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Chongdee Aojanepong, M.D.*, Kongsawate Khaogate, M.D.*, Adisak Wongkajornsilp, M.D., Ph.D.**, Sunisa
Duangsa-ard, Ph.D.**, Kanda Kasetsinsombat, Ph.D.**
*Division of Plastic Surgery, Department of Surgery, **Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University,
Bangkok 10700, ailand.
Keratinocyte Culture: Siriraj’s Experience
ABSTRACT
Objective: Cell-based therapy is gaining increasing prominence in medicine, where it has the potential to replace
or repair damaged tissue using new engineered cells. Skin cell engineering, also known as keratinocyte culture
or cultured epithelial autogra (CEA), is a promising eld in cell-based therapy. CEA is now used in many parts
of the world as an alternative treatment for some diseases that require large defects to be covered, such as severe
and major burn patients and congenital melanocytic nevus. e use of CEA in conjunction with acellular skin
substitution is rapidly expanding.
Materials and Methods: is study is an initiative aimed at supporting the production and use of keratinocyte
cultures at Siriraj Hospital. is is the rst stage of developing sheet keratinocyte culture in vitro.
Results: Our study yielded very promising results. As feeder cells, we used irradiated 3T3 murine broblasts, as per
the standard protocol for keratinocyte culture. e growth duration was four weeks: 2 weeks for the 3T3 murine
broblasts and 2 weeks for the keratinocytes. e keratinocytes grew rapidly and formed sheets with irradiated
3T3 murine broblasts. e retrieval of the cell sheets was straightforward thanks to the temperature-response cell
culture dish and halo-ring cell recovery sheet. Flow cytometry revealed that the cells had a very high viability and
purity. H&E staining revealed the sheets comprised two to four layers of stratied epithelial tissue.
Conclusion: From this study, our method of manufacturing the CEA can oer a promising result. is can be use
in the treatment which require large skin coverage. However, we aim to initiate animal and human trial phase next.
Keywords: Keratinocyte culture; keratinocyte culture in Siriraj Hospital; cultured epithelium autogra; CEA; cultured
epithelium autogra in Siriraj Hospital; CEA in Siriraj Hospital (Siriraj Med J 2022; 74: 274-283)
Corresponding author: Kongsawate Khaogate
E-mail: kongsawate.kha@gmail.com
Received 23 December 2021 Revised 17 February 2022 Accepted 21 February 2022
ORCID ID: https://orcid.org/0000-0002-0128-7483
http://dx.doi.org/10.33192/Smj.2022.34
All material is licensed under terms of
the Creative Commons Attribution 4.0
International (CC-BY-NC-ND 4.0)
license unless otherwise stated.
Aojanepong et al.
INTRODUCTION
e treatment workhorse for covering large wounds,
such as in burn victims or aer cancer resection surgery,
is the skin gra. Skin gras are classied into three
categories based on the origin of the tissue: autogras
(from the patient), allogras (from another person), and
xenogras (from other species, such as pigs).
Generally, using autologous tissue is the best option;
however, in some cases, such as severe burns or aer the
removal of a large tumor, an autologous gra may not
be sucient. As a temporary dressing, an allogra or
xenogra may be used, but must be later removed due
to gra rejection.
The field of regenerative medicine and tissue
engineering has grown in recent years. In North America
and some European countries, autologous skin culture
(keratinocyte culture) is now commercially available.
is keratinocyte culture is extremely useful in covering
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large skin defects.
1-3
ere are three types of keratinocyte
culture
4
: sheet, suspension, and spray. However, none
of these cultures are available yet in ailand.
is study is an initiative to support the production
and use of keratinocyte cultures at Siriraj Hospital. is
is the rst stage of developing sheet keratinocyte culture
ex vivo. Animal and human phases will follow later.
MATERIALS AND METHODS
This study was conducted at the Plastic and
Reconstructive Surgery Unit Department of Surgery,
and Department of Pharmacology, Faculty of Medicine
Siriraj Hospital, Bangkok, ailand. is study protocol
(Si 122/2020) was approved by Ethics Committee of the
Siriraj Institutional Review Board. e subjects understood
the protocol and gave informed consent prior to the
participation.
Preparation of human skin
Aer receiving informed consent, the human skin
used in this study was obtained from patients who had
surgical debridement or from a skin gra that was le
over aer skin gra transplantation. e skin was harvest
from thigh using Zimmer dermatome (Zimmber biomet
company, Ohio, USA) with 0.010 inches thickness. e
sample skin was cleansed with 100 ml of normal saline
and wrapped in a sterile gauze soaked in normal saline.
e skin was then transferred to a laboratory room in a
sterile plastic bag. e skin was washed in phosphate-
buered saline (PBS) with 50 µg/mL streptomycin and
50 unit/mL penicillin G before being transferred to a
new sterile 10.0 cm diameter dish (Fig 1).
