www.blackwellpublishing.com/EXD
Methods
An improved method of human keratinocyte culture
from skin explants: cell expansion is linked to markers
of activated progenitor cells
Aihua Guo and Colin A. B. Jahoda
Department of Biological Sciences, University of Durham, Durham, UK
Correspondence: Colin Jahoda, PhD, Department of Biological Sciences, University of Durham, South Road, Durham, DH1 3LE, UK,
Tel.: 0191 334 1338, Fax: 0191 334 1201, e-mail: colin.jahoda@durham.ac.uk
Accepted for publication 7 April 2009
Abstract: Human keratinocyte primary cultures are commonly
established by tissue dissociation and often rely on feeder cell
supports and culture medium that is not defined. Further,
contamination by unwanted fibroblasts can be problematic. Here,
we developed a skin explant method for growing primary
keratinocytes that was rapid, simple, and reliably generated
keratinocyte cultures free of fibroblast contamination. The process
capitalized on the observation that fibroblasts migrate out of adult
skin explants later than epidermal cells, allowing the early
harvesting of keratinocytes by trypsinization. When grown
subsequently in defined medium in the absence of feeder cells, the
explant-derived cells grew rapidly and could be cultured for
multiple passages. Immunofluorescence microscopy revealed that a
high percentage of cells harvested from the explant outgrowths
expressed K15, while very few expressed the differentiation marker
K10. Cells that were stained while migrating out from explants
strongly expressed markers associated with progenitor cells,
including p63, K15 and CD133, and displayed intense K6
expression, indicative of activated keratinocytes in wound-healing
epidermis. By replenishing the explants with fresh medium after
harvesting, further epidermal outgrowths could be obtained,
offering the possibility of greatly increased keratinocyte yields for
clinical applications.
Key words: explant – keratinocytes – primary culture – progenitor
cell – wound healing
Please cite this paper as: An improved method of human keratinocyte culture from skin explants: cell expansion is linked to markers of activated progenitor
cells. Experimental Dermatology 2009; 18: 720–726.
Introduction
A reliable source of cultured keratinocytes is essential as a
component of skin substitutes to treat burns and wounds
and for laboratory testing. The epidermis is one of only a
few tissues from which it is possible to propagate its principal cell type (the keratinocyte) and to use these cultured
cells to reconstitute a stratified and fully differentiated
human tissue in vitro (1–4). As Medawar (5) successfully
separated a pure epidermal sheet from human skin by
trypsinization, it has been possible to readily obtain epidermal cells for expansion in tissue culture. In the years that
have passed, continuing improvements have been made on
the original culture method. In particular, Rheinwald and
Green (6) used a feeder layer of X-irradiated 3T3 cells to
support keratinocytes in culture. In this method, however,
the activation of 3T3 cells must be kept under control, and
contamination must be avoided. Kitano and Okada (7)
modified the process by introducing a milder protease
known as dispase to separate the epidermal sheet from the
720
underlying dermis of the skin. Then, Boyce and Ham (8)
adopted a serum-free medium for primary keratinocyte
culture, in which the 3T3 feeder layer is no longer needed
and therefore has benefits for use in clinical applications.
Among several varieties of keratinocyte serum-free medium
currently available, EpilifeTM (Invitrogen Ltd, Paisley, UK)
is one commercial form which we employed for this study.
This medium contains various human recombinant growth
factors and has a low calcium concentration at 0.06 mM.
While the dissociation method of keratinocyte primary
culture is well established, attempts to acquire purified
adult stem-cell like ⁄ progenitor keratinocytes from whole
human skin are ongoing in many laboratories. In particular,
different techniques are currently used to achieve high purity or homogeneous primary cultures enriched for keratinocyte progenitor ⁄ stem cells. These include filtration,
density gradient centrifugation, fluorescence-activated cell
sorting with cell surface antibodies as well as differential
adhesion to enrich for cells that rapidly attach to particular
substrates (9). However, despite the efforts to characterize
ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 720–726
16000625, 2009, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0625.2009.00900.x by INASP/HINARI - INDONESIA, Wiley Online Library on [12/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DOI:10.1111/j.1600-0625.2009.00900.x
and purify keratinocyte cells from intact human skin, there
still is a need for improved reliable techniques to isolate
high quality progenitor keratinocytes and propagate them
in culture, preferably in the absence of a feeder layer.
Cultures of adult skin explants have been used to model
adult skin epidermal growth and behaviour (10–12). A similar culture method has been previously used with cervical
uteri explants to test factors affecting serial cultivation of cervical epithelial cells (13). However, as fibroblasts grow out
from these explants, it is difficult to separate the fibroblasts
from the keratinocytes once the two cell types are mixed.
