Bioengineering of endothelial grafts using femtosecond Laser cut corneal lamellae or collagen lenticules, endothelialized with immortalized or hIPSC-derived endothelial cells

Purpose The bioengineering of endothelial grafts appears as a realistic solution to reduce the global scarcity for corneal donation worldwide. We focused our strategies of endothelial bioengineering on the endothelialization of human corneal stromal lamellae with hiPSC-derived endothelial cells. Methods Human corneas unsuitable for graft but with a normal stroma, were cut into 4 to 7 lamellae of <100 μm thickness with a femtosecond laser (FsL), decellularized from their keratocytes using an optimized protocol (Ethanol/DNAse/SDS) and stored at +4°C. They were compared with collagen I compressed thin discs (RAFT) already described as carrier. Endothelialization was performed with immortalized endothelial cells (ECs) (BAG12) and hIPSC-derived ECs (also presented during this congress). Efficiency was assessed by determining endothelial cell density (ECD), morphology after alizarin red staining and SEM and immunolabeling for Na/K-ATPase expression. Transparency was measured using our customized transparometer Results both carriers were evenly endothelialized by B4G12 with similar alizarin red staining and Na/K ATPase expression patterns but higher ECD for stromal lamellae. Transparency was better for stromal lamellae. hIPSC-derived ECs gave similar characteristics Conclusion Decellularized Fsl cut stromal lamellae seem better than collagen I compressed lenticules for the endothelial bioengineering using hISPC-derived ECs. Unsuitable human cornea could be therefore “recycled” to increase the pool of available grafts. Grants: ABM2013, AIRE2013