ARTICLE Normal
human melanocyte (NHM) grows poorly in culture unless stimulated by serum
and several other mitogens. In 1982, Eisinger and Marko successfully isolated
pure NHM in a medium that has been supplemented with 5 % fetal bovine
serum, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)
and cholera toxin [1]. Likewise, Tsuji and Karasek obtained NHM in a medium
containing, in addition to 10 % fetal calf serum, cholera toxin, cAMP
elevator and 5-fluorouracil [2]. The elimination of keratinocytes from these
cultures has been attributed to the toxic effects of TPA and the cytotoxic
drug. Nevertheless, because of the high concentration of serum, fibroblasts
were still a major contaminant [3]. Furthermore, another major disadvantage
of both culture models was the use of serum and potentially mutagenic agents
that render cells obtained inappropriate for either research or therapy
[4]. Gilchrest et al. overcame this problem and successfully isolated
NHM in a hormone-supplemented medium that had been enriched with minimal
serum supplementation [5]. Nevertheless, keratinocyte and fibroblast elimination
and the low yield of NHM were also obvious problems in this culture system
[3, 5].
This report describes a modification of this culture system, which completely
avoids the problem of keratinocyte and fibroblast contamination and supports
good melanocyte proliferation in the absence of tumor precursors and without
serum supplementation. In addition, the relative significance of each
mitogen on melanocyte proliferation has been examined.
Material and methods
Melanocyte culture and morphological characteristics
Melanocyte culture was obtained from newborn foreskin and adult truncal
skin, obtained from adults undergoing elective operations (3 different
donors each, type IV skin). Specimens were transported to the laboratory
in tissue culture medium. The steps of skin processing and obtaining cell
suspensions were followed as previously described [5, 6]. Briefly, deep
dermis and subcutaneous fat are cut short (to minimize the risk of contaminating
melanocyte cultures with fibroblasts), remaining skin is cut into 4-5 mm3
pieces and incubated in 0.25 % trypsin (Sigma, Deisenhofen, Germany)
for 16 h at 4 °C. During this time, separation of the epidermis
from the dermis is observed. Fetal calf serum (FCS) (10 %) (Biochrom,
Berlin, Germany) is added to the specimens to inactivate the trypsin and
separation of the epidermis from the dermis is completed with the aid
of forceps. The dermis is discarded whereas the epidermis is vigorously
pipetted to obtain a single cell suspension. The epidermal cells are counted
in a hemocytometer chamber and inoculated at a density of 1.25 x 105
cells/ml in melanocyte growth medium (MGM) and maintained in 8 %
CO2 and 92 % air. The medium is changed 2-3 times
weekly. The complete MGM consisted of modified MCDB 153 (MCDB 153 + amino
acids + antibiotics) (Biochrom), cholera toxin (10 — 9
M) (Calbiochem, Bad Soden, Gemany), basic fibroblast growth factor (bFGF)
(2 ng/ml) (Boehringer, Mannheim, Germany), epidermal growth factor
(EGF) (10 ng/ml), crude bovine pituitary extract (BPE) (70 mu
g/ml) (Clonetics, San Diego, CA, USA), calcium hydrochloride (2 mM)
(Merck, Darmstadt, Germany), insulin (10 >mu g/ml), transferrin (10 mu
g/ml) (Sigma, Deisenhofen, Germany), hydrocortisone (1.4 x 10 — 6
M) (Serva, Heidelberg, Germany) and triiodothyronine(10 — 9
M) (Sigma). FCS (2 %) was added only during the first 2 days
of the primary culture and the first 24 hours of each subculture.
Cells (6-well culture plate) were monitored by inverted phase contrast
microscopy (Olympus, Tokoyo, Japan). Photographs were taken to document
and monitor microscopic alterations after cultures were washed once with
Ca + + and Mg + + free phosphate buffer
saline (PBS) (Biochrom).
Proliferation assays
First passage pure NHM cultures were used in this experiment (obtained
from each of the 3 newborn foreskins). Proliferation assays of cells
were carried out (each in triplicate) in tissue culture plates (96 flat
bottomed wells) (Becton and Dickenson, Mountain view, CA) by incubating
1 x 103 cell/well in 0.2 ml of medium by the highly sensitive
fluorimetric microassay using 4-methylumbelliferyl heptanoate. Fluorimetric
values, expressed as absolute fluorescence units, were measured by a Titertec
Fluoroscan II (Flow Lab Meckenheim, Germany) as previously described [7].
Each supplement was omitted from the complete MGM and the proliferation
rates were measured one day after inoculation (day 0) and every 3 days
afterwards for 3 weeks.
