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Human Skin Models for Research Applications in Pharmacology and Toxicology: Introducing NativeSkin ® , the “Missing Link” Bridging Cell Culture and/or Reconstructed Skin Models and Human Clinical Testing

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  • ALTEXA Development Monaco

Abstract and Figures

The use of human skin models in pharmacology and toxicology has become a widely accepted approach for the evaluation of dermato-cosmetic products, both finished formulations and active ingredients. In particular, in vitro–reconstructed human skin models consisting on human keratinocytes and/or fibroblasts are used globally in both industrial and academic research laboratories, and regulatory bodies endorse the use of several of them as valid alternatives to animal testing requirements. Despite the recent progress that has been made in the reconstruction of more complex skin models containing other cell types, such models still have inherent limitations, as they are not an exact copy of human skin in vivo, and hence the predictability of the human in vivo response remains limited. Here we describe a new testing approach based on the use of NativeSkin Ò , a standardized ex vivo model of human skin, maintaining physiological skin biology, skin barrier function, and metabolism during 7 days of culture in tissue culture inserts. NativeSkin models are cultivated in quality-controlled and assured conditions , and demonstrate high reproducibility among donors. Where initial product testing is often performed on skin cell cultures and in vitro–reconstructed models, we propose the use of NativeSkin being the most complete skin model available, as highly predictive and cost-effective ''last-line screen in laboratory conditions'' prior to clinical evaluation in humans.
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ORIGINAL ARTICLES
Human Skin Models for Research Applications
in Pharmacology and Toxicology:
Introducing NativeSkin
!
, the ‘Missing Link’ Bridging
Cell Culture and/or Reconstructed Skin Models
and Human Clinical Testing
Bart De Wever,
1
Sandrine Kurdykowski,
2
and Pascal Descargues
2
Abstract
The use of human skin models in pharmacology and toxicology has become a widely accepted approach for the
evaluation of dermato-cosmetic products, both finished formulations and active ingredients. In particular,
in vitro–reconstructed human skin models consisting on human keratinocytes and/or fibroblasts are used globally
in both industrial and academic research laboratories, and regulatory bodies endorse the use of several of them as
valid alternatives to animal testing requirements. Despite the recent progress that has been made in the recon-
struction of more complex skin models containing other cell types, such models still have inherent limitations,
as they are not an exact copy of human skin in vivo, and hence the predictability of the human in vivo response
remains limited. Here we describe a new testing approach based on the use of NativeSkin
"
, a standardized ex
vivo model of human skin, maintaining physiological skin biology, skin barrier function, and metabolism during
7 days of culture in tissue culture inserts. NativeSkin models are cultivated in quality-controlled and assured con-
ditions, and demonstrate high reproducibility among donors. Where initial product testing is often performed on
skin cell cultures and in vitro–reconstructed models, we propose the use of NativeSkin being the most complete
skin model available, as highly predictive and cost-effective ‘last-line screen in laboratory conditions’ prior to
clinical evaluation in humans.
Key words: dermal absorption, drug delivery, ex vivo, human skin model, in vitro, skin explant.
Human Skin Models Based on Tissue Reconstruction
In Vitro: Applications and Limitations
I
N VITRO RECONSTRUCTED HUMAN SKIN MODELS consisting of
human keratinocytes, some of which are cocultured with
human fibroblasts, are commercially available by a variety
of suppliers: the mostly used ones are the EpiSkin
"
model
(Episkin s.a., Lyon, France), the EpiDerm
"
models (MatTek
Corporation, Ashland, MA), the RHE or Reconstructed
Human Epidermis (Episkin s.a.), the epiCS
"
skin model
(CellSystems, Troisdorf, Germany), the Phenion
"
Full-
Thickness Skin Model and OS-REp (Open Source Recon-
structed Epidermis) model (Henkel, Du
¨
sseldorf, Germany),
and the Labcyte
TM
skin model ( Japan Tissue Engineering
Co, Gamagori, Japan). The characteristics of these skin mod-
els as well as testing applications have extensively been de-
scribed in scientific literature elsewhere.
1
Although in vitro
reconstructed skin models have proven useful in toxicity,
pharmacology, efficacy, and drug transport studies, and
clearly offer advantages compared to the use of human ca-
daver and/or animal skin in terms of reproducibility, avail-
ability in large numbers, metabolism, and typical ‘tissue
response,’ these models are not exact copies of human
skin in vivo. In fact their biggest limitation is still their
relatively weak barrier function, which limits their applica-
bility for dermal penetration studies and sometimes leads
to false-positive results in other tests.
2
Morphologically,
in vitro–reconstructed human skin models form a multilay-
ered epithelium, displaying characteristic epidermal struc-
ture and expressing markers of epidermal differentiation.
3,4
1
ATERA SAS, Nice, France.
2
Genoskin, Toulouse, France.
APPLIED IN VITRO TOXICOLOGY
Volume 1, Number 1, 2015
ª Mary Ann Liebert, Inc.
DOI: 10.1089/aivt.2014.0010
26
On the ultrastructural level, the stratum granulosum and
stratum corneum display keratohyalin granules, lamellar
bodies, and lamellar structures filled with epidermal lipids.
Although the stratum corneum contains multiple lipid lamel-
lae located in the intercellular spaces between keratinized
cells and the epidermal barrier is associated with a calcium
gradient similar to that found in native human skin,
5
it has
been established that there is a difference in the lipid organi-
zation. Lipids in human in vitro –reconstructed skin models
pack in the stratum corneum in a hexagonal pattern, whereas
in native skin in vivo, the lipid organization is orthorhombic.
6
This difference presumably contributes to the 5–50-fold
higher penetration rate observed in human skin models
for most of the substances tested.
7
In addition, the Scientific
Committee on Consumer Safety (SCCS) guidelines for der-
mal absorption of cosmetic ingredients
8
consider the
in vitro–reconstructed human skin models to be inadequate
as alternative membranes for use in dermal absorption
study because of this impaired barrier function. Also, it
was recently shown that nonphototoxic concentrations of es-
sential oils, when tested in an in vitro skin model, provoked
phototoxic reactions when tested in vivo with humans and
that a safety factor 10 should be used when extrapolating
in vitro skin model data to human risk.
