Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells

摘要

Purpose: To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs). Methods: Posterior lamellae of ten bovine corneas were decellularized using ethylene diamin tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/mL) and 0.3% sodium dodecyl sulphate (SDS). Hematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining was done to confirm the absence of bovine cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using a DNA Purification Kit. HCECs were harvested from human donor eyes and seeded on the Descemet's membrane of the DBCs. Cell morphology was assessed after 6h of incubation, and at days 1, 4, 7, 10 and 14. Expression of zonula occludens-1 (ZO-1), connexin-43 (CX-43), Na +/K +-adenosine triphosphatase (Na +/K +-ATPase), natrium hydrogen carboanhydrase (Na +/HCO3 -), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry. Results: HE staining and DAPI staining showed that bovine cells were substantially removed from the stroma and Descemet's membrane. A significant DNA reduction (mean before decelluraziation 365.3±88.6ng/mg, mean after decelluarization 23.2±7.9ng/mg, p0.001) was observed. HCECs formed a continuous, viable, predominantly polygonal monolayer with a mean cell density of 2380±179 cells/mm 2 on DBCs. Immunohistochemistry analysis demonstrated positive staining for AE5, collagen type VIII, ZO-1, CX-43, Na +/HCO3 -, and Na +/K +-ATPase. Conclusions: Phenotypical properties of HCECs on DBCs imply that the HCEC sheets are capable of maintaining an intact barrier and ionic pump function in vitro. DBCs might, therefore, be a promising scaffold for ex vivo expansion of HCECs. This xenogeneic substrate might be used for therapy of isolated corneal endothelial diseases.