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• W2024546590 abstract "To the Editor: Cadaver skin has been used in drug transport studies but its limited availability and large variation between specimens have increased interest in organotypic culture models with permeability characteristics resembling that in native skin. Other benefits of live organotypic cultures include their metabolic activity, which affects the permeability of some drugs (Kao and Hall, 1987Kao J. Hall J. Skin absorption and cutaneous first pass metabolism of topical steroids: in vitro studies with mouse skin in organ culture.J Pharmacol Exp Ther. 1987; 241: 482-487PubMed Google Scholar), and their potential for research on irritation, toxicity, and keratinocyte differentiation (Slivka et al., 1993Slivka S.R. Landeen L.K. Zeigler F. Zimber M.P. Bartel R.L. Characterization barrier function, and drug metabolism of an in vitro skin model.J Invest Dermatol. 1993; 100: 40-46Abstract Full Text PDF PubMed Google Scholar;Vicanova et al., 1998Vicanova J. Boelsma E. Mommaas A.M. et al.Normalization of epidermal calcium distribution profile in reconstructed human epidermis is related to improvement of terminal differentiation and stratum corneum barrier formation.J Invest Dermatol. 1998; 111: 97-106Crossref PubMed Scopus (88) Google Scholar;Boelsma et al., 2000Boelsma E. Gibbs S. Faller C. Ponec M. Characterization and comparison of reconstructed skin models: morphological and immunohistochemical evaluation.Acta Derm Venereol. 2000; 80: 82-88PubMed Google Scholar); however, culture models with barrier properties comparable of human skin and yet suitable for simple, large-scale research such as permeability tests of topically applied drugs and their vehicles, have not been established (Ernesti et al., 1992Ernesti A.M. Swiderek M. Gay R. Absorption and metabolism of topically applied testosterone in an organotypic skin culture.Skin Pharmacol. 1992; 5: 146-153Crossref PubMed Scopus (40) Google Scholar;Nolte et al., 1993Nolte C.J. Oleson M.A. Bilbo P.R. Parenteau N.L. Development of a stratum corneum and barrier function in an organotypic skin culture.Arch Dermatol Res. 1993; 285: 466-474Crossref PubMed Scopus (68) Google Scholar;Regnier et al., 1993Regnier M. Caron D. Reichert U. Schaefer H. Barrier function of human skin and human reconstructed epidermis.J Pharm Sci. 1993; 82: 404-407Crossref PubMed Scopus (36) Google Scholar). A keratinocyte cell line derived from newborn rat skin (rat epidermal keratinocyte; REK) retains its ability to form stratified cultures with basal, spinous, and granular cells, and multiple orthokeratinized layers of corneocytes when grown at the air–liquid interface on collagen gel inserts (MacCallum and Lillie, 1990MacCallum D.K. Lillie J.H. Evidence for autoregulation of cell division and cell transit in keratinocytes grown on collagen at an air–liquid interface.Skin Pharmacol. 1990; 3: 86-96Crossref PubMed Scopus (19) Google Scholar;Tammi et al., 2000Tammi R.H. Tammi M.I. Hascall V.C. Hogg M. Pasonen S. MacCallum D.K. A preformed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte “organotypic” cultures grown on collagen matrices.Histochem Cell Biol. 2000; 113: 265-277Crossref PubMed Scopus (46) Google Scholar). We explored whether the naturally high differentiation potential of REK cultures could be developed into a model of epidermal permeability barrier research. To prepare the supports for differentiating cultures, type I collagen (from rat tail, Becton Dickinson Labware, Bedford, MA) was mixed with Earle's balanced salt solution (10 × EBSS, Life Technologies, Paisley, Scotland), 7.5% sodium bicarbonate (Life Technologies), and 1 M sodium hydroxide solution, at a volume ratio of 8:1:0.3:0.2, respectively, on an ice bath. The collagen solution (800 µL) was pipetted onto 24 mm diameter tissue culture inserts (3. 