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Reproducible subcutaneous transplantation of cell sheets into recipient mice

Nature Protocols volume 6, pages 1053–1059 (2011)Cite this article

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A Retraction to this article was published on 25 February 2016

This article has been updated

Abstract

Perfecting tissue engineering and cell sheet transplantation is an important step toward realizing regenerative medicine and is a growing area of research. Before being applied to clinical settings, it is important that these approaches are evaluated in vivo. Here we provide a detailed protocol for handling thin cell sheets, for a simple and highly reproducible subcutaneous transplantation of cell sheets into mice, and for the histological examination of regenerated tissues. Various aspects of transplants can be assessed, such as maintenance, differentiation and proliferation. An emphasis is placed on surgical precision and reproducibility. The resulting consistency between surgeries helps minimize artifacts from surgical variation and therefore enables researchers to not only observe and compare the interactions between host tissues but also to compare transplants among different host animals. A single transplantation can be carried out within ∼10 min.

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Figure 1: Preparation of transplantable cell sheets.
Figure 2: Subcutaneous transplantation.
Figure 3: Tissue preparation for histology.
Figure 4: Histology of regenerated epidermal tissue in subcutaneous space.
Figure 5: Quantitative examination of inflammatory cell reaction.

Change history

  • 13 January 2016

     Masayuki Yamato, Satoshi Tsuneda and Teruo Okano would like to retract this protocol after concerns were raised by the community about some of the figures. Specifically, concerns were raised that the fourth graph in Figure 5a and the first graph in Figure 5b look very similar, and some of the error bars look unevenly positioned. Masayuki Yamato, Satoshi Tsuneda and Teruo Okano have been unable to locate some of the raw data to verify these figures and are no longer confident in the paper's results. Given that these results are key to demonstrating the reliability and reproducibility of the protocol, these authors wish to retract the protocol, and they sincerely apologize for the adverse consequences that may have resulted from its publication. Haruko Obokata could not be reached by the journal for comment on the retraction.

References

  1. Mori, G.G., Moraes, I.G., Nunes, D.C., Castilho, L.R. & Poi, W.R. Biocompatibility of acetazolamide pastes in the subcutaneous tissue of rats. Braz. Dent. J. 20, 17–21 (2009).

    Article  Google Scholar 

  2. Vaisman, B., Motiei, M., Nyska, A. & Domb, A.J. Biocompatibility and safety evaluation of a ricinoleic acid-based poly(ester-anhydride) copolymer after implantation in rats. J. Biomed. Mater. Res. A 92, 419–431 (2010).

    PubMed  Google Scholar 

  3. Boennelycke, M., Christensen, L., Nielsen, L.F., Everland, H. & Lose, G. Tissue response to a new type of biomaterial implanted subcutaneously in rats. Int. Urogynecol. J. Pelvic Floor Dysfunct. 22, 191–196 (2011).

    Article  Google Scholar 

  4. Kim, S.W., Dobratz, E.J., Ballert, J.A., Voglewede, A.T. & Park, S.S. Subcutaneous implants coated with tissue-engineered cartilage. Laryngoscope 119, 62–66 (2009).

    Article  CAS  Google Scholar 

  5. Dressel, R. et al. The tumorigenicity of mouse embryonic stem cells and in vitro differentiated neuronal cells is controlled by the recipients' immune response. PLoS One 3, e2622 (2008).

    Article  Google Scholar 

  6. Aleckovic, M. & Simon, C. Is teratoma formation in stem cell research a characterization tool or a window to developmental biology? Reprod. Biomed. Online 17, 270–280 (2008).

    Article  Google Scholar 

  7. Elloumi-Hannachi, I., Yamato, M. & Okano, T. Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine. J. Intern. Med. 267, 54–70 (2010).

    Article  CAS  Google Scholar 

  8. Okano, T., Yamada, N., Sakai, H. & Sakurai, Y. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J. Biomed. Mater. Res. 27, 1243–1251 (1993).

    Article  CAS  Google Scholar 

  9. Nishida, K. et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N. Engl. J. Med. 351, 1187–1196 (2004).

    Article  CAS  Google Scholar 

  10. Obokata, H. et al. Subcutaneous transplantation of autologous oral mucosal epithelial cell sheets fabricated on temperature-responsive culture dishes. J. Biomed. Mater. Res. A 86, 1088–1096 (2008).

    Article  Google Scholar 

  11. Pirraco, R. et al. Development of osteogenic cell sheets for bone tissue engineering applications. Tissue Eng. Part A 17 published online, doi:10.1089/ten.tea.2010.0470 (2011).

  12. Akizuki, T. et al. Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs. J. Periodontal Res. 40, 245–251 (2005).

    Article  Google Scholar 

  13. Asakawa, N. et al. Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering. Biomaterials 31, 3903–3909 (2010).

    Article  CAS  Google Scholar 

  14. Hasegawa, M., Yamato, M., Kikuchi, A., Okano, T. & Ishikawa, I. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model. Tissue Eng. 11, 469–478 (2005).

