DIFFERENTIATED AIRWAY EPITHELIAL CULTURES

I期研究表明，生产高度分化的、无血清的、商业上可行的气道上皮细胞培养模型是现实的。原代正常人支气管上皮细胞（NHBE）在单层培养中扩增并冷冻保存，以提供大量同质来源的正常NHBE细胞。接下来，开发了无血清分化培养基，其诱导NHBE形成分层的分化组织，其表现出体内样的粘膜纤毛表型。所得组织的特征在于形态，超微结构，活力，和粘蛋白的生产。在所有情况下，体内和体外支气管组织之间具有良好的对应性。最后，对生产这种组织所涉及的成本的经济评估表明，这种分化的气道上皮产品似乎是商业上可行的。II期研究将在I期结果的基础上进一步发展三维分化支气管上皮模型，使其成为准确、有用和科学严谨的产品。最初的第二阶段研究将集中在优化分化培养基。培养基优化完成后，将根据纤毛形成的程度、紧密连接的形成、趋化因子/细胞因子分泌、表面粘附分子、对粘蛋白分泌的控制和再现性对组织进行表征。最后，将研究使用该组织作为可能引起职业性哮喘的试剂的筛选，以及使用该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气道上皮组织培养模型。这种模式预计将有广泛的商业应用，以帮助公司确定其挥发性产品是否可能导致不良呼吸道反应。此外，这种体外组织将为研究人员提供研究呼吸道各种疾病状态和发育的有价值的工具。

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.