Immunohistochemistry
First passage NHMs from both newborn and adults were labeled with alkaline
phosphatase-antialkaline phosphatase (APAAP) technique as previously described
[8, 9]. Briefly, cells were fixed with pre-cooled methanol (— 4 °C)
for 5 min and were exposed to primary antibody (30 min) diluted
in 0.1 M (1:100). As a secondary antibody, anti-mouse IgG (Immunotech,
Marsielle, France) diluted in 1:100 in PBS was used with APAAP complex
(Dako, Glostrup, Denmark). The incubation steps with the secondary antibody
and the APAAP complex were repeated twice (30 min each) to intensify
the labeling reaction. Naphthol AS BI sodium salt (Sigma) and new fuchsin
(Merck) were used for visualization. Lastly, counterstaining with Meyer’s
hemalaun solution (Merck) was done. The slides were mounted with Kaiser’s
glycerol gelatin (Merck) and examined by light microscopy. Fixed cells
not exposed to the primary antibody served as negative control. Positive
staining appears bright red, whereas negative staining acquires the counterstain
as a faint blue color.
Statistical analysis
The mean and SD values obtained from the three newborn foreskins (each
in triplicate) were calculated and the percentage decrease of proliferation
was determined. Student’s t test was used for statistical evaluation
and p values of < 0.05 were considered significant.
Results
Melanocyte culture characteristics and morphological
features
Microscopic examination of the culture immediately after inoculation
of the desegregated epidermal cells revealed rounded refractile cells
floating in the medium. However, as early as 12 h of inoculation
two populations of cells, namely small dark bipolar and tripolar cells
were seen in between flat small rounded and polygonal cells that represent
the keratinocytes (Fig. 1).
On subsequent days, large sheets of keratinocytes are formed and the number
of dendritic cells progressively increased. The presumed dendritic melanocytes
were almost always seen in contact with adjacent keratinocytes via their
dendrites [5, 10] (Fig. 2).
After 3-4 weeks in adult culture and 4-5 weeks in newborn culture,
keratinocytes detached from the culture plate and the dendritic cells
changed their morphology to bipolar or fibroblasts-like spindle shaped
cells reminiscent of histopathological feature of intradermal melanocytic
nevus (Fig. 3). By
the 5th week, almost all keratinocytes have been detached from the culture
plate and only one population of cells remained. The presumed melanocytes
could be passaged up to 6 times in newborn and 4 times in adult
skin (average 5 weeks), after which cells underwent senescence, became
clumped and degenerated regardless of any mitogen, including serum, added
to them (Fig. 4).
No striking difference between melanocytes of adults and newborns was
observed, though newborn NHM were more slender and tended to be less dendritic.
Removal of each mitogen did not have any striking effect on melanocyte
morphology as cells retained their characteristic fibroblast-like shape
that is seen with the complete MGM, though the density of NHM decreased
progressively in the absence of mitogens. NHM incubated with plain medium
were almost absent from culture plates from day 6 through day 21 of
incubation.
Proliferation assays
Proliferation assay in the absence of each mitogen one day after inoculation
(day 0) revealed no significant results not only with the complete MGM
medium but also with cells incubated with plain medium (p > 0.05).
Interestingly, 3 days after incubation, a significant increase of
growth of NHM incubated not only with the complete MGM medium but also
with the plain and supplement deficient media when compared with day 0 was
detected (< 0.001). Absence of cholera toxin, EGF, Ca + +,
transferrin, hydro cortisone, and triiodothyronine did not significantly
affect NHM proliferation (p > 0.05). However, absence
of bFGF, BPE and insulin was associated with a significant NHM growth
retardation (p < 0.01). Similarly, NHM incubated with
plain medium showed a significant decrease of proliferation (p < 0.001).
Six days after incubation revealed similar results, though there was a
significant drop of proliferation rate values that reached almost less
than 50 % of day 3 (p < 0.001). Proliferation
rate of NHM on subsequent days, i.e., 9, 12, 15, 18 and 21 revealed
a significant decrease of proliferation in the absence of each mitogen
when compared with the complete MGM (p < 0.001) (Table
II). From day 12 through day 21, no further significant increase
of proliferation rate of the NHM incubated with the complete MGM was detected
as the proliferation rate reached to a plateau. Virtually, no NHM proliferation
was detected in culture plates incubated with plain medium on day 6 and
thereafter (Fig. 5).
Immunohistochemical findings
All cultured cells showed strong positive reaction to MAbs detecting
differentiated melanocytes and melanin producing cells, namely HMB 45 and
K.1.2.58. No significant number of cells showing negative labeling was
detected. The characteristic fibroblast-like morphology was well preserved
and could be easily visualized. The cytoplasm of the cells contained fine
granules showing the strong labeling, which represents the labeling with
the melanosomal antigen targeted by the activation marker HMB-45 [11]
(Fig. 6a). Similarly,
all cultured cells showed negative reaction to MAbs specific for other
epidermal cells, such as basal or suprabasal keratinocytes and Langerhans
cells (Fig. 6b). No difference
in antigen expression was detected between newborn and adult skin NHM.
