DIFFERENTIATED AIRWAY EPITHELIAL CULTURES

第一阶段研究表明，生产一种高度分化的、无血清的、具有商业可行性的呼吸道上皮细胞培养模型是可行的。原代正常人支气管上皮细胞(NHBE)在单层培养中扩增，并进行冷冻保存，以提供大量的正常NHBE细胞。接下来，开发了一种无血清分化介质，可以诱导NHBE形成分层的分化组织，该组织表现出类似活体的粘液纤毛表型。从形态、超微结构、活性和粘蛋白产生等方面对所形成的组织进行了表征。在所有病例中，体内和体外的支气管组织之间有很好的一致性。最后，对生产这种组织所涉及的成本进行的经济评估表明，这种分化的呼吸道上皮产品似乎在商业上是可行的。第二阶段研究将在第一阶段结果的基础上扩大，以进一步将三维、分化的支气管上皮模型开发成准确、有用和科学严谨的产品。初步的第二阶段研究将集中在优化分化培养基上。在培养基优化完成后，将从纤毛形成的程度、紧密连接的形成、趋化因子/细胞因子的分泌、表面黏附分子、粘液分泌的控制和重复性等方面对组织进行表征。最后，将研究利用组织作为筛选可能导致职业性哮喘的因素，以及利用组织作为研究支气管细菌感染的模型。拟议的商业应用：拟议的研究将产生一个非常有用的呼吸道上皮组织培养模型。这样的模型有望在帮助公司确定其挥发性产品是否会导致不良呼吸道反应方面具有广泛的商业应用。此外，这种体外组织将为研究人员研究呼吸道的各种疾病状态和发育过程提供宝贵的工具。

Phase I research demonstrated that the production of a highly differentiated, serum-free, commercially viable cell culture model of the airway epithelium is realistic. Primary, normal human bronchial epithelial cells (NHBE) were expanded in monolayer culture and cryopreserved to provide a large homogenous source of normal NHBE cells. Next, a serum-free differentiation medium was developed which induced the NHBE to form a stratified, differentiated tissue, which exhibited an in vivo-like, mucociliary phenotype. The resultant tissue was characterized in terms of morphology, ultrastructure, viability, and mucin production. In all cases, there was good correspondence between the in vivo and in vitro bronchial tissue. Finally, an economic assessment of the costs involved in producing such tissues showed that such a differentiated airway epithelial product appears to be commercially feasible. Phase II studies will expand on the Phase I results to further develop the three-dimensional, differentiated bronchial epithelial model into an accurate, useful, and scientifically rigorous product. Initial Phase II research will focus on optimizing the differentiation medium. After medium optimization is complete, the tissue will be characterized in terms of the extent of cilia formation, the formation of tight junctions, chemokine/cytokine secretion, surface adhesion molecules, control over mucin secretion, and reproducibility. Finally, the use of the tissue as a screen for agents which are likely to cause occupational asthma, and the utilization of the tissue as a model to study bacterial infection of the bronchia, will be investigated. PROPOSED COMMERCIAL APPLICATION: The proposed research will produce a highly useful tissue culture model of the airway epithelium. Such a model is anticipated to have broad commercial application in assisting companies determine whether their volatile products may cause adverse respiratory reactions. In addition, such an in vitro tissue will provide researchers with a valuable tool in studying various disease states of the respiratory tract and in developing.