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# A bladder blueprint to build better models for understanding homeostasis and disease

Léa Deltourbe, Méline Durand, Ariana Costas, Molly Ingersoll

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Léa Deltourbe, Méline Durand, Ariana Costas, Molly Ingersoll. A bladder blueprint to build better models for understanding homeostasis and disease. Nature Reviews Urology, 2025, 22 (8), pp.496-508. 10.1038/s41585-025-01013-x. pasteur-05451385

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Submitted on 15 Jan 2026

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A bladder blueprint to build better models for understanding homeostasis and
disease

Léa G. Deltourbe<fnref n="1" />, Méline Durand<fnref n="1" />, Ariana Costas<fnref n="1" />,<fnref n="2" />, Molly A. Ingersoll<fnref n="1" />*

<footnote n="1">¹Mucosal Inflammation and Immunity Team, Université Paris Cité, CNRS, Inserm, Institut Cochin, 75014 Paris, France & Department of Immunology, Institut Pasteur, Paris 75015 France</footnote>

<footnote n="2">²Australian Institute for Microbiology and Infection, University of Technology Sydney, ULTIMO, Australia</footnote>

<footnote>*Email: [molly.ingersoll@pasteur.fr](mailto:molly.ingersoll@pasteur.fr)</footnote>
**Abstract**

The bladder is a complex organ that can be affected by various pathologies, such as cancer or infection. It has a specific tissue structure composed of many different cell types and layers, including urothelial and endothelial cells, but also a muscle layer controlling stretch and contraction to void urine. The bladder has constitutive and induced immune responses to infection or damage and harbours a microbiome. Each of these features can be influenced by factors including age and biological sex, which makes modelling homeostasis and disease in the bladder quite complex and challenging. To model diseases that affect the bladder, mouse models are an invaluable tool to understand the bladder *in situ*. However, stark differences exist between mice and humans, and so mouse models of human disease have limitations. Thus, models that more closely approximate human physiology would be expected to contribute to improved understanding of bladder biology. As technology advances, improvements in model development and creation of 3D bladder structures are enabling scientists to recapitulate essential aspects of human bladder physiology to gain increased understanding of bladder homeostasis and diseases.
[H1] Introduction

Most knowledge concerning tissue and organ systems comes from the study of diverse models, such as cell culture, human or animal samples, and organisms such as flies, fish, or rodents. Performing human studies or having access to intact organs or tissues from healthy individuals is challenging, necessitating the use of representative model systems¹. Typically, animal models (such as mice or fish) or animal tissues are used to study anatomy, physiology, or disease mechanisms, therefore, much of what we know is extrapolated from sources that are not intact humans, which complicates understanding of homeostatic and pathological processes²,³. Additionally, researchers are facing increasing pressure to apply the ‘3Rs’ of animal research: to refine, reduce, or replace animal models⁴,⁵. Thus, innovative approaches and sophisticated tools are needed to understand human biology in healthy and disease-perturbed scenarios. These tools and approaches must approximate human physiology as closely as possible to limit our need for extrapolation and to improve understanding of the physiology and pathology of different cell types and physical structures. With more relevant, physiologically representative models, our capacity to understand human biology and develop improved therapeutics will be greatly enhanced.

One organ that is critically understudied, despite the prevalence and diversity of diseases that affect it, is the bladder. For example, in human and animal studies, in which a ‘pan-organ’ approach (that, is investigating ‘all organs’ by transcriptomics) is undertaken, the bladder is often overlooked⁶. Thus, understanding of bladder biology lags behind that of other mucosal organs, such as the gut or the lung. Indeed, only in the last six years have publications describing organoids, differentiated multilayer Transwell systems, and *ex vivo* approaches to study the bladder in healthy and disease states started to address this crucial need⁷⁻¹². However, these approaches are still new, and many questions concerning them arise. These questions include the accuracy of recapitulating intact organs in these systems, the features that should or could be incorporated into these models to improve tissue representation, the balance of the need for reproducible models versus reductionist approaches to understand biological interactions at the whole-organ level, and inclusion of features that arise from sexual dimorphism.

To create improved model systems of the bladder that approach human physiology, including
sexual dimorphism, consideration of what is known about this tissue, what is already available, and how bladder models can be further optimized for study is needed. In this Review, we present the development, structure and microenvironment, and defence mechanisms of the bladder, which together create a blueprint that can be used to optimize current models described here or create new approaches. We focus primarily on models used for infection studies; however at least some models presented can and have been used to study bladder cancer and other bladder malignancies.

## [H1] Components of the bladder

When designing model systems, understanding how an organ forms and what components are present in or as part of the tissue is crucial. This understanding is key to replicate complex tissue architecture, particularly in a 3D structure, and it might also help guide the choice of growth factors and media, extracellular matrix materials, or physical support needed to build a model. Fundamentally, understanding how the bladder develops and what components, such as local immune cells or urine, are essential to its functioning will be crucial to create a representative experimental model.

## [H2] Bladder development

Urogenital tract development is initiated in the first trimester of pregnancy in humans and is nearly complete by the end of the second trimester¹³. In the first trimester, the kidneys, ureters, and bladder develop without divergent sex-specific physical characteristics or morphology¹⁴. Throughout gestation, the thickness of the bladder wall increases, irrespective of the sex of the foetus¹⁵. The bladder has an overall uniform thickness; however, women and men have different bladder thicknesses, averaging 3.0 mm in women and 3.3 mm in men¹⁶. Precisely when this sex difference emerges is unclear and the implications of these differences are not known, but, increased thickness might be associated with prostate disease in men¹⁶. To fully understand divergent thicknesses between the sexes or to assess the impact of bladder wall thickening in disease, multi-compartmental models that include muscle and other stroma will be required.
[H2] Bladder structure

