JP2010502175A - Method for producing Langerhans cells and / or dermis / stromal dendritic cells from CD14 + monocytes - Google Patents

Abstract

The present invention is a method for producing Langerhans cells (LC), or dermal / stromal dendritic cells (IDC), or LC and IDC from CD14 + monocytes derived from living peripheral blood, particularly humans, CD14 + monocytes are placed in the presence of a cellular environment containing epithelial cells such as keratinocytes and / or mesenchymal cells such as dermal fibroblasts, and CD14 + monocytes are differentiated into LC, IDC, or LC and IDC. It is related with the method characterized by this. The present invention also relates to a cell or tissue model obtained by the present method and comprising at least LC and / or IDC, and optionally macrophages and endothelial cells, and uses thereof.
[Selection figure] None

Description

  The present invention relates to a method for producing dendritic cells, in particular Langerhans cells and / or stromal dendritic cells / dermal dendritic cells, from CD14 + monocytes isolated from peripheral circulating blood, It relates to a method for creating a tissue model, in particular for testing active ingredients and / or studying related biological / biochemical phenomena.

  Dendritic cells (DC) are cells that have antigens that are indicative of the immune system. In fact, such cells are present almost everywhere, for example, in peripheral tissues such as the thymus, systemic circulatory system and second lymphoid organs, skin and single-layer or multi-layer mucous membranes. Although there are very few dendritic cells in an organism, DC is the center that triggers a specific immune response by controlling the specificity, strength, and type of immune response, and between innate and acquired immunity It is located in the middle. In addition to the function that causes an immune response, DC also has a role in causing peripheral tolerance.

  Like many immune system cells and blood cells, DC precursors are derived from the differentiation of CD34 + hematopoietic progenitor cells. They move through the skin and mucous membranes by blood flow, differentiate and settle there as immature DCs. This immature state has a characteristic phenotype and has the ability to capture antigens very effectively. Two different phenotypes of peripheral DC are known, depending on their location within the cell.

-Langerhans cells (LC) are present in the epithelium of malbigi tissue (skin and mucous membranes), where they specifically secrete Langerin, a C-type lectin. Langerin is involved in the formation of a cytoplasmic substance, Barbeck, which is a reference label specific to LC having an ultrafine structure. LC also expresses characteristic labels such as CD1a and HLA-DR.

-Interstitial DC (IDC) is found in the mucosal layer and also in the dermis of the skin. In the latter case, these are described as skin DC (DDC). These cells have many similarities and share a common label with the monocytes / macrophage cell line. In the dermis of the skin, DCC specifically expresses DC-SIGN and also expresses characteristic labels such as HLA-DR, FXIIIa, MMR, CD1a. In the following, interstitial dendritic cells (IDC) in general will be described, regardless of whether they are present in the mucosal layer or the dermal dermis.

  After capturing the antigen, the LC and IDC move towards the lymph node and pass the antigen information to the T lymphocytes. This movement with the activation of LC and IDC appears as a phenotypic change and a functional change. For example, “activated” LC and IDC will express the costimulatory labels CD80 and CD86, and will express the CCR7 receptor, which is essential for skin migration of these cells. In lymph nodes, activated LCs and IDCs acquire a “mature” phenotype, or they form “entangled” DCs by interaction with T lymphocytes. This maturation condition is synonymous with the expression of characteristic phenotypes such as CD83 and DC-LAMP, and is synonymous with the ability to induce strong external stimuli, ie T lymphocyte proliferation. LC and IDC cause many skin pathologies, such as contact allergies, due to their ability to move exoantigens to nearby lymph nodes after capture and have recently been the first target of human immunodeficiency virus (HIV) Yes.

Interesting uses of LC and IDC, particularly of LC and IDC in combination with skin or mucosal fibroblast-type epithelial cells or mesenchymal cells, include the following three-dimensional organ types: To be in culture:
-"Regenerated epidermis" model and "regenerated mucosa" model using only LC (eg, vagina and oral model) (Regnier et al, JID 1997; 109: 510-2; Patent EP 0789074 of L'OREAL; Sivard et al, Exp. Dermatol. 2003; 12: 346-55),
“Regenerative dermis” and “regenerative chorion” models using DCC or IDC (Guironet et al, JID 2001; 116: 933-9 and Dumont et al., AIDS Res. Hum. Retroviruses 2004; 20: 383-97 ).

