Bioengineering 3D airway tubes to study epithelial morphogenesis

我的长期研究计划旨在阐明推动人类肺发育的基本细胞和分子机制。人类胚胎肺的发育在很大程度上仍不清楚，特别是肺内气流对上皮细胞行为的压力，这让人想起出生后生命的早期阶段。模型生物和当前的体外细胞模型不能用于研究驱动人类肺细胞命运决定的基本机制，因为存在着遗传、细胞和/或超微结构的复杂性，这些复杂性在任何当前的系统中都没有概括。重要的是，产生具有细胞、细胞外基质和生物力学力量的管状结构来模拟呼吸道还没有实现。以前开发“呼吸道管”的尝试使用的是不能自组织和/或多细胞分化的细胞。因此，在发育过程中，细胞在3D呼吸道管道中形成时，驱动细胞行为的机制仍不清楚。在这项建议中，我们的目标是开发一种新型的3D模拟气道，具有适当的细胞外基质(ECM)和生理气流，以研究在生理相关压力(如气流)下上皮细胞的命运决定和模式。我们推测，3D模拟呼吸道将诱导上皮形态发生，其空间分布、细胞组成和功能与天然组织相似。目的1.使用已定义的生物细胞外基质优化3D试管生物打印。我们将首先确定支持上皮细胞存活、扩张和极化所需的适当的底物形态和细胞外基质。目的2.优化微流控装置中双向气流灌流，以维持上皮细胞的完整性。在我们预制的微流控装置中，我们将确定暴露于定义的双向管腔气流中的人支气管上皮细胞的活性。气流对上皮形态发生(分化和空间组织)的影响将进行细胞活性、上皮完整性测试，并评估与天然组织类似的形态和离子转运功能。我们还将与静止的充满液体的管子进行比较，这让人联想到宫内的肺。目的3.探讨生理性气流作用下的上皮形态发生。在这里，我们将利用多细胞分化的多潜能干细胞来源的基础干细胞。我们将通过实时成像来监测上皮细胞的形态发生，并确定这些衍生物的细胞分布和细胞命运。纤毛跳动，一种衡量功能活动和纤毛细胞分化的指标，是近端呼吸道的一个关键标志，将在3D管中使用荧光珠跟踪进行评估。意义重大。我们将利用最先进的生物打印技术和干细胞工程创造第一个人性化的可灌流3D呼吸道模拟物，以研究驱动人类呼吸道发育的基本机制。

My long-term research program aims to elucidate the fundamental cellular and molecular mechanisms driving human lung development. Human fetal lung development remains largely unknown especially the pressures of air flow in the lungs on epithelial cell behaviour, reminiscent of the early period of post-natal life. Model organisms and current in-vitro cell models cannot be used to study fundamental mechanisms driving human lung cell fate decisions as there are genetic, cellular and/or ultrastructural complexities that are not recapitulated in any of the current systems. Importantly, generating tubular structures to mimic the airways with cellular, extracellular matrix, and biomechanical forces has not been achieved. Previous attempts at developing `airway tubes' have used cells incapable of self-organization and/or multicellular differentiation. Therefore, the mechanisms driving cellular behaviour as they form in 3D airway conduits during development remain unclear. In this proposal, we aim to develop a novel 3D airway mimetic with proper extracellular matrices (ECM) and physiological airflow to study epithelial cell fate decisions and patterning under physiologically relevant stresses such as air flow. We hypothesize that the 3D airway mimetic will induce epithelial morphogenesis with spatial distribution, cellular composition and function similar to native tissues. Aim 1. To optimize 3D tube bioprinting using defined biological extracellular matrices. We will first determine the appropriate substratum topography and ECM that is needed to support epithelial cell viability, expansion and polarization. Aim 2. To optimize bidirectional air flow perfusion in a microfluidic device that will sustain integrity of the epithelium. We will determine the viability of human bronchial epithelial cells in the tubes exposed to defined bidirectional lumenal air flow in our prefabricated microfluidic device. The influence of airflow on epithelial morphogenesis (differentiation and spatial organization) will be tested for cellular viability, epithelial integrity and assessed for morphological and ion transport function similar to native tissue. We will also compare to static fluid-filled tubes reminiscent of lungs in-utero. Aim 3. To investigate epithelial morphogenesis under physiological airflow. Here, we will utilize pluripotent stem cell-derived basal stem cells capable of multicellular differentiation. We will monitor epithelial morphogenesis by live imaging and determine the cellular distribution and cell fate of the derivatives. Cilia beating, a measure of functional activity and ciliated cell differentiation, a key hallmark of the proximal airways, will be assessed in the 3D tubes using fluorescent bead tracking. Significance. We will create the first humanized perfusable 3D airway mimetic using state-of-the art bioprinting technology and stem cell engineering to study fundamental mechanisms driving human airway development.