Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes

Abstract

Background:
There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required.

Aim:
In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients.

Methods:
The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed.

Results:
Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model.

Conclusion:
We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.

Full text

599Indian Journal of Dermatology, Venereology, and Leprology | September-October 2012 | Vol 78 | Issue 5
Development of melanocye-keratinocyte co-culture
model for controls and vitiligo to assess regulators
of pigmentation and melanocytes
Ravinder Kumar, Davinder Parsad, Amrinderjit Kanwar, Deepak Kaul
1
Original
Article
ABSTRACT
Background: There is a need to develop an in vitro skin models which can be used as
alternative system for research and testing pharmacological products in place of laboratory
animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo,
reliable in vitro skin pigmentation models are required. Aim: In this study, we used primary
cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and
vitiligo patients. Methods: The skin grafts were taken from control and patients of vitiligo.
In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes.
Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase
assay, MTT assay and melanin content assay were performed. Results: Melanocytes and
keratinocytes were successfully cultured from control and vitiligo patients and after that
co-culture models were prepared. After treatment of co-culture model with melanogenic
stimulator we found that tyrosinase activity, cell proliferation and melanin content increased
whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and
melanin content decreased. We also found some differences in the control co-culture model
and vitiligo co-culture model. Conclusion: We successfully constructed in vitro co-culture
pigmentation model for control and vitiligo patients using primary cultured melanocytes and
keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over
the use of transformed cells. The only limitation of these models is that these can be used
for screening small numbers of compounds.
Key words: Coculture, keratinocytes, melanocytes, skin models
Departments of Dermatology,
and 1Experimental Medicine
and Biotechnology,
Postgraduate Institute of
Medical Education and
Research, Chandigarh, India
Address for correspondence:
Dr. Davinder Parsad,
Department of Dermatology,
Postgraduate Institute of
Medical Education and
Research,
Chandigarh-160012, India.
E-mail: parsad@mac.com
How to cite this article: Kumar R, Parsad D, Kanwar A, Kaul D. Development of melanocye-keratinocyte co-culture model for controls and
vitiligo to assess regulators of pigmentation and melanocytes. Indian J Dermatol Venereol Leprol 2012;78:599-604.
Received: November, 2011. Accepted: July, 2012. Source of Support: IADVL-Loreal Grant. Conict of Interest: None declared.
INTRODUCTION
The use of animals in research and clinical trials for
testing the chemical for safety use is decreasing now
days because of social and political pressure. To study
the biology of pigmentation and pathophysiology of skin
disease vitiligo, it is necessary to develop convenient
experimental skin models that are versatile and allow
translatability. Therefore research requires reliable
in vitro and experimental animal models to investigate
novel therapies and biologic and pathophysiologic
pathways.[1] Many models have been developed,[2] but
in vitro and in vivo models may show poor consistency
with clinical situations.[3] In vitro human models help
investigate skin pigmentation physiology in a more
equivalent fashion. The advantage of in vitro models
is the opportunity to analyze environmental changes,
substrate and dose-response interactions in a standard
fashion.[2,4]
Melanocytes are the cells which are responsible for
skin color and melanin synthesis. Melanocytes are
highly responsive cells that modulate their levels of
melanogenesis or proliferation according to extrinsic
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Kumar, et al. Melanocye-keratinocyte co-culture model
Indian Journal of Dermatology, Venereology, and Leprology | September-October 2012 | Vol 78 | Issue 5600
signals and factors derived from keratinocytes.
The melanin is produced in the melanocytes and
transferred to the keratinocytes.[5] Skin and hair color
depends on the amount, size, and type of melanin
produced by melanocytes, the subsequent transfer
of the pigment to the keratinocytes, and its eventual
