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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
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  • C12N2502/00—Coculture with; Conditioned medium produced by
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  • C12N2502/1121—Dendritic cells
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
  • C12N2502/1157—Monocytes, macrophages
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1323—Adult fibroblasts
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/24—Genital tract cells, non-germinal cells from gonads
  • C12N2502/243—Cells of the female genital tract, non-germinal ovarian cells

Definitions

• a method for producing Langerhans cells and/or dermal/interstitial dendritic cells from CD 14+ monocytes A method for producing Langerhans cells and/or dermal/interstitial dendritic cells from CD 14+ monocytes
• the invention relates to a method for producing dendritic cells, and notably Langerhans cells and/or interstitial dendritic cells/dermal dendritic cells, from CD 14+ monocytes isolated from peripheral circulatory blood, notably by preparing tissue models, notably with the purpose of testing active ingredients and/or studying biological/biochemical phenomena involved at these models.
• DC Dendritic cells
• the DCs are cells having antigens described as sentries of the immune system. Indeed, they have a quasi-ubiquitous localization, i.e., in the thymus, systemic circulation, secondary lymphoid organs and also peripheral tissues such as the skin and the mono-stratified or pluri-stratified mucosae.
• the DCs are at the centre of the triggering of specific immune responses, by exerting a control on the specificity, the intensity and the nature of the immune response and are placed at the interface between innate and acquired immunity. In addition to their function for igniting the immune response, the DCs also play a role in inducing peripheral tolerance.
• the precursors of DCs stem from the differentiation of CD34+ haematopoietic progenitors just as many populations of the immunitary system and blood cells. They are conveyed via the blood at the skin and the mucosae in order to differentiate and reside their as immature DCs. This immature state is expressed by a characteristic phenotype and a strong functional capability of capturing antigens.
• Two types of peripheral DCs are described according to their in vivo localization:
• the Langerhans cells are localized at the epithelia of the malpighian type (skin and mucosae) where they specifically express langerine which is a lectin of types C. Langerine is involved in the formation of an intracytoplasmic organite, Birbeck's granule which is the specific ultrastructural and reference label of the LCs. LCs also express characteristic labels such as CDIa and HLA-DR.
• Interstitial DCs as for them are found at the lamina of mucosae like at the dermis of the skin where they are also described as dermal DCs (DDCs). These cells share many similarities and common labels with the cell line of monocytes/macrophages. In the dermis of the skin, DCCs specifically express DC-SIGN but also characteristic labels such as HLA-DR, FXIIIa, MMR and CDIa. The description which follows generally refers to interstitial dendritic cells (IDCs) independently of the fact that they are located at the lamina of the mucosae or at the dermis of the skin.
• IDCs interstitial dendritic cells
• the LCs and IDCs migrate towards the lymph nodes in order to present the antigenic information to the lymphocytes T.
• This migration which is correlated with activation of LCs and IDCs is expressed by phenotypical and functional changes.
• the « activated » LCs and IDCs acquire the expression of CD80 and CD86 which are co-stimulation labels and acquire the expression of the CCR7 receptor which is absolutely necessary to skin migration of the cells.
• the activated LCs and IDCs acquire a phenotype of
• LCs and IDCs notably combined with epithelial cells or mesenchymatous cells of the fibroblast type, stemming from skin or human mucosae, consists of integrating them into three-dimensional organotypical cultures such as:
• This patent generally defines the precursors of LCs as being able to express CDIa+, however only the use of CD34+ haematopoietic cells as CD34+ precursors of LCs is described therein.
• the CD34+ haematopoietic cells are however not very numerous in the peripheral blood and do not allow development of an industrially satisfactory differentiation method.
• the use of precursors stemming from umbilical chord blood is unsatisfactory in so far that this blood is not available in a large amount.
• the invention mainly consists of generating LCs, or IDCs or LCs and IDCs simultaneously, or IDCs and macrophages and endothelial cells simultaneously or LCs and IDCs and macrophages and endothelial cells simultaneously, from a unique living precursor stemming from peripheral circulatory blood.
• the invention notably consists of providing epidermis, epithelium, dermis, chorion, skin or mucosa models containing the aforementioned cells (LC, IDC/DCC, etc.), said models being of the best possible quality in order to reproduce an epidermis, an epithelium, a dermis, a chorion, a skin or a mucosa of a living being, in particular of a mammal, and notably of a human being.
• the object of the present invention further is to provide epithelial and/or conjunctive sheets as well as immunocompetent equivalents of skin or of mucosae.
• the present invention notably consists of providing a method for differentiating monocytes stemming from peripheral circulatory blood in order to obtain the aforementioned cells (LC, IDC/DCC, etc.)
• the present invention also consists of providing models described above as alternative methods to animal experimentation, notably for testing the irritating and/or sensitizing potency of a cosmetic ingredient.
• the object of the present invention is also to provide models as described above for testing active ingredients notably in cosmetics, in dermo-pharmacy, and in pharmacy, notably for assessing their activity and/or their toxicity or pharmacotoxicity.
• the object of the present invention is also to provide models as described above for testing molecules or chemicals notably for assessing their toxicity.
• the present invention also consists of providing models as described above for investigating biological/biochemical phenomena at intercellular and intracellular level.
