Walshe Jennifer, Abdulsalam Najla Al Khaled, Suzuki Shuko, Chirila Traian V, Harkin Damien G
Queensland Eye Institute;
King Faisal University.
J Vis Exp. 2020 Feb 6(156). doi: 10.3791/60762.
Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet's membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.
角膜内皮细胞培养物在细胞间接触丧失后有发生上皮-间充质转化（EMT）的倾向。EMT对细胞是有害的，因为它会降低细胞形成成熟且功能正常的细胞层的能力。在此，我们介绍一种建立和传代培养人及羊角膜内皮细胞培养物的方法，该方法可将细胞间接触的损失降至最低。从供体角膜获取角膜内皮/后弹力层的外植体，并在允许细胞集体迁移到培养表面的条件下置于组织培养中。一旦建立了培养物，就将外植体转移到新的培养皿中以启动新的培养。使用 Dispase II 轻轻将细胞团从组织培养皿中提起以进行传代培养。使用该方案建立的角膜内皮细胞培养物适用于转移到生物材料膜上，以生产用于动物试验移植的组织工程细胞层。描述了一种在组织培养过程中支撑生物材料膜的定制装置，并展示了一个组织工程移植物的实例，该移植物由 I 型胶原膜两侧的一层角膜内皮细胞和一层角膜基质细胞组成。
