Human epidermis reconstructed on synthetic membrane: Influence of experimental conditions on terminal differentiation (original) (raw)
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Histology and histopathology, 1999
Culture of keratinocytes in conventional medium without a mesenchyme-derived feeder layer leads to poor growth and impaired differentiation; however, the exact pathway and degree of differentiation achieved in such conditions is unclear. We have cultured normal human keratinocytes in Rheinwald and Green's medium, on plastic without a feeder layer, in order to investigate the degree of differentiation that they achieve in these conditions. Intermediate filament proteins, tonofibrils and desmosomes were assumed as markers of differentiation and their expression was analyzed by immunohistochemistry and electron microscopy. Before reaching confluence, keratinocytes expressed keratin molecules, as well as vimentin, and formed tonofibrils and desmosomes. The expression of these markers was progressively reduced until confluence and was totally lost thereafter, while cultures could be propagated for at least six passages. On the contrary, reseeding on a feeder layer after the first pas...
In Vitro Cellular & Developmental Biology - Animal, 1992
We have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into a morphologica/ly differentiated epidermis.
Serum-Free Serial Culture of Adult Human Keratinocytes From Suction-Blister Roof Epidermis
Journal of Investigative Dermatology, 1987
Coatin g cell culture fl as ks with natural extracellul ar matrix (ECM) en hanced the culture of adult hum an keratinocytes from suction-blister roof epidermis in an en vironment without fetal ca lf serum (FCS), bo vin e pituitary ex tracts or cellul ar feeder layers. A hi g her in ciden ce of cell attachm ent on natural ECM was observed than 011 coll agen and human fibro nectins(HFN)-coated plas tic di shes , and natural ECM was necessa ry for g rowth and proliferation of attached cells under th e culture condition s used. Cells in T he suction-blister m eth od is convenient and suitable to ob tain the epidermis from in fa nts and adu lts beca use it ca uses no pain and it produces no scar on the exa mined subjects. The ro ofs of the suction blisters are reported to be composed of pure epiderma l cells lack in g an y derm al cells [1]. Although there have been many tec hnica l ap proaches to the culture of hum an keratinocytes, m ost culture m ethods need a cellular feeder la yer and /or se rum. The presence of serum or other cell types, ho wever, prohibits the interpretation of certain types or-experiments on cellular attachm ent or of protein synthes is. and m ay chan ge the morphology and perh aps the biology of the cultured cells [2]. Recently. m eth od s for serum-free culture of hum an keratinocytes have been reported [2-5]. in most of w hi ch coll agen and other matrix-coated gro wth substrata are used. or in w hi ch pituitary extracts (PE) replace fetal calf se rum (FCS). Accumulatin g evidence o n the bio lo gic fe atures of culfured keratinocytes in serum-free env iron m ents has provided new insig hts in bas ic in ves ti ga ti ve dermatology and so m e striking clinical ap pli ca tions. It has been a lo ng-range goal of many investigators to isolate and to grow human epidermal keratinocy tes from both norm al and diseased hum an skin in suffi cient quantities for va ri o us biological studies [6]. This has not been feasible to date beca use Manuscript
Developmental Biology, 1987
Embryonic and fetal human epidermis differentiates in organ culture in an age dependent, though accelerated, manner. The older the specimen the less time is required for epidermal differentiation. Morphological markers of epidermal differentiation, including the different epidermal strata, keratohyalin granules, lamellar granules, and cornified cell envelopes, are formed in a manner that is faithful to development in viva. The high molecular weight, "differentiation specific" (67 and 56.5 kDa) keratins are also expressed in these cultures, even in the absence of morphological evidence for keratinization. Unstratified, embryonic epidermis was found to stratify overnight in culture. The time course of cell surface changes, detected by lectin binding, and cytoskeletal changes, detected by expression of the high molecular weight keratins, was followed in these cultures. Peanut agglutinin (PNA) binding sites appeared overnight in culture coincidently with epidermal stratification while expression of the 67 and 56.5 kDa keratins was not detected until the third day of culture. The possible significance of these results is discussed.
Expression of Desmosomal Proteins in Rat Keratinocytes during In Vitro Differentiation
Journal of Veterinary Medical Science, 2002
The keratinocyte, the major component of the epidermis, expresses several proteins that characterize the keratinization during the differentiation. Proliferation and differentiation of cultured human keratinocytes are known to be regulated by the Ca 2+ concentration in the culture medium. However, informations about the rat keratinocyte are relatively limited and their physiology is still an open question. To elucidate the characteristics of the rat keratinocyte, we established rat keratinocyte culture system and examined effects of extracellular calcium concentration on the expression of differentiation-related proteins. Keratinocytes were isolated from the newborn rat skin with 0.25% trypsin, followed by separation with a Percoll density gradient. The separated cells were grown in MCDB153 medium containing several growth factors and Ca 2+-free fetal bovine serum, then stimulated with Ca 2+. Immunoblotting demonstrated strong expression of β1 integrin in unstimulated cells, suggesting that the primary culture of rat keratinocytes was successfully established. Expression of desmoglein and transglutaminase was increased by Ca 2+ stimulation, whereas β1 integrin expression was decreased in response to increasing concentrations of Ca 2+. These observations indicate that cultured rat keratinocytes maintain the ability to differentiate in vitro, which is similar to that of the basal keratinocytes in the epidermis.
