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Peer-reviewed article
TESTING METHODS
Class
Rueffer
In vitro methods demonstrate effects
on skin self-renewal and
homeostasis in 3D skin models
CLAAS RÜFFER*, LISA BÖCKELMANN, WERNER VOSS
*Corresponding author
Dermatest GmbH Engelstrasse 37, 48143, Germany
KEYWORDS: Skin homeostasis; cell renewal; proliferation; viability.
ABSTRACT: In vitro 3D skin models are able to recapitulate the
major events in the process of skin homeostasis very well. Two cell
biological assays have been applied on Phenion®-FT-skin models
and demonstrated their usefulness for describing cellular activities
in the process of skin renewal and homeostasis in vitro. A cell
proliferation assay was applied to study the general proliferative
responsiveness of basal keratinocytes and dermal fibroblasts,
whereas the MTT-viability-assay was used to analyse the overall
improvement of epidermal and dermal tissue viability. For
demonstration purpose, single experiments have been performed
in which skin models were systematically stimulated with known
cosmetic and medical actives (atRA & 13cRA), whose time and
dose dependent effectiveness are well characterized (18, 21).
INTRODUCTION
Skin homeostasis
Skin maintains its capability of self renewal throughout a
whole human life. The continuum of keratinocyte birth,
differentiation and desquamation comprises the coreprinciple of a physiological and physical skin barrier that
holds fluids inside the body and acts as a protective shield
against biological, chemical and physical environmental
knoxes to the outside (3). Although skin continuously
accumulates chronological and photo-induced signs of
ageing, it still manages to keep up its skin barrier maintenance
a life-long (6). The origin of interfollicular epidermal self
regenerative capacity is located within a single, basal layer
of keratinocytes, which is anchored to the papillar dermis
via a range of connective structural proteins (6, 7). The first
effective step in interfollicular regeneration takes place in
the basal layer of epidermal keratinocytes. Undifferentiated
basal keratinocytes undergo constantly acts of mitosis. Newborn daughter cells detach from the underlying basement
membrane and migrate into suprabasal epidermal layers.
From that point on they withdraw from cell cycle and
start the highly organized sequencial program of terminal
differentiation and desquamation. Terminal differentiation
comprises a complex process of cornification in which
granular cells transform into keratin-enriched cell remnants
in order to form a multilayered interconnected protective
envelope (3). Desquamation is a finely tuned and timed
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sequence where outer layers of cornified keratinocytes are
enzymatically detached from the skin surface. Therefore
cell proliferation at the basal epidermal layer and the
desquamation process of the stratum corneum are finely
balanced processes which keep the constantly rejuvenating
epidermis in a regulated state and the skin barrier properties
permanently maintained (3, 6).
Interfollicular Stem cells
The homeostasis of interfollicular epidermis is characterized
through a high cellular turn-over propelled by cell divisions
of stem cells within the basal layer of keratinocytes. A well
accepted scientific model today suggests the presence
of distinct populations of stem cells that locate within the
interfollicular epidermis. Epidermal stem cells have the ability
to produce committed progenitor cells, also known as transient
amplifying stem cell daughters (TA cells). TA cells undergo cell
divisions up to 5 times before they differentiate. Therefore it
is supposed that stem cells in their specific niches are rather
non-dividing, mostly quiescent cells which leave most of the
epidermal regenerative activity to committed and frequently
activated TA cells (10, 14, 20). With ageing epidermis becomes
more fragile and predisposed for trauma. A significantly
reduced homeostatic activity and decline in healing rate of
elder skin most probably originates from a drastic reduction
of stem cell’s proliferative capacity and mobility. In general
adult stem cells show the same susceptibility to chronological
ageing as non-dividing cells. The chronological accumulation
of intrinsic- as well as stem cell niche specific impairments is
discussed to be relevant for the general and steady decline in
stem cell activity (4, 11, 16, 20).
In vitro skin regeneration
In vitro skin equivalents are composed of a highly
differentiated, stratified epithelium which resides on top of a
dermal matrix with incorporated living fibroblasts. Despite their
simplified model-like character, organotypic skin equivalents
are capable of a far-reaching recapitulation of the in vivo
keratinocyte differentiation program (17). In contrast to real
skin in vitro 3D skin models are quite short-lived. Nevertheless,
for a limited time period skin models are capable of sustaining
a constant pool of skin cells to keep their epidermal structure
maintained (13, 17). Analogue to the in vivo skin situation, only
cells within the single layer of basal keratinocytes are able to
pass through the cell cycle to fuel the cellular balance. The
model`s full thickness design allows possible mutual molecular
dermal-epidermal crosstalk within the skin equivalent.
