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Determining the origin of cells in tissue engineered skin substitutes: a pilot study employing in situ hybridization

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Abstract

Background

Definitive and high-quality coverage of large and, in particular, massive skin defects remains a significant challenge in burn as well as plastic and reconstructive surgery because of donor site shortage. A novel and promising approach to overcome these problems is tissue engineering of skin. Clearly, before eventual clinical application, engineered skin substitutes of human origin must be grafted and then evaluated in animal models. For the various tests to be conducted it is indispensable to be able to identify human cells as such in culture and also to distinguish between graft and recipient tissue after transplantation. Here we describe a tool to identify human cells in vitro and in vivo.

Methods

In situ hybridization allows for the detection and localization of specific DNA or RNA sequences in morphologically preserved cells in culture or tissue sections, respectively. We used digoxigenin-labeled DNA probes corresponding to human-specific Alu repeats in order to identify human keratinocytes grown in culture together with rat cells, and also to label split and full thickness skin grafts of human origin after transplantation on immuno-incompetent rats.

Results

Digoxigenin-labeled DNA probing resulted in an intensive nuclear staining of human cells, both in culture and after transplantation onto recipient animals, while recipient animal cells (rat cells) did not stain.

Conclusion

In situ hybridization using primate-specific Alu probes reliably allows distinguishing between cells of human and non-human origin both in culture as well as in histological sections. This method is an essential tool for those preclinical experiments (performed on non-primate animals) that must be conducted before novel tissue engineered skin substitutes might be introduced into clinical practice.

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Acknowledgments

This work was supported by a grant from the European Union (EuroSTEC: LSHB-CT-2006-037409) and by the University of Zurich. We are particularly grateful to the Fondation Gaydoul, and the sponsors of DonaTissue (Therese Meier and Robert Zingg), the Vontobel Foundation and the Werner Spross Foundation for their financial support and their interest in our work.

Conflict of interest

The authors declare that none of them has any conflict of interest.

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Authors and Affiliations

  1. Tissue Biology Research Unit, Department of Surgery, University Children’s Hospital Zurich, Steinwiesstrasse 75, 8032, Zurich, Switzerland

    Andreas Daniel Weber, Clemens Schiestl, Martin Meuli & Ernst Reichmann

  2. Tissue Biology Research Unit, Department of Surgery, University Children’s Hospital Zurich, August Forel Strasse 7, 8008, Zurich, Switzerland

    Luca Pontiggia, Thomas Biedermann & Ernst Reichmann

Authors
  1. Andreas Daniel Weber
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  2. Luca Pontiggia
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  3. Thomas Biedermann
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  4. Clemens Schiestl
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  5. Martin Meuli
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  6. Ernst Reichmann
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Corresponding author

Correspondence to Ernst Reichmann.

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Weber, A.D., Pontiggia, L., Biedermann, T. et al. Determining the origin of cells in tissue engineered skin substitutes: a pilot study employing in situ hybridization. Pediatr Surg Int 27, 255–261 (2011). https://doi.org/10.1007/s00383-010-2776-1

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  • DOI: https://doi.org/10.1007/s00383-010-2776-1

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