Isolation, expansion and characterization of porcine urinary bladder smooth muscle cells for tissue engineering

Abstract

BackgroundA 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.MethodFive 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.ResultsEnzymatic 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.ConclusionThe 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.

A Novel in situ Approach to Studying Detrusor Smooth Muscle Cells in Mice

“…For dose-response and time to response curves, data were fitted to a sigmoidal function using the following equation: f(x) = a/(1 + exp (−(x − x°)/b)). To calculate relative activity of individual SMCs, time series were adjusted to fit the 2 8 range, displayed as one pixel row per SMC, and further used for statistical analysis. A smoothing spline function was used to create fit for the SMC relative activity curve.…”

“…In these methods, the urinary bladder is dissected and the urothelial and the serous layer are usually removed 4,5 , since responses to pharmacological stimuli can be altered in the presence of mucosa 6 . Bladder tissue of different species has been investigated using this approach 7,8 .…”

Single-cell Profiling Reveal Types of Interstitial Cells in Human and Rat Bladder

“…This is mainly because of the di cult process of tissue digestion. A large amount of connective tissues are present in the bladder, so it is likely that biases are introduced by the different tissue dissociation protocols used [21,22]. We used a combination of enzymatic dissociation that can avoid biases of the cell populations captured for scRNA-sEq.…”

Innervation: the missing link for biofabricated tissues and organs

“…3m). Engineering a functional bladder would require culture of urothelial cells along with the bladder smooth muscle cells, which can be harvested from different layers of bladder tissue from rodent, porcine, or human sources [190][191][192][193][194] . Moreover, both urothelial cells 195,196 and bladder smooth muscle cells 197,198 can be induced from human PSCs.…”