Optimization of porcine urothelial cell cultures: Best practices, recommendations, and threats

Pokrywczyńska, Marta; Czapiewska, Monika; Jundziłł, Arkadiusz; Bodnar, Magdalena; Balcerczyk, Daria; Kloskowski, Tomasz; Nowacki, Maciej; Marszałek, Andrzej

doi:10.1002/cbin.10614

Cited by 14 publications

(13 citation statements)

References 13 publications

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“…Nevertheless, the applied composition of growth factors failed to improve regeneration quality. In this regard, a decellularised scaffold such as BAM or SIS with naturally incorporated active factors within the ECM had a better ability to induce regeneration (Pokrywczynska et al, ). Therefore, it is important to recognise the various signalling pathways regulating specific stages of regeneration.…”

Section: Tissue Engineering Strategies For Urinary Tract Reconstructionmentioning

confidence: 99%

Reconstructive urology and tissue engineering: Converging developmental paths

Adamowicz

Kuffel

Breda

et al. 2019

J Tissue Eng Regen Med

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Reconstructive urology is a complex and demanding branch of modern urology.Complicated cases, necessity of microsurgical approach, and constant exposure to urine make urinary reconstruction especially difficult. With impaired healing, excessive scarring, and recurring fibrosis, functional results are still not satisfying.For better, more successful outcomes, a novel tissue engineering technology-based solutions are being gradually investigated. The use of tissue engineering is the most promising strategy to improve results of reconstructive urology procedures due to possibility of designing organ-specific grafts. Moreover, targeted modification of healing environment by stem cells and growth factors is a unique opportunity that might bring reconstructive urology on molecular level. This review defined limitations and problems encountered in reconstructive urology and discussed relevant tissue engineering-based achievements in the field of urethra, urinary bladder, and ureter regeneration. The background justifying tissue engineering approach to urethra, urinary bladder, and ureter reconstruction was discussed. Then, the wide range of experimental methods utilising biomaterials and cell seeding was deliberated to show readers the current tools offered by tissue engineering. At the end, we characterised major challenges that are needed to be addressed before tissue entering would become standard technology in urological departments.

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Section: Tissue Engineering Strategies For Urinary Tract Reconstructionmentioning

confidence: 99%

Reconstructive urology and tissue engineering: Converging developmental paths

Adamowicz

Kuffel

Breda

et al. 2019

J Tissue Eng Regen Med

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“…On the other hand, collagenase type I has been used for broblast isolation [31] and culture of small intestinal epithelial cells [32]. Dispase II is a collagenase type IV with a mild proteolytic activity that has been used for isolation of labile primary cells such as porcine urothelial cells, lymphatic and embryonic endothelial cells, stem cells and Schwan cells [33][34][35][36]. In addition, protease inhibitors (STI and BSA) is recommended to protected cells from non-speci c proteolytic activity [37] and antioxidant (DTT) preserved in a cellular redox environment [38].…”

Section: Discussionmentioning

confidence: 99%

A Novel Methodology For Isolation and Primary Culture of Swine Gastric Epithelial Cells.

Bautista-Amorocho

Silva-Sayago

Goyeneche-Patino

et al. 2020

Preprint

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Background: Culture of primary epithelial cells has a great advantage over tumor-derived or immortal cells lines since functional phenotype and genetic makeup are preserved. Swine model has proved to be helpful and reliable as a surrogate model in human diseases. Several porcine cell lines have been established from a variety of tissues and shown to extensively contribute to the current understanding of several pathologies, including cancer. However, few protocols for the isolation and culture swine gastric epithelial cells with phenotype preservation have been described. Therefore, the objective of this research was to develop a new methodology for establishing a primary cell culture from the fundic gland area of the porcine stomach.Results: Enzymatic disaggregation of gastric tissue by using a combination of collagenase type I and dispase II, protease inhibitors (soybean trypsin inhibitor and bovine serum albumin), and antioxidants (Dithiothreitol) allowed the isolation of gastric epithelial cells from the fundic gland area with viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM HG, and DMEM/F12 media did not lead to cell adhesion, cluster formation and cell proliferation. By contrast, Williams’ medium supplemented with growth factors supports the confluence and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37oC in a 5% CO2 incubator. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining as well as the expression of MUC1 and MUC20 genes by RT-PCR and DNAc sequencing. Swine Gastric epithelial cells also showed origin-specific markers such as epithelial membrane antigen (EMA), cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) detected by immunohistochemistry and immunofluorescence, respectively. Conclusions: A new methodology was successfully established for the isolation of primary gastric epithelial cells from the fundic gland area in a swine model, based on a combination of tissue specific proteases, protease inhibitors, and antioxidants. The formulation of Williams’ medium with growth factors for epithelial cells supports the adherence and maintains the functional phenotype of primary cells, which was confirmed by mucin production and expression of typical epithelial markers in the long term.

