Development of a human 3D co-culture model of the airway blood barrier to investigate cell:cell cross talk

屏障的完整性对于维持组织稳态至关重要。在肺中，上皮细胞形成吸入环境的屏障，内皮细胞形成循环的屏障。这些不同细胞类型之间的细胞串扰是高度协调的，并维持组织的稳态。吸入环境因子对上皮的损伤导致炎症反应，以遏制入侵因子，防止进一步的组织损伤并启动组织修复。严格调节炎症对于预防慢性炎症和纤维化至关重要，慢性炎症和纤维化会影响肺功能和呼吸系统健康。关于细胞串扰维持人体气道屏障稳态的机制知之甚少。这主要是由于缺乏合适的人体外气道模型，以准确地概括复杂的体内情况。为了更详细地了解细胞串扰的机制，对体外三维人体气道组织共培养模型的需求尚未得到满足。为了解决这个问题，南安普顿大学的跨学科研究开发了一种微流体平台，可以长期培养人类气道细胞来创建离体3D组织结构。该技术基于一个微流控平台，该平台再现了模拟体内细胞环境的间质流动。上皮细胞在与广泛接受的Transwell系统相似的材料上培养。这包括一个薄的无孔聚酯支架，具有有限的孔隙率，位于空气-液体表面，以支持组织结构并帮助上皮细胞分化。然而，该系统不允许免疫细胞渗透到组织结构中。该博士项目将通过以下方式改进气道-血液屏障的复杂3D体外模型：(i)开发一种生物相容性膜支持材料，提供更具生理代表性的屏障模型，该模型允许高孔隙度支持比常用支架更高程度的细胞-细胞通信。（二）利用微流体将免疫细胞（中性粒细胞）纳入复杂的3D共培养模型，研究引发炎症的细胞串扰。(i)对于体外上皮屏障的形成，上皮细胞目前生长在细胞外基质包裹的纳米孔膜上，这有助于它们的极化。然而，这些膜是坚硬的，小孔径阻碍了它们用于评估免疫细胞迁移。这些合成膜，以及许多天然和合成的生物材料，都是有限的，因为它们不能模仿机械性能，包括气道粘膜的强度和刚度。该项目将研究一系列新的生物可降解聚合物的适用性，这些聚合物具有高强度和韧性的机械可调性。利用这些可生物降解的聚合物，上皮细胞极化和内皮细胞屏障的形成将被确定，并将研究它们对环境挑战的反应。（二）组织损伤后，中性粒细胞是在数小时内浸润组织的第一批免疫细胞。上皮屏障完整性的丧失可以启动上皮和内皮屏障之间的细胞串扰，以调节炎症起始阶段中性粒细胞流入组织。中性粒细胞浸润的动力学在许多水平上受到调节，特别是通过趋化因子和脂质介质的释放。在我们目前的3D模型中，中性粒细胞不能迁移到组织结构中。博士项目的目标是在3D模型中加入可生物降解的支架，使上皮和内皮屏障形成，并直接免疫细胞浸润。在环境挑战后，将实时监测中性粒细胞的粘附和内流，并确定内皮粘附分子的时间介质释放和表达。

Barrier integrity is crucial for maintaining tissue homeostasis. In the lung, epithelial cells form a barrier to the inhaled environment and endothelial cells to the circulation. Cellular crosstalk between these different cell types is highly coordinated and maintains tissue homeostasis. Damage of the epithelium by inhaled environmental agents results in an inflammatory response in order to contain intruding agents, prevent further tissue damage and initiate tissue repair. Tight regulation of inflammation is essential to prevent chronic inflammation and fibrosis that can affect lung function and respiratory health. There is little knowledge about the mechanisms of cellular crosstalk to maintain barrier homeostasis in the human airway. This is mainly due to a lack of suitable human in vitro models of the airway which recapitulate the complex in vivo situation accurately. There is an unmet need for in vitro 3D tissue co-culture models of the human airway in order to understand the mechanisms of cellular crosstalk in detail. To address this, interdisciplinary research at Southampton has developed a microfluidic platform that enables the long term culture of human airway cells to create an ex-vivo 3D tissue construct. The technology is based on a microfluidic platform which recapitulates the interstitial flow mimicking the in-vivo cellular environment. The epithelial cells are cultured on materials similar to the widely accepted Transwell system. This consists of a thin nonporous polyester scaffold with limited porosity that sits at the air-liquid surface to support the tissue construct and aid epithelial cell differentiation. However, this system does not allow the infiltration of immune cells into the tissue construct. This PhD project will refine this complex 3D in vitro model of the airway-blood barrier by(i)Developing a biocompatible membrane support material that delivers a more physiological representative barrier model which allows for a high porosity supporting a much higher extent of cell-cell communication than in commonly used scaffolds.(ii)Incorporating immune cells (neutrophils) into the complex 3D co-culture model using microfluids to investigate cellular cross talk that initiates inflammation.(i)For in vitro epithelial barrier formation, epithelial cells are currently grown on extracellular matrix coated nanoporous membranes which aids their polarisation. However, these membranes are stiff and the small pore size hinders their use for the assessment of immune cell migration. These synthetic membranes, as well as many natural and synthetic biomaterials, are limited as they cannot mimic the mechanical properties, including strength and stiffness of the airway mucosa. This project will investigate the suitability of a new range of biodegradable polymers which are mechanically tunable with high strength and toughness. Using these biodegradable polymers, epithelial cell polarisation and endothelial cell barrier formation will be determined and their response to environmental challenges will be investigated. (ii)After tissue injury neutrophils are the first immune cells infiltrating the tissue within hours. The loss of epithelial barrier integrity can initiate cellular crosstalk between the epithelial and endothelial barriers to regulate the influx of neutrophils into the tissue during the initiation phase of inflammation. The kinetics of neutrophil infiltration is regulated at many levels, in particular through the release of chemokines and lipid mediators. In our current 3D model, neutrophils cannot transmigrate into the tissue construct. The PhD project aims to incorporate biodegradable scaffold that allows the formation of epithelial and endothelial barriers and direct immune cell infiltration the 3D model. Following environmental challenge, the adhesion and influx of neutrophils will be monitored in real time, temporal mediator release and expression of endothelial adhesion molecules will be determined.