Corneal endothelium produced by tissue engineering

Abstract

Engineered corneal endothelium (ECE) must assure cell morphology and physiology. To do so, a biocompatible, transparent, scaffold is the best option. The purpose of this thesis is to produce an ECE with a collagen scaffold and corneal endothelium cells (CECs) harvested in a two-phase system that resembles healthy corneal endothelium (CE) characteristics. Collagen based scaffolds were produced in two steps: 1) Gelification; ~2 µl/mm2 of collagen type I, HEPES solutions, and fetal bovine serum mixture per well were placed on a 12 well plate, 37°C, 5% CO2 for 2 hrs. 2) Vitrification in a Matryoshka System: sealed desiccation chamber with a saturated solution of K2CO3 was placed in an oven set to 40°C. Collagen gels were left inside for 37 days to decrease relative humidity up to 40%. Scaffolds were characterized with confocal microscopy, SEM and spectrophotometry. CECs were isolated from young New Zealand rabbits and from human donor corneas independently. Descement’s membrane was peeled from cornea, digested and, CECs obtained were cultured until confluence in proliferative media (OptiMEM I, FBS 8%, nerve growth factor 20 ng/ml, endothelial growth factor 5ng/ml, CaCl2 200 µg/ml, ascorbic acid 20 µg/ml, chondroitin sulfate 0.08%, antibiotic 1%). Passages 1-2 were carried out in resting media (OptiMEM I 8%FBS). At passage 3, ~24,000 CECs were planted onto 8 mm Ø CV membranes. The alternate use of Proliferative and Resting media conforms the two phase culture system. SEM and confocal microscopy tests showed CV membranes yielded a ~4 µm thickness and smooth surface upon 20 min hydration. SEM also showed collagen fibers merge to form a mesh-like laminar structure. Spectrophotometric scan from 450-700 nm showed a 94-95.5% transmittance. CECs seeded on CV membranes showed adhesion and proliferation at 24 hours; 72 hours served to reach confluence in a ~5 mm Ø. Culture on scaffolds reached canonical CE shape. We produced 12 rabbit and 5 human ECE with desired morphology and specific molecular marker expression. In conclusion, our collagen membrane synthesis method, along with the two phase CECs culture system, offers an option to produce ECE with healthy endothelium characteristics.