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A non-enzymatic method for dissection of mouse bladder urothelial tissue

Nature Protocols volume 14, pages 1280–1292 (2019)Cite this article

Abstract

Urothelial cells contribute to bladder functions, including urine storage, urine emptying, and innate immune response. Functional studies of urothelial cells usually use either freshly isolated cells or cultured cells. Most methods of isolating urothelial cells require enzymes; however, these techniques remove proteins that connect the cells and disrupt the orientation of the cells within the multilayered urothelium. In addition, PCR or immunoblot results obtained from homogenates of bladder mucosa or whole bladder do not represent pure urothelial cells. We describe a dissection process that does not require enzymes and is able to obtain pure urothelial tissues from mice and humans. This method can isolate single urothelial cells for electrophysiology in situ and can also isolate pure urothelial tissue for PCR, microarray, and immunoblot procedures. The time required to obtain urothelial tissue from one mouse bladder is 15–20 min. This method is simple and time efficient as compared with alternative methods and therefore facilitates our understanding of urothelial biology.

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Fig. 1: A cross-section of mouse bladder with H&E staining.
Fig. 2: Adjustment of the dissection forceps.
Fig. 3: Reagents and instruments for perfusion and operation should be positioned so they are all within easy reach during the dissection.
Fig. 4: Mucosa jutting from edge of a bladder piece.
Fig. 5: Dissection of different bladder compartments.
Fig. 6: Dissected urothelial tissue under a microscope.
Fig. 7: Removal of single cell from a sheet of urothelial tissue.
Fig. 8: Uroplakin subunit-Ib (UPIb) is expressed only in urothelial tissue.
Fig. 9: Microscope light sources for the dissection process.

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Acknowledgements

We thank L. Sanders for editing the manuscript, L. Alvarez-Lugo for RT–PCR assistance, J. Lu for narration of the video, and K. Dong for help in taking H&E images. This paper was supported by Yale University institutional funding.

Author information

Authors and Affiliations

  1. Department of Urology, Yale University School of Medicine, New Haven, CT, USA

    Ming Lu, Peter G. Schulam & Toby C. Chai

  2. Department of Urology, Qilu Hospital of Shandong University, Jinan, China

    Kejia Zhu & Toby C. Chai

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Contributions

M.L. and T.C.C. conceived and designed the research. M.L. demonstrated the dissection and edited the video. M.L. and K.Z. performed the experiments. M.L. and T.C.C. drafted the manuscript. M.L., P.G.S., and T.C.C. edited and revised the manuscript. All the authors approved the final version of the manuscript.

Corresponding authors

Correspondence to Ming Lu or Toby C. Chai.

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The authors declare no competing interests.

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Journal peer review information: Nature Protocols thanks David Klumpp and Hiroshi Miyamoto for their contribution to the peer review of this work.

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Related links

Key reference(s) using this protocol

Li, Y., Lu, M., Alvarez-Lugo, L., Chen, G. & Chai, T. C. Neurourol. Urodyn. 36, 1020–1025 (2017): https://doi.org/10.1002/nau.23057

Lu, M. et al. Am. J. Physiol. Cell Physiol. 314, C643–C653 (2018): https://doi.org/10.1152/ajpcell.00339.2017

Acevedo-Alvarez, M. et al. Neurourol. Urodyn. 37, 2398–2405 (2018): https://doi.org/10.1002/nau.23592

Integrated supplementary information

Supplementary Figure 1 Dissected sheet of human urothelial tissue under a microscope.

A, B and C are the same dissected sheet of urothelial tissue from a human bladder at different magnifications. The basal cells are shown facing up. The underlining intermediate cells and umbrella cells could not be seen without removal of the basal cells. Scale bars in: A = 400 µm; B = 200 µm; C = 100 µm. (Human bladder obtained from cystectomy specimens. The Yale IRB approved the protocol.).

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1

Reporting Summary

Supplementary Video 1

How to dissect urothelial tissue from mouse bladder. Part 1: making the dissection ice dish. Supplementary Video 1 © The Author(s), under license to Springer Nature America, Inc. 2019.

Supplementary Video 2

How to dissect urothelial tissue from mouse bladder. Part 2: perfusing the mouse with PBS. Supplementary Video 2 © The Author(s), under license to Springer Nature America, Inc. 2019.

Supplementary Video 3

How to dissect urothelial tissue from mouse bladder. Part 3: dissecting urothelial tissue. Supplementary Video 3 © The Author(s), under license to Springer Nature America, Inc. 2019.

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Lu, M., Zhu, K., Schulam, P.G. et al. A non-enzymatic method for dissection of mouse bladder urothelial tissue. Nat Protoc 14, 1280–1292 (2019). https://doi.org/10.1038/s41596-019-0142-x

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