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Skin reconstruct models and melanoma seeded together onto the dermis equiv a l e n t .
A l t e r n a t ive l y, co-cultures of ke r a t i n o cytes and m e l a n o cytes can be used. In general, approximately5 x 1 05 cells per cm2 dermis equivalent are seeded and U n d i fferentiated ke r a t i n o cytes are used for the left under submerged culture conditions in low calcium, reconstruction of the epidermis equivalent. Melanocytes low serum medium for 3 to 6 days. Subsequent exposure are mixed with ke r a t i n o cytes in a ratio 1:3 to 1:20 and to air induces a differentiation process in thekeratinocytes leading to the formation of multiple layerspiling up on top of each other. The different layers of anormal stratified epithelium are described in Fig. 2A. Amacroscopic view of a pigmented skin reconstruct after2 weeks of culture is demonstrated in Fig. 2C. Themelanocytes are found in the basal layer as single cellssurrounded by the neighboring ke r a t i n o cytes (Fig. 2D).
The melanocytic dendrites extend into the upper layersof the epidermis and pigment is transferred to thekeratinocytes (Archambault et al., 1995). Recently, it hasbeen shown that melanocytes in skin reconstructs protectepidermal basal cells from UVB-induced apoptosis(Cario-Andre et al., 2000). Grafting of skin reconstructs
Skin reconstructs are being used in medicine for covering wounds of patients (Kirsner, 1998). While skinreconstructs in vitro h ave a maximum life-span ofapproximately one month, survival is increased tos everal months or years when grafted to living hosts.
Long-term studies have become possible, which isparticularly of interest for carcinogenesis studies( J avaherian et al., 1998). Fig. 2E shows a pigmentedhuman skin reconstruct on an immunodeficient albinomouse. Histological and immunohistochemical analysesrevealed that: i) host vessels and immune cells infiltratedthe dermis of the grafts, ii) a papillary morphology of theupper dermis developed, iii) the differentiation of theepidermis improved, and iv) the basement membranematured (Fig. 2F). Obviously, not yet defined diffusiblefactors from the host's microenvironment support themorphology and viability of the grafted skinreconstructs. Melanoma in skin reconstructs
When melanoma cells are incorporated into skin reconstructs, a progression stage-related growth patterncan be observed (Bechetoille et al., 2000; Eves et al.,2000; Meier et al., 2000). While cells derived from RGPprimary melanomas or non-aggressive melanoma celllines are found as single cells or clusters throughout theepidermis, cells derived from VGP primary melanomasor aggressive metastatic melanoma cell lines traverse thebasement membrane and invade the dermis equiva l e n t(Fig. 2G,H). Competence for metastasis and long-termg r owth properties may be tested when melanoma-containing skin reconstructs are grafted to living hosts,for example, to immunodeficient mice. Induced expression of invasion-related adhesion Fig. 1. Schematic of the sequential steps of skin reconstruction using
type I collagen with embedded fibroblasts as dermis equivalent.
molecules, such as melanoma cell adhesion molecule

Skin reconstruct models and melanoma Fig. 2. Examples
of human skin
reconstructs.
A. Normal human
skin reconstruct
after 13 days of
culture. H&E,
x 400. B. Normal
human skin
reconstruct made
of de-epidermized
sterilized human
dermis cultured
on top of type I
collagen with
embedded
normal human
skin fibroblasts,
which migrated
up into the
dermis, after 16
days of culture.
H&E, x 100.
C. Macroscopic
view of a
pigmented normal
human skin
reconstruct; the
dermis is made of
type I collagen
with embedded
normal human
skin fibroblasts.
D. Normal human
skin reconstruct
with melanocytes
(arrows) in the
basal layer of the
epidermis after 11
days of culture.
S100 stain,
x 200.
E. Pigmented
normal human
skin reconstruct
20 days after
grafting to a SCID
mouse.
F. Pigmented
normal human
skin reconstruct
39 days after
grafting to a SCID
mouse. H&E,
x 100. G. Non-
invasive human
melanoma in a
human skin
reconstruct (S100
stain, x 400). H.
Invasive human
melanoma in a
human skin
reconstruct.
