Periodic Reporting for period 1 - Micro-SENSE (A novel approach for modelling the human nose-brain axis in vitro)
Período documentado: 2024-01-01 hasta 2025-12-31
Context: Although mostly underestimated, the human nose significantly contributes to maintaining human health. The respiratory barrier, formed by tight junctions (zonula occludens-1, occludin), harbours the nasal microbiome. It serves as a primary gatekeeper, primarily controlling systemic absorption of external molecules into the human body. In the upper nasal cavity, the neuronal epithelium contains olfactory nerves for smell perception and the facial trigeminal nerve, which transmits tactile, noxious and thermal signals. In addition, these neurons provide a direct route to the brain, making the nose an attractive pathway for non-invasive drug delivery, while changes in smell perception have been suggested as an early indicator of neurodegenerative diseases. Gender differences in olfactory performance, together with variations in nasal microbiome activity, further complicate human nasal physiology.
Motivation: Up to now, the full potential of the human nose-brain axis for diagnosis and disease prevention has not been fully exploited due to an incomplete understanding of its mechanisms. Needs: The current use of nasal cell lines in combination with stiff, rigid materials limits the translation of in vitro findings to in vivo outcomes. Moreover, conventional cell-based assays are mainly invasive and endpoint, further challenging the understanding of in vitro outcomes.
Thus, the overall objectives of the Micro-SENSE project have focused on establishing a novel nose-on-chip platform that bridges conventional 2D in vitro nose models and healthy human nasal physiology of men and women. For this, electrical readouts were combined with cell-based assays to investigate a patient-derived in vitro nose model:
I. Respiratory nasal barriers from patient-derived cells were established on conventional cell culture inserts and novel customised tissue-like Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) scaffolds. This material is electroconductive enabling the non-invasive electrical monitoring in real-time of impedance as a measure of epithelial barrier integrity. Moreover, the material’s porosity for nutrient and oxygen exchange, together with its soft, tissue-like properties, promotes the establishment of complex in vitro models.
II. Nasal neuroepithelium from patients coupled to microelectrode arrays aimed to study neuronal transport to the brain. Action potentials serve as functional electrical outcome measures to monitor cellular responses triggered by molecule absorption.
III. Integration of the nasal microbiome into bioelectronic in vitro nose model to improve physiological translation to humans.
On an individual level, the project impacts the fellow’s scientific independence on using patient samples to conduct studies using advanced human in vitro models. This was shown by presenting data at national and international conferences and their current preparation for a peer-reviewed manuscript as a shared first author. This impact is substantial to the fellow as she aims to be an independent group leader in studying sensorial sciences with advanced human in vitro models. At the societal level, using patient-derived material from men and women, consideration of the microbiome, and integration of novel technologies, the project is expected to optimise pre-clinical intranasal drug development and guide the selection of candidates for human testing.
Motivation: Up to now, the full potential of the human nose-brain axis for diagnosis and disease prevention has not been fully exploited due to an incomplete understanding of its mechanisms. Needs: The current use of nasal cell lines in combination with stiff, rigid materials limits the translation of in vitro findings to in vivo outcomes. Moreover, conventional cell-based assays are mainly invasive and endpoint, further challenging the understanding of in vitro outcomes.
Thus, the overall objectives of the Micro-SENSE project have focused on establishing a novel nose-on-chip platform that bridges conventional 2D in vitro nose models and healthy human nasal physiology of men and women. For this, electrical readouts were combined with cell-based assays to investigate a patient-derived in vitro nose model:
I. Respiratory nasal barriers from patient-derived cells were established on conventional cell culture inserts and novel customised tissue-like Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) scaffolds. This material is electroconductive enabling the non-invasive electrical monitoring in real-time of impedance as a measure of epithelial barrier integrity. Moreover, the material’s porosity for nutrient and oxygen exchange, together with its soft, tissue-like properties, promotes the establishment of complex in vitro models.
II. Nasal neuroepithelium from patients coupled to microelectrode arrays aimed to study neuronal transport to the brain. Action potentials serve as functional electrical outcome measures to monitor cellular responses triggered by molecule absorption.
III. Integration of the nasal microbiome into bioelectronic in vitro nose model to improve physiological translation to humans.