The bladder is composed of layers, including the serosa, the muscle, the lamina propria, and the urothelium<sup>17</sup>. The serosa is a thin connective tissue layer composed of epithelial cells that partially encases the bladder, is contiguous with the peritoneum, and covers the muscle layer (Figure 1)<sup>18</sup>. Serosal tissues secrete a lubricating fluid that minimizes friction between organs as they slide over each other. The lamina propria is adjacent to the muscle layer and the urothelium is the innermost layer, facing the lumen<sup>19</sup>. Within the lamina propria are blood and lymphatic vessels, immune cells, and structure components of the bladder, including fibroblasts and extracellular matrix proteins<sup>20</sup>. The urothelium is a stratified epithelium composed of small basal cells adjacent to the lamina propria that give rise to intermediate cells (Figure 1). The intermediate layer undergoes differentiation into binucleated superficial umbrella cells<sup>21</sup>. These cells, also known as facet cells, are large (~100 µM), hexagonal, and highly polarized<sup>21,22</sup>. Umbrella cells form a single layer in direct contact with the lumen of the bladder. Different layers of the urothelium can be identified by the presence or absence of many molecules, such a cytokeratins, the patterns of which vary depending on the species<sup>22</sup>. Notably, mouse or rat models are used in many laboratories, but rodent bladders have only one or two intermediate cell layers compared with up to five intermediate layers in humans<sup>2,23</sup>. Differences in the intermediate cell layer between species might affect the rate at which umbrella cells differentiate, turn over, or respond to damage, which would affect studies of tissue regeneration or, potentially, development of non-infectious malignancies, such as bladder cancer. The number of intermediate cell layers might also affect structural aspects of the urothelium, including thickness or stiffness, which could influence how pathogens interact with the bladder surface. For example, increased tissue stiffness supports uropathogen invasion<sup>24</sup>.

The urothelium serves as the impermeable barrier between the host and metabolic waste before elimination and limits interactions between the bladder and pathogens, toxins, or other threats<sup>22</sup>. Barrier function is maintained though expression of four major uroplakin proteins, which form membrane plaques; tight junction proteins, such as claudins and ZO-1; and adherens junction proteins, including E-cadherin<sup>25-28</sup>. The bladder is covered with a glycosaminoglycan (GAG) layer, as well as proteoglycans and glycoproteins, which contribute to its impermeable nature (Figure 1)<sup>29-32</sup>. The GAG layer protects the urothelium from urinary solutes and pathogens.
Damage to the GAG layer contributes to the pathogenesis of interstitial cystitis and recurrent urinary tract infections (UTI)²⁹,³³. Removal of this layer through acid treatment in rabbit bladders, leads to increased colonization of the urothelium by uropathogens³⁴⁻³⁶. However, this approach is non-targeted and probably challenging to restrict to the GAG layer of the bladder. Thus, these studies limit our understanding of the contribution of the GAG layer to protection of the bladder surface or prevention of bacterial colonization.

Different anatomical spaces within the bladder remain poorly characterized, and as such, difficult to model. Whether the bladder serosa has a more precise or specialized function is not known and whether and how it should be incorporated into a bladder model is unclear. Incorporating all the different cell types of the lamina propria is challenging, but including a space that contains vessels would assist study of how immune cells infiltrate the bladder or how resident immune cells exit the bladder via blood or lymphatic vessels. Moreover, whether and how divergent tissue architecture between humans and rodents affects disease manifestation or extrapolation of animal studies to humans is largely unknown and merits study. For example, in a model in which the number of intermediate cell layers that develop could be controlled, it might be possible to test phenotypes such as bacterial invasion or umbrella cell turn-over to determine whether they differ between species. This knowledge would help in interpreting results from animal models. Interestingly, therapies to replace or replenish the GAG layer show promise for individuals with chronic inflammatory bladder diseases, such as interstitial cystitis³⁷⁻³⁹. Thus, efforts to develop a GAG layer in a humanized bladder model might help to elucidate its function in health and disease.

## [H2] Bladder function

The urothelium must stretch during urine accumulation and contract to expel urine without losing barrier function. These tissue dynamics induce stress and damage to the urothelium²². To maintain barrier function, uroplakins and other surface molecules cycle in and out of the cell via fusiform vesicles derived from the cell membrane⁴⁰. With respect to modelling the bladder, one key component is barrier function, mediated by junction and uroplakin protein expression⁴⁰. Barrier function can be confirmed by measuring resistance (transepithelial/transendothelial electrical resistance, or TEER) or performing functional assays such as fluorescein
isothiocyanate (FITC) exclusion⁴¹. Given that a lack of barrier would increase the access of uropathogens or molecules to inner cell layers in a 3D system, inclusion of barrier function will be key in a representative bladder model.

## [H2] Bladder immune components

The bladder is often described as having a multilayered defence system, from passive micturition to active macrophage phagocytosis of pathogens and expression of inflammatory cytokines⁴²⁻⁴⁴. As would be expected of any surface exposed to the external environment, mouse bladders contain a diverse resident immune cell population⁴²,⁴⁵,⁴⁶. The most numerous immune cells in C57BL/6 mice are resident macrophages, which make up a network situated directly under the urothelium and throughout the muscle layer in the bladder⁴⁴,⁴⁵. Macrophages have an important role in the immune response against UTI; however, probably also have key roles in the maintenance of barrier function and homeostasis, as they do in other organs⁴⁷⁻⁴⁹. Different types of T cells, including resident memory and gamma delta (γδ) T cells also reside in the mouse bladder and potentially provide rapid local protection against uropathogens⁴⁶. Notably, knowledge regarding the immune cell types and proportions in the human bladder is lacking.

The bladder responds to damage or disease through the production of soluble mediators, such as antimicrobial peptides, secretory IgA (sIgA), or cytokines⁴³,⁵⁰. The expression of these mediators can be constitutive, or they can be upregulated by damage or infection⁵⁰. One example is Tamm-Horsfall protein (THP), an anti-microbial protein also known as uromodulin that is produced in the kidneys and accumulates to become the most abundant protein in urine⁵¹. THP can bind bacteria in the lumen and prevent their interactions with uroplakin 1a and 1b on urothelial cells⁵². Higher concentrations of THP are found in urine from women than urine from men; however this difference is only apparent when THP is normalized to urine creatinine levels, which are substantially higher in men⁵³. Whether these small differences contribute to sex differences in susceptibility in UTI is unknown and would require a study specifically designed to test this hypothesis. In humans, sIgA is produced by plasma cells in the lamina propria and the main function of sIgA is to limit the access of uropathogens to the urothelium⁵⁴. Whether sIgA is present in the mouse bladder is uncertain as it is undetectable in healthy mice and very few, if any B cells, are present in young mice⁴². Indeed, intriguingly, sIgA and other antibodies might
contribute to bladder immunity with increasing age, as their presence increases over time in
humans and the number of B1a B cells increases with age in the human and mouse urinary
tract⁵⁵,⁵⁶.

Cytokine production is induced in part by signalling via Toll-like receptors (TLRs) (Figure 1). In total, 13 TLRs are expressed in the bladder: humans express TLR1–10, whereas mice express TLR1–9 and TLR11–13⁵⁷. The use of gene-deficient mouse models shows that TLR2, TLR4, and TLR5 have key roles in the initiation of inflammation in UTI and are the best studied TLR molecules in this infection model⁴³,⁵⁰. Divergent TLR expression might affect how the bladder responds to threats, such as pathogens, between the species, which would affect extrapolation of immune mechanisms from mice to humans. However, the evident substantial overlap in TLR expression between the species might support comparison of immune mechanisms across species.

