Skip to main content
SpringerLink
Log in

A Method to Investigate the Epidermal Permeability Barrier In Vitro

  • Protocol
  • First Online:
Molecular Dermatology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2154))

Abstract

The epidermal permeability barrier serves as a multifunctional partition to protect its host from the external environment. Most epidermal permeability barrier studies have been conducted using in vivo human and experimental animals, although some studies have used in vitro cultured cells. There currently is an increased demand for these cultured models, thus avoiding the use of laboratory animals. Here, we first summarize required features that need to be recaptured in cultured keratinocytes for an epidermal permeability barrier study and second, we describe a method for culturing these cells. We also introduce methods to analyze epidermal permeability barrier function using cultured keratinocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Uchida Y (2017) Skin lipids. In: Sakamoto K, Lochhead RY, Maibach H, Yamashita Y (eds) Cosmetic science and technology: theoretical principles and applications. Elsevier, UK, pp 685–698

    Chapter  Google Scholar 

  2. Uchida Y, Park K (2016) Stratum corneum. Immunology of the skin. Springer, Tokyo, pp 15–30

    Book  Google Scholar 

  3. Elias PM (2012) Structure and function of the stratum corneum extracellular matrix. J Invest Dermatol 132:2131–2133

    Article  CAS  Google Scholar 

  4. Elias PM, Menon GK (1991) Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv Lipid Res 24:1–26

    Article  CAS  Google Scholar 

  5. Elias PM, Gruber R, Crumrine D, Menon G, Williams ML, Wakefield JS, Holleran WM, Uchida Y (2014) Formation and functions of the corneocyte lipid envelope (CLE). Biochim Biophys Acta 1841:314–318

    Article  CAS  Google Scholar 

  6. Holleran WM, Takagim Y, Uchida Y (2006) Epidermal sphingolipids: metabolism, function, and role(s) in skin disorders. FEBS Lett 23:5456–5466

    Article  Google Scholar 

  7. Horikoshi T, Igarashi S, Uchiwa H, Brysk H, Brysk MM (1999) Role of endogenous cathepsin D-like and chymotrypsin-like proteolysis in human epidermal desquamation. Br J Dermatol 141:453–459

    Article  CAS  Google Scholar 

  8. Igarashi S, Takizawa T, Yasuda Y, Uchiwa H, Hayashi S, Brysk H, Robinson JM, Yamamoto K, Brysk MM, Horikoshi T (2004) Cathepsin D, but not cathepsin E, degrades desmosomes during epidermal desquamation. Br J Dermatol 151:355–361

    Article  CAS  Google Scholar 

  9. Kishibe M (2019) Physiological and pathological roles of kallikrein-related peptidases in the epidermis. J Dermatol Sci. in press 95:50

    Article  CAS  Google Scholar 

  10. Miyai M, Matsumoto Y, Yamanishi H, Yamamoto-Tanaka M, Tsuboi R, Hibino T (2014) Keratinocyte-specific mesotrypsin contributes to the desquamation process via kallikrein activation and LEKTI degradation. J Invest Dermatol 134:1665–1674

    Article  CAS  Google Scholar 

  11. O’Regan GM, Kemperman PM, Sandilands A, Chen H, Campbell LE, Kroboth K, Watson R, Rowland M, Puppels GJ, McLean WH, Caspers PJ, Irvine AD (2010) Raman profiles of the stratum corneum define 3 filaggrin genotype-determined atopic dermatitis endophenotypes. J Allergy Clin Immunol 126:574–580.e571

    Article  Google Scholar 

  12. Sun R, Celli A, Crumrine D, Hupe M, Adame LC, Pennypacker SD, Park K, Uchida Y, Feingold KR, Elias PM, Ilic D, Mauro TM (2014) Lowered humidity produces human epidermal equivalents with enhanced barrier properties. Tissue Eng Part C Methods 21:15–22

    Article  Google Scholar 

  13. Uchida Y, Behne M, Quiec D, Elias PM, Holleran WM (2001) Vitamin C stimulates sphingolipid production and markers of barrier formation in submerged human keratinocyte cultures. J Invest Dermatol 117:1307–1313

    Article  CAS  Google Scholar 

  14. Hanley K, Jiang Y, Elias PM, Feingold KR, Williams ML (1997) Acceleration of barrier ontogenesis in vitro through air exposure. Pediatr Res 41:293–299

    Article  CAS  Google Scholar 

  15. Prunieras M, Regnier M, Woodley D (1983) Methods for cultivation of keratinocytes with an air-liquid interface. J Invest Dermatol 81:28s–33s

    Article  CAS  Google Scholar 

  16. Williams ML, Hanley K, Elias PM, Feingold KR (1998) Ontogeny of the epidermal permeability barrier. J Invest Dermatol Symp Proc 3:75–79

    Article  CAS  Google Scholar 

  17. Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ (2015) TEER measurement techniques for in vitro barrier model systems. J Lab Autom 20:107–126

