Swine pericardium as dermal substrate for human keratinocyte culture

Background Synthetic skin analogues or living allogeneic or autologous cells are used as dressings for the care of skin wounds, as well as temporary or permanent substitutes for damaged epithelia. Objectives To evaluate if keratinocyte growth on a swine pericardium substrate mimics the natural epithelial layers compared with cultures on allogeneic dermis, which is accepted as having appropriate physical and chemical properties for growth and differentiation. Methods Keratinocytes were cultured on a swine pericardium substrate and allogeneic dermis, either submerged or at the air–liquid interface. At 7, 14 and 21 days postseeding the cultures were evaluated by light microscopy after both haematoxylin and eosin staining and immunohistochemistry. Results Cell–substrate interactions led to growth, stratification and differentiation of cells, with the definition of epithelial layers. The submerged system showed a continuous growth rise on both composites, but this was more prominent with the swine pericardium substrate. An increase in the number of layers at the air–liquid interface with the dermis composites, in contrast to the submerged cultures, occurred only from days 7 to 14. The pattern of keratinocyte growth on swine pericardium substrate was much better in the submerged than in the air–liquid interface cultures. Conclusions The results indicate that swine pericardium is a better substrate than allogeneic dermis for keratinocyte cultures in submerged but not in air–liquid interface cultures. Swine pericardium as a substrate opens one more possibility for skin restoration after trauma or burns.     

A putative in vitro organotypic model of molting with human skin explants

We report finding a simple method to partially reproduce the characteristic process of molting that takes place in invertebrates using human skin explants in vitro. In this method, human skin explants discarded from regular plastic surgery procedures were kept, submersed, in regular growth medium for 10 days at 4°C. After that period, the skin explants were cultured at the air–liquid interface for another 10 days. Histological analysis of the skin revealed the formation of one full epidermal structure and an additional intermediate epidermal structure containing a putative stratum corneum, superimposed one of top of the other, in which we consider an equivalent model of “molting” or “ecdysis”. Basic analysis of cell proliferation and differentiation of the explants at different stages of the process are briefly presented. We believe this model can be used in the study of certain human skin diseases as well as in comparative animal physiology.     

Reconstruction of a human skin equivalent using a spontaneously transformed keratinocyte cell line (HaCaT)

Reconstruction of a skin equivalent using an immortalized human keratinocyte line, HaCaT, was investigated in an attempt to generate an in vitro system representative for human skin. Three different substrates were used to establish air-exposed cultures of HaCaT cells: de-epidermized dermis, collagen gels, and filter inserts. Effects of variations in culture conditions on tissue morphology, on the expression of proliferation-specific and differentiation-specific protein markers, and on lipid profiles were investigated. When grown at the air-liquid interface HaCaT cells initially developed a multilayered epithelium, but during the course of culture marked alterations in tissue architecture were observed. Ultrastructurally, a disordered tissue organization was evident as judged from the presence of rounded cells with abnormally shaped nuclei. Keratins K1 and K10 were irregularly expressed in all cell layers, including stratum basale. Staining of K6/K16 was evident in all cell layers. Locally, basal and suprabasal cells were positive for K4 and additionally expressed K13 and K19. The cornified envelope precursors were expressed only in older cultures (>2 wk after air exposure), except for transglutaminase and small proline rich protein 1, which were irregularly expressed in both early and older cultures. In addition, HaCaT cells showed an impaired capacity to synthesize lipids that are necessary for a proper barrier formation as indicated by the absence of free fatty acids and a very low content and incomplete profile of ceramides. Our data demonstrate that the ultimate steps of terminal differentiation in HaCaT cells do not occur irrespective of the type of substrate or the culture conditions.     

In vitro acantholysis by captopril and thiopronine

The possible acantholytic property of captopril and thiopronine has been investigated using in vitro tissue cultures. Normal human breast skin explants have been cultured in Hanks' balanced salt solution containing 40% normal inactivated human serum with the addition of L-cysteine, or reduced glutathione (GSH), or captopril, or thiopronine, at four different concentrations (1, 5, 10, 15 mM). Patterns of diffuse, mainly suprabasal acantholysis, with formation of bullae, were observed in the skin explants cultured with captopril or thiopronine at a 15-mM concentration after 5 days of culture; intraepidermal splits were present also at a 10-mM concentration. Focal acantholysis was seen in specimens cultured with L-cysteine or GSH at a 15-mM concentration. No lesions occurred in the samples treated with lower concentrations of the above substances, nor in controls. The results show a biochemical acantholytic potential of both captopril and thiopronine, resembling that of penicillamine in similar experimental conditions, and consonant with clinical observations of pemphigus induced by drugs containing thiol groups in their molecule (SH drugs).     

