Full-Thickness Human Skin Equivalent Models of Atopic Dermatitis

  • Gopu SriramEmail author
  • Paul Lorenz Bigliardi
  • Mei Bigliardi-Qi
Part of the Methods in Molecular Biology book series (MIMB, volume 1879)


Atopic dermatitis is a chronic inflammatory skin disease caused by complex multifactorial etiology. In the recent years, there have been significant advances in tissue engineering and the generation of in vitro skin models representative of healthy and diseased states. This chapter describes the methodology for the fabrication of in vitro human skin equivalent (HSE) from human keratinocytes and fibroblasts using a fibrin-based dermal matrix and serum-free culture conditions. Modification of the culture conditions with the supplementation of Th2 cytokines such as interleukin-4 induces the development of atopic dermatitis-like skin model. The chapter also describes the histological and immunohistochemical tools for characterization of the HSE model. The reconstruction of tissue-engineered HSE models that recapitulate the essential features of atopic dermatitis provides powerful tools for deeper understanding of the underlying pathological mechanisms on epidermal level, identification and testing of novel treatment options, and safety and toxicological evaluation in a pathophysiologically relevant system.


3D tissue constructs Atopic dermatitis Fibrin Full-thickness Human skin equivalent Organoids Organotypic culture Tissue engineering 



This work was partially supported by grants from Singapore Ministry of Education, NUS Start Up Grant (R221000118133) to G.S., and A*STAR’s Joint Council Office grant, Singapore [1334K00081] to P.L.B., and M.B. The authors thank Dr. J. Rheinwald (Harvard Medical School, Boston, MA) for his kind gift of immortalized human N/TERT-1 keratinocytes. The authors thank Skin Bank, Institute of Medical Biology, A*STAR for provision of human primary foreskin-derived dermal fibroblasts.