Similarly, no striking difference in antigens expression was detected
in the absence of any mitogen.
Discussion
Serum is a potent growth factor for all cells and melanocytes are no
exception. However, because of its presence, the melanocytes obtained
may not be optimal for examination of factors that may influence melanocyte
function; as serum may contain substances having structural or functional
similarities with those being tested and hence their effects could be
masked. Also, because of the presence of TPA, a potential carcinogenic
agent, melanocytes obtained are not suitable for therapeutic purposes
[4, 12]. Results of the present study show that melanocytes, indeed, can
be easily grown in a serum- and TPA- free medium.
Fetal calf serum (2 %) is added to cultures at time of inoculation
for 48 hrs in primary culture and with every passage for first 24 hrs
only. Addition of serum has been found to be responsible for attachment
of the cultivated cells to culture plates and for stimulation of their
growth [5]. Although serum was essential for attachment of adult melanocytes,
it was not essential for attachment of melanocytes of newborn origin,
however, melanocyte yield in the absence of serum was low (20-30 %
of the 2 % serum-containing complete MGM medium) (data not shown).
Keratinocytes and melanocytes grow usually together, however, after the
differentiation, full keratinization and separation, the melanocytes continue
to grow and pure melanocyte cultures are obtained. The minor differences
of NHM morphology between adults and newborns are most probably due to
the relative higher proliferative capacity of the latter.
A constant observation in this study and others is the close relationship
of NHM and keratinocytes in which NHMs were almost always seen in contact
with the adjacent keratinocyte colonies with dendritic projections. Pure
melanocytes in culture were fibroblast like or spindle shaped cells. Removal
of each mitogen did not have any striking effect on melanocyte morphology.
Indeed, it has been shown that contact with differentiated keratinocytes
is the signal for dendrite formation [10, 13]. Although pure melanocytes
could be obtained after several weeks, the presence of keratinocytes was
of benefit due to the supporting nature and the paracrine stimulatory
effects of these cells [10, 14]. This was also the rational for the use
of MCD153 medium with added supplements as it supports growth of
both melanocytes and keratinocytes but is suboptimal for fibroblasts [15].
Although alpha-melanocyte stimulating hormone can replace some of the
added mitogens e.g., BPE, it was not used in the present work as it is
not known to support keratinocyte proliferation [16]. Calcium is usually
added to medium to induce keratinocyte differentiation and keratinization
rather than as a mitogen [17].
Proliferation assay in the absence of each mitogen revealed significant
results that could be detected as early as 3 days of incubation.
The initial increase of proliferation that was observed is most likely
due to the effect of serum that has been removed on day 0 of assessment.
In favor of this assumption is the drastic drop of proliferation on day
6; by that time the effect of serum has disappeared. Nevertheless, absence
of bFGF, insulin and BPE resulted in a significant decreased NHM proliferation
that was also observed on day 6. As most experimental or therapeutic works
are done within one week of NHM passage, it is evident that they are the
most significant mitogens [18-20]. Absence of all added supplements revealed
significant NHM growth retardation from day 9 and afterwards, indicating
that long term NHM cultures require several mitogens for survival and
proliferation. It has been shown that confluence or growth factors depletion
caused reversible, G1 phase-specific, NHM cycle growth arrest; thus providing
an explanation for the almost non significant change in NHM proliferation
from day 9 through day 21 of incubation [20]. The main mitogenic
component in BPE is bFGF [21]; results of the present work, however, indicate
that factors in pituitary extract other than bFGF are also essential for
melanocyte proliferation, alpha MSH is a possible candidate [12].
The identity of the melanocytes is confirmed by the positive labeling
with specific MAbs. NHMs in culture but not in situ express melanoma
associated antigens. Nevertheless, this phenomenon does not reflect the
genetic and biologic properties, since the cultured NHMs have a diploid
karyotype, are non-tumorigenic and do not spontaneously transform in culture
[22]. The almost complete absence of other epidermal and dermal cells
as shown immunohistochemically is a clear advantage of this culture system.
From the above data, it is obvious that NHM can be selectively cultivated
in a serum free medium and without the use of tumor promoters. It is also
concluded that bFGF, insulin and BPE are of marked significance for melanocyte
growth from the early phases of pure melanocyte culture, whereas the other
supplements, although stimulating melanocyte proliferation, are not essential.
It is also recommended that examining factors that may influence melanocyte
proliferation should be carried out in the absence of all or any of these
mitogens according to experimental design.
CONCLUSION Article
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