Biomimetic Urothelial Tissue Models for the in Vitro Evaluation of

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Biomimetic urothelial tissue models for the in vitro evaluation of barrier physiology and bladder drug efficacy Simon Charles Baker, Saqib Shabir, and Jennifer Southgate Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500065m • Publication Date (Web): 03 Apr 2014 Downloaded from http://pubs.acs.org on April 10, 2014

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Molecular Pharmaceutics

Biomimetic urothelial tissue models for the in vitro evaluation of barrier physiology and bladder drug efficacy.

Simon C. Baker*, Saqib Shabir and Jennifer Southgate

Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, Heslington, York YO10 5DD, UK.

*Correspondence to S.C. Baker, Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, Heslington, York YO10 5DD, UK. E-mail: [email protected]

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Abstract The bladder is an important tissue in which to evaluate xenobiotic drug interactions and toxicities due to the concentration of parent drug and hepatic/enteric-derived metabolites in the urine as a result of renal excretion. Breaching of the barrier provided by the bladder epithelial lining (the urothelium) can expose the underlying tissues to urine and cause harmful effects (eg cystitis or cancer). Human urothelium is most commonly represented in vitro as immortalised or established cancer-derived cell lines, but the compromised ability of such cells to undergo differentiation and barrier formation means that non-immortalised, normal human urothelial (NHU) cells provide a more relevant cell culture system. The impressive capacity for urothelial self-renewal in vivo can be harnessed in vitro to generate experimentallyuseful quantities of NHU cells, which can subsequently be differentiated to form a functional or “biomimetic” urothelium. When seeded onto permeable membranes, these barrierforming human urothelial tissue models enable the modelling of serum and luminal (intravesical) exposure to drugs and metabolites, thus supporting efficacy/toxicity assessments. Biomimetic human urothelial constructs provide a potential step along the preclinical trail and may support the extrapolation from rodent in vivo data to determine human relevance. Early evidence is beginning to demonstrate that human urothelium in vitro can provide information that supersedes conventional rodent studies, but further validation is needed to support widespread adoption.


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Keywords: Urinary Tract, Ureter, Toxicology, Pharmacology, Drug Delivery



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Introduction The bladder is an important tissue in which to evaluate xenobiotic drug interactions and toxicities. Parent drug and hepatic/enteric-derived metabolites circulate through the body in the vasculature and are ultimately excreted and concentrated via the kidneys into the urine as the major elimination pathway. The urine can thus contain high concentrations of drug/metabolites that may be stored in contact with the bladder’s epithelial lining (the urothelium) for several hours, potentially causing harmful effects (eg cystitis or cancer). A recent example of this unanticipated problem is the dual-PPARα+γ agonists, known as “glitazars”, which at one stage were being widely developed for the treatment for metabolic syndrome.

The development of these drugs has been largely abandoned due to the

frequent development of urothelial tumours in rodents (reviewed1, 2). In such cases, where controversy exists about drug reactions, mode of action and relevance to man, the availability of suitable ex vivo human urothelial cell/tissue culture systems may be useful for generating specific insight. The urothelium is a transitional epithelium that lines the bladder and associated urinary tract where it functions primarily to prevent the reabsorption of noxious compounds from urine.

As a consequence of tissue specialisations acquired through the process of

cytodifferentiation, the urothelium generates a tight urinary barrier. The paracellular and transcellular components of this barrier are comprised, respectively, of well-developed intercellular tight junctions3 and unique uroplakin-containing plaques of asymmetric unit membrane4, 5 in the lumen-facing superficial cells. Bladder pathologies require targeted therapies and a new generation of intravesicallyadministered drugs is under development,6,

7

along with the relicensing of off-patent


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compounds for bladder instillation.8

