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Engineering a Multilayered Skin Equivalent: The Importance of Endogenous Extracellular Matrix Maturation to Provide Robustness and Reproducibility

  • Lydia Costello
  • Nicola Fullard
  • Mathilde Roger
  • Steven Bradbury
  • Teresa Dicolandrea
  • Robert Isfort
  • Charles Bascom
  • Stefan PrzyborskiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1993)

Abstract

Human skin equivalents (HSEs) are a valuable tool for both academic and industrial laboratories to further the understanding of skin physiology and associated diseases. Over the last few decades, there have been many advances in the development of HSEs that successfully recapitulate the structure of human skin in vitro; however a main limitation is variability due to the use of complex protocols and exogenous extracellular matrix (ECM) proteins. We have developed a robust and unique full-thickness skin equivalent that is highly reproducible due to the use of a consistent scaffold, commercially available cells, and defined low-serum media. The Alvetex® scaffold technology allows fibroblasts to produce their own endogenous ECM proteins within the scaffold, which alleviates the need for exogenous collagen, and supports the differentiation and stratification of the epidermis. Our full-thickness skin equivalent is generated using a detailed step-by-step protocol, which sequentially forms the multilayered structure of human skin in vitro. This model can be adapted for many downstream applications such as disease modeling and testing of active compounds for cosmetics.

Key words

Skin equivalent Organotypic culture Alvetex® scaffold Skin tissue engineering Endogenous extracellular matrix deposition 

References

  1. 1.
    Proksch E, Brandner JM, Jensen JM (2008) The skin: an indispensable barrier. Exp Dermatol 17(12):1063–1072CrossRefGoogle Scholar
  2. 2.
    Brenner M, Hearing VJ (2008) The protective role of melanin against UV damage in human skin. Photochem Photobiol 84(3):539–549CrossRefGoogle Scholar
  3. 3.
    Merkel F (1875) Tastzellen und Tastkörperchen bei den Hausthieren und beim Menschen. Arch Mikrosk Anat 11(1):636–652CrossRefGoogle Scholar
  4. 4.
    Streilein JW, Bergstresser PR (1984) Langerhans cells: antigen presenting cells of the epidermis. Immunobiology 168(3–5):285–300CrossRefGoogle Scholar
  5. 5.
    Eckert RL, Rorke EA (1989) Molecular biology of keratinocyte differentiation. Environ Health Perspect 80:109–116CrossRefGoogle Scholar
  6. 6.
    Rice RH, Green H (1979) Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell 18(3):681–694CrossRefGoogle Scholar
  7. 7.
    Briggaman RA, Wheeler CE (1975) The epidermal-dermal junction. J Investig Dermatol 65(1):71–84CrossRefGoogle Scholar
  8. 8.
    Cotta-Pereira G, Rodrigo G, Bittencourt-Sampaio S (1976) Oxytalan, elaunin, and elastic fibers in the human skin. J Investig Dermatol 66(3):143–148CrossRefGoogle Scholar
  9. 9.
    Wong VW, Sorkin M, Glotzbach JP, Longaker MT, Gurtner GC (2011) Surgical approaches to create murine models of human wound healing. Biomed Res Int 2011:969618Google Scholar
  10. 10.
    Gerber PA, Buhren BA, Schrumpf H, Homey B, Zlotnik A, Hevezi P (2014) The top skin-associated genes: a comparative analysis of human and mouse skin transcriptomes. Biol Chem 395(6):577–591CrossRefGoogle Scholar
  11. 11.
    EU (2009) Regulation (EC) No. 1223/2009 of the European parliament and of the council of 30 November 2009 on cosmetic products (recast). Off J Eur Union L342:59–209Google Scholar
  12. 12.
    Bikle DD, Xie Z, Tu CL (2012) Calcium regulation of keratinocyte differentiation. Expert Rev Endocrinol Metab 7(4):461–472CrossRefGoogle Scholar
  13. 13.
    Rehder J, Souto LRM, Issa CMBM, Puzzi MB (2004) Model of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermis. Sao Paulo Med J 122(1):22–25CrossRefGoogle Scholar
  14. 14.
    Carlson MW, Alt-Holland A, Egles C, Garlick JA (2008) Three-dimensional tissue models of normal and diseased skin. Curr Protoc Cell Biol:19–19Google Scholar
  15. 15.
    Mieremet A, Rietveld M, van Dijk R, Bouwstra JA, El Ghalbzouri A (2018) Recapitulation of native dermal tissue in a full-thickness human skin model using human collagens. Tissue Eng A 24(11–12):873–881CrossRefGoogle Scholar
  16. 16.
    Knight E, Murray B, Carnachan R, Przyborski S (2011) Alvetex®: polystyrene scaffold technology for routine three dimensional cell culture. In: 3D cell culture. Humana Press, New York, NY, pp 323–340CrossRefGoogle Scholar
  17. 17.
    Hill DS, Robinson ND, Caley MP, Chen M, O'Toole EA, Armstrong JL et al (2015) A novel fully humanized 3D skin equivalent to model early melanoma invasion. Mol Cancer Ther 14(11):2665–2673CrossRefGoogle Scholar
  18. 18.
    Ng W, Ikeda S (2011) Standardized, defined serum-free culture of a human skin equivalent on fibroblast-populated collagen scaffold. Acta Derm Venereol 91(4):387–391CrossRefGoogle Scholar
  19. 19.
    Reijnders CM, van Lier A, Roffel S, Kramer D, Scheper RJ, Gibbs S (2015) Development of a full-thickness human skin equivalent in vitro model derived from TERT-immortalized keratinocytes and fibroblasts. Tissue Eng A 21(17–18):2448–2459CrossRefGoogle Scholar
  20. 20.
    Roger M, Fullard N, Costello L, Bradbury S, Markiewicz E, O’Reilly S, Nelson G (2019) Bioengineering the microanatomy of human skin. J Anat 234(4):438–455CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Lydia Costello
    • 1
  • Nicola Fullard
    • 1
  • Mathilde Roger
    • 1
  • Steven Bradbury
    • 1
  • Teresa Dicolandrea
    • 2
  • Robert Isfort
    • 2
  • Charles Bascom
    • 2
  • Stefan Przyborski
    • 1
    • 3
    Email author
  1. 1.Department of BiosciencesDurham UniversityDurhamUK
  2. 2.Mason Business Centre, Procter & GambleMasonUSA
  3. 3.Reprocell EuropeSedgefieldUK

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