Generation of a Functional, Differentiated Porcine Urothelial Tissue In Vitro

Abstract

BackgroundThe primary function of urothelium is to serve as a physical urinary barrier. This function is dependent on features expressed at the molecular level that are acquired during cytodifferentiation. Urothelial cells lose differentiated and functional characteristics when propagated in vitro. ObjectiveTo investigate methods of inducing molecular and functional differentiation of normal porcine urothelial (NPU) cells in vitro. Design and MeasurementsNPU cells were isolated from normal porcine bladders and propagated in a low-calcium keratinocyte serum-free medium. Effects of 5% fetal bovine serum (FBS) and exogenous calcium were investigated. Molecular differentiation was assessed by immunolabelling for urothelial differentiation-associated proteins (UPIIIa, CK20, ZO-1), and barrier function was assessed by measurement of transepithelial electrical resistance (TER). ResultsNPU cell cultures grew as monolayers in low-calcium, serum-free medium. Supplementation with 5% FBS and/or physiological calcium resulted in stratification into basal, intermediate, and superficial cell zones. Superficial cells were positive for UPIIIa, CK20, and ZO-1. TER measurement showed that NPU cells grown with FBS had significantly enhanced barrier function (6720ohms·cm2±1312 SD) compared with cells grown without FBS (102ohms·cm2±34 SD; p<0.001). LimitationsImportantly, our study demonstrates that expression of differentiation-associated immunohistochemical markers by cultured urothelial cells can be regarded as evidence of only morphological differentiation and does not represent a surrogate marker of function. ConclusionsWe have shown that normal porcine bladder urothelium has many cell biological properties equivalent to normal human urothelium, making it an excellent research substitute for difficult-to-obtain tissue. A differentiated, functional barrier urothelium has been produced from porcine bladder urothelial cells propagated in vitro and displays molecular and functional properties equivalent to native urothelium. This tissue has application in developing tissue-engineered bladders with urinary barrier properties and as a research tool for understanding the relationship between molecular and functional tissue differentiation.