Isolation of human skin keratinocytes
e sample skin was nely chopped into small
pieces (approximately 2 × 2 mm
2
). The pieces were
then transferred into a 50 mL tube. e tube was then
incubated in a water bath for 20 minutes with 5 mL of
0.25% trypsin-EDTA at 37°C. e mixture was washed
twice through centrifuged at 1000 rpm for 5 minutes at
room temperature. e supernatant was discarded, and
the cell pellets were reconstituted in 5 mL of Keratinocyte
culture medium (KCM).
Irradiated 3T3 broblast preparation (Feeder cell)
e frozen cryotube of 3T3 broblasts (murine
broblast) was then removed from the cryopreserved
tank, and 70% ethanol was used to clean the outside of the
tube. e frozen cryotube of 3T3 broblasts was thawed
in a 37°C water bath. When the cell-preservative medium
had nearly completely defrosted, the cell suspension was
quickly mixed into 5 ml pre-warmed Fibroblast derived
matrix (FDM) in a 15 ml tube. The cell suspension
solution (approximately 6 ml) was then divided equally
and added to each of the two 75 cm
2
asks. e next
day, the culture medium was changed was completely
changed to remove the remaining cryoprotectant (Fig 2).
Note, the more 75 cm
2
asks there are, the larger the cell
expansion possible.
All of the cultured cells from the 75 cm
2
asks were
collected and placed in a 50 mL tube aer two passages.
e tube was transferred for two cycles of 34 Gy radiation.
Note, the preparation of the irradiated 3T3 broblasts
took about one to two weeks, and so must be planned
ahead of time when needed.
Fig 1. Skin sample was retrieved from a split-thickness skin gra leover (1A), Primary keratinocytes prepared from the skin samples on
day 7 were visualized under 10× objective lens (1B)
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Fig 2. 3T3-murine broblasts (feeder cell) were visualized under 10× objective lens.
Plating the human keratinocytes over irradiated 3T3
broblasts (Feeder layer)
We used an UpCell dish, which is a specialized
culture dish. is UpCell dish has the unique property
that when the temperature is reduced, the cultured cells
automatically li o the surface.
Irradiated 3T3 broblasts were seeded onto the
dish rst, covering the entire surface overnight. e
irradiated-broblasts were then seeded with a suspension
of human keratinocytes at concentrations 2.0×10
5
and
4.0×10
5
cells in a 3.5 cm UpCell dish. e dish was then
placed in a CO
2
incubator and incubated at 37°C.
Keratinocyte sheet liing
Every day, the culture medium [keratinocyte
medium (KCM)] was changed. Also, the cultured cells
were examined every day under a microscope. The
keratinocyte sheet was ready to be lied o once the
cell conuence reached 100%, which took about one to
two weeks. e keratinocyte sheet was lied from the
dish’s surface by lowering the temperature from 37°C
to 20°C over 30 minutes. (Fig 3)
A specialized doughnut-shaped paper called a
halo-ring cell recovery sheet was used to retrieve the
keratinocyte sheet. e halo-ring sheet’s outer diameter
was smaller than the dish’s diameter, so that when the
halo-ring sheet was placed over the keratinocyte sheet,
the keratinocyte sheet’s edge was larger than that of the
halo-ring sheet’s. Next, by folding the keratinocyte sheet’s
edge over the edge of the halo-ring sheet, the halo-ring
sheet and the keratinocyte sheet could be lied o the
surface of the dish together (Fig 4).
RESULTS
Duration of keratinocyte sheet culture
e preparation of the irradiated 3T3 broblasts
took about one to two weeks in this study. It then took
two weeks from the time the keratinocytes were seeded
to the formation of a keratinocyte sheet. As a result, the
entire process took three to four weeks overall.
Characteristics of the cultured keratinocyte sheets
Keratinocyte cells were found to grow on irradiated
3T3 broblasts in explant culture. At days 5, 7, and 14, the
conuence rates were 20%, 80%, and 100%, respectively
(Fig 5). Keratinocytes with typical morphological features,
such as a polygonal cobblestone shape, were observed
to have proliferated.
