title: Optimization of porcine urothelial cell cultures: Best practices, recommendations, and threats.
abstract: Many experimental approaches have been conducted in order to isolate urothelial cells from bladder tissue biopsies, but each method described has utilized different protocols and sources of bladder tissue. In this study, we compared the different methods of urothelial cell isolation available in literature together with standardized methods in order to obtain more unified results. Five methods for primary porcine urothelial culture establishment were compared: tissue explants and four enzymatic methods utilizing collagenase II, dispase II, combination of dispase II and trypsin, and trypsin alone. The average number of isolated cells, cell morphology, success of established culture, average number of cells from the first passage, expression of p63 and pancytokeratin and the characterization of urothelial cell growth, and aging were analyzed during the in vitro culture. The method utilizing dispase II was the most efficient and reproducible method for the isolation and culture of porcine urothelial cells when compared to the other tested methods. Urothelial cells obtained by this method grew considerably well and the cultures were established with high efficiency, which enabled us in obtaining a large quantity of cells with normal morphology. Contamination with fibroblasts in this method was the lowest. The utilization of a proper method for urothelial cell isolation is a critical step in the urinary tract regeneration when using tissue engineering techniques. In summary, this study demonstrated that by utilizing the described method with dispase II, a suitable number of cells was achieved, proving the method useful for tissue regeneration.
authors.affiliation: Chair of Urology, Department of Regenerative Medicine, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Karlowicza 24 Street, Bydgoszcz, 85-092, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Pokrywczynska
authors.affiliation: Chair of Urology, Department of Regenerative Medicine, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Karlowicza 24 Street, Bydgoszcz, 85-092, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Czapiewska
authors.affiliation: Chair of Urology, Department of Regenerative Medicine, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Karlowicza 24 Street, Bydgoszcz, 85-092, Poland.
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Jundzill
authors.affiliation: Department of Clinical Pathomorphology, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Bodnar
authors.affiliation: Chair of Urology, Department of Regenerative Medicine, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Karlowicza 24 Street, Bydgoszcz, 85-092, Poland.
authors.forename: Daria
authors.initials: D
authors.lastname: Balcerczyk
authors.affiliation: Chair of Urology, Department of Regenerative Medicine, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Karlowicza 24 Street, Bydgoszcz, 85-092, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Kloskowski
authors.affiliation: Chair of Urology, Department of Regenerative Medicine, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Karlowicza 24 Street, Bydgoszcz, 85-092, Poland.|Chair and Department of Surgical Oncology, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Oncology Centre Prof, Franciszek Łukaszczyk Memorial Hospital in Bydgoszcz, Bydgoszcz, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Nowacki
authors.affiliation: Department of Clinical Pathomorphology, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland.|Department of Pathology, Poznan University of Medical Sciences, Poznan, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Marszalek
authors.affiliation: Chair of Urology, Department of Regenerative Medicine, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Karlowicza 24 Street, Bydgoszcz, 85-092, Poland.|Department of General and Oncological Urology, Nicolaus Copernicus Hospital, Torun, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Drewa
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods* / Q000592:standards; D002469:Cell Separation / Q000379:methods; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D007150:Immunohistochemistry; D008297:Male; D013552:Swine; D023822:Tissue Engineering / Q000379:methods; D001743:Urinary Bladder / Q000166:cytology; D019459:Urothelium / Q000166:cytology*
keywords: isolation; primary culture; tissue engineering; urothelial cells