TESTING METHODS
Such cross-talk has recently been shown to favour in vitro
epidermal regeneration, formation of stratum corneum and
model longevity (17). Most interestingly full thickness skin models
permit the analysis of dermal influences on the epidermal
tissue rejuvenation and differentiation process (13, 17). A
popular cell source for building 3D skin models are neonatal
human foreskin-derived epidermal keratinocytes which reveal
a vast proliferative capacity (8). A comparative analysis
revealed that populations of human foreskin keratinocytes
can be subdivided into three phenotypic and functionally
distinct subgroups, which were roughly characterized as
stem cell candidate-, transient amplifying- and as basal and
suprabasal epidermal cells (11, 16). Experiments to test their in
vitro regenerative capacity demonstrated that the stem cell
fraction showed superior capabilities followed by the other
two populations (11, 16). Further data revealed that besides
intrinsic cellular capabilities, extrinsic or microenvironmental
factors such as extracellular matrix and dermal regulatory
components can act as effective restorative elements on the
epidermal regenerative capacity (16).
3D Skin Models as a tool to study effects on skin renewal and
epidermal homeostasis
Human native skin biopsies are very close to the in vivo
situation and therefore quite ideal analytical in vitro tools to
evaluate endpoints of skin safety and efficacy. Because of
their very limited availability and donor specific variability
skin biopsies do not represent a solution for industrial high
throughput applications. Therefore organotypic 3D skin models
become increasingly important in medical and cosmetic skin
research but also for substrate and product safety evaluations
(REACH). Todays organotypic skin equivalents definitely reveal
the necessary structural and physiological complexity to
recapitulate important steps in the process of skin homeostasis.
In this article Dermatest GmbH demonstrates two in vitro
assays, which can be used to characterize the influence and
effectiveness of single cosmetic and medical actives as well
as whole formulations on the processes of skin renewal and
revitalization.
MATERIAL & METHODS
In vitro 3D culture
Phenion® FT skin models were Incubation at 37°C, 95 percent
H2O, 5 percent CO2. Media components were purchased
from Life Technologies, unless otherwise stated. Basic culture
media: DMEM with Glutamax/Ham`s F12 medium (3:1)
supplemented with 100 IU/ml of penicillin and 100 mg/ml of
streptomycin. Supplements: DMEM 1.6 mg/ml of bovine serum
albumin (Sigma, Germany), 0.4 mg/ml of hydrocortisone
(Sigma, Germany), 0.12 IU/ml of insulin, 1 mM ascorbic acid
2-phosphat (Sigma, Germany).
In vitro applications of atRA, 13cRA, IL-1a
10 mM stock solutions of atRA 13cRA in DMSO were prepared.
1000, 500 and 100 nM of atRA and 1000 and 100 nM 13cRA
were applied to the basal medium. Pro-inflammatory cell
culture conditions were generated by application of 5 pg/ml
of cytokine IL-1a (R&D-Systems). Cultivation media was daily
renewed. For topical application of 13cRA, Isotrex Crème 0,05
percent (Stiefel,UK) was utilized. Histological Analyisis: 3x4 mm
biopsies from center of skin models were embedded in OCT at
-80°C. 5 mm cryo tissue sections were mounted on Superfrost®
Plus microscope slides (Menzel GmbH, Germany) and fixed
in 4 percent formalin solution (Sigma, Germany). Proliferation
Assay: Cell proliferation was detected for the last 24h of total
incubation period by applying Click-iT® EdU Imaging Alexa
598 Kit (Life-Technologies, Germany), EdU-positive nuclei
were evaluated from 400-1000 counted DAPI-positive nuclei.
Detected tissue sections were embedded in Vectorshield
(Vector Lbt, USA) before microscopic analysis (Zeiss). Viability
Assay: Skin models were incubated with 1 mg/ml DuraliQ stable
MTT solution (Dojindo) in basic DMEM for 3h in the dark at 37°C,
95 percent H2O, 5 percent CO2. For analysis, model triplicates
per endpoint were rinsed thoroughly, separated into its model
components via enzymatic thermolysin activity (2.4 U/ml, 3h
at 4°C) and lysed separately overnight at RT in isopropanol
(epidermis: 1 ml; dermis: 5 ml, Karl Roth) on a plate shaker. ODs
of lysates were measured in triplicates at 570 nm. Parameters of
result acceptance are defined as follows: OD570 of formazan
extracts from negative control must be greater than 0.8. For
experimental exceptance, CV of identical treated tissues had
to be less than 30 percent, with exception of cases with OD
below 0.3. Calculation of viability: 100*(OD570 Exp/(OD570 control).
