A Novel 3-Dimensional Culture Model of the Anterior Cornea

描述（由申请人提供）：上皮-基质相互作用被认为在维持正常角膜稳态和调节角膜损伤和屈光手术后伤口愈合中起关键作用。例如，在PRK之后，受伤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间质细胞之间的串扰的重要性是明确的，但要更详细地研究特定细胞因子在介导上皮-间质相互作用中的作用，需要一个可以严格调节培养条件的体外模型。不幸的是，目前的体外模型不能充分模拟体内角膜组织的三维几何形状和力学特性。成功组织工程体外前角膜构建的两个关键障碍是：(1)具有高机械刚度的3-D细胞外基质的发展，但支持维持静止的基质细胞（角质细胞）表型；(2)角膜上皮细胞系的发展，在体外分化和分层。我们最近进行了试点实验，其中细胞种子的3-D胶原蛋白基质被压缩以获得高刚度组织当量。这些结构支持角膜角质细胞的分化，并有几个独特的性质，可能是理想的间质组织工程。我们还开发了一种独特的人类端粒酶永生化角膜上皮细胞系（hTCEpi），该细胞系在分层、空气培养条件下表达关键的分化标记。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结合起来，产生一种新的3d模型，用于研究体内平衡和伤口愈合过程中上皮-基质的相互作用。为了实现这一目标，我们建议：(1)确定压缩胶原基质是否可以在体外无血清条件下用于支持角膜上皮的分化和分层，(2)研究对这些结构进行诱导损伤以模拟角膜前创面愈合的可行性。完成这些目标将建立使用压缩胶原基质作为模型系统来研究体内平衡和伤口愈合过程中上皮-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，其中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-基质相互作用在正常角膜功能和损伤和屈光手术后角膜伤口愈合的调节中起关键作用。提出的研究目标是开发一种新的，多层的三维培养模型，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.