A Novel 3-Dimensional Culture Model of the Anterior Cornea

描述(申请人提供)：上皮-基质的相互作用被认为在维持正常的角膜动态平衡和调节损伤和屈光手术后的角膜伤口愈合中起着关键作用。例如，PRK后，受损的上皮细胞释放几种细胞因子和生长因子，调节基质细胞的死亡、增殖和分化。虽然角膜上皮细胞和基质细胞之间的相互作用的重要性是显而易见的，但要更详细地研究特定细胞因子在介导上皮细胞和基质细胞相互作用中的作用，需要建立一个体外模型，在该模型中可以严格控制培养条件。遗憾的是，目前的体外模型并不能很好地模拟体内角膜组织的三维几何形状和力学性质。组织工程学成功构建体外前角膜的两个关键障碍是：(1)三维细胞外基质的发展，这种细胞外基质具有高的机械硬度，但支持静止的基质细胞(角质细胞)表型的维持；(2)体外分化和分层的角膜上皮细胞系的发展。我们最近进行了初步实验，在这些实验中，细胞种子三维胶原基质被压缩，以获得高硬度的组织等效物。这些结构支持角膜基质细胞的分化，并具有一些独特的特性，可能是基质组织工程的理想选择。我们还开发了一种独特的人端粒酶永生化角膜上皮细胞系(HTCEpi)，它在分层、空气提升的培养条件下表达关键分化标志。这项拟议研究的目的是测试我们是否可以结合这两种独特的技术来产生一种新的3D模型，该模型可以用于研究动态平衡和伤口愈合过程中的上皮-间质相互作用。为此，我们建议：(1)确定压缩的胶原基质能否在体外无血清条件下支持角膜上皮细胞的分化和复层；(2)研究诱导这些结构损伤的可行性，以模拟角膜前部伤口的愈合。这些目标的实现将建立使用压缩的胶原基质作为模型系统来研究动态平衡和伤口愈合过程中的上皮-基质相互作用的总体可行性，从而为更详细的研究提供基础，在此基础上可以研究介导这些关键相互作用的特定因素。公共卫生相关性：上皮-基质的相互作用在正常的角膜功能和调节角膜损伤和屈光手术后的伤口愈合中起着关键作用。这项研究的目标是开发一种新颖的、多层的三维培养模型，该模型可以用于在受控的体外条件下研究这些相互作用。

DESCRIPTION (provided by applicant): Epithelial-stromal interactions are thought to play a critical role in maintaining normal corneal homeostasis, and in regulation of corneal wound healing following injury and refractive surgery. For example, following PRK, injured epithelial cells release several cytokines and growth factors that modulate stromal cell death, proliferation, and differentiation. While the importance of the cross-talk between corneal epithelial and stroma cells is clear, a more detailed investigation of the role of specific cytokines in mediating epithelial-stromal interactions requires an in vitro model in which culture conditions can be tightly regulated. Unfortunately, current in vitro models do not adequately mimic the in vivo 3-D geometry and mechanical properties of corneal tissue. Two of the key barriers to successful tissue engineering of an in vitro anterior corneal construct are: (1) the development of a 3-D extracellular matrix which has high mechanical stiffness, yet supports maintenance of the quiescent stromal cell (keratocyte) phenotype, and (2) the development of a corneal epithelial cell line that differentiates and stratifies in vitro. We have recently performed pilot experiments in which cell-seeded 3-D collagen matrices were compressed to achieve high stiffness tissue equivalents. These constructs support differentiation of corneal keratocytes, and have several unique properties which may be ideal for stromal tissue engineering. We have also developed a unique human telomerase-immortalized corneal epithelial cell line (hTCEpi), which expresses key differentiation markers under stratified, air-lifted culture conditions. The purpose of the proposed research is to test whether we can combine these two unique technologies to produce a novel 3-D model that can be used for studying epithelial-stromal interactions during homeostasis and wound healing. To accomplish this, we propose to: (1) determine whether compressed collagen matrices can be used to support the differentiation and stratification of corneal epithelium under serum-free conditions in vitro, and (2) investigate the feasibility of inducing injuries to these constructs in order to simulate anterior corneal wound healing. Accomplishing these aims would establish the overall feasibility of using compressed collagen matrices as a model system for studying epithelial-stromal interactions during homeostasis and wound healing, and thus provide a foundation for more detailed studies in which the specific factors mediating these critical interactions can be investigated. PUBLIC HEALTH RELEVANCE: Epithelial-stromal interactions play a critical role in normal corneal function and in regulation of corneal wound healing following injury and refractive surgery. The goal of the proposed research is to develop a novel, multi-layered 3-dimensional culture model that can be used to investigate these interactions under controlled, in vitro conditions.