  1. 1.
    Bieber T (2010) Atopic dermatitis. Ann Dermatol 22(2):125–137. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Matsunaga MC, Yamauchi PS (2016) IL-4 and IL-13 inhibition in atopic dermatitis. J Drugs Dermatol 15(8):925–929PubMedGoogle Scholar
  3. 3.
    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):577–591. CrossRefPubMedGoogle Scholar
  4. 4.
    Ewald DA, Noda S, Oliva M, Litman T, Nakajima S, Li X, Xu H, Workman CT, Scheipers P, Svitacheva N, Labuda T, Krueger JG, Suarez-Farinas M, Kabashima K, Guttman-Yassky E (2017) Major differences between human atopic dermatitis and murine models, as determined by using global transcriptomic profiling. J Allergy Clin Immunol 139(2):562–571. CrossRefPubMedGoogle Scholar
  5. 5.
    MacNeil S (2007) Progress and opportunities for tissue-engineered skin. Nature 445(7130):874–880. CrossRefPubMedGoogle Scholar
  6. 6.
    Ponec M (2002) Skin constructs for replacement of skin tissues for in vitro testing. Adv Drug Deliv Rev 54(Suppl 1):S19–S30CrossRefGoogle Scholar
  7. 7.
    Sriram G, Bigliardi PL, Bigliardi-Qi M (2015) Fibroblast heterogeneity and its implications for engineering organotypic skin models in vitro. Eur J Cell Biol 94(11):483–512. CrossRefPubMedGoogle Scholar
  8. 8.
    De Vuyst E, Charlier C, Giltaire S, De Glas V, de Rouvroit CL, Poumay Y (2014) Reconstruction of normal and pathological human epidermis on polycarbonate filter. Methods Mol Biol 1195:191–201. CrossRefPubMedGoogle Scholar
  9. 9.
    Wille JJ, Burdge JJ, Park JY (2014) Methods for the preparation of an autologous serum-free cultured epidermis and for autografting applications. Methods Mol Biol 1195:203–218. CrossRefPubMedGoogle Scholar
  10. 10.
    Auxenfans C, Fradette J, Lequeux C, Germain L, Kinikoglu B, Bechetoille N, Braye F, Auger FA, Damour O (2009) Evolution of three dimensional skin equivalent models reconstructed in vitro by tissue engineering. Eur J Dermatol 19(2):107–113. CrossRefPubMedGoogle Scholar
  11. 11.
    Niehues H, Bouwstra JA, Waheb El Ghalbzouri A, Brandner JM, Zeeuwen P, van den Bogaard EH (2018) 3D skin models for 3R research: the potential of 3D reconstructed skin models to study skin barrier function. Exp Dermatol. CrossRefGoogle Scholar
  12. 12.
    Thakoersing VS, Gooris GS, Mulder A, Rietveld M, El Ghalbzouri A, Bouwstra JA (2012) Unraveling barrier properties of three different in-house human skin equivalents. Tissue Eng Part C Methods 18(1):1–11. CrossRefPubMedGoogle Scholar
  13. 13.
    Benny P, Badowski C, Lane EB, Raghunath M (2015) Making more matrix: enhancing the deposition of dermal-epidermal junction components in vitro and accelerating organotypic skin culture development, using macromolecular crowding. Tissue Eng A 21(1–2):183–192. CrossRefGoogle Scholar
  14. 14.
    Tan KK, Salgado G, Connolly JE, Chan JK, Lane EB (2014) Characterization of fetal keratinocytes, showing enhanced stem cell-like properties: a potential source of cells for skin reconstruction. Stem Cell Rep 3(2):324–338. CrossRefGoogle Scholar
  15. 15.
    van Kilsdonk JW, van den Bogaard EH, Jansen PA, Bos C, Bergers M, Schalkwijk J (2013) An in vitro wound healing model for evaluation of dermal substitutes. Wound Repair Regen 21(6):890–896. CrossRefPubMedGoogle Scholar
  16. 16.
    Rossi A, Appelt-Menzel A, Kurdyn S, Walles H, Groeber F (2015) Generation of a three-dimensional full thickness skin equivalent and automated wounding. J Vis Exp (96).
  17. 17.
    Egles C, Garlick JA, Shamis Y (2010) Three-dimensional human tissue models of wounded skin. Methods Mol Biol 585:345–359. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Bernard FX, Morel F, Camus M, Pedretti N, Barrault C, Garnier J, Lecron JC (2012) Keratinocytes under fire of proinflammatory cytokines: bona fide innate immune cells involved in the physiopathology of chronic atopic dermatitis and psoriasis. J Allergy 2012:718725. CrossRefGoogle Scholar
  19. 19.
    Jean J, Lapointe M, Soucy J, Pouliot R (2009) Development of an in vitro psoriatic skin model by tissue engineering. J Dermatol Sci 53(1):19–25. CrossRefPubMedGoogle Scholar
  20. 20.
    Cornelissen C, Marquardt Y, Czaja K, Wenzel J, Frank J, Lüscher-Firzlaff J, Lüscher B, Baron JM (2012) IL-31 regulates differentiation and filaggrin expression in human organotypic skin models. J Allergy Clin Immunol 129(2):426–433.e428CrossRefGoogle Scholar
  21. 21.
    Hönzke S, Wallmeyer L, Ostrowski A, Radbruch M, Mundhenk L, Schäfer-Korting M, Hedtrich S (2016) Influence of Th2 cytokines on the cornified envelope, tight junction proteins, and β-defensins in filaggrin-deficient skin equivalents. J Invest Dermatol 136(3):631–639CrossRefGoogle Scholar