Interestingly, previous work has suggested that the keratinocytes which migrate out from whole skin explants apparently
undergo little or no cell division over the first few days in
culture (14). Moreover, transmission and scanning electron
microscopy studies (15) have shown that these early migrating cells originate from the basal layer of the epidermis.
It has previously been shown that fibroblasts do not
grow out from adult human skin explants until several days
after the appearance of keratinocytes. We have taken
advantage of this time lag between the migration of keratinocytes versus fibroblasts to develop a quick and easy
method to produce keratinocyte cultures from skin
explants. This method avoids fibroblast contamination,
provides rapid cell growth and offers great simplicity and
adaptability to specific experimental needs. In addition, we
examined the expression of molecular markers in these cells
during early migration. We found that cells in the outgrowth, expressed a range of progenitor and wound healing
markers, suggesting they are potentially enriched for activated progenitor cells. Explant-derived keratinocytes could
be grown rapidly to multiple passages using any of the current methods of culture, and importantly, the original
explants could be recycled and used as a continuing source
of keratinocytes.
Methods
Keratinocyte cell culture from explants
Primary cultures of human keratinocytes were established
using discarded healthy skin from patients of both genders
and various ages with informed consent and Ethics Committee approval to CABJ. (C. Jahoda, NHS; Ethics submission version 1, 23 ⁄ 9 ⁄ 2005). Skin samples were soaked in
minimal essential medium (MEM, M4655; Sigma–Aldrich,
Dorset, UK) with double strength antibiotics (1.250 lg ⁄ ml
amphotericin B, 200 IU ⁄ ml penicillin and 200 lg ⁄ ml streptomycin) overnight at 4C. The skin was then flattened in
a 60-mm sterile Petri dish with the epidermis facing up
in fresh MEM (above). Strips of split thickness skin about
2-mm wide were obtained by using sharp scissors and fine
forceps (Fig. 1a). Each strip was then cut into pieces
between 1 and 2 mm in length placed onto the bottom of
ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 720–726
35-mm diameter culture dishes [(Primaria; Falcon, Becton
Dickinson, Oxford, UK) or NunclonTM Surface, Nalge
Nunc International, Roskilde, Denmark] at a density of
around 30 pieces ⁄ dish (Fig. 1b). At this stage, it was imperative that the explants were oriented with the epidermis
facing up. The culture dish with explants were incubated at
37C in 5% CO2 for 30 min to 1 h (according to the
amount of liquid associated with the specimens) to secure
attachment of the explants to the culture dish. At this
point, the specimens were still moist. About 1.5–2 ml of
the MEM containing 20% fetal bovine serum (FBS, F7524;
Sigma) and antibiotics (0.625 lg ⁄ ml amphotericin B,
100 IU ⁄ ml penicillin and 100 lg ⁄ ml streptomycin) were
then carefully added to cover the explants and the dishes
were returned to the incubator. Cultures were checked for
cell growth the next day. On day 3 or 4 of culture the cells
were subcultured. The culture dishes with explants were
washed with calcium- and magnesium-free phosphatebuffered saline (PBS), and then incubated with 0.25%
Trypsin–EDTA (Invitrogen Ltd, Paisley, UK) until the
sheets of cells surrounding the explants had been dissociated. The trypsin was inactivated with serum-containing
medium, and the cell suspension was spun at 1000 rpm for
5 min. The cell pellet was resuspended in EpilifeTM growth
medium (Invitrogen Ltd, Paisley, UK) supplemented with
human keratinocyte growth supplement (Invitrogen Ltd,
Paisley, UK, 5 ml ⁄ 500 ml) and seeded into T25 ⁄ T75 flasks.
Routinely, the explants were re-fed with fresh medium of
MEM containing 20% fetal calf serum (FCS) and incubated
further to check their further capacity for keratinocyte
growth. Keratinocyte cultures established from explant
cultures were serially subcultured in EpilifeTM medium. To
examine whether keratinocytes produced from explants
were capable of normal growth under conventional conditions, some cultures were transferred to Rheinwald and
Green medium (6) and grown on 3T3 feeder layers.