• the main object of the invention is to provide a model/tool for pharmacotoxicological investigation, for example with the purpose of conducting tests in vitro for predicting the allergizing/irritating/sensitizing power of external agents.
• the object of the present invention is still to provide a model/tool for investigating substances having immunomodulating properties.
• the invention also relates to the use of the models as described above in tissue or cell engineering, notably for repairing at least one portion of the tissues of a living being.
• peripheral circulatory blood » is meant according to the invention the blood of any living being having a blood system in which the blood accomplishes a circuit, notably at the periphery, in particular in animals, in mammals, preferably in a human being.
• active ingredient » any substance, product or composition potentially capable of having an interesting activity in industry, in particular in the agro-feeding, food, dermo- pharmaceutical, pharmaceutical, cosmetics industry, etc.
• addition of exogenous cytokine is meant addition of at least one cytokine in addition to the cytokines synthesized by the cells present in the relevant cell medium.
• the invention therefore relates to differentiation of CD 14+ monocytes, in the presence of epithelial cells and/or mesenchymatous cells, for example of the fibroblast type, into:
• IDCs and macrophages and/or endothelial cells simultaneously, - LCs and IDCs and macrophages and/or endothelial cells simultaneously.
• the invention notably relates to differentiation of CD 14+ monocytes into LCs and/or IDCs (without prior growing under conditions promoting their differentiation, and notably without prior growing with exogenous cytokines) in the presence of epithelial cells and/or mesenchymatous cells, for example of the fibroblast type, in order to obtain a cell model comprising LCs and/or IDCs, their culture being preferably performed essentially without significant addition of exogenous cytokines, i.e. without adding any cytokine in addition to the endogenous cytokines synthesized by the cells present in the medium.
• the invention notably relates to a method for differentiating CD 14+ monocytes into LCs and/or IDCs, comprising the following steps:
• CD 14+ monocytes from circulatory blood, notably of a human being
• CD 14+ monocytes into contact in a cell environment comprising epithelial cells, for example keratinocytes, and/or mesenchymatous cells, for example fibroblasts, under conditions which do not promote differentiation of CD 14+ monocytes into LCs, or IDCs, or LCs and IDCs.
• epithelial cells for example keratinocytes
• mesenchymatous cells for example fibroblasts
• the step for maintaining CD 14+ monocytes under conditions which do not promote their differentiation into CDs is preferably limited in time in order to notably carry out sowing of the CD 14+ monocytes shortly or even directly after they have been collected from the circulatory blood.
• conditions which do not promote differentiation of CD 14+ monocytes into DCs are obtained under culture conditions which do not comprise any exogenous cytokine.
• the purpose here is notably that the CD 14+ monocytes do not engage in DC differentiation paths.
• DCs generated in vitro are very sensitive and fragile cells.
• the first parameter to be checked is the yield in the obtained cells. It is obvious, but not quantifiable, that the loss and/or cell mortality of DCs is significant when the latter are sown in any cell culture model.
• the CD 14+ monocytes which have initiated their differentiation generate LCs and DCCs which do not all have the same differentiation stage. This imperfection may be dispensed with by avoiding this differentiation step.
• the monocytes grown in the present invention are influenced by the cell, cytokine and matrix environment of the cell reconstructions, which forms a more physiological environment for differentiation of DCs.
• DCs generated in vitro have the capability of « maturating » spontaneously, which is a major problem as mature DCs may no longer be activated and stimulated.
• the inventors control the immature conditions of these cells in vitro, the risk of spontaneous maturity in vitro may be dispensed with by directly using monocytes as precursors of LCs and DCCs.
• the advantageous use of cell or tissue culture models according to the invention as a system for differentiation of CD 14+ monocytes into DCs is more physiological as it better reproduces the natural conditions for differentiation of DCs in the skin and in the mucosae and immature DCs comparable to their homologs in vivo may thereby be obtained.
• CD 14+ monocytes as precursors of DCs, as opposed to the use of DCs generated beforehand, it is possible to generate in the present models for growing phenotypically more immature and functionally more sensitive LCs and DCCs.
• DCs derived from CD34+ progenitors and then grown in a reconstructed chorion in the presence of exogenous cytokines has a more satisfactory immaturity condition, in terms of replication of the HIV virus, than its homologs grown in the presence of exogenous cytokines (Sandy DUMONT et ah, When integrated in a subepithelial mucosal layer equivalent, dendritic cells keep their immature stage and their ability to replicate type R5 HIV-I strains in absence of T cell subsets. AIDS Res. and Hum. Retroviruses 20: 383-397, 2004).
• the obtained DCs are very different from those described previously and notably in French Patent FR 2 833 271Bl, filled by Coletica on December 10 th 2001.
• CD 14+ monocytes as precursors of skin DCs is a more physiological process as compared with the process described earlier. Indeed, in vivo in the organism, blood monocytes colonize the skin, where the cell, cytokine and matrix environment controls their differentiation. Integration of DCs differentiated beforehand in the present culture models was an interesting alternative which today is less satisfactory than directly using their precursors which are the CD 14+ monocytes. This improvement of the system is immunohistologically expressed by a more homogenous cell distribution of LCs and DCCs in the present three-dimensional culture models.