On an individual level, the project impacts the fellow’s scientific independence on using patient samples to conduct studies using advanced human in vitro models. This was shown by presenting data at national and international conferences and their current preparation for a peer-reviewed manuscript as a shared first author. This impact is substantial to the fellow as she aims to be an independent group leader in studying sensorial sciences with advanced human in vitro models. At the societal level, using patient-derived material from men and women, consideration of the microbiome, and integration of novel technologies, the project is expected to optimise pre-clinical intranasal drug development and guide the selection of candidates for human testing.
1. Respiratory nasal barriers:
Activities performed: Ethics procedure was approved by the Liguria Territorial Ethics Committee to work with fresh nasal specimens from male and female patients undergoing surgery. Protocol setup on cell isolation and culturing on conventional cell culture inserts and customised devices.
Main achievements: Isolation of patient cells and successful formation of a nose barrier. Functionality was assessed by inducing a transient loss of tight junctions with a permeability enhancer. Barrier formation and tight junction reassembly were detected by impedance and immunohistochemistry.
2. Activities performed: Ethics procedure to work with fresh nasal biopsies of the neuroepithelium from male and female patients undergoing surgery was also approved by the Liguria Territorial Ethics Committee. Thus, fresh human tissue specimen was coupled to microelectrode arrays to monitor neuron activity. Additionally, characterisation of nasal epithelial cells using patch-clamp technique was conducted.
Main achievements: Ethics approval for work with fresh human neuroepithelium in vitro. A protocol was developed for patient sample collection and limited assessment of spontaneous neuronal firing via multielectrode arrays.
3. Activities performed: The influence of the bacterial metabolite butyrate on neuronal firing was examined in a brain organoid model developed by the host.
Main achievements: A 1-hour treatment with butyrate led to an increase in burst frequency in brain organoids, suggesting a modulatory effect on neuronal signalling.
Activities performed: Ethics procedure was approved by the Liguria Territorial Ethics Committee to work with fresh nasal specimens from male and female patients undergoing surgery. Protocol setup on cell isolation and culturing on conventional cell culture inserts and customised devices.
Main achievements: Isolation of patient cells and successful formation of a nose barrier. Functionality was assessed by inducing a transient loss of tight junctions with a permeability enhancer. Barrier formation and tight junction reassembly were detected by impedance and immunohistochemistry.
2. Activities performed: Ethics procedure to work with fresh nasal biopsies of the neuroepithelium from male and female patients undergoing surgery was also approved by the Liguria Territorial Ethics Committee. Thus, fresh human tissue specimen was coupled to microelectrode arrays to monitor neuron activity. Additionally, characterisation of nasal epithelial cells using patch-clamp technique was conducted.
Main achievements: Ethics approval for work with fresh human neuroepithelium in vitro. A protocol was developed for patient sample collection and limited assessment of spontaneous neuronal firing via multielectrode arrays.
3. Activities performed: The influence of the bacterial metabolite butyrate on neuronal firing was examined in a brain organoid model developed by the host.
Main achievements: A 1-hour treatment with butyrate led to an increase in burst frequency in brain organoids, suggesting a modulatory effect on neuronal signalling.
• Incorporating cells from both male and female patients enhances insights into gender-dependent variability in nasal physiopathology, crucial for optimising intranasal drug development.
• Incorporating patient cells and tissues into the nose platform improves in vitro translation and supports the 3Rs principles, helping to reduce and refine animal experimentation.
• The adoption of this patient-derived approach requires further research, since in vitro work with human tissue is constrained by ethical approval processes and the irregular availability of samples, unlike studies using commercial cell models.
• The nose-on-chip platform provides a tool to investigate intranasal drug repurposing. Insulin, for instance, exerts systemic metabolic effects and has been shown to enhance cognition when administered intranasally in humans.
• Incorporating patient cells and tissues into the nose platform improves in vitro translation and supports the 3Rs principles, helping to reduce and refine animal experimentation.
• The adoption of this patient-derived approach requires further research, since in vitro work with human tissue is constrained by ethical approval processes and the irregular availability of samples, unlike studies using commercial cell models.
• The nose-on-chip platform provides a tool to investigate intranasal drug repurposing. Insulin, for instance, exerts systemic metabolic effects and has been shown to enhance cognition when administered intranasally in humans.