Antibodies and immunofluorescent microscopy
To investigate the expression profiles of keratinocyte cells,
the cells were subcultured and grown for a 3-day period in
Epilife in 35-mm culture dishes. To study patterns of
expression during initial explant outgrowth, the cultures of
skin explants from breast and abdominal sites were stained
after 1 and after 3 days in culture in MEM containing 20%
FBS. Briefly, the culture dish with cells ⁄ explants were
washed with PBS and then fixed with 95% ethanol and 5%
acetone for 10 min, and again washed in PBS before blocking with10% donkey serum (D9663, Sigma) in PBS. Cells
were then incubated with primary antibodies overnight at
4C followed by Alexa-Red or fluorescein isothiocyanateconjugated secondary antibodies with 4¢-6-diamidino-2phenylindole at room temperature for 2 h. The dishes were
then washed and mounted.
721
16000625, 2009, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0625.2009.00900.x by INASP/HINARI - INDONESIA, Wiley Online Library on [12/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Explant method for culturing primary human keratinocytes
(b)
(a)
(f)
(e)
(d)
(c)
(h)
(g)
(i)
(j)
(k)
(l)
(m)
Figure 1. Preparation of explants of human skin for keratinocyte
culture and keratinocyte outgrowth from skin explants. Strips of skin
were obtained by catching the tissue with fine forceps and then cutting
with sharp-angled scissors. The depth of cut was below the basal layer
of epidermis but relatively shallow in relation to the dermis (a). Each
strip was cut into pieces 1–2 mm in length and placed in the bottom of
a 35 mm diameter culture dishes at a density of 20–30 pieces per dish
(b). Cells started growing out of the explants between 24 to 36 h (c).
Outgrowths resembled a ring and continuously expanded surrounding
the explants by day 3–4 (d). Cells with fibroblast or melanocyte
morphology were first observed beyond the fringe of the keratinocyte
outgrowths on day 5 (e). Early growth comparison between
keratinocytes harvested from explant culture. Light microscopic views of
cells subcultured from explants (after 4 days) and grown for a further
1 day (f), 2 days (g) and 6 days (h). Expression of K5, K10 and K15 in
passage 1 cells derived from explant culture. Dual immunofluorescent
labeling was performed for K5 (green) and K10 (red) in (i) and for K5
(green) and K15 (red) in (j). Thus, yellow colour indicates areas of
K5 ⁄ K10 or K5 ⁄ K15 colocalization. 4¢-6-Diamidino-2-phenylindole (DAPI)
was labeled as in blue. Repeated keratinocyte outgrowth from skin
explants. Continuous keratinocyte outgrowth was observed after
resupplying the explants with medium and returning dishes into culture
under the same conditions as before. Cell outgrowth at 2 days from a
recycled explant has a similar but faster growth pattern to the initial
culture. Both the inner (k) and outer (l) regions of a keratinocyte
outgrowth are free of fibroblasts. Keratinocyte cells harvested for a
third time from the same explant growing in EpilifeTM 3 days after
subculture (m). Scale bars: 60 lm.
722
The primary antibodies included: human basal epidermal
marker K5 (goat anti-cytokeratin 5, Santa Cruz Biotechnology, Inc, Heidelberg, Germany); suprabasal and differentiating keratinocyte marker K10 (mouse anti-cytokeratin 10
monoclonal antibody; Chemicon International, Herts, UK);
a6b1integrin (mouse anti-rat monoclonal antibody; TCS
Biologicals Ltd, Buckingham, UK); cytokeratin15 (mouse
monoclonal keratin 15 antibody; Labvision, Cheshire, UK);
CD34 (mouse monoclonal to CD34; Abcam, Cambridge,
UK); CD133 (purified mouse monoclonal; Abgent, Abingdon, UK); K6 (mouse monoclonal to cytokeratin 6; Abcam,
Cambridge, UK); P63 (mouse monoclonal, clone 4A4;
Labvision, Cheshire, UK); DSG3 (mouse anti-human
monoclonal to desmoglein 3, gifted by Dr Jim Wahl,
University of Nebraska, Nebraska, USA). The secondary
antibodies used were Alexa Fluor 594 donkey anti-mouse
IgG and Alexa Fluor 488 donkey anti-goat IgG (both
from Invitrogen). Cells and tissue samples were then
examined and images obtained using a Zeiss Axio Imager.M1 (Zeiss, Oberkochen, Germany) fluorescence microscope (or Zeiss LSM 510 Confocal microscope from Carl
Zeiss, Germany). To investigate expression in explantderived cells after subculture, cells were stained with K5,
K10 and K15 antibodies as described above and images
were captured at low (10· lens) magnification. The percentage of positively expressing cells in each of four randomly chosen fields of view was then counted, and a
mean percentage figure obtained.