9
Besides their poor barrier function, most of today’s com-
mercially available reconstructed human skin models are
composed of only epidermal keratinocytes and/or dermal fi-
broblasts. Only a few specialized models contain other cell
types such as melanocytes. However, human skin is com-
posed of many other cell types, including Langerhans cells,
Merkel cells, dermal dendritic cells, resident T cells, and en-
dothelial cells, not to mention the skin appendages such as se-
baceous glands and hair follicles, which all are inherent active
constituents that influence the normal physiological reactions
taking place in the skin, including immunomodulative re-
sponses, regulation of skin surface temperature, as well as
somato-sensory effects among others.
10
NativeSkin
!
: The Next-Generation Ex Vivo Skin Model
Human ex vivo skin and skin explant culture have a long
tradition in investigative dermatology to study the physiol-
ogy and pathophysiology of human skin and epidermal ap-
pendages in vitro.
11–13
In addition, human ex vivo skin is
described as one of the tissues of choice for regulatory
dermal absorption experiments.
14
However, due to the un-
optimized conditions in which human skin is being main-
tained in culture, their applicability has so far been limited.
For instance, it was recently demonstrated that the experi-
mental conditions in which human skin is used influenced
the outcome of metabolism experiments: when investigating
the biotransformation of prednicarbate, a three times higher
transformation was observed when human ex vivo skin was
used in inserts compared to Franz cells, presumably due to
the higher stress level of skin maintained in Franz cells
resulting in lower enzyme activity. Furthermore, the authors
also showed that cryopreservation of human skin almost
completely degraded steroidogenic enzymes, indicating the
importance of maintaining physiological tissue viability
while using human ex vivo skin.
15
NativeSkin
"
is a patented ready-to-use ex vivo skin model
(skin explant) that consists of a full-thickness round human
skin biopsy, which is embedded in a proprietary nourishing
and solid matrix. The tissues are fixed into cell culture inserts
and loaded into a multiwell culture plate allowing easy cul-
turing and manipulation with a forceps for screening pur-
poses (Fig. 1). The epidermal surface of NativeSkin is in
direct contact with the air, making topical application of for-
mulations and/or active ingredients very easy. Furthermore,
a flat ring of silicon is firmly fixed at the periphery of the
skin surface to delimit a constant dosing area for topical ap-
plication. In addition, this hermetic seal prevents any un-
wanted lateral diffusion of topically applied products. At
the same time, the semisolid matrix, similarly like a sponge,
constantly nourishes the dermal part of the skin tissues with
an optimized, serum- and hydrocortisone-free, and chemi-
cally defined cell culture medium (Fig. 1c). NativeSkin
skin models are obtained from donors who have undergone
an abdominoplasty surgery and having given their informed
consent. Moreover, the NativeSkin tissue models are pro-
duced in a quality-controlled and assured way and can be
shipped by conventional express airfreight to any destination
around the world.
One of the unique features of the NativeSkin skin explant
models is that they can be cultured ex vivo up to 7 days at
37#C with 5% CO
2
and maximal humidity. After 7 days of
culture, NativeSkin demonstrates normal histological struc-
tures of both epidermal and dermal compartments (hematox-
ylin and eosin staining), normal dermal collagen content
(Masson trichrome staining), as well as normal cell viability
(Methyl green Pyronin Y) as shown in Figure 2. In addition,
the persistence of cell viability in the NativeSkin model cul-
tured during 7 days could be demonstrated by MTT assay
analysis (Fig. 3). Epidermal differentiation markers, includ-
ing keratin-10 and 14, involucrin, loricrin, and fillagrin, are
expressed similar to human skin in vivo (Fig. 4). Similarly,
proteins of the dermo–epidermal junction, as well as dermal
markers, are normally present after 7 days of ex vivo culture
(data not shown). Furthermore, some Langerhans cells persist
in the epidermis (Fig. 5), resident T-cells are clearly found
around blood vessels in the dermis, and melanocytes are still
able to produce melanin, even after several days of culture.
Recently, we could show that NativeSkin models repro-
duce hallmark features found in human skin when topically
treated with Tretinoin cream 0.05%, a well-known com-
mercial retinoid cream (Fig. 6). Treated NativeSkin mod-
els demonstrated a slight increase in epidermal thickness
and suprabasal keratinocytes enlargement. In addition, pro-
collagen I was slightly increased and MMP1 markedly de-
creased in treated NativeSkin models compared to untreated
controls (Fig. 6). Interestingly, similar results were previ-
ously obtained with standard skin explant cultures or with
skin equivalents, but only by adding the retinoid in the cul-
ture medium.
16–19
Thus, NativeSkin models demonstrated,
for the first time, the feasibility to repeat topical applications
of a retinoid cream ex vivo.
NativeSkin models are produced from the same body site,
the abdomen, and demonstrate high reproducibility among
a same donor. However, even between different donors,
the barrier function of the NativeSkin models is quite repro-
ducible (data not shown). On the contrary, it is important to
mention that the NativeSkin model is reflecting the genetic
and biological characteristics of the donor used. This
means that similar genetic and biological differences that
NATIVESKIN
!
, THE ‘MISSING LINK’ 27
FIG. 2. Histological characterization of NativeSkin after 7 days of ex vivo culture. (a, c, and e) 5 lm skin cross sections of
fresh human skin fixed with 10% buffered formalin and embedded in paraffin wax. (b, d, and f) 5 lm skin cross sections of
NativeSkin fixed, after 7 days of ex vivo culture, with 10% buffered formalin and embedded in paraffin wax. (a and b) Hem-
atoxylin and eosin staining. (c and d) Masson trichrome staining. (e and f) Methyl green-pyronin Y staining. Magnification is
20 · . Color images available online at www.liebertpub.com/aivt
FIG. 1. Presentation of NativeSkin
"
, a ready-to-use ex vivo skin model. (a and b) Top and profile views of the NativeSkin
model, respectively. An overhanging transwell contains a human skin biopsy embedded in a proprietary nourishing and solid
matrix. The system is easy to handle with forceps. The skin surface is left in direct contact with the air, enabling topical ap-
plication of formulations. A flat silicon ring delimits a working area of 0.5 cm
2
and prevents lateral diffusion of topically
applied products (1, overhanging transwell; 2, human skin biopsy; 3, flat silicon ring; 4, nourishing and solid matrix). (c)
Picture of a NativeSkin kit consisting of four 12-well plates loaded with NativeSkin models provided together with a ded-
icated culture medium, chemically defined, and free of serum, hydrocortisone, growth factors, and phenol red. Color images
available online at www.liebertpub.com/aivt
28
exist between different people (diversity) can be found in dif-
ferent NativeSkin batches.