Delivering a drug intravesically allows direct

administration, minimising hepatic metabolism, reducing side effects and increasing target tissue exposure. In this context, it is clear that human tissue-specific in vitro “models” of the urothelium could provide critical early information on the selection, efficacy and toxicity of intravesical therapies in advance of clinical trials. However, future evaluation of specific targets using in vitro models will require careful validation to ensure the in situ pathway is recapitulated. Non-immortalised normal human urothelial cells grown as monolayer cultures on tissue culture-treated plastics have been employed to test the reactions to a host of xenobiotic compounds.9,

10

However, once in culture, human urothelial cells rapidly lose their

quiescent, differentiated characteristics, taking on a highly proliferative, non-specialised phenotype that dictates their response.11-13

There is therefore a need to develop

physiologically-representative barrier-forming systems that can be used to provide greater insight into human urothelial tissue biology.

Urothelial culture systems Organ/Explant Culture

The simplest and arguably most biologically-relevant in vitro representation of the urothelium is provided by organ culture.14 The ex vivo culture of small, intact tissue pieces of urothelium with intact stroma can retain much of the in situ tissue architecture for extended periods, including maintenance of a differentiated urothelial phenotype (see Figure 1). Whereas the widespread adoption of organ culture for human studies is largely prevented by the limited supply of tissue,15,

16

the more abundant availability of animal 5

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tissues has enabled organ culture to be used successfully, particularly for exploring epithelial:stromal interdependencies.17, 18 By its nature, organ culture typically represents a model of tissue repair and maintenance rather than an approach to expand the tissue bulk by cell propagation and de novo differentiation. Organ culture of rodent bladder tissue is particularly useful,19 as isolated normal rodent urothelial cells are notoriously difficult to propagate20-23 and as a result, de novo functional differentiation in vitro has proven elusive. As the best in vitro representation of the rodent bladder, organ culture studies provide an important link in bridging the gap between in vivo studies and the understanding of risk to man.24 Although rodent in vivo studies cannot be directly extrapolated to humans due to underlying species differences, this gap may in part be “bridged” through effective combination of in vitro cell/organ culture models from the two species to identify common/unique mechanistic aspects to the biology in question. The basis of this “bridging” concept lies in changing only a single variable at a time: firstly findings from rodent in vivo studies can be translated to a rodent in vitro model, if the effect persists in vitro then species differences can be assessed by transferring to a human in vitro model and finally, assuming the mechanism is conserved, these human in vitro findings can be used to support likely relevance to man. Explant culture is an alternative approach by which primary cultures are established from multiple, finely-chopped fragments of intact tissue.25

Following attachment to the

substrate, primary epithelial cell outgrowth proceeds around each explant until growth becomes established, at which point the explant may be removed. As for organ culture, the explant approach tends to preserve architectural organisation and interrelationships


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between epithelium and stroma, at least in the primary culture. Such cultures may survive passage to secondary cultures and beyond, but the tendency to rely on the use of serumcontaining growth media means that problems of stromal cell overgrowth usually prevail with time. Where secondary (urothelial) cultures have been derived from primary explant cultures of porcine bladder, thicker urothelial tissues were reported to develop in submerged cultures, whereas culture at an air-liquid interface led to a greater extent of apical uroplakin expression.26 Based on these observations, the authors proposed a multistep approach to develop a biomimetic urothelium whereby thick stratified urothelial tissues are first developed in submerged culture, followed by removal of the apical medium to enhance expression and polarity of differentiation markers.26 Air-liquid interface culture has been commonly adopted in bladder organ culture models27, but since the lumen of the bladder is not exposed to air, the physiological relevance of this approach to mechanisms of differentiation/polarisation in situ is unclear. Finally, it is important to remember that any characterisation and interpretation of results must necessarily account for the presence of mixed (urothelial and stromal) cell types in organ/explant cultures. For this latter reason, the development of “pure” urothelial cell cultures, usually incorporating a propagation or expansion phase, is preferred.

