The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications.     

Rat epidermal keratinocytes as an organotypic model for examining the role of Cx43 and Cx26 in skin differentiation

In order to characterize connexin expression and regulation in the epidermis, we have characterized a rat epidermal keratinocyte (REK) cell line that is phenotypically similar to basal keratinocytes in that they have the ability to differentiate into organotypic epidermis consisting of a basal cell layer, 2-3 suprabasal cell layers, and a cornified layer. RT-PCR revealed that REK cells express mRNA for Cx26, Cx31, Cx31.1, Cx37, and Cx43, which mimics the reported connexin profile for rat tissue. In addition, we report the expression of Cx30, Cx30.3, Cx40, and Cx45 in rat keratinocytes, highlighting the complexity of the connexin complement in rat epidermis. Furthermore, 3-dimensional analysis of organotypic skin revealed that Cx26 and Cx43 are exquisitely regulated during the differentiation process. The life-cycle of these connexins including their expression, transport, assembly into gap junctions, internalization, and degradation are elegantly depicted in organotypic epidermis as keratinocytes proceed from differentiation to programmed cell death.     

Synchronization of oscillations of proliferation of keratinocytes in psoriatic skin by external periodic force: a mathematical model

A mathematical model of mitotic activity of epidermis in normal skin and skin afflicted with psoriasis is presented as a system of two autonomous nonlinear differential equations. Its qualitative analysis was carried out and numerical solutions were obtained at the parameter values corresponding to these states. It was shown that in the norm, a single stable equilibrium of a "focus" type exists in the system; whereas in psoriasis, owing to an increase in the growing fraction, hyperproliferation, and enhanced migration of interacting keratinocytes, a stable limit cycle arises from the state of unstable focus. In this paper we also report on the results of computer modeling of synchronization of self-excited oscillations of keratinocyte population density in psoriatic lesions by an external periodic force. This synchronization is viewed as a possible mechanism of the clinically observed dependence of psoriasis course on some natural factors of cyclic nature.     

Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.     

Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model

The stratified squamous epithelia differ regionally in their patterns of morphogenesis and differentiation. Although some reports suggested that the adult epithelial phenotype is an intrinsic property of the epithelium, there is increasing evidence that subepithelial connective tissue can modify the phenotypic expression of the epithelium. The aim of this study was to elucidate whether the differentiation of cutaneous and oral epithelia is influenced by underlying mesenchymal tissues. Three normal skin samples and three normal buccal mucosa samples were used for the experiments. Skin equivalents were constructed in four ways, depending on the combinations of keratinocytes (cutaneous or mucosal keratinocytes) and fibroblasts (dermal or mucosal fibroblasts), and the effects of subepithelial fibroblasts on the differentiation of oral and cutaneous keratinocytes were studied with histological examinations and immunohistochemical analyses with anti-cytokeratin (keratins 10 and 13) antibodies. For each experiment, three paired skin equivalents were constructed by using single parent keratinocyte and fibroblast sources for each group; consequently, nine (3 x 3) organotypic cultures per group were constructed and studied. The oral and cutaneous epithelial cells maintained their intrinsic keratin expression. The keratin expression patterns in oral and cutaneous epithelia of skin equivalents were generally similar to their original patterns but were partly modified exogenously by the topologically different fibroblasts. The mucosal keratinocytes were more differentiated and expressed keratin 10 when cocultured with dermal fibroblasts, and the expression patterns of keratin 13 in cutaneous keratinocytes cocultured with mucosal fibroblasts were different from those in keratinocytes cocultured with cutaneous fibroblasts. The results suggested that the epithelial phenotype and keratin expression could be extrinsically modified by mesenchymal fibroblasts. In epithelial differentiation, however, the intrinsic control by epithelial cells may still be stronger than extrinsic regulation by mesenchymal fibroblasts.     

Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.     