Currently these are rarely used. This is for the following reason.
1. There is no fast, simple and inexpensive industrial method for producing LC and IDC in vitro.
2. An incomplete 3D model that has only epithelial or connected portions of regenerative tissue and never has portions that include both related tissue portions.

The reports of Regnier et al., Sivard et al., And Dumont et al. Deal with CD34 + precursors from umbilical cord blood and contain the following significant drawbacks:
1. Since the amount of umbilical cord blood used for extraction is small, the number of CD34 + progenitor cells to be separated is small, and industrial utilization is difficult.
2. Differentiation of cells into LC or IDC requires pre-culture (6-12 days) of CD34 + progenitor cells before incorporation into the 3D model.
3. The described method requires the addition of exogenous cytokines to the medium in order to cultivate regenerated epidermis, regenerated mucosa, regenerated chorion containing LC or partial IDC.

In the report of Guironnet et al., The authors generate DCC from blood monocytes and incorporate it into a regenerated dermis model. Similarly, there are two drawbacks:
1. Differentiation into DCC requires monocyte pre-culture (6 days) before incorporation into regenerated dermis.
2. In order to culture regenerated dermis containing DCC, it is necessary to add exogenous cytokines to the medium.
In the same report, the authors report that monocytes taken directly into the dermal equivalent without added cytokines do not differentiate into DCs.

  In L'Oreal, US Pat. No. 6,130,482, keratinocytes and LC precursors are co-cultured and then incorporated into a suitable nutrient medium, especially in the epidermis model, so that the precursors differentiate into LC. The product is co-cultured to evaluate the stimulating activity or allergic activity of a product. However, in this patent application, Caux et al., Nature, 1992 Nov. 19, 360 (6401): 258-61, CD34 + hematopoietic progenitor cells derived from umbilical cord blood have been used. The method described in this report includes culturing CD34 + hematopoietic progenitor cells in the presence of exogenous cytokines. This patent generally allows the precursor of LC to express CD1a +, but only describes the use of CD34 + hematopoietic cells as the CD34 + precursor of LC. However, the number of CD34 + hematopoietic cells in peripheral blood is small, and it is difficult to develop an industrially satisfactory differentiation method. The use of umbilical cord blood-derived precursors is not preferred because this blood is not available in large quantities.

Recently, BASF Beauty Care Solution France (Coletica, French patent FR 2833271B1, filed on December 1, 2001) differentiated CD14 + monocytes into LC, IDC, a small amount of LC and a small amount of DCI. Is cultured in the presence of human skin or mucosal epithelial cells and / or mesenchymal cells. The following are described in this patent.
-"Regenerated epidermis" model containing only LC and "Regenerated mucosa"
A “regenerated dermis” model and a “regenerated chorion” model containing DCC or IDC
-"Regenerative skin" model and "Regenerative mucosa" model containing LC and IDC simultaneously.

  As mentioned above, the disadvantages of existing methods are that in the presence of exogenous cytokines, monocytes prior to incorporation into the 3D model are pre-cultured (6 days) to place the cells in LC, IDC, or LC. It is necessary to differentiate the IDC at the same time.

  For the purpose of providing LC and / or IDC to develop an immunocompetent tissue model that approximates normal human skin, to date, the differentiation of monocytes into LC and / or IDC has been described by Engel. Only the method developed by Hardlion, namely patent FR 2833271B1. However, this method can also be further improved using simpler, cheaper and faster differentiation methods.

US Patent US 6,130,482 French patent FR 2833271B1

Nature, 1992 Nov. 19, 360 (6401): 258-61

  The present invention overcomes each of the above technical problems for the first time in a safe, reliable and reproducible manner, industrially and commercially, in particular for agricultural feed and / or medical and / or drug and / or cosmetic applications. Available on an industrial scale.