In the context of disease, such as infection or cancer, diverse populations of immune cells
infiltrate the bladder, including neutrophils, monocytes, eosinophils, and T cells<sup>43,50,58–61</sup>.
Recruitment is mediated by chemokines and damage-associated signals from resident immune
cells and the urothelium<sup>45,62–64</sup>. The quantity and kinetics of cellular infiltration depend on local
and systemic signals and the microenvironment of the bladder<sup>45,62–64</sup>. During UTI, CD4<sup>+</sup> and
CD8<sup>+</sup> T cells accumulate in the bladder, take on a resident phenotype, and protect against
recurrent UTI<sup>60</sup>. These T cells, called tissue-resident memory T cells, depend on the presence of
sufficient antigen in the early stages of infection and are both necessary and sufficient to provide
protection against recurrent UTI<sup>60</sup>. Notably, the signals that support their development are not
known, but probably include local contributions within the bladder.

Excluding *in vivo* approaches, most current bladder models lack immune cells or only include a
single type of immune cell. This omission leaves out key components of the bladder, which are
the diverse resident immune cell populations that are present from before birth<sup>42,45</sup>. How these
cells contribute to tissue homeostasis or determine disease susceptibility is poorly understood.
Incorporating immune cells into the very fabric of a model will help to unravel how they
contribute to bladder function. However, this incorporation will be challenging, as although
immune cell populations in mouse bladder are well-described, a detailed analysis of the composition of the healthy human bladder immune compartment has not been performed. Thus, creating a bladder model with immune cells can only draw information from mouse studies. Whether the same immune cells are present in humans, and in the same proportions, anatomical locations, or activation state is not known. Additionally, given increases in B cell populations over time, incorporation of B cells into a bladder model might help specifically address aging immunity to bladder disease. Finally, whether sex differences in homeostatic immune cell populations exist in humans is unknown. In C57BL/6 mice, very few resident immune cell populations exhibit sex differences in their proportions or numbers, with the exception of γδ T cells, which are present in higher numbers in male mice than female mice⁴⁶. The implications of this difference are not known and require further study.

## [H2] Bladder microbiome

The bladder microbiome is often overlooked in bladder biology and modelling. Interestingly, the human bladder microbiome, composed of microbiota resident in the bladder that might or might not be found in the urine, is influenced by sex, age, and, in women, menopausal status (Figure 1)⁵⁹. For example, women have a more heterogenous mix of bacteria than men⁶⁵,⁶⁶, which might affect susceptibility to infection or potentially shape local immune responses and tolerance differently between women and men⁶⁷. In addition, the diversity of the microbiome decreases with age in women but it increases with age in men⁶⁵, which might influence disease susceptibility or even cancer development over time. Transcriptomic analyses of germ-free and specific pathogen-free mice revealed that 97 genes are differentially expressed between germ-free and specific pathogen-free mice, supporting a role for the bladder microbiota in controlling gene expression and bladder physiology⁶⁸.

The functional role of the urinary microbiome is unknown, but dysbiosis is often associated with disease or susceptibility to infection⁶⁹. However, whether dysbiosis is caused by or causes pathological states remains unclear. For example, patients with recurrent UTI take multiple courses of antibiotics, which probably affect the composition of the microbiome, in addition to the gut microbiome. Patients with neuropathic bladder have a reduced proportion of *Lactobacillus* in women and *Staphylococcus haemolyticus* in men compared with healthy
individuals and increased proportions of other microorganisms, such as *Enterococcus faecalis*, *Proteus mirabilis*, and *Klebsiella pneumoniae*⁵⁹,⁷⁰⁻⁷³. These patients are at higher risk of UTI than healthy individuals, and are often infected with multi-drug resistant pathogens, including ESKAPE (*Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa* and *Enterobacter* spp) pathogens. Thus, inclusion of diverse microbiota in bladder models might provide clues as to how a healthy microbiome interacts with and potentially protects the urothelium. The gut microbiota and vaginal microbiome also influence bladder health and disease and could potentially be considered when building an improved bladder model²,⁷⁴⁻⁷⁶.

## [H2] Urine

Urine, produced by the kidneys, is a complex matrix of water, urea, uric acid, creatinine, and other metabolic waste products and solutes⁷⁷,⁷⁸. The total amount of protein in urine from healthy individuals is under 150 mg/day amounting to 0-14 mg/dl⁷⁸,⁷⁹. In healthy individuals, 70% of the urinary proteome originates from the kidney and the urinary tract, whereas the remaining 30% is composed of proteins filtered by the glomerulus⁸⁰. It is mainly composed of shed cells, plasma proteins that pass the glomerular barrier, and proteins shed by cells within the urogenital system⁸⁰,⁸¹. Over six thousand proteins have been identified by deep profiling of the healthy human urinary proteome⁸². In healthy individuals, THP is the most abundant protein in urine, followed by albumin and immunoglobulins⁷⁸,⁸³. Urine can also contain glucose, bilirubin or urobilinogen, and bacteria, despite the common belief that urine is sterile⁷⁷. Changes in the composition of urine can reflect alterations in homeostasis, pathologies, or pharmacological treatment and may impact the health of the urothelium⁷⁸,⁸⁰,⁸⁴.

Urine has high interindividual differences but also intraindividual variations, which might affect physiology and disease outcome. Urine also has species-specific differences, including the ‘major urinary proteins” (MUPs) present in mouse urine but not found in human urine⁸³. Species-specific differences in urine composition could influence urothelial cell function, host-pathogen interactions, and disease development or progression. Species or sex differences, such as those reported for urinary metabolites⁸⁵,⁸⁶, probably also influence the biology of the bladder,
and are an important parameter to consider in bladder models. How this variability can be
incorporated into tissue models remains unclear.

Overall, certain aspects of the bladder can be integrated into complex tissue models, such as a multi-layered urothelium, but many elements, such as microbiota or urine, are often left out, potentially because of their complexity. Bladder structure and cellular composition are clearly key aspects to include in a bladder model. Additional considerations include sex differences and the role of steroid hormones, and aging, although the best way to build these aspects into bladder models requires further study. Importantly, incorporation of any or all of these elements into a model will depend on the research question to be addressed (Figure 1).