RESULTS
In vitro assay to evaluate in vitro cell proliferation
Basal epidermal keratinocytes and dermal fibroblasts of 3D
skin models undergo mitosis. Before entering the phase of
active cell division or mitosis (M-Phase), cells traverse a prepreparative Interphase (I-Phase), during which pre-mitotic
cells start to grow and to fully replicate their genomic DNA (S
Phase). EdU® is a detectable nucleotide base analogue which
is specifically inserted into replicating DNA strands during
S-phase of cell cycle. By utilising the Click-iT® EdU detection
kit, replicated genomic DNA in nuclei of proliferative cells
could be visualized in tissue slides via immunofluorescence
microscopy (Figure 1A).
In vitro Modulation of cell proliferative activity
The following experimental approaches revealed that the
proliferative activity of basal keratinocytes and fibroblasts
can be modulated by external stimulation. When grown in
basal medium without growth stimuli, cells in epidermis and
dermis showed a basic cell cycle activity. Explicit stimulation
of cell growth could be triggered by application of growth
TESTING METHODS
supplements into culture media, which lead to a noticeable
increase of cell proliferation activity in epidermis and dermis.
Otherwise the increased proliferative activity of stimulated
epidermal and dermal cells could be silenced again when an
explicit pro-inflammatory milieu was created by addition of
Interleukin-1 alpha (IL-1a); (12) into the culture media (Figure
1A; dermis not shown).
the incubation period. Compared to basal media controls, in atRA
supplemented media cultivated skin models showed an elevated
proliferative activity of epidermal keratinocytes but no effect on
dermis. The topically treatment of skin models with 0,05 percent
13cRA for 24 hours showed at least an increase of proliferative activity
in epidermis by trend whereas the increase of proliferative activity
of dermal fibroblasts was noticeable. For control the cultivation of
skin models in media supplemented
with ascorbic acid 2-phosphate,
insulin and hydrocortisone increased
epidermal and dermal proliferation
likewise (Figure 1A, B, C).
Figure 1. Cell proliferation. (A) 3D skin models were cultivated under basal (a, d) and under proproliferative (supplemented) media conditions (b, e). To reverse the pro-proliferative effects again, 5
pg/ml of hu-IL-1a was added to the supplemented 3D skin model cultures (c, f). (B) Effects on
proliferation described in A) were quantified via Immunefluorescence (IF)-microscopy. EdU®-positive
cells were counted and expressed as percentage. (C) The Click-iT®-EdU Kit was applied to tag DNA of
proliferating epidermal and dermal cells during S-Phase of the cell cycle. Histological tissue slides from
3D skin models have been prepared and tagged DNA was visualized via IF analysis. Cell nuclei of
proliferating cells appear red. The universal nuclei stain is blue (DAPI). The 3D skin tissue structures were
visualized via DIC microscopy. (D) 3D skin models were systemically treated with 500 nM atRA or
creamed with 0,05 percent 13cRA. Epidermal and dermal cell proliferation was evaluated in parallel.
As expected, the data revealed that the proliferative cell
activity in skin equivalents reacts dynamically according to
the type of stimulus.
atRA- and 13cRA treatment of in vitro 3D Models
Skin models were incubated in parallel with single or combinations
of different pro-proliferative actives for 4 days. Only the topical
application of Isotrex® (0,05 percent of 13cRA) was time-limited to
the last 24 hours of the total cultivation period in order to avoid strong
irritating side effects on epidermis. 13-cis-retinoic acid (13cRa) is an
isomeric derivative of all-trans retinoic acid (atRA) and often used for
medical skin applications (18). Epidermal and dermal proliferative
cell activity was marked with Click iT® EdU for the last 24 hours of
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The MTT viability assay
Viability is defined as the overall
capacity of living. That summarizes
all cellular metabolic activities which
are needed for house-keeping,
growth and division (1). A wellestablished method to measure
the viability of tissues and cells is the
MTT-assay. 3-(4,5-Dimethylthiazol-yl)2,5-diphenyl-tetrazoliumbromide or
short MTT is a yellow, water-soluble
tetrazolium salt, which is reduced
via tetrazolium-ring cleavage and
transformed into water-insoluble
purple-blue formazan. The waterinsoluble,
crystalline
formazan
precipitates into the cellular cytosol
and the amount of precipitate
correlates quantitatively with the
degree of overall cell viability. After
stopping the MTT reaction, formazan
is released via chemical tissue lysis
and its dissolution`s optical density
is measured. The MTT transforming
reaction is mediated by enzymes
associated with the endoplasmatic
reticulum and the mitochondria.
