Chip URInary Engineered System for modelling bladder biomechanics and disease

Project Information

CURIES

Grant agreement ID: 101106199

DOI

10.3030/101106199

Project closed

EC signature date 21 April 2023

Start date 10 May 2024

End date 9 May 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 230 774,40

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - CURIES (Chip URInary Engineered System for modelling bladder biomechanics and disease)

Reporting period: 2024-05-10 to 2026-05-09

Summary of the context and overall objectives of the project

Antimicrobial Resistance (AMR) remains a serious healthcare issue in Europe, which currently costs the EU/EEA €11.7 billion every year. The urinary tract is a leading site for chronic bacterial infections, with 1 in 4 women versus 1 in 10 in men having suffered from Urinary Tract Infections (UTI) in their lifetimes. Treatment regimens remain broad and insubstantial. AMR can never be combatted without effective human tissue modelling pipelines upon which to develop more targeted strategies. A gateway towards establishing more targeted pipelines is to advance in vitro systems through organ-on-a-chip technologies. These systems empower a vision to increase complexity within bioengineered tissues, giving fourth dimensions of biophysical events over time to more traditional and static three-dimensional culture systems. An in vitro model of the specialised bladder epithelium (the urothelium), that undergoes cycles of stretch and release to simulate the urinary cycle, has more power as a pipeline for modelling UTI than in a static culture system. Constructing such as system has potential to better understand the connection between chronic UTIs and how the homeostatic biomechanics of the urothelium are targeted in chronic infections.

In this project, the aim was to construct a state-of-the-art stem cell derived urothelium-on-a-chip in combination with a unique microfluidic system to impose isotropic stretch with increased pressures. Chip URInary Engineered System for modelling bladder biomechanics and disease (CURIES) was conceived to bridge the gap between current infection models and traditionally static engineered urothelia. Characterisation with omics and live-imaging, seeking molecular and biophysical alterations between stretched and voided states, was established to benchmark for normal human urothelial function. The ultimate goal was to create an infection model within the cycling urothelium to explore how each biophysical state of the urothelium, stretched and voided, becomes altered in infections conditions in infected urothelia. The outcome will likely advance the current understanding of how damaged urothelium acts under infection conditions.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

An in vitro model of the urothelium was first reciprocated from published protocols, and further characterised to reveal it to be architecturally biomimetic, polarised, functional and self-renewing. This provided a biologically relevant foundation for subsequent biomechanical investigations. The cellular model was then repurposed into custom microfluidic devices, unique in their design to impose stretch upon the model isotropically via increased pressure delivered apically. Therefore the in vitro model is stretched much like the urothelium experiences expansive and reductive forces when the bladder fills and voids. This required not only careful optimisation to reproduce the urothelium in vitro, but also fabrication of a microfluidic system to simulate pressure-driven stretch during bladder expansion. In a true state-of-the-art combination of tissue engineering and bioengineering, the two were combined to successfully implement CURIES. Evidence of stretch-compliance included secretion of the urinary cycle biomarker ATP in response to increasing pressure. To investigate whole tissue changes, reporter lines were derived that could enable individual cell and whole cellular compartment responses to deformation. Live imaging revealed coordinated displacement across multiple cell layers that reform after cycles of high pressure, demonstrating that the model is stretch compliant. Bulk RNA sequencing reveals a modest change in genes associated actin nucleation, cytoskeletal modulation, cellular adhesion and vesicle transportation, consistent with adaptive cellular responses to expansion and mechanical stress. The project ended by simulating the damage response of UTI with careful dose titration of approved pharmacological compounds with known urothelial irritative and exfoliative side effects. These compounds were shown to significantly impair the barrier function of the model. These studies established a reproducible experimental workflow for inducing urothelial damage in a simulation of how UTIs impact normal urothelial function.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

CURIES has established a state-of-the-art experimental platform for advanced modelling of biomechanical events in the urothelium. The project was founded on the hypothesis that hypersensitive bladder disorders associated with urinary tract infections (UTIs) arise, at least in part, from impaired urothelial mechanotransduction—the ability of the urothelium to sense and decode mechanical tension. Existing microfluidic systems that model stretching cellular systems simulate expansive processes through a uniaxial pulling of a flexible substrate to which cellular models are adhered. While valuable, these approaches do not accurately reproduce the physiological forces experienced by the urothelium in vivo. CURIES overcomes this limitation through a pressure-driven microfluidic design that applies isotropic stretching from the apical direction, closely mimicking bladder filling dynamics. We propose that this mode of stimulation is essential for activating physiologically relevant mechanosensory pathways and signalling networks in urothelial cells.

With the validation of the model, showing it to be amenable to controlled stretching and to respond biomimetically, future work will be to use it for discovery, as there exist no tractable experimental system to study human urothelial biomechanics. Profiling the proteome and phosphoproteome, will be a powerful route to identify mechanosensors, signalling intermediates and downstream effectors. From these data benchmarks of critical reference framework of healthy urothelial biomechanics can be set, against which to decipher how they are then impacted by serious disease. While the current single-unit prototype is well suited to exploratory studies and the identification of potential therapeutic targets, widespread adoption across academic and industrial research environments will necessitate the development of a scalable, parallelised microfluidic platform capable of supporting higher-throughput experimentation. Furthermore, the platform provides a foundation for the development of in vitro pipelines to optimise bacteriophage-based therapies targeting antimicrobial-resistant pathogens. By integrating physiologically relevant host-tissue models with infection biology, the technology will be used to accelerate the identification and refinement of phage candidates and support the development of precision antimicrobial strategies.