8073 - ISSN 2307 Culture technique of rabbit primary epidermal keratinocytes
The epidermis is the protective covering outer layer of the mammalian skin. The epidermal cells are stratified squamous epithelia which undergo continuous differentiation of loss and replacement of cells. Ninety per cent of epidermal cells consist of keratinocytes that are found in the basal layer of the stratified epithelium called epidermis. Keratinocytes are responsible for forming tight junctions with the nerves of the skin as well as in the process of wound healing. This article highlights the method of isolation and culture of rabbit primary epidermal keratinocytes in vitro. Approximately 2cm x 2cm oval shaped line was drawn on the dorsum of the rabbit to mark the surgical area. Then, the skin was carefully excised using a surgical blade and the target skin specimens harvested from the rabbits were placed in transport medium comprising of Dulbecco's Modified Eagle Medium (DMEM) and 1% of antibiotic-antimycotic solution. The specimens were transferred into a petri dish containing 70% ethanol and washed for 5 min followed by a wash in 1 x Dulbecco's Phosphate Buffered Saline (DBPS). Then, the skin specimens were placed in DMEM and minced into small pieces using a scalpel. The minced pieces were placed in a centrifuge tube containing 0.6% Dispase and 1% antibiotic-antimycotic solution overnight at 4°C in a horizontal orientation. The epidermis layer (whitish, semi-transparent) was separated from the dermis (pink, opaque, gooey) with the aid of curved forceps by fixing the dermis with one pair of forceps while detaching the epidermis with the second pair. The cells were cultured at a density of 4 x 10 4 cells/cm 2 in culture flask at 37°C and 5% CO 2. The cell morphology of the keratinocytes was analyzed using inverted microscope.
Virchows Archiv. B, Cell pathology including molecular pathology, 1983
The present study was undertaken to characterize primary epithelial cultures obtained from human skin explants as experimental systems for studies of the differentiation process. When human skin explants were incubated at 34-35 degrees C, fibroblastic growth was strongly inhibited, whereas the epithelial growth proceeded unchanged. The lateral growth of the epithelial cells could be divided into two phases - a migratory and a proliferative one. Only cultures incubated at 35 degrees C or below completed the morphological differentiation process before sloughing, whereas no qualitative difference in protein synthesis was observed between cultures incubated at temperatures from 33-37 degrees C. Cultured epidermal cells were labelled with 3H-thymidine and analysed by flow cytometry and cell sorting. Cells sorted from the S- and G2-phase populations were further analysed by autoradiography and a considerable heterogeneity as to the nuclear labelling was disclosed. A large fraction of S-p...
Journal of Dermatological Science, 1999
We devised a simple method to maintain an immortalized newbown rat keratinocyte cell line at the air-liquid interface using a tissue culture insert fitted with a microporous membrane. The cells formed stratified layers of flattened and anucleated cells resembling stratum corneum of the epidermis. Deiminated proteins, which are localized in the cornified layer of epidermis as the reaction products of peptidylarginine deiminase were detected immunohistochemically in the differentiated cells. Western blot analyses revealed that major deiminated proteins were type I keratins K10 and K14. Deiminated products of type II keratin K5 were found as minor components. Our observations show that deimination of keratins might be correlated with terminal differentiation of the immortalized keratinocyte cell line.
The Journal of Cell Biology, 1978
A population of neonatal mouse keratinocytes (epidermal basal cells) was obtained by gentle, short-term trypsin separation of the epidermal and dermal skin compartments and discontinuous Ficoll gradient purification of the resulting epidermal cells. Over 4--6 wk of culture growth at 32--33 degrees C, the primary cultures formed a complete monolayer that exhibited entire culture stratification and upper cell layer shedding. Transmission and scanning electron microscopy demonstrated that the keratinocyte cultures progressed from one to two cell layers through a series of stratification and specialization phenomena to a six to eight cell layer culture containing structures characteristic of epidermal cells and resembling in vivo epidermal development. The temporal development of primary epidermal cell culture specialization was confirmed by use of two histological techniques which differentially stain the specializing upper cell layers of neonatal mouse skin. No detectable dermal fibroblast co-cultivation was demonstrated by use of the leucine aminopeptidase histochemical technique and routine electron microscope surveillance of the cultures. Incorporation of [3H]thymidine ([3H]Tdr) was greater than 85% into DNA and was inhibited by both 20 micron cytosine arabinoside (Ara-C) and low temperature. Autoradiography and 90% inhibition of [3H]Tdr incorporation by 2 mM hydroxyurea indicated that keratinocyte culture DNA synthesis was scheduled (not a repair phenomenon). The primary keratinocytes showed an oscillating pattern of [3H]Tdr incorporation into DNA over the initial 23--25 days of growth. Autoradiography demonstrated that the cultures contained 10--30% proliferative stem cells from days 2-25 of culture. The reproducibility of both the proliferation and specialization patterns of the described primary epidermal cell culture system indicates that these cultures are a useful tool for investigations of functioning epidermal cell homeostatic control mechanisms.