Three‐Dimensional Tissue Models of Normal and Diseased Skin

Mark W. Carlson1, Addy Alt‐Holland1, Christophe Egles1, Jonathan A. Garlick2

1 School of Dental Medicine, Tufts University, Boston, Massachusetts, 2 School of Engineering, Tufts University, Medford, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.9
DOI:  10.1002/0471143030.cb1909s41
Online Posting Date:  December, 2008
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Abstract

Over the last decade, the development of in vitro, human, three‐dimensional (3D) tissue models, known as human skin equivalents (HSEs), has furthered understanding of epidermal cell biology and provided novel experimental systems. Signaling pathways that mediate the linkage between growth and differentiation function optimally when cells are spatially organized to display the architectural features seen in vivo, but are uncoupled and lost in two‐dimensional culture systems. HSEs consist of a stratified squamous epithelium grown at an air‐liquid interface on a collagen matrix populated with dermal fibroblasts. These 3D tissues demonstrate in vivo–like epithelial differentiation and morphology, and rates of cell division, similar to those found in human skin. This unit describes fabrication of HSEs, allowing the generation of human tissues that mimic the morphology, differentiation, and growth of human skin, as well as disease processes of cancer and wound re‐epithelialization, providing powerful new tools for the study of diseases in humans. Curr. Protoc. Cell Biol. 41:19.9.1‐19.9.17. © 2008 by John Wiley & Sons, Inc.

Keywords: organotypic culture; three‐dimensional model; human skin; wound repair; intraepithelial neoplasia

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction of a Three‐Dimensional Model of Normal Human Skin
  • Support Protocol 1: Inculding Specialized Substrates For Growth of Human Skin Equivalents on Basement membrane or Extracellular Matrix Proteins
  • Alternate Protocol 1: Fabrication of Three‐Dimensional Model of Human Skin Cancer
  • Basic Protocol 2: Fabrication of Three‐Dimensional Wound Healing Model of Human Skin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction of a Three‐Dimensional Model of Normal Human Skin

  Materials
  • Human foreskin fibroblasts
  • Fibroblast culture medium (see recipe)
  • Acellular and cellular collagen matrices (see recipe)
  • Feeder layer of mitotically inactivated mouse 3T3 fibroblasts (e.g., see Rheinwald and Green, )
  • Human neonatal foreskin keratinocytes
  • Keratinocyte culture medium (see recipe)
  • Trypsin/EDTA (see recipe)
  • EDTA/PBS (see recipe)
  • PBS (phosphate‐buffered saline; Invitrogen, cat. no. 14190; also see appendix 2A)
  • Human skin equivalent (HSE) culture media (see Table 19.9.1) including:
    • Epidermalization medium I
    • Epidermalization medium II
    • Cornification medium
  • 10% (v/v) formalin
  • 2 M sucrose
  • Embedding medium: Tissue‐Tek optimal cutting temperature (OCT) medium (Ted Pella)
  • Liquid nitrogen
  • Pipets, chilled 15 min at −20°C before use
  • 10‐cm2 cell culture plates
  • 6‐well tissue culture plate with 3‐µm porous polycarbonate membrane inserts (Organogenesis, cat. no. MS‐10‐3‐5)
  • 15‐ml centrifuge tubes
  • Centrifuge capable of 500‐1000 × g
  • Scalpel
  • Tissue processing cassette
  • Aluminum foil
  • ∼2‐cm bottle or cap (to use as a mold form)
  • Narrow‐tip forceps
  • Metal rack (for embedded tissue)
  • Styrofoam box
  • Additional reagents and equipment for trypsinizing (unit 1.1) and counting (unit 1.1) cells
    Table 9.9.1   Materials   Supplements for HSE Organotypic Media a   Supplements for HSE Organotypic Media

    Ingredient Stock concentration Final concentration Epidermalization I (ml) Epidermalization II (ml) Cornification (ml)
    O1O medium b 363 363 237
    Ham's F12 supplements 120 120 237
    L‐glutamine 200 mM 4 mM 10 10 10
    Adenine b 18 mM 40 µM 1 1 1
    Hydrocortisone b 500× 1 µM 1 1 1
    Triiodothyronine (T3) b 500× 20 nM 1 1 1
    Transferrin c 5 mg/ml 10 µg/ml 1 1 1
    Insulin b 5 mg/ml 10 µg/ml 1 1 1
    Progesterone b 2 µM 2 nM 0.5 0.5 0
    PES c 500× 1 1 1
    Calcium chloride 0.5 M 1.8 mM 0 1.8 1.8
    FBS d 0.5 0.5 10

     aMedia construction for epidermalization I, epidermalization II, and cornification media, including stock concentrations, final concentration, and volume of each supplement for a 500 ml total volume.
     bSee recipe.
     cBiosource.
     dFetal bovine serum (Hyclone, cat. no. 30071). The user is highly encouraged to test different FBS lots to find the optimal one. Lots used successfully by the authors include: ALG14153, AQC23532, AMA15402, and APB20546.

