Fernández-Pérez Julia, Madden Peter W, Ahearne Mark
Department of Mechanical, Manufacturing and Biomedical Engineering, School of Engineering, Trinity College Dublin, University of Dublin, Dublin, Ireland.
Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, University of Dublin, Dublin, Ireland.
Bio Protoc. 2021 Mar 20;11(6):e3963. doi: 10.21769/BioProtoc.3963.
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. .
组织工程已成为一种应对人类角膜移植供体短缺的策略。目前，合成角膜替代品无法支持人类细胞的正常表型，因此已采用脱细胞动物角膜来更紧密地提供促进细胞附着和功能的地形和生化线索。尽管全层脱细胞角膜可以支持角膜细胞，但细胞在支架上的填充速度较慢，且密度从表面开始下降。为避免这些问题，本方案描述了将脱细胞猪角膜片层与载有细胞的胶原水凝胶交替层堆叠以制备角膜构建体的方法。这些片层通过对猪角膜进行冷冻切片、用去污剂和核酸酶使其脱细胞，最后风干以储存并便于制造而获得。然后将角膜基质细胞封装在I型胶原溶液中，并浇铸在这些片层之间。本方案提出了一种仅使用组织衍生材料就能确保整个构建体具有高细胞密度的快速方法。