A New Testing Approach Based on the Use of
NativeSkin: Bridging Cell Culture and/or Reconstructed
Skin Models and Human Clinical Testing
Despite the usefulness of in vitro–reconstructed human
skin models as alternatives to animal experiments and their
adoption in several OECD guidelines as valid methods for
corrosion and skin irritation testing, as well as their utility
in other toxicological evaluations, such as phototoxicity,
20
sen-
sitization,
21
and genotoxicity testing,
22
none of them mimic the
three-dimensional organization of human skin in vivo and are
still far from reproducing the complexity of this organ. In par-
ticular, they miss minority cell types, such as Merkel cells,
Langerhans cells, and dermal immune cells, and they lack
epidermal appendage structures and the physiological barrier
function, which explains their limited applicability in der-
mato-cosmetic research and development (Table 1).
Due to its inherent characteristics of ex vivo human skin, in
terms of cell population, tissue viability, structure, and me-
tabolism, as well as cutaneous barrier properties, NativeSkin
skin models are the relevant model of choice for a variety of
testing applications especially where in vitro–reconstructed
skin models often fail, such as dermal absorption, drug deliv-
ery, and pharmacology testing, allowing a more accurate pre-
diction of cutaneous effects prior to final clinical evaluation
in humans.
For instance, it was recently demonstrated that glucocorti-
coid-induced skin atrophy in in vitro–reconstructed human
skin models resulted in marked decrease in collagen I and re-
duction of the epidermal layers in the Phenion
"
FT skin
model. The authors relate these responses due to the im-
paired barrier function of the reconstructed skin model,
whereas repeated daily topical application using the Native-
Skin skin model up to 7 days did not alter the skin morphol-
ogy at the epidermal level.
23
In another study, the dermal diffusion of caffei ne and four
parabens was evaluated on the NativeSkin skin model, dem-
onstrating the strong barrier function and metabolic capacity
FIG. 3. Persistence of cell viability in NativeSkin. Cell vi-
ability was established by measuring, with a spectrophotom-
eter, the reduction of a tetrazolium dye MTT solution
incubated at 37#C with samples from fresh human skin
(day 0) and NativeSkin cultured during 7 days (day 7). All
samples analyzed were produced from six donors. The data
are presented as a percentage of cell viability relative to
cell viability measured at day 0 on fresh skin. Color images
available online at www.liebertpub.com/aivt
FIG. 4. Normal epidermal differentiation markers’ expression in NativeSkin. (a, c, and e) 5 lm skin cross sections of fresh
human skin fixed with 10% buffered formalin and embedded in paraffin wax. (b, d, and f) 5 lm skin cross sections of Nati-
veSkin fixed, after 7 days of ex vivo culture, with 10% buffered formalin and embedded in paraffin wax. (a and b) Immuno-
fluorescence with anti-(pro)-FLG antibody. (c and d) Immunofluorescence with anti-cytokeratins 1,2,10 antibody (clone
E2106). (e and f) Immunofluorescence with anti-cytokeratin 14 antibody. Magnification is 20 · . Color images available
online at www.liebertpub.com/aivt
NATIVESKIN
!
, THE ‘MISSING LINK’ 29
of the model,
24
whereas diffusion of caffeine through recon-
structed skin models has been reported repeatedly as very
fast.
25
Also, the high tolerability of the NativeSkin skin model’s
stratum corneum allows repeat daily topical application of
different vehicles, including gels, creams, and other solu-
tions. In addition, due to its robustness, the NativeSkin
model can also potentially be used for other applications:
its usefulness in tape stripping procedures, dermal patch
drug delivery application, and even intradermal injec-
tions in vitro is currently being investigated (personal
communications).
FIG. 5. Persistence of Langerhans
cells and skin-resident T cells in
NativeSkin. (a and b) Isolated epi-
dermal sheets made from fresh
human skin and NativeSkin models
cultured 4 days, fixed with 10%
buffered formalin. (b and d) 5 lm
skin cross sections of NativeSkin
fixed, after 4 days of ex vivo culture,
with 10% buffered formalin and
embedded in paraffin wax. (a
and b) Immunofluorescence with
anti-CD1a antibody to specifically
stain Langerhans cells. (c and d)
Immunofluorescence with anti-CD3
antibody to stain the T cells. Mag-
nification is 20 · . Color images
available online at www.liebertpub
.com/aivt
FIG. 6. Repeated topical applications of retinoic acid on NativeSkin. NativeSkin models prepared with abdominal skin
samples from a male donor of 38 years old were analyzed 7 days after ex vivo culture and 6 consecutive daily topical appli-
cations of 10 lL of Adapalene cream. (a, c, and e) Untreated controls. (b, d, and f) Adapalene-treated samples. All samples
were fixed in formalin and embedded in paraffin wax before performing 5 lm skin cross sections. (a and b) Hematoxylin and
eosin (H&E) staining. (c and d) Anti-pro-COLA1 immunofluorescence. (e and f) Anti-MMP1 immunofluorescence. Magni-
fication is 10 · for H&E and 20 · for immunofluorescence. Color images available online at www.liebertpub.com/aivt
30 DE WEVER ET AL.
Conclusions and Future Perspectives
In vitro–reconstructed human skin models, both epidermis
and full-thickness, have been adopted by cosmetic, pharma-
ceutical, and chemical laboratories as reliable alternatives to
animal experimentation for several decades. Not only do
these models comply with the demands of regulatory author-
ities, animal welfare organizations, and industry, but also
they provide a valuable tool to improve and extend our
knowledge on skin biology. However, the fact that these
models are not exact copies of human skin in vivo limits
their applications in toxicology, pharmacology, and absorp-
tion studies. The use of skin organ culture models offers an
interesting alternative, however, only if such ex vivo skin
model can be maintained in culture for several days in a
quality-controlled way.
