelial and goblet cells. The artificial conjunctival epithelium can be manipulated and sutured, and it is currently being studied in vivo in a rabbit model of conjunctival injury. Reference 1. McIntosh Ambrose W, etc. (2009) J Biomed Mater Res B Appl Biomater 90:818-31.
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