promoting factors into the area of the wound. If this isthe case then short term biological dressings incorpo-rating suitable human cell types, free from known viralpathogens, on a substratum compatible with thewound may provide a valuable adjunct to conventionalgrafting procedures, with the added advantage thatsuch material can survive cryopreservation and bereconstituted for use in large amounts and at shortnotice.

The Institute of Cancer Research is supported by fundsfrom the Cancer Research Campaign and the MedicalResearch Council.

1 Green H, Kehinde 0, Thomas J. Growth of cultured human epidermal cellsinto multiple epithelium suitable for grafting. Proc Natl Acad Sci USA1979;76:5665-8.

2 Clarke JA, Burt AM, Eldad A. Cultured skin for burn injury. Lancet1986;ii:809.

3 Eisinger M, Monden M, Raaf JH, Fortner JG. Wound coverage by a sheet ofepidermal cells grown in vitro. Surgery 1980;88:287-93.

4 Gallico GG III, O'Connor NE, Compton CC, Kehinde 0, Green H.

Permanent coverage of large burn wounds with autologous cultured humanepithelium. N EnglJ Med 1984;311:448-5 1.

5 Hefton JM, Madden MR, Finkelstein JL, Shires GT. Grafting ofbum patientswith allografts of cultured epidermal cells. Lancet 1983;iu:428-30.

6 Leigh IM, Purkis PE. Culture grafted leg ulcers. Clin Exp Dermatol1986;11:650-2.

7 Leigh IM, Purkis PE, Navsaria HA, Phillips TJ. Treatment of chronic venousulcers with sheets of cultured allogenic keratinocytes. -Br J Dennatol1987;117:591-7.

8 Morhenn VB, Benike CJ, Cox AJ, Charron DJ, Engleman EG. Culturedhuman epidermal cells do not synthesize HLA-DR. J Invest Dermatol1982;78:32-7.

9 Thivolet J, Faure M, Demidem A, Mauduit G. Cultured human epidermalallografts are not rejected for a long period. Arch Dermatpl Res 1986;278:252-4.

10 Burns J, Chan VTW, Jonasson JA, Fleming KA, Taylor S, McGee JO'D.Sensitive system for visualising biotinylated DNA probes hybridised in situ:rapid sex determination on intact cells. J7 Clin Pathol 1986;38:1085-92.

1 1 Cooke HJ, Schmidtke J, Gosden JR. Characterisation of a human Ychromosome repeated sequence and related sequences in higher primates.Chromosomea 1982;87:491-502.

12 Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic labelling ofmonoclonal antibodies using immune complexes of aLkaline phosphataseand monoclonal anti-alkaline phosphatase (APAAP complexes).J7 HistochemCytochem 1984;32:219-29.

(Accepted 30anuary 1989)

Survival of cultured allogeneic keratinocytes transplanted to deepdermal bed assessed with probe specific for Y chromosome

Anne Brain, Patricia Purkis, Philip Coates, Michael Hackett, Harshad Navsaria, Irene Leigh

North East ThamesRegional Plastic Unit, StAndrew's Hospital,Billericay, EssexAnne Brain, FRCS, registrar inplastic surgeryMichael Hackett, FRCS,consultant plastic surgeon

London Hospital, LondonEl 1BBPatricia Purkis, chieftechnicianHarshad Navsaria, MSC,technicianIrene Leigh, MRCP, seniorlecturer in dermatology

Department ofHistopathology, StBartholomew's Hospital,London EClA 7BEPhilip Coates, PHD, researchscientist

Correspondence andrequests for reprints to:Ms Anne Brain, Departmentof Dermatology,The London Hospital,London El 1BB.

BrMedJf 1989;298:917-9

AbstractTo determine the survival of cultured allogeneickeratinocytes transplanted to a deep dermal bed24 tattoos that had been removed by deep shaveexcision in 19 patients were grafted with sheets ofcultured aliogeneic keratinocytes from donors of theopposite sex. Celis carrying the Y chromosome wereidentified in biopsy specimens taken from the graftsite by in situ DNA hybridisation with a biotinylatedY probe (pHY 2.1) and visualised with a techniqueusing immunoperoxidase. The cultured allograftsites were biopsied one, two, and three weeks aftertransplantation. No male cells were identified in anybiopsy specimen from female patients who weregiven transplants of male cultured keratinocytes,and all biopsy specimens from male patients, whoreceived female cultured keratinocytes, showedpercentages of male cells within the normal range formale skin.The beneficial effects of cultivated allogeneic

keratinocytes result from effects on wound healingother than forming a successful graft that "takes."

IntroductionOrgans transplanted across a histocompatibility

barrier are usually rejected within 10-14 days unlessthe host's immune system is suppressed. Transplantedorgans contain passenger leucocytes, which include theantigen presenting cells that present antigen to helperT cells and may initiate the rejection of a transplant byexpressing class II histocompatibility molecules andsecreting cytokines. Eliminating such cells from allo-grafts may reduce the graft's immunogenicity andprolong its survival. Culturing endocrine tissues suchas thyroid' and pancreatic islet cells2 both eliminatesantigen presenting cells and prolongs the survival ofthe cultured cells when they are transplanted as acultured allograft.

In skin grafts the main antigen presenting cells areLangerhans cells, which form 5% of the total mixedepidermal cell population. Seven to 10 days after asingle cell suspension of mixed epidermal cells iscultured in vitro the Langerhans cells are lost,3 as

determined by the expression of HLA-DR antigens.4The mixed epidermal cell lymphocyte reaction in lostin parallel, which seems to confirm the loss of theLangerhans cells.5 Other cells with the potential abilityto express class II antigens, such as endothelialcells, which are particularly important in rejectionreactions,6 are also lost during the culture of keratino-cytes from mixed suspensions.

Sheets of cultured autologous keratinocytes havebeen used as skin grafts in patients with severe burns,7leg ulcers,8 and congenital skin diseases such asepidermolysis bullosa.9 The delay of three to fourweeks in producing expanded sheets of cells froma donor specimen, however, remains a practicalproblem, especially for patients with burns. Thuscultured allogeneic keratinocytes were used as grafts inpatients with burns and clinically the allograft seemedto be successful (to "take").'0 This failure of rejectionwas attributed to the loss of the passenger leucocytes-that is, Langerhans cells. In previous studies oftransplantation the survival of skin grafts was pro-longed when skin that had been irradiated withultraviolet light was used; such irradiation also reducesthe numbers of Langerhans cells. "

Subsequent studies of the fate of cultured allogeneickeratinocytes have been controversial, with little proofthat the allograft took rather than accelerated woundhealing. To distinguish the two processes requires abiological technique giving unequivocal results-forexample, HLA analysis, DNA fingerprinting, ordetecting the sex of cells. In 1982 Cooke et al character-ised a sequence that was repeated in tandem with 2000copies on the Y chromosome.'2 This constitutes a fifthof the DNA and is located on the tip of the long arm. Asimple reproducible method to detect this multiplecopy human gene has been established.'3To establish whether cultured allografts take

biologically we used in situ hybridisation with abiotinylated probe to the Y chromosome to detectthe sex of cells in skin biopsy specimens taken fromdeep clean surgical wounds that had been graftedwith sheets of cultured allogeneic keratinocytesfrom unrelated donors of the opposite sex to thepatients.

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MethodsRedundant normal skin from female patients having

plastic surgery (who had been screened after counsel-ling for infection with HIV and hepatitis B) or frominfants having circumcision was the source of thecultured allogeneic keratinocytes. The skin sampleswere prepared and cultured as previously described.'4Cultures were confluent at 10-14 days and left tostratify for two to three weeks, when multilayeredsheets of poorly differentiated layers of keratinocyteswere obtained. Sheets were separated by treatmentwith 0-1% dispase for one to two hours and thenbacked with a dressing (Johnson's NA (non-adherentdressing)) and separated from the plastic substratewith a rubber policeman, ensuring that the graft wasoriented with basal cells next to the wound bed.

Nineteen patients (six men, 13 women) asking forthe removal of decorative tattoos of a size that requiredgrafting (n=24) gave their informed consent to beincluded in the study. The patients were treated asoutpatients. The tattoos were injected with localanaesthetic (0 5% lignocaine and adrenaline 1/200 000)and removed by serial shave excision until all thepigment was removed. Thus the depth of the woundvaried according to clinical need from the deep dermisto the borders of subcutaneous fat (five to six shaves of1 mm). After haemostasis the wound was covered witha sheet of cultured allogeneic keratinocytes from anunrelated donor of the opposite sex. The wound wasdressed and the arm splinted. Dressings were per-formed weekly until the wound was completely healed,and each time the wound was dressed a punch biopsyspecimen (3 mm) was taken from its centre.The biopsy specimens were snap frozen in liquid

nitrogen. Sections (4 rim) were cut, mounted onmultiwell slides, and fixed in Carnoy's fixative for 15minutes. Cells carrying the Y chromosome were identi-fied by in situ hybridisation with a biotinylated Y probe(pHY 2.1)12 and visualised by a technique usingimmunoperoxidase staining.'3 The samples were pro-cessed in duplicate, each with two concentrations ofprobe (0- 1 and 0-2 ng/I0 jt hybridisation medium) andtwo negative controls (hybridisation medium alone andpBR322). 3 The sections were counterstained withhaematoxylin. The number of cells containing the Ychromosome, as detected with the Y probe, wascounted as a percentage of the epidermal nuclei seenunder high magnification (x 1000) and the mean oftwoto five high power fields per section was taken to coverthe whole 3 mm punch biopsy specimen.

Biopsy specimen from male patient given female donor keratinocytes two weeks after grafting. Labellingof cells with Yprobe (dot in nucleus) confirms that cells arefrmn male recipient

Control samples of normal male skin and sheets ofmale keratinocytes were processed and counted. Skinbiopsy specimens were studied after routine stainingwith haematoxylin and eosin, and the lowest shave ofthe excision was studied to assess the presence ofappendageal epithelium in later biopsy specimens.

ResultsClinical-Three out of 21 grafts were clinically

healed at one week and 13 by two weeks; all grafts werehealed by three weeks. Three patients could not beassessed because of non-compliance with the changingof dressings.

Analysis ofbiopsy specimens-No cells carrying the Ychromosome were seen in any biopsy specimens aftermale keratinocytes were transplanted to femalerecipients (table). When female cells were transplanted

Mean percentages of cells carrying Y chromosome in culturedallogeneic keratinocytes transplanted to receipient of opposite sex todonor

Female donor, male recipient Male donor, female recipient

Week after No of biopsy% Cells carrying No of biopsy% Cells carryingtransplant specimens Y chromosome specimens Y chromosome

1 9 52 7 02 7 45 5 03 10 59 5 0

to male recipients the percentage of cells with a Ychromosome was within the normal range for male skinin the control samples and sheets of keratinocytes (50-70% of cells labelled) (figure).

Histopathological results-Epidermal cells werepresent in 10 out of 16 biopsy specimens at one week,in 11 out of 12 at two weeks, and in all 13 at threeweeks. In the early biopsy specimens the epidermiswas poorly cornified and there was no evidence of anormal stratum corneum. The epidermis was hyper-plastic with poor rete peg formation. Extensiveimmunohistochemical studies will be reported else-where. There were no follicular remnants seen in anyof the biopsy specimens, and sweat glands were seen inonly two specimens. There were no remnants of theappendage-that is, sweat glands and hair follicles-inthe deep shave sections. Biopsy specimens at twoand three weeks showed increasing morphologicaldifferentiation with the development of an orthokera-totic stratum corneum. A mixed cell chronic inflamma-tory infiltrate was seen in the dermis with no evidenceof intraepidermal lymphocytes.

DiscussionWe found that all donor cells were lost from wounds

treated with cultured allogeneic keratinocytes by sevendays after grafting despite considerable epithelialisa-tion (fig 1). We therefore have no evidence ofthese cellstaking on to a deep surgical wound. This contrasts withreports from Mauduit et al, who used culturedallogeneic keratinocytes to graft 22 surgical woundsand claimed that they took completely on to superficialdonor sites, which were clinically healed after a week. 1'The recipient's failure to express his or her blood groupantigen was used as a cell marker of the allograft'ssurvival, but the expression of blood group antigens isaltered during wound healing. Another study usingHLA antigens as markers in four patients showedprogressive replacement of the allograft from recipientcells on grafting in the appendages from 14 daysonwards.'6 The controls for HLA markers should alsobe examined during normal wound healing because ofmodulation of membrane proteins during the normalhealing process. An assay system based on DNA, such

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as one using a Y probe or DNA fingerprinting, istherefore better than one based on membrane antigens.We treated wounds at a deep level of the dermis with noremaining hair follicles and few sweat glands; thereforethe epithelial keratinocytes largely arose and migratedfrom the edge of the wound and there was little if anycontribution from the appendages.