NativeSkin, the next-generation ex vivo skin model, fea-
tures a native skin tissular composition for at least 7 days
in culture without loss of tissue integrity, viability, and
barrier properties. Being of abdominal origin, the Native-
Skin models are processed in a quality-controlled and as-
sured way. They are cultivated in tissue culture inserts
mounted in multiwell transport plates, by use of a propri-
etary matrix that surround and constantly nourishes the
dermal part of the skin model using a serum- and hydro-
cortisone-free culture medium. In addition , each Native-
Skin skin model is sealed topically with a silicon ring,
allowing topical application of compounds in a controlled
way. As such, the NativeSkin skin model has been shown
to be a very useful and essential complementary test sys-
tem for testing applications where in vitro–reconstructed
skin models fail, including dermal absorption, drug deliv-
ery, and pharmacology, and where specialized testing,
such as transdermal patch testing, tape stripping, and in-
tradermal injections, is required.
The model is also useful for the final pharmacotoxicolog-
ical evaluation of active ingredients and formulations, ini-
tially performed on cell cultures and reconstructed skin
models, prior to clinical trials in humans. In the future, Nati-
veSkin skin models replicating pathological conditions, in-
cluding psoriasis, dermatitis, and skin cancer, will become
available for compound research and development and effi-
cacy testing in dermatological diseases.
Author Disclosure Statement
No competing financial interests exist.
References
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Table 1. Characteristics of the NativeSkin
"
Model,
Compared to IN VITRO–RECONSTRUCTED SKIN MODELS
In vitro–reconstructed
skin models Ex vivo NativeSkin
Partial copy of human
skin based on 2–3
cell types
Complete culture of
human skin including
all cell types and
appendices
Impaired cutaneous
barrier function
Physiological barrier
function
Fragile stratum corneum Robust (native) stratum
corneum
Foreskin origin Abdominal origin
Pooled donors Individual donor(s)
Early screening applications
and regulatory toxicity
testing
Final testing of selected
formula prior to clinical
testing in humans
NATIVESKIN
!
, THE ‘MISSING LINK’ 31
20. Justine P, Christiaens F, Bonaventure N, et al. The EpiSkin
phototoxicity assay (EPA): development of an in vitro tiered
strategy using 17 reference chemicals to predict phototoxic
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Europe strategy for animal-free genotoxicity testing: project
status up-date. Toxicol In Vitro 2014:28;18–23.
23. Norsgaard H, Kurdykowski S, Descargues P, et al. Calcipo-
triol counteracts betamethasone-induced decrease in extra-
cellular matrix components related to skin atrophy. Arch
Dermatol Res 2014:306;719–729.
24. Rolland P, Duracher L, Kurdykowski S, et al. Skin delivery
of caffeine and parabens through Nativeskin
"
: a newly de-
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ing, Valbonne, France, 2013.
25. Dreher F, Fouchard F, Patouillet C, et al. Comparison of cu-
taneous bioavailability of cosmetic preparations containing
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Skin Physiol 2002:15 Suppl 1;40–58.
Address correspondence to:
Pascal Descargues
Genoskin
1 Place Pierre Potier
31100 Toulouse
France
E-mail: pascal.descargues@genoskin.com
32 DE WEVER ET AL.
... La présence de cellules de Langerhans et de cellules T résidentes a également été démontrée, révélant une certaine immunocompétence du modèle. Enfin, des études ont prouvé que les mélanocytes présents dans le modèle conservaient leur capacité à produire de la mélanine même après plusieurs jours de culture (De Wever et al., 2015). ...
... Dans les modèles de peau ex vivo, certaines étapes de ce processus ne peuvent pas avoir lieu, en raison de l'absence de circulation sanguine, telles que le recrutement de cellules inflammatoires présentes dans le sang par exemple. Cependant, la présence de cellules immunitaires résidentes a été démontrée pour le modèle NativeSkin® (De Wever et al. 2015 ;Jardet et al. 2020). La réaction inflammatoire à un corps étranger pourrait donc tout de même être caractérisée dans nos modèles suite à l'implantation du cathéter, en étudiant la dégranulation des mastocytes présents dans le tissu, ou la sécrétion de cytokines pro-inflammatoires comme l'IL-6, l'IL8 ou l'IL-33 par exemple. ...
Thesis
Les modèles organotypiques tels que les explants de peau humaine sont les modèles les plus complexes et parmi les plus représentatifs de la peau in vivo existants à ce jour pour tester l’efficacité ou l’innocuité de molécules d’intérêts thérapeutiques au stade des études pré-cliniques. Cependant, l’absence de circulation sanguine et lymphatique dans ces modèles reste une limite importante dans l’homéostasie du tissu, notamment pour prédire les réponses de la peau à un traitement. De plus, les échanges en nutriments et en oxygène n’étant possibles que par diffusion, la durée de vie de ces modèles reste limitée. Différentes stratégies ont été mises en place afin de contrôler les mécanismes de transports moléculaires au sein de tissus biologiques. La microfluidique offre un fort potentiel pour contrôler la convection et la diffusion permettant l’échange de composés dans ces modèles de peau.L’objectif de ce projet est de développer, caractériser et valider un modèle de peau humaine ex vivo perfusé. Le but de cette perfusion intra-tissulaire est de favoriser les échanges de nutriments, d’oxygène ou de médicaments, mais également d’améliorer l’élimination des déchets métaboliques.Le premier objectif de mes travaux a consisté à mettre en place un flux intra-tissulaire dans un explant de peau humaine, et à développer un procédé permettant de maintenir l’explant perfusé en culture pendant plusieurs jours. Pour cela, un dispositif poreux a été implanté dans le derme du modèle ex vivo de peau humaine NativeSkin, développé par la société Genoskin, puis relié à un système microfluidique permettant l’infusion de composés au sein du tissu.Le deuxième objectif a consisté à développer des méthodes d’analyse de la diffusion de composés dans des explants de peau. Quatre méthodes ont été développées : l’évaluation macroscopique et qualitative de la diffusion à l’aide d’un colorant, l’étude de la diffusion en temps réel par radiographie à rayons X, l’étude de la diffusion en trois dimensions par tomographie à rayons X, et enfin l’analyse de la diffusion de dextrans fluorescents de différents poids moléculaires, sur coupes histologiques. Un modèle numérique permettant de simuler la diffusion dans le modèle de peau a également été développé sur le logiciel COMSOL, permettant de prédire le profil de diffusion d’un composé.Le troisième et dernier objectif a consisté à déterminer les paramètres de perfusion permettant une bonne diffusion des composés dans l’explant de peau, sans toutefois endommager le tissu. Une première série d’expériences (8 donneurs) a été réalisée sur des modèles perfusés à flux constant (2,5µL/min) avec du milieu de culture, pendant 10 jours. Les résultats ont montré qu’à l’issue de la culture, les modèles de peau ne présentent pas d’altération de la viabilité cellulaire ni de l’intégrité du tissu, avec un maintien de la prolifération et du métabolisme cellulaire. Cependant, la caractérisation de la diffusion dans le modèle a démontré un manque de reproductibilité dans les expériences, avec d’importantes variabilités inter et intra-donneurs. De plus, la perfusion de dextrans de différents poids moléculaires a démontré que la diffusion de composés de hauts poids moléculaires était limitée. Afin de pallier ces limites, nous avons proposé une nouvelle méthode de perfusion basée sur une modulation de la pression au sein du dispositif. L’application d’une légère surpression au sein du dispositif poreux permet d’améliorer la reproductibilité et l’efficacité des échanges moléculaires au sein de l’explant.Les résultats obtenus positionnent le modèle FlowSkin ainsi développé comme un nouvel outil pertinent pour évaluer l’efficacité ou la toxicité de molécules administrées par voie intraveineuse, directement sur de la peau humaine. De plus, la perfusion de transporteurs d’oxygène via ce système pourrait permettre de prolonger la durée de vie et donc d’améliorer encore la pertinence du modèle de peau ex vivo.