[H1] The bladder blueprint

Using current knowledge of the components of the bladder, a blueprint of the elements and features that would ideally be included in a bladder model can be created. These elements include material of human origin, 3D structure of the urothelium, immune cells or the capacity for immune cell infiltration, urine tolerance, tissue stretch, fluid flow, and, regardless of the number of elements incorporated, reproducibility (Figures 1 and 2). Currently used models contain these elements to varying degrees and might serve as platforms to build better models.

[H1] Modelling the bladder

The bladder has a specific tissue architecture, which varies to some extent between rodents and humans — such as the number of urothelial cell layers; between the sexes — such as specific immune cell populations in mice; and during aging — such as B cell accumulation⁵⁵,⁸⁷⁻⁸⁹. Additionally, it is a complex and dynamic organ, subject to physical, chemical, and biological stresses during homeostasis and disease¹⁹,⁵⁰. To understand these functions and how the bladder responds in a disease context, such as infection, two major approaches have been used for many years, animal models and *in vitro* cell culture. These approaches have advantages and caveats, and neither completely include the major components of the bladder blueprint: material of human origin, 3D structure, immune cell inclusion, urine tolerance, tissue stretch, fluid flow and reproducibility (Figure 2).
[H2] Animal models

In vivo animal models are a valuable tool for investigating physiology and disease pathophysiology in a systemic setting. Mouse, and to a lesser extent, rat models are commonly used for bladder infection studies because the physiopathology of UTI in these animals is similar to that observed in humans⁹⁰ (Figure 2A). These models have enabled investigation of host-pathogen interactions, host response to uropathogens, and the influence of biological sex on disease progression and immunity⁵⁰,⁵⁹,⁹¹. In terms of the bladder blueprint, in vivo models have 3D structure, immune cell inclusion, urine tolerance, tissue stretch and fluid flow, but they clearly are not derived of human materials, and reproducibility can be a challenge, for example, owing to variable conditions in different animal facilities.

A wide variety of mouse strains are used to investigate UTI, including the C57BL/6, CBA, BALB/c, and C3H backgrounds⁹². As the choice of mouse strain influences disease outcome, different strains of mice, with genetic variability, can be used to address specific research questions⁹²,⁹³. For example, C3H/HeJ mice have a susceptibility locus that increases vesico-uretic reflux compared with C57BL/6 mice, leading to induction of pyelonephritis in this strain⁹⁴. Indeed, both the C3H/HeJ and CBA/J mouse backgrounds have increased reflux compared with BALB/cJ, C57BL/6J, and A/J strains, which have little to no reflux, supporting the idea that studies of pyelonephritis, or other kidney-related diseases that require interaction with the bladder are best carried out in C3H or CBA strains⁹⁴. As a second example, C3H/HeJ mice have a mutation in *Tlr4*, and inflammation is not induced in response to Gram-negative bacterial infection in these mice⁹⁵,⁹⁶. In C3H/HeJ mice, uropathogenic *Escherichia coli* (UPEC) infection reaches very high bacterial titres because neutrophils do not infiltrate the bladder⁹⁷. C3H/HeN mice, which do not have this mutation, are a natural wild-type control to the C3H/HeJ mouse to investigate the innate host response to Gram-negative infection⁹⁵,⁹⁶. In addition to natural variance within these inbred strains, transgenic mice can be genetically engineered for the study of host-specific factors, including the addition, deletion, or alteration of host proteins.

In some ways, mice reflect human urinary tract physiology, but species differences do exist. Urothelial thickness and urine concentration and composition (for example, the presence of MUP in mouse but not human urine) are different between the species⁹⁸. Furthermore, mice void urine
more frequently than humans do, owing to a lack of storage function<sup>99-101</sup>. The implications of these differences are not clear, and pose challenges to understanding healthy bladder physiology, but some might be overcome using transgenic mice, such as the MUP-knockout mouse<sup>83</sup>.

*In vivo* models can capture both local and systemic phenotypes, but they do not encompass all aspects of bladder disease. Bacterial instillation via catheterization in mouse UTI models bypasses the biology of bacterial ascension via the urethra and, therefore, does not permit study of this important first step of infection<sup>2,102</sup>. This mechanism could potentially be recapitulated in a 3D tissue model by placing bacteria at the opening of a channel lined with urothelial cells and observing bacterial migration in the presence or absence of fluid flow. A second major open question is regarding the quantity of bacteria necessary to induce a symptomatic infection in humans. Consistent infection in mouse models requires intravesical instillation of a minimum quantity of bacteria (for example, 10<sup>5</sup> colony-forming units of UPEC in C57BL/6 mice<sup>42,60</sup>). Whether human infection results from a large bolus of bacteria entering the bladder or develops as bacteria divide in the bladder is unknown and challenging to answer without experimental infection of humans. Related to this question, limiting antigen during infection — by using a lower inoculum or treating mice with antibiotics — abrogates development of adaptive immunity<sup>42,60</sup>. Thus, interestingly, varying the quantity of bacteria used for *in vivo* infection could help to capture some of the mechanisms that drive these differences, although whether antigen load affects immunity the same way in humans is unclear. Another challenge of *in vivo* models is that analyses of early bacterial adhesion and colonization are difficult owing to the technical challenge of isolating tissues very quickly after infection. Finally, studying the interindividual variability of the human immune response to infection<sup>103</sup> in mice that all have the same genetic background is quite difficult. Using multiple mouse strains to compare host responses might model the variability of the human immune system to some extent, but this approach quickly becomes very expensive and challenging to manage.

## [H2] *In vitro* 2D human approaches

At the opposite spectrum to *in vivo* animal models are 2D *in vitro* models with primary cells or immortalized cell lines from human donors. Some mouse urothelial cells do lines exist, but these will not be discussed as they are not as relevant with respect to a discussion of human-oriented
models. Human cell lines and primary cell approaches are widely used for their simplicity, easy maintenance, and accessibility (Figure 2B, C). One example is the human urothelial cell line 5637 (ATCC HTB9). This cell line was used to show that when bladder cells are in an undifferentiated state, actin microfilament distribution is key in the formation of quiescent intracellular UPEC reservoirs, which might be responsible for the resurgence of UPEC in recurrent UTI¹⁰⁴. 5637 cells were also used to demonstrate that oestrogen induces the expression of antimicrobial peptides and cell-cell contact associated proteins, promoting urothelial defense¹⁰⁵. However, this cell line comes from a grade II carcinoma of an elderly male donor, which does not recapitulate many aspects of a healthy or homeostatic bladder owing to genetic abnormalities. Additionally, aged male carcinoma cells probably do not provide good representation of the population primarily afflicted by UTI — healthy young women. Finally, these cells do not possess characteristics of terminally differentiated umbrella cells, which are targeted by uropathogens for invasion¹⁰⁴. This major difference suggests that these cells might not be ideal to study host-pathogen binding and invasion interactions.

