Multilayered Fabrication Assembly Technique to Engineer a Corneal Stromal Equivalent

Tissue engineering has emerged as a strategy to combat the donor shortage of human corneas for transplantation. Synthetic corneal substitutes are currently unable to support the normal phenotype of human cells and so decellularized animal corneas have been deployed to more closely provide the topographical and biochemical cues to promote cell attachment and function. Although full thickness decellularized corneas can support corneal cells, the cells are slow to populate the scaffold and density declines from the surface. To avoid these problems, this protocol describes the stacking of alternate layers of decellularized porcine corneal sheets and cell-laden collagen hydrogel to produce a corneal construct. The sheets are obtained by cryosectioning porcine corneas, decellularizing them with detergents and nucleases and finally air drying for storage and ease of manufacture. Corneal stromal cells are then encapsulated in a collagen type I solution and cast between these sheets. This protocol presents a rapid method to ensure high cellularity throughout the construct using tissue-derived materials alone. Graphic abstract: Overview of main process to obtain corneal stromal equivalents.