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Sonja Mertsch, Christina Neumann, Gerd Geerling, Stefan Schrader; Decellularized human keratocyte matrices for ocular surface reconstruction. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2250.
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© ARVO (1962-2015); The Authors (2016-present)
The limitations of substitute tissues such as human amniotic membrane for ocular surface reconstruction leads to a necessity for the development of alternative matrices. This in vitro study evaluates decellularized human corneal keratocytes matrices (DCS) as an alternative matrix for ocular surface reconstruction.
Human keratocytes were isolated from donor corneas. By supplementation with vitamin C, cells synthesize extracellular matrix and generate a cell sheet (CS). After 12 month in culture, CS were decellularized (DCS). Complete cell removal was confirmed by HE and DAPI staining and DNA measurement using a fluorescence DNA assay. The amount of collagen in the CS and DCS was assessed by a Sircol Assay. Preservation of collagen ultrastructure was analyzed by transmission electron microscopy. Cell viability of hLESCs on the matrices was quantified using WST-1 assay and by IHC staining. Furthermore, cellular influence on matrix stability was assessed by gel zymography. Surgical feasibility of the CS and DCS was tested on enucleated porcine eyes.
Human keratocytes generated a stable collagen matrix (CS) after 4 month in culture. After 12 month, the CS showed a 2.7 times higher collagen content (91.17±4.5 µg/sheet) compared to 4 month old CS (33.86±2.52 µg/sheet). Decellularisation process reduced the collage amount to 44.96±4.3 µg/sheet, but cell sheet stability was not influenced. DNA measurement of DCS showed a nearly absent residual DNA content of 31.3±15.6 ng/sheet (compared to 235.6±44.7 ng/sheet in 12 month CS and 206.4±38.2 ng/sheet in 4 month CS). Cellular viability measurement using WST-1 revealed no significant differences of cells growing on CS or DCS. Measurement of active MMP-9 and MMP-2 using zymography showed no sig. difference between the two matrices. Furthermore, surgical feasibility of 12 month old CS and DCS was comparably with 18.52±3.4 min sewing time for CS and 17.01±3.07 min sewing time for DCS.
The results of this study show, that it is possible to generate a mechanically stable collagen matrix using corneal keratocytes. After decellularisation a cell free substitute tissue is obtained, which is a suitable matrix for LESC expansion and shows good surgical feasibility. Therefore, DCS is a promising new substitute tissue; however future in vivo studies are necessary to further elucidate its potential for ocular surface reconstruction.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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