Collagen IV stain,
x 200
Skin reconstruct models and melanoma (Mel-CAM) or the beta 3 integrin subunit of the Bessou S., Surleve-Bazeille J.E., Pain C., Donatien P. and Taieb A.
vitronectin receptor, can convert non-aggressive non- (1996). Ex vivo study of skin phototypes. J. Invest. Dermatol. 107, i nvading melanoma cells into aggressive invading ones as can be demonstrated in skin reconstructs Boyce S.T., Christianson D.J. and Hansbrough J.F. (1988). Structure of ( S a t y a m o o r t hy et al., unpublished data). Thus, a new a collagen-GAG dermal skin substitute optimized for cultured human model for mechanistic studies of human melanoma epidermal keratinocytes. J. Biomed. Mater. Res. 22, 939-957.
biology has become available and is expected to be of Cario-Andre M., Pain C., Gall Y., Ginestar J., Nikaido O. and Taieb A.
increasing use by various research groups. (200 0). Stud ies on ep ide rmis reco nstructed with an d without melanocytes: melanocytes prevent sunburn cell formation but not appearance of DNA damaged cells in fair-skinned caucasians. J.
Invest. Dermatol. 115, 193-199.
O rganotypic culture models as opposed to Cooper M.L., Hansbrough J.F., Spielvogel R.L., Cohen R., Bartel R.L.
monolayer cultures mimic the three-dimensional and Naughton G. (1991). In vivo optimization of a living dermal structure of the respective reconstructed organ. They substitute employing cultured human fibroblasts on a biodegradable combine different orga n - s p e c i fic cell types and va r i o u s polyglycolic acid or polyglactin mesh. Biomaterials 12, 243-248.
d i fferentiation stages of the same cell type with Dekker S.K., van Doorn R., Kempenaar J., Gruis N.A., Vermeer B.J.
s u p p o r t ive matrix. Studies of cell-cell and cell-matrix and Ponec M. (2 000). Skin equivalent: an attractive model to interactions have become possible and culture artifa c t s , evaluate early melanoma metastasis. Melanoma Res. 10, 127-140.
which are common in cells grown in monolayer are Eves P., Layton C., Hedley S., Dawson R.A., Wagner M., Morandini R., absent or diminished in organotypic cultures. The Ghanem G. and Mac Neil S. (2000). Characterization of an in vitro reconstruction of human skin is most advanced and has model of human melanoma invasion based on reconstructed human reached the stage of successful transplantation to skin. Br. J. Dermatol. 142, 210-222.
patients to cover skin defects of ulcers or burns. More Ghosh M.M., Boyce S., Layton C., Freedlander E. and Mac Neil S.
r e c e n t l y, melanocytes and melanoma cells have been (1997). A comparison of methodologies for the preparation of human introduced into the organotypic cultures and their epidermal-dermal composites. Ann. Plast. Surg. 39, 390-404.
biological behavior resembles the in vivo situation to a Guldberg P., Kirkin A.F., Gronbaek K., thor Straten P., Ahrenkiel V. and great extent. Long-term studies have become possible Zeuthen J. (1997). Comp lete scanning of the C DK4 gene by by grafting these skin reconstructs to immunodefi c i e n t denaturing gradient gel electrophoresis: a novel missense mutation laboratory animals. The time has now come to focus on but low overall frequency of mutations in sporadic metastatic more detailed studies of gene candidates and cell-cell as malignant melanoma. Int. J. Cancer 72, 780-783.
well as cell-matrix interactions of relevance to Herlyn M., Clark W.H., Rodeck U., Mancianti M.L., Jambrosic J. and melanocyte transformation and melanoma biology.
Koprowski H. (1987). Biolo gy of tumor p rogre ssion in human melanocytes. Lab. Invest. 56, 461-474.
Hsu M.Y., Wheelock M.J., Johnson K.R. and Herlyn M. (1996). Shifts in Acknowledgements. We are grateful to Dr. Jonathan Garlick, who provided us with invaluable technical support for the skin reconstruction.
melanomas. J. Invest. Dermatol. Symp. Proc. 1, 188-194.
We also want to thank Emma D eJesus and Ren a Finko for th eir Hsu M.Y., Meier F.E., Nesbit M., Hsu J.Y., Van Belle P., Elder D.E. and Herlyn M. (2000). E-cadherin expression in melanoma cells restores experiments, and Elsa Aglow and Katerina Chruma for their excellent histological work. The studies were supported by National Institutes of expression of invasion-related adhesion receptors. Am. J. Pathol.
Health grants CA 80999, CA 25874, and CA 10815 to M.H. and a 156, 1515-1525.
postdoctoral research fellowship BE 2189/1-1 from the Deutsche Javaherian A., Vaccariello M., Fusenig N.E. and Garlick J.A. (1998).
Forschungs-gemeinschaft to C.B.
progression in stratified epithelium. Cancer Res. 58, 2200-2208.
Kamb A., Shattuck-Eidens D., Eeles R., Liu Q., Gruis N.A., Ding W., References
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Smola H., Stark H.J., Thiekotter G., Mirancea N., Krieg T. and Fusenig Accepted February 16, 2001

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