Automatic supplementation of minerals in fed-batch culture to high cell mass concentration

Automatic constant-value control of mineral ions was attempted in semibatch culture of high cell mass concentration (more than 150 g dry cell/L) with ethanol and ammonia feeds. Equations were derived from the mass balance principle to calculate the required concentration of each mineral ion in the mineral feed solution, taking into account both the decrease in the volume of the culture supernatant as a proportion of the whole culture broth and the increase in the volume of the whole culture broth during the cultivation. The mineral solution was supplied automatically, linked either with ethanol feed or ammonia water feed. The actual concentrations of mineral ions could be kept within small variations. To adjust the supplementation in accordance with the culture change from oxygen sufficiency (early growth phase) to oxygen deficiency (later growth phase), the concentration of each mineral ion was altered stepwise when the dissolved oxygen concentration fell to zero. The mineral supplementation gave better results coupled with ethanol feed than with ammonia feed. The mineral ions studied were K(+), Mg(2+), Na(+), Fe(2+), Zn(2+), Ca(2+), Co(2+), Cu(2+), Mn(2+), NH(+) (4), PO(4) (3-) and SO(4) (2-).     

Actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human epidermal keratinocytes in culture

Summary In humans, the skin is a particularly sensitive target for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and certain halogenated analogs. Reported lesions include a thickening of the epidermis (acanthosis), hyperkeratosis, and squamous metaplasia of the epithelial lining of the sebaceous glands. In this report we describe ongoing studies on the actions of TCDD on cultured human epidermal cells. This system has been established as an in vitro model for interfollicular epidermal hyperkeratinization. Treatment of newly confluent cultures with TCDD results in enhanced differentiation as judged by histologic examination of the cultures, a decrease in the number of basal proliferating cells, and an increase in the number of envelope competent (differentiating) cells and terminally differentiated cells with highly cross-linked cornified envelopes. Changes in the differentiation program are preceded by a decrease in epidermal growth factor (EGF) binding. The concentration dependence and stereospecificity for these responses suggest the involvement of theAh receptor. We propose that TCDD modulates normal patterns of epidermal differentiation through direct actions on proliferating basal cells, modulating the responsiveness of these cells to growth factors such as EGF. This paper was presented at the Session-In-Depth on In Vitro Applications in Toxicology at the 34th Annual Meeting of the Tissue Culture Association, Orlando, FL, June 12–16, 1983. Rosemarie Osborne was a Chemical Industry Institute of Toxicology Postdoctoral Fellow.     

Physical characterization of the stratum corneum of an in vitro psoriatic skin model by ATR-FTIR and Raman spectroscopies

The stratum corneum is an important permeability barrier for the skin. The disorganization of the skin protective barrier characterizes some skin diseases such as psoriasis. Indeed, psoriatic skin is known to be more permeable than normal human skin. An in vitro human skin substitute may be obtained by the auto-assembly method. This method was adapted to produce psoriatic substitutes. FTIR spectroscopy is a well-established method to evaluate the order of hydrocarbon chains in terms of population of trans and gauche conformers. Using ATR-FTIR, we have compared the physicochemical properties of the stratum corneum in skin models derived from uninvolved and involved psoriatic cells with those derived from normal cells. Our results suggest that the stratum corneum of involved psoriatic skin substitutes is less organized than that of normal skin substitutes. Also, it seems that the properties of uninvolved psoriatic skin may vary with seriousness of the disease. The development of a new psoriatic skin model would be helpful in the design of new treatments and to increase the understanding of the mechanisms of this pathology.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Purification of new calcium activated protease (low calcium requiring form) and comparison to high calcium requiring form

A new calcium activated protease which requries low concentration of calcium was purified to almost homogeneity from porcine heart muscle. The protease was composed of two polypeptide chains of approximately 90 K and 30 K. The 90 K subunit was larger than the large subunit of the high calcium requring form of calcium activated protease, therefore we concluded that the low calcium requiring form is different from the high calcium requring form and its auto-digested protease. The low calcium reqiring form of calcium activated protease was also activated by manganese and balium, and was very stable even at pH 9.0     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: lipid composition and thermal phase behavior of the stratum corneum.