... NativeSkin is a patented human skin model from GENOSKIN. 26 In the NativeSkin model, round human skin biopsies are embedded in a solid matrix supplemented with culture medium for cosmetic testing, which can maintain and nourish the skin alive for 7 days (https://www.genos kin. ...
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Background: Cultured human skin models have been widely used in the evaluation of dermato-cosmetic products as alternatives to animal testing and expensive clinical testing. The most common in vitro skin culture approach is to maintain skin biopsies in an airlifted condition at the interface of the supporting culture medium and the air phase. This type of ex vivo skin explant culture is not, however, adequate for the testing of cleansing products, such as shampoos and body washes. One major deficiency is that cleansing products would not remain confined on top of the epidermis and have a high chance of running off toward the dermal side, thus compromising the experimental procedure and data interpretation. Materials and methods: Here, we describe an improved ex vivo method for culturing full-thickness human skin for the effective testing and evaluation of skin care products by topical application. Results: This newly developed ex vivo human skin culture method has the ability to maintain healthy skin tissues for up to 14 days in culture. Importantly, the model provides a quick and safe way to evaluate skin care products at different time points after single or repetitive topical applications using a combined regimen of leave-on and wash-off. We found that the results obtained using the new skin culture method are reproducible and consistent with the data collected from clinical testing. Conclusion: Our new ex vivo skin explant method offers a highly efficient and cost-effective system for the evaluation and testing of a variety of personal care products and new formulations.
... However, as they are based only on keratinocytes, they lack crosstalk with the dermis, which limits their application (Wang et al., 2016). More complex full-thickness skin models (FTSm) are also commercially available, namely, Phenion ® , Epiderm-FT ® , Stratatest, ® and T-Skin ® (Ackermann et al., 2010;Rasmussen et al., 2010;De Wever et al., 2015a). These models are created using hydrogel-based technologies, usually with animal-derived collagen, into which fibroblasts are integrated. ...
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There is a global trend towards the development of physiologically relevant in vitro skin models to reduce or replace animal testing in the evaluation of therapeutic drug candidates. However, only commercial reconstructed human epidermis models (RHEms) have undergone formal validation. Although these commercial models are suitable for a wide range of applications, they are costly, lack flexibility, and the protocols used to generate them are not transparent. In this study, we present an open-source full-thickness skin model (FTSm) and assess its potential for drug testing. The FTSms were developed using endogenous extracellular matrix to recreate the dermal compartment, avoiding animal-derived hydrogels. An RHEm based on an open-source protocol was also evaluated in parallel. The integrity of the skin barrier was analysed by challenging the surface with detergents and measuring cell viability as well as through trans-epithelial electrical resistance (TEER) measurements. Skin irritation studies were performed based on OECD guidelines and complemented with an evaluation of the impact on the skin barrier using TEER. The comparative permeation study of the developed models and a commercial membrane (Strat-M®) was performed using Franz diffusion cells and an infinite dose approach. The FTSm demonstrated structural and barrier properties comparable to the native human skin. Although the RHEm showed an overall better performance during drug testing, the FTSm presented better barrier properties than commercial models reported in the literature. These skin models can be a valuable contribution to accelerating the development and dissemination of alternatives to animal testing, circumventing the limitations of commercial models.
... et l'imperméabilité de sa barrière(De Wever, Kurdykowski, and Descargues 2015). En outre, les mélanocytes présents dans l'épiderme du modèle conservent leur capacité à produire de la mélanine après plusieurs jours de culture(Calapre et al. 2016), et des études ont révélé une certaine immunocompétence du modèle en culture, avec la présence de cellules de Langerhans dans l'épiderme et de lymphocytes T résidents dans le derme.Différentes études réalisées sur ce modèle ont démontré sa pertinence dans le cadre de l'évaluation d'effets de molécules pharmaceutiques appliquées à sa surface(Norsgaard et al. 2014), mais également pour des études de défaut de barrière cutanée(Duracher et al. 2015), ou nécessitant la réalisation d'injections intradermiques (données non publiées).IntroductionPartie 1 : Présentation générale de la peau ...
