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ARUNI MAKULOLUWA, Rachel Williams, Stephen B Kaye, Rosalind M K Stewart; The role of extracellular matrix (ECM) protein composition on the adhesion and growth of conjunctival epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3025.
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© ARVO (1962-2015); The Authors (2016-present)
There is a need to understand how the ECM may affect cell behaviour. This will guide development of substrates for conjunctival reconstruction. We carried out a laboratory study to establish the ECM deposited by conjunctival cells and determine the behaviour of these cells when seeded onto pre-adsorbed proteins modelling the natural ECM composition.
ECM proteins deposited by a conjunctival cell line (HCjE-Gi) were assessed by immunofluorescence (IF) at days 5-42. Maximum adsorption of fibronectin, collagen IV and laminin 111 onto tissue culture plastic (TCP) was determined by ELISA. Adhesion and growth of HCjE-Gi cells seeded onto uncoated TCP and pre-adsorbed proteins up to the saturation points were assessed by DAPI staining. HCjE-Gi-secreted ECM was isolated using 1% NH4OH to remove the cells at days 5-42. Cellular growth on this secreted ECM was determined until 7 days by DAPI staining and counted using Image J. Statistical analysis was performed using 1-way ANOVA and independent t-tests.
Laminin α3 was present abundantly from day 5 onwards, fibronectin was present earlier but reduced with time. Collagen IV was only detected from day 21. Maximum adsorption of fibronectin and collagen IV onto TCP were reached from a 5μg/mL solution and laminin 111 from a 0.5μg/mL solution. Adhesion of cells was increased by pre-adsorption of fibronectin (p<0.05) and collagen IV (p<0.05), however, this was unaffected by pre-adsorption of laminin 111. Cellular growth was not affected by pre-adsorption of fibronectin or laminin 111, but was increased by both pre-adsorption of collagen IV (p<0.05), or ECM secreted (p<0.05) for greater than 5 days.
The presence of secreted ECM or collagen IV alone enhances the growth of conjunctival cells. Substrates or substrate coating with these components could enhance ex-vivo expansion of conjunctival cells for ocular surface reconstruction.
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