• the present invention notably relates to a unique cell precursor which, when it is co- cultivated:
• the present invention relates to a method for preparing LCs, or IDCs, or LCs and IDCs, from CD 14+ monocytes stemming from peripheral circulatory blood of a living being, and notably of a human being, comprising differentiation of CD 14+ monocytes into LCs or into IDCs or into LCs and IDCs by putting CD 14+ monocytes in presence with a cell environment comprising epithelial cells, such as keratinocytes, and/or mesenchymatous cells, such as dermal fibroblasts.
• differentiation of CD 14+ monocytes allows to obtain IDCs and macrophages and endothelial cells, or LCs, IDCs, macrophages and endothelial cells.
• the distribution of the cell population of LCs and IDCs is a function of the cell type which is jointly grown with monocytes.
• the use of keratinocytes promotes differentiation into LCs and the use of fibroblasts promotes differentiation into IDCs.
• the differentiation of the monocytes is carried out without significantly adding any exogenous cytokine.
• no exogenous cytokine is added.
• keratinocytes promotes differentiation of the monocytes into immature and functional LCs.
• immature » the inventors notably mean: that they do not or only very slightly express activation labels (CD80, CD86, CCR7) and maturation labels (CD83, DC-LAMP).
• immature LCs express CCR6.
• functional » the inventors notably mean: provided with antigene internalization capacities (immature condition), cell migration capacities (immature and activated condition), and antigenic presentation capacities (mature condition).
• the culture of monocytes with a mesenchymatous cell environment promotes differentiation of the monocytes into immature and functional IDCs.
• a mesenchymatous cell environment such as dermal fibroblasts
• culture of monocytes with a cell environment comprising epithelial cells, such as keratinocytes, and mesenchymatous cells, such as dermal fibroblasts promotes differentiation of the monocytes into typical LCs and IDCs.
• epithelial cells such as keratinocytes
• mesenchymatous cells such as dermal fibroblasts
• the proportion of epithelial cells and/or mesenchymatous cells, relatively to the CD 14+ monocytes, used for the culture (for the differentiation) depends on the cell distribution between LCs and/or IDCs and epithelial cells and/or mesenchymatous cells, which one wishes to obtain.
• the invention according to a second aspect relates to a method for growing CD 14+ monocytes, the growing method comprising the integration into a cell or tissue model of CD 14+ monocytes stemming from peripheral circulatory blood of a living being, and notably of a human being, said cell or tissue model comprising epithelial cells, such as keratinocytes and/or mesenchymatous cells, such as dermal fibroblasts, in order to obtain differentiation of CD 14+ monocytes within the model into LCs, or IDCs, or into LCs and IDCs, by growing CD 14+ monocytes in the presence of epithelial cells, such as keratinocytes and/or mesenchymatous cells, such as dermal fibroblasts.
• epithelial cells such as keratinocytes and/or mesenchymatous cells, such as dermal fibroblasts
• the growing of monocytes is carried out without adding any exogenous cytokine.
• the cell or tissue model is selected from the group consisting in an epidermis model, an epithelium model, a dermis model, a chorion model, a skin model or a model of a mucosa, in particular of a gingival or vaginal mucosa.
• the three-dimensional culture model comprises a dermal or chorion matrix support selected from the group consisting of: a collagen- or fibrin-based gel or film comprising mesenchymatous cells, in particular fibroblasts, - a porous matrix which is made from collagen which may contain one or more glycosaminoglycans and/or optionally chitosan, these porous matrices either integrating mesenchymatous cells, in particular fibroblasts, or not,
• an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a semi-permeable nylon membrane, a polytetrafluoroethylene (Teflon®, PTFE), a membrane or sponge, a semi-permeable polycarbonate or polyethylene terephthalate (PET) membrane, an inorganic membrane with a capillary porous structure of aluminum oxide (semi-permeable Anopore membrane), a semipermeable polyester membrane, said inert support either containing mesenchymatous cells, in particular fibroblasts, or not. an inert support treated for polycarbonate- or polystyrene-based culture, said inert support either containing mesenchymatous cells, in particular fibroblasts, or not.
• a semi-permeable synthetic membrane in particular a semi-permeable nitrocellulose membrane, a semi-permeable nylon membrane, a polytetraflu
• the tissue model used comprises said dermal or chorion support on which epithelial cells, in particular keratinocytes, have been deposited at the surface.
• the cell or tissue model comprises at least one additional cell type, for example nerve cells and/or endothelial cells and/or melanocytes and/or lymphocytes and/or adipous cells and/or cutaneous annexes, such as bristles, hairs, sebaceous glands.
• a portion of the CD 14+ monocytes differentiates into endothelial cells and macrophages, in particular when they are put into a cell or tissue model comprising at least mesenchymatous cells.
• the method mainly comprises LCs, or IDCs, or a mixture of LCs and IDCs, or a mixture of LCs, IDCs, endothelial cells and macrophages, or a mixture of IDCs, endothelial cells and macrophages.
• the cell or tissue model comprises an epithelial portion and a conjunctive matrix and is characterized in that the LCs are essentially localized in the epithelial portion, and in that the IDCs, the macrophages and the endothelial cells are essentially localized in the conjunctive matrix.
• the invention according to a third aspect, relates to a cell model comprising at least one of said LC and/or IDC populations, and further optionally comprising a population of macrophages and/or endothelial cells, capable of being obtained according to a method as defined earlier.