Results
Keratinocyte cell outgrowth from skin explants –
growth, expression and maintenance after
passaging
Keratinocytes were first observed growing out of the explants in a continuous sheet between 24 and 36 h. By day
2, the outgrowth resembled a ring surrounding the explants
(Fig. 1c) and over day 3 and 4 (Fig. 1d) this outgrowth
continued to expand. After 5 days in culture, cells with
spindle-shaped fibroblast or melanocyte morphology were
visible outside of the keratinocyte ring (Fig. 1e). Sufficient
numbers of cells for harvesting were produced by 30
explants in 3–4 days. Comparison of numbers of cells
obtained from six individual 35-mm dishes derived from
skin explants from a single donor revealed a mean of
1.6 ± 0.36 · 105 cells per dish. As an estimate, therefore,
1cm2 of skin would yield approximately 5.45 · 105 cells on
the first harvest. When subcultured in EpilifeTM, these cells
exhibited exemplary attachment and growth capabilities.
After one day, cells were attached and were growing
(Fig. 1f), and between 2 and 3 days, colonies larger than 32
cells were common (Fig. 1g). Importantly, no fibroblast
contamination was observed in these cultures. Confluent
ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 720–726
16000625, 2009, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0625.2009.00900.x by INASP/HINARI - INDONESIA, Wiley Online Library on [12/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Guo and Jahoda
keratinocyte monolayers were generated 5 or 6 days after
passaging (Fig. 1h). We next characterized explant-derived
keratinocytes with specific markers after subculture. Cultures had high cytokeratin 5 expression, but less than 5%
of cells expressed the differentiation marker keratin 10
(Fig. 1i), while a much higher percentage (43%) expressed
the basal marker keratin 15 (Fig. 1j). Levels of K10 staining
increased slightly after passaging but remained low (data
not shown). Explant-derived cultures have been obtained
from adult specimens of different ages and sex and various
body sites, including haired regions incorporating terminal
hair follicles. Cells initiated from explants and cultured in
EpilifeTM were routinely grown to passage 5 and beyond,
and one cell strain was grown up to passage 10 (Table 1)
without evidence of differentiation. Several of the cultures
were also frozen and successfully recovered. The K5, K10
and K15 expression profile described above remained stable when cells were stained after four passages, following
cryopreservation and recovery (data not shown). We also
tested if explant-derived keratinocytes could be switched
to conventional Rheinwald and Green (6) growth medium with 3T3 support layers. With a mitomycin-C-treated 3T3 feeder layer, the keratinocytes exhibited a similar
growth pattern as in EpilifeTM. Cells settled after 1 day
produced colonies by 3 days, reached confluence at
around 9 days and could be subcultured successfully
(data not shown).
We then investigated the potential of the original to produce more keratinocytes. Interestingly, those explant cultures
that were resupplied with medium in the same dish after
enzymatic removal of the initial keratinocyte outgrowth produced a second, robust outgrowth of epithelial cells. These
cells migrated out in a similar manner and even more rapidly
than the first outgrowth, and could also be harvested by
trypsinization after 2–3 days, at which point the cultures
were still free of fibroblast contamination (Fig. 1k–m and
Video Clip S1). The keratinocytes obtained could be subcultured and showed a similar growth pattern to those from the
first outgrowths. We have been able to repeat this process up
to five times from the same original explants, although some
fibroblast contamination was observed on the third and subsequent outgrowths (data not shown).
Keratinocytes from initial explant outgrowths
express stem cell and wound healing markers
We then examined what markers cells were expressing as
they grew out from the explants. After 24 to 36 h, dual
staining revealed that the cells were K5 positive, and coexpressed K15 throughout. In contrast, cells co-expressing
K5 and K10 were restricted to the innermost cells of the
outgrowth, closest to the explants, while no a6b1 integrin
labeling was observed (data not shown).
At 3 days cultured explants, cells on the leading edge
strongly expressed K15, while those closer to the explant
were weakly labelled (Fig. 2a). Meanwhile, K10 expression
was present but was restricted to a relatively small circle of
cells closest to the explant (Fig. 2b). a6b1 Integrin was
expressed by cells throughout the outgrowth, but a distinctive inner ring of very brightly labelled cells was visible
close to the explant (Fig. 2c). All the remaining markers
labeled the cell outgrowths positively but showed different
patterns of expression. When stained with the p63 antibody, cells throughout the outgrowth showed distinct
nuclear staining, with the strongest labeling located towards
the leading edge (Fig. 2d). Desmoglein 3 and CD34 were
both expressed strongly in cells through most of the outgrowths apart from cells at the leading edge (Fig. 3a and
c). CD133 labeling was strongest in a band closest to the
explant (Fig. 3b) in a pattern similar to that shown by
a6b1 integrin (Fig. 2c). In contrast, fluorescence labeling
with the antibody to K6 was also widespread but strongest
towards the outer edge of the outgrowths (Fig. 3d) more
akin to the expression seen with K15 (Fig. 2a). A schematic
diagram summarizing the localization and strength of marker expression in keratinocyte outgrowths at 3 days is
shown in Fig. 3e.