Deranged epidermal differentiation in kl/kl mouse and the effects of βKlotho siRNA on the differentiation of HaCaT cells

Mice deficient in the klotho gene (kl/kl mice) display the phenotypes of human ageing. We found that the expression of epidermal differentiation‐associated factors (keratin 1, keratin 10, filaggrin and loricrin) was lower in the skin of kl/kl mice than that of wild‐type mice. In vitro experiments showed that the expression of βKlotho, a family of klotho gene‐encoded protein, was induced concomitantly with the differentiation of an immortalized human epidermal keratinocyte cell line (HaCaT cells) when they were cultured in an air–liquid interface. βKlotho knockdown by small interfering ribonucleic acid suppressed the expression of the above differentiation‐associated factors in HaCaT cells. βKlotho small interfering ribonucleic acid increased the expression of keratin 14, which is expressed in mitotically active basal layer cells, and activated p44/p42 mitogen‐activated protein kinase in the HaCaT cells grown in the air–liquid interface. These findings suggest that the epidermal differentiation is deranged in kl/kl mice, and βKlotho is required for the differentiation of human epidermal keratinocytes.     

CD 2394, a novel synthetic retinoid, initiates an embryonic type of differentiation in hyperproliferative skin

In human skin, there are 2 types of epidermal differentiation: normal differentiation, characterized by keratin 10 expression, and alternative differentiation. Alternative differentiation may be regeneration-associated differentiation (keratin 6 and 16) or re-induction of embryonic differentiation (expression of keratin 13, 15 and 19). The purpose of this study was to investigate the effect of the novel synthetic retinoid CD 2394 on hyperproliferative human skin, with respect to embryonic differentiation in particular. The effects of CD 2394 were compared with untreated and vehicle-treated skin 48 h after tape-stripping. In a multiparameter flow cytometric assay, parameters of proliferation, normal differentiation, embryonic differentiation and inflammation were assessed. With respect to proliferation, treatment with CD 2394 resulted in a decreased number of cells in the G2M-phase. Normal differentiation was decreased in CD 2394 treated skin. Furthermore, most of the CD 2394 treated samples showed expression of keratin 13, which was not seen in the otherwise treated skin. A correlation between keratin 10 and keratin 13 expression could not be demonstrated. This study showed that CD 2394 is capable of inducing an embryonic pathway of differentiation, which is distinct from normal differentiation or regeneration-associated differentiation.     

Expression of proteoglycans and glycosaminoglycans in angiofibroma and fibrous plaque skin lesions from patients with tuberous sclerosis

Tuberous sclerosis complex (TSC) is a disorder of cell lineage, migration, proliferation and differentiation, characterized by the development of widespread benign hamartomas, which are particularly evident in hamartomatous lesions of the skin. The aim of this study was to investigate differences in gene expression of certain proteoglycans (PGs) and to characterize glycosaminoglycans (GAGs) in tissue specimens of normal skin, fibrous plaques and angiofibromas from patients with TSC. The expression of PG mRNA was determined by semiquantitative RT-PCR analysis. Total GAGs were isolated from tissue specimens after lipid extraction and extensive digestion with Pronase and DNase and characterized by treatment with GAG-degrading enzymes followed by electrophoresis on polyacrylamide gradient gels and cellulose acetate membranes. Normal skin specimens express versican, decorin and aggrecan and contain hyaluronic acid and dermatan sulphate. In angiofibroma specimens aggrecan is not expressed while versican splice variant with two EGF-like domains and decorin are downregulated. Furthermore, angiofibromas differ from normal skin in that they additionally contain keratan, heparan and chondroitin sulphates and do not contain dermatan sulphate. In fibrous plaque specimens gene expression of PGs was similar to that in normal skin, but with respect to GAGs, they contained a single acidic glycan population that did not share common structural features with known GAGs. The variations of the above ECM molecules between normal and TSC skin may be attributed to TSC-related mutations and, overall, support the TSC-associated pathological manifestations of cell migration, proliferation and differentiation.     