Studies in animals by Hammond et al using culturedallogeneic keratinocytes in a transplantation chamberthat ensured the physical separation of donor andrecipient cells showed survival of donor keratinocytes(free of Langerhans cells) for 70 days compared with 14days for intact allografts.'7 This also supports theconcept that donor keratinocytes are tolerated afterthey have been cultured. In this system, however,there are no competing host epithelial cells to replacethe allograft. In contrast, our results are supported byAubock et al, who found rejection of cultured allo-geneic keratinocytes in 11 patients 14 days after theyhad received the allograft.'8 Levick and Clarke et alwere also unable to find convincing evidence that theallograft had taken.9 120The loss of keratinocytes so soon after transplanta-

tion could be explained by mechanisms other thanhyperacute rejection, and certainly we saw no lysis ofthe allograft clinically or intraepidermal infiltration bylymphocytes histologically (although in rejection, theresponse of lymphocytes is mixed and diagnosticmarkers difficult to define2). Mackenzie and Hill alsoused the transplantation chamber system with auto-logous mouse keratinocytes,22 and Mackenzie hasrecently shown differences between the ability ofsuperficial and deep connective tissue to supportepidermal differentiation-epidermal cells retain asimple epithelial phenotype when transplanted on todeep connective tissue (unpublished data). Thus ourresults could reflect the failure of deep connectivetissue to support allogeneic keratinocytes. Clinicalreports of autologous cells grafted on to deep fasciahave, however, claimed clinical take, although thewounds were unstable with blistering.23 Our earlybiopsy specimens from the allograft did show substan-tial epithelialisation that was poorly differentiated butcertainly multilayered. These features suggest rapidwound healing, which may mean that there is physicalreplacement of the allograft rather than rejection, andaccelerated wound healing could account for the mainclinical benefits reported previously.' 13

Controlled trials of cultured allogeneic keratinocytesagainst simple dressings in leg ulcers and againsthydrocolloid dressings in donor sites are in progressthat will address the question of accelerated woundhealing. Activated keratinocytes in culture havemultiple biological effects, which could enhancewound healing. These include the production ofbasement membrane proteins (collagen types IV-VII,laminin, fibronectin, basic protein antigen) to enhancecell migration; physical scaffolding; protection; andthe production of growth promoting agents. Culturedkeratinocytes have recently been shown to producetransforming growth factor alpha,24 which accelerateswound healing in pigs.25 As biological take does notseem to occur after transplantation from sheets ofcultured allogeneic keratinocytes on to deep surgicalwounds further studies are needed to examine theeffects of the depth of the wound, the state of

keratinocyte culture, the immunosuppression of therecipient, and the provision of a substrate on thesuccess of allografts. Controlled studies of woundhealing are also required to establish that woundhealing is accelerated and whether it occurs because ofa diffusible product or a different biological effectrequiring contact with live cells. The possibility existsof producing a keratinocyte product or products byrecombinant technology in a form that could helpwound healing without live cells.

This work was supported by the Billericay Burn ResearchFund and the Skin Disease Research Fund.

1 Lafferty KJ, Bootes A, Dart G, et al. Effect of organ culture on the survival ofthyroid allografts in mice. Transplantation 1976;22:138-49.

2 Kedinger M, Haffen K, Grenier J, Eloy R. In vitro culture reducesimmunogenicity of pancreatic endocrine islet cells. Nature 1977;270:736-8.

3 Rheinwald JC, Green H. Serial cultivation of strains of keratinocytes: theformation of keratinising colonies from single cells. Cell 1975;6:331-43.

4 Morhenn VB, Benike CJ, Cox AJ, Charron DJ, Engelman EG. Culturedhuman epidermal cells do not synthesise HLA-DR. J Invest Dermatol1982;78:32-7.

5 Hefton JM, Amberson JB, Biozes DG, Weksler ME. Loss of HLA-DRexpression by human epidermal cells after growth in culture. J InvestDermatol 1984;83:48-50.

