Epithelial barrier model: in silico modelling and high throughput assessment

该项目旨在通过提高通量、可重复性和信心来增强我们现有技术的功能，以生产复杂的分层上皮器官型筏，以取代口腔黏膜和皮肤研究中的动物模型。人体皮肤和口腔黏膜受多层复合体上皮的保护，免受外界环境的影响。这些上皮形成抵抗磨损、毒素、感染因子、水分流失和紫外线辐射的屏障，因此是维持组织功能健康的关键。当上皮屏障受损或减少时（即在衰老，化疗期间或炎症组织中），这可能导致感染，癌症或自身免疫性疾病。上皮细胞（也称为角质形成细胞）在靠近结缔组织的层内繁殖，然后向组织表面迁移。在这种迁移过程中，角质形成细胞分化，增加细胞间相互作用和脂质，角蛋白和其他蛋白质的产生，在称为成熟的过程中加强屏障结构。其结果是多层，坚固和不断补充的组织与严格控制的渗透性。了解上皮屏障如何在健康组织中形成和维持，对于确定疾病状态和治疗非常重要。这种知识不能通过使用标准的细胞培养方法来提高，在这种方法中，细胞在单层中或作为球体生长。上皮生物学的关键方面，包括分化、信号传导、屏障功能、基因和蛋白质表达、感染和伤口愈合，只能在动物模型或体外复制分层上皮组织的3D器官型培养中得到充分解决。通常，在这个3D模型（上皮筏）中，角化细胞被鼓励形成多层分化组织，通过将它们维持在含有成纤维细胞的胶原蛋白（结缔组织当量）上的气液界面（ALI）。上皮器官型筏培养目前有两个方面阻碍了其更广泛的应用，以及限制了动物模型的使用。首先，对这些模型中上皮屏障的分层和发育的理解还远远不够。在这里，我们将利用一种新技术（单细胞rna测序）来逐层模拟上皮分层，并在口腔上皮等效组织成熟过程中跟踪屏障相关基因的表达。这种新颖的方法将为未来模型的发展提供参考。其次，缺乏可靠的高吞吐量格式阻碍了它们在需要大量复制和控制的应用程序中的使用。高通量的格式很难执行，因为ALI的维护是耗费人力的，并且依赖于操作人员，ALI的任何变化都会导致不一致的生长和分层。为了解决这些问题，我们最近开发并测试了一种浮力上皮培养装置（BECD），其中ALI通过漂浮在培养基顶部的筏体自动维持，大大简化了上皮组织的生成，从而降低了成本，提高了可重复性，并得到了更广泛的应用。在这个项目中，我们打算将becd发展成半高通量系统，并使其适应一系列评估上皮完整性和屏障功能的方法。最后，为了尽量减少动物源性试剂的使用，我们将尝试用合成的杂交水凝胶来替代胶原蛋白，这些水凝胶经过修饰，可以复制胶原蛋白凝胶的物理和机械性能。通过提高我们对体外产生的上皮组织的理解，并提高其评估的吞吐量，该项目旨在增加在基础和转化研究中使用这些模型的信心，使其有利于动物模型。

The project aims to enhance the functionality of our established technology to produce complex stratified epithelial organotypic rafts by increasing throughput, reproducibility and confidence to replace animal models in oral mucosa and skin research.Human skin and oral mucosa are protected from the external environment by multi-layered complex epithelia. These epithelia form a barrier against abrasion, toxins, infectious agents, water loss and UV radiation, and as such are key for maintaining functional healthy tissues. When epithelial barrier is compromised or decreased (i.e. in ageing, during chemotherapy, or in inflamed tissues) this can lead to infections, cancer or autoimmune conditions. Epithelial cells (also known as keratinocytes) multiply within a layer close to underlying connective tissue and then migrate towards the tissue surface. During this migration keratinocytes differentiate, increase cell-cell interactions and production of lipids, keratins and other proteins reinforcing the barrier structure in the process called maturation. The result is multi-layered, strong and constantly replenished tissue with tightly controlled permeability. Understanding how the epithelial barrier is formed and maintained in healthy tissues is very important for addressing disease states and treatments. This knowledge cannot be advanced by using standard cell culture methods in which cells are grown in a single layer or as spheroids. Key aspects of epithelial biology including differentiation, signalling, barrier function, gene and protein expression, infections and wound healing can only be fully addressed in either animal models or 3D organotypic cultures that replicate stratified epithelial tissue in vitro. Typically, in this 3D models (epithelial rafts) keratinocytes are encouraged to form multi-layered differentiating tissue by maintaining them at air-liquid interface (ALI) on top of fibroblast-containing collagen (connective tissue equivalent).Two aspects of epithelial organotypic rafts cultures currently prevent their wider application as well as ability to limit the use of animal models. Firstly, understanding of stratification and development of epithelial barrier in these models is far from complete. Here we will take advantage of a novel technology (single cell RNA-sequencing) to model epithelial stratification layer by layer and track barrier-related gene expression during maturation in tissue equivalents of oral epithelium. This novel approach will form a reference for future model developments. Secondly, the lack of reliable high throughput formats prevents their use in applications requiring large numbers of replicates and controls. The high-throughput formats are difficult to execute as ALI maintenance is labour-consuming and operator-dependent, with any variation in ALI leading to inconsistent growth and stratification. To address these issues, we recently developed and tested a Buoyant Epithelial Culture Device (BECD) in which ALI is maintained automatically by the raft floating on top of media, considerably simplifying epithelial tissue generation thus reducing costs and improving reproducibility and wider adoption. In this project, we intend to evolve the BECDs towards a semi-high throughput system and adapt it to an array of methodologies to assess epithelial integrity and barrier function. Finally, to minimize the use of animal-derived reagents, we will attempt to replace collagen with synthetic hybrid hydrogels modified to replicate physical and mechanical properties of collagen gels. Through advancing our understanding of in vitro produced epithelial tissues and enabling higher throughput for their assessment, the project aims to increase confidence in using these models in basic and translational research, putting it in favour of animal models.