    Article  CAS  Google Scholar 

  15. Hayashida, Y. et al. Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface. Invest. Ophthalmol. Vis. Sci. 46, 1632–1639 (2005).

    Article  Google Scholar 

  16. Shimizu, T. et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ. Res. 90, e40 (2002).

    Article  CAS  Google Scholar 

  17. Shiroyanagi, Y., Yamato, M., Yamazaki, Y., Toma, H. & Okano, T. Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps. BJU Int. 93, 1069–1075 (2004).

    Article  CAS  Google Scholar 

  18. Yamato, M. et al. Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature. Tissue Eng. 7, 473–480 (2001).

    Article  CAS  Google Scholar 

  19. Sekine, H. et al. Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts. Circulation 118, S145–S152 (2008).

    Article  CAS  Google Scholar 

  20. Mimura, T. et al. Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest. Ophthalmol. Vis. Sci. 45, 2992–2997 (2004).

    Article  Google Scholar 

  21. Ang, L.P. et al. Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency. Invest. Ophthalmol. Vis. Sci. 51, 758–764 (2010).

    Article  Google Scholar 

  22. Ronford, V.B.H., Mitchell, V., Galizia, J.P., Hochart, D., Chambon, E., Pellerin, P. & Huart, J.J. Use of human keratinocytes cultured on fibrin glue in the treatment og burn wounds. Burns 17, 181–184 (1991).

    Article  Google Scholar 

  23. Barrandon, Y., Li, V. & Green, H. New techniques for the grafting of cultured human epidermal cells onto athymic animals. J. Invest. Dermatol. 91, 315–318 (1988).

    Article  CAS  Google Scholar 

  24. Zbrodowski, A., Marty, F.M., Gumener, R. & Montandon, D. Blood supply of the subcutaneous tissue of the upper limb and its importance in the subcutaneous flap. J. Hand. Surg. Br. 12, 189–193 (1987).

    Article  CAS  Google Scholar 

  25. Zbrodowski, A., Gumener, R., Gajisin, S., Montandon, D. & Bednarkiewicz, M. Blood supply of subcutaneous tissue in the leg and its clinical application. Clin. Anat. 8, 202–207 (1995).

    Article  CAS  Google Scholar 

  26. Alexander, M., Chaudry, I.H. & Schwacha, M.G. Relationships between burn size, immunosuppression, and macrophage hyperactivity in a murine model of thermal injury. Cell Immunol. 220, 63–69 (2002).

    Article  CAS  Google Scholar 

  27. Hudson-Peacock, M.J., Matthews, J.N. & Lawrence, C.M. Relation between size of skin excision, wound, and specimen. J. Am. Acad. Dermatol. 32, 1010–1015 (1995).

    Article  CAS  Google Scholar 

  28. Kayisli, U.A., Berkkanoglu, M., Zhang, L., Kizilay, G. & Arici, A. The broad-spectrum chemokine inhibitor NR58-3.14.3 suppresses the implantation and survival of human endometrial implants in the nude mice endometriosis model. Reprod. Sci. 14, 825–835 (2007).

    Article  Google Scholar 

  29. Shimizu, T. et al. Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. FASEB J. 20, 708–710 (2006).

    Article  CAS  Google Scholar 

  30. Ohashi, K. et al. Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat. Med. 13, 880–885 (2007).

    Article  CAS  Google Scholar 

  31. Kamath, S., Bhattacharyya, D., Padukudru, C., Timmons, R.B. & Tang, L. Surface chemistry influences implant-mediated host tissue responses. J. Biomed. Mater. Res. A 86, 617–626 (2008).

    Article  Google Scholar 

  32. Tang, L. & Eaton, J.W. Inflammatory responses to biomaterials. Am. J. Clin. Pathol. 103, 466–471 (1995).

    Article  CAS  Google Scholar 

  33. Dunning, K. & Safo, A. The ultimate Wright-Giemsa stain: 60 years in the making. Biotech. Histochem. 86, 69–75 (2010).

    Article  Google Scholar 

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Acknowledgements

This study was partially supported by: Grants-in-Aid for JSPS Fellows; the Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology; and the Global COE program. All these grants are from from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Authors and Affiliations

  1. Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan

    Haruko Obokata & Satoshi Tsuneda

  2. Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), Tokyo, Japan

    Haruko Obokata, Masayuki Yamato & Teruo Okano

Authors
  1. Haruko Obokata
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  2. Masayuki Yamato
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  3. Satoshi Tsuneda
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  4. Teruo Okano
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Contributions

H.O. designed and conducted the experiments, analyzed the data and wrote the paper. M.Y. designed experiments, analyzed data and wrote the paper. S.T. and T.O. supervised the project.

Corresponding author

Correspondence to Teruo Okano.

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The authors declare no competing financial interests.

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Obokata, H., Yamato, M., Tsuneda, S. et al. Reproducible subcutaneous transplantation of cell sheets into recipient mice. Nat Protoc 6, 1053–1059 (2011). https://doi.org/10.1038/nprot.2011.356

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