distribution in the skin and hairs.[6-8] Melanin also
protects the individual from various environmental
assaults and potential cellular injury.[9,10] Melanin
prevents consequential DNA damage by effectively
absorbing ultraviolet light (UV) penetrating the
skin. Melanin is an effective scavenger of free
radicals too.[11-15]
Artificial skin pigmentation models are required
for the research on various pigment skin diseases
and to understand the regulation of pigmentation.
Also the artificial skin is required to test the
chemicals and skin care products for any type of
irritation, allergies, hypo-pigmentation and hyper-
pigmentation. Currently the demand for artificial
skin is not satisfied, and the experiments expenses
are growing due to the limited amounts of artificial
skin available. Many melanocyte or skin equivalent
models have been used to evaluate the potential
efficacy of melanogenic compounds to regulate
pigmentation. Variation in results has been found
in those skin equivalent models mainly because of
the use of different cell lines or because of the use
of keratinocytes and melanocytes alone. In vivo,
melanocytes are present mainly with keratinocytes.
Therefore testing of the compounds on melanocytes
alone in culture does not mimic the exact conditions
as found in actual skin. Keratinocytes also secretes
many compounds which act on melanocytes. The
melanocyte culture model alone does not reflect the
effects of compounds on keratinocytes that might
indirectly affect melanocyte function.
Therefore, there is a need to develop a melanocyte–
keratinocyte co-culture protocol that allows testing
of compounds for potential effects on pigmentation
in a more physiologically relevant context. Similarly
there is need for eliciting the role of various growth
promoters of melanocytes in vitiligo. For this, there
is a need to develop the melanocyte keratinocyte
co-culture model for vitiligo subjects using vitiligo
patients’ keratinocytes and melanocytes. Therefore
we decided to use primary cultured melanocytes and
keratinocytes to prepare the skin co-culture model in
the present study.
METHODS
Clinical samples
Patients with unstable vitiligo with no ongoing
treatment for their disease for the previous 3 weeks
were selected at the outpatient clinic of the Department
of Dermatology, Postgraduate Institute of Medical
Education and Research, Chandigarh, India. A graft
(2cmX2cm) was taken on clinically active perilesional
skin from five patients with vitiligo and five controls
with their written informed consent and this study was
approved by the ethics committee at the Postgraduate
Institute of Medical Education. Control samples were
obtained from the healthy control visiting skin OPD who
agreed to give skin graft or Plastic Surgery department
from the normal skin of patients undergoing graft
surgery. Grafts were taken from lateral thigh regions
of the subjects. The age range of unstable vitiligo
patients and controls was 18-40 years. The mean age
of patients with unstable vitiligo was 24.2 years, and
of the controls was 27.6 years. Unstable vitiligo was
defined as appearance of new lesions or increase in
size of existing lesion over last 2 months.
Melanocyte and Keratinocyte culture
Primary culture of melanocytes and keratinocytes were
established by the following procedure: Fresh biopsy
specimens were obtained under aseptic conditions in
phosphate buffer saline with antibiotics (penicillin
and streptomycin). Skin graft was cut into small pieces
(5 x 5mm) and incubated with trypsin-EDTA (0.25%
trypsin and 0.02% EDTA) at 4°C overnight. Trypsin
activity was required to separate the epidermis from
dermis. Next day trypsin activity was neutralized by
adding FBS (Fetal bovine serum) in 1:1 ratio and replaced
by phosphate buffer saline solution. The epidermis
was separated from the dermis with sterile forceps.
Thorough pipetting was done to separate the cells and
cell rich suspension was formed. The solid waste of
tissue was removed and suspension was centrifuged at
1000 rpm for five minutes. Cell pellet was resuspended
in serum free medium containing melanocyte basal
medium with supplements and for keratinocyte
culture the pellet was resuspended in keratinocyte
culture media with supplements. The cell number was
adjusted to 2.5 x104 cells per cm2. The cultures were
maintained at 37°C in a humidified, 95% air and 5%
CO2 atmosphere. The medium was changed at 3-4 days
intervals. The cultures were routinely examined for the
contamination as well as for the outgrowth of the cells.
Cells were split at confluence by trypsin treatment for 5
minutes at room temperature.