Recent data showed that MTT
reduction predominantly involves
NAD(P)H-dependend
enzymes
of the endoplasmatic reticulum
which are involved in glycolysis.
Whereas in contrary to previous
beliefs the mitochondrial succinate
dehydrogenase activity, involved in
cell respiration, adds a much smaller
contribution to the overall MTT
turnover in cultured human cells (1).
Measuring epidermal and dermal
cell viability
3D skin models offer the opportunity
for a differential analysis of dermal and epidermal viability (Figure
2). For assay demonstration, skin models have been systemically
incubated with concentrations of 1000 and 100 nM of atRA and
13cRA (18, 19). Recent growth response studies on epidermal
keratinocytes have demonstrated that both Vitamin A substances
in named concentrations have a markly positive influence on
tissue viability (18). After four and seven days of atRA and 13cRA
treatment, viability of skin models was tested via MTT-assay (Figure
2A, B). A subsequent analysis of epidermal and dermal equivalents
revealed that in correlation with recent data both Vitamin A
substances showed an positive effect on skin model`s epidermal or
dermal cell viability after seven but not after four days (not shown)
of incubation in a dose-dependent manner (Figure 2A).
TESTING METHODS
via tuned and coordinated
enzymatic
activity
inside
the native stratum corneum
(3).
Such
functional
microenvironmental
conditions seem to be strongly
disturbed in reconstructed skin
models (2).
3D skin models as a useful
tool to study cosmetic and
medical actives
Skin
models
allow
the
correlating
analysis
of
substance
and
dose
Figure2. MTT-viability: (A) 3D skin models were cultivated over 7 days in media with 1000 and 100 nM of atRA
dependent
epidermaland 13cRA applied. The status of organotypic epidermal and dermal tissue viability was measured in
parallel. (B) Preparation of MTT-treated Phenion® FT skin models. An enzymatic separation of the 3D skin
and dermal cell responses.
model in its epidermal and dermal equivalents allows a differential MTT-analysis.
Cell proliferation of basal
epidermal
keratinocytes
and
dermal
fibroblasts
DISCUSSION
are important episodes in the process of skin renewal and
homeostasis (3). A characteristic morphology and a clearly
Participation of hair follicles in epidermal homeostasis and
defined localization make basal keratinocytes and dermal
regeneration
fibroblasts good study objects for a microscopic analysis (13).
Under physiological conditions the activity of hair follicles and
The stimulation with pro-proliferative retinoic actives in different
their stem cells are nearly dispensable for regular epidermal
ways of application revealed a noticeable susceptibility of in
processes of skin homeostasis (9). Under conditions of severe
vitro skin cells for proliferative stimuli, which is adjustable through
trauma and wounding, when larger amounts of lost cell
the definition of medium parameter. As a result pro- as well as
material have to be replaced in due time, hair follicles and
anti-proliferative, agent and dose-dependent effects could
their stem cells especially assist in the acute phase of rebe studied likewise. Proliferation assays reveal a high sensitivity
epithelialization to accelerate the start of wound closure (5, 9).
in a quite short time of incubation (Figure1). Even with relative
Recent research data revealed that optimal wound healing
low doses of actives and relative short periods of stimulation
involves an epidermal and dermal response and definitely
clear effects should be detectable especially on retinoid
depends on intact hair follicles, whereas the final wound
sensitive basal keratinocytes (18). Another option for studying
closure is accomplished by the regenerative capacity of the
effects on skin homeostasis offers the MTT-assay, which allows
interfollicular epidermis (9).
the measurement of whole tissue viability (1, Figure 2). Besides
quantification of direct effects on skin renewal it also offers the
Skin models recapitulate the key processes of terminal
possibility of studying substances and formulations which act
differentiation but not of desquamation
over a longer time period indirectly on cell viability via DNA3D skin models can be characterized as reconstructs of
protective or anti-inflammatory activities, which support tissue
interfollicular skin which are able to recapitulate structural
longevity by enhancing cell survivability instead of increasing
and molecular processes of skin homeostasis (13, 17). In
cellular turnover (1).
comparison to in vivo skin in vitro constructs are quite shortlived but reveal nevertheless a good regenerative potential
of the basal kerationocytes for a limited time period (13, 17).
REFERENCES AND NOTES
The decline in tissue regenerative potential in vitro is related to
a quite high degree of replicative ageing of the proliferative
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M.V. Berridge, et al., Biochemica, 4, pp. 14-19 (1996).
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E. Candi, et al., Nature molcellbio., 6, pp. 328-340 (2005).
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(2007).
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