Normal human urothelial (NHU) cell culture

Presumptive NHU cell cultures have been derived from exfoliated cells collected in voided patient/donor urine samples28; however, caution must be taken as these cultures represent an epithelial cell population derived primarily from the kidney, but also other regions of the



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urinary tract.29, 30 Indeed, as a long-lived, low turnover epithelium, it is exceedingly unlikely that normal urothelial cells are voided in any useful number in the urine. The best sources of urothelium are surgical resection or cold cut cystoscopic biopsy specimens16 from which the viable urothelium can be isolated efficiently following incubation in EDTA.31, 32 NHU cell culture methods not requiring stromal feeder cells have been adapted from protocols originally established for keratinocytes; these typically use low calcium [0.09mM] keratinocyte serum free medium (KSFM) containing bovine pituitary extract, epidermal growth factor and may be additionally supplemented with cholera toxin to enhance initial cell plating efficiencies.33 Cultured in this artificial environment where there is an absence of differentiation-inducing cues, NHU cells rapidly lose the expression of differentiation markers,32, 34 whilst acquiring a highly proliferative phenotype.35 Thus, NHU cells can be expanded as finite (ie non-immortalised) cell lines with basal epithelial cell characteristics. Such finite cell lines can be sustained in monolayer culture through multiple serial passages (typically around 12); although for experimental purposes, we recommend using lines within 6 passages. The advantage of this approach is that the isolation of the urothelium and subsequent propagation through serial passages in serum-free medium results in the generation of large numbers of NHU cells that are uncontaminated by any non-urothelial (stromal) cell types. As discussed in the next section, an important attribute is that these NHU cells retain the capacity for functional redifferentiation and interestingly, NHU cells adapted to these culture conditions are homogeneous and exhibit the same growth and differentiation potentials irrespective of whether cells are derived from basal or suprabasal compartments in vivo.36


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Functional or “Biomimetic” Urothelium

The advent of permeable membrane culture supports has been crucial in the development of functionally-relevant or “biomimetic” in vitro epithelial models. Culture of epithelia on such supports allows nutrient supply from the basal aspect to replicate delivery via the capillary bed in situ and supports ion channel formation, stratification and the development of polarity (see Figure 1). From a drug efficacy/toxicity viewpoint, membrane cultures allow the modelling of two different routes of epithelial exposure: serum by addition of drug to the basal compartment and luminal/intravesical by adding the compound to the apical chamber. Membrane cultures are inherently more costly and labour intensive to generate and maintain than monolayer cultures, but in the case of biomimetic epithelia, they currently provide the gold standard. Monolayer NHU cell cultures may be stratified in vitro by increasing the exogenous calcium concentration of the medium to near-physiological [2mM]. Following long-term culture of human urothelial cells on permeable membranes under stratifying conditions, expression of a subgroup of urothelial differentiation-associated markers, including cytokeratin 20, has been reported.37-39 By contrast, other reports suggest this approach does not lead to functional urothelial barrier formation, as illustrated by the failure to develop a high transepithelial electrical resistance (TEER) or to limit permeability to molecules such as dextran.40 Although commonly indicated as a marker of urothelial barrier function, the assessment of water/urea permeability needs to be considered in the light of recent evidence of salt-modulated aquaporin expression by differentiated urothelium.11, 41 The differentiation of in vitro-propagated NHU cells to generate barriers with measured TEER of >2kΩ.cm2 has to-date only been achieved reproducibly by variations of one



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method.42 Reported in 2005 by Cross et al., this approach first expands NHU cell numbers by serial growth and passage in serum-free medium, before exposing confluent cultures to serum for several days, prior to passaging cells onto permeable membranes and raising the calcium concentration to 2mM (near physiological).40 By this route, the created urothelium recapitulates the tight epithelial barrier observed in situ, exhibiting high TEER (typically >2kΩ.cm2) and low permeability to dextran and urea.11, 40 This process has also been shown effective with porcine urothelial cells,43 whereas application to rodent urothelial cells in vitro has proved elusive, mainly due to their inefficient sub-culture that necessitates the use of primary cells in culture - often within 96 hours of isolation.20-23 Preliminary studies have demonstrated the successful in vivo transplantation of autologous biomimetic porcine urothelium generated by these techniques in a novel surgical technique for bladder reconstruction, called “composite enterocystoplasty”.44 In the absence of a reliable source of human tissue, porcine urothelial cells provide a useful alternative model which show similar expansion and functional differentiation potential albeit with unknown differences to man. Immortalised human urothelial cell cultures

