含有角膜基质细胞的新型压缩胶原三维角膜模型的体外重建

For the safety concern and the fast development of the new pharmacitical eye drops, scientists are focused on establishing a convient in vitro corneal toxicity model to take the current animal test instead. Previous work has demonstrated that Mesenchymal stem cells grown on surfaces of different stiffness will differentiate into different lineages. This highlights that simply growing cell on tissue culture dishes which have stiffness’ over 100,000x more than the in vivo is likely to have a profound effect on the cells. By seeding the cells in a gel we put them in a system which has similar properties to that of normal tissue and they should therefore be a better model of the in vivo system. In order to further mimic the normal corneal stroma, keratocytes were embedded into the collagen scaffold. Polyvinyl alcohol (PVA) was incorporated into collagen hydrogels to further increase the mechanical stability of the compressed scaffold. We will put limbal stem cells on the traditional collagen gel (UC ) and compressed collagen gel(CC) with or without keratocyte inside the gel to establish the different type of in vitro corneal models. Modulating the Rho pathways has been shown to modulate stress fibre accumulation. Inhabitor of Rho kinase effector, ROCK, Y27632 will be applied to the different model to observe the effect for the highly differential cells on different stiffness. Well known eye irritates will be applied to different models in evaluate the efficacy of the in vitro model. Our previously experiment shows the tight junction found between the cells in the CC while negative in UC, which highlights the way the we can establish a more robust real 3D model using CC within keratocytes. The condition for storaging the in vitro model is also focused in this study. At the same time, investigate the effect of different levels of compression affect the corneal stromal progenitor cells growth, proliferation and differentiation which were embedded in collagen scaffold, in order to provide references for the individual collagen scaffold design and production.

药品开发的迅速发展和对安全性的重视，使人们越来越关注寻找一个能方便快捷，并准确替代现有活体实验的体外测试模型。近年来发现生长在不同刚度的表面上的间质干细胞分化成不同的谱系，而目前广泛应用的体外细胞培养模型将细胞培养在刚度比在体内超过100000 X多倍的组织培养皿上是不严谨的。申请者以往研究发现压缩胶原构建的体外模型可能构建出优于现在广泛接受的体外角膜毒性测试模型。本研究在此基础上拟以 I 型胶原溶液为主要材料，并添加聚乙烯醇和角膜基质祖细胞，通过机械压缩联合毛细原理，构建力学强度高的微米级压缩胶原支架，承载角膜上皮干细胞后通过评估经典的化学添加剂毒性测试，是真正包含了角膜基质细胞的三维体外模型，探讨毒性对基质细胞的作用，优选出目前可以替代的体外角膜毒性测试模型。 探讨不同程度压缩对包埋在胶原支架中角膜基质祖细胞生长、增殖及分化，丰富不同刚性支架对已分化细胞的影响的理论依据

药品开发的迅速发展和对安全性的重视，使人们越来越关注寻找一个能方便快捷，并准确替代现有活体实验的体外测试模型。近年来发现生长在不同刚度的表面上的间质干细胞分化成不同的谱系，而目前广泛应用的体外细胞培养模型将细胞培养在刚度比在体内超过100000 X多倍的组织培养皿上是不严谨的。针对此问题，本研究在以I型胶原溶液为主要材料，并添加聚乙烯醇和角膜基质祖细胞，通过机械压缩联合毛细原理，构建力学强度高的微米级压缩胶原支架，承载角膜上皮干细胞后通过评估经典的化学添加剂毒性测试，是真正包含了角膜基质细胞的三维体外模型，探讨毒性对基质细胞的作用，优选出目前可以替代的体外角膜毒性测试模型。同时探讨了不同程度的压缩（UC，MC，CC）对包埋在胶原支架中角膜基质祖细胞生长、增殖及分化，丰富不同刚性支架对已分化细胞的影响的理论依据。