LETTERS TO THE EDITOR

Formation of Permeability Barrier in Epidermal OrganotypicCulture for Studies on Drug Transport

To the Editor:

Cadaver skin has been used in drug transport studies but its limitedavailability and large variation between specimens have increasedinterest in organotypic culture models with permeability charac-teristics resembling that in native skin. Other bene®ts of liveorganotypic cultures include their metabolic activity, which affectsthe permeability of some drugs (Kao and Hall, 1987), and theirpotential for research on irritation, toxicity, and keratinocytedifferentiation (Slivka et al, 1993; Vicanova et al, 1998; Boelsma etal, 2000); however, culture models with barrier properties com-parable of human skin and yet suitable for simple, large-scaleresearch such as permeability tests of topically applied drugs andtheir vehicles, have not been established (Ernesti et al, 1992; Nolteet al, 1993; Regnier et al, 1993).

A keratinocyte cell line derived from newborn rat skin (ratepidermal keratinocyte; REK) retains its ability to form strati®edcultures with basal, spinous, and granular cells, and multipleorthokeratinized layers of corneocytes when grown at the air±liquid interface on collagen gel inserts (MacCallum and Lillie, 1990;Tammi et al, 2000). We explored whether the naturally highdifferentiation potential of REK cultures could be developed into amodel of epidermal permeability barrier research.

To prepare the supports for differentiating cultures, type Icollagen (from rat tail, Becton Dickinson Labware, Bedford, MA)was mixed with Earle's balanced salt solution (10 3 EBSS, LifeTechnologies, Paisley, Scotland), 7.5% sodium bicarbonate (LifeTechnologies), and 1 M sodium hydroxide solution, at a volumeratio of 8:1:0.3:0.2, respectively, on an ice bath. The collagensolution (800 mL) was pipetted onto 24 mm diameter tissue cultureinserts (3. 0 mm pore size; Transwellâ, Costar, Cambridge, MA)and incubated overnight at 37°C in a humidi®ed atmosphere. Theinserts covered by a gel composed of reconstituted native collagen®brils were washed with the culture medium before use. Recentlycon¯uent stock cultures of REK with no morphologic evidence ofstrati®cation were trypsinized and 300 000 cells in 2 ml Dulbecco'smodi®ed Eagles medium (4500 mg per L glucose) (LifeTechnologies) were applied to the collagen mats on the cultureinserts. After 3 d with culture medium present both in the wellbeneath the insert as well as on the surface of the cells, the uppermedium was removed and the amount of the lower medium(~1.5 ml) was adjusted to the level of the collagen gels. Newmedium with a supplement of 40 mg per ml L-ascorbic acid (Sigma,St. Louis, MO) was changed every 2 d for the ®rst week and dailythereafter.

For light microscopy, cultures were ®xed with Histochoiceâ

(Amresco, Solon, OH) overnight, dehydrated in graded ethanol,and embedded in paraf®n. Vertical 3 mm thick sections were usedfor hematoxylin and eosin and immunohistologic stainings. Forelectron microscopy, cultures were ®rst ®xed in 0.1 M cacodylate-buffered (pH 7.4) 2.5% glutaraldehyde overnight at 4°C and then

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc.

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Manuscript received September 19, 2000; revised July 3, 2001; acceptedfor publication July 27, 2001.

Reprint requests to: Dr. Sanna Pasonen-SeppaÈnen, MSc, Department ofAnatomy, University of Kuopio, Kuopio, Finland. Email: Sanna.Pasonen@uku.®

Figure 1. The morphology of 3 wk old organotypic REKcultures. (a) Paraf®n embedded, hematoxylin and eosin stained culturesshow numerous keratohyalin granules (arrow in a) and a prominentorthokeratotic stratum corneum (SC). To visualize the SC intercellularlipids, the cultures were post®xed with ruthenium tetroxide (b±d).Numerous ovoid lamellar bodies are seen in granular cells (b). At thestratum granulosum (SG)/stratum corneum (SC) interface lamellar bodylipid contents are extruded (arrows in c). The intercorneocyte lamellarlipid layers (asterisk in c) show the characteristic alternating electrondense and electron lucent repeating pattern (d). Scale bars: (a) 30 mm, (b)200 nm, (c) 100 nm, (d) 50 nm.

post®xed in 0.4% ruthenium tetroxide with 0.2% aqueous potas-sium ferrocyanide for 2 h at 4°C. The specimens were thendehydrated in graded ethanol and propylene oxide before embed-ding in Epon. Ultrathin sections were stained with uranyl acetateand lead citrate.