The burden of ethics, finite lifespan and donor variability associated with the use of normal (non-transformed) cells can cause problems for large scale or industry-based studies. As a result, the immortalisation of urothelial cells has been an attractive goal achieved by incorporating viral oncogenes. However, some caution needs to be taken in interpreting the “normality” of immortalised cell behaviour, as the potential to differentiate and form a functional barrier urothelium appears to be an aspect of normal cell behaviour that is lost as a result of the immortalisation process.45, 46


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Overexpression of human telomerase reverse transcriptase (hTERT) has been proposed to maintain the in situ representation of primary cells whilst combining it with the in vitro immortality of cancer cell lines.

Two groups have independently produced hTERT-

immortalised human urothelial cells, but in both cases it was shown that although immortalisation was achieved without gross karyotypic changes, the differentiation capacity of the cells was compromised, thereby reducing their biological relevance and usefulness.45, 46

Perrone and colleagues described the first barrier-forming urothelial cell culture in 1996 using SV40 immortalised cells from an interstitial cystitis patient.47 The cultures were reported to achieve a trans-epithelial electrical resistance (TEER) of 500-1000 Ω.cm2, which is above the 500 Ω.cm2 threshold used to characterise epithelia as “tight”.48 This result is perhaps surprising on two counts. Firstly, due to the above ascribed loss of differentiation capacity that typically accompanies immortalisation and secondly, because as a chronic inflammatory condition of the bladder, it is considered that an inherently compromised urinary barrier may contribute to the aetiopathology of interstitial cystitis.49 The UROtsa line was also generated by SV40-immortalisation, in this case from paediatric uretericderived urothelial cells and is commonly used to represent “normal” urothelium50,

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,

despite a lack of any functional evidence to support it representing normal human urothelium in situ. The TEU-2 cell line was created by immortalising normal human ureteric cells with an amphotrophic retrovirus encoding the E6 and E7 oncoproteins of human papillomavirus type 16.52 These cells have been shown to express many of the tight junctional proteins associated with barrier function,53 but at the present time functional barrier resistance data is unavailable.



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Summary of urothelial culture systems for drug evaluation

The compromised capacity for differentiation observed in immortalised urothelial cell lines means that at present normal human urothelial (NHU) cells remain the most representative system available. NHU cells have an impressive capacity for self-renewal in vitro which can be harnessed to enable small amounts of urothelium harvested from biopsies (such as those taken by flexible cystoscopy) to be expanded to generate useful quantities of cells for experimental and potentially for tissue engineering purposes.32 Furthermore, the capability retained by in vitro-propagated NHU cells to undergo differentiation supports the practicality of developing biomimetic human urothelial tissue models for targeted drug studies. Despite developing an impressively tight barrier, urothelial constructs created by the Cross method still lack some of the organised features of urothelium in situ, indicating the potential for methodological improvements in promoting tissue polarity and differentiation. In addition, the Cross method is reliant on serum, which is not ideal for proposed clinical applications44 and serum components may also interact with study compounds to modify their efficacy in unpredictable ways. Despite such limitations, recent characterisation of the biomimetic urothelial models has revealed how differentiation-status radically alters cellular phenotype,11-13 opening the potential to use these systems to investigate urothelial-drug interactions.


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Biomimetic Urothelia for Barrier Modulation Studies In situ, the urothelium has a resistance of 2-3 kΩ.cm2 in most mammalian species.4 Maintaining a physical urinary barrier is the primary functional property of urothelium and modulation of barrier tightness may be a useful therapeutic approach in certain pathologies. In cystitis, rapid restitution of the compromised barrier is critical to reducing the pain caused by urine penetration. Conversely, the efficacy of intravesical drug therapies might be improved if the urinary barrier could be temporarily lowered to enhance drug penetration into the urothelium.

