title: Isolation, expansion and characterization of porcine urinary bladder smooth muscle cells for tissue engineering.
abstract: BACKGROUND
  A key requirements for therapy utilizing the tissue engineering methodologies is use of techniques which have the capability to yield a high number of cells, from small tissue biopsy in a relatively short time. Up to date there was no optimal methods of isolation and expansion of urinary bladder smooth muscle cells (UB-SMCs). The aim of this study was to compare isolation and expansion techniques of UB-SMCs to select the most repeatable and efficient one.
  
  
  METHODS
  Five protocols of porcine UB- SMCs isolation including enzymatic and explant techniques and three expansion techniques were compared. Isolation effectiveness was evaluated using trypan blue assay. Cell phenotype was confirmed by immunofluorescence staining. Proliferation rate was analyzed using MTT and X- Celligence system. Cellular senescence was assessed measuring β-galactosidase activity.
  
  
  RESULTS
  Enzymatic methods using collagenase with dispase (method I) or collagenase only (method III) allowed to isolate much larger number of cells than the methods using trypsin with collagenase (method II) and collagenase after digestion with trypsin (method IV). The success rate of establishment of primary culture was the highest when the isolated cells were cultured in the Smooth muscle Growth Medium-2 (SmGM-2). Expression of the smooth muscle markers- alpha smooth muscle actin and smoothelin was the highest for cells isolated by enzymatic method I and cultured in SmGM-2. There was no significant signs of cell senescence until the 8th passage.
  
  
  CONCLUSIONS
  The most efficient method of establishment of porcine UB-SMCs culture is enzymatic digestion of urinary bladder tissue with collagenase and dispase and culture of isolated cells in SmGM-2. This method was up to 10 times more efficient than other methods used for isolation and culture of UB-SMCs. This is an easy and consistent method for obtaining high numbers of urinary bladder smooth muscle cells.
authors.affiliation: Department of Regenerative Medicine, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Chair of Urology, Karlowicza 24 Street, 85-092 Bydgoszcz, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Pokrywczynska
authors.affiliation: Department of Regenerative Medicine, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Chair of Urology, Karlowicza 24 Street, 85-092 Bydgoszcz, Poland.
authors.forename: Daria
authors.initials: D
authors.lastname: Balcerczyk
authors.affiliation: Department of Regenerative Medicine, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Chair of Urology, Karlowicza 24 Street, 85-092 Bydgoszcz, Poland.
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Jundzill
authors.affiliation: Chair of Histology and Embryology, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College, Bydgoszcz, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Gagat
authors.affiliation: Department of Regenerative Medicine, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Chair of Urology, Karlowicza 24 Street, 85-092 Bydgoszcz, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Czapiewska
authors.affiliation: Department of Regenerative Medicine, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Chair of Urology, Karlowicza 24 Street, 85-092 Bydgoszcz, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Kloskowski
authors.affiliation: Department of Regenerative Medicine, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Chair of Urology, Karlowicza 24 Street, 85-092 Bydgoszcz, Poland ; Chair of Surgical Oncology, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College, Bydgoszcz, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Nowacki
authors.affiliation: Department of Regenerative Medicine, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Chair of Urology, Karlowicza 24 Street, 85-092 Bydgoszcz, Poland.
authors.forename: Agata M
authors.initials: AM
authors.lastname: Gastecka
authors.affiliation: Department of Clinical Pathomorphology, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College, Bydgoszcz, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Bodnar
authors.affiliation: Chair of Histology and Embryology, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College, Bydgoszcz, Poland.
authors.forename: Alina
authors.initials: A
authors.lastname: Grzanka
authors.affiliation: Department of Clinical Pathomorphology, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College, Bydgoszcz, Poland ; Department of Pathology, Poznan University of Medical Sciences, Poznan, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Marszalek
authors.affiliation: Department of Regenerative Medicine, Nicolaus Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz, Chair of Urology, Karlowicza 24 Street, 85-092 Bydgoszcz, Poland ; Department of Urology, Nicolaus Copernicus Hospital, Torun, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Drewa
keywords: Isolation; Primary culture; Smooth muscle cells; Tissue engineering