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Juliane Teichmann, Monika Valtink, Stefan Gramm, Mirko Nitschke, Carsten Werner, Katrin Engelmann; Generation of Human Corneal Endothelial Cell Sheets Under Serum-Free Conditions Using Reactive And Temperature-Responsive Cell Culture Carriers. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6466.
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Generation of temperature-responsive cell culture carriers with tunable biochemical and mechanical properties for the fabrication of transplantable human corneal endothelial cell sheets under serum-free conditions.
Human corneal endothelial cells (HCEC) were cultured on electron beam cross-linked thin films of poly(vinyl methyl ether) (PVME, temperature-responsive component) blended with 1 wt% or 10 wt% of the alternating copolymer of PVME and maleic anhydride (PVME-alt-MA, protein/peptide-binding component). Film thickness and degree of cross-linking were varied to tune the mechanical properties while MA units allowed for the covalent attachment of laminin/ chondroitin sulfate, collagen type IV, fibronectin, or cyclic RGD peptide (cRGD). Cell adhesion, proliferation and detachment after lowering the temperature were monitored by microscopy and were compared for HCEC cultured with and without serum-containing growth medium (immortalized population HCEC-12 and clonal cell line HCEC-B4G12, respectively).
First experiments showed that an initial coating with cRGD gave best results in both culture systems. HCEC-12 cultured in serum-supplemented medium showed very good cell adhesion, monolayer formation and cell sheet detachment upon temperature reduction on 1 wt% PVME-alt-MA containing surfaces with various thicknesses and a medium cross linking degree as well as on thicker 10 wt% PVME-alt-MA containing surfaces. In contrast, serum-free cultured HCEC-B4G12 showed good adhesion, proliferation and detachment behavior only on thicker, high cross-linked 10 wt% PVME-alt-MA containing coatings.
Temperature-responsive cell culture carriers based on electron beam cross-linked MA functionalized PVME are suitable for the generation of HCEC sheets under serum-supplemented and serum-free culture conditions.
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