First report on perfusion flow on urogenital epithelial cells for urethral tissue engineering purposes

De Graaf P1, Hollis L1, Casademont-Roca A1, Van Dijk C2, Cheng C2, De Kort L1

Research Type

Pure and Applied Science / Translational

Abstract Category

Urethra Male / Female

Best in Category Prize: Urethra Male / Female
Abstract 5
Best Urology
Scientific Podium Session 1
Thursday 8th September 2022
10:05 - 10:20
Hall D
Stem Cells / Tissue Engineering Cell Culture Basic Science Genital Reconstruction
1. UMCU, Department of Urology, Utrecht, the Netherlands, 2. UMCU, Department of Nephrology, Utrecht, the Netherlands
In-Person
Presenter
Links

Abstract

Hypothesis / aims of study
For urethral reconstruction in urethral stricture or hypospadias, over 300 surgical techniques are known. A wide variety of grafts, including autologous skin and oral mucosa, have been used. However, these substitutes are inferior to native urethral tissue and can lead to (recurrent) stricture and/or graft failure. 
Tissue engineering may be a solution for restoration of the urethra in complex stricture or hypospadias cases. This project will focus on the luminal epithelial cells. Previous research has revealed that urethra epithelium is different from bladder epithelium [1]. However, as urethral biopsies to extract urethral cells may lead to stricture formation, bladder cells may be an alternative in urethral tissue engineering.
Epithelium in the urethra is exposed to fluid flow, unlike bladder epithelium, were fluid is more constantly present. Biomechanical cues influence the behavior and differentiation of cells, and our hypothesis is that fluidic flow induced shear stress may stir cells towards different organization. To investigate whether bladder epithelial cells may be used as a substitute for more difficult to obtain urethra epithelial cells for tissue engineering purposes, we compared the cell response to fluidic flow induced shear stress, comparing urethral epithelial cells with bladder epithelial cells.
Study design, materials and methods
Primary cells from both bladder and urethra were isolated from male pigs [2]. Microfluidic devices were used to study effects of shear stress. Pump driven systems (ibidi GmbH, Gräfelfing, Germany) were compared with the F300R microfluidic device (Finnadvance, Oulo, Finland) on the rocking system (WAVE) (FinnAdvance, Oulo, Finland). The bladder and urethra derived epithelial cells were exposed for 72 hours to flow, causing fluidic shear stress (FSS) of τmax= 10.0 dyn/cm2 and τmax= 20.0 dyn/cm2 in a ibidi flow system. 
The F300R microfluidic device was placed on the platform of the rocking machine with the following settings: set timer: 0,00 min; set hold time: 0,29 min; set speed: 30 rpm; and set angle: -18,43º / 18,43º. These settings together with a volume of 240 μL for each channel, results in a FSS of 0,049 dyne/cm2, which is the maximum FSS that the set-up consisting of the F300R microfluidic device and the rocker can generate. 
For both systems, cell elongation, cell alignment and actin fiber alignment were analyzed.
Results
In an ibidi flow system, both urethral cells as well as bladder cells aligned to FSS. No significant difference was observed between urethral and bladder cells in cell elongation and orientation. 
In a FinnAdvance microfluidic system, we could not achieve similar high FSS as in the ibidi systems. Nevertheless we saw that even low FSS improved the formation of a tight monolayer. However no cell alignment in the direction of the flow was observed.
Interpretation of results
Both bladder and urethral epithelial cells similarly adapted to FSS: they both elongate and align in the direction of the flow. So in this respect, both cell types could potentially be used in tissue engineering for urethral reconstruction. This is important information, because the harvesting of bladder epithelium is much easier than of urethra epithelium: bladder epithelial cells can be isolated from urine or bladder washout or may be obtained by biopsy, in contrast to urethral epithelial cells. Next steps in our approach would be creating intermitted flow to mimic natural voiding in patients as well as comparing other sources of cells (like oral keratinocytes) to these urogenital epithelial cells.
Concluding message
Although from different origin, epithelial cells derived from the urethra and bladder behave similarly in vitro under fluid induced shear stress, mimicking urethral fluid flow. Bladder cells, which are more easy to obtain than urethral epithelial cells, may be used as a cell source in tissue engineering for urethral reconstruction.
References
  1. de Graaf P, van der Linde EM, Rosier PFWM, Izeta A, Sievert KD, Bosch JLHR, de Kort LMO. Systematic Review to Compare Urothelium Differentiation with Urethral Epithelium Differentiation in Fetal Development, as a Basis for Tissue Engineering of the Male Urethra. Tissue Eng Part B Rev. 2017 Jun;23(3):257-267. doi: 10.1089/ten.TEB.2016.0352. Epub 2016 Dec 2. PMID: 27809709
  2. Southgate, J., J.R.W. Masters, and L.K. Trejdosiewicz, Culture of Human Urothelium, in Culture of Epithelial Cells, Second Edition, R.I. Freshney and M.G. Freshney, Editors. 2002, Wiley-Liss. p. 381-399.
Disclosures
Funding None Clinical Trial No Subjects None
Citation

Continence 2S2 (2022) 100195
DOI: 10.1016/j.cont.2022.100195

25/10/2024 21:53:42