|Ann. Medit. Burns Club - vol. V71 - n. 4 - December 1994
CULTURED SKIN CELLS FOR TREATMENT OF BURNS
Brychta R*, Adler J.**, Rihovd R*, Suchdnek V, Komdrkovd
* Burns Centre, Brno, Czech Republic
** Tissue Culture Laboratory, Masaryk University, Brno
SUMMARY. After considering the history of
research into the cultivation in vitro of skin cells, the authors describe their
experience at the Brno Burns Centre with this technique in the treatment of burn patients.
So far about 10,000 sq cm of cultured epidermal grafts have been applied. The results and
costs are described. Keratinocytes cultured in vitro can also be used to test the
cytotoxicity of various synthetic bandages. The importance of in vitro cultured
skin cells is destined to increase, both on the theoretical level and in clinical
The history of in vitro skin cell
cultivation is not long. In the 1950s Billingharn succeeded in dividing the epidermis from
the dermis enzymatically using trypsin, without destroying the vitality of the epithelial
cells. The next important milestones were in the early 1960s, when Karasek demonstrated
that keratinocytes could survive in tissue culture, and in 1975, when Rheinwald and Green
published their work (1) on the growth and proliferation of dermal cells in vitro up to
the point at which a larninar, keratinizing, transplantable skin epithelium was achieved.
Finally O'Connor et al. (2), in 1981, reported the first success with the
autotransplantation of cultured epidermal grafts in man.
Many other researchers and institutions soon realized the importance of this method, which
allows an expansion of the epithelium of up to several thousand times for use in the
treatment of burns, and the cultivation of keratinocytes and their application began to be
intensively performed. In the Brno Burns Centre we have also followed with interest
developments in in vitro cultivation of skin cells, and since 1991 we have used the
method ourselves. We have so far applied about 10,000 sq cm of cultured epidermal grafts.
About twice as much was actually cultivated. We use a slightly modified version of the
Rheinwald-Green method using 3T3, sublethally inhibited with mitomycin. In cases of
extensive bums we take a skin sample 2 to 6 cm square at full thickness, or using a Watson
knife in the form of a split-thickness skin graft. Disinfection is carried out using 76%
ethanol or 3% iodonal. Any area not affected by bums can be used. We also use skin
fragments obtained during plastic surgery, the best being skin from the eyelids or from
the back of the ears. Samples are rinsed in Ringer solution, placed in transfer medium and
immediately taken to the laboratory for cultivation.
In the laboratory the skin is cleaned of the subcutis and placed in 0.2% trypsin solution,
which separates the epidermis from the dermis in 16 to 20 hours. The keratinocytes are
mechanically loosened, flushed, centrifuged and resuspended in a nutrient medium. The
primary culture is established from cells prepared in this fashion. Each plastic flask
(capacity 80 sq cm) is inoculated with about 2 X 106 cells. Subcultures from the primary
culture are established on about day 10, and on about day 18 to 21 growth of multilayers
can be observed on the base of the flasks. The upper parts are then sliced off using thin
wire. The grafts are released from the flasks using Dispase, prefixed to the carrier,
which is usually Grasolind, and then either transferred in a transfer medium back to the
operating theatre or cryopreserved for later use.
In the operating theatre the grafts are applied to the wound after disinfection with
antibiotic solution (not normal antiseptics) according to sensitivity, and flushed with
Ringer solution. They are fixed with metal clamps, or bandages if the area is a small one.
We apply cultured keratinocytes to areas of second- and third-degree burns, either as
autografts or as allografts.
All donors for keratinocyte cultivation are screened according to a standard protocol to
eliminate the possibility-of infection from HIV 1/2, HBsAg, BWR and CMV a~lografts. The
cost of cultured epidermal grafts is 193 crowns per sq cm and 208 crowns per sq cm in the
case of cryoconservation (i.e. US$ 6.45 and 6.93 respectively). In third-degree burns we
achieved only 18% take, which is relatively very low. The cause in all cases was bacterial
contamination of the burn wounds and insufficient use of cadaver dermoepidermal
allografts. Conversely, with the application of cultured epidermal grafts as
allotransplants in very deep second-degree bums, we recorded excellent results. The
patients experienced alleviation of pain within a few hours and on rebandaging after 48
hours the wound exhibited a dry, pain-free, epithelialized surface. It had already been
proved that these allografts were also subject to rejection and that the best results were
achieved by maximum stimulation of the remaining cell elements through apocrine production
of various cytokines and hormones in the donor keratinocytes and fibroblasts, of which
about 30 have so far been identified.
