Skip to main content

Advertisement

SpringerLink
Log in

How to isolate urothelial cells? Comparison of four different methods and literature review

  • Research Article
  • Published:
Human Cell Aims and scope Submit manuscript

Abstract

The aim of this study is to present the comparison of four different methods for urothelial cell isolation and culture and compare them to methods cited in the literature. Four different techniques were examined for urothelium isolation from rat bladders. Isolation effectiveness was calculated using trypan blue assay. Confirmation of isolated cell phenotype and comparison with native bladder tissue was confirmed using immunohistochemical (IHC), immunocytochemical (ICC) and immunofluorescence (IF) analysis. The method with bladder inversion and collagenase P digestion resulted in the highest number of isolated cells. These cells showed positive expression of cytokeratin 7, 8, 18, α6-integrin and p63. Our results and the literature review showed that the best method for urothelium bladder isolation is dissection of the epithelium layer from other bladder parts and digestion of mechanically prepared tissue in a collagenase solution.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Birder LA, de Groat WC. Mechanisms of disease: involvement of the urothelium in bladder dysfunction. Nat Clin Pract Urol. 2007;4:46–54.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  2. Nagele U, Maurer S, Feil G, Bock C, Krug J, Sievert KD, et al. In vitro investigations of tissue-engineered multilayered urothelium established from bladder washings. Eur Urol. 2008;54:1414–22.

    Article  PubMed  Google Scholar 

  3. van der Kwast TH, van Rooy H, Mulder AH. Establishment and characterization of long-term primary mouse urothelial cell cultures. Urol Res. 1989;17:289–93.

    Article  PubMed  Google Scholar 

  4. Kreft ME, Romih R, Sterle M. Antigenic and ultrastructural markers associated with urothelial cytodifferentiation in primary explants outgrowths of mouse bladder. Cell Biol Int. 2002;26:63–74.

    Article  PubMed  CAS  Google Scholar 

  5. Pariente JL, Bordenave L, Bareille R, Baquey Ch, Guillou ML. Cultured differentiated human urothelial cells in the biomaterials field. Biomaterials. 2000;21:835–9.

    Article  PubMed  CAS  Google Scholar 

  6. Remmele W, Stegner HE. Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue. Pathologe. 1987;8:138–40.

    PubMed  CAS  Google Scholar 

  7. Romih R, Jezernik K, Masera A. Uroplakins and cytokeratins in the regenerating rat urothelium after sodium saccharin treatment. Histeochem Cell Biol. 1998;109:263–9.

    Article  CAS  Google Scholar 

  8. Drewa T, Adamowicz J, Sharma A. Tissue engineering for the oncologic urinary bladder. Nat Rev Urol. 2012;9:561–7.

    Article  PubMed  CAS  Google Scholar 

  9. Subramaniam R, Hinley J, Stahlschmidt J, Southgate J. Tissue engineering potential of urothelial cells from diseased bladders. J Urol. 2011;186:2014–20.

    Article  PubMed  CAS  Google Scholar 

  10. Bajek A, Drewa T, Joachimiak R, Marszałek A, Gagat M, Grzanka A. Stem cells for urinary tract regeneration. Cen Eur J Urol. 2012;65:7–10.

    Article  CAS  Google Scholar 

  11. Rahman Z, Reedy EA, Heatfield BM. Isolation and primary culture of urothelial cells from normal human bladder. Urol Res. 1987;15:315–20.

    Article  PubMed  CAS  Google Scholar 

  12. Petzoldt JL, Leigh IM, Duffy PG, Masters JRW. Culture and characterization of human urothelium in vivo and in vitro. Urol Res. 1994;22:67–74.

    Article  PubMed  CAS  Google Scholar 

  13. Wu Y, Parker L, Binder N, Beckett M, Sinard J, Griffiths C, et al. The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia. Cell. 1982;31:693–703.

    Article  PubMed  CAS  Google Scholar 

  14. Truschel ST, Ruiz WG, Shulman T, Pilewski J, Sun TT, Zeidel ML, et al. Primary uroepithelial cultures. J Biol Chem. 1999;274:15020–9.

    Article  PubMed  CAS  Google Scholar 

  15. Sugasi S, Lesbros Y, Bisson I, Zhang YY, Kucera P, Frey P. In vitro engineering of human stratified urothelium: analysis of its morphology and function. J Urol. 2000;164:951–7.

    Article  PubMed  CAS  Google Scholar 

  16. Zhang YY, Ludwikowski B, Hurst R, Frey P. Expansion and long-term culture of differentiated normal rat urothelial cells in vitro. In Vitro Cell Dev Biol Anima. 2001;37:419–29.

    Article  CAS  Google Scholar 

  17. Southgate J, Masters JRW, Trejdosiewicz LK. Culture of human urothelium. In: Culture of epithelial cells, 2nd edn. Wiley-Liss, New York, 2002.

  18. Fraser M, Thomas DFM, Pitt E, Harnden P, Trejdosiewicz LK, Southgate J. A surgical model of composite cystoplasty with cultured urothelial cells; a controlled study of gross outcome and urothelial phenotype. BJU Int. 2003;93:609–16.

    Article  Google Scholar 

  19. Magnan M, Berthod F, Champigny MF, Soucy F, Bolduc S. In vitro reconstruction of a tissue-engineered endothelialized bladder from a single porcine biopsy. J Pediatr Urol. 2006;2:261–70.

    Article  PubMed  Google Scholar 

  20. Adelöw CAM, Frey P. Synthetic hydrogel matrices for guided bladder tissue regeneration. In: Methods in molecular medicine, Tissue engineering, 2nd edn. Humana, Totowa, 2007.