Support Protocol 1: Inculding Specialized Substrates For Growth of Human Skin Equivalents on Basement membrane or Extracellular Matrix Proteins

  Materials
  • AlloDerm (de‐epidermalized basement membrane; LifeCell, cat. no. 102‐009)
  • PBS (phosphate‐buffered saline; Invitrogen, cat. no. 14190; also see appendix 2A)
  • 6‐well cell culture PET membrane inserts coated with the appropriate material:
    • Collagen I (Becton‐Dickinson, cat. no. 354442)
    • Laminin I (Becton‐Dickinson, cat. no. 354446)
    • Fibrillar Collagen I (Becton‐Dickinson, cat. no 354472)
    • Fibronectin/collagen I (Becton‐Dickinson, cat. no 354633)
    • Fibronectin (Becton‐Dickinson, cat. no 354440)
    • Collagen IV (Becton‐Dickinson, cat. no 354544)
  • Serum‐free, low‐glucose Dulbecco's modified Eagle medium (DMEM; Invitrogen)
  • 1.4‐cm stainless steel dermatological punch (Delasco, cat. no. KP‐14)
  • Scalpel

Alternate Protocol 1: Fabrication of Three‐Dimensional Model of Human Skin Cancer

  • Cancer cell line of interest

Basic Protocol 2: Fabrication of Three‐Dimensional Wound Healing Model of Human Skin

  Materials
  • Human skin equivalent (HSE; see protocol 1, steps 1‐18) with keratinocyte cultures at day 7
  • Contracted collagen gel (prepared 1 week in advance of the wounding; protocol 1 steps 1‐9)
  • Cornification medium (see Table 19.9.1)
  • 10‐cm2 sterile tissue culture dish
  • 1.4‐cm stainless steel dermatological punch (Delasco, cat. no. KP‐14), optional
  • Forceps
  • Dental mirror
  • Scalpel with #22 blade, sterile
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Figures

Videos

Literature Cited

Literature Cited
   Andriani, F., Margulis, A., Lin, N., Griffey, S., and Garlick, J.A. 2003. Analysis of microenvironmental factors contributing to basement membrane assembly and normalized epidermal phenotype. J. Invest. Dermatol. 120:923‐931.
   Andriani, F., Garfield, J., Fusenig, N.E., and Garlick, J.A. 2004. Basement membrane proteins promote progression of intraepithelial neoplasia in 3‐dimensional models of human stratified epithelium. Int. J. Cancer 108:348‐357.
   Bissell, M.J. and Radisky, D. 2001 Putting tumours in context. Nat. Rev. Cancer JID 101124168 1:46‐54.
   Carlson, M.W., Iyer, V.R., and Marcotte, E.M. 2007. Quantitative gene expression assessment identifies appropriate cell line models for individual cervical cancer pathways. BMC Genomics 8:117.
   Garlick, J.A. and Taichman, L.B. 1994a. Effect of TGF‐beta 1 on re‐epithelialization of human keratinocytes in vitro: An organotypic model. J. Invest. Dermatol. 103:554‐559.
   Garlick, J.A. and Taichman, L.B. 1994b. Fate of human keratinocytes during re‐epithelialization in an organotypic culture model. Lab. Invest. 70:916‐924.
   Garlick, J.A., Parks, W.C., Welgus, H.G., and Taichman, L.B. 1996. Re‐epithelialization of human oral keratinocytes in vitro. J. Dent. Res. 75:912‐918.
   Hagios, C., Lochter, A., and Bissell, M.J. 1998. Tissue architecture: The ultimate regulator of epithelial function? Philos. Trans. R. Soc. Lond. B. Biol. Sci. 353:857‐70.
   Javaherian, A., Vaccariello, M., Fusenig, N.E., and Garlick, J.A. 1998. Normal keratinocytes suppress early stages of neoplastic progression in stratified epithelium. Cancer Res. 58:2200‐2208.
   Karen, J., Wang, Y., Javaherian, A., Vaccariello, M., Fusenig, N.E., and Garlick, J.A. 1999. 12‐O‐tetradecanoylphorbol‐13‐acetate induces clonal expansion of potentially malignant keratinocytes in a tissue model of early neoplastic progression. Cancer Res. 59:474‐481.
   Kolodka, T.M., Garlick, J.A., and Taichman, L.B. 1998. Evidence for keratinocyte stem cells in vitro: Long‐term engraftment and persistence of transgene expression from retrovirus‐transduced keratinocytes. Proc. Natl. Acad. Sci. U.S.A. 95:4356‐4361.
   Margulis, A., Zhang, W., Alt‐Holland, A., Crawford, H.C., Fusenig, N.E., and Garlick, J.A. 2005. E‐cadherin suppression accelerates squamous cell carcinoma progression in three‐dimensional, human tissue constructs. Cancer Res. 65:1783‐1791.
   Mudgil, A.V., Segal, N., Andriani, F., Wang, Y., Fusenig, N.E., and Garlick, J.A. 2003. Ultraviolet B irradiation induces expansion of intraepithelial tumor cells in a tissue model of early cancer progression. J. Invest. Dermatol. 121:191‐197.
   Rheinwald, J.G. and Green, H. 1975. Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6:331‐344.
   Segal, N., Andriani, F., Pfeiffer, L., Kamath, P., Lin, N., Satyamurthy, K., Egles, C., and Garlick, J. 2008. The basement membrane microenvironment directs the normalization and survival of bioengineered human skin equivalents. Matrix Biol. 27:163‐170.
   Vaccariello, M., Javaherian, A., Wang, Y., Fusenig, N.E., and Garlick, J.A. 1999. Cell interactions control the fate of malignant keratinocytes in an organotypic model of early neoplasia J. Invest. Dermatol. 113:384‐391.
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