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T2) at a slightly lower temperature (68 degrees C) than human skin (74 degrees C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

Response of plants to calcium concentration in flowing solution culture with chloride or sulphate as the counter-ion

Summary Solution calcium concentrations required for the growth of a range of plant species, including both monocotyledons and dicotyledons, were determined in two experiments in which plants were grown in flowing solution culture at constantly maintained calcium concentrations ranging from 0.5 to 3000 μM. Calcium chloride was used as the calcium source in the first experiment, calcium sulphate was used in the second. At calcium concentrations of 10 μM and below, all species developed calcium deficiency symptoms. The severity of the deficiency was more pronounced in the dicotyledons than in the monocotyledons. However, cassava was much more tolerant than all other dicotyledons and equally as tolerant as rice, the most tolerant monocotyledon. Solution calcium concentrations required for 90% of maximum yield were generally lower for monocotyledons (3 to 20 μM) than for dicotyledons (7 to 720μM) when calcium chloride was used as the calcium source. When calcium sulphate was used, 7 out of 11 species, including 3 monocotyledons, required external calcium concentrations of 1200 μM and above. The results are discussed in relation to effects of solution composition and the choice of counter-ions on plant response to calcium and other macronutrient cations. It is concluded that yield depressions due to toxicity of excesses of chloride, and possibly other counter-ions, can lead to serious underestimation of limiting external cation concentrations for plant growth.     

Differential effects of arsenic on calcium signaling in primary keratinocytes and malignant (HSC-1) cells

Arsenic is highly toxic to living cells, especially skin, and skin cancer is induced by drinking water containing arsenic. The molecular mechanisms of arsenic-induced cancer, however, are not well understood. To examine the initial processes in the development of arsenic-induced cancer, we analyzed calcium signaling at an early stage of arsenic treatment of human primary cells and compared the effects with those observed with arsenic treatment in carcinoma-derived cells. We found that arsenic inhibited inositol trisphosphate receptor (IP3R) function in the endoplasmic reticulum by inducing phosphorylation, which led to decreased intracellular calcium levels. Blockade of IP3R phosphorylation by the serine/threonine protein kinase Akt inhibitor wortmannin rescued calcium signaling. In contrast, arsenic treatment of cells derived from a carcinoma (human squamous carcinoma; HSC-1) for 1h had no obvious effect. Taken together, these results suggest that arsenic-induced reduction in calcium signaling is one of the initial mechanisms underlying the malignant transformation in the development of skin cancer.     

A morphofunctional analysis of the hemocytes in the cricket Gryllus bimaculatus (Orthoptera: Gryllidae) normally and in acute microsporidiosis due to Nosema grylli

Microsporidians (M) are supposed to be ancient eukaryotic parasites with a broad range of animal hosts, being especially abundant in Arthropoda. They are supposed to pass a long way of adaptation to parasitism, that usually means inhibiting or avoiding host immune reactions alongside with the reduction of pathogenicity. However M, unlike other eukaryotic obligate parasites, preserved a high pathogenicity, comparable with one of viruses, and thus they could be expected to possess a unique mode of interactions with their hosts. The goal of the present work is to assess how M influence the cellular immune response of an insect host. Experiments were performed on the host-parasite system Gryllus bimaculatus (Orthoptera, Gryllidae)--Nosema grylli (Microsporidia, Nosematidae); coccidia Adelina grylli--infected crickets were used to compare the host cellular response against two pathogens. Haemocytes (H) were observed using phase contrast and electron microscope. H smears were stained for a phenoloxidase (PO), esterase activities and "respiratory burst" reaction. Five H type can be distinguished in the cricket haemolymph. (1) Prohaemocytes, relatively small (13-30 microns) cells with large nuclei, are observed both in control and infected insects. (2) Plasmatocytes, round (30-35 microns in diameter) or fusiod (40-63 x 13-38 microns) cells, can hardly be distinguished from (1) ultrastructurally; during the coccidian infection of the cricket fat body these H infiltrate the infected organ and turn into amebocytes with laciniate nuclei, they usually contain electron dense granules, that release during the formation of a capsule around the coccidian oocytes. (3) Granulocytes (Gr, 20-33 microns in diameter) are cells with the extremely refractive cytoplasm when observed in phase contrast microscope, they contain vacuoles with typical crystal needle-like inclusions. The transitional forms between the mentioned above three cell types can be observed. The next two H types also observed on H monolayers are supposed to be the specialized forms of Gr: (4) coagulocytes, cells with the fragile cytoplasm that are easily disintegrated after a contact with a pathogen; they have been described in Orthoptera for the first time now; (5) spherulocytes, giant cells filled with electron lucid granules and small, often eccentrically located nucleus. Both H types were observed only after infection with A. gryllus in the vicinity of encapsulated oocysts. Infection with M does not cause such intensive concentration of haemocytes near the infected organ, or so abundant nodule formation, until the acute stage of the disease when M spores are liberated from the destroyed cells and contact the insect haemolymph. Thereafter, the number of granulocytes significantly increases. In the presence of M spores, haemocytes produce long cytoplasm protrusions and form clapms. Some spores adhere to the haemocyte surface and are phagocytized. Giant round cells loaded with spores, can be observed in the host lymph. They are surrounded by a sheath composed of flattened cells and resemble xenomas, described for fish microsporidiosis. A. grylli caused the increase in the quote of PO-positively stained cells up to 80% from 40-50% in control, that well corresponds to the host immune reactions activation and melanization of infected tissue, while microsporidiosis significantly reduced quote of PO+ cells. Carboxyl esterase activity expressed as quote of positively stained cells was 40-60% in naive and coccidia-parasitized samples, M decrease this number to 10-20%. "Respiratory burst" reaction, detected by reducing of NBT, did not alter significantly in microsporidia-infected insects. From the presented data in can be concluded: 1) M do not suppress such cellular reactions as a clamp formation and phagocytosis of spores, liberated from the infected tissue; 2) at the same time they suppress activities of enzymes involved in immune response.     

Interferon-inducible protein 9 (CXCL11)-induced cell motility in keratinocytes requires calcium flux-dependent activation of mu-calpain

Keratinocyte migration is critical to reepithelialization during wound repair. The motility response is promoted by growth factors, cytokines, and cytokines produced in the wound bed, including those that activate the epidermal growth factor (EGF) receptor. The Alu-Leu-Arg-negative CXC chemokine interferon-inducible protein 9 (IP-9; also known as CXCL11, I-TAC, beta-R1, and H-174) is produced by keratinocytes in response to injury. As keratinocytes also express the receptor, CXCR3, this prompted us to examine the role and molecular mechanism by which IP-9 regulates keratinocyte motility. Unexpectedly, as CXCR3 liganding blocks growth factor-induced motility in fibroblasts, IP-9 alone promoted motility in undifferentiated keratinocytes (37 +/- 6% of the level of the highly motogenic EGF) as determined in a two-dimensional in vitro wound healing assay. IP-9 even enhanced EGF-induced motility in undifferentiated keratinocytes (116 +/- 5%; P < 0.05 compared to EGF alone), suggesting two separate mechanisms of action. IP-9-increased motility and -decreased adhesiveness required the intracellular protease calpain. The increases in both motility and calpain activity by IP-9 were blocked by pharmacological and molecular inhibition of phospholipase C-beta3 and chelation of calcium, which prevented an intracellular calcium flux. Molecular downregulation or RNA interference-mediated depletion of mu-calpain (calpain 1) but not M-calpain (calpain 2) blocked IP-9-induced calpain activation and motility. In accord with elimination of IP-9-induced de-adhesion, RNA interference-mediated depletion of calpain 1 but not calpain 2 prevented cleavage of the focal adhesion component focal adhesion kinase and disassembly of vinculin aggregates. In comparison, EGF-induced motility of the same undifferentiated keratinocytes requires the previously described extracellular signal-regulated kinase to the M-calpain pathway. These data demonstrate that while both EGF- and IP-9-induced motility in keratinocytes requires calpain activity, the isoform of calpain triggered depends on the nature of the receptor for the particular ligand. Interestingly, physiological nonapoptotic calcium fluxes were capable of activating mu-calpain, implying that the calcium requirement of mu-calpain for activation is attained during cell signaling. This is also the first demonstration of differential activation of the two ubiquitous calpain isoforms in the same cell by different signals.     

Human breast milk stimulates low density lipoprotein (LDL) binding to human skin fibroblasts in culture

Human breast milk incorporated at 1% concentration into the culture medium significantly (p less than 0.05) increased the binding of 125I-LDL to receptors of human skin fibroblasts in culture. Homogenized cows milk and infant formula (Similac) also possessed this stimulating property. The stimulating activity of milk persisted after dialysis and extraction with cold acetone. These preliminary studies suggest that milk might contain potent factor(s) influencing cholesterol metabolic process in early life.