Detection of human herpesvirus 8 sequences in cutaneous cherry angiomas

Cherry angiomas (CAs) are common vascular benign skin tumors, characterized by abnormal angiogenesis, whose etiology is still unclear and poorly studied. We investigated the presence of HHV8 in CAs due to virus ability of inducing neoangiogenesis in endothelial cells. A total of 29 patients were enrolled in a randomized, controlled, blinded analysis of skin specimens including various vascular lesions. All clinical samples were anonymized and analyzed by three different biomolecular assays to minimize the risk of false positive/negative results. Results showed that 53 % of eruptive CAs harbor HHV8 sequences, with the highest viral loads in samples derived from immunosuppressed patients. By contrast, no paucilesional CAs were positive for HHV8. Considering HHV8 prevalence in the Mediterranean population (10–15 %), results obtained in eruptive CAs are significant and suggest for the first time a possible involvement of HHV8 in eruptive cherry angiomas development, particularly in the context of immunosuppression, similar to that recognized for major HHV8-induced pathologies.     

A fully differentiating epidermal model with extended viability: development and partial characterization

A highly differentiated porcine skin organ culture model has been developed for future investigations of membrane-coating granules (MCG) and their role in epidermal differentiation. In contrast to many previous systems, cultures do not undergo necrosis of the upper epidermis or display dermo-epidermal separation, but survive for at least 3 weeks, at which time mitotic cells are still evident. Although rete projections are gradually smoothed out and the viable epidermis thins at a rate of approximately 0.35 cells per day, the stratum corneum gains approximately 1.5 corneocytes per day. Furthermore, at 3 weeks all the major differentiation markers are expressed, including keratohyalin granules, MCG, and an orthokeratotic stratum corneum. The system is inexpensive, simple to establish, and does not require elevated oxygen levels. The main requirements are 1) the use of Dulbecco's minimal essential medium supplemented with 2) hydrocortisone (100 micrograms/ml), 3) growth at an air/liquid interface, and 4) attached connective tissue. The further addition of vitamin C (300 micrograms/ml) and/or bovine serum albumin (2 mg/ml) offered no obvious advantage. Degeneration of organ cultures in standard cell culture media was discovered to be caused by fetal bovine serum (FBS). FBS-induced degeneration was not prevented by adding any of the supplements tested, or the inclusion of 3T3 fibroblasts, even when culturing at an air/liquid interface. Complete submersion rapidly killed specimens, presumably through oxygen starvation. The ability to maintain a fully keratinizing system for several weeks, in a totally chemically defined medium, will prove valuable for research not only into the role(s) of MCG in epidermal biology but also studies of desquamation and epidermal differentiation.     

Immunohistochemical localization of proliferating cell nuclear antigen/cyclin in human skin

Summary Expression of proliferating cell nuclear antigen/cyclin (PCNA/cyclin) in skin tissue specimens and cultured keratinocytes was studied using a monospecific antibody, obtained from a patient with systemic lupus erythematosus, and a monoclonal antibody. Indirect immunofluorescent staining revealed that cultured keratinocytes obtained from human foreskins expressed PCNA/cyclin as variable nuclear patterns in 15–30% of the cells. In normal human skin tissue specimens, PCNA/cyclin was demonstrated in only a few basal cells. Interestingly, PCNA/cyclin was expressed strongly in almost all the cells of the lowest layer of the epidermis adjacent to squamous cell carcinomas, whereas the tumor aggregates themselves had no positive staining. In contrast, no such characteristic staining was demonstrated in specimens of basal cell carcinoma. The staining pattern of PCNA/cyclin was different from that of Ki-67 in the skin tissue specimens. Our results suggest that PCNA/cyclin could be a useful marker of cell proliferation.     

Transglutaminase inhibitors induce hyperproliferation and parakeratosis in tissue-engineered skin

