Epithelial barrier model: in silico modelling and high throughput assessment

上皮屏障模型：计算机建模和高通量评估

• 批准号: NC/X002322/1
• 负责人: Malgorzata Wiench
• 金额: $ 25.78万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FX002322%2F1
• 关键词: Epithelial; barrier; model; silico; modelling

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.

该项目旨在通过提高生产量、可重复性和可信度来增强我们已建立的技术的功能，以生产复杂的分层上皮器官型筏，以取代口腔粘膜和皮肤研究中的动物模型。人类皮肤和口腔粘膜由多层复合上皮保护免受外部环境的影响。这些上皮细胞形成了抵抗磨损、毒素、感染因子、水分流失和紫外线辐射的屏障，因此是维持功能健康组织的关键。当上皮屏障受损或减少时（即在老化，化疗期间或发炎组织中），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）在接近下层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-细胞相互作用，并产生脂质，角蛋白和其他蛋白质，在称为成熟的过程中加强屏障结构。其结果是多层的，强大的和不断补充的组织与严格控制的渗透性。了解健康组织中上皮屏障是如何形成和维持的，对于解决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，其中细胞以单层或球状体生长。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器官型培养物中完全解决。通常，在该3D模型（上皮筏）中，通过将角质形成细胞保持在含成纤维细胞的胶原蛋白（结缔组织等效物）的顶部上的气液界面（ALI）处，促进角质形成细胞形成多层分化组织。首先，对这些模型中上皮屏障的分层和发育的理解还远未完成。在这里，我们将利用一种新的技术（单细胞RNA测序）来模拟上皮分层层，并跟踪口腔上皮组织等同物成熟过程中屏障相关基因的表达。这种新方法将为未来的模型开发提供参考。第二，缺乏可靠的高通量格式阻止了它们在需要大量重复和对照的应用中的使用。高通量形式难以执行，因为ALI维持是消耗劳动力且依赖于操作者的，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细胞培养装置（BECD），其中ALI通过漂浮在培养基顶部的筏自动维持，大大简化了上皮组织的产生，从而降低了成本，提高了重现性和更广泛的采用。在这个项目中，我们打算将BECD发展成一个半高通量系统，并使其适应一系列方法来评估上皮完整性和屏障功能。最后，为了最大限度地减少动物源性试剂的使用，我们将尝试用经修饰的合成混合水凝胶代替胶原蛋白，以复制胶原蛋白凝胶的物理和机械性质。通过推进我们对体外产生的上皮组织的理解，并使其评估具有更高的通量，该项目旨在提高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。