Manufacturing of human corneal endothelial grafts

Purpose: Human corneal endothelial cells (HCECs) play a significant role in maintaining visual function. However, these cells are notorious for their limited proliferative capacity in vivo. Current treatment of corneal endothelial dysfunction resorts to corneal transplantation. Herein we describe an ex vivo engineering method to manufacture HCEC grafts suitable for transplantation through reprogramming into neural crest progenitors. Methods: HCECs were isolated by collagenase A from stripped Descemet membrane of cadaveric corneoscleral rims, and induced reprogramming via knockdown with p120 and Kaiso siRNAs on collagen IV-coated atelocollagen. Engineered HCEC grafts were released after assessing their identity, potency, viability, purity and sterility. Phase contrast was used for monitoring cell shape, graft size, and cell density. Immunostaining was used to determine the normal HCEC phenotype with expression of N-cadherin, ZO-1, ATPase, acetyl-& alpha;-tubulin, & gamma;-tubulin, p75NTR, & alpha;-catenin, & beta;-catenin, and F-actin. Stability of manufactured HCEC graft was evaluated after transit and storage for up to 3 weeks. The pump function of HCEC grafts was measured by lactate efflux. Results: One HCEC graft suitable for corneal transplantation was generated from 1/8th of the donor corneoscleral rim with normal hexagonal cell shape, density, and phenotype. The manufactured grafts were stable for up to 3 weeks at 37 degrees C or up to 1 week at 22 degrees C in MESCM medium and after transcontinental shipping at room temperature by retaining normal morphology (hexagonal, >2000 cells/mm2, >8 mm diameter), phenotype, and pump function.Conclusions: This regenerative strategy through knockdown with p120 and Kaiso siRNAs can be used to manufacture HCEC grafts with normal phenotype, morphology and pump function following prolonged storage and shipping.