Characterization of a collagen-based engineered corneal endothelium

Abstract

Purpose : Corneal endothelium replacement must assure cell morphology and physiology. In order to do so, a biocompatible, transparent, scaffold is the best option. The purpose of our project was to characterize a construct produced by seeding corneal endothelial cells (CEC) on collagen vitrigel (CV) membranes.

Methods : CV membranes were produced modifying a previous methodology 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: a 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%. CV membranes were characterized with confocal microscopy, SEM and spectrophotometry to analyze surface, thickness and transparency respectively. CEC were isolated from young New Zealand rabbits. Descement’s membrane was peeled from rabbit cornea, digested and, CEC obtained were cultured until confluence in mitotic 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 and 2 were carried out in resting media (OptiMEM I 8%FBS). At passage 3, ~24,000 CEC were planted onto 8 mm Ø CV membranes.

Results : 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. CEC seeded on CV membranes showed adhesion and proliferation at 24 hours; 72 hours served to reach confluence in a ~5 mm Ø. Culture on CV membranes reached canonical corneal endothelium shape. We produced 12 constructs made of collagen vitrigel and culture-expanded CEC from one cornea.

Conclusions : In conclusion, CV synthesis modified methodology offers a robust option to produce an scaffold with optical properties that can be easily stablished in every laboratory and can be scaled up. CEC cultured in a two-phase system adhere and proliferate over the CV membrane maintaining their canonical morphology.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Corneal endothelium cells (CEC) on collagen-vitrigel membrane, 48 hours after seeding. The CEC were isolated from a rabbit, second passage expansion under a two phase culture system.