The glycosaminoglycan (GAG) layer

Polysaccharides on the luminal surface of the urothelium play an important role as part of the physical barrier, modulating the movement of both charged and uncharged small molecules.54,

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Damage to the GAG layer (possibly due to infection or by chemical

exposure) has been hypothesised to underlie the increased permeability observed in inflammatory uropathies, including interstitial cystitis, and therapy based on the concept of repairing the GAG layer with intravesical instillation (of pentosan polysulphate56, sodium hyaluronate57 or chondroitin sulphate58, alone or in combination59, 60) is common in clinical practice albeit lacking evidence from randomised prospective clinical trials. Intravesical GAG administration is reportedly effective in many cases, with for example sensitivity to potassium reduced in 60% of interstitial cystitis patients administered pentosan polysulphate, suggesting barrier tightness was enhanced.56 The full mechanism of instilled GAG therapies remains to be elucidated since robust in vitro studies using barrier-forming models are only beginning now to be published.



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A recent study of urothelial GAGs suggested chondroitin sulphate was the key luminal GAG on porcine and human urothelium in situ.61

In addition, the study showed TEER

measurements of primary porcine urothelial cultures were reduced by 27% following enzymatic digestion of chondroitin sulphate using chondroitin ABCase, but unfortunately did not include sufficient controls to demonstrate the specificity of the enzymatic digestion or the causal influence of chondroitin sulphate production on TEER.61 This is an exciting preliminary study demonstrating the power of in vitro models of urothelium in this field; however, further investigation is required.

In particular, no existing model of the

urothelium has been capable of surviving the application of urine to its apical aspect for more than a few hours and as GAGs are proposed to provide the first line of defence, it seems right to question whether the current in vitro models truly recapitulate the in situ GAG layer.

Reducing the barrier via actin depolymerisation

TEER is closely regulated by rapid transportation of tight junction proteins (that constitute the paracellular barrier) to and from the membrane, mediated by the actin cytoskeleton. In Madin-Darby canine kidney (MDCK) cells, the induction of actin depolymerisation by latrunculin A (which binds globular actin) led to a drop in TEER caused by dynamin IImediated vesicle endocytosis of tight junction components (including occludin) that could be seen in minutes.62 Since actin polymerisation can be driven by the action of Rhoassociated protein kinase (ROCK) on cofilin; the inhibition of ROCK is an ideal route for temporary disruption of the actin cytoskeleton and the urinary barrier. In support of this concept, preliminary work in our group has shown the ROCK family inhibitors Y27632 and H-


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1152 both inhibit the formation of a urothelial barrier (Figure 2A) and can reduce the TEER of a mature barrier in vitro (Figure 2B). In both cases, these effects are transient and normal barrier function was restored following removal of the drug suggesting this approach has potential as an adjuvant for intravesical therapies that warrants further research.

Inhibition of tight junction formation

Epithelial barrier function might potentially be regulated directly using anti-claudin peptides which mimic a critical extracellular loop. Using a T84 colorectal carcinoma cell model which generates TEERs of ~2kΩ.cm2, Mrsny and colleagues showed that micromolar concentrations of peptides targeting the first extracellular loop of claudin 1 could reversibly rearrange key tight junction proteins to prevent TEER exceeding the low tens of ohms.63 This claudin 1-targeting peptide was also shown to bind to claudin 3,63 suggesting it might have more general utility as a tight junction inhibitor. As claudin 3 is expressed specifically in urothelium in association with the superficial cell terminal tight junction,3 this suggests a specific potential use in the bladder as an adjunct to intravesical drug delivery.