0 µm pore size; Transwell®, Costar, Cambridge, MA) and incubated overnight at 37°C in a humidified atmosphere. The inserts covered by a gel composed of reconstituted native collagen fibrils were washed with the culture medium before use. Recently confluent stock cultures of REK with no morphologic evidence of stratification were trypsinized and 300 000 cells in 2 ml Dulbecco's modified Eagles medium (4500 mg per L glucose) (Life Technologies) were applied to the collagen mats on the culture inserts. After 3 d with culture medium present both in the well beneath the insert as well as on the surface of the cells, the upper medium was removed and the amount of the lower medium (∼1.5 ml) was adjusted to the level of the collagen gels. New medium with a supplement of 40 µg per ml L-ascorbic acid (Sigma, St. Louis, MO) was changed every 2 d for the first week and daily thereafter. For light microscopy, cultures were fixed with Histochoice® (Amresco, Solon, OH) overnight, dehydrated in graded ethanol, and embedded in paraffin. Vertical 3 µm thick sections were used for hematoxylin and eosin and immunohistologic stainings. For electron microscopy, cultures were first fixed in 0.1 M cacodylate-buffered (pH 7.4) 2.5% glutaraldehyde overnight at 4°C and then postfixed in 0.4% ruthenium tetroxide with 0.2% aqueous potassium ferrocyanide for 2 h at 4°C. The specimens were then dehydrated in graded ethanol and propylene oxide before embedding in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. In permeability studies, the cadaver skin samples or organotypic cultures were clamped between 3 ml phosphate-buffered saline-filled, stirred, thermostatted (37°C) chambers of a diffusion cell apparatus (Side-Bi-Side, Crown Glass Company, Somerville, NJ) with an effective diffusional area of 0.64 cm2. Tritiated corticosterone or mannitol (NEN Life Science Products, Boston, MA) 20 000–40 000 dpm per 10 µL was added to the donor chamber. Aliquots were withdrawn at predetermined time intervals from both chambers, counted for radioactivity, and replaced with phosphate-buffered saline to maintain a constant volume. Experiments were continued long enough to ensure that the steady-state phase was attained. The permeability coefficients (P, cm per s) for the probe permeants were calculated at steady state under sink conditions by dividing the steady-state flux (dpm per s cm2) 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 Delfin-SWL system (Delfin Technologies Oy, Kuopio, Finland). In this system, sensors inside the evaporimeter probehead measure the increasing humidity of the air inside the probehead that is kept in contact with the culture surface or skin for 1 min. The cultures were allowed to equilibrate with ambient air for 1 h at room temperature before the measurement, which was carried out at 21–23°C. Control values of TEWL of normal human skin were obtained from the forearm of nine healthy Caucasian people (aged from 18 to 42). By 3 wk on collagen at the air–liquid interface, the cultures showed complete morphologic differentiation of the epidermis, including cuboidal basal cells, 1–2 spinous cell layers, granular cells with numerous keratohyalin granules, and a well-developed stratum corneum displaying the typical “basketweave” separation of corneocytes when embedded in paraffin Figure 1a. Immunohistologic studies revealed the regular presence and localization of differentiation markers keratin 10, involucrin, and filaggrin (data not shown). The light microscopic structure stayed similar for at least 6 wk, except that the height of the cornified layer increased continuously. The epidermal barrier function, critically dependent on proper arrangement of the intercellular SC lipids (Elias and Menon, 1991Elias P.M. Menon G.K. Structural and lipid biochemical correlates of the epidermal permeability barrier.Adv Lipid Res. 1991; 24: 1-26Crossref PubMed Google Scholar;Bouwstra et al., 1996Bouwstra J.A. Gooris G.S. Cheng K. Weerheim A. Bras W. Ponec M. Phase behavior of isolated skin lipids.J Lipid Res. 1996; 37: 999-1011PubMed Google Scholar), was specifically studied by electron microscopy of ruthenium tetroxide postfixed cultures. At 3 wk, the