  The present invention mainly produces LC or IDC from biological precursors derived from peripheral circulation, simultaneous generation of LC and IDC, simultaneous generation of IDC and macrophages and endothelial cells, or simultaneous LC and IDC, macrophages and endothelial cells. Consists of generation.

  In particular, the present invention relates to an epidermis, epithelium, dermis, chorion, skin or mucosa model containing the above cells (LC, IDC / DCC, etc.) It provides the best quality model for regenerating skin or mucous membranes. It is another object of the present invention to provide epithelial and / or connecting sheets and skin or mucosal immunocompetent equivalents.

  In particular, the present invention comprises providing a method for differentiating peripheral circulating blood-derived monocytes to obtain the above cells (LC, IDC / DCC, etc.).

  The present invention also consists of providing the above model as an alternative to animal testing, especially methods for testing the irritancy and / or sensitization of cosmetic ingredients.

  Another object of the present invention is to provide the above model for the testing of active ingredients, in particular cosmetics, dermatological drugs, medicines, in particular for testing their activity and / or toxicity or drug toxicity.

  Another object of the present invention is to provide the above model for molecular or chemical testing, especially for these toxicity tests.

  The present invention also provides the above model for the assessment of biological / biochemical phenomena in the cell and at the intracellular level.

  The main object of the present invention is to provide a model / tool for toxicological tests for the purpose of conducting in vitro tests to predict the allergenicity / irritation / sensitization of external reagents, for example. is there.

  Yet another object of the present invention is to provide a model / tool for investigating substances with immunomodulatory activity.

  The invention also relates to the use of the above model for tissue engineering or cell engineering, in particular for tissue engineering or cell engineering for repairing at least part of a biological tissue.

  Unless otherwise specified, “cells” are always “live cells” within the scope of the present invention.

  The “peripheral circulating blood” of the present invention is the blood of any organism having a blood system in which the blood forms a circuit, in particular peripheral blood, in particular animal blood, mammals, preferably human blood.

  An “active ingredient” is any substance, product or composition that has an interesting activity in a potentially industrial aspect, particularly in the aspects of agricultural feed, food, dermatologic drugs, drugs, cosmetics industry, and the like.

  “Addition of exogenous cytokine” means adding at least one cytokine in addition to the cytokine synthesized by the cells present in the cell culture medium concerned.

The present invention thus relates to the −LC of CD14 + monocytes in the presence of epithelial cells and / or mesenchymal cells such as fibroblasts,
-IDC,
-LC and IDC,
-IDC and macrophages and / or endothelial cells,
-Regarding LC, IDC and differentiation into macrophages and / or endothelial cells.

  In particular, the present invention relates to differentiation of CD14 + monocytes into LC and / or IDC (differentiation without preculture under differentiation promoting conditions, in particular without preculture using exogenous cytokines), in particular LC and / or Differentiation in the presence of epithelial cells and / or mesenchymal cells of fibroblast type, for example, in order to obtain a cell model comprising IDC, preferably added with a substantial amount of exogenous cytokines Without differentiation, ie without adding any cytokines other than endogenous cytokines synthesized by cells present in the medium.

In particular, the present invention comprises the following steps:
Taking CD14 + monocytes from circulating blood, in particular human circulating blood;
Maintaining CD14 + monocytes under conditions that do not promote differentiation into DC; and-CD14 + monocytes under epithelium such as keratinocytes under conditions that do not differentiate CD14 + monocytes into LC, or IDC, or LC and IDC. The present invention relates to a method for differentiating CD14 + monocytes into LC and / or IDC, comprising the step of contacting a cell environment containing mesenchymal cells such as cells and / or fibroblasts.

  In particular, in order to disseminate CD14 + monocytes in a short time or immediately after collection from circulating blood, the step of maintaining CD14 + monocytes under conditions that do not promote differentiation into CD should preferably be limited in time.

  The conditions that do not promote the differentiation of CD14 + monocytes into DCs are preferably those obtained under culture conditions that do not contain any exogenous cytokines. In particular, the purpose is that CD14 + monocytes do not enter the DC differentiation pathway.

  In particular, the present invention allows the pre-culture of CD14 + monocytes with a certain level of cell death to be avoided, thus increasing the precursor / cell ratio.

  DC generated extracellularly is a very sensitive and fragile cell. The first variable that needs to be examined is the yield of cells obtained. It is clear that when DCs are seeded in either cell culture model, the amount of DC and / or cell mortality is large, although the amount cannot be determined. Utilizing the CD14 + monocytes of the present invention as a DC precursor can overcome this problem and lead to an increase in cell mass.

  According to the present invention, monocytes can be differentiated into LC or IDC in 6 days of culture, so that the time can be greatly reduced.

  According to the present invention, since there is preferably no use of exogenous cytokines, it is particularly advantageous in terms of reactants.

  According to the present invention, it is particularly advantageous in terms of the quality of the produced cells. The generation of DC from monocytes in vitro is related to the phase of cell differentiation under the influence of soluble immune mediators, cytokines. Recently, a report (Nicolas Becheoil et al., Journal of Leukocytes Biology: a mixture of monocyte-derived Langerhans cells and stromal / dermal dendritic cell subsets is prone to inflammatory stimuli in Th2-mediated inflammatory conditions. According to each of the different reactions, it is stated that the LC and DDC DCs derived from monocytes are not “synchronized” during the cell differentiation process. In other words, CD14 + monocytes that have begun to differentiate generate LC and DCC that are not all in the same differentiation stage. By avoiding this differentiation stage, this imperfection can be eliminated. The monocytes cultured in the present invention are affected by the cells and the environment of cytokines and matrix of cells regenerated, and form a more physiological environment for DC differentiation.

  It is well known that DCs generated in vitro have the ability to “mature” spontaneously, but this is a major problem because mature DCs are no longer activated or stimulated. is there. The present inventors control the immature conditions of these cells in vitro, but eliminate the risk of spontaneous maturation in vitro by using monocytes directly as precursors of LC and DCC. Can do. In addition, the advantage of using the cell or tissue culture model according to the present invention as a differentiation system for CD14 + monocytes into DC is physiological. This is because the natural conditions of DC differentiation in the skin and mucous membranes can be reproduced better, and immature DCs similar to homologs in vivo can be obtained.

  In the same sense, by using CD14 + monocytes as precursors to DCs rather than using pre-generated DCs, phenotypically more immature and functionally more sensitive LCs and DCCs in the current model Can be cultured. Indeed, in the same sense, we have recently compared to DCs derived from CD34 + progenitor cells and cultured in regenerated chorion in the presence of exogenous cytokines compared to homologs cultured in the presence of exogenous cytokines. Have shown that they have more satisfactory non-mature conditions in terms of HIV virus replication. (Sandy Domont et al., When incorporated into the submucosal mucosal equivalent, dendritic cells remain immature and have the ability to replicate R5 HIV-1 species in the absence of T cell subsets. AIDS Res. And Hum.Retroviruses 20: 383-397, 2004). The obtained DC is very different from that already reported, and particularly different from that of the French patent FR 2833271B1 (filed on Dec. 10, 2001).

  Furthermore, the use of CD14 + monocytes directly as a precursor of skin DC provides a more physiological method compared to previously reported methods. In fact, in living organisms, blood monocytes invade the skin, where cells, cytokines, and matrix environments control their differentiation. Although pre-differentiated DCs can be incorporated into this culture model, they are still unsatisfactory at present compared to methods using their precursor CD14 + monocytes directly. This improved method means, from an immunohistological perspective, a more uniform cell distribution of LC and DCC in this three-dimensional culture model.

  According to the present invention, freshly isolated monocytes may be frozen prior to use in the model.

  According to the present invention, a cell having substantially the same phenotype and the same function as a homologue in a living body can be generated.

The present invention, particularly when co-cultured,
-Phenotypically and / or functionally differentiated into LC in the epithelial environment,
-In a connected environment, it relates to unique cell precursors that differentiate phenotypically and / or functionally into IDCs, macrophages and endothelial cells.

  Thus, according to a first aspect, the present invention provides a method for producing LC, or IDC, or LC and IDC, from CD14 + monocytes from an organism, particularly human peripheral circulation, comprising CD14 + monocytes Characterized by differentiating CD14 + monocytes into LC, or into IDC, or into LC and IDC by placing the cell in a cellular environment containing epithelial cells such as keratinocytes and / or mesenchymal cells such as dermal fibroblasts On how to do.

  Conveniently, differentiation of CD14 + monocytes results in IDC and macrophages and endothelial cells or LC and IDC, macrophages and endothelial cells. Conveniently, the distribution of LC and IDC cell populations varies with the type of cells cultured with monocytes. The use of keratinocytes increases the differentiation to LC, and the use of fibroblasts increases the differentiation to IDC.

  Conveniently, monocyte differentiation can be caused without the addition of any exogenous cytokines. In certain embodiments, no exogenous cytokine is added.

  Conveniently, monocytes growing in an epithelial cell environment such as keratinocytes promote the differentiation of monocytes into immature functional LC. As intended by the present inventors, “immature” means that these monocytes do not express or express an activation label (CD80, CD86, CCR7) or a mature label (CD83, DC-LAMP). Even very small amounts. On the other hand, immature LCs express CCR6.

  As intended by the present inventors, “functional” means having an antigen internalization ability (immature condition), a cell migration ability (immature activation condition), and an antigen presentation ability (maturation condition). Say.

  In certain embodiments, culturing monocytes in a mesenchymal cell environment such as dermal fibroblasts promotes the differentiation of monocytes into immature functional IDCs. See definition above. However, immature IDC does not express CCR6.

  Conveniently, culturing monocytes in an environment containing epithelial cells such as keratinocytes and mesenchymal cells such as dermal fibroblasts promotes differentiation of monocytes into typical LC and IDC. In our intent, “typical” means that they are close to their intracellular homologues in terms of immature phenotype and function, i.e. in terms of their ability to react after stimulation or stress. Say.

  The ratio of epithelial cells and / or mesenchymal cells used for culture (differentiation) to CD14 + monocytes depends on the desired cell distribution between LC and / or IDC and epithelial cells and / or mesenchymal cells is doing.

  According to a second aspect, the present invention provides a method of culturing CD14 + monocytes comprising a fusion of CD14 + monocytes derived from the peripheral circulating blood of an organism, in particular a human, within a cell or tissue model, Or the tissue model includes epithelial cells such as keratinocytes and / or mesenchymal cells such as dermal fibroblasts, and in which CD14 + monocytes are differentiated into LC, or into IDC, or into LC and IDC Therefore, the present invention relates to a method comprising culturing the CD14 + monocytes in the presence of epithelial cells such as keratinocytes and / or mesenchymal cells such as dermal fibroblasts.

  It is preferred to culture monocytes without adding any exogenous cytokines.

  This cell or tissue model is selected from the group consisting of epidermis model, epithelial model, dermis model, chorion model, skin model or mucosal model, and particularly preferably gingival or vaginal mucosa.

  This three-dimensional culture model is preferably a skin or chorionic matrix support selected from the group consisting of:

-Collagen-based or fibrin-based gels or films containing mesenchymal cells, in particular fibroblasts,
A collagenous porous matrix which may contain one or more glycosaminoglycans and / or chitosan if necessary and may or may not contain mesenchymal cells, in particular fibroblasts. ,
An inert support comprising any of the following groups of materials, which may or may not contain mesenchymal cells, in particular fibroblasts: semipermeable synthetic membranes, in particular semipermeable nitrocellulose Membrane, semipermeable nylon membrane, polytetrafluoroethylene (Teflon (R), PTFE) membrane or sponge, semipermeable polycarbonate or polyethylene terephthalate (PET) membrane, inorganic having a capillary porous structure of aluminum oxide Membrane (semipermeable anopore membrane), semipermeable polyester membrane,
An inert support treated for polycarbonate-based or polystyrene-based culture, which may or may not contain mesenchymal cells, in particular fibroblasts.

  The tissue model to be used is preferably the above-mentioned skin or chorion support on which epithelial cells, particularly keratinocytes are mounted.

