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Rachelle Palchesko, James L. Funderburgh, Adam Feinberg; In Vitro Expansion Of Corneal Endothelial Cells On Biomimetic Substrates. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6003.
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The corneal endothelium is responsible for maintaining the clarity of the cornea and loss of endothelial cells leads to impaired vision and the need for corneal transplantation. Corneal endothelial cells (CECs) are non-proliferative in vivo with minimal proliferation in vitro,making expansion of these cells for therapeutic application difficult. This study tests the hypothesis that culturing CECs on substrates that mimic the biomechanics and protein composition of native Descemet’s membrane can enhance proliferation and maintain phenotype of CECs in vitro.
CECs were isolated from fresh bovine eyes and cultured in DMEM with 10% FBS on three different surfaces: (1) tissue culture-treated polystyrene (TCPS), (2) collagen type IV coated TCPS (Col4-TCPS), and (3) collagen type IV coated polydimethylsiloxane (Col4-PDMS), a surface with more flexibility than rigid polystyrene. After confluence, cell density was determined by image analysis and cultures were expanded 1:3. At passages 1, 5, 8, and 10 fibronectin, laminin, collagen IV, ZO-1 and F-actin were imaged by immunofluorescent staining.
CECs cultured on collagen (Col4-PDMS and Col4-TCPS) reached confluence more rapidly than on TCPS. Cell density on the compliant Col4-PDMS substrate was approximately 1.5 times greater than on rigid TCPS at each passage. CECs on Col4-PDMS maintained a cuboidal cellular morphology and high cell density up to passage 8, whereas CECs on the rigid Col4-TCPS and TCPS lost proliferative capacity and phenotypic markers at earlier passages.
A biomimetic substrate matching the mechanical properties and collagen type IV composition of Descemet’s membrane was able to enhance in vitro expansion of CECs. An approximate 3000-fold expansion of bovine CECs was achieved while maintaining high expression of endothelial phenotypic markers. The results suggest that substrata mimicking the composition and compliance of Descemet’s membrane may be useful in expanding human CECs in order to bioengineer corneal endothelial sheets suitable for implantation.
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