The PD07i cell line, which is a human papillomavirus type 16-immortalized paediatric human urothelial cell line, was used to investigate UPEC-induced apoptosis of urothelial cells, intracellular bacterial community formation mechanisms, the regulation of UPEC virulence factors (such as fimH, hlyA, cnf1, fliC, kpsD, and yfaL) and the role of uroplakins in the host response to UTI¹⁰⁶⁻¹⁰⁸. It was also used to illustrate the dynamic morphological changes bacteria undergo during the infection cycle¹⁰⁹⁻¹¹¹. This cell line is virus-immortalized, but it retains a number of important characteristics of primary urothelial cells, such as cytokeratin and uroplakin expression, tolerance to urine exposure, inflammatory responses, and capacity to undergo apoptosis¹⁰⁷,¹⁰⁸,¹¹²,¹¹³. However, human papillomavirus type 16 immortalization has modified the ability of this cell line to differentiate, potentially affecting invasion or the response to infection¹¹⁴. In addition, these cells come from an infant donor, which could be a good model to study specific mechanisms of paediatric UTI, but might not entirely recapitulate the physiology of adult bladder⁵⁹.

Additional cell lines used include the human bladder epithelial cell lines J82 (58-year-old male transitional cell carcinoma virus-immortalized cell line), T24 (82-year-old female transitional
cell carcinoma virus-immortalized cell line) UROtsa (12-year-old female normal human urothelium Simian Virus 40-immortalized cell line) and TEU-1/TEU-2 (human ureteral papillomavirus type 16-immortalized cell lines). Overall, these human bladder cell lines have variable resemblance to primary urothelial cells in terms of size, protein expression, or function, might detract from the advantages of *in vitro* cell culture models or could potentially help to model different scenarios, such as young or old bladders (Figure 2B).

Fresh, healthy human bladder cells would undeniably be the best source for 2D *ex vivo* studies. For example, exfoliating cells from donor urine were used to show that oestrogen treatment increases antimicrobial peptide expression and cell-cell contact¹⁰⁵. However, the number of cells that can be collected and cultured from urine is quite limited and this approach probably yields only umbrella cells in a healthy context. Additionally, whether exfoliated cells are actually healthy representatives of the urothelium is unclear¹¹⁵. Nevertheless, this approach does provide a means to have human cells from the bladder in a non-invasive manner (Figure 2C).

Regarding the bladder blueprint, 2D cell culture approaches certainly incorporate material of human origin and can be highly reproducible, particularly to study aspects of UTI such as attachment, invasion, bacterial morphology, and intracellular immune responses. In contrast to genetically uniform mice, using diverse sources of human material, such as urothelial progenitors, might help to capture the influence of genetic variability, as well. The strengths of cell culture systems are that they are simple and inexpensive, particularly in comparison to animal models. They are ideal for high-resolution microscopy studies of intracellular or molecular dynamics¹⁰⁹⁻¹¹¹. However, they come with the caveat that they cannot be used to understand 3D cellular interactions and they do not include immune cells. Incorporating fluid flow or shear stress *in vitro* is possible using a parallel plate flow chamber or other microfluidics materials¹⁰⁹⁻¹¹¹. In one type of set-up, T24 cells are cultured on a slide, which is then covered with a gasket and a 'parallel plate' with a fluid inlet and outlet. Fluorescent bacteria can be flowed over the cells and binding can be imaged in real time to assess the influence of shear stress and flow rate on bacterial adhesion¹¹⁶. Results of studies using this approach show that bacteria maintain tight interactions with the urothelium under continuous or discontinuous flow conditions¹¹⁶. Thus, although these models have revealed important information about cell and
bacterial dynamics, these simple models do not recapitulate the complex bladder microenvironment, including different cell types, cellular interactions with the external environment, urine tolerance, tissue stretch, or fluid flow. These interactions are important as they are responsible for the differentiation of cells, expression of proteins, secretion of soluble factors, and how the organ responds to stimuli.

## [H2] Building a better model

In addition to animal models and human cell culture systems, diverse 3D-models, preferably derived from human material, will be key to the study of bladder physiology and pathophysiology of diseases, including infection or cancer. *In vivo* rodent models do not always recapitulate the essential bladder biophysical environment, and *in vitro* human approaches do not reach the systemic level or complexity achieved when studying UTI in an animal model (Figure 2A-C). Only a few human models exist owing to challenges in incorporating diverse features of the bladder. However, advances have enabled researchers to recapitulate essential aspects of bladder physiology for improved understanding of human bladder homeostasis and disease, while also refining, reducing, or replacing animal studies.

## [H3] Ex vivo mouse models

A major drawback of working *in vivo* is the challenge of capturing events that happen quickly, such as the initiation of infection or extracellular bacterial behaviour. To overcome this challenge, *ex vivo* models have been developed to study specific events during infection (Figure 2D). Most of these models are rodent based because obtaining healthy human tissue is difficult as collection is invasive and not common from organ donors. Thus, these models enable the study of early or rapid events, but they do not always address the 3Rs of animal research. In addition, regarding the bladder blueprint, they have 3D structure, immune cell inclusion, urine tolerance, and in some cases, fluid flow, but they lack dynamic tissue stretch and are obviously of rodent rather than human origin.

One such *ex vivo* mouse bladder model employs an organ chamber to hold an explanted mouse bladder, which enables confocal imaging and simultaneous cytokine measurements¹¹⁷. This model system has an organ chamber sealing the bladder between coverslips and three inlet
compartments that provide media and solutions containing antibiotics or bacteria. This model maintains all layers of the urothelium and intact muscle and contains resident immune cells. Intracellular bacterial communities, infiltrating immune cells, and phagocytosis of bacteria can also be studied if bladders are infected before removal from the mouse. Bacterial interactions with the urothelium and phagocytosis by immune cells can be analysed by live imaging, which is exceedingly difficult to do in animal models. When the tissue is infected ex vivo, the concentration of MCP, IL-6, and TNF-α increases compared with uninfected bladders. Interestingly, this model incorporates fluid flow but with culture medium rather than urine. The lack of urine might reflect the choice to provide the tissue with a nutrient-rich medium compared with nutrient-poor urine, or because collecting large volumes of urine from mice is challenging. This set-up permits imaging and study of early infection events, but it does not entirely recapitulate the bladder environment and the absence of urine could result in alterations of different stages of the bacterial pathogenic cycle, including the morphological process of filamentation, which requires urine flow<sup>118</sup>. Another limitation of this model is that it does not include stretch, which is an important element of the bladder capacity to fill with and void urine.