Histological examination revealed that all the
manufactured cell sheets with a 2-4 stratied structure
were made up of epithelial cells (Fig 6). e results
showed that the keratinocyte cells could be cultured on
temperature-responsive cell culture inserts and that the
cell sheets could stratify (Table 1).
Liing the cultured keratinocyte sheets
Aer 14 days of culture on the temperature-responsive
cell culture dish, all the cells were successfully harvested
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Fig 3. e culture epithelial sheet retrieving method
Fig 4. Aer 14 days, the culture epithelium sheet was growing all over the temperature-responsive UpCell dish (le). For the liing, the
temperature was lowered from 37°C to 20°C over 30 min; then a recovery ring sheet was used for liing the cultured epithelial cell sheets
(right), culture epithelial sheet (arrow).
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278
a
c
b
d
Fig 5. Culture dish at 10x microscopic view on the 1
st
day showing a low keratinocyte : broblast ratio (a). During the culture, the keratinocytes
continuously grew in number while the broblasts decreased, as can be seen on the 5
th
day (b), 7
th
day (c), and nally, on the 14
th
day, by
which time the keratinocytes were conuence. (d)
Fig 6. e Cross section of the cell sheet was stained with H&E and viewed under 10× objective lens: keratinocyte at 2.2×10
4
cells/cm
2
(above), and 4.4×10
4
cells/cm
2
(below).
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TABLE 1. Physical characteristics of the cultured keratinocyte sheet seeding at 2.2×10
4
cells/cm
2
and 4.4×10
4
cells/cm
2
.
Cell sheet 2.2×10
4
cells/cm
2
4.4×10
4
cells/cm
2
Cell morphology Full conuence Conrmed Conrmed
keratinocytes and cobble
stone-like morphology
Cell sheet recovery Harvesting w/o any damage Conrmed Conrmed
Total cell number Over 1.0×10
5
cells 8.9×10
5
cells 17.3×10
5
cells
Cell viability Over 60.0% 92.8% 95.6%
Karatinocytes purity Over 80.0% 96.5% 98.2%
Degree of stratication More than 2 layers More than 2–4 layers More than 2–4 layers
as contiguous transplantable cell sheets by lowering
the incubation temperature from 37°C to 20°C over 30
minutes and by using a halo-ring cell recovery sheet.
Validation of the viability of the culture
Flow cytometry was used to validate the cultured
cell sheets. e results showed that the total cell counts
in the cell sheets using keratinocyte at cell seedings of
2.0×10
5
(2.2×10
4
cells/cm
2
) and 4.0×10
5
(4.4×10
4
cells/
cm
2
) were 8.9×10
5
and 17.3×10
5
cells, respectively. e
viability rates were 92.8% and 95.6%, respectively (Figs
7&8).
Purity of the cultured keratinocyte sheets
Cell purity was 96.5% and 98.2%, respectively, in
the above cultures.
DISCUSSION
Cultured epithelial autogra (CEA) was rst developed
30 years ago by Green and Rheinwald based on murine
3T3 broblasts.
5,6
Because of the high cost and time
required for processing, subsequent progress in this
eld has been very slow.
In the new millennium, cell-based therapy has gained
increasing prominence in medicine; particularly in the
elds of tissue engineering, regenerative medicine, and
stem cell therapy, and is widely recognized to oer the
potential to replace or repair damaged tissue using new
engineered cells.
Skin cell engineering, also known as keratinocyte
culture or cultured epithelial autogra (CEA), is a promising
eld in cell-based therapy. CEA is now used in many
countries as an alternative treatment for large wounds.
1
e indication is still within the controversy, such as
major burn greater than 30% of total body surface area.
e lack of skin donor is still a major problem in
numerous cases such as severe burn, large post-oncologic
resection, or congenital melanocytic nevus in pediatric.
In these cases, we can use mesh or meek technique
for expand the gra tissue 2 to 6 times. However, the
wider mesh/meek is needed to facilitate larger areas of
cover, result in the poorer donor site’s scar outcome.
Re-harvesting of the donor sites normally used, but
is associated with a delay overall healing time, as the
donor sites require time to heal between procedure. e
CEA may play an important role in these cases. is
technology has capability to expand the tissue more
than the previous strategy we utilized in the past and
use fewer tissue donor.
ere are currently three types of CEA available:
the sheet, suspension, and spray forms.
1,4,7-9
Morimoto et al.
2,3
demonstrated the use of CEA for
accelerating wound healing in neonates with complicated
wounds.
e ReCELL spray-on skin system
4,10,11
oers the
use of a spray form of CEA combined with an animal-
derived enzyme for less complicated wounds.