Discussion
Culturing epidermal cells from both animal and human
skin has traditionally involved one of two approaches: splitting of the epidermis from the dermis followed by epidermal cell dissociation or explant culture. As Rheinwald and
Green (6) first described a protocol to achieve single keratinocytes from skin epidermal sheets, this strategy has
Table 1. Details of serial cultivation of human adult skin specimens after explant culture
Gender
Male
Age (years)
Donor site
Longevity
68
Beard
p10
Female
Na
Beard
C at p3
R to p6
43
Beard
p7 O
67
Leg
p9
55
Breast
C at p4
R to p7
27
Breast
C at p2
25
Breast
E for staining
43
Breast
C at p2
44
Breast
C at p1
42
Abdomen
E for staining
44
Scar tissue
p6 O
p, passage number; C, cryofrozen; R, recovered and cultured further; E, used for experimental analysis; O, ongoing culture.
ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 720–726
723
16000625, 2009, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0625.2009.00900.x by INASP/HINARI - INDONESIA, Wiley Online Library on [12/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Explant method for culturing primary human keratinocytes
(b)
(a)
(c1)
(c2)
(d)
(c3)
(c4)
Figure 2. Expression of K5, K10 and K15, a6b1 integrin and P63
markers by explant outgrowth cells after 3 days in culture. Dual
immunofluorescent labeling was performed for K5 (green) and K15
(red) in a; for K5(green) and K10(red) in b; K5 (green) and a6b1
integrin (red) in c; for K5 (green) and P63 (red) in d. Thus, yellow colour
indicates areas of K5 ⁄ K10; K5 ⁄ K15 co-localization; K5 ⁄ a6b1 integrin
and K5 ⁄ P63 co-localization. 4¢-6-Diamidino-2-phenylindole was labeled
as in blue. The purple arrows show the direction of outgrowth, and E
indicates the location of the explants. Scale bar: 30 lm except c2
(15 lm).
become the method of choice by most practitioners. Much
human work has been performed on new born foreskin,
whose cells have a very different replicative profile to keratinocytes from older skin (16). Here, we developed a skin
explant method for primary keratinocyte culture from adult
human skin that was quick, simple and reliably generated
keratinocytes without fibroblast contamination. The cells
we produced by this means had robust growth characteristics. This led us to further define the nature of explant outgrowth, in which we observed the presence of putative
stem cell and wound healing markers in the cell outgrowths.
Explant culture wherein a small piece of skin will settle on
a culture dish and produce a sizeable outgrowth of cells has
long been employed as a model of wound healing or adult
skin epidermal outgrowth rather than a source of keratinocytes for clinical or experimental purposes (10–12,17–20).
The major limitation is due to the fact that fibroblasts also
grow out from the same explants, and will eventually outgrow the keratinocytes. In our study, keratinocytes emanated
from tissue explants after approximately 24 h, which is consistent with some previous reports (17,21). However, rather
than let them continue until fibroblasts began to appear at
about 5 days, we removed the epidermal cells from the dish
after 3–4 days as an alternative method of obtaining keratinocyte primary cultures free of fibroblasts. As no fibroblast
outgrowth was observed until at least 5 days, it was possible
to obtain fibroblast-free populations of keratinocytes.
724
What is the biological basis underpinning the robust
growth characteristics and longevity of explant-derived
keratinocytes? Previous work has suggested that outgrowing
epidermal cells have relatively little thymidine incorporation during the first few days in explant culture. This was
interpreted as meaning that migration, rather than cell
division, is the main mechanism of outgrowth in early
explant cultures (15). Importantly, transmission and scanning electron microscopy by the same group determined
that the outgrowing cells originated from the basal layer of
the epidermis which conventionally is believed to contain
the stem and transient amplifying cell populations. The
basal epidermis is conventionally believed to contain at
least two distinct subpopulations of keratinocyte progenitors: keratinocyte stem cells, which constitute a minor subpopulation of relatively quiescent or slow-cycling cells with
great proliferative potential and an unlimited capacity for
self-renewal, and transit amplifying (TA) cells which are
the progeny of the stem cells and are believed to have a
limited proliferative capacity (22–24). In mature epidermis,
the undifferentiated stem cells and proliferative TA cells are
both contained in the basal layer. However, recent appreciation of plasticity ⁄ multipotency in a number of tissues has
lead to a blurring of the distinction between TA and stem
cells, and tissues of the quiescent nature of stem cells (25–
28). This concept probably applies even more in the context of wound healing epidermis where the environment is
transformed. As the explant cultures initially migrate out
maintaining contact with each other and with the underlying basement membrane, we postulate that the outgrowing cells are undergoing an active process of self-selection
of cells with high replicative potential. By preserving the
basement membrane, it is likely that the keratinocytes
recruited in the outgrowths emanate from both the stem
cell and TA cell compartments, and are mobilized in a way
that mimics ‘activated keratinocytes’ (29) during the
wound healing process in vivo.