    Article  CAS  Google Scholar 

  18. Chen S, Einspanier R, Schoen J (2015) Transepithelial electrical resistance (TEER): a functional parameter to monitor the quality of oviduct epithelial cells cultured on filter supports. Histochem Cell Biol 144:509–515

    Article  CAS  Google Scholar 

  19. Schmuth M, Man MQ, Weber F, Gao W, Feingold KR, Fritsch P, Elias PM, Holleran WM (2000) Permeability barrier disorder in Niemann-Pick disease: sphingomyelin-ceramide processing required for normal barrier homeostasis. J Invest Dermatol 115:459–466

    Article  CAS  Google Scholar 

  20. Honari G, Maibach H (2014) Skin structure and function. In: Dermatotoxicology, 8th edn. Elsevier, UK, pp 1–10

    Google Scholar 

  21. Pinnagoda J, Tupker RA, Agner T, Serup J (1990) Guidelines for transepidermal water loss (TEWL) measurement. A report from the Standardization Group of the European Society of Contact Dermatitis. Contact Dermatitis 22:164–178

    Article  CAS  Google Scholar 

  22. Imhof RE, De Jesus ME, Xiao P, Ciortea LI, Berg EP (2009) Closed-chamber transepidermal water loss measurement: microclimate, calibration and performance. Int J Cosmet Sci 31:97–118

    Article  CAS  Google Scholar 

  23. Crotty Alexander LE, Drummond CA, Hepokoski M, Mathew D, Moshensky A, Willeford A, Das S, Singh P, Yong Z, Lee JH, Vega K, Du A, Shin J, Javier C, Tian J, Brown JH, Breen EC (2018) Chronic inhalation of e-cigarette vapor containing nicotine disrupts airway barrier function and induces systemic inflammation and multiorgan fibrosis in mice. Am J Physiol Regul Integr Comp Physiol 314:R834–R847

    Article  CAS  Google Scholar 

  24. Panisset F, Treffel P, Faivre B, Lecomte PB, Agache P (1992) Transepidermal water loss related to volar forearm sites in humans. Acta Derm Venereol 72:4–5

    CAS  PubMed  Google Scholar 

  25. Hatano Y, Terashi H, Arakawa S, Katagiri K (2005) Interleukin-4 suppresses the enhancement of ceramide synthesis and cutaneous permeability barrier functions induced by tumor necrosis factor-alpha and interferon-gamma in human epidermis. J Invest Dermatol 124:786–792

    Article  CAS  Google Scholar 

  26. Kuntsche J, Bunjes H, Fahr A, Pappinen S, Ronkko S, Suhonen M, Urtti A (2008) Interaction of lipid nanoparticles with human epidermis and an organotypic cell culture model. Int J Pharm 354:180–195

    Article  CAS  Google Scholar 

  27. Pittelkow MR, Scott RE (1986) New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns. Mayo Clin Proc 61:771–777

    Article  CAS  Google Scholar 

  28. Ponec M, Weerheim A, Kempenaar J, Mommaas AM, Nugteren DH (1988) Lipid composition of cultured human keratinocytes in relation to their differentiation. J Lipid Res 29:949–961

    Article  CAS  Google Scholar 

  29. Shin KO, Choe SJ, Uchida Y, Kim I, Jeong Y, Park K (2018) Ginsenoside Rb1 enhances keratinocyte migration by a sphingosine-1-phosphate-dependent mechanism. J Med Food 21:1129–1136

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Ms. Joan Wakefield for superb editorial assistance. This study was supported by UCSF grant #129594A, Leo Pharma sponsor “Full thickness skin models from human pluripotent stem cells for identification and test effectiveness of personalized therapies in atopic dermatitis” UCSF HDFCCC Laboratory for cell analysis (NIH P30CA082103 and S10OD021818-01) to A.C.

Author information

Authors and Affiliations

  1. Northern California Institute for Research and Education, San Francisco, CA, USA

    Yoshikazu Uchida

  2. Department of Dermatology, University of California, San Francisco, CA, USA

    Yoshikazu Uchida & Anna Celli

Authors
  1. Yoshikazu Uchida
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anna Celli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yoshikazu Uchida .

Editor information

Editors and Affiliations

  1. Monasterium Laboratory Skin and Hair Research Solutions GmbH, Münster, Germany

    ​Natalia V. Botchkareva

  2. Centre for Skin Sciences, University of Bradford, Bradford, West Yorkshire, UK

    Gillian E. Westgate

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Uchida, Y., Celli, A. (2020). A Method to Investigate the Epidermal Permeability Barrier In Vitro. In: Botchkareva, ​.V., Westgate, G.E. (eds) Molecular Dermatology. Methods in Molecular Biology, vol 2154. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0648-3_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0648-3_7

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0647-6

  • Online ISBN: 978-1-0716-0648-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation