Annals of Burns and Fire Disasters - vol. X - n. 4 - December 1997


Burt AM., Clarke J.A.

Stephen Kirby Skin Bank, Burns Unif
Queen Mary's University Hospital, London, Great Britain

SUMMARY. A method has been developed to transfer autologous keratinocytes to a wound bed within 5-7 days of receipt of biopsy. With the development by Smith and Nephew of a polymer that supports cell growth it has been found that actively dividing (undifferentiated) keratinocytes will invert and grow on a suitable wound bed. Keratinocytes were grown in flasks in serum containing medium for 48 h, fed with serum-free medium, and when actively growing were transferred to the polymer. This was then incubated for 2-3 days to produce a subconfluent cell layer (70-90% confluent) which was easily inverted on to the wound bed in theatre or at the bedside. When placed on a suitable wound bed, healing was evident as a translucent film after 5-7 days.


Since the development of keratinocyte cultures in Howard Green's' department in Boston and their use by O'Connor' to graft severely burned patients, there has been worldwide interest in wound healing by this method. The Burns Unit at Queen Mary's Hospital, Roehampton has been using keratinocytes cultured by this method since 1985 .4,5 However, it was found that the length of time required from biopsy to graft often extended rather than reduced the healing time.
The use of a polymer film (developed by Smith and Nephew)' compatible with keratinocyte culture has enabled us to reduce the time required for cell growth, and allowed easier transfer of cells to the patient than the original method.

Materials and methods

The original culture method for keratinocyte grafting has been modified. 15 A thin split-skin biopsy, about 5 sq. cm, was taken under general anaesthetic and transferred to the laboratory for culture.
The sample was washed well in saline and incubated in Dispase (Collaborative Biochemicals Ltd., Bedford, Ma.) at 37 'C for 30-40 mins. The epidermis was then removed to a standard trypsin/Versene solution and incubated at 37 'C for 10 min. The cell suspension was removed and the trypsin neutralized with serum-containing medium before centrifugation. (The remaining sample was trypsinized a second time and treated like the first.) The resulting pellets were kept separate (to limit contamination), each being resuspended in serum-containing growth medium.
The cells were counted in a haemocytometer. Culture flasks were inoculated at 3-5 x 101 cells per ml in growth medium containing serum, and incubated at 36.5 'C for 48 h. (This medium, based on 3:1 DMEM/1712 with added growth factors and 10% foetal calf serum, was designed by Green' for keratinocyte growth.) Cultures showing growth at this time were fed with serum-free medium by replacing half the supernatant with MCDB 153' (Clonetics Corp., La Jolla, Ca.)
After a further 2-3 days growth, or 48 h before required for grafting, the keratinocytes were trypsinized and transferred to polymer discs (35 mm diameter) in 6well plates at 3-5 x 101 cells per disc in 2 ml serum-free medium. The plates were incubated at 36.5 'C in 5% C02 in a humid incubator.
When the wound had been prepared the cultures were taken to theatre or bedside. The polymer discs were lifted with forceps and inverted on to the wound. The area was covered with Jelonet and a dry dressing, and bound firmly with cr&pe bandage.
The sources of media and other solutions were as follows: foetal calf serum and bovine pituitary extract, First Link UK, West Midlands, UK; Dulbeccos Modified Eagles Medium (DMEM), Hams F12; Imperial Laboratories, Hampshire, UK; Dispase; Collaborative Biomedical Products, Bedford Ma., USA; Trypsin/EDTA and Medium Additives, Sigma Biosciences Company, Dorset, UK.
This method differs from that of Green in several respects:

  • the epidermis is separated from the dermis with Dispase before trypsinization
  • no feeder layer is used, and the number of cells in the inoculum is increased
  • after 48 h the cultures are gradually weaned on to serum-free medium
  • the cultures are subcultured before reaching confluence in order to maximise growth
  • the cultures are easy to transfer. The polymer discs need only to be lifted from the dish with forceps and inverted on to the wound


Case 1
A three-year-old victim of a house fire sustained burns to the right side of her head, arm and leg. Following resuscitation procedures and excision, the leg was grafted with meshed split-skin from the father. This was left for two weeks before removal of the epidermis, thorough washing with saline, and coverage of the wound with polymer discs. Autologous keratinocytes grown on the polymer discs were 60% confluent. At the same time other wounds were treated with meshed autograft.
At the first dressing change there was evidence of infection in both areas. Topical antibiotics were applied and washed off with saline after three days. Further cultured keratinocytes on polymer discs were then applied to the wound.
After a further five days the dressings were removed, showing distinct opaque areas of epidermis where some of the discs had been placed. Some of the discs had slipped and further cultures were therefore used to cover the remaining wound on the girl's leg.
The patient was allowed home wearing a Jobst garment and she healed without further incident (Figs. 1-4).

Fig. I - Case 1. De-epidermidized allografted

Fig. 2 - Case 1. Wound after second culture graft,

Fig. I - Case 1. De-epidermidized allografted
wound bed prior to first culture graft.
Fig. 2 - Case 1. Wound after second culture graft,
5 days post-grafting, showing opaque healed area
beneath disc.

Fig. 3 - Case 1. Patient ready for release after 3 weeks

Fig. 4 - Case 1. Appearance of wound after 3 years.

Fig. 3 - Case 1. Patient ready for release after 3 weeks Fig. 4 - Case 1. Appearance of wound after 3 years.

Case 2
This patient was transferred to our Burns Unit three weeks post-burn. The injury had taken place as she was cooking over an open fire. Ten days later, after excision, twelve polymer disc autocultures (of 60-90% confluence) were grafted on to full-thickness wounds on both legs. Seven days later the discs were removed with the dressings. The biopsies taken showed considerable epithelialization (Figs. 5-6). Unfortunately, the patient died two weeks later.

Fig. 5 - Case 2. Right leg 10 days post-grafting showing site of biopsy (x).

Fig. 6 - Case 2. Biopsy section showing narrow epidermal layer (y) loosely attached to fascia (z) of wound bed.

Fig. 5 - Case 2. Right leg 10 days post-grafting showing site of biopsy (x). Fig. 6 - Case 2. Biopsy section showing narrow epidermal layer (y) loosely attached to fascia (z) of wound bed.

Case 3
This patient, aged two years, had 30% skin loss owing to meningococcal septicaemia. Following excision he was grafted with glycerol-preserved allograft and five days later this was de-epidermidized and keratinocyte cultures placed on an allodermal area on each lower leg (Fig. 7).
After a further five days the discs were removed and biopsies taken from each site. The right leg was infected and both wounds were split-skin grafted. The biopsies showed that full epidermis had grown on the left leg.

Fig. 7 - Case 3. Biopsy 5 days post-grafting on de-epidermidized homograft (stain ME), showing re-epithelialization. This was not visible macroscopically as differentiation was incomplete.

Fig. 7 - Case 3. Biopsy 5 days post-grafting on de-epidermidized homograft (stain ME), showing re-epithelialization. This was not visible macroscopically as differentiation was incomplete.


Novel synthetic polymer films produced by Smith and Nephew have been shown to support the growth of human keratinocytes. These have been used to transfer actively growing cells to burn wounds in patients. The film, made in 3.5 cm diameter circles, was impermeable to air and non-adherent to the wound bed.
The theories behind this technique were:

  • that cells in a subconfluent culture show more mitotic activity and a reduced tendency to differentiate than in confluent cultures and cell sheets
  • that actively dividing, undifferentiated cells are able to invert and transfer from the polymer and grow on a suitable wound bed

By the use of serum-free low-calcium medium, growth was enhanced and differentiation minimized, so that cells were able to grow on to the wound bed. This method enabled autologous cells to be grafted within days of receiving the biopsy instead of the more usual 3-4 weeks' delay of previous methods. First passage cells were grafted before confluence occurred and they therefore underwent fewer divisions before application. Differentiation took place on the wound bed, protected by the polymer, until epidermis was formed and the disc dropped off during a dressing change.
There were other advantages using this method. Unlike the previous technique, according to which the cell sheet must be removed from the base of the culture vessel and mounted on a dressing before transfer to theatre or bedside, transfer of the discs was quick and easy, requiring little skill to lift (with forceps) from culture dish to wound bed, with no previous preparation. Problems with transportation over long distances and viability maintenance are thus minimized using this system, the cultures being kept in growth medium until grafting has taken place.
From the small number of patients grafted in this trial it has been demonstrated that cells will migrate from the polymer surface and form areas of healthy epidermis on suitable wound beds. It is unlikely that this is a 'dressing effect' which stimulates epidermal migration from adnexal elements or the wound edge, as discrete areas of healthy epidermis were observed beneath the centre of the polymer discs five days after grafting over areas of previously allografted, full-thickness wound.
The major causes of graft failure in this study were displacement of the polymer, and microbial infection entering round the edge of the disc. The condition of the wound bed at the time of grafting and the presence or absence of dermal elements also affected the 'take' or failure. We hope to combine this polymer/epidermal system with allodermis from banked skin from the new Stephen Kirby Skin Bank or dermal equivalents currently being developed.


RESUME. Les Auteurs ont développé une méthode pour permettre de transférer des kératinocytes autologues jusqu'au site d'une lésion entre 5-7 jours après la biopsie. Grâce au développement par Smith et Nephew d'un polymer qui supporte la croissance des cellules, ils ont trouvé que les kératinocytes (non différenciés) qui se divident activement peuvent invertir et croître sur une lésion. Les kératinocytes ont été cultivés dans des éprouvettes dans un milieu qui contenait du sière pendant 48 h, alimentés avec milieu sans sérum, et transférés, une fois en phase de croissance active, jusqu'au polymer. Celui-ci a été incubé pendant 2-3 jours pour produire une strate cellulaire subconfluente (confluence 70-90%) qui était facile à invertir sur le site de la lésion en salle opératoire ou au chevet du lit. Après la mise en place sur une lésion appropriée, la guérison était évidente, comme une pellicule translucide, après 5-7 jours.


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  8. Ben-Bassat IL, Eldad A., Chacmat M. et al.: Structural and functional evaluation of modifications in the composite skin graft: cryopreserved dermis and cultured keratinocytes. Plast. Reconstr. Surg., 89: 510-20, 1992.


This paper was received on 20 August 1997.

Address correspondence to: Dr Avril J. Burt
Stephen Kirby Skin Bank, Burns Unit
Queen Mary's University Hospital
Roehampton Lane, London SW15 5PN, Great Britain.


Acknowledgements. We wish to thank both Smith and Nephew for their support and provision of the polymer and Dr Y. Barlow during her employment with them. Thanks also for the help of the nursing staff of the Burns Unit at Queen Mary's Hospital, Roehampton.


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