This was reflected in our short-term cultures showing
that outgrowing keratinocytes expressed not only stem
cell-associated markers, including p63 (30) and K15 (31)
but also expressed K6, an early marker of wound healing
epidermis (32,33). Strong expression of a6 (34) and b1
(35) integrins have separately been identified as putative
stem cell markers. For the current state of the art, high
a6 but not b1 is a good indicator for stemness. However,
the sublocalization of a6b1 expressing cells within the
explant outgrowths may also be a reflection of differences
in their motility status as the brightly labelled cells were
those close to the explant and not the migratory cells at
the periphery. Downregulation of bright desmoglein 3
expression, which is also a putative marker of keratinocyte
stemness (36,37) was not observed. Quite surprisingly, the
keratinocytes were also labelled by CD34 and CD133 anti-
ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 720–726
16000625, 2009, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0625.2009.00900.x by INASP/HINARI - INDONESIA, Wiley Online Library on [12/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Guo and Jahoda
(a)
(b)
(c)
(d)
(e)
Figure 3. Expression of K5, K6, CD133, CD34 and DSG 3 markers by explant outgrowth cells after 3 days in culture. Dual immunofluorescent
labeling was performed for K5 (green) and DSG3 (red) in a; for K5 (green) and CD133(red) in b; K5 (green) and CD34 (red) in c; for K5 (green) and
K6 (red) in d. Thus, yellow colour indicates areas of K5 ⁄ K6; K5 ⁄ CD133; K5 ⁄ DSG3 or K5 ⁄ CD34 co-localization. 4¢-6-Diamidino-2-phenylindole (DAPI)
was labeled as in blue. The purple arrows show the direction of outgrowth and E indicates the location of the explants. Scale bar: 60 lm.
bodies neither of which has been observed in this context.
The CD34 antibody clone that we used has previously
been shown to delineate cells in the outer root sheath of
the human hair follicle (38) but not interfollicular epidermis. CD133 labels epithelial stem cells in the prostate (39)
but only one isoform AC133-2 has been shown to be
expressed in cultured keratinocytes (40). After subculture,
the explant-derived cultures continued to have few K10
positively expressing cells, and expressed high levels of
K15 – which may be an indicative marker of progenitor
cells (41).
In conclusion, we show that keratinocyte cells obtained
from explant culture display progenitor markers and grow
for multiple passages when transferred to serum-free culture. From the clinical and practical standpoint, this
method provides a simple means of growing large numbers
of keratinocytes from only a small biopsy quickly and in
the absence of feeder layers, which could be important in
relation to patients with limited donor skin availability.
Elegant strategies can be used to isolate and grow enriched
stem and TA cells from dissociated epidermis, however,
ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 720–726
these usually involve cell sorting (34,42), and are thus more
time consuming and require equipment that might not be
readily available in all laboratories, or in clinical settings
where simple, rapid isolation of cells with robust growth
characteristics is a priority. Although the method reported
here currently involves FBS for a few days, we have confirmed the possibility of replacing FCS with human serum
if required in a clinical context.
The finding that serial outgrowths of keratinocytes could
be harvested from the same explants further enhances the
utility of this method as it provides the possibility of
obtaining much larger numbers of keratinocytes from a
single source. It also has parallels in split thickness grafting
where a donor site can be repeatedly used, highlighting the
capacity of the wound stimulated epidermis activity to serially renew itself. Finally, this observation is significant in
relation to dermal–epidermal interactions in the skin
explants. The absence of fibroblasts during the second or
subsequent keratinocyte outgrowth suggests that fibroblast
activation or motility is being inhibited by the epidermal
cells by a process worthy of future investigation.