  22. 22.
    Dancik Y, Sriram G, Rout B, Zou Y, Bigliardi-Qi M, Bigliardi PL (2018) Physical and compositional analysis of differently cultured 3D human skin equivalents by confocal Raman spectroscopy. Analyst 143(5):1065–1076. CrossRefPubMedGoogle Scholar
  23. 23.
    Pageon H (2010) Reaction of glycation and human skin: the effects on the skin and its components, reconstructed skin as a model. Pathol Biol 58(3):226–231. CrossRefPubMedGoogle Scholar
  24. 24.
    Bernerd F, Asselineau D (2008) An organotypic model of skin to study photodamage and photoprotection in vitro. J Am Acad Dermatol 58(5 Suppl 2):S155–S159. CrossRefPubMedGoogle Scholar
  25. 25.
    Toh PP, Bigliardi-Qi M, Yap AM, Sriram G, Stelmashenko O, Bigliardi P (2016) Expression of peropsin in human skin is related to phototransduction of violet light in keratinocytes. Exp Dermatol 25(12):1002–1005. CrossRefPubMedGoogle Scholar
  26. 26.
    Leong C, Bigliardi PL, Sriram G, Au VB, Connolly J, Bigliardi-Qi M (2018) Physiological doses of red light induce IL-4 release in cocultures between human keratinocytes and immune cells. Photochem Photobiol 94(1):150–157. CrossRefPubMedGoogle Scholar
  27. 27.
    Chen CO, Smith A, Liu Y, Du P, Blumberg JB, Garlick J (2017) Photoprotection by pistachio bioactives in a 3-dimensional human skin equivalent tissue model. Int J Food Sci Nutr 1–9. CrossRefGoogle Scholar
  28. 28.
    Commandeur S, van Drongelen V, de Gruijl FR, El Ghalbzouri A (2012) Epidermal growth factor receptor activation and inhibition in 3D in vitro models of normal skin and human cutaneous squamous cell carcinoma. Cancer Sci 103(12):2120–2126. CrossRefPubMedGoogle Scholar
  29. 29.
    Li L, Fukunaga-Kalabis M, Herlyn M (2011) The three-dimensional human skin reconstruct model: a tool to study normal skin and melanoma progression. J Vis Exp (54).
  30. 30.
    Vorsmann H, Groeber F, Walles H, Busch S, Beissert S, Walczak H, Kulms D (2013) Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing. Cell Death Dis 4:e719. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Jannasch M, Groeber F, Brattig NW, Unger C, Walles H, Hansmann J (2015) Development and application of three-dimensional skin equivalents for the investigation of percutaneous worm invasion. Exp Parasitol 150:22–30. CrossRefPubMedGoogle Scholar
  32. 32.
    Haisma EM, de Breij A, Chan H, van Dissel JT, Drijfhout JW, Hiemstra PS, El Ghalbzouri A, Nibbering PH (2014) LL-37-derived peptides eradicate multidrug-resistant Staphylococcus aureus from thermally wounded human skin equivalents. Antimicrob Agents Chemother 58(8):4411–4419. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Haisma EM, Rietveld MH, de Breij A, van Dissel JT, El Ghalbzouri A, Nibbering PH (2013) Inflammatory and antimicrobial responses to methicillin-resistant Staphylococcus aureus in an in vitro wound infection model. PLoS One 8(12):e82800. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Pellegrini G, Ranno R, Stracuzzi G, Bondanza S, Guerra L, Zambruno G, Micali G, De Luca M (1999) The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. Transplantation 68(6):868–879CrossRefGoogle Scholar
  35. 35.
    Ronfard V, Rives JM, Neveux Y, Carsin H, Barrandon Y (2000) Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix. Transplantation 70(11):1588–1598CrossRefGoogle Scholar
  36. 36.
    Sriram G, Alberti M, Dancik Y, Wu B, Wu R, Zhaou F, Ramasamy S, Bigliardi P, Bigliardi-Qi M, Wang ZP (2017) Full thickness human skin-on-chip with enhanced epidermal morphogenesis and barrier function. Mater Today. CrossRefGoogle Scholar
  37. 37.
    Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, Louis DN, Li FP, Rheinwald JG (2000) Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 20(4):1436–1447CrossRefGoogle Scholar
  38. 38.
    Smits JPH, Niehues H, Rikken G, van Vlijmen-Willems I, van de Zande G, Zeeuwen P, Schalkwijk J, van den Bogaard EH (2017) Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models. Sci Rep 7(1):11838. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    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):2448–2459. CrossRefGoogle Scholar
  40. 40.
    van Drongelen V, Danso MO, Mulder A, Mieremet A, van Smeden J, Bouwstra JA, El Ghalbzouri A (2014) Barrier properties of an N/TERT-based human skin equivalent. Tissue Eng A 20(21–22):3041–3049. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  • Gopu Sriram
    • 1
    Email author
  • Paul Lorenz Bigliardi
    • 2
  • Mei Bigliardi-Qi
    • 2
  1. 1.Faculty of DentistryNational University of SingaporeSingaporeSingapore
  2. 2.Department of DermatologyUniversity of MinnesotaMinneapolisUSA

Personalised recommendations