• the invention according to the fourth aspect, relates to a tissue model, comprising at least one of said LC and/or IDC populations, and further optionally comprising a population of macrophages and/or endothelial cells, capable of being obtained according to a method as defined earlier, said tissue model being selected from the group consisting of an epidermis model, an epithelium model, a dermis model, a chorion model, a skin model, or a model of a mucosa, in particular of a gingival or vaginal mucosa.
• the cell or tissue model described above is immunocompetent.
• the tissue model comprises an epithelial portion comprising epithelial cells, such as keratinocytes, and a conjunctive matrix comprising mesenchymatous cells, such as dermal or chorion fibroblasts, said model being characterized in that the LCs are essentially localized in the epithelial portion, and in that the present IDCs and macrophages and/or endothelial cells are essentially localized in the conjunctive matrix.
• epithelial cells such as keratinocytes
• mesenchymatous cells such as dermal or chorion fibroblasts
• Said model is characterized in that the obtained DCs are different from those obtained according to the method described earlier, the main differences being concerned with the more homogenous cell distribution of LCs and DCCs in the present three-dimensional culture models.
• the invention according to a fifth aspect relates to frozen CD 14+ monocytes, isolated from the peripheral circulatory blood of a living being, in particular of a human being.
• the invention relates to the use of at least one cell or tissue model as defined earlier as an investigation model in the field of cosmetics, dermo-pharmacy or pharmacy, and/or for active ingredient selection.
• Another interesting use of LCs and/or IDCs consists of using them for assessing the irritating versus the sensitizing potency of new molecules.
• European directive 2003/15/EC which prohibits since 2005 the use of animals for assessing the toxicity of a cosmetic finished product, strongly urges public and industrial laboratories to develop in vitro or in silico predictive methods for predicting the sensitizing potency of new molecules.
• the use of cutaneous DCs is today an axis of development.
• the invention according to a seventh aspect relates to the use of at least one cell or tissue model as defined earlier, for investigating phenomena occurring in the organism's defense/infection processes, and activity, in particular the immuno-stimulating or immunosuppressive activity, of an active ingredient or for assessing or for inducing immunomodulation (imrnunotolerance or immunoactivation) by said active ingredient, or for conducting in vitro tests for predicting the allergizing/irritating/sensitizing potency of external agents or for investigating toxicity of molecules or chemicals.
• the invention according to an eighth aspect relates to the use of at least one cell or tissue model as defined earlier, for investigating the physiopathology of epithelial barriers; irritation of the skin and mucosae; aggressions of a biological nature such as for example viruses, retroviruses, such as HIV, bacteria, fungi, micro-organisms, particle antigens; phototoxicity; photoprotection; the effect of active ingredients, in particular of cosmetic or pharmaceutical ingredients; the effect of finished products, in particular of cosmetic or pharmaceutical products: the effect of molecules or chemicals; the mechanisms for infection by a pathogenic agent.
• a biological nature such as for example viruses, retroviruses, such as HIV, bacteria, fungi, micro-organisms, particle antigens
• phototoxicity photoprotection
• the effect of active ingredients in particular of cosmetic or pharmaceutical ingredients
• the effect of finished products in particular of cosmetic or pharmaceutical products: the effect of molecules or chemicals
• the mechanisms for infection by a pathogenic agent for investigating the physiopathology of epithelial barriers; irritation of the skin
• this invention relates to investigating toxicity of active ingredients or other substances. Notably this investigation is carried out by studying cell labels, notably including those of DCs.
• the invention according to a ninth aspect relates to the use of at least one cell or tissue model as defined earlier, for investigating mechanisms involved in the phenomena of viral infection, replication and transmission of viruses, including retroviruses like HIV, or for investigating and developing alternative therapeutic methods, including the administration of vaccines or drugs.
• the invention according to a tenth aspect relates to the use of at least one cell or tissue model as defined earlier for detecting the presence of a pathogenic agent such as for example viruses, retroviruses, such as HIV, bacteria, fungi, micro-organisms, particle antigens.
• the invention according to an eleventh aspect relates to the use of at least one cell or tissue model as defined earlier, for a medical, biomedical, or cosmetic application, in particular for modulating the immune or tolerance response in vitro or in vivo, as a result of an environmental aggression, in particular of the physical type, such as UV irradiation, of the chemical type, such as irritating/allergizing/sensitizing agents, of the biological type, in particular with a preventive or curative therapeutic purpose.
• the invention according to a twelfth aspect relates to the use of at least one cell or tissue model as defined earlier, for cell or tissue engineering applications, for medical or biomedical applications, for example in anti-cancer cell therapy, for example by a DCs injection capable of stimulating the immune response; for example in cell therapy in the case of an auto-immune disease, for example by creating an immunotolerance stimulation, for example by producing anergic T cells; for example in gene therapy of diseases affecting the immunitary system; or for developing and making vaccines.
