Reuse of bladder mucosa explants provides a long lasting source of urothelial cells for the establishment of differentiated urothelia
- 108 Downloads
Organ explant cultures are well-established in vitro models that are used to study normal cell biological and regeneration processes as well as carcinogenesis. Primary urothelial cultures from bladder mucosa explants are highly differentiated and are thus broadly used as in vitro experimental equivalents of native urothelial tissue. Since experiments on differentiated urothelial cultures from bladder mucosa explants currently allow only a single use of explants, establishment of sufficient quantities of cultures requires large numbers of sacrificed animals. There is thus a great need for a cheaper approach with less ethical dilemmas. Herein, we demonstrate that mouse bladder mucosa explants can be reused. Reused explants produce outgrowths with highly differentiated urothelia, just like primary explants. Even after being recycled ten times, urothelial outgrowths have the supramolecular and ultrastructural features that are comparable to the native urothelium. Ten times reused explants produce superficial urothelial cells that express uroplakins in the apical plasma membrane, claudin-8 in the tight junctions, and have a subapical network of cytokeratin 20. Basal urothelial cells in urothelial outgrowths of ten times reused explants express p63 which indicates that these urothelial outgrowths have a persistent proliferative capacity. Using our approach, one can perform experiments that were previously not feasible due to low quantities of donor tissue. The method also offers opportunity for effective use of scarce healthy human urothelial tissue.
KeywordsExplant cultures Urothelial model Donor tissue Urinary bladder Microscopy Immunofluorescence
The authors acknowledge the financial support from the Slovenian Research Agency (research core Funding No. P3-0108, Young-researcher funding, and Project No. J3-7494). Thanks also to Sanja Čabraja, Nada Pavlica Dubarič, Linda Štrus, and Sabina Železnik for technical assistance.
This study was funded by Slovenian Research Agency (research core Funding No. P3-0108, Young-researcher funding, and Project No. J3-7494).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
- Acharya P, Beckel J, Ruiz WG, Wang E, Rojas R, Birder L, Apodaca G (2004) Distribution of the tight junction proteins ZO-1, occludin, and claudin-4, -8, and -12 in bladder epithelium. Am J Physiol Renal Physiol 287(2):F305–F318. https://doi.org/10.1152/ajprenal.00341.2003 CrossRefPubMedGoogle Scholar
- Kachar B, Liang F, Lins U, Ding M, Wu XR, Stoffler D, Aebi U, Sun TT (1999) Three-dimensional analysis of the 16 nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation. J Mol Biol 285(2):595–608. https://doi.org/10.1006/jmbi.1998.2304 CrossRefPubMedGoogle Scholar
- Karni-Schmidt O, Castillo-Martin M, Shen TH, HuaiShen T, Gladoun N, Domingo-Domenech J, Sanchez-Carbayo M, Li Y, Lowe S, Prives C, Cordon-Cardo C (2011) Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression. Am J Pathol 178(3):1350–1360. https://doi.org/10.1016/j.ajpath.2010.11.061 CrossRefPubMedPubMedCentralGoogle Scholar
- Kreft ME, Hudoklin S, Sterle M (2005a) Establishment and characterization of primary and subsequent subcultures of normal mouse urothelial cells. Folia Biol (Praha) 51(5):126–132Google Scholar
- Kreft ME, Di Giandomenico D, Beznoussenko GV, Resnik N, Mironov AA, Jezernik K (2010a) Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells. Biol Cell 102(11):593–607. https://doi.org/10.1042/BC20100024 CrossRefPubMedGoogle Scholar
- Višnjar T, Chesi G, Iacobacci S, Polishchuk E, Resnik N, Robenek H, Kreft M, Romih R, Polishchuk R, Kreft ME (2017) Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models. Sci Rep 7(1):12842. https://doi.org/10.1038/s41598-017-13103-x CrossRefPubMedPubMedCentralGoogle Scholar
- Zupančič D, Romih R, Robenek H, Žužek Rožman K, Samardžija Z, Kostanjšek R, Kreft ME (2014) Molecular ultrastructure of the urothelial surface: insights from a combination of various microscopic techniques. Microsc Res Tech 77(11):896–901. https://doi.org/10.1002/jemt.22412 CrossRefPubMedGoogle Scholar