upper, vital keratinocytes showed a number of lamellar bodies with a regular internal stacking organization Figure 1b. At the interface between the live epidermis and the stratum corneum the lamellar bodies fused with the plasma membrane and extruded their contents into the intercellular space Figure 1c. The secreted lipid stacks were subsequently transformed into lamellar lipid layers with a typical repeating pattern of alternating electron dense and electron lucent bands Figure 1d (Landmann, 1986Landmann L. Epidermal permeability barrier: transformation of lamellar granule-disks into intercellular sheets by a membrane-fusion process, a freeze-fracture study.J Invest Dermatol. 1986; 87: 202-209Abstract Full Text PDF PubMed Scopus (168) Google Scholar;Madison et al., 1987Madison K.C. Swartzendruber D.C. Wertz P.W. Downing D.T. Presence of intact intercellular lipid lamellae in the stratum corneum.J Invest Dermatol. 1987; 88: 714-718Abstract Full Text PDF PubMed Google Scholar;Swartzendruber et al., 1989Swartzendruber D.C. Wertz P.W. Kitko D.J. Madison K.C. Downing D.T. Molecular models of the intercellular lipid lamellae in mammalian stratum corneum.J Invest Dermatol. 1989; 92: 251-257Abstract Full Text PDF PubMed Google Scholar). These structural features, similar to those in normal human skin, prompted us to study the barrier function of organotypic REK cultures by measuring the transepidermal water loss (TEWL) and the permeability for corticosterone, a frequently used indicator substance. TEWL was relatively high in 1-wk-old cultures Table I but was reduced to 24% of that at 2 wk, and did not significantly change thereafter Table I. The permeability of corticosterone was high at 1 wk, but was considerably reduced by 2 and 3 wk Table I. At 3 wk the corticosterone permeability reached a value close to normal human cadaver skin Table I. Prolongation of the culturing time up to 6 wk did not cause further change in the corticosterone permeation (data not shown). For comparison, we also measured the permeability of mannitol, a tracer more polar than corticosterone. Like TEWL, it showed permeation values close to, but somewhat higher than those of normal cadaver skin Table I. Furthermore, we have preliminary data to suggest that REK cultures can predict the human skin permeability of a number of other drugs and chemicals. The permeability in REK cultures corresponds to those of a human skin equivalent model (Asbill et al., 2000Asbill C. Kim N. El-Kattan A. Creek K. Wertz P. Michniak B. Evaluation of a human bio-engineered skin equivalent for drug permeation studies.Pharm Res. 2000; 17: 1092-1097Crossref PubMed Scopus (59) Google Scholar). The convenience of a continuous cell line, independent of fibroblast support, makes REK organotypic cultures an easily maintained and reproducible model for studies on transepidermal barrier function, with permeation values close to normal human skin.Table IThe TEWL and permeability of corticosterone and mannitol in organotypic REK cultures (1–3 wk) and human skinaData are presented as means ±SD in the number of experiments (n) shown in parenthesis.1 wk2 wk3 wkHuman skinTEWL (g m2 per h)91.20 ± 8.70 (n = 10)22.0 ± 6.20 (n = 11)19.44 ± 7.90 (n = 11)10.07 ± 7.90bNormal human skin in vivo. (n = 9)Corticosterone (P ×10-6 cm per s)29.10 ± 19.43 (n = 8)1.60 ± 0.60 (n = 8)0.22 ± 0.08 (n = 15)0.18 ± 0.11cCadaver skin, n.d. not determined. (n = 11)Mannitol (P ×10-6 cm per s)n.d.n.d.0.59 ± 0.14 (n = 12)0.17 ± 0.16cCadaver skin, n.d. not determined. (n = 10)a Data are presented as means ±SD in the number of experiments (n) shown in parenthesis.b Normal human skin in vivo.c Cadaver skin, n.d. not determined. Open table in a new tab This study was financially 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." @default.
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• W2024546590 title "Formation of Permeability Barrier in Epidermal Organotypic Culture for Studies on Drug Transport" @default.
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