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

尽管人类鼻子的主要功能往往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，鼻子与微生物群之间存在着与健康和疾病相关的复杂相互作用。年龄和性别对鼻子的生理机能影响最大。鼻腔主要由呼吸屏障组成，它保护鼻腔不受外界物质（如病毒、污染物）的侵入，并抑制病原体定植的共生鼻腔细菌。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，这些嗅觉神经元是化学传感器，在气味剂（如肉桂）与嗅觉受体（ORs）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，鼻腔宿主与微生物相互作用的复杂性就更加突出了。到目前为止，由于缺乏合适的人体体外模型，微生物群-鼻-脑轴的机制尚不清楚。市售的鼻腔体外细胞系RPMI2650通常用于2D Transwell插入物，用于研究屏障完整性。虽然这是一种非常有价值的高通量筛选工具，但其对人类的翻译是有限的：RPMI2650细胞来自鼻腔上皮的不同区域，具有更紧密的屏障特征。嗅觉的机制通常在过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，我们还不知道人类是如何只用400个ORs就能感知1万亿种气味的。我们的目标是建立一个迫切需要的，先进的人类鼻子-脑轴的3D体外模型，以开发健康人类鼻子的全部潜力。由于这个人类鼻子平台将提高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能够减少用于研究鼻脑轴的小鼠数量，这仍然是黄金标准。我们的合作伙伴Rishi Sharma博士（阿登布鲁克医院耳鼻喉外科医生）将提供来自不同年龄和性别患者的鼻鼻部手术中获得的新鲜人体微生物组和组织活检（呼吸和嗅觉）。生物电子平台将连接最近开发的用于测量屏障模型电阻的e-跨膜装置和允许记录神经元自发放电的微电极阵列。鼻类器官将在整合到e-跨膜装置中的PEDOT:PSS电活性支架上培养。支架由仿生聚合物组成，具有组织样结构。与Transwell等刚性2D设备相比，这些设备促进了复杂3D模型的托管。此外，这种支架将装置划分为上下两个腔室，允许研究药物和代谢物进入呼吸和嗅觉上皮的吸收。将底部腔室的气流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。通过鉴定细菌及其代谢产物（代谢组）和RNA转录物（转录组）的完整集合，进行相关分析，揭示电信号与微生物之间的构效关系。CNBio（一家器官芯片公司）和Symrise AG（香料生产商）已经宣布对我们的模型感兴趣。

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.