  21. Cheng Y, Mansfield KJ, Sandow SL, Sadananda P, Burcher E, Moore KH. Porcine bladder urothelial, myofibroblast, and detrusor muscle cells: characterization and ATP relese. Front Pharmacol. 2011;2:1–9.

    Article  CAS  Google Scholar 

  22. Woodman JR, Mansfield KJ, Lazzaro VA, Lynch W, Burcher E, Moore KH. Immunocytochemical characterization of cultures of human bladder mucosal cells. BMC Urol. 2011;11:5.

    Article  PubMed Central  PubMed  Google Scholar 

  23. Shi JG, Fu WJ, Wang XX, Xu YD, Li G, Hong BF, et al. Transdifferentiation of human adipose-derived stem cells into urothelial cells: potential for urinary tract tissue engineering. Cell Tissue Res. 2012;347:737–46.

    Article  CAS  Google Scholar 

  24. Drewa T, Gałązka P, Wolski Z, Prokurat AI, Sir J. Isolation of urothelial cells for tissue engineered bladder augmentation. Ann Diag Pediatr Pathol. 2004;8:95–8.

    Google Scholar 

  25. Kurzrock EA, Lieu DK, de Graffenried LA, Isseroff RR. Rat urothelium: improved techniques for serial cultivation, expansion, freezing and reconstitution onto acellular matrix. J Urol. 2005;173:281–5.

    Article  PubMed  Google Scholar 

  26. Wang CS, Goulet F, Tremblay N, Germain L, Auger F, Tetu B. Selective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer. Pathol Res Pract. 2001;197:175–81.

    Article  PubMed  CAS  Google Scholar 

  27. Masson-Gadais B, Fugere C, Paquet C, Leclerc S, Lefort NR, Germain L, et al. The feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3. J Cell Physiol. 2006;206:831–42.

    Article  PubMed  CAS  Google Scholar 

  28. Zhang YY, Ludwikowski B, Hurst R, Frey P. Expansion and long-term culture of differentiated normal rat urothelial cells in vitro. In Vitro Cell Dev Biol. 2001;37:419–29.

    Article  CAS  Google Scholar 

  29. Thangappan R, Kurzrock EA. Three clonal types of urothelium with different capacities for replication. Cell Prolif. 2009;42:770–9.

    Article  PubMed  CAS  Google Scholar 

  30. Drewa T, Szmytkowska K, Włodarczyk Z, Sir J, Kierzenkowska-Mila C. Does the presence of unwanted dermal fibroblasts limit the usefulness of autologous epidermal keratinocyte grafts? Transplant Proc. 2006;38:3088–91.

    Article  PubMed  CAS  Google Scholar 

  31. Scriven SD, Booth C, Thomas DFM, Trejdosiewicz LK, Southgate J. Reconstitution of human urothelium from monolayer cultures. J Urol. 1997;158:1147–52.

    Article  PubMed  CAS  Google Scholar 

  32. Mudge CS, Klumpp DJ. Induction of the urothelial differentiation program in the absence of stromal cues. J Urol. 2005;174:380–5.

    Article  PubMed  Google Scholar 

  33. Kirk D. Serum free cell culture of normal human urothelium. J Tissue Cult Method. 1985;9:37–42.

    Article  Google Scholar 

  34. Dubeau L, Jones PA. Growth of normal and neoplastic urothelium and response to epidermal growth factor in a defined serum-free medium. Cancer Res. 1987;47:2107–12.

    PubMed  CAS  Google Scholar 

  35. Liao W, Yang S, Song C, Li Y, Meng L, Li X et al. Tissue-engineered tubular graft for urinary diversion after radical cystectomy in rabbits. J Surg Res. 2012; S0022-4804(12)00905-5.

  36. Liao WB, Song C, Li YW, Yang SX, Meng LC, Li XH. Tissue engineered conduit using bladder acellular matrix and bladder epithelial cells for urinary diversion in rabbits. Chin Med J (Engl). 2013;126:335–9.

    Google Scholar 

  37. Fossum M, Gustafson CJ, Nordenskjöld A, Kratz G. Isolation and in vitro cultivation of human urothelial cells from bladder washings of adult patients and children. Scand J Plast Reconstr Surg Hand Surg. 2003;37:41–5.

    Article  PubMed  Google Scholar 

Download references

Acknowledgment

This work was supported by research task within framework of the statutory activities no. 585 from Nicolaus Copernicus University.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Tissue Engineering, Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruń, Karlowicza Str. 24, 85-092, Bydgoszcz, Poland

    T. Kloskowski, M. Uzarska, N. Gurtowska, J. Olkowska, R. Joachimiak, A. Bajek & T. Drewa

  2. Department of Histology and Embryology, Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland

    M. Gagat & A. Grzanka

  3. Department of Clinical Pathomorphology, Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland

    M. Bodnar & A. Marszałek

  4. Urology Department, Nicolaus Copernicus Hospital, Toruń, Poland

    T. Drewa

Authors
  1. T. Kloskowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Uzarska
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. N. Gurtowska
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Olkowska
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. R. Joachimiak
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. Bajek
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M. Gagat
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. Grzanka
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. M. Bodnar
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. A. Marszałek
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. T. Drewa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T. Kloskowski.

Additional information

T. Kloskowski and M. Uzarska have contributed equally to this paper.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kloskowski, T., Uzarska, M., Gurtowska, N. et al. How to isolate urothelial cells? Comparison of four different methods and literature review. Human Cell 27, 85–93 (2014). https://doi.org/10.1007/s13577-013-0070-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13577-013-0070-y

Keywords

Search

Navigation