Femtosecond Laser Cutting of Human Crystalline Lens Capsule and Decellularization for Corneal Endothelial Bioengineering.

The bioengineering of corneal endothelial grafts consists of seeding in vitro cultured corneal endothelial cells onto a thin, transparent, biocompatible, and sufficiently robust carrier which can withstand surgical manipulations. This is one of the most realistic alternatives to donor corneas, which are in chronic global shortage. The anterior capsule of the crystalline lens has already been identified as one of the best possible carriers, but its challenging manual preparation has limited its use. In this study, we describe a femtosecond laser cutting process of the anterior capsule of whole lenses in order to obtain capsule discs of 8 mm diameter, similar to conventional endothelial grafts. Circular marks made on the periphery of the disc indicate its orientation. Immersion in water for 3 days is sufficient to completely remove the lens epithelial cells and to enable the seeding of corneal endothelial cells, which remain viable after 27 days of culture. Therefore, this method provides a transparent, decellularized disc ready to form viable tissue engineered endothelial grafts.

Design and validation of a custom-made system to measure transepithelial electrical impedance in human corneas preserved in active storage machine.

Corneal epithelial barrier represents one of the major limitations to ocular drug delivery and can be explored non-invasively through the evaluation of its electrical properties. Human corneas stored in active storage machine (ASM) could represent an interesting physiological model to explore transcorneal drug penetration. We designed a new system adapted to human corneas preserved in ASM to explore corneal epithelial barrier function ex-vivo. A bipolar set-up including Ag/AgCl electrodes adaptors to fit the corneal ASM and a dedicated software was designed and tested on freshly excised porcine corneas (n = 59) and human corneas stored 14 days in ASM (n = 6). Porcine corneas presented significant and proportional decrease in corneal impedance in response to increasing-size epithelial ulcerations and acute exposure to benzalkonium chloride (BAC) 0.01 and 0.05%. Human corneas stored 14 days in ASM presented a significant increase in corneal impedance associated with the restoration of a multi-layer epithelium and an enhanced expression of tight junctions markers zonula occludens 1, claudin 1 and occludin. These results support the relevance of the developed approach to pursue the exploration and development of human corneas stored in ASM as a physiological pharmacological model.