Conclusions Physiologically-representative, barrier-forming in vitro models of human urothelia are beginning to be available from research studies. The need to develop relevant human systems to replace the use of animals in drug and toxicity testing is widely prescribed by policy directives, with recent adoption of in vitro skin models in irritation studies64, 65 paving the way for use of in vitro models in other areas of toxicology. Early evidence is beginning to demonstrate that a human urothelial in vitro approach can indeed provide information



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that supersedes conventional rodent studies,12,

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but nevertheless, further validation,

leading to endorsement by drug agencies, will be required before widespread adoption. An intermediate position may be to carry out parallel studies using rodent in vivo and organ culture models with biomimetic human urothelial constructs as a bridge to extrapolate in vivo to in vitro and from rodent to man.


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37. Feil, G.; Maurer, S.; Nagele, U.; Krug, J.; Bock, C.; Sievert, K. D.; Stenzl, A. Immunoreactivity of p63 in monolayered and in vitro stratified human urothelial cell cultures compared with native urothelial tissue. Eur Urol 2008, 53, (5), 1066-72. 38. Nagele, U.; Maurer, S.; Feil, G.; Bock, C.; Krug, J.; Sievert, K. D.; Stenzl, A. In vitro investigations of tissue-engineered multilayered urothelium established from bladder washings. Eur Urol 2008, 54, (6), 1414-22. 39. Sugasi, S.; Lesbros, Y.; Bisson, I.; Zhang, Y. Y.; Kucera, P.; Frey, P. In vitro engineering of human stratified urothelium: analysis of its morphology and function. The Journal of urology 2000, 164, (3 Pt 2), 951-7. 40. Cross, W. R.; Eardley, I.; Leese, H. J.; Southgate, J. A biomimetic tissue from cultured normal human urothelial cells: analysis of physiological function. Am J Physiol Renal Physiol 2005, 289, (2), F459-68. 41. Rubenwolf, P. C.; Georgopoulos, N. T.; Clements, L. A.; Feather, S.; Holland, P.; Thomas, D. F.; Southgate, J. Expression and localisation of aquaporin water channels in human urothelium in situ and in vitro. Eur Urol 2009, 56, (6), 1013-23. 42. Cross, W. R.; Southgate, J. Biomimetic Urothelium. US2005233445 (A1) 2003. 43. Turner, A. M.; Subramaniam, R.; Thomas, D. F.; Southgate, J. Generation of a functional, differentiated porcine urothelial tissue in vitro. Eur Urol 2008, 54, (6), 1423-32. 44. Turner, A.; Subramanian, R.; Thomas, D. F.; Hinley, J.; Abbas, S. K.; Stahlschmidt, J.; Southgate, J. Transplantation of autologous differentiated urothelium in an experimental model of composite cystoplasty. Eur Urol 2011, 59, (3), 447-54. 45. Chapman, E. J.; Hurst, C. D.; Pitt, E.; Chambers, P.; Aveyard, J. S.; Knowles, M. A. Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway. Oncogene 2006, 25, (36), 5037-45. 46. Georgopoulos, N. T.; Kirkwood, L. A.; Varley, C. L.; MacLaine, N. J.; Aziz, N.; Southgate, J. Immortalisation of normal human urothelial cells compromises differentiation capacity. Eur Urol 2011, 60, (1), 141-9. 47. Perrone, R. D.; Johns, C.; Grubman, S. A.; Moy, E.; Lee, D. W.; Alroy, J.; Sant, G. R.; Jefferson, D. M. Immortalized human bladder cell line exhibits amiloride-sensitive sodium absorption. Am J Physiol 1996, 270, (1 Pt 2), F148-53. 48. Fromter, E.; Diamond, J. Route of passive ion permeation in epithelia. Nat New Biol 1972, 235, (53), 9-13. 49. Southgate, J.; Varley, C. L.; Garthwaite, M. A.; Hinley, J.; Marsh, F.; Stahlschmidt, J.; Trejdosiewicz, L. K.; Eardley, I. Differentiation potential of urothelium from patients with benign bladder dysfunction. BJU international 2007, 99, (6), 1506-16. 50. Rossi, M. R.; Masters, J. R.; Park, S.; Todd, J. H.; Garrett, S. H.; Sens, M. A.; Somji, S.; Nath, J.; Sens, D. A. The immortalized UROtsa cell line as a potential cell culture model of human urothelium. Environ Health Perspect 2001, 109, (8), 801-8. 51. Petzoldt, J. L.; Leigh, I. M.; Duffy, P. G.; Sexton, C.; Masters, J. R. Immortalisation of human urothelial cells. Urological research 1995, 23, (6), 377-80. 52. Klumpp, D. J.; Weiser, A. C.; Sengupta, S.; Forrestal, S. G.; Batler, R. A.; Schaeffer, A. J. Uropathogenic Escherichia coli potentiates type 1 pilus-induced apoptosis by suppressing NFkappaB. Infect Immun 2001, 69, (11), 6689-95. 53. Rickard, A.; Dorokhov, N.; Ryerse, J.; Klumpp, D. J.; McHowat, J. Characterization of tight junction proteins in cultured human urothelial cells. In Vitro Cell Dev Biol Anim 2008, 44, (7), 261-7. 54. Lilly, J. D.; Parsons, C. L. Bladder surface glycosaminoglycans is a human epithelial permeability barrier. Surg Gynecol Obstet 1990, 171, (6), 493-6. 55. Parsons, C. L.; Boychuk, D.; Jones, S.; Hurst, R.; Callahan, H. Bladder surface glycosaminoglycans: an epithelial permeability barrier. The Journal of urology 1990, 143, (1), 139-42.