• the invention according to a thirteenth aspect relates to a method for making a tissue model comprising: - isolation of CD 14+ monocytes from peripheral circulatory blood of a living being,
• CD 14+ monocytes either simultaneously or not with skin or mucosal cells
• - growing the reconstructed tissue comprising CD 14+ monocytes and skin or mucosal cells under conditions allowing differentiation of CD 14+ monocytes into LCs, into a mixture of IDCs, endothelial cells and macrophages, into a mixture of LCs, IDCs, endothelial cells and macrophages
• the skin cells being epidermal keratinocytes when the reconstructed tissue is an epidermis model
• the skin cells being dermal fibroblasts when the reconstructed tissue is a dermis model
• the mucosal cells being epithelial mucosal cells when the reconstructed tissue is an epithelium model
• the mucosal cells being mucosal fibroblasts when the reconstructed tissue is a chorion model.
• the epithelial cells are for example isolated from at least one skin tissue. Sowing of the skin or mucosal cells may be carried out before sowing or after having sown CD 14+ monocytes. Preferably, sowing of CD 14+ monocytes is carried out simultaneously with the sowing of skin or mucosal cells.
• the invention according to a fourteenth aspect relates to a method for making a reconstructed skin or a reconstructed mucosa comprising an epithelial portion comprising keratinocytes or epithelial mucosal cells, optionally in the presence of Merkel cells and/or melanocytes, and a conjunctive matrix comprising dermal or mucosal fibroblasts, said method comprising:
• keratinocytes or epithelial mucosal cells possibly in the presence of Merkel cells and/or melanocytes, at the surface of a dermis or chorion model, comprising IDCs and optionally endothelial cells and macrophages, said dermis or chorion model being able to be obtained by a method for making a dermis or chorion model as defined above,
• CD 14+ monocytes isolated from peripheral circulatory blood of a living being in the presence of keratinocytes or epithelial mucosal cells under conditions allowing differentiation of CD 14+ monocytes into LCs.
• the cells are cells of a human being.
• these methods for making tissue models do not comprise any addition of exogenous cytokine.
• each example has a general scope.
• all the percentages are given by weight, unless stated otherwise, and the temperature is expressed in degrees Celsius, unless stated otherwise, and the pressure is atmospheric pressure, unless stated otherwise.
• Example 1 Methods for separating monocytes from peripheral circulatory blood
• peripheral circulatory blood was harvested by sampling venous blood on one or more human donors, preferably in vacutainers or bags supplemented with usual anti-coagulant products such as lithium heparin. Separation of monocytes from circulatory blood may advantageously be performed according to the following protocols:
• a specific antibody of the monocytes such as an anti-CD 14 antibody, coupled with magnetic beads; after passing over a magnetic column, only the labeled monocytes are retained in the column. After elution from the column, the labeled monocytes are recovered.
• monocytes or labeled with a specific antibody of the monocytes, such as an anti-CD 16 antibody, coupled with a fluorochrome, such as phycoerythrin. After cell sorting in flux cytometry, only the labeled monocytes are recovered. 2/ The monocytes are recovered by proceeding with any physical separation method well-known to one skilled in the art and notably by sedimentation or centrifugation and they are eluted as such for subsequent cultures.
• Example 2 Method for freezing monocytes isolated from peripheral circulatory blood
• the monocytes as obtained in Example 1, are suspended in a nutritious medium, for example RPMI medium, supplemented with serum and a cryoprotective agent, such as DMSO ⁇ dimethyl sulfoxide), and then frozen.
• a nutritious medium for example RPMI medium, supplemented with serum and a cryoprotective agent, such as DMSO ⁇ dimethyl sulfoxide
• Example 3 A pericellular monolayer model of keratinocytes and LCs in a co-culture Obtaining the monocytes: see Example 1 or 2.
• 1 to 2.10 6 human keratinocytes and 1 to 2.10 6 human monocytes are jointly grown in a nutritious medium, for example of the K-SFM type, in culture dishes, for example of the 6-well plate type.
• the joint culture is maintained for 6 days in a nutritious medium, for example of the K-SFM type, without adding any exogenous cytokine.
• the cells are then recovered by an enzymatic method well-known to one skilled in the art and notably by trypsination.
• 2.10 5 cells of the mixed cell suspension consisting of keratinocytes and monocytes are incubated with a monoclonal anti-Langerine antibody, and then analyzed in flux cytometry. We observe up to 40% of Langerine+ LCs.
• Example 4 Pericellular monolayer model of fibroblasts and DDC in a co-culture Obtaining the monocytes: see Example 1 or 2.
• 1 to 2.10 6 human fibroblasts and 1 to 2.10 human monocytes are jointly grown in a nutritious medium, for example of the FBM type, in culture dishes for example of the 6-well plate type.
• the joint culture is maintained for 6 days in a nutritious medium, for example of the FBM type, without adding any exogenous cytokine.
• the cells are then recovered by an enzymatic method well-known to one skilled in the art and notably by trypsination. 2.10 s cells of the mixed cell suspension consisting of fibroblasts and monocytes are incubated with a monoclonal anti-DC-SIGN antibody, and then analyzed in flux cytometry. We observe up to 60% of DC-SIGN+ DCCs.
• Example 5 The use of monolayer pericellular models described in Examples 3 and 4 for investigating the profile of cytokines secreted under the effect of an active ingredient
• Retinol is then added into the culture medium at a final concentration of 0.05% for 3 days.
• the culture supernatants are then recovered and analyzed. It is observed that retinol causes stimulation of secretion of pro-inflammatory cytokines.