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“…For urothelial cell isolation we used the previously optimized protocol (Pokrywczynska et al, 2016). For urothelial cell isolation we used the previously optimized protocol (Pokrywczynska et al, 2016).…”

Section: Urothelial Cell Isolationmentioning

confidence: 99%

“…In our study p63 protein was chosen as one of the markers, because actually it is the best candidate to show the existence of stem cells in the urothelium (Pignon et al, 2013;Pokrywczynska et al, 2016). Authors hypothesized that observed in vitro organization of urothelial colonies can represent in vivo relationships.…”

Section: Expression Of Selected Markers On Urothelial Holoclonal Mermentioning

confidence: 99%

The different expression of key markers on urothelial holoclonal, meroclonal, and paraclonal cells in in vitro culture

Bühl

Kloskowski

Jundziłł

et al. 2019

Cell Biology International

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Urothelial cell populations which differ in morphology and proliferation capacities can be isolated from the urinary bladder. The goal of this study was to analyze a clonal, proliferative, and self-renewing potential of porcine urothelial cells and to compare expression of selected adhesion and tight junction molecules, urothelial and stem cell markers for the urothelial clone types. Urothelial cells were isolated from 10 porcine urinary bladders. Three different clone types: holoclone-, meroclone-and paraclonelike colonies were identified based on their morphology. To characterize and compare the urothelial clones the immunofluorescent stains were performed. Expression of pancytokeratin (PanCK), Ki-67 and p63 was higher for holoclone-like cells compared to meroclone-and paraclone-like cells (P < 0.05). Meroclone-like cells expressed higher levels of p63 compared to paraclone-like cells (P < 0.05). The level of Ki-67 and PanCK for meroclone-and paraclone-like cells was comparable (P > 0.05). b1 and b4 integrins were not expressed. Expression of zonula occludens-1 (ZO-1) in cell-cell junctions for paraclone-, meroclone-and holoclone-like cells was 17.6 AE 0.6, 14.7 AE 0.5, and 16.1 AE 0.4, respectively. The results of actin filaments (F-actin) expression were 253,634 AE 6,920 for meroclone-like cells, 198,512 AE 7,977 for paraclone-like cells and 133,544 AE 3,169 for holoclone-like cells. Three urothelial cell types with differing features can be isolated from the bladder. Holoclone-like cells are the richest in stem cells and should be used in further studies for construction of neo-bladder or neo-conduit using tissue engineering methods. Supporting InformationAdditional supporting information may be found in the online version of this article at the publisher's web-site. Figure S1. Representative fluorescence signals of integrins b4 (a, d, g), b1 (b e, h) and Merge (c, f, i) in the holoclonelike (a-c), meroclone-like (d-f) and paraclone-like (g-i) cells. Laser scanning confocal microscopy; bar 25 mm.The porcine urothelial cell populations M. Buhl et al.

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Optimization of porcine urothelial cell cultures: Best practices, recommendations, and threats

Cited by 14 publications

References 13 publications

Reconstructive urology and tissue engineering: Converging developmental paths

Reconstructive urology and tissue engineering: Converging developmental paths

A Novel Methodology For Isolation and Primary Culture of Swine Gastric Epithelial Cells.

The different expression of key markers on urothelial holoclonal, meroclonal, and paraclonal cells in in vitro culture

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