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56. Parsons, C. L.; Forrest, J.; Nickel, J. C.; Evans, R.; Lloyd, L. K.; Barkin, J.; Mosbaugh, P. G.; Kaufman, D. M.; Hernandez-Graulau, J. M.; Atkinson, L.; Albrecht, D. Effect of pentosan polysulfate therapy on intravesical potassium sensitivity. Urology 2002, 59, (3), 329-33. 57. Morales, A.; Emerson, L.; Nickel, J. C.; Lundie, M. Intravesical hyaluronic acid in the treatment of refractory interstitial cystitis. The Journal of urology 1996, 156, (1), 45-8. 58. Nickel, J. C.; Egerdie, R. B.; Steinhoff, G.; Palmer, B.; Hanno, P. A multicenter, randomized, double-blind, parallel group pilot evaluation of the efficacy and safety of intravesical sodium chondroitin sulfate versus vehicle control in patients with interstitial cystitis/painful bladder syndrome. Urology 2010, 76, (4), 804-9. 59. Cervigni, M.; Natale, F.; Nasta, L.; Padoa, A.; Voi, R. L.; Porru, D. A combined intravesical therapy with hyaluronic acid and chondroitin for refractory painful bladder syndrome/interstitial cystitis. Int Urogynecol J Pelvic Floor Dysfunct 2008, 19, (7), 943-7. 60. Porru, D.; Leva, F.; Parmigiani, A.; Barletta, D.; Choussos, D.; Gardella, B.; Dacco, M. D.; Nappi, R. E.; Allegri, M.; Tinelli, C.; Bianchi, C. M.; Spinillo, A.; Rovereto, B. Impact of intravesical hyaluronic acid and chondroitin sulfate on bladder pain syndrome/interstitial cystitis. Int Urogynecol J 2012, 23, (9), 1193-9. 61. Janssen, D. A.; van Wijk, X. M.; Jansen, K. C.; van Kuppevelt, T. H.; Heesakkers, J. P.; Schalken, J. A. The distribution and function of chondroitin sulfate and other sulfated glycosaminoglycans in the human bladder and their contribution to the protective bladder barrier. The Journal of urology 2013, 189, (1), 336-42. 62. Shen, L.; Turner, J. R. Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis. Mol Biol Cell 2005, 16, (9), 3919-36. 63. Mrsny, R. J.; Brown, G. T.; Gerner-Smidt, K.; Buret, A. G.; Meddings, J. B.; Quan, C.; Koval, M.; Nusrat, A. A key claudin extracellular loop domain is critical for epithelial barrier integrity. The American journal of pathology 2008, 172, (4), 905-15. 64. Cotovio, J.; Grandidier, M. H.; Portes, P.; Roguet, R.; Rubinstenn, G. The in vitro skin irritation of chemicals: optimisation of the EPISKIN prediction model within the framework of the ECVAM validation process. Altern Lab Anim 2005, 33, (4), 329-49. 65. Spielmann, H.; Hoffmann, S.; Liebsch, M.; Botham, P.; Fentem, J. H.; Eskes, C.; Roguet, R.; Cotovio, J.; Cole, T.; Worth, A.; Heylings, J.; Jones, P.; Robles, C.; Kandarova, H.; Gamer, A.; Remmele, M.; Curren, R.; Raabe, H.; Cockshott, A.; Gerner, I.; Zuang, V. The ECVAM international validation study on in vitro tests for acute skin irritation: report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test. Altern Lab Anim 2007, 35, (6), 559-601. 66. Shabir, S.; Cross, W.; Kirkwood, L. A.; Pearson, J. F.; Appleby, P. A.; Walker, D.; Eardley, I.; Southgate, J. Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium. American journal of physiology. Renal physiology 2013, 305, (3), F396-406.