A model to investigate host-pathogen interactions while avoiding the requirement for specialized equipment has been developed (Figure 2D)¹². In this simple model, a whole mouse bladder is cut into four equal pieces and the muscle and urothelial layers are separated¹⁷. The muscle is discarded, and the remaining urothelial ‘sheet’ has a homeostatic layered urothelial structure with resident immune cells. Urothelial sheets can be used to assess bacterial interactions with the urothelium from minutes to up to 120 hours. This technique is easily applicable to female and male mice, which supports the study of sex-specific mechanisms of host-bacteria interactions. Overall, this model is highly reproducible, can be performed with bladders from any mouse strain and in any medium or urine, but comes with the caveat that the tissue does not retain the stiffness present in an intact organ. This factor is important because increased tissue stiffness enhances bacterial invasion²⁴. Infected bladders can be isolated for this approach, but the strength of this model is that ex vivo infections can be synchronized across many samples, and very early interactions can be studied. Furthermore, if bladders are sourced from colleagues sacrificing mice for other reasons, this approach addresses the 3Rs of animal research by reducing the number of animals used for experimentation.
*Ex vivo* models have many advantages, such as maintaining the 3D structure of the bladder, tolerance to urine, and the presence of resident immune cells. They are useful to study the homeostatic organization of the bladder and host-bacterial interactions without the effects of the infiltrating immune cells. Sourcing mouse bladders from colleagues who would otherwise discard the tissue contributes to a reduction in the number of animals used for experimentation. However, *ex vivo* models also have limitations: some models require specialized equipment, which may not be universally available<sup>117</sup>, and interspecies differences make it unclear how much we can extrapolate observations in these models to human physiology. Overall, *ex vivo* models are reproducible and have 3D structure, immune cell inclusion, and urine tolerance, but they do not always recapitulate tissue stretch and fluid flow. Despite these limitations, their use can answer many questions related to early or rapid events in bladder disease and potentially treatment.

### [H3] 3D-organoid and organoid-derived models

Novel 3D-urothelial models, using immortalized human urothelial cells or mouse urothelial cells, better recreate bladder structure than 2D tissue culture models and provide platforms for the development of new treatments against infection or cancer (Box 1: Organoids). Regarding the bladder blueprint, these models can use material of human origin and have 3D-structure and urine tolerance. However, they do not always have immune cell inclusion, dynamic tissue stretch, or fluid flow.

Healthy mouse organoids with lumens were developed to investigate host-pathogen interactions (Figure 2E)⁷,⁹. These organoids form after culture of mouse bladder cells in hydrogel domes⁷,⁹. Once mature, bacteria can be injected into the organoid lumen, where they invade umbrella-like cells and proliferate to form intracellular communities⁷. In this system, neutrophils isolated from blood and added to the organoid cultures are recruited to infected tissue and migrate out of the lumen once it is sterilized⁷. Infiltration of the organoids is specific to those that are infected, but one caveat of the system is that neutrophils do not extravagate from vessels, and potentially do not receive signals needed for their activation via this mechanism. Thus, whether neutrophils in this system resemble those that respond to infection in mice or humans remains to be determined.
Mouse organoids have been used for infection studies, but human bladder organoids are used more often for bladder cancer studies (Figure 2F)⁹, although human organoids would have great utility in infection studies. This preference for human organoids might reflect the standard of care for bladder cancer, which includes resecting malignant tissue that could be available for research purposes.

Mouse assembloids have also been developed with a multilayered urothelial organoid covered with a stroma, an endothelial cell layer, and an outer muscle layer (Figure 2G)¹¹⁹. These assembloids mimic *in vivo* tissue in terms of cell type proportion, gene expression, and contractile response to acetylcholine and noradrenaline. This complex model was also reproduced with human tumour-adjacent bladder tissue and induced pluripotent stem cells to obtain smooth muscle cells (Figure 2H). Indeed, models from tissue-adjacent biopsies maintain the ability to differentiate, stratify, and develop a robust barrier function *in vitro*⁹,¹¹⁹. However, tumour-adjacent tissue is never completely mutation free and might carry abnormalities that modify the response to bladder diseases or therapies¹²⁰. Of the current 3D models, assembloids most accurately capture the complex structure of the bladder, as they contain different cell types. However, importantly, they do not have all the different cell types of the lamina propria, or intact vessels, which would be ideal to study immune cell migration into and out of the bladder. Another drawback is that they are challenging to make, as primary tissue for each layer is not readily available.

A human multi-layered 3D-tissue model using spontaneously immortalized human urothelial cells (HBLAK, from an 88-year-old man) grown on Transwell inserts has been created to study infection (Figure 2I)⁸,¹⁰,⁴¹. This approach, called the 3D urine-tolerant human urothelial model (3D-UHU), requires urine for terminal urothelial differentiation, which suggests that the cells used in the model might undergo differentiation pathways that are like those in human bladder development. This possibility remains to be shown, but this line of study would be interesting for understanding healthy bladder development. The 3D-UHU model has three different urothelial cell types organized in seven layers, urothelial protein expression, and barrier integrity⁸. Upon UPEC infection, this model secretes pro-inflammatory cytokines and chemokines and exhibits reduced barrier function⁴¹. One drawback of this model is that these cells are not primary human
cells; however, it recapitulates many aspects of bladder biology and is useful to answer specific human-based questions of immunity and tissue damage.

The bladder organoid models developed in the past few years are the first to recapitulate 3D-structure of the bladder. Comparing mouse organoids with human cell-derived organoids to understand how the structures differ from each other would be interesting. Similarities and differences between organoids from these species might help to identify what aspects of mouse-based studies can be extrapolated to humans. However, like any model, organoid models come with limitations, such as the lack of resident immune cells. In addition, the structure of the organoids themselves come with challenges. Round organoids lack an entry point into the bladder, therefore, require needles to mimic a urethra and initiate bacterial entry. The 3D-UHU model has an entirely open ‘lumen’ in the upper chamber of the Transwells, which probably exposes the urothelial surface to a higher oxygen concertation than might be present in the bladder. All organoid and organoid-derived models described here also lack tissue stretch and fluid flow but owing to their simplified structures can be quite reproducible.