Background The transglutaminase (TG) family consists of eight distinct isoforms. TG types 1, 3 and 5 play a major role in normal skin development, with TG2 also being elevated during dermal wounding. TG1, 3 and 5 are responsible for the cross‐linking of keratin precursors and formation of the cornified envelope during keratinocyte differentiation. TG2 may play a role in keratinocyte basement membrane cross‐linking. Abnormal TG expression has been demonstrated in Darier disease, Netherton syndrome, psoriasis and lamellar ichthyosis. During a recent investigation of skin contraction in tissue‐engineered skin, transglutaminase inhibitors were found to produce hyperproliferation and parakeratosis. Objectives Accordingly, this study was designed to study the effect of pan‐transglutaminase inhibition on morphology of tissue‐engineered skin and expression of keratinocyte differentiation and proliferation‐associated antigens. Methods We used a tissue‐engineered model of human skin, based on de‐epidermized acellular human dermis, seeded with normal keratinocytes and dermal fibroblasts and cultured at an air–liquid interface. The pan‐transglutaminase inhibitors putrescine, NTU283 (1‐dimethyl,2‐[(oxopropyl)thio]imidazolium) and NTU285 (N‐benzyloxycarbonyl‐l ‐glutaminyl‐6‐dimethylsulfonium‐5‐oxo‐l ‐norleucine) were added to the culture medium. After 28 days, histology and immunohistochemistry for collagen IV, involucrin and cytokeratins 6, 10 and 16 were performed. Results Keratinocyte hyperproliferation and parakeratosis were seen in response to transglutaminase inhibition. Inhibition of transglutaminase also resulted in loss of basement membrane collagen IV. Involucrin and cytokeratins 6 and 16 were confined to the basal layers in control composites but expressed throughout the epidermis in response to transglutaminase inhibition. A distinct band of expression of cytokeratin 10 was seen in the upper stratum granulosum of control composites but only patchy expression was seen after transglutaminase expression. Conclusions Pan‐transglutaminase inhibition inhibits terminal differentiation of keratinocytes, leading to a hyperproliferative epidermis with parakeratosis and enhanced expression of involucrin and cytokeratins 6 and 16. Expression of the differentiation‐associated cytokeratin, cytokeratin 10, is reduced. Basement membrane integrity is also lost as a result of transglutaminase inhibition.     

The deoxyribonucleic acid (DNA) skin test in systemic lupus erythematosus.

A clinical evaluation of the intradermal DNA-test was carried out on a series of patients with untreated or with treated definite systemic lupus erythematosus (SLE), or with suspected SLE with or without circulating antinuclear factors. A saline solution of a commercially available DNA-preparation was used. The course of the DNA reaction was followed for 24–48 h after the injection. All nine cases of untreated definite SLE had a positive DNA test 6 h after the injection, and eight cases a positive result at 24 h. All seven patients with definite SLE who were on low-dosage systemic steroid treatment had a clinically positive DNA test at 6 h. In all except one of these cases the test was still positive at 24 h. All five patients with definite SLE on antimalarial treatment had a positive test at 6 h which had become negative at 24 h after the injection. Eleven of the twelve patients with suspected SLE and circulating antinuclear factors had a positive DNA test at 6 h, which in nine cases persisted for 24 h. On the other hand, of the eleven ANF negative patients with various connective tissue diseases five had a positive test at 6 h. In only one of these cases it persisted for 24 h. Two of the eighteen control patients with various dermatoses exhibited a positive test at 6 h, and in one of these the positive reaction persisted for 24 h. It is concluded that the intradermal skin test using native DNA is a useful diagnostic tool for the detection of SLE when the reaction is followed up for 24 h. Antimalarial treatment seems to decrease skin reactivity to native DNA.     

儿童头皮及光滑皮肤组织对体外培养犬小孢子菌PQ-LRP表达水平的影响

目的探讨儿童头皮与光滑皮肤组织对体外培养犬小孢子菌膜绑定蛋白编码基因(PQ-LRP)表达水平的影响。方法以18SrRNA基因为内参,应用RT-PCR方法检测SDA培养基中自患儿皮损处分离的头癣株、体癣株第1代及第5代PQ-LRP的mRNA水平;检测体外经儿童头皮及光滑皮肤组织诱导培养后头癣株及体癣株PQ-LRP的mRNA水平。结果 IODPQ-LRP/IOD18S rRNA(SDA第1代头癣株)为1.0352±0.11279,IODPQ-LRP/IOD18S rRNA(SDA第5代头癣株)为1.1157±0.11530,二者差异无统计学意义(P>0.05);IODPQ-LRP/IOD18S rRNA(头皮组织诱导后头癣株)为2.1375±0.11093,与未经诱导者相比差异有统计学意义(P<0.01);IODPQ-LRP/IOD18S rRNA(SDA第1代体癣株)为0.5921±0.12025,IODPQ-LRP/IOD18S rRNA(SDA第5代体癣株)为0.6712±0.12177,二者差异无统计学意义(P>0.05);IODPQ-LRP/IOD18S rRNA(光滑皮肤诱导后体癣株)为1.4734±0.13125,与未经诱导者相比差异有统计学意义(P<0.01)。结论 PQ-LRP在犬小孢子菌中有活性表达,且在头癣株中表达活性明显高于体癣株。SDA培养基中犬小孢子菌PQ-LRP呈稳定表达状态,而儿童头皮组织与光滑皮肤组织对PQ-LRP表达水平存在诱导作用,以头皮组织尤为显著。     

