A novel approach for modelling the healthy nose-brain axis in vitro

一种体外模拟健康鼻脑轴的新方法

• 批准号: NC/X001903/1
• 负责人: Roisin Owens
• 金额: $ 25.72万
• 依托单位: University of Cambridge
• 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%2FX001903%2F1
• 关键词: novel; approach; modelling; healthy; nose

Although the main functions of the human nose are often reduced to delivering air to the lungs and the perception of smell, increasing evidence reveals a complex nose-microbiome interplay related to health and disease. Age and gender are suggested to have the most impact on nose physiology. The nasal cavity predominantly consists of a respiratory barrier that protects from the entry of external substances (e.g. viruses, pollutants) and commensal nasal bacteria that suppress colonization by pathogens. A smaller surface area above the respiratory epithelium is covered by olfactory epithelium including olfactory neurons that are chemosensors and are activated after binding of odorants (e.g. cinnamon) to olfactory receptors (ORs). The complexity of the nasal host-microbiome interactions is even more emphasized considering that olfactory neurons can sense metabolites, small molecules produced by the bacteria. Up to now, mechanisms of the microbiome-nose to brain axis are not well understood, mainly due to a lack of suitable human in vitro models. The commercially available nasal in vitro cell line RPMI2650 is commonly used on 2D Transwell inserts for studying barrier integrity. Although this is a highly valuable tool for high throughput screening, its translation to humans is limited: RPMI2650 cells derive from a different area of the nasal epithelium with much tighter barrier characteristics. The mechanism of smell is often studied in engineered cell lines overexpressing one receptor protein or in animals that express 1,000 - 2,000 olfactory receptors. Up to now, it is not known how humans can perceive 1 trillion odors with only 400 ORs. We are aiming to build an urgently needed, advanced 3D in vitro model of the human nose-brain axis for exploiting the full potential of the healthy human nose. Since this human nose platform will improve predictions on barrier integrity and neuron activity, this will be a next-generation non-animal technology development. We expect that our platform will reduce the number of mice used for studying the nose-brain axis, which is still the gold standard. Our collaborator Dr Rishi Sharma (ENT surgeon, Addenbrooke's Hospital) will provide freshly obtained human microbiome and tissue biopsies (respiratory & olfactory) from sinonasal surgery from patients of different ages and gender. The bioelectronic platform will connect the recently developed e-Transmembrane device for measuring the electrical resistance of barrier models and microelectrode arrays allowing to record the spontaneous firing of neurons. Nasal organoids will be cultured on PEDOT:PSS electroactive scaffolds integrated into the e-Transmembrane device. The scaffold consists of biomimetic polymers, with a tissue-like structure. The devices promote the hosting of complex 3D models compared to rigid 2D counterparts such as the Transwell. In addition, this scaffold compartmentalizes the device into a top and bottom chamber, allowing the study of the uptake of drugs and metabolites into the respiratory and olfactory epithelium. Transferring the flow-through from the bottom chamber onto the whole olfactory tissue fixed on microelectrode arrays allows for the recording of the spontaneous firing of neurons. By identifying the complete set of bacteria and metabolites (metabolome) as well as RNA transcripts (transcriptome), correlation analysis will reveal the structure-activity relationship between the electrical signal and the microbes. CNBio (an organ-on-chip company) and Symrise AG (producer of flavors and fragrances) have already declared their interest in our model.

尽管人类鼻子的主要功能通常被简化为向肺部输送空气和感知气味，但越来越多的证据表明，鼻子-微生物组之间存在着与健康和疾病相关的复杂相互作用。年龄和性别被认为对鼻子的生理影响最大。鼻腔主要由呼吸屏障组成，其保护免受外部物质（例如病毒、污染物）和抑制病原体定殖的鼻腔细菌的进入。呼吸上皮上方较小的表面积被嗅觉上皮覆盖，包括嗅觉神经元，其是化学传感器并且在气味剂（例如肉桂）与嗅觉受体（OR）结合后被激活。考虑到嗅觉神经元可以感知细菌产生的代谢物和小分子，鼻宿主-微生物组相互作用的复杂性更加突出。到目前为止，微生物组-鼻-脑轴的机制还不清楚，主要是由于缺乏合适的人体体外模型。市售鼻体外细胞系RPMI 2650通常用于2D Transwell插入物，以研究屏障完整性。尽管这是高通量筛选的非常有价值的工具，但其对人类的转化是有限的：RPMI 2650细胞来自具有更紧密屏障特征的鼻上皮的不同区域。嗅觉的机制通常在过度表达一种受体蛋白的工程细胞系或表达1,000 - 2,000种嗅觉受体的动物中进行研究。到目前为止，人们还不知道人类是如何用400个OR感知1万亿种气味的。我们的目标是建立一个迫切需要的，先进的三维体外模型的人鼻脑轴，以充分利用健康的人鼻子的潜力。由于这种人类鼻子平台将改善对屏障完整性和神经元活动的预测，这将是下一代非动物技术的发展。我们希望我们的平台将减少用于研究鼻脑轴的小鼠数量，这仍然是黄金标准。我们的合作者Rishi Sharma博士（Addenbrooke's Hospital的耳鼻喉科外科医生）将从不同年龄和性别的患者的鼻窦手术中提供新鲜获得的人类微生物组和组织活检（呼吸和嗅觉）。该生物电子平台将连接最近开发的e-Transmembrane设备，用于测量屏障模型的电阻和微电极阵列，以记录神经元的自发放电。鼻类器官将在集成到e-Transmembrane装置中的PEDOT：PSS电活性支架上培养。该支架由具有组织样结构的仿生聚合物组成。与Transwell等刚性2D对应物相比，这些设备促进了复杂3D模型的托管。此外，这种支架将装置分隔成顶部和底部腔室，允许研究药物和代谢物进入呼吸和嗅觉上皮的摄取。将来自底室的流通液转移到固定在微电极阵列上的整个嗅觉组织上，可以记录神经元的自发放电。通过识别细菌和代谢物（代谢物组）以及RNA转录物（转录物组）的完整集合，相关性分析将揭示电信号与微生物之间的结构-活性关系。CNBio（器官芯片公司）和Symrise AG（香精香料生产商）已经宣布对我们的模型感兴趣。