Optimizing Functional Recellularization of Acellular Human Lung Scaffolds

优化非细胞人肺支架的功能再细胞化

• 批准号: 9250807
• 负责人: DANIEL J WEISS
• 金额: $ 38.13万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9250807
• 关键词: 3-Dimensional; AGTR2 gene; Address; Adult; Alveolar; Biological Assay; Biomedical Engineering; Blood Vessels; Cell Differentiation process; Cell physiology; Cells; Complex; Data; Detergents; Development; Dissection; Distal; Engineering; Environmental air flow; Epithelial; Epithelial Cells; Family suidae; Goals; Growth Factor; Human; Individual; Lung; Maintenance; Mechanical ventilation; Mechanics; Modeling; Perfusion; Periodicity; Play; Pleural; Primates; Protocols documentation; Publishing; Residual state; Rodent; Rodent Diseases; Rodent Model; Role; Scaffolding Protein; Scheme; Stem cells; Stretching; Structure of parenchyma of lung; Supporting Cell; Techniques; Transformed Cell Line; Vascular Endothelial Cell; base; cell growth; cell type; combinatorial; fetal; flexibility; induced pluripotent stem cell; innovation; novel; perfusion imaging; progenitor; public health relevance; scaffold; stem; surfactant production; tool

 DESCRIPTION: Despite exciting recent progress in ex vivo lung bioengineering utilizing decellularized whole lung scaffolds, a number of hurdles remain. These include schemes for functional recellularization with combinations of appropriate cell types, long term maintenance of recellularizing lungs, and incomplete understanding of optimal vascular perfusion and cyclic mechanical stretch influences on proliferation, differentiation, and appropriate function of cells inoculated into the scaffolds. Further, most progress to date has been made in rodent models. This in part reflects the relative difficulties in working with human as compared to rodent lungs including a more limited supply of human lungs and the practical aspects of handling larger more cumbersome lungs. This has hampered progress in assessing the multiple combinatorial conditions that must be evaluated for development of functional human lung tissue. To address these hurdles, we have developed novel and innovative techniques for studying recellularization of acellular human lung scaffolds. These include; a) More optimal detergent- based decellularization protocols; b) Infrared perfusion imaging and sophisticated mass spectrometric assessment of residual scaffold proteins; and c) Novel high throughput approaches for studying decellularized human lungs. This latter technique involves dissection of multiple small 1cm3 segments, each with a cannulatable bronchovascular bundle, from whole decellularized lungs. We have further developed a flexible artificial pleural coating for use with these segments that allows study of ventilation and perfusion of the recellularizing human lung segments. We have also developed a functional assay for surfactant production by assessing changes in lung mechanics in recellularizing scaffolds. Using these techniques, we have made significant advances in recellularizing acellular scaffolds produced from normal and diseased rodent, pig, primate, and human lungs. In particular, we have found significant effects of perfusion and of cyclic mechanical stretch on survival and differentiation, respectively, of pulmonary vascular endothelial cells and of type 2 alveolar epithelial cells in the scaffolds. The goal of the current application is to continue to develop advanced translational applicable schemes for recellularization of human lung scaffolds. Focus will be on combinations of relevant human lung cell types, including endogenous airway progenitor cells and iPS-derived lung epithelial cells (Specific Aim 1), developing appropriate perfusion schemes and perfusates for long term maintenance or recellularizing scaffolds (Specific Aim 2), and developing optimal mechanical ventilation schemes that will maximize influence of cyclic mechanical stretch on development of functional lung tissue (Specific Aim 3). Importantly, we have built an outstanding collaborative team with which to achieve these goals.

 描述：尽管在利用脱细胞全肺支架进行体外肺生物工程方面取得了令人振奋的最新进展，但仍然存在一些障碍。这些包括结合适当细胞类型的功能性再细胞化方案，再细胞化肺的长期维持，以及对接种到支架中的细胞的增殖、分化和适当功能的最佳血管灌流和周期性机械拉伸影响的不完全了解。此外，迄今为止最大的进展是在啮齿动物模型上取得的进展。这在一定程度上反映了与啮齿动物肺相比，与人类合作的相对困难，包括人类肺的供应更有限，以及处理更大、更笨重的肺的实际方面。这阻碍了评估多种组合条件的进展，这些条件必须评估才能发育成有功能的人类肺组织。为了解决这些障碍，我们开发了新的和创新的技术来研究无细胞人肺支架的再细胞化。这些包括：a)更优化的基于洗涤剂的脱细胞方案；b)红外灌注成像和对残留支架蛋白的复杂的质谱学评估；以及c)用于研究脱细胞人肺的新的高通量方法。后一种技术包括从整个脱细胞的肺中解剖多个1cm3的小段，每个段都有一个可插管的支气管血管束。我们进一步开发了一种灵活的人工胸膜涂层，用于这些节段，允许研究再细胞化的人类肺段的通风和灌流。我们还开发了一种通过评估再细胞化支架中肺力学变化来产生表面活性物质的功能分析方法。利用这些技术，我们在对正常和患病的啮齿动物、猪、灵长类动物和人肺产生的无细胞支架进行再细胞处理方面取得了重大进展。特别是，我们发现灌流和周期性机械拉伸分别对支架内肺血管内皮细胞和2型肺泡上皮细胞的存活和分化有显著影响。当前的目标是 应用是继续开发先进的可翻译适用方案，用于人体肺支架的再细胞化。重点将放在相关人类肺细胞类型的组合上，包括内源性气道祖细胞和诱导性肺上皮细胞(特定目标1)，开发用于长期维护或重新细胞化支架的适当灌流方案和灌流液(特定目标2)，以及开发最优机械通风方案，使周期性机械拉伸对功能肺组织的发育产生最大影响(特定目标3)。重要的是，我们已经建立了一支出色的协作团队，与之一起实现这些目标。