725
16000625, 2009, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0625.2009.00900.x by INASP/HINARI - INDONESIA, Wiley Online Library on [12/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Explant method for culturing primary human keratinocytes
Acknowledgements
We are very grateful to the British Skin Foundation and the MRC (grant
number G0300353 to CABJ) for support. We are very thankful to Mr Clifford Lawrence, Dr Reika Taghizadeh, Mr Andrew Owens and Mr Martin
Coady for their invaluable help with the provision of skin samples; Dr Trevor Booth for assistance with some confocal microscopy; and Dr James
Waters for his helpful suggestions on the manuscript.
References
1 Bell E, Ehrlich H P, Buttle D J, Nakatsuji T. Living tissue formed in vitro and
accepted as skin-equivalent tissue of full thickness. Science 1981: 211: 1052–
1054.
2 El Ghalbzouri A, Jonkman M F, Dijkman R, Ponec M. Basement membrane
reconstruction in human skin equivalents is regulated by fibroblasts and ⁄ or
exogenously activated keratinocytes. J Invest Dermatol 2005: 124: 79–86.
3 Gangatirkar P, Paquet-Fifield S, Li A, Rossi R, Kaur P. Establishment of 3D
organotypic cultures using human neonatal epidermal cells. Nat Protoc 2007:
2: 178–186.
4 Yannas I V, Burke J F, Orgill D P, Skrabut E M. Wound tissue can utilize a polymeric template to synthesize a functional extension of skin. Science 1982:
215: 174–176.
5 Medawar P B. Sheets of pure epidermal epithelium from human skin. Nature
1941: 148: 783.
6 Rheinwald J G, Green H. Serial cultivation of strains of human epidermal
keratinocytes: the formation of keratinizing colonies from single cells. Cell
1975: 6: 331–344.
7 Kitano Y, Okada N. Separation of the epidermis sheet by dispase. Br J Dermatol 1983: 108: 555–560.
8 Boyce S T, Ham R G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial
culture. J Invest Dermatol 1983: 81: 33–40.
9 Kaur P, Li A. Adhesive properties of human basal epidermal cells: an analysis
of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells. J Invest Dermatol 2000: 114: 413–420.
10 Halprin K M, Lueder M, Fusenig N E. Growth and differentiation of postembryonic mouse epidermal cells in explant cultures. J Invest Dermatol 1979: 72:
88–98.
11 Hammar H. Stimulated mouse ear epidermis in explant culture – the effect of
retinoic acid and hexadecane. Arch Dermatol Res 1981: 270: 469–481.
12 Stoll S W, Kansra S, Elder J T. Keratinocyte outgrowth from human skin
explant cultures is dependent upon p38 signaling. Wound Repair Regen 2003:
11: 346–353.
13 Stanley M A, Parkinson E K. Growth requirements of human cervical epithelial
cells in culture. Int J Cancer 1979: 24: 407–414.
14 Taylor V R, Halprin K M, Levine V, Woodyard C. Effect of methotrexate in vitro on epidermal cell proliferation. Br J Dermatol 1983: 108: 45–61.
15 Van Der Schueren B, Cassiman J J, Van Den Berghe H. Morphological characteristics of epithelial and fibroblastic cells growing out from biopsies of human
skin. J Invest Dermatol 1980: 174: 29–35.
16 Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 1987: 84: 2302–2306.
17 Karasek M A. In vitro culture of human skin epithelial cells. J Invest Dermatol
1966: 47: 533–540.
18 Koeper L M, Schulz A, Ahr H J, Vohr H W. In vitro differentiation of skin sensitizers by cell signaling pathways. Toxicology 2007: 242: 144–152.
19 Lewis S R, Pomerat C M, Ezell D. Human epidermal cells observed in tissue
culture with phase-contrast microscopy. Anat Rec 1949: 104: 487–503.
20 Mutasim D F, Vaughan A, Supapannachart N, Farooqui J. Skin explant culture:
a reliable method for detecting pemphigoid antibodies in pemphigoid sera
that are negative by standard immunofluorescence and immunoblotting. J
Invest Dermatol 1993: 101: 624–627.
21 Flaxman B A, Lutzner M A, VanScott E J. Cell maturation and tissue organization in epithelial outgrowth from skin and buccal mucosa in vitro. J Invest Dermatol 1967: 49: 322.
22 Bickenbach J R, McCutecheon J, MacKenzie I C. Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissues. Cell Tissue Kinet 1986: 19:
325–333.
23 Morris R J, Fischer S M, Slaga T J. Evidence that the centrally and peripherally
located cells in the murine epidermal proliferative unit are two distinct cell
populations. J Invest Dermatol 1985: 8: 277–281.
24 Potten C S, ed. Stem Cells: Their Identification and Characterization. London:
Churchill Livingston, 1983: 200–232.