Thesis
Le mélanome métastatique est le cancer de la peau le plus agressif. Bien que son taux d’incidence soit inférieur à 1%, plus de 75% des décès associés à un cancer de la peau lui sont attribués. Au cours des dernières années, de nouvelles stratégies thérapeutiques ont permis d’améliorer la survie des patients. Cependant, des mécanismes de résistance à ces traitements se développent dans la majorité des cas, conduisant à une phase de rechute, et une survie à 5 ans inférieure à 20%. Des modèles d’étude expérimentaux sont nécessaires afin de comprendre les mécanismes impliqués dans l’apparition de ces résistances et développer de nouvelles stratégies thérapeutiques. Différents modèles in vitro sont actuellement utilisés pour le développement de drogues anti-tumorales, tels que celui du sphéroïde. Bien qu’il permette de reproduire l’organisation tridimensionnelle d’une tumeur, l’absence de microenvironnement tumoral empêche l’étude des interactions entre les cellules tumorales et celui-ci alors que ces facteurs jouent un rôle primordial dans la croissance tumorale et le développement de métastases. Dans ce contexte, mes travaux ont porté sur le développement et la caractérisation d’un modèle ex vivo de mélanome humain complet permettant l’étude de l’évolution d’une tumeur dans le tissu sain et l’évaluation de composés pharmacologiques. Les travaux réalisés ont tout d’abord conduit au développement d’un modèle de cancer cutané basé sur la combinaison d’un modèle de sphéroïde de lignée cellulaire de mélanome humain et du modèle de peau humaine ex vivo NativeSkin®, développé par la société Genoskin. Une procédure a été développée et validée pour permettre l’implantation reproductible d’un sphéroïde dans le derme des explants de peau. Parallèlement, j’ai développé une approche d’imagerie in situ par microscopie à feuille de lumière après transparisation des modèles. J’ai également développé une stratégie d’analyse d’images permettant la caractérisation quantitative de l'évolution du sphéroïde implanté en 3 dimensions et de suivre la dispersion des cellules du tumorales au sein de l’explant de peau. La caractérisation histologique du modèle implanté a révélé de façon très inattendue une perte progressive de l’intégrité du sphéroïde après implantation, associée à une diminution rapide de la prolifération des cellules le constituant et l’apoptose massive des cellules situées à sa périphérie. Ce phénomène a été observé de façon similaire lors de l’implantation de sphéroïdes produits à partir de différents types cellulaires. Afin de comprendre ces résultats, j’ai étudié l’implication potentielle de différents paramètres dans l’induction de la mortalité cellulaire observée tels que les conditions d’implantation, les facteurs synthétisés par le modèle et la contrainte mécanique exercée par le derme. Les résultats obtenus suggèrent que les facteurs sécrétés par les modèles après implantation du sphéroïde ont un effet antiprolifératif sur les sphéroïdes de mélanome et qu’ils induisent la mortalité des cellules situées à sa périphérie. Par ailleurs, l’application d’une contrainte mécanique extérieure sur les sphéroïdes de mélanome entraîne la perte de la cohésion de leur structure. Enfin, l’implantation de sphéroïdes dans le derme de biopsies de peau préalablement desséchées, induisant une perte de la viabilité cellulaire, a conduit à des résultats opposés à ceux observés avec de la peau normale : la structure des sphéroïdes reste cohésive et la prolifération des cellules est maintenue en périphérie du sphéroïde sans qu’aucune apoptose massive ne soit observée. L'ensemble de ces travaux semble suggérer que la mortalité du sphéroïde pourrait être, en partie, la conséquence d’une contrainte mécanique exercée par la peau sur le sphéroïde et/ou de facteurs produits par la peau durant sa culture. Ces données ouvrent des perspectives intéressantes dans le domaine de l’ingénierie tissulaire pour l’évaluation pharmacologique de composés thérapeutiques.
... Néanmoins, la préparation et la conservation de la peau modifie les capacités métaboliques(Abd et al., 2016;Dumont et al., 2015;OECD, 2004a). La commercialisation d'explants de peau humaine complète standardisés nommés NativeSkin® avec des conditions de culture optimisées, a permis d'élargir le champs d'application(De Wever et al., 2015). A cause des contraintes éthiques, économiques et d'approvisionnement en résidus chirurgicaux, la peau ex vivo d'origine animale est parfois utilisée. ...
Thesis
La dermatite de contact allergique (DCA) est une réaction exacerbée du système immunitaire cutané vis-à-vis d’un allergène de contact. La prévalence de la DCA étant de 20 % au sein de la population mondiale, il est important d’identifier les composés allergisants. Différentes réglementations européennes, telles que le règlement REACh ou la directive cosmétique, interdisent l’utilisation de test sur l’animal. C’est dans ce contexte que différentes méthodes alternatives ont été développées pour évaluer la sensibilisation cutanée. La stratégie actuelle d’évaluation du potentiel sensibilisant consiste à réaliser un ensemble de tests alternatifs, chacun mimant un évènement clé du mécanisme : l’hapténisation, l’activation des kératinocytes ou des cellules dendritiques.Cependant, ces tests utilisent principalement des monocultures et ne prennent donc pas en compte les interactions cellulaires qui peuvent avoir lieu in vivo. De plus, les évaluations de la pénétration et du métabolisme cutanés sont négligées dans les tests développés.Afin de mimer la fine orchestration des événements intervenant lors de la sensibilisation cutanée, nous proposons un modèle d’épiderme humain reconstruit (RhE) co-cultivé avec la lignée cellulaire THP-1, servant de substitut aux cellules dendritiques. Nous avons caractérisé, et étudié la pertinence de ce modèle à l’aide de molécules chimiques de référence. Ce travail a permis l’identification de biomarqueurs, tels que CD54, IL-8 et CCL3, spécifiques à l’évaluation in vitro de la sensibilisation cutanée des xénobiotiques.
... The appearance of senescent cells in burn wounds has to be verified in in vivo experiments, however, the model enables a bottom up approach that can stepwise be evaluated in more complex systems. Potential modifications include the co-cultivation of different cell types (fibroblasts, keratinocytes and immune cells) and an extension to existing skin models (De Wever et al., 2015;Nguyen and Pentoney, 2017). ...