Kumar, et al. Melanocye-keratinocyte co-culture model
601Indian Journal of Dermatology, Venereology, and Leprology | September-October 2012 | Vol 78 | Issue 5
Co-culture and treatment
Cultured keratinocytes were harvested by treatment
with trypsin/EDTA and seeded at 2.5 x105 cells/well in
six-well plates. After two days, cultured melanocytes
(5 x104) were collected by trypsin/EDTA and added to
each well containing the keratinocytes. Co-cultures
of melanocytes and keratinocytes were maintained
in culture media (keratinocyte media + melanocyte
media), the initial seeding ratio of keratinocytes to
melanocytes being 5:1. After one day of co-culture,
fresh medium containing melanocyte inhibitors and
stimulators were added. Again after two days fresh
medium with fresh compounds at the appropriate
concentrations were added; two days after that,
cultures were photographed and then harvested with
trypsin/EDTA. The cells were dislodged with trypsin/
EDTA; media was added to inactivate the trypsin.
100 μl cell suspension was then seeded into flat-bottom
96-well tissue culture plates for the MTT proliferation
assay. The remaining cell suspension was centrifuged
at 1500 g for 5 min, washed with PBS, and then
solubilized in 200 μl extraction buffer at 4°C for 1 hour
and various assays were then conducted [Figure 1].
Tyrosinase assay
The cells were washed with ice-cold 1XPBS. 150 μl
lysis buffer (1% Triton X-100 in 0.1 M phosphate buffer)
was added to each 6-well plate. Cells were scraped and
transferred to a 1.5 ml tube, lysed through 3~5 times
‘freezing and thawing’ cycle, in liquid nitrogen and
centrifuged at 15,000 rpm for 5~10 min at 4oC. The
samples (300~500 ug/80 μl) were added to a new 96
well plate on the ice. 20 μl of 5 mM L-DOPA (L-3,4-
dihydroxyphenylalanine) was added to the each well.
It is then incubated at 37oC and measured absorbance
at 475 nm.
MTT assay
The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, a yellow tetrazole)
assay was used to determine cell proliferation.[16] After
treatment, 100 μl aliquots of cells were harvested as
detailed above and plated in flat-bottom 96-well plates.
Cells were allowed to attach and grow overnight at
37°C. MTT assay was performed, according to the
manufacturer’s instructions. The formazan precipitates
were quantitated by absorbance at 562 nm in an ELISA
plate reader.
Melanin content assay
Melanin content was determined using a standard
protocol.[17] Cells were lysed with 200μl 1 N NaOH
and pipetted repeatedly to homogenize them. After
homogenization, the cell extract was transferred
into 96-well plates. Relative melanin content was
determined by absorbance at 405nm in an ELISA plate
reader.
Statistical analysis
Statistical analysis was performed using SPSS 14.0
(SPSS Inc., Chicago, IL, U.S.A.). The data was analyzed
by ANOVA followed by Post hoc test Bonferroni.
P values < 0.05 were considered as significant.
RESULTS
Melanocytes culture
Melanocytes were successfully cultured from controls
[Figure 2a] and unstable vitiligo patients [Figure 2b].
We found that melanocytes cultured from unstable
vitiligo patients were confluent in 15 to 25 days
whereas the control melanocytes took 10 to 15 days to
confluency. We observed some significant differences
in the morphology of melanocytes between control
and unstable vitiligo patients. Melanocytes from
control were normal in perinuclear region and size.
On the other hand melanocytes from unstable vitiligo
patients showed bigger perinuclear region. Dendrites
of perilesional unstable vitiligo patients were small
with clubbed ends. Dendrites of these melanocytes
seem to be retracted where as the dendrites of control
were normal in shape and size as mentioned in our
previous paper.[18]
Keratinocytes culture
We found no significant difference in the time taken
by the keratinocytes to confluency between control
Figure 1: Flow chart showing coculture, treatment with stimulators
(Latanoprost, Stem cell factor) and inhibitors (Kojic acid,
Hydroquinone) and various assay performed
Primary Cultured melanocytes
(5 10 cells/well)4
Primary Cultured
Keratinocytes
(2.5 10 cells/well)5
Primary Coculture
Treatment withstimulators
1. Latanoprost
2. Stem cell factor
1. Kojic acid
2. Hydroquinone
Treatment withInhibitors
Melanin AssayMTT AssayTyrosinase Assay