Reorganization of hair follicles in human skin organ culture induced by cultured human follicle-derived cells

Studies of human hair follicle (HF) induction by follicle-derived cells have been limited due to a lack of suitable test systems. In this study, we established a skin organ culture system which supports HF formation by follicle-derived cells. Long-term skin organ cultures were set up from human retroauricular skin specimens and maintained in culture for up to 8 weeks. In vitro expanded human HF-derived cells from the dermal papilla (DP) and the outer root sheath (ORS) were injected together into the skin specimens and evaluated for their ability to induce reorganization of HFs. Macroscopic analysis of the cultured skin specimens demonstrated the growth of velus-like hair after 4 weeks in culture. Histologic evaluation of the cultured skin specimens after 8 weeks of culture revealed multiple miniaturized HFs with sebaceous glands. In addition, cell clusters of various differentiation stages could be demonstrated in serial sections of the cultured skin specimens. Labeling of HF-derived cells with the fluorescence dye CFDA-1 prior to injection suggested a de novo reorganization of HFs out of the injected cells. In conclusion, the study demonstrated HF formation by HF-derived cells in an in vitro skin organ culture model.     

Suppression of UV‐induced damage by a liposomal sunscreen: a prospective,open‐label study in patients with cutaneous lupus erythematosus and healthy controls

This study aimed to determine whether a broad‐spectrum liposomal sunscreen with a very high sun protection factor (Daylong actinica) can prevent damage induced by ultraviolet (UV) irradiation in patients with cutaneous lupus erythematosus (CLE) and healthy controls (HCs) under standardised conditions. In 20 patients with CLE and 10 HCs, defined areas of sunscreen‐untreated and sunscreen‐treated skin on the upper back were irradiated with combined UVA/UVB light. Disease‐specific skin lesions were induced by UVA/UVB light in the untreated areas of nine patients with CLE; no specific eruptions or any sun damage was observed in the sunscreen‐treated areas in any of the CLE patients, nor in the HCs. Histological analysis of skin biopsy specimens confirmed the clinical results. In conclusion, the use of a high‐protection, broad‐spectrum sunscreen can prevent UV‐induced damage in patients with CLE and HCs.     

A novel human skin chamber model to study wound infection ex vivo

Wound infections with multi-drug resistant bacteria increase morbidity and mortality and have considerable socioeconomic impact. They can lead to impaired wound healing, resulting in rising treatment costs. The aim of this study was to investigate an ex vivo human wound infection model. Human full-thickness skin from the operating room (OR) was placed into the Bo-Drum^® and cultivated for 7 days in an air–liquid interphase. On day 8, the skin was inoculated with either (1) Pseudomonas aeruginosa, (2) Staphylococcus aureus (10⁵ CFU, n = 3) or (3) carrier control. 1, 3 and 7 days after inoculation colony forming units in the tissue/media were determined and cytokine expression was quantified. A reliable and reproducible wound infection could be established for 7 days. At this timepoint, 1.8 × 10⁸ CFU/g tissue of P. aeruginosa and 2 × 10⁷ CFU/g tissue of S. aureus were detected. Immunohistochemical analysis demonstrated bacterial infection and epidermolysis in infected skin. RT-PCR analysis exhibited a significant induction of proinflammatory cytokines after infection. The BO-drum^® is a robust, easy-to-use, sterilizable and reusable ex vivo full-skin culture system. For investigation of wound infection, treatment and healing, the BO-drum^® presents a convenient model and may help to standardize wound research.     

Presence of melanophages in the normal Japanese skin

Human skin samples were obtained from the normal peripheral portion of specimens removed from persons with various cutaneous and systemic diseases. A portion of each specimen was embedded in paraffin and another part in water-soluble embedding medium, and some was frozen in liquid nitrogen for light microscopy and histochemistry. Some specimens were also investigated by electron microscopy. In 31 of 32 specimens, cells containing brown pigment were observed in the superficial dermis. Because both acid phosphatase and Masson-Fontana staining were positive and the 3,4-dihydroxyphenylalanine reaction negative, the cells were considered to be melanophages. Electron microscopic examination revealed that these cells contained melanosome-laden phagosomes. Some fibroblastlike cells were also observed with intracellular single or multiple melanosomes. This study documents the occurrence of melanophages in the normal skin of Japanese subjects.     