6 Fabre JW, Milton AD, Spencer S, Settaf A, Houssin D. Regulation ofalloantigen expression in different tissues. Transplant Proc 1987;19:45-9.

7 Gallico GG 3d, O'Connor NE, Cofiipton CC, Kehinde N, Green H. Permanentcoverage of large bum wounds with cultured autologous human epithelium.N Engl' Med 1984;311:448-5 1.

8 Leigh IM, Purkis PE. Culture grafted leg ulcers. Clin Exp Dermatol1986;11:650-2.

9 Carter DM, Lin AN, Varghese MC, Caldwell D, Pratt LA, Eisenger M.Treatment of junctional epidermolysis bullosa with epidermal autografts.J7 Am Acad Dermatol 1987;172:246-50.

10 Hefton JM, Madden MR, Finkelstein JL, Shires GT. Grafting of burnspatients with allografts of cultured epidermal cells. Lancet 1983;ii:428-9.

11 Kripke ML. Immunological unresponsiveness induced by ultravioletradiation. Immunol Rev 1984;80:87-102.

12 Cooke HJ, Schmidtke J, Gosden JR. Characterisation of a human Y repeatedsequence and related sequences in higher primates. Chromosoma 1982;87:49 1-502.

13 Bums J, Chan VTW, Jonasson JA, Fleming KA, Taylor S, McGee JO'D.Sensitive system for visualising biotinylated DNA probes hybridised in situ:rapid sex determination of intact cells. J Clin Pathol 1985;38:1085-92.

14 Leigh IM, Purkis PE, Navsaria HA, Phillips TJ. Treatment of chronic venousulcers with sheets of cultured allogenic keratinocytes. Br J Dermatol1987;117:591-7.

15 Mauduit G, Faure M, Demiderm A, Kanitakis J, Thivolet J. Cultured humanepidermis used as allografts-studies on their differentiation. J7 InvestDermatol 1986;87:1546.

16 Gielen V, Faure M, Mauduit G, Thivolet J. Progressive replacement of humancultured epithelial allografts by recipient cells as evidenced by HLA class Iantigen expression. Dermatologica 1987;175:166-70.

17 Hammond EJ, Ng R, Stanley MA. Prolonged survival of allogenic culturedkeratinocyte sheets in the mouse without imunosuppression. J InvestDermatol 1986;87:143.

18 Aubock J, Fritsch P. Autologous versus allogenic cultivated epidermis forwound grafting. In: Teepe R, ed. Proceedings symposium on "clinical use ofcultured epithelium in surgery and dermatology," Leiden. Wheathampstead:Medical and Scientific Conferences Ltd, 1987:59-64.

19 Levick PL. Generation of autografts-current techniques and results. In: TeepeR, ed. Proceedings symposium on "clinical use of cultured epithelium in surgeryand dermatology," Leiden. Wheathampstead: Medical and ScientificConferences Ltd, 1987:15-8.

20 Clarke JA, Burt AM, Eldad A, Yardeni P, Gusterson B. Allograft culturedepithelium and meshed allograft skin. In: Teepe R, ed. Proceedingssymposium on "clinical use of cultured epithelium in surgery and dermatology,Leiden. Wheathampstead: Medical and Scientific Conferences Ltd, 1987:33-6.

21 Hall BM. Cellular infiltrates in allografts. Transplant Proc 1987;19:50-6.22 Mackenzie IC, Hill MW. Connective tissue influences on patterns of epithelial

architecture and keratinisation in skin and oral mucosa of adult mouse.Cell Tissue Res 1984;235:551-9.

23 Gallico GG, O'Connor NE, Briggs SM, Compton CC, Kehinde 0, Green H.Cultured epithelial autografts: indications and long term results. In: TeepeR, ed. Proceedings symposium on "clinical use of cultured epithelium in surgeryand dermatology," Leiden. Wheathampstead: Medical and ScientificConferences Ltd, 1987:19-24.

24 Coffey RJ, Derynck R, Wilcox JN, et al. Production and autoinduction oftransforming growth factor alpha in human keratinocytes. Nature 1987;328:817-20.

25 Schultz GS, White M, Mitchell R, et al. Epithelial wound healing enhanced bytransforming growth factor alpha and vaccinia growth factor. Science1987;235:350-2.

(Accepted 303anuary 1989)

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