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Figure Legends Figure 1 – Illustrates a human ureteric organ culture and a “biomimetic” urothelium developed from human ureteric urothelial cells. (A) Organ cultures are routinely performed at an air-liquid interface and maintain the interactions of the stroma with the urothelium. Urothelial outgrowth occurs from the edges of the tissue to cover the exposed substrate. Explant culture is a variation on this method where the tissue is minced into many smaller pieces and the outgrowth used to establish subcultures. The haematoxylin and eosin stained image shows a piece of opened human ureter that was organ cultured for 17 days. (B) “Biomimetic” urothelium is routinely cultured submerged and develops a multi-layered, barrier forming epithelium with morphological similarities to the native tissue. Scale bar denotes 100µm and applies to both haematoxylin and eosin images. Figure 2 – Effect of Rho kinase inhibitors on barrier formation (A) and a mature tight barrier (B) developed following the Cross method.40 Both Rho kinase inhibitors prevented barrier development whilst cells were exposed but this inhibition was rapidly relieved following removal of the drug. Both Rho kinase inhibitors also reduced the tightness of a mature (>3 kΩ.cm2) barrier.



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TOC Figure 30x10mm (300 x 300 DPI)


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Figure 1 – Illustrates a human ureteric organ culture and a “biomimetic” urothelium developed from human ureteric urothelial cells. (A) Organ cultures are routinely performed at an air-liquid interface and maintain the interactions of the stroma with the urothelium. Urothelial outgrowth occurs from the edges of the tissue to cover the exposed substrate. Explant culture is a variation on this method where the tissue is minced into many smaller pieces and the outgrowth used to establish subcultures. The haematoxylin and eosin stained image shows a piece of opened human ureter that was organ cultured for 17 days. (B) “Biomimetic” urothelium is routinely cultured submerged and develops a multi-layered, barrier forming epithelium with morphological similarities to the native tissue. Scale bar denotes 100µm and applies to both haematoxylin and eosin images. 90x44mm (300 x 300 DPI)



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Figure 2 – Effect of Rho kinase inhibitors on barrier formation (A) and a mature tight barrier (B) developed following the Cross method.40 Both Rho kinase inhibitors prevented barrier development whilst cells were exposed but this inhibition was rapidly relieved following removal of the drug. Both Rho kinase inhibitors also reduced the tightness of a mature (>3 kΩ.cm2) barrier. 152x152mm (300 x 300 DPI)


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Biomimetic Urothelial Tissue Models for the in Vitro Evaluation of

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