Kumar, et al. Melanocye-keratinocyte co-culture model
Indian Journal of Dermatology, Venereology, and Leprology | September-October 2012 | Vol 78 | Issue 5602
[Figure 2c] and vitiligo unstable patients [Figure 2d].
The only difference we found was that the number of
vacuolated keratinocytes cultured from vitiligo were
more as compared to the control keratinocytes.
Co-culture of Keratinocytes and Melanocytes
After successfully culturing the melanocytes and
keratinocytes we prepared co-culture model for
control [Figure 3a] and vitiligo patients [Figure 3b].
For co-culture model we used cultured melanocytes
to keratinocytes in 1:5 ratio. These co-cultures were
maintained up to 6 days. First the keratinocytes
were seeded in the culture plate and after two days
melanocytes were added. One day later the co-cultures
were treated with melanogenic compounds, which
were replenished every two days.
Effects of Melanogenic Stimulators
For the present research work, two melanogenic
stimulators compounds stem cell factor and latanoprost
which are known to stimulate melanogenesis were
examined on this co-culture system and the effects
on control co-culture model was compared with
the effect on vitiligo co-culture model. Our results
confirmed that tyrosinase activity, melanin content
and proliferation increased significantly in dose
dependent manner in the control co-culture model
after treatment with stem cell factor and latanoprost
[Figure 4a and b and Figure 5a and b]. On the other
hand we found a small increase (insignificant) in the
tyrosinase activity, melanin content and proliferation
in vitiligo vulgaris co-culture model [Figure 6a and b
and Figure 7a and b].
Effects of Melanogenic Inhibitors
In this study, we examined the effects of two
melanogenic inhibitor compounds (Kojic acid
and Hydroquinone) used in the treatment of
hyperpigmentary disorders. We measured effects
on tyrosinase activity, melanin content, and cell
proliferation in melanocytes keratinocytes co-culture
model from control and vitiligo patients. Tyrosinase
activity, melanin content and proliferation decreased
significantly in the co-culture model [Figure 4c and d
and Figure 5c and d] after treatment with Kojic acid
and Hydroquinone. In vitiligo vulgaris co-culture
model [Figure 6c and d and Figure 7c and d], we
found that the tyrosinase activity, melanin content
and proliferation decreased more significantly as
compared to control co-culture model.
DISCUSSION
Many skin equivalent models have been used for
evaluating the efficacy of melanogenic compounds,
but each has significant shortcomings when
considered as a physiologically relevant model.
Pigmented melanoma cell lines have sometimes
been used to assess the efficacy of melanogenic
regulators,[17,19-21] despite the fact that transformed
cells are not really appropriate since they are
abnormal melanocytes by definition and since their
proliferative potential and disrupted intracellular
signaling interfere with the normal regulation of
melanin production.
Lei et al.,[17] developed a melanocyte-keratinocyte
Figure 2: Representative pictures of the cultured primary
melanocytes from control (a) and Vitiligo vulgaris patients (b)
and cultured primary keratinocytes from control (c) and Vitiligo
vulgaris patients (d)
ab
cd
Figure 3: Representative pictures of the cocultured primary
melanocytes and keratinocytes from control (a) and Vitiligo
vulgaris patients (b)
ab