体外重建皮肤与正常人皮肤基本特征的对比研究

目的 用正常人的皮肤角质形成细胞和黑素细胞在体外重建皮肤,为将来用于临床、生物及药理学研究奠定基础.方法 从正常儿童的包皮分离、培养角质形成细胞、黑素细胞和成纤维细胞.用成纤维细胞和I型鼠尾胶原制备真皮类似物.将角质形成细胞和黑素细胞接种到真皮类似物表面进行培养,制备表皮类似物.将重建的皮肤类似物和正常人皮肤进行对比研究.结果 皮肤类似物的组织形态包括基底层、基底上层、角质层与正常人皮肤相似.与基底膜形成有关的标志,在皮肤类似物和正常人皮肤中的表达是相似的.角蛋白10、泛角蛋白在皮肤类似物和正常人皮肤中的表达也是相似的.结论 组织形态和所检测的各种标志的表达显示,该皮肤类似物与正常人皮肤相似.     

HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin-embedded tissue specimens

AIM: This study was undertaken to assess whether heat-shock protein (HSP)47 is a useful cell marker for skin fibroblasts in formalin-fixed, paraffin-embedded skin specimens. BACKGROUND: HSP47, a 47-kDa HSP, is a collagen-specific molecular chaperone localized in the endoplasmic reticulum. HSP47 plays an essential role in collagen biosynthesis in skin fibroblasts. METHODS: Immunohistochemistry was performed to detect HSP47 in skin fibroblast cultures and skin tissue sections. RESULTS: Immunostaining for HSP47 clearly detected skin fibroblasts in paraffin tissue sections as well as in fibroblast cultures and frozen tissue sections. HSP47 staining on paraffin sections from diseased skin specimens revealed that skin ulcer, keloid, nodular fascitis, spindle cell lipoma, and dermatofibroma had strong signals for HSP47 compared with the signals obtained from normal skin. Dermatofibrosarcoma protuberans had many HSP47 positive cells, but signals on individual cells were not as strong as those seen from the above benign proliferative disease samples. In neurofibroma, a small number of faintly positive cells were detected. Our double-immunostaining studies also demonstrated that HSP47 staining distinguished skin fibroblasts from CD68-positive histiocytes/macrophages, factor VIII-related antigen-positive endothelial cells, or factor XIIIa-positive dermal dendritic cells. CD34-positive interstitial cells coexpressed HSP47 in spindle cell lipoma. CONCLUSIONS: These findings indicate that HSP47 staining can detect skin fibroblasts in routine, paraffin-embedded specimens. A panel approach using HSP47 and other cell markers on paraffin sections may help the identification of the cell type involved with mesenchymal proliferative disorders.     

The cytokeratin expression of cultured human foetal keratinocytes

Skin samples taken from foetuses of 15-19 weeks gestational age and keratinocytes were cultured by the 3T3 feeder method or in serum-free MCDB 153 medium on 16 mm coverslips. Keratinocytes taken from paediatric circumcisions and patients undergoing plastic surgery were also cultured using the 3T3 feeder method. A panel of monoclonal antibodies against a number of cytokeratins and differentiation markers were used in the PAP technique to analyse the cells. Cryostat sections taken from the donor skin samples were stained simultaneously. Foetal skin expressed the cytokeratins 7, 13 and 19 that were not observed postnatally. This cytokeratin expression as maintained in both culture conditions and also observe in paediatric and adult keratinocytes. Cytokeratin 7 was expressed on a greater proportion of foetal cells than in vivo, whereas the expression of 13 and 19 decreased. All keratinocytes expressed vimentin, transferrin receptor and Ki67 (proliferating cell antigen), while a small proportion expressed involucrin throughout culture, indicating their high level of differentiation and proliferation. No differences were observed between low and high density cultures. Foetal keratinocytes cultured in MCDB 153 did not reach confluence and stronger staining of differentiation markers was observed in these cells. These results show that the in vivo differences in cytokeratin expression of foetal and adult keratinocytes disappear in the culture.