• Example 6 The use of pericellular monolayer models described in Examples 3 to 4 for investigating the immunoactivating or immunosuppressive activity of an active ingredient
• the active ingredient is then added into the culture medium at various concentrations and for 3 days.
• the cells are then recovered by trypsination. 2.10 5 cells of the mixed cell suspension consisting of keratinocytes and LCs or of fibroblasts and DCCs are incubated in an antibody battery: anti- CCR7, anti-HLA-DR, anti-CD80, anti-CD83, anti-CD86, anti-DC-LAMP.
• the immunoactivating (induction and/or increase of the expression of labeled molecules) or immunosuppressive profile (inhibition and/or suppression of the expression of labeled molecules) of the tested active ingredients may be established.
• Example 7 The use of pericellular monolayer models described in Examples 3 to 4 for investigating the immunomodulating activity of an active ingredient
• the model is made according to the protocol described in Example 3 or 4. 300 ⁇ l of TNP (2,4,6-trinitrobenzene-sulfonic acid) are then added at a concentration of 5 niM for 30 min at 37°C.
• the active ingredients tested at various concentrations are added into the culture medium for 2 days.
• the culture media comprising the cells are then recovered in order to investigate the secretion of immune cytokines, for example IL- 12; the cells are recovered and then incubated either with an anti-Langerine antibody or with an anti-DC-SIGN antibody in order to sort the LCs or the DCCs in flux cytometry, respectively.
• the sorted LCs or DCCs are then sown in migration chambers of the Boyden type (porosity of the membrane from 5 to 8 ⁇ m either covered with MATRIGELTM or not) and then incubated for up to 72 hrs at 37°C. -
• the number of LCs or DCCs having migrated is quantified, for example by counting in optical microscopy.
• Example 8 Three-dimensional pericellular reconstructed epidermis model containing LCs Obtaining the monocytes: see Example 1 or 2.
• the model is made according to the following protocol: - 0.5 to 1.10 6 keratinocytes of normal human skin are sown in inserts of the Boyden chamber type (membrane with porosity 0.4 ⁇ m) and then grown in a nutritious medium, for example of the DMEM-Glutamax type supplemented with calf serum, ascorbic acid and preferably a final concentration of 1 niM of EGF ⁇ epidermal growth factor) and preferably at a final concentration of 10 ng/mL, hydrocortisone and preferably at a final concentration of 0.4 ⁇ g/mL, umulin and preferably at a final concentration of 0.12 IU/mL, isuprel and preferably at a final concentration of 0.4 ⁇ g/mL, triiodothyronine and preferably at a final concentration of 2.10 "
• the cultures are then placed at the air-liquid interface for a further period of 10 days in the same medium used for the immersion culture, except for calf serum, hydrocortisone, umulin, isuprel and triiodothyronine.
• the cultures are then embedded in an amorphous resin, such as Tissue-Teck ® , and directly frozen in liquid nitrogen.
• the monocytes integrated into the reconstructed epidermis model differentiate into DCs of the LC type as demonstrated by observation of the lectin, Langerine.
• Example 9 Three-dimensional pericellular pigmented and/or nervous reconstructed epidermis model containing LCs
• the model is made according to the protocol described in Example 8, by simultaneously sowing together 0.5 a 1.10 5 melanocytes and/or Merkel cells stemming from normal human skin with keratinocytes.
• labeling of the melanocytes (HMB45) and a DOPA reaction are carried out in order to detect melanin as well as labeling with an anti- keratin 20 antibody for identifying Merkel cells.
• the monocytes integrated into the pigmented and/or nervous reconstructed epidermis model differentiate into DCs of the LC type as demonstrated by observation of the lectin, Langerine.
• Example 10 Three-dimensional pericellular epithelium model of gingival and vaginal reconstructed mucosae containing LCs
• the model is made according to the protocol described in Example 8 with the following changes: - Normal human keratinocytes are replaced with epithelial cells of normal human gingival and vaginal mucosae.
• the percentage of serum in the culture medium is 1 %. Growing is totally performed with immersion in the medium. The achieved labelings show the presence of LCs (Langerine+ cells).
• the monocytes integrated into the epithelium model of gingival and vaginal reconstructed mucosae differentiate into DCs or the LC type as demonstrated by observation of the lectin Langerine.
• Example 11 Three-dimensional pericellular reconstructed skin model
• This model is the association of a reconstructed dermis culture on which an additional culture of a reconstructed epidermis is then performed.
• the reconstructed dermis model is made according to the following protocol:
• fibroblasts of normal human skin are sown on a matrix substrate based on cross- linked collagen with diphenyl-phosphoryl azide, and then grown in a nutritious medium, for example DMEM-Glutamax supplemented with 10% calf serum, ascorbic acid and preferably at a final concentration of 1 mM, EGF ⁇ epidermal growth factor) and preferably at a final concentration of 10 ng/mL, normocin and preferably at a final concentration of 100 ⁇ g/mL, for
• the reconstructed skin model is made according to the following protocol:
• 0.5 to 1.10 6 normal human keratinocytes are sown on the dermal equivalent, and then grown in a nutritious medium, for example DMEM-Glutamax/Ham F- 12 (ratio 3/1 v/v) supplemented with calf serum, ascorbic acid and preferably at a final concentration of 1 mM, EGF ⁇ epidermal growth factor) and preferably at a final concentration of 10 ng/mL, hydrocortisone and preferably at a final concentration of 0.4 ⁇ g/mL, umulin and preferably at a final concentration of 0.12 IU/mL, isuprel and preferably at a final concentration of 0.4 ⁇ g/mL, triiodothyronine and preferably at a final concentration of 2.10 "9 M, adenine and preferably at a final concentration of 24.3 ⁇ g/mL, normocin and preferably at a final concentration of 100 ⁇ g/mL.