Kumar, et al. Melanocye-keratinocyte co-culture model
603Indian Journal of Dermatology, Venereology, and Leprology | September-October 2012 | Vol 78 | Issue 5
co- culture protocol that allows testing of compounds
for potential effects on pigmentation but they used
immortalized melanocyte and keratinocyte. On the
other hand we used primary cultured melanocytes
and keratinocyte to developed in vitro co-culture
model. We developed in vitro co-culture model for the
unstable vitiligo which is a pigmentary disease and
we found some significant melanocyte keratinocyte
morphological differences between healthy controls
and unstable vitiligo patients.
Primary melanocytes and keratinocytes would be an
optimal co-culture model to test compounds. Therefore
we decided to use primary cultured melanocytes
and keratinocytes to prepare the skin co-culture
model in the present study. We have successfully
designed a melanocytes-keratinocytes co-culture
model using primary cultured skin melanocytes
and keratinocytes. Melanocytes and keratinocytes
from vitiligo patients were morphologically different
from control melanocytes and keratinocytes. The co-
culture pigmentation model of vitiligo patients also
shows morphological differences to that of control co-
culture model, which were confirmed after treatment
with pigmentation stimulators and inhibitors and
performing melanin assay, MTT assay and tyrosinase
assay. After treatment with melanogenic stimulators
melanin content, cells proliferation and tyrosinase
activity significantly increased but the increase
was not significant in vitiligo co-cultured model
as compared to control co-cultured model. After
Figure 4: Representative morphologies of control melanocyte–
keratinocyte cocultures exposed for four days to (a) Stem cell
factor (10ng/ml), (b) latanoprost (20µg/ml), (c) Kojic acid (0.2mg/ ml)
and (d) Hydroquinone (0.2mg/ml)
ab
cd
Figure 5: Tyrosinase activity, Melanin content and MTT assay in
control melanocytes keratinocyte coculture after treatment with
the Melanogenic Stimulators (Stem cell factor (a) and latanoprost
(b)) and Melanogenic Inhibitors (Kojic acid (c) and Hydroquinone
(d)). Data is presented as mean ± SD. (Statistical signicance is
shown by *P<0.05 vs. control
ab
cd
Figure 6: Representative morphologies of vitiligo melanocyte–
keratinocyte cocultures exposed for four days to (a) Stem cell
factor (10ng/ml), (b) latanoprost (20µg/ml), (c) Kojic acid (0.2mg/
ml) and (d) Hydroquinone (0.2mg/ml)
ab
cdFigure 7: Tyrosinase activity, Melanin content and MTT assay in
vitiligo melanocytes keratinocyte coculture after treatment with
the Melanogenic Stimulators (Stem cell factor (a) and latanoprost
(b)) and Melanogenic Inhibitors (Kojic acid (c) and Hydroquinone
(d)). Data is presented as mean ± SD. (Statistical signicance is
shown by *P<0.05 vs. control.
ab
cd

Kumar, et al. Melanocye-keratinocyte co-culture model
Indian Journal of Dermatology, Venereology, and Leprology | September-October 2012 | Vol 78 | Issue 5604
treatment with melanogenic inhibitors melanin
content, cells proliferation and tyrosinase activity
decreased which was more significant in vitiligo co-
cultured model as compared to control co-cultured
model.
We conclude that we successfully constructed
co- culture pigmentation model for control and vitiligo
patients using primary cultured melanocytes and
keratinocytes. Pigmentation of the skin is because of
melanin synthesis which is regulated by tyrosinase
activity. These pigmentation models can be used
for testing of compounds for potential effects on
pigmentation in a more physiologically relevant
context. Developed in vitro co-culture models can
be used for screening small numbers of compounds
as we are using primary cultured melanocytes
and keratinocytes, as compared the models using
immortalized melanocytes and keratinocytes. But
the use of primary melanocytes and keratinocytes is
appropriate over the use of transformed cells as the
transformed cells are abnormal melanocytes and
disrupted intracellular signaling interferes with the
normal regulation of melanin production.
The in vitro vitiligo pigmentation model can be used
to study the pathogenesis of vitiligo and effect of
various compounds. We have shown this system to
be a suitable model for testing known melanogenic
inhibitors or stimulators.
ACKNOWLEDGMENTS
Financial support for this research work was received from
IADVL-Loreal Grant.
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