Engineered In Vitro Cochlea for Drug Discovery and Regeneration Studies

描述（由申请人提供）：我们的目标是使用工程支架上的内耳祖细胞创建仿生耳蜗。我们的目标是复制内耳感觉上皮细胞在体外的药物筛选和再生研究中使用的方面。这种新的工程方法将利用模拟基底膜细胞外基质的天然微环境的机械和化学线索的基板。除了重述原生环境的关键要素，我们将设计一种微流体装置，提供精确的空间和剂量控制的启动子化合物的交付，以指导祖细胞分化成内耳上皮细胞，导致毛细胞和支持细胞的方向在模仿基底膜上的细胞行的模式。这些仿生耳蜗将是一个有用的工具，研究再生和药物筛选。该设备将加速药物发现，并促进改善干细胞在听力损失治疗中的使用。据估计，超过10%的美国人口患有听力损失，随着人口老龄化，这一数字将继续上升。虽然在理解听力损失的细胞机制方面取得了重大进展，许多学术和商业研究人员正在努力开发治疗方法，但治疗方法尚未转化为临床。缺乏稳定的，良好控制的体外模型，我们的目标是开发一个组织工程测试平台，以加速药物发现和再生研究。为了测试和验证我们的方法，我们将优化工程支架，以促进内耳祖细胞的附着，粘附和分化。微流体递送系统将使细胞暴露于LY411575的交替区域，LY411575是一种Notch抑制剂，显示出诱导分化成毛细胞，可溶性锯齿状1是一种支持细胞诱导剂。我们选择这些作为示例性化合物是因为它们已被证实的反应。我们将开发一个用于体外药物测试的细胞平台，我们预计该工具在生理学，病理学和再生研究中的许多未来用途。该工程系统将能够进行多种支架改变，以适应其他细胞类型的偏好，并能够使用新型微流体方法以前所未有的空间，时间和剂量控制递送广泛的化合物。内耳祖细胞是在治疗听力损失方面显示出希望的靶点，但难以以受控方式培养和分化，使其成为这种方法益处的理想证明。我们预计我们的平台将被研究人员用于更好地了解和治疗人类听力损失的原因。因此，拟议的工作重点是小鼠祖细胞，但直接应用于治疗人类未满足的临床需求。我们的方法是独特的，因为它结合了一个专门优化的工程支架和微流体输送系统与耳蜗细胞培养和操纵，这是以前从未实现过的。我们的系统的一个重要方面是，工程方法已经在其他组织类型中得到了很好的研究，并且在听觉领域的应用中具有理想的可调性和控制性。工程开发将分阶段进行，以允许对支架和药物输送进行平行的细胞研究，提供持续的反馈以指导器械开发。我们将通过各种生化和生理结果评估仿生耳蜗，并探索其作为药物发现和再生研究的生物工具的用途。

DESCRIPTION (provided by applicant): Our objective is to create a biomimetic cochlea using inner ear progenitor cells on an engineered scaffold. We aim to replicate aspects of the inner ear sensory epithelium in vitro for use in drug screening and regeneration studies. This novel engineered approach will utilize substrates with mechanical and chemical cues that mimic the natural microenvironment of the basilar membrane extracellular matrix. In addition to recapitulating critical elements of the native environment, we will design a microfluidic device providing precise spatial and dose control over delivery of promoter compounds to direct the differentiation of progenitor cells into inner ear epithelial cells, resulting in hair cells and supporting cells oriented in a pattern mimicking the rows of cells on the basilar membrane. These biomimetic cochleae will be useful as a tool to study regeneration and for drug screening. This device will accelerate drug discovery and facilitate the improved use of stem cells in therapies for hearing loss. Estimates of over 10% of the U.S. population suffer from hearing loss, and this number will continue to rise with an aging population. Although significant strides have been made in understanding the cellular mechanisms of hearing loss and numerous academic and commercial researchers are working to develop treatments, therapies have not translated to the clinic. Stable, well-controlled in vitro models are lacking, and we aim to develo a tissue engineered test platform to accelerate drug discovery and regeneration studies. For testing and validation of our approach, we will optimize an engineered scaffold to encourage attachment, adhesion and differentiation of inner ear progenitor cells. A microfluidic delivery system will expose the cells to alternating regions of LY411575, a Notch inhibitor shown to induce differentiation into hair cells, and soluble jagged1, a supporting cell inducer. We have selected these as exemplary compounds due to their proven response. We will develop a cellular platform for in vitro drug testing, and we anticipate many future uses for the tool for physiological, pathological and regenerative studies. The engineered system will be capable of numerous scaffold alterations to accommodate the preferences of other cell types and enable the delivery of a broad range of compounds with unprecedented spatial, temporal, and dose control using a novel microfluidic approach. Inner ear progenitor cells are targets that have shown promise in treating hearing loss but are difficult to culture and differentiate in a controlld manner, making them the ideal demonstration of the benefits of this approach. We foresee our platform being used by researchers to better understand and treat the causes of hearing loss in humans. Thus the proposed work focuses on mouse progenitor cells, yet has direct application to the treatment of an unmet clinical need in humans. Our approach is unique in that it combines a specifically optimized engineered scaffold and a microfluidic delivery system with cochlear cell culture and manipulation that has never before been realized. An important aspect of our system is that the engineering approaches have been well-studied in other tissue types and are ideal in their tunability and control for application in the auditory field. The engineering development will be staged to allow parallel cellular studies of scaffolds and drug delivery, providing continuous feedback to guide device development. We will evaluate the biomimetic cochleae through various biochemical and physiological outcomes and explore its use as a biological tool for drug discovery and regenerative studies.