A Novel 3-Dimensional Culture Model of the Anterior Cornea

描述（由申请方提供）：上皮-基质相互作用被认为在维持正常角膜稳态以及调节损伤和屈光手术后的角膜伤口愈合方面发挥关键作用。例如，在炎症后，受损的上皮细胞释放几种细胞因子和生长因子，其调节基质细胞死亡、增殖和分化。虽然角膜上皮细胞和基质细胞之间的串扰的重要性是明确的，更详细的调查，在介导上皮-基质相互作用的特定细胞因子的作用，需要在体外模型中，培养条件可以严格调节。不幸的是，目前的体外模型不能充分模拟角膜组织的体内3-D几何形状和机械特性。体外前角膜构建体的成功组织工程的两个关键障碍是：（1）具有高机械刚度但支持静止基质细胞（角膜细胞）表型的维持的3-D细胞外基质的开发，和（2）体外分化和分层的角膜上皮细胞系的开发。我们最近进行了试点实验，其中细胞接种的3-D胶原蛋白基质被压缩，以实现高刚度的组织等效物。这些构建体支持角膜基质细胞的分化，并且具有几种独特的性质，这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），该细胞系在分层、气升培养条件下表达关键分化标志物。这项研究的目的是测试我们是否可以将这两种独特的技术联合收割机产生一种新的3-D模型，用于研究稳态和伤口愈合过程中上皮-基质的相互作用。为了实现这一点，我们建议：（1）确定压缩的胶原基质是否可以用于支持角膜上皮在体外无血清条件下的分化和分层，以及（2）研究诱导这些结构损伤的可行性，以模拟前角膜伤口愈合。实现这些目标将建立一个整体的可行性，使用压缩的胶原蛋白基质作为模型系统，用于研究在稳态和伤口愈合过程中的上皮-基质的相互作用，从而提供了一个基础，更详细的研究，其中介导这些关键的相互作用的具体因素可以进行调查。公共卫生相关性：上皮-基质相互作用在正常角膜功能和调节损伤和屈光手术后的角膜伤口愈合中起关键作用。拟议研究的目标是开发一种新型的多层三维培养模型，可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.