Summary
The objective of this study is to establish a reliable cell culture system for the long-term culture of rat urothelial cells (RUC), in which the cells multiply in vitro and form stratified polarized urothelium. Urothelial cells were harvested by the enzymatic digestion of the urothelium exposed by the eversion of resected rat bladders. Primary cultures were initiated in keratinocyte serum-free medium (KSFM) for selective proliferation of urothelial cells. Subsequently, the cells were propagated in a mixture of conditioned medium (CM) derived from Swiss 3T3 cell culture supernatant and KSFM (CM-KSEM). Mean population doubling time was 13.8±0.9 h. RUC were successfully maintained for 18 passages over a period of 4–5 mo. Detailed investigations of culture conditions showed that CM-KSFM yielded a differentiated multilayer structure. The stratified urothelial sheets measuring 4×6 cm2 could be formed and then detached using dispase. Cytokeratin pattern in both the cultured urothelial monolayer and engineered stratified layers was similar to those seen in vivo, as assessed with monoclonal antibody against cytokeratin 17. Ultrastructural morphology showed microvilli, basal cell layer, and desmosomes between adjacent cells in the stratified urothelium.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Atala, A.; Freeman, M. R.; Vacanti, J. P.; Shepard, J.; Retik, A. B.. Implantation in vivo and retrieval of artificial structures consisting of rabbit and human urothelium and human bladder muscle. J. Urol. 150:608–612; 1993.
Baskin, L.; Howard, P. S.; Macarak, E.. Effect of physical forces on bladder smooth muscle and urothelium. J. Urol. 150:601–607; 1993.
Chlapowski, F. J. Long term growth and maintenance of stratified rat urothelium in vitro. Cell Tissue Kinet. 22:245–257; 1989.
Fujiyama, C.; Masaki, Z.; Sugihara, H. Reconstruction of the urinary bladder mucosa in three-dimensional collagen gel cultures fibroblast-extracellular matrix interactions on the differentiation of transitional epithelial cells. J. Urol. 153:2060–2067; 1995.
Green, H.; Kehinde, O.; Thomas, J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Natl. Acad. Sci. USA 76:5665–5668; 1979.
Guan, Z. C.; Ricard, G.; Charest-Boule, L.; Neilson, K.; Kuruluta, G. Augmentation cystoplasty in rats: development of an animal model. J. Urol. 144:461–465; 1990.
Guhe, C.; Follmann, W. Growth and characterization of porcine urinary bladder epithelial cells in vitro. Am. J. Physiol. 266:F298-F308; 1994.
Herz, F.; Gazivoda, P.; Papenhausen, P. R.; Katsuyama, J.; Koss, L. G. Normal human urothelial cells in culture. Subculture procedure, flow cytometric and chromosomal analyses. Lab. Invest. 53:571–574; 1985.
Howlett, A. R.; Hodges, G. M.; Rowlatt, C. Epithelial-stromal interactions in the adult bladder: urothelial growth, differentiation, and maturation on culture facsimiles of bladder stroma. Dev. Biol. 118:403–415; 1986.
Hultman, C. S.; Brinson, G. M.; Siltharm, S.; deSerres, S.; Cairns, B. A.; Peterson, H. D.; Meyer, A. A. Allogeneic fibroblasts used to grow cultured epidermal autografts persist in vivo and sensitize the graft recipient for accelerated second-set rejection. J. Trauma 41:51–58; 1996.
Hutton, K. A. R.; Trejdosiewicz, L. K.; Thomas, D. F. M.; Southgate, J. Urothelial tissue culture for bladder reconstruction: an experimental study. J. Urol. 150:721–725; 1993.
Johnson, M. D.; Bryan, G. T.; Reznikoff, C. A. Serial cultivation of normal rat bladder epithelial cells in vitro. J. Urol. 133:1076–1081; 1985.
Leighton, J. Structural biology of epithelial tissue in histophysiologic gradient culture. In Vitro Cell. Dev. Biol. 28A:482–492; 1992.
Ludwikowski, B.; Zhang, Y. Y.; Frey, P. Long-term culture of porcine urothelial cells and induction of urothelial stratification. Br. J. Urol. 84: 507–514; 1999.
Malczak, H. T. The urinary bladder epithelium of the rat. Scanning electron-microscopic study. Acta Anat. 108:89–93; 1980.
Negrete, H. O.; Lavelle, J. P.; Berg, J.; Lewis, S. A.; Zeidel, M. L. Permeability properties of the intact mammalian bladder epithelium. Am. J. Physiol. 271:F886-F895; 1996.
Noguchi, S.; Yura, Y.; Sherwood, E. R.; Kakinuma, H.; Kashihara, N.; Oyasu, R. Stimulation of stromal cell growth by normal rat urothelial cell-derived epidermal growth factor. Lab. Invest. 62:538–544; 1990.
Peter, S. the junctional connections between the cells of the urinary bladder in the rat. Cell Tissue Res. 187:439–448; 1978.
Roszell, A. J.; Douglas, C. J.; Irving, C. C. Polyamine-stimulated growth of rat urinary bladder epithelial cells. Cancer Res. 37:239–243; 1977.
Southgate, J.; Hutton, K. A. R.; Thomas, D. F. M.; Trejdosiewicz, L. K. Methods in laboratory investigation. Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab. Invest. 71:583–594; 1994.
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. J. Urol. 164:951–957; 2000.
Tomson, A. M.; Scholma, J.; Blaauw, E. H. The effect of detachment of cultured epithelial sheets on cellular ultrastructure and organisation. Epithelial Cell Biol. 4:43–51; 1995.
Zhang, Y. Y.; Kropp, B.; Moore, P.; Cowan, R.; Furness III, P. M.; Kolligian, M. E.; Frey, P.; Cheng, E. Y. Co-culture of bladder urothelial and smooth muscle cells on small intestinal submucosa (SIS): potential applications for tissue engineering technology. J. Urol. 164:928–935; 2000.
Zhang, Y. Y.; Leuba, A.; Frey, P. Tissue-engineered human urothelium for urinary tract reconstruction. J. Urol. 161(4) (Suppl.):238; 1999.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zhang, Y.Y., Ludwikowski, B., Hurst, R. et al. Expansion and long-term culture of differentiated normal rat urothelial cells in vitro. In Vitro Cell.Dev.Biol.-Animal 37, 419–429 (2001). https://doi.org/10.1290/1071-2690(2001)037<0419:EALTCO>2.0.CO;2
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1290/1071-2690(2001)037<0419:EALTCO>2.0.CO;2