Article
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Thermal injuries cause severe damage on the cellular and tissue level and are considered especially challenging in the clinical routine. Complex interactions of different cell types and pathways dictate the formation of burn wounds. Thus, complications like burn wound progression, where so far viable tissue becomes necrotic and the size and depth of the wound increases, are difficult to explain, mainly due to the lack of simple model systems. We tested the behavior of human fibroblasts after heat treatment. A prominent response of the cells is to activate the heat shock response (HSR), which is one of the primary emergency mechanisms of the cell to proteotoxic stress factors such as heat. However, after a powerful but not lethal heat shock we observed a delayed activation of the HSR. Extending this model system, we further investigated these static cells and observed the emergence of senescent cells. In particular, the cells became β-galactosidase positive, increased p16 levels and developed a senescence-associated secretory phenotype (SASP). The secretion of cytokines like IL-6 is reminiscent of burn wounds and generates a bystander effect in so far non-senescent cells. In agreement with burn wounds, a wave of cytokine secretion enhanced by invading immune cells could explain complications like burn wound progression. A simple cell culture model can thus be applied for the analysis of highly complex conditions in human tissues.
Chapter
3D skin equivalents were one of the first TE organs used for clinical trials. In early skin models, fibroblastsFibroblastsand keratinocytesKeratinocytes were incorporated into a nylon meshMesh to mimic the dermal matrix. Since then, the evolution of scaffolds for skin cell culture has spurred the development of more sustainable and biologically accurate biomaterials that allow for the mimicking of properties and prediction of skin behaviour.
Article
Skin models are used for many applications such as research and development or grafting. Unfortunately, most lack a proper microenvironment producing poor mechanical properties and inaccurate extra-cellular matrix composition and organization. In this report we focused on mechanical properties, extra-cellular matrix organization and cell interactions in human skin samples reconstructed with pure collagen or dermal decellularized extra-cellular matrices (S-dECM) and compared them to native human skin. We found that Full-thickness S-dECM samples presented stiffness two times higher than collagen gel and similar to ex vivo human skin, and proved for the first time that keratinocytes also impact dermal mechanical properties. This was correlated with larger fibers in S-dECM matrices compared to collagen samples and with a differential expression of F-actin, vinculin and tenascin C between S-dECM and collagen samples. This is clear proof of the microenvironment's impact on cell behaviors and mechanical properties. Statement of Significance In vitro skin models have been used for a long time for clinical applications or in vitro knowledge and evaluation studies. However, most lack a proper microenvironment producing a poor combination of mechanical properties and appropriate biological outcomes, partly due to inaccurate extra-cellular matrix (ECM) composition and organization. This can lead to limited predictivity and weakness of skin substitutes after grafting. This study shows, for the first time, the importance of a complex and rich microenvironment on cell behaviors, matrix macro- and micro-organization and mechanical properties. The increased composition and organization complexity of dermal skin decellularized extra-cellular matrix populated with differentiated cells produces in vitro skin models closer to native human skin physiology.
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Native human skin has been reported in the literature as being an important experimental model for studying skin biology. Studies performed by our group have shown that ex vivo skin, from elective plastic surgery, maintains the biological characteristics of native skin under specific culture conditions. As such, it might be a feasible model for the safety and efficacy testing of topical substances. While Brazil is at the forefront of global regulation implementation, Brazilian researchers are not always able to transfer certain widely used protocols to their laboratories, particularly protocols that involve the use of reconstructed tissues with limited viability, such as those for skin corrosion (OECD TG 431) and irritation testing (OECD TG 439). In this study, we investigated the applicability of the ex vivo skin model to the evaluation of irritation and corrosion potential of a number of proficiency substances described in TG 431 and TG 439. The skin fragments were standardised in size and diameter, and placed into cell culture inserts. The experimental protocol was conducted according to TG 431 and TG 439. The results obtained show that ex vivo skin could represent a promising tool for the evaluation of irritation and corrosion potential of substances (subject to inclusion and exclusion criteria), as recommended by OECD guidelines. While this is a proof-of-concept study, the use of ex vivo skin should be considered for such testing.
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The calcipotriol/betamethasone dipropionate fixed-combination gel is widely used for topical treatment of psoriasis vulgaris. It has been hypothesized that calcipotriol counteracts glucocorticoid-induced skin atrophy which is associated with changes in the extracellular matrix (ECM). To elucidate the combined effects of calcipotriol and betamethasone on key ECM components, a comparative study to the respective mono-treatments was carried out. The effect on collagen I synthesis, matrix metalloproteinase (MMP) secretion, and hyaluronic acid (HA) production was investigated in primary human fibroblast and keratinocyte cultures as well as in a human skin explant model. We show that calcipotriol counteracts betamethasone-induced suppression of collagen I synthesis. Similarly, calcipotriol and betamethasone have opposing effects on MMP expression in both fibroblasts and keratinocytes. Moreover, calcipotriol is able to restore betamethasone-impaired HA synthesis in keratinocytes and prevent betamethasone-induced epidermal thinning in minipigs upon treatment with the calcipotriol/betamethasone gel. In summary, our results show for the first time in primary human skin cultures that calcipotriol reduces early signs of betamethasone-induced skin atrophy by modulation of key ECM components. These results indicate that the calcipotriol component of the fixed-combination gel counteracts the atrophogenic effects of betamethasone on the skin. Electronic supplementary material The online version of this article (doi:10.1007/s00403-014-1485-3) contains supplementary material, which is available to authorized users.
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The objective of this review is to introduce Merkel cells (MCs), to provide a basic overview on the theoretical background of function, development and clinical importance of MCs. The origin of human MCs have been controversial. Some investigators believe that it is a neural crest derivate, whereas others have proposed that it is a differentiation product of the fetal epidermal keratinocytes. MCs are cells primarily localized in the epidermal basal layer of vertebrates and concentrated in touch-sensitive areas in glabrous, hairy skin and in some mucosa. In routine light microscopy, human MCs can hardly be identified. Cytokeratin 20 (CK20) is a reliable marker with highest degree of specificity. MCs can be also distinguished by electron microscopy. MC carcinoma (MCC) is an uncommon and often aggressive malignancy and found mainly in elderly patients. It occurs most frequently in the head and neck region. Diagnosis is based on typical histological presentation on hematoxylin and eosin (H and E) stained slides together with the results of immunohistochemistry. Histologically, MCC has been classified into three distinct subtypes: Trabecular, intermediate and small cell type.
