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

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.

Although the human nose plays a crucial role in human health, its mechanisms along the nose-brain axis are still not fully understood. The nasal epithelial barrier is formed by tight junction proteins, regulating the entry of external substances into the human body. In addition, the olfactory epithelium on the top of the nasal cavity enables a direct and almost non-invasive route to the brain, particularly interesting for the treatment of central nervous system disease. Commensal nasal microbes are assumed to induce health-promoting effects as the gut or skin microbiome does. Both, barrier integrity and microbiome change with age, making the elderly often more susceptible to infectious diseases like Sars-CoV-2. Our limited understanding of nose physiology is mainly traced back to currently available over-simplified 2D in vitro models as well as animal in vivo studies, both limited in replicating the complex human environment. Thus, we are proposing to build an advanced bioelectronic 3D model of the human nose-brain axis urgently needed for reducing the number of in vivo studies. This newly developed next-generation non-animal technology will connect the recently developed e-Transmembrane device hosting a nasal organoid and Multielectrode Arrays with affixed olfactory epithelium. The e-Transmembrane device consists of an electroactive conducting polymer scaffold as a highly biocompatible soft and tissue-like structure which promotes the hosting of complex 3D models, in contrast to rigid materials currently used to host 2D models. Integration of bioelectronic sensors enables non-invasive monitoring of nasal host-microbiome interactions by measuring epithelial barrier integrity and olfactory neuron firing in real-time. Moreover, sampling of age and gender-dependent nasal microbiomes as well as tissue from sinonasal surgery (Addenbrooke's Hospital), allows the development of new preventive strategies (e.g. nasal probiotics) for maintaining a person's overall health.