In permeability studies, the cadaver skin samples or organotypiccultures were clamped between 3 ml phosphate-buffered saline-®lled, stirred, thermostatted (37°C) chambers of a diffusion cellapparatus (Side-Bi-Side, Crown Glass Company, Somerville, NJ)with an effective diffusional area of 0.64 cm2. Tritiated corticos-terone or mannitol (NEN Life Science Products, Boston, MA)20 000±40 000 dpm per 10 mL was added to the donor chamber.Aliquots were withdrawn at predetermined time intervals fromboth chambers, counted for radioactivity, and replaced withphosphate-buffered saline to maintain a constant volume.Experiments were continued long enough to ensure that thesteady-state phase was attained. The permeability coef®cients (P,cm per s) for the probe permeants were calculated at steady stateunder sink conditions by dividing the steady-state ¯ux (dpm per scm2) through the skin by the concentration of the test substance(dpm per cm3) in the donor phase. The transepidermal water loss(TEWL) (g m2 per h) was measured by using a Del®n-SWL system(Del®n Technologies Oy, Kuopio, Finland). In this system, sensorsinside the evaporimeter probehead measure the increasing humid-ity of the air inside the probehead that is kept in contact with theculture surface or skin for 1 min. The cultures were allowed toequilibrate with ambient air for 1 h at room temperature before themeasurement, which was carried out at 21±23°C. Control values ofTEWL of normal human skin were obtained from the forearm ofnine healthy Caucasian people (aged from 18 to 42).

By 3 wk on collagen at the air±liquid interface, the culturesshowed complete morphologic differentiation of the epidermis,including cuboidal basal cells, 1±2 spinous cell layers, granular cellswith numerous keratohyalin granules, and a well-developedstratum corneum displaying the typical ``basketweave'' separationof corneocytes when embedded in paraf®n (Fig 1a).Immunohistologic studies revealed the regular presence andlocalization of differentiation markers keratin 10, involucrin, and®laggrin (data not shown). The light microscopic structure stayedsimilar for at least 6 wk, except that the height of the corni®ed layerincreased continuously.

The epidermal barrier function, critically dependent on properarrangement of the intercellular SC lipids (Elias and Menon, 1991;Bouwstra et al, 1996), was speci®cally studied by electronmicroscopy of ruthenium tetroxide post®xed cultures. At 3 wk,the upper, vital keratinocytes showed a number of lamellar bodieswith a regular internal stacking organization (Fig 1b). At theinterface between the live epidermis and the stratum corneum thelamellar bodies fused with the plasma membrane and extruded theircontents into the intercellular space (Fig 1c). The secreted lipidstacks were subsequently transformed into lamellar lipid layers witha typical repeating pattern of alternating electron dense and electron

lucent bands (Fig 1d) (Landmann, 1986; Madison et al, 1987;Swartzendruber et al, 1989).

These structural features, similar to those in normal human skin,prompted us to study the barrier function of organotypic REKcultures by measuring the transepidermal water loss (TEWL) andthe permeability for corticosterone, a frequently used indicatorsubstance. TEWL was relatively high in 1-wk-old cultures(Table I) but was reduced to 24% of that at 2 wk, and did notsigni®cantly change thereafter (Table I). The permeability ofcorticosterone was high at 1 wk, but was considerably reduced by 2and 3 wk (Table I). At 3 wk the corticosterone permeabilityreached a value close to normal human cadaver skin (Table I).Prolongation of the culturing time up to 6 wk did not cause furtherchange in the corticosterone permeation (data not shown). Forcomparison, we also measured the permeability of mannitol, atracer more polar than corticosterone. Like TEWL, it showedpermeation values close to, but somewhat higher than those ofnormal cadaver skin (Table I). Furthermore, we have preliminarydata to suggest that REK cultures can predict the human skinpermeability of a number of other drugs and chemicals. Thepermeability in REK cultures corresponds to those of a human skinequivalent model (Asbill et al, 2000). The convenience of acontinuous cell line, independent of ®broblast support, makes REKorganotypic cultures an easily maintained and reproducible modelfor studies on transepidermal barrier function, with permeationvalues close to normal human skin.

This study was ®nancially supported by the Academy of Finland, the Technology

Development Center of Finland (TEKES) and Orion Corporation Orion Pharma,

and by a grant from Juliana von Wendt Foundation.

Sanna Pasonen-SeppaÈnen, T. Marjukka Suhonen,*Merja Kirjavainen,* Merja Miettinen,² Arto Urtti,*

Markku Tammi, Raija TammiDepartment of Anatomy and *Department of Pharmaceutics,

University of Kuopio, Kuopio, Finland²Department of Oncology, Kuopio University Hospital,

Kuopio, Finland

REFERENCES

Asbill C, Kim N, El-Kattan A, Creek K, Wertz P, Michniak B: Evaluation of ahuman bio-engineered skin equivalent for drug permeation studies. Pharm Res17:1092±1097, 2000

Boelsma E, Gibbs S, Faller C, Ponec M: Characterization and comparison ofreconstructed skin models: morphological and immunohistochemicalevaluation. Acta Derm Venereol 80:82±88, 2000

Bouwstra JA, Gooris GS, Cheng K, Weerheim A, Bras W, Ponec M: Phase behaviorof isolated skin lipids. J Lipid Res 37:999±1011, 1996

Elias PM, Menon GK: Structural and lipid biochemical correlates of the epidermalpermeability barrier. Adv Lipid Res 24:1±26, 1991

Ernesti AM, Swiderek M, Gay R: Absorption and metabolism of topically appliedtestosterone in an organotypic skin culture. Skin Pharmacol 5:146±153, 1992

Table I. The TEWL and permeability of corticosterone and mannitol in organotypic REK cultures (1±3 wk) and humanskina

1 wk 2 wk 3 wk Human skin

TEWL(g m2 per h)

91.20 6 8.70(n = 10)

22.0 6 6.20(n = 11)

19.44 6 7.90(n = 11)

10.07 6 7.90b

(n = 9)Corticosterone(P 3 10±6 cm per s)

29.10 6 19.43(n = 8)

1.60 6 0.60(n = 8)

0.22 6 0.08(n = 15)

0.18 6 0.11c

(n = 11)Mannitol(P 3 10±6 cm per s)

n.d. n.d. 0.59 6 0.14(n = 12)

0.17 6 0.16c

(n = 10)

aData are presented as means 6 SD in the number of experiments (n) shown in parenthesis.bNormal human skin in vivo.cCadaver skin, n.d. not determined.

VOL. 117, NO. 5 NOVEMBER 2001 LETTERS TO THE EDITOR 1323

Kao J, Hall J: Skin absorption and cutaneous ®rst pass metabolism of topical steroids:in vitro studies with mouse skin in organ culture. J Pharmacol Exp Ther241:482±487, 1987

Landmann L: Epidermal permeability barrier: transformation of lamellar granule-disks into intercellular sheets by a membrane-fusion process, a freeze- fracturestudy. J Invest Dermatol 87:202±209, 1986

MacCallum DK, Lillie JH: Evidence for autoregulation of cell division and cell transitin keratinocytes grown on collagen at an air±liquid interface. Skin Pharmacol3:86±96, 1990

Madison KC, Swartzendruber DC, Wertz PW, Downing DT: Presence of intactintercellular lipid lamellae in the stratum corneum. J Invest Dermatol 88:714±718, 1987

Nolte CJ, Oleson MA, Bilbo PR, Parenteau NL: Development of a stratum corneumand barrier function in an organotypic skin culture. Arch Dermatol Res 285:466±474, 1993

Regnier M, Caron D, Reichert U, Schaefer H: Barrier function of human skin andhuman reconstructed epidermis. J Pharm Sci 82:404±407, 1993

Slivka SR, Landeen LK, Zeigler F, Zimber MP, Bartel RL: Characterization barrierfunction, and drug metabolism of an in vitro skin model. J Invest Dermatol100:40±46, 1993

Swartzendruber DC, Wertz PW, Kitko DJ, Madison KC, Downing DT: Molecularmodels of the intercellular lipid lamellae in mammalian stratum corneum. JInvest Dermatol 92:251±257, 1989