Treatment of deep dermal bums using cultured epidermal allografts is of great importance
in infants, as these account for over 90% of burn cases in hospitalized children. These
injuries are usually 5-40% TBSA in extent, and treatment is a major problem. A comparison
was made between the cost of typical child burn treatment, i.e. second-degree scalds in
about 10% BSA, with application of cultured epidermal allografts and with conservative
treatment using Flamazine. The conservatively treated group consisted of ten infants and
the cultured allograft group of three typical patients. The cost of conservative treatment
was US$ 1672 (average hospital stay: 18.4 days per patient). With cultured allografts the
cost was US$ 1037 (average hospital stay: 7.3 days).
A totally different application of the use of keratinocytes cultured in vitro is
the testing of cytotoxicity of various synthetic bandages or temporary skin covers. We use
the elution method and we have so far tested 15 synthetic materials in the context of HeLa
and LEP (human embryonic lung) cells. It is interesting that some commercially available
synthetic bandages were cytotoxic for human keratinocytes.
In order to follow current trends in the study of cultured skin cells we adopted three
basic lines in our practical experience. Substitution of the dermal component of skin and
its recombination with cultured human keratinocytes in vitro. The best material in
vitro appears to be cadaver dermis, trypsinized and meshed in the ratio of 1 to 1.5.
Surface-cultured keratinocytes form a good multilayer and dermoepidermal junction. Another
suitable dermal substitute is the collagen lattice, contracted by human fibroblasts and
subsequently set with cultured human keratinocytes. In this case, however, a problem
arises because the cultivation time is rather long and the price comparatively high. It is
also difficult to control continuing collagen contraction.
In an animal experiment we created a circular fullthickness skin defect on the back of
rats (diameter 3 cm), using a plastic ring which prevented spontaneous healing from the
edges of the wound. The rats were non-inbred, and we exchanged their corium with epidermis
removed by trypsination. To this corium we applied the keratinocytes themselves, which had
meanwhile been cultivated in vitro to a multilayer. Wound healing took 25 days,
while in the control group there was no healing before removal of the ring.
We have achieved very well formed recombined skin, consisting of allogenous dermis and
native cultured keratinocytes, including a very well formed structure of the dermis and
A similar approach was used in the clinical experiment. In a male child with extensive
burns to whom classical dermoepidermal allografts were applied, taken from the father, we
covered part of the burn wound with the socalled second-cut-dermis, while the epidermis
was returned to the donor site of his father. The healing of the donor site was naturally
very fast and less painful than in the case of other areas, and the cosmetic effect was
There was likewise no problem in the take of the meshed dermis on the burn wound. However,
subsequent application of autologous cultured keratinocytes was not successful owing to
secondary rejection caused by massive contamination of all wound surfaces with
methicillin-resistant Staphyloccus aureus.
This is a modification of the method
presented by Hickerson (3), who after the dermoepidermal allografts have taken removes the
epidermis with a dermatome and substitutes it with autologous cultured keratinocytes on
day 7- 10 after transplantation.
Take is over 90%, but removal of the epidermis is difficult and it cannot be returned to
the donor. At present we are trying to compare the two methods. Another direction of our
studies is the use of the above-mentioned effects of cultured epidermal allografts to
prepare so-called active wound dressing, in which the optimum effects of synthetic cover
is recombined with the secretory effects of cultured skin cells. In this case the cells
need not be applied as a multilayer, but on a suitable car~ rier. We are trying various
forms of collagen and silicone.
It is very interesting to compare keratinocytes and fibroblasts taken from adults with the
saine cells from embryonic and fetal sources. The generally higher metabolic activity of
embryonic and fetal keratinocytes can be seen in the faster confluence and the formation
of multilayer (13-16 days), and there is a striking increase in the speed of contraction
of the collagen lattice by embryonic fibroblasts (4-7 days).
Morphology in the electron microscope shot shows a thick, reticular nucleolus, very well
developed mitochondria and multiple keratin fibrils. This indicates the high metabolic
activity of embryonic and fetal skin cells.
In the clinical experiment there.was also rapid epithelialization of the wound as compared
to use of the classical bandage. In conclusion we would express the conviction that the
importance of cultured skin cells in vitro will increase, both on the theoretical level
and in clinical practice.
RESUME. Après avoir considéré l'histoire
des recherches dans le champ de la cultivation in vitro des cellules cutanées, les
auteurs décrivent leur expérience au Centre des Brûlés de Brno avec cette technique
dans le traitement des patients brûlés. Jusqu'ici ils ont appliqué environ 10.000 cm'
de greffes épidermiques cultivées. Les résultats et les coûts sont discutés. On peut
aussi utiliser les kératinocytes cultivés in vitro pour vérifier la cytoxicité de
divers pansements synthétiques. L'importance des cellules cutanées cultivées in vitro
est destinée à augmenter non seulement sur le plan théorique mais aussi dans la
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