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This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50% cytotoxicity (EE-EC50) or the 2-fold increase in IL-1α (IL-1α2x). Four laboratories received 13 coded sensitizers. Reproducible results were obtained in each laboratory. A binary prediction model, EC50 ≥ 7 mg/ml = weak to moderate sensitizer and EC50 < 7 mg/ml = strong to extreme sensitizer had an accuracy of 77%. A superior EE (EC50 and IL-1α2x) correlation was observed with human in vivo DSA05 data compared to LLNA-EC3 data. Human in vivo NOEL and LLNA-EC3 data correlated to a similar extent to in vitro EE data. Our results indicate that this easily transferable EE potency assay is suitable for testing chemical allergens of unknown potencies and may now be ready for further validation, providing complementary potency information to other assays already undergoing validation for assessing skin sensitization potential.
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The Cosmetics Europe (formerly COLIPA) Genotoxicity Task Force has driven and funded three projects to help address the high rate of misleading positives in in vitro genotoxicity tests: The completed "False Positives" project optimized current mammalian cell assays and showed that the predictive capacity of the in vitro micronucleus assay can was improved dramatically by selecting more relevant cells and more sensitive toxicity measures. The on-going "3D skin model" project has been developed and is now validating the use of human reconstructed skin (RS) models in combination with the micronucleus (MN) and Comet assays. These models better reflect the in use conditions of dermally applied products, such as cosmetics. Both assays have demonstrated good inter and intra-laboratory reproducibility and are entering validation stages. The completed "Metabolism" project investigated enzyme capacities of human skin and RS models. The RS models were shown to have comparable metabolic capacity to native human skin, confirming their usefulness for testing of compounds with dermal exposure. The program has already helped to improve the initial test battery predictivity and the RS projects have provided sound support for their use as a follow-up test in the assessment of the genotoxic hazard of cosmetic ingredients in the absence of in vivo data.
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Reconstructed human epidermis (RHE) is used in non-animal testing for hazard analysis and reconstructed human skin (RHS) gains growing interest in preclinical drug development. RHE and RHS have been characterized regarding their barrier function, but knowledge about biotransformation capacity in these constructs and in human skin remains rather poor. However, metabolizing enzymes can be highly relevant for the efficacy of topical dermatics as well as genotoxicity and sensitization. We have compared the esteratic cleavage of the prednisolone diester prednicarbate and the enzyme kinetic parameters (V(max) and S(0.5)) of the model substrate fluorescein diacetate (FDA) in commercially available RHS and RHE with excised human skin and monolayer cultures of normal and immortalized human keratinocytes and of fibroblasts. Formation of the main metabolite prednisolone and of fluorescein ranked as: RHS ∼ RHE > excised human skin and keratinocytes > fibroblasts, respectively. Because of the aromatic probe, however, V(max) of FDA cleavage did not show a linear relationship with prednicarbate metabolism. In conclusion, RHE and RHS may be useful to quantitatively address esterase activity of human skin in drug development and hazard analysis, although an increased activity compared to native human skin has to be taken into account.
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Skin morphogenesis and physiology (hormone and vitamin actions) are the fields that mostly benefit from the skin organ culture techniques. The few papers within toxicology and pharmacology, however, demonstrate that these research areas can successfully exploit skin organ cultures. The lack of established and relevant parameters for quantitative toxicologic studies has delayed a more general adoption of this model, but Smith and Holland and Kao and coworkers suggest some alternatives for its use. It has been claimed that organ cultures are not suitable for biochemical work because of small tissue quantities. Skin, however, can be cultured in sufficient amounts for metabolic labeling and biochemical analysis, as shown by the studies on keratins and glycosaminoglycans. On the other hand, quantitative morphologic methods, morphometry and quantitative histochemistry, offer an alternative by which numerical data can be collected, data that can be readily analyzed with modern statistical methods. In past decades, the techniques introduced by Robison and Fell and modernized by Trowell enabled the numerous studies evaluated in this review. We consider the foundations built and recently widened to facilitate even stronger and more active future use of skin organ culture in several aspects of dermatologic research.
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The physiological status of native skin is suffering from large inter-individual variations, especially in terms of inorganic ions content. For this reason, together with the advent of ethic laws on animal experimentation, reconstructed skin or epidermis models are extensively employed nowadays in penetration studies for cosmetic or pharmacological applications.It has been already verified that reconstructed human epidermis (RHE) has similar physiological mechanisms to native human skin, but until now, there are few studies where the elemental concentrations of both skins, reconstructed and native, are compared. In this work, freeze-dried thin sections of human native skin obtained from surgery have been characterized using PIXE, RBS and STIM at the CENBG nuclear microprobe. RHE samples were treated and analyzed in the same conditions for comparison. The combination of the different imaging and analysis techniques made possible a clear delimitation and identification of skin ultrastructure. The elemental concentrations of P, S, Cl, K and Ca were measured in the different strata. For both skins, concentrations have been compared and significant differences in terms of elemental concentrations have been determined using statistical approaches. Similar physiological characteristics were pointed out in both skin models, in particular the Ca gradient presumably involved in the regulation of the barrier effect.
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In diabetes, foot ulceration may result from increased skin fragility. Retinoids can reverse some diabetes-induced deficits of skin structure and function, but their clinical utility is limited by skin irritation. The effects of diabetes and MDI 301, a nonirritating synthetic retinoid, and retinoic acid have been evaluated on matrix metalloproteinases (MMPs), procollagen expression, and skin structure in skin biopsies from nondiabetic volunteers and diabetic subjects at risk of foot ulceration using organ culture techniques. Zymography and enzyme-linked immunosorbent assay were utilized for analysis of MMP-1, -2, and -9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) and immunohistochemistry for type I procollagen protein abundance. Collagen structure parameters were assessed in formalin-fixed, paraffin-embedded tissue sections. The % of active MMP-1 and -9 was higher and TIMP-1 abundance was lower in subjects with diabetes. Type 1 procollagen abundance was reduced and skin structural deficits were increased in diabetes. Three μM MDI 301 reduced active MMP-1 and -9 abundance by 29% (P < .05) and 40% (P < .05), respectively, and increased TIMP-1 by 45% (P = .07). MDI 301 increased type 1 procollagen abundance by 40% (P < .01) and completely corrected structural deficit scores. Two μM retinoic acid reduced MMP-1 but did not significantly affect skin structure. These data indicate that diabetic patients at risk of foot ulceration have deficits of skin structure and function. MDI 301 offers potential for repairing this skin damage complicating diabetes.