Human epidermis reconstructed on synthetic membrane: Influence of experimental conditions on terminal differentiation

Abstract

Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham's F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham's F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no proper stratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham's F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)

Barrier Function of Reconstructed Epidermis at the Air-Liquid Interface: Influence of Dermal Cells and Extracellular Components

“…The results of this study show that epider mis reconstructed in DM resembles native epidermis with respect to its morphology and architecture and, like other reconstructed skin models [11][12][13][14][15][16][17][18][19][20][21], provides a significant barrier for water and estradiol. However, like other models, it was respectively 10 and 5 times more permeable for water and estradiol than human native epidermis incubated overnight at 37°C in a humidified atmosphere [19,24], The increased permeability of reconstructed epidermis is to some extent due to an in creased hydration provoked by the medium through the membrane, as well as the humidi ty of atmosphere [23], Furthermore, air- Table 6.…”

“…Recent studies suggest that the structural organization of stratum corneum lipids may be as important for barrier function as is the lipid composition itself [1][2][3][4][5][6][7][8][9][10], Cultured skin systems must thus ensure not only synthesis of both comeocytes and intercorneocyte lipid constituents, but also intercellular lipid organization. Recent progress in epithelial culture tech niques has led to the development of culture systems in which reconstructed epidermis ex hibits in vivo-like morphological and bio chemical differentiation [11][12][13][14][15][16][17][18][19][20][21], but barrier function remains subnormal [8,9,18,19,[22][23][24][25][26][27], Our laboratory has recently developed a culture model in which human kératinocytes proliferate and terminally differentiate on a synthetic porous membrane. Epidermal cells are exposed at the air-liquid interface (a pre requisite for terminal differentiation) and the reconstructed epidermis possesses the major structural characteristics of native epidermis [21].…”

Interleukin-4 Induces Activation of Mitogen-activated Protein Kinase and Phosphorylation of Shc in Human Keratinocytes

“…Primary human keratinocytes (PHK) were isolated from human foreskin of a 1-year-old donor as described by Noël-Hudson et al (14) and were grown in keratinocyte serum-free medium supplemented with EGF (1 ng/ml) and bovine pituitary extracts. Cells were seeded at 10 5 cells/ 100-mm Petri dish and grown for 8 days.…”

Custom-Designed Wells and Flow Chamber for Exposing Air–Liquid Interface Cultures to Wall Shear Stress

“…Cells cultured under air-liquid interface (ALI) conditions were utilized in a variety of in vitro studies of human keratinocytes, 11,12,19 mouse gastric epithelial cells, 13,14 human placental chorionic villi, 5 human buccal epithelial cells, 8 bovine conjunctival epithelial cells, 15 and human and rat respiratory epithelial cells. 3,9,10,18 In in vitro studies with airway epithelial cells the use of ALI conditions enables cellular polarization so that the cultured cells undergo mucociliary differentiation, and demonstrate mucus secretion.…”