Engineered salivary gland tissue chips

翻译后摘要：对于超过550，000例患者，每年诊断为头颈部癌症，严重 唾液腺功能的丧失（口干症）是放射治疗的不可避免的结果。有 目前还没有可靠和安全的药物治疗方法来解决或预防辐射诱导的 口干症。努力研究辐射敏感性，以发现有效的辐射防护和再生策略 由于分泌腺泡的丧失， 细胞表型该计划的主要里程碑是设计功能性人类唾液腺组织 芯片来克服这个障碍。我们的实验室率先使用水凝胶封装培养唾液 体外培养腺细胞。我们已经成功地证明了唾液腺细胞存活长达1个月后， 封装此外，细胞组织成具有顶基极性的结构，并表达分泌性 腺泡标志物，包括Mist1。虽然这些数据是有希望的，但分泌标志物表达减少 与天然腺体相比。此外，水凝胶的宏观性质排除了其高- 吞吐量使用。因此，我们将利用我们的微泡（MB）阵列技术作为一种高通量，模块化 用于组织芯片的平台。MB是在聚二甲基硅氧烷（PDMS）中模制的微米级球形腔。 MB具有与腺体的分泌腺泡单位相似的长度尺度和曲率的优点， 提供促进细胞-细胞接触的小生境，并提供促进细胞-细胞接触的自分泌和旁分泌因子的浓度， 已经显示出增强组织组装。此外，MB可以与其它模块集成。 微生理系统，如内皮、神经和免疫系统芯片。在UG3阶段， 在本项目中，通过/不通过标准将是使用MB平台开发人类腺体组织模拟物 具有长期分泌功能。特别是对于UG3，Aim 1将使用基因标记的小鼠腺泡 和导管细胞来鉴定使腺组织模拟功能最大化的培养特征。腺泡和导管 细胞接种比例和密度将在"空白"、细胞外基质蛋白官能化的和 水凝胶都在MB内。目的2（UG3）将验证人唾液腺细胞的细胞分化能力， 类似地，在我们先前开发的大凝胶和MB中的水凝胶中组织和维持功能， 在Aim 1中的小鼠细胞。我们的目标是在MB阵列中展示功能性人类腺体模拟物的发育 在UG3结束时。如果成功，UH3阶段将研究水凝胶微环境线索，以进一步 促进腺体拟态组织和功能。最后，目标3将证明腺体模拟物的效用 通过筛选FDA批准的药物来识别有效的辐射防护剂。这些化合物将 导管后注射到小鼠中以验证辐射防护潜力。唾液组织发育成功 芯片将具有变革性;通过对功能性腺体模拟物进行体外分析，我们能够追求 放射防护和再生的治疗策略将得到显著改进。

Abstract: For more than 550,000 patients annually diagnosed with head and neck cancers worldwide, severe loss of salivary gland function (xerostomia) is an unavoidable outcome of radiation therapy. There are presently no reliable and safe pharmacologic treatments for the resolution or prevention of radiation-induced xerostomia. Efforts to study radiosensitivity to discover effective radioprotective and regenerative strategies have been hampered by the inability to culture salivary gland mimetics in vitro, due to loss of secretory acinar cell phenotype. The principal milestone of this proposal is to engineer functional human salivary gland tissue chips to overcome this obstacle. Our labs have pioneered the use of hydrogel encapsulation to culture salivary gland cells in vitro. We have successfully demonstrated salivary gland cell survival up to 1 month post- encapsulation. Furthermore, cells organize into structures with apicobasal polarity and express secretory acinar markers, including Mist1. Although these data are promising, secretory marker expression is reduced compared to the native gland. Furthermore, the macroscale nature of hydrogels precludes their high- throughput use. Thus, we will utilize our microbubble (MB) array technology as a high-throughput, modular platform for the tissue chips. MBs are micron-scale spherical cavities molded in polydimethylsiloxane (PDMS). MBs have the advantage of length scales and curvatures similar to the secretory acinar unit of glands, providing a niche that promotes cell-cell contact and the concentration autocrine and paracrine factors that have been shown to enhance tissue assembly. Furthermore, MBs can be integrated with other microphysiological systems such as endothelial, nerve, and immune system chips. During the UG3 phase of this project, the go/no-go criteria will be the use of the MB platform to develop human gland tissue mimetics capable of long-term secretory function. Specifically for UG3, Aim 1 will use genetically labeled mouse acinar and duct cells to identify culture characteristics that maximize gland tissue mimetic function. Acinar and duct cell seeding ratios and densities will be varied in ‘blank’, extracellular matrix protein-functionalized, and in hydrogels all within MBs. Aim 2 (UG3) will validate the ability of human salivary gland cells to cellularly organize and maintain function in our previously developed macrogels and in within hydrogels in MBs, similarly to mouse cells in Aim 1. Our goal is to demonstrate functional human gland mimetic development in MB arrays by end of UG3. If successful, the UH3 phase will investigate hydrogel microenvironmental cues to further promote gland mimetic organization and function. Finally, Aim 3 will demonstrate the utility of gland mimetics by screening FDA-approved drugs to identify effective radioprotective agents. These compounds will be retroductally injected into mice to validate radioprotective potential. Successful development of salivary tissue chips will be transformative; by enabling in vitro analysis of functional gland mimetics, our ability to pursue therapeutic strategies, radioprotective and regenerative, will be dramatically improved.