Nowadays, the sheet form of CEA is classified
as a skin substitute. Skin substitution is divided into
two types: cellular (composed of living cells, such
as CEA) and non-cellular or acellular (composed of
biocompatible or biodegradable materials). Acellular
skin substitution is further subdivided into allogenic
(made up of a decellularized extracellular matrix from the
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control
Fig 7. e ow cytometry was used to characterize cultured keratinocytes sheet seeding at 2.2 × 10
4
cells/cm
2
. e cluster of putative
keratinocytes was gated based on front scattering and side scattering (le panels). e viability was assessed using 7-amino actinomycin D
(7-AAD) assay in PerCP-Cy5.5 channel. e histogram illustrated 92.8% live cells and 6.48% dead cells (top). e purity was assessed using
anti-cytokeratin 5 + 8 in FITC (uorescein isothiocyanate) channel to stain keratinocytes. e histograms illustrated minuscule autouorescence
(1.63%) in the unstained group (middle), and overwhelmingly 96.5% positive cells in the stained group (bottom).
Aojanepong et al.
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control
Fig 8. e characteristics of cultured keratinocyte sheet seeding at 4.4 × 10
4
cells/cm
2
based on ow cytometry were similar to those with
lower cell density. ere were 95.6% live cells and 3.52% dead cells (top). e autouorescence in FITC channel was 1.89% (middle). e
purity of keratinocytes was 98.2% (bottom).
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same species, such as a human cadaver) and xenogenic
(composed of a decellularized extracellular matrix from
dierent species, such as bovine or porcine).
e disadvantage of CEA is the thinning of the tissue,
as it consists with a couple layers of stratied keratinocyte.
In case the wound is deeper to subcutaneous tissue,
utilizing this CEA alone will result in loss contour of the
area. In the deep wound, acellular skin substitution is very
useful, as it is designed to stimulate neodermis formation
for 3-4 weeks resulting in the tissue fullness. It can be
used as an intermediate step for split- or full-thickness
graing in patients with both small and large defects.
Additionally, it can be used in the wound that exposed
bone or tendon which cannot be graed primarily.
ere have been numerous reports on these acellular
skin substitutes being used as scaolds in conjunction
with the sheet form of CEA for complex wounds.
7,12-15
Matsumura et al.
16
reported the successful use of combined
CEA and acellular skin substitution in severe burn patients.
Our research yielded very promising results. As
feeder cells, we used irradiated 3T3 murine broblasts, as
per the standard protocol for keratinocyte culture. In our
protocol, we use partial thickness of skin (around 0.010
inches) for isolate human keratinocytes instead of full
thickness skin donor. We found out that it can shorten
time in cell isolation process and reducing cell damage,
as normally it must use thermolysin and incubation
overnight for separating epidermis from dermis. e
overall growth duration was four weeks: 2 weeks for the
3T3 murine broblasts and 2 weeks for the keratinocytes.
e keratinocytes grew rapidly and formed sheets with
irradiated 3T3 murine broblasts. In the retrieval of
the cell sheets, we used the temperature-response cell
culture dish and halo-ring cell recovery sheet. Normally,
enzymatic treatment (for example: dispase) is typically
used in the collection of epithelial keratinocyte sheets, but
it tends to break the adhesion and basement membrane
proteins. We assume that using harvesting technique
by temperature dish can lowering cell damage result in
improve the survival outcome of epithelial sheet. e ow
cytometry revealed that the cells had very high viability
and purity. H&E staining revealed two to four layers of
stratied epithelial tissue. Following these promising
results, animal and human trial phases will be initiated.
In our practice, we usually use acellular skin substitutes
in conjunction with the split-thickness skin gra especially
in cosmetic area or exposed bone or tendon wound. It
will take times approximately 3-4 week for the tissue to
be vascularized and good adhere to wound bed. Next,
the patient will undergo the second operation for skin
graing. In our perspective, this research is giving more
benet to the patient. During the time for waiting the
revascularized process, we aim to prepare the culture
keratinocyte sheet and utilize for the second stage operation.
CONCLUSION
e future of CEA is very promising in the treatment
of some diseases that require large defects to be covered,
such as severe and major burn patients and congenital
melanocytic nevus. e use of CEA in conjunction with
acellular skin substitution is rapidly expanding globally,
and will hopefully be an option in ailand soon too.
ACKNOWLEDGEMENTS
is work was funded by Aojanepong C. We would
like to express sincere gratitude to Siriraj’s Laboratory
for supporting the equipment of this study.
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