Tammi RH, Tammi MI, Hascall VC, Hogg M, Pasonen S, MacCallum DK: Apreformed basal lamina alters the metabolism and distribution of hyaluronan inepidermal keratinocyte ``organotypic'' cultures grown on collagen matrices.Histochem Cell Biol 113:265±277, 2000

Vicanova J, Boelsma E, Mommaas AM, et al: Normalization of epidermal calciumdistribution pro®le in reconstructed human epidermis is related toimprovement of terminal differentiation and stratum corneum barrierformation. J Invest Dermatol 111:97±106, 1998

p53 Patches are Not Increased in Patients with MultipleNonmelanoma Skin Cancers

To the Editor:

Mutation of p53 tumor suppressor gene is considered to be afrequent early event in nonmelanoma skin cancer (NMSC)development. NMSC includes basal cell carcinoma (BCC) andsquamous cell carcinoma (SCC). Up to 40% of patients withNMSC develop a second primary tumor; however, recognition ofthis individual risk is currently impossible. UV irradiation is themajor carcinogen for NMSC. Chronically sun-exposed epidermisharbors numerous mutated p53 clones that are detected as p53patches by immunohistochemistry (Jonason et al, 1996; Ren et al,1996). But whether they are precancerous remains open to debate.

We hypothesized that if p53 patches are precancerous lesions,their prevalence would increase with the number of NMSC andcould serve as an individual risk marker for tumor development. Ina retrospective study of 250 cases (1996±98, Hospital St-Louis,Paris, France) we scoped the presence or absence of p53 patches in6±8 sections of normal peritumoral skin in patients treated forexcision of NMSC on chronically sun-exposed skin (i.e., face,neck, and hands).

Patients were sorted into a solitary (no previous history ofNMSC) or multiple group (two or more NMSC, simultaneous orsuccessive). For each group the number of patients was 125 and themedian age 71. For the solitary NMSC group, ages ranged from 26to 97, male gender was 66%, and there was BCC in 93 patients(74%) and SCC in 32 (26%). For the multiple NMSC group, agesranged from 32 to 100, male gender was 72%, and there was BCCin 106 patients (85%) and SCC in 19 (15%). Skin samples ®xed in10% formalin were deparaf®nized and stained after antigen retrievalin a microwave oven 15 mn, at 450 W, in tris buffer pH 7.3, usingmonoclonal antibody D-O7 (Dako, code M7001, Denmark), withthe avidin-biotin-coupled immunoperoxidase staining method anddiaminobenzidine. P53 patches are de®ned as a well-demarcatedcompact pattern of strong immunostaining of basal and suprabasalkeratinocytes nuclei in nondysplastic nontumoral epidermis(Fig 1). Mayer's hematoxylin was used for counterstaining. Allslides were blind scoped for the presence of p53 patches by threeindependent observers (FP, AJ, NBS). Fisher's exact test was usedto compare percentages between groups. Two sided tests were

computed, and p values of 0.05 or less were considered statisticallysigni®cant (SAS software, SAS Institute, Cary, NC).

There was no signi®cant difference between the prevalence ofp53 patches of solitary versus multiple NMSC groups (66% vs 70%,respectively, p = 0.59), neither was there a signi®cant differencebetween the prevalence of p53 patches in the different age groups(Table I).

Manuscript received May 23, 2001; revised July 9, 2001; accepted forpublication July 17, 2001.

Reprint requests to: Dr. N. Basset-Seguin, Institut de Recherche sur laPeau, Pavillon Bazin, HoÃpital Saint-Louis, 1 avenue Claude Vellefaux75010 Paris France. Email: nbs@chu-stlouis.fr

Figure 1. A typical p53 patch. P53 immunostaining 3 Scale bar: 40 mm.

Table I. P53 patch prevalences in normal skin aroundnonmelanoma skin cancers, a series of 250 patients

Patients'

p53 patches/total

characteristics N/N %

Age (y)Overall 169/250 68%<50y 9/18 50%50±59 y 24/34 71%60±69 y 41/57 72%70±79 y 43/63 68%80±89 y 43/65 66%> 90 y 9/13 69%

Tumor typeBCC 132/199 66%SCC 37/51 73%Multiple NMSC 87/125 70%Solitary NMSC 82/125 66%

1324 LETTERS TO THE EDITOR THE JOURNAL OF INVESTIGATIVE DERMATOLOGY