  This cell or tissue model can be applied to at least one other cell type, such as nerve cells and / or endothelial cells and / or melanocytes and / or lymphocytes and / or fat cells and / or skin such as bristles, hair, sebaceous glands. It is preferable to have a kimono.

  It is preferred that some CD14 + monocytes differentiate into endothelial cells and macrophages when taken into a cell or tissue model containing at least mesenchymal cells.

  Preferably, the method comprises mainly LC, or IDC, or a mixture of LC and IDC, or a mixture of LC, IDC, endothelial cells and macrophages, or a mixture of IDC, endothelial cells and macrophages.

  Preferably, the cell or tissue model has an epithelial portion and a connecting matrix, the LC is substantially localized in the epithelial portion, and the IDC, macrophage and endothelial cells are substantially localized in the connecting matrix. Features.

  According to a third aspect, the present invention relates to a cell model obtained by the above method, comprising at least one LC and / or IDC cell, and optionally macrophages and / or endothelial cells.

  The model is characterized in that the obtained DC is different from that obtained by the above method, and the main difference is in the synchronism of LC and IDC cell differentiation processes.

  According to a fourth aspect, the present invention provides an epidermis model obtained by one of the above methods, comprising at least one of the LC and / or IDC, and, if necessary, macrophages and / or endothelial cells. And a tissue model selected from the group consisting of an epithelial model, a dermis model, a chorionic model, a skin model and a mucosal model, particularly a tissue model of the gingiva or vaginal mucosa.

  The cell or tissue model is preferably immunocompetent.

  The tissue model includes an epithelial portion containing epithelial cells such as keratinocytes and a connected matrix containing mesenchymal cells such as between skin or chorionic fibroblasts, and LC is substantially localized in the epithelial portion, IDC, It is preferred that macrophages and / or endothelial cells are substantially localized in the connected matrix.

  The above model is characterized in that the obtained DC is different from that obtained by the above method, and the main difference is in the synchronization of the cell differentiation process of LC and IDC in this three-dimensional culture model.

  According to a fifth aspect, the invention relates to frozen CD14 + monocytes isolated from the peripheral circulating blood of an organism, in particular humans.

  In particular, according to the present invention, it is possible to prepare a plurality of DC cell groups having different functionalities that can explain the entire phenomenon involved in the defense / infection process of organisms, such as stimulation, allergy, and sensitization.

  Thus, according to a sixth aspect, the present invention provides a test model for cosmetics, dermatology, pharmaceutics and / or selection of active ingredients of at least one cell model or tissue model as defined above. Regarding usage.

  Another interesting use of LC and / or IDC is to use them to assess the ratio of stimulating and sensitizing properties of new molecules. EU Directive 2003/15 / EC, which prohibits the use of animals for toxicological testing of cosmetic end products since 2005, is an in vitro evaluation method and in vitro evaluation for sensitization testing of new molecules to public test organs and industrial testing institutions. Seeking the development of a method. Due to its important role in contact allergy, the use of dermal DC as an alternative to animal testing is currently the mainstay of development.

  According to a seventh aspect, the present invention relates to an assessment of the phenomena and activities that occur in the defense / infection process of an organism, in particular the immunostimulation of specific active ingredients or the at least one cell model or tissue model as defined above. For the evaluation of immunosuppressive activity, for the evaluation and induction of immune modulation by the above active ingredients, for in vitro testing for predicting allergic, stimulating, sensitizing activity of external reagents, or for molecules or chemicals In particular for use in conducting toxicity studies.

  According to an eighth aspect, the present invention relates to the physiopathology of the epithelial barrier of at least one cell model or tissue model as defined above; irritation of the skin and mucous membranes; biological attack such as viruses and Attacks of retroviruses such as HIV, bacteria, filamentous fungi, microorganisms, particle antigens; phototoxicity; photoprotection; and effects of active ingredients, especially cosmetic or pharmaceutical active ingredients; end products, especially cosmetic or pharmaceutical products Effect; relating to use in testing mechanisms of infection by pathogenic substances.