## [H3] Microfluidic models

To study the urothelium under fluid flow and mechanical stretch conditions, which are two key biomechanical properties of the bladder, an increasing number of options are available¹⁹. Similar in concept to the parallel plate model, the Cellix Vena8 Fluoro+ Biochip model is a simple system that uses cultured cells that can be grown with extracellular matrix components. It has parallel chambers for imaging multiple flow conditions at the same time. This approach was used to demonstrate that residues E50 and T530 are necessary for FimH-mediated adhesion of *E. coli* to urothelial and bladder endothelial cell lines under flow conditions¹²¹. The CellASIC ONIX microfluidics system is another microscopy-friendly apparatus that also supports the application of fluid flow conditions to 3D cultures¹⁰⁹. This system and the PD07i cell line were used to follow bacterial morphological changes induced by urine composition and pH, which influence intracellular bacterial community development during infection¹⁰⁹. Many observations made with these models would have potentially been impossible to see in *ex vivo* models without fluid flow. Whether the physics of flow are comparable to that *in vivo* remains to be confirmed.
Stretch also occurs in the bladder during filling and voiding. The Emulate Chip S-1 system is a microfluidic chip with two fluid flow channels separated by a porous membrane (Box 2: Organ-on-Chip). This system was used to grow a monolayer of 5637 cells (human urothelial from a male bladder carcinoma) in the upper channel and HMVEC-Bd, which are human bladder microvascular endothelial cells, in the lower channel. The channels have independent inlet and outlet reservoirs, which enabled culture of the 5637 cells in diluted urine and the endothelial cells in medium, under flow conditions (Figure 2J)¹¹. In this model, cyclical stretch was applied to the chip itself to mimic slow filling and rapid voiding of the bladder. During infection of this chip, UPEC enters the urothelial cells and forms intracellular communities, similar to those observed in mice, that persist in the face of antibiotic administration¹¹. To study the role of neutrophils in the response to infection in this model, these cells were isolated from fresh human blood and added to the endothelial cell channel. Neutrophils extravasated from the endothelial side of the chip to the urothelial side and migrated towards bacteria and infected cells¹¹. This model does not completely recapitulate the 3D-structure of a bladder, especially in terms of the number of cell layers and cell types, but it provides evidence that key physiological features, such as stretch and flow, can be incorporated into tissue models. Indeed, this model ticks nearly all the bladder blueprint boxes: material of human origin, immune cell inclusion, urine tolerance, tissue stretch, fluid flow, and reproducibility¹¹.

In summary, microfluidic models can be made using material of human origin. They might be useful to study the influence of tissue stretch or fluid flow on the homeostatic maintenance of bladder tissue. They have been used to assess bacterial behaviour, host interactions, and the response to antibiotics. However, these microfluidic bladder models are simplistic in terms of urothelial composition compared with the more complex structures of organoids and assembloids. In addition, modelling the degree or frequency of stretch or the rate of flow is challenging as these parameters are not currently well understood in humans. One key challenge for these models will be the inclusion of resident and infiltrating immune cells. The inclusion of infiltrating neutrophils has been explored¹¹, but incorporation of bladder-resident immune cells has not been reported.
[H1] Challenges ahead

Each of the models described has brought new knowledge and observations for understanding bladder biology and, particularly, response to infection. However, challenges remain to be surmounted. First, with the exception of the assembloids model and unpeeled *ex vivo* bladder models, inclusion of non-urothelial cell layers in bladder 3D models is rare¹¹⁹. This omission limits our understanding of how different layers interact with the urothelium. The lamina propria and muscle probably have a major role in providing structural support, signals, and nutrients from distal sites. The lamina propria also includes substantial vasculature, such as blood vessels and lymphatics. Including vessels in a model in some form would provide a means to analyse how immune cells infiltrate or exit the bladder. Inclusion of endothelial cells in multi-channel microfluidic models is a reasonable approach to begin to bring this aspect to a bladder model. Inclusion of a muscle layer would be integral to understand how physical forces of contraction affect urothelial integrity, for example. How these characteristics could be incorporated in a fluidics or multichannel model is not clear. As an added challenge, sourcing primary human cells and tissues would be ideal compared with cells lines, as primary material would enable investigation into human variability, but also provide a potential future path for personalized medicine approaches.

Second, simulating stretch is a unique challenge for bladder models, as bladder filling and voiding is not regular and uniform, such as in the lung. This movement probably requires diverse structural and cellular contributions, and potentially some form of innervation to truly recapitulate the mechanism. Cyclic stretch, in which microfluidic control systems are connected to compressed air line and diaphragm vacuum pumps for positive and negative pressure, respectively, attempt to mimic filling and voiding of the bladder, but to date, this approach is too regular to fully recapitulate the unique dynamics of the bladder¹¹.

Third, a microbiome is notably lacking in bladder models. To date, current models, such as the 3D-UHU model, have monocolonised their systems with non-pathogenic bacteria, finding that non-pathogenic *E. coli* can invade the urothelium similarly to UPEC¹⁰. Incorporating a microbiome in bladder models will be especially challenging, as understanding of the bladder microbiome, in health and disease, and between women and men, is still incomplete.
Additionally, atmospheric incubator conditions used to grow and culture most models might not support viability of some or all bladder microbiome-associated-microorganisms, owing to differences between laboratory and intra-tissular conditions.

The fourth major question is how to include immune cells in models, such as Transwell systems, organoids, or organ-on-chip models. These populations are integral to understanding how the bladder responds in disease settings. Neutrophils might be the most straight-forward to include, as they are recruited rapidly to sites of infection and can be easily isolated from human blood. Inclusion of monocytes faces similar challenges, but provided the chemotactic signals are present, they would also be expected to enter organoids or other tissue models. A much larger challenge, particularly for human-based tissue models, is the inclusion of disease-experienced cells, such as T cells, which very probably require signals from the bladder for their development.

Finally, owing to the limited diversity of human material, establishing models to specifically address the influence of biological sex or age on bladder homeostasis and response to disease is challenging. Testing the influence of sex on host response is not impossible, but, by definition, doubles the material needed for experimentation. The addition of steroid hormones, such as testosterone, to experimental set-ups might be a means to circumvent some of these challenges but considering that cells derived from female and male sources probably express different levels of steroid hormone receptors is important.

Given these challenges, concluding that animal models are better for experimental approaches than other models might be tempting, as they enable investigation of physiology and disease physiopathology in a systemic context that has yet to be achieved in 2D or 3D tissue models. However, understanding that animal models cannot fully recapitulate diverse human responses and findings from animal studies will still need to be tested in a human context is important.

