Optimization of porcine urothelial cell cultures: Best practices, recommendations and threat
Authors
Marta Pokrywczynska, Monika Czapiewska, Arkadiusz Jundzill, Magdalena Bodnar, Daria Balcerczyk, Tomasz Kloskowski, Maciej Nowacki, Andrzej Marszalek, Tomasz Drewa
Institution
Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz
Country
Poland
Year
2016
Journal
Cell Biology International
Abstract
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 1st 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 .