Abstract
Purpose:
Corneal endothelial cells (CECs) are non-proliferative in vivo with minimal proliferation in vitro, making expansion of these cells for therapeutic application difficult. Our previous work has shown that culturing cells on a biomimetic substrate that mimics the mechanical and biochemical properties of Descemet’s membrane enables the expansion of CECs >3000-fold compared to 139-fold on tissue culture polystyrene (TCPS). Here, we demonstrate that the biomimetic substrate also maintains CEC phenotype and prevents transition to senescence or a fibroblast-like phenotype.
Methods:
CECs were isolated from bovine corneas and cultured on one of three different surfaces: the biomimetic substrate consisting of collagen type IV coated polydimethylsiloxane soft elastomer (COL4-PDMS) and two controls, TCPS and collagen type IV coated TCPS (COL4-TCPS). CECs were cultured for 8 passages and immunofluorescently labeled at passages 1, 5, and 8 for fibronectin (FN), zonal occludins (ZO-1) and F-actin to analyze changes in extracellular matrix production, cell-cell coupling and polygonal morphology. At these same time points qRT-PCR was used to quantify mRNA expression of COL4A2, COL8A1, and SLC4A4 as CEC markers and COL3A1 as the fibroblastic gene marker.
Results:
CECs cultured on the COL4-PDMS produced short, immature FN fibrils at all time points where as CECs on TCPS and COL4-TCPS produced large fibrils at passages 5 and 8 similar to those produced by fibroblasts. On COL4-PDMS CECs grew at higher density, had more continuous ZO-1 staining and maintained a polygonal morphology. Gene expression followed a similar pattern with fibroblast associated FN and COL3A1 genes higher on TCPS and COL4-TCPS and CEC associated COL8A1 and SLC4A4 genes maintained at levels comparable to CECs in vivo on COL4-PDMS.
Conclusions:
We have demonstrated that a biomimetic substrate which recapitulates the mechanical stiffness and collagen type IV composition of Descemet’s membrane significantly enhances expansion and maintains phenotypic stability of CECs in vitro compared to TCPS and COL4-TCPS controls. Current efforts are focused on extending this system to the expansion of human CECs to enhance its clinical relevance. The ability to expand CECs is crucial to obtain the cell numbers necessary for bioengineering a corneal endothelium suitable for implantation, a future goal of this research project.
Keywords: 481 cornea: endothelium