• the cultures are then placed at the air-liquid interface for 14 additional days in the same medium as the immersion culture, except for calf serum, hydrocortisone, isuprel, triiodothyronine and umulin.
• Example 12 Three-dimensional pericellular reconstructed dermis model containing DCCs, macrophages and endothelial cells
• the cultures are then embedded in an amorphous resin, such as Tissue-Teck ® , and then directly frozen in liquid nitrogen.
• an amorphous resin such as Tissue-Teck ®
• Immunohistochemical investigations are carried out on histological sections in order to characterize the cell types present by means of specific antibodies.
• the performed labelings show the simultaneous presence of DDC (DC-SIGN+ cells), macrophages (CD68+ cells) and endothelial cells (CD31+ and CD36+ cells).
• the monocytes integrated and then grown in our reconstructed dermis model are capable of simultaneously differentiating into DCs of the DDC type, into macrophages and endothelial cells.
• Example 13 Three-dimensional pericellular reconstructed chorion model containing IDCs, macrophages and endothelial cells
• the fibroblasts are fibroblasts stemming from normal human gingival and vaginal mucosae. - The performed labelings show the simultaneous presence of IDCs (DC-SIGN+ cells), macrophages (CD68+ cells) and endothelial cells (CD31+ and CD36+ cells).
• the monocytes integrated and then grown in our reconstructed chorion model of the gingival and vaginal type are capable of simultaneously differentiating into DCs of the IDC type, into macrophages and endothelial cells.
• Example 14 Three-dimensional pericellular reconstructed skin model containing LCs
• the culture of the reconstructed skin model is performed according to Example 11.
• the model is made according to the following protocol:
• Example 15 Three-dimensional pericellular pigmented and/or nervous reconstructed skin model containing LCs
• the model is made according to the following protocol: - 1 to 5.10 4 melanocytes and/or Merkel cells stemming from normal human skin are jointly sown with keratinocytes and monocytes on the reconstructed dermis.
• labeling of the melanocytes (HMB45) and a DOPA reaction are carried out in order to detect melanin, as well as labeling with an anti- keratin 20 antibody for identifying Merkel cells.
• Example 16 Three-dimensional pericellular reconstructed mucosa model containing LCs Obtaining the monocytes: see Example 1 or 2.
• the model is made according to the protocol described in Example 14 with the following changes: Keratinocytes are replaced with epithelial cells of normal human gingival and vaginal mucosae, and the fibroblasts are fibroblasts stemming from normal human gingival and vaginal mucosae.
• the percentage of calf serum in the culture medium for the epithelialization step is 1%.
• Growing is totally accomplished in immersion in the medium - The performed labelings show the presence of LCs (Langerine+ cells and Birbeck+ granules) in the epithelial compartment of the cultures.
• Example 17 Three-dimensional pericellular reconstructed skin model containing DCCs, macrophages and endothelial cells
• the model is made according to the following protocol:
• the performed labelings show the simultaneous presence of DCCs (DC-SIGN+ cells), macrophages (CD68+ cells) and endothelial cells (CD31+ and CD36+ cells).
• Example 18 Three-dimensional pluricellular pigmented and/or nervous reconstructed skin model containing DCCs, macrophages and endothelial cells
• the model is made according to the protocol described in Example 17, by simultaneously sowing together 1 to 5.10 melanocytes and/or Merkel cells stemming from normal human skin with keratinocytes.
• Example 19 Three-dimensional pericellular model of reconstructed mucosae containing IDCs, macrophages and endothelial cells
• Example 1 Obtaining the monocytes: see Example 1 or 2.
• the model is made according to the protocol described in Example 17 with the following changes:
• the keratinocytes are replaced with epithelial cells of normal human gingival and vaginal mucosae, and the fibroblasts are fibroblasts stemming from normal human gingival and vaginal mucosae.
• the percentage of calf serum in the culture medium for the epithelialization step is 1%. Growing is totally performed in immersion in the medium.
• the performed labelings show the simultaneous presence of IDCs (DC-SIGN+ cells), macrophages (CD68+ cells) and endothelial cells (CD31+ cells and CD36+).
• Example 20 Three-dimensional pericellular reconstructed skin model containing LCs, DCCs, macrophages and endothelial cells
• the reconstructed dermis model containing DCCs, macrophages and endothelial cells is made according to Example 12.
• the reconstructed skin model containing LCs is made according to Example 14.
• Birbeck+ granules in the epidermal compartment of the cultures, and of DCCs (DC-SIGN+ cells), macrophages (CD68+ cells) and endothelial cells (CD31+ and CD36+ cells) in the dermal compartment of the cultures.
• DCCs DC-SIGN+ cells
• macrophages CD68+ cells
• endothelial cells CD31+ and CD36+ cells
• Example 21 Three-dimensional pericellular pigmented and/or nervous reconstructed skin model containing LCs, DCCs, macrophages and endothelial cells Obtaining the monocytes: see Example 1 or 2.