726
25 Ghazizadeh S, Taichman L B. Organization of stem cells and their progeny in
human epidermis. J Invest Dermatol 2005: 124: 367–372.
26 Jaks V, Barker N, Kasper M et al. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nature Genet 2008: 40: 1291–1299.
27 Jones P H, Simons B D, Watt F M. Sic transit gloria: farewell to the epidermal
transit amplifying cell? Cell Stem Cell 2007: 1: 371–381.
28 Majo F, Rochat A, Nicolas M, Jaoudé G A, Barrandon Y. Oligopotent stem
cells are distributed throughout the mammalian ocular surface. Nature 2008:
456: 250–254.
29 Morasso M I, Tomic-Canic M. Epidermal stem cells: the cradle of epidermal
determination, differentiation and wound healing. Biol Cell 2005: 97: 173–
183.
30 Pellegrini G, Dellambra E, Gosliano O et al. p63 identifies keratinocyte stem
cells. Proc Natl Acad Sci USA 2001: 98: 3156–3161.
31 Webb A, Li A, Kaur P. Location and phenotype of human adult keratinocyte
stem cells of the skin. Differentiation 2004: 272: 387–395.
32 Machesney M, Tidman N, Waseen A, Kirby L, Leigh I. Activated keratinocytes
in the epidermis of hypertrophic scars. Am J Pathol 1998: 152: 1133–1141.
33 Tyner A L, Fuchs E. Evidence for posttranscriptional regulation of the keratins
expressed during hyperproliferation and malignant transformation in human
epidermis. J Cell Biol 1986: 103: 1945–1955.
34 Li A, Simmons P J, Kaur P. Identification and isolation of candidate human
keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci
USA 1998: 95: 3902–3907.
35 Jones P H, Watt F M. Separation of human epidermal stem cells from transit
amplifying cells on the basis of differences in integrin function and expression.
Cell 1993: 73: 713–724.
36 Wan H, Stone M G, Simpson C et al. Desmosomal proteins, including desmoglein 3, serve as novel negative markers for epidermal stem cell-containing population of keratinocytes. J Cell Sci 2003: 116: 4239–4248.
37 Wan H, Yuan M, Simpson C et al. Stem ⁄ progenitor cell-like properties of Desmoglein 3dim cells in primary and immortalized keratinocyte lines. Stem Cells
2007: 25: 1286–1289.
38 Poblet E, Jimenez-Acosta F, Rocamora A. QBEND ⁄ 10 (anti-CD34 antibody) in
external root sheath cells and follicular tumors. J Cutan Pathol 1994: 21: 224–
228.
39 Richardson G D, Robson C N, Lang S H, Neal D E, Maitland N J, Collins A T.
CD133, a novel marker for human prostatic epithelial stem cells. J Cell Sci
2004: 117: 3539–3545.
40 Yu Y, Flint A, Dvorin E L, Bischoff J. AC133-2, a novel isoform of human
AC133 stem cell antigen. J Biol Chem 2002: 277: 20711–20716.
41 Kaur P. Interfollicular epidermal stem cells: identification, challenges, potential.
J Invest Dermatol 2006: 126: 1450–1458.
42 Li A, Kaur P. FACS enrichment of human keratinocyte stem cells. Methods
Mol Biol 2005: 289: 87–96.
43 Drewa T, Szmytkowska K, Wlodarczyk Z, Sir I, Kierzenkowska-Mila C. Does
the presence of unwanted dermal fibroblasts limit the usefulness of autologous epidermal keratinocyte grafts? Transplant Proc 2006: 38: 3088–3091.
44 Linge C, Green M R, Brooks R F. A method for removal of fibroblasts from
human tissue culture systems. Exp Cell Res 1989: 185: 519–528.
Supporting Information
Additional Supporting Information may be found in the online version of
this article:
Video Clip S1. This video shows the outside of a typical explant after
initial enzymatic removal of the keratinocyte outgrowth, and following the
addition of fresh medium to the dish. At the start of the video rounded
cells from enzyme treatment are visible at the edge of the explant. Over the
48 hours of the image capture period, a second, robust outgrowth of
epithelial cells takes place. These cells migrated out in a similar manner to
the first outgrowth, and could also be harvested by trypsinization after 2–3
days, at which point the cultures were still free of fibroblast contamination.
Time lapse images were taken with Live Cell Imaging system by Zeiss Axiovert(·10 object), video was edited with Axiovision software.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries
(other than missing material) should be directed to the corresponding
author for the article.
ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 720–726
16000625, 2009, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1600-0625.2009.00900.x by INASP/HINARI - INDONESIA, Wiley Online Library on [12/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Guo and Jahoda