DIFFERENTIATED AIRWAY EPITHELIAL CULTURES

I期研究表明，生产高度分化，无血清，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，以提供大量均匀的正常NHBE细胞来源。接下来，开发了一种无血清分化培养基，诱导NHBE形成分层、分化的组织，该组织表现出体内样粘膜纤毛表型。所得组织在形态、超微结构、活力和粘蛋白产生方面进行了表征。在所有病例中，体内和体外支气管组织之间有良好的对应关系。最后，对生产这种组织的成本进行的经济评估表明，这种分化的气道上皮产品在商业上是可行的。II期研究将在I期研究结果的基础上进一步发展三维、分化支气管上皮模型，使之成为一种准确、有用、科学严谨的产品。初期II期研究将集中于分化培养基的优化。培养基优化完成后，将从纤毛的形成程度、紧密连接的形成、趋化因子/细胞因子的分泌、表面粘附分子、对粘蛋白分泌的控制以及可重复性等方面对组织进行表征。最后，将研究使用该组织作为筛选可能引起职业性哮喘的药物，并利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。预计这种模型在协助公司确定其挥发性产品是否可能引起不良呼吸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呼吸道的各种疾病状态和发展提供有价值的工具。

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.