• the model is made according to the following protocol: -
• the reconstructed dermis model containing DCCs, macrophages and endothelial cells is made according to Example 12.
• the pigmented and/or nervous reconstructed skin model containing LCIs is made according to
• the performed labelings detect simultaneous presence of LCs (Langerine+ cells and Birbeck granules) in the epidermal compartment of the cultures, and of DCCs (DC-SIGN+ cells), macrophages (CD68+ cells) and endothelial cells (CD31+ and CD36+ cells) in the dermal compartment of the cultures.
• LCs Longerine+ cells and Birbeck granules
• DC-SIGN+ cells DC-SIGN+ cells
• macrophages CD68+ cells
• endothelial cells CD31+ and CD36+ cells
• Example 22 Three-dimensional pericellular reconstructed mucosa model containing LCs, IDCs, macrophages and endothelial cells
• the model is made according to the following protocol:
• the reconstructed chorion model containing IDCs, macrophages and endothelial cells is made according to Example 13.
• the reconstructed mucosa model containing LCs is made according to Example 16.
• the performed labelings show the simultaneous presence of LCs (Langerine+ cells and Birbeck+ granules) in the epithelial compartment, and the presence of IDCs (DC-SIGN+ cells), macrophages (CD68+ cells) and endothelial cells (CD31+ and CD36+ cells) in the conjunctive compartment of the cultures.
• the monocytes successively cultivated in the conjunctive compartment and then with epithelial cells in the reconstructed mucosa model of the gingival of vaginal type differentiate into DCs of the LC type in the epithelial compartment and into DCs of the IDC type, into macrophages and endothelial cells in the conjunctive compartment simultaneously.
• Example 23 Use of the three-dimensional pericellular reconstructed skin model described in Example 20 for investigating the influence of solar UV radiation
• the reconstructed skins are irradiated, for example at a single dose of
• Example 24 The use of the three-dimensional pericellular reconstructed skin model described in Example 20 for investigating the efficiency of active ingredients
• the reconstructed skins are irradiated, for example with a unique dose of 526 kJ/cm 2 of solar energy corresponding to 2 J/cm 2 of UVA and 0.5 J/cm 2 of UVB (solar irradiator Suntest CPS+, ATLAS).
• Caprylate, ENGELHARD or not, are applied on the reconstructed skins for 10 days.
• the reconstructed skins are grown for a further period of 48 hours in the immersion medium and then the efficiency of the anti-oxidant treatment is assessed by: 1/ analysis of cell viability (methylthiazoletetrazolium - MTT test), 2/ flux cytometry dosage (CBA - Cytometric Bead Array) of the secretion of cytokines in the culture supernatants.
• Example 25 Use of the three-dimensional pericellular reconstructed skin model described in
• the model is made according to the protocol described in Examples 14, 17 and 20.
• the active ingredient is directly applied on the reconstructed skins by means of a swab (topical application) or the active ingredient is introduced into the culture medium (systemic application) for 10 days.
• the cultures are successively digested by 2 hr enzymatic treatments at 37°C with trypsin (1 mg/niL) and with collagenase (1 mg/mL) in order to recover the LCs and/or DCCs.
• the LCs and/or DCCs are grown for 3 days with allogenic naive T lymphocytes in an RPMI culture medium supplemented with 10% human AB serum. A range of LCs and/or DCs between 125 and 8,000 cells is achieved and grown with 10 5 naive T lymphocytes. On the 3 rd day of the mixed lymphocyte culture, 20 ⁇ l of 5 mCi tritiated thymidine are added for a period of 18 hrs. The results are expressed on a graph with the number of LCs and/or DCCs in abscissae and in ordinates the incorporation of tritiated thymidine into allogenic naive T lymphocytes expressed in cpm ⁇ counts per minute).
• the LCs and/or DCCs After treatment with our active X, the LCs and/or DCCs strongly stimulate proliferation of T lymphocytes (between 5.10 4 and 7.10 4 cpm) as compared with the untreated LCs and/or DCCs which only induce weak proliferation of naive T lymphocytes (between 1.10 and 4.10 cpm).
• Example 26 Use of three-dimensional pericellular models for performing screening of active ingredients capable of modulating allergic reactions
• the immunomodulating effect of an active ingredient after inducing an irritating, sensitizing and allergizing stress is investigated according to the following protocol:
• the model is made according to the protocol described in Example 12. - After 21 days of culture, the irritant ⁇ sodium dodecyl sulphate) preferably at the final concentration of 2% and the sensitizer DNCB (1, 4-chloro-dinitrobenzene) preferably at a final concentration of 0.25% are introduced into the culture medium for 24 hrs.
• the culture supernatants are recovered for dosing IL-10 and IL- 12 for example, and an immunohistochemical study is developed with an antibody battery: anti- CD80, anti-CD83, anti-CD86, anti-CCR7, anti-DC-LAMP, anti-HLA-DR.
• the immunomodulating profile of the tested active ingredients may be established.
• Our pericellular model is therefore a predictive tool with which it is possible to assess the potential immmunomodulating effect of an active ingredient by screening DCCs which have cytokine secretion, activation and maturation capabilities.

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