Interestingly, bladder tissue from donors with recurrent UTI has been assessed in a few studies⁵⁶,¹²²,¹²³. In one study, biopsies from female patients were obtained and high bacterial burden in the bladder of donors was observed to be associated with an increased risk of recurrent
UTI within 6 months¹²². These biopsies maintained different aspects of the bladder blueprint, including the tissue structure of the lamina propria and urothelium. What elements of the host immune system were present in the samples is unclear; however, imaging or flow cytometry could be employed in future studies to obtain this information. This human *ex vivo* approach is certain to bring new knowledge of bladder biology; however, these types of samples are not available to all researchers and require close clinical collaboration.

[H1] Conclusions

Overall, the techniques and approaches discussed include viable alternatives to animal models and capture specific key aspects of human physiology, such as integration of multiple cell types of human origin, 3D structure, immune cell inclusion, dynamic fluid flow and tissue stretch, local microbiome, and sexual dimorphism. The key to truly dissecting the biology of the bladder will depend upon incorporating as many elements of the bladder as possible, but also recognizing when these efforts hamper the development of a consistent and robust model. With time invested in optimizing these new models, the research community will have improved options to address specific research questions, and the ability to reduce or replace animal experimentation with a more physiologically relevant model system. Future efforts to build these models will provide researchers with new resources to ask a variety of questions, and potentially, support personalized medicine with the possibility to study disease phenotypes and drug responses in a patient-specific manner

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## Key Points

* The bladder has a complex structure with different cells layers and specific functions.

* The bladder is a dynamic organ that undergoes irregular tissue stretch and has fluid flow.

* The bladder contains immune cells and a microbiome, which are key components in the maintenance of homeostasis.
* Many models exist to model specific aspects of healthy and diseased bladders. New models, including organoids and microfluidic devices help to recapitulate aspects of the bladder that are understudied.

* Although great efforts have been invested, physiological human models that include components such as a microbiome or urine, or parameters such as biological sex and age, are still lacking.

* A ‘bladder blueprint’, which considers key parameters of this tissue, will help guide development of more sophisticated models to understand homeostatic and disease-associated mechanisms.

## Acknowledgements

LD received funding from the Agence nationale de la recherche (ANR)-supported Labex Milieu Intérieur (ANR-10-LABX-69-01) and Labex Who Am I (ANR-11-LABX-0071). MD was supported by funding from the ANR (ANR-19-CE15-0015 and ANR-20-PAMR-0001). AC is supported by a PhD research fellowship from the University of Technology Sydney and the PHC FASIC Franco-Australian Hubert Curien Program, implemented jointly by the Ministry of Europe and Foreign Affairs (MEAE) and the Ministry of Higher Education and Research (MESR) in France. MAI is supported by ANR funding (ANR-19-CE15-0015, ANR-20-PAMR-0001, ANR-21-CE15-0006, ANR-22-CE15-0022).

## Author contributions

The authors contributed equally to all aspects of the article.

## Competing interests

The authors declare no competing interests.

## Peer review information

*Nature Reviews Urology* thanks William Kim, Stephane Bolduc, Tahlita Zuiverloon and Vandana Solanki for their contribution to the peer review of this work.
**Figure 1: Specific components to consider when building a bladder model.** (a) The bladder is composed of several distinct layers, including a serosa, muscle layer, and lamina propria, which surround the urothelium. (b) The scheme illustrates the urothelial structure, with a stratified urothelium composed of umbrella, intermediate, and basal cells on a stromal support. The presence of Toll-like receptors, a GAG layer, and tight cell-cell junctions are also important components to incorporate in a model. (c) Additional features to include in a bladder model, depending on the research question, are urine, a bladder microbiome, and components of the immune system. The sex and age of the cells used might affect specific phenotypes and should be considered when interpreting results. Steroid hormone levels also influence the physiology of the bladder, such as the expression of tight junction molecules.

**Figure 2: Advantages and limitations of bladder models.** The models presented were evaluated with respect to how closely they reflect human physiology for the: origin of the cells, presence of a 3D urothelial structure, presence of immune cells, urine tolerance, tissue stretch and fluid flow, and inclusion of biological sex as a variable. Reproducibility was evaluated based on the availability of a detailed protocol and the published use of the protocol or similar models by others.

## Box 1: Organoids

Organoids are cell-based, self-organized 3D tissue models, which recapitulate functional, structural, and biological elements of the corresponding organ or tissue<sup>124</sup>. Organoids can be used for fundamental mechanistic studies of development or for translational and clinical applications in diagnostics and biomarker research, disease modelling, drug discovery, or personalized medicine<sup>125</sup>. Organoids have even been tested for their ability to replace damaged or diseased tissue<sup>126</sup>. To date, organoids for almost all organs and tissues have been developed with defined physicochemical conditions<sup>124</sup>. Organoids are typically evaluated by assessment of their cellular composition, structure, and function. A variety of engineering strategies have emerged to support organoid culture, growth, proliferation, differentiation, and maturation<sup>124</sup>. However, organoids also come with limitations, including that they often lack rare cell types, or
that they do not reflect tissue organization *in vivo*. As more advanced technologies supporting their development arise, organoids will become critical players in studies of homeostasis and disease.

**Box 2: Organ-on-chip**

Organ-on-chips (OoC) are microfluidic devices with networks of hair-fine microchannels¹²⁷. They contain miniature tissues grown with cells cultured in minute volumes of media or other liquids. The cell microenvironment can be entirely controlled, including the speed of fluid flow. Depending on the flexibility of the material and the type of support, microchannels can be stretched to mimic activities such as breathing¹²⁸,¹²⁹. OoC recapitulate one or more tissue-specific functions while being less complex than tissues or organs, making them an ideal model to replace certain aspects of in vivo animal experimentation. The OoC field has grown exponentially, with the development of a large panel of organs to study physiology and pathophysiology of disease and therapeutic approaches¹²⁸⁻¹³³. By chaining chips together, new multi-organ and even body-on-a-chip, and breathing lung-on-a-chip models have been developed¹²⁸⁻¹³³. Given the abundance of models, and the relative cost compared with animal experimentation, OoC technologies are a valuable tool not only to research communities, but also cosmetic, food, and chemical industries. All these applications stand to benefit with respect to production or testing, as society seeks humanized in vitro alternatives to animal testing.

In this Review, the authors present the development, structure and microenvironment, and defence mechanisms of the bladder, which together create a blueprint that can be used to optimize current models or create new approaches.