  Preferably, the present invention relates to the evaluation of toxicity of active ingredients and the like. In particular, this test is performed by measuring the labeling of cells, in particular the labeling of DC.

  According to a ninth aspect, the invention relates to the testing of the mechanism of the infection, replication, transmission phenomenon of viruses, including retroviruses such as HIV, of at least one cell model or tissue model as defined above, or It relates to the search and development of new therapies, including the administration of vaccines and drugs.

  According to a tenth aspect, the present invention provides a pathogenic substance, for example a virus, a retrovirus such as HIV, a bacterium, a filamentous fungus, a microorganism, a particle antigen, etc. of at least one cell model or tissue model as defined above. Related to the detection of the presence of

  According to an eleventh aspect, the invention relates to the use of at least one cell model or tissue model as defined above for medical, biomedical or cosmetic applications, in particular the regulation of an immune or tolerance response in vitro or in the body. For the prevention or treatment of immune or tolerance responses, particularly as a result of environmental attacks, especially physical attacks such as UV radiation, chemical attacks such as irritants / allergies / sensitizers and biological attacks Regarding use for adjustment.

  According to a twelfth aspect, the invention relates to the use of at least one cell model or tissue model as defined above for tissue engineering and cell engineering applications, medical or biomedical applications, in particular immune Anti-cancer cell therapy by DC injection method that can stimulate response; use in cell therapy in case of autoimmune disease, induction of immune tolerance stimulation by production of, for example, anergic T cells; gene therapy for diseases affecting immune system Use for, or use for manufacturing vaccine development.

  According to a thirteenth aspect, the present invention relates to a method for manufacturing a tissue model comprising the following steps.

Isolating CD14 + monocytes from the peripheral circulating blood of the organism,
-Seeding skin or mucosal cells on a support and culturing in nutrient medium to obtain regenerated tissue;
-Seeding the regenerated tissue with CD14 + monocytes simultaneously with or not with skin or mucosal cells;
-Differentiating the regenerated tissue containing CD14 + monocytes and skin or mucosal cells into LC, a mixture of IDC and endothelial cells and / or macrophages, and a mixture of LC and IDC and endothelial cells and / or macrophages. (If the regenerated tissue is an epidermis model, the skin cells are epidermal keratinocytes, and if the regenerated tissue is a dermis model, the skin cells are skin fibroblasts and the regenerated tissue is epithelial. In the case of a model, the mucosal cells are epithelial mucosal cells, and in the case where the regenerated tissue is a chorionic model, the mucosal cells are mucosal fibroblasts).

  These epithelial cells are isolated from at least one type of skin tissue, for example.

  The seeding of these skin or mucosal cells may be performed before or after seeding of CD14 + monocytes.

  CD14 + monocytes are preferably seeded simultaneously with the skin or mucosal cell seeding.

  According to a fourteenth aspect, the present invention comprises a regeneration comprising keratinocytes or epithelial mucosal cells, and optionally an epithelial portion comprising Merkel cells and / or melanocytes and a connected matrix comprising dermis or chorionic fibroblasts. The present invention relates to a method for producing skin or regenerated mucosa, comprising the following steps.

The surface of a dermis or chorionic model containing keratinocytes or epithelial mucosal cells in a nutrient medium, optionally in the presence of Merkel cells and / or melanocytes, and containing IDC and optionally endothelial cells and / or macrophages In the presence of the keratinocytes or epithelial mucosal cells, wherein the dermis or chorion model is obtained by the method for producing a dermis or chorion model as defined above, and A step of seeding and culturing CD14 + monocytes isolated from the peripheral circulating blood of an organism at the same time or not simultaneously with keratinocytes or epithelial mucosal cells under the condition that the cells differentiate into LC.

  The methods for producing these tissue models preferably do not require the addition of exogenous cytokines.

  Other objects, features and advantages of the present invention will become more apparent to those skilled in the art after reading the description of the embodiments. In addition, these Examples are provided for the purpose of explanation and do not limit the scope of the present invention.

  These examples are an important part of the present invention, and as a whole from the specification, any feature that appears to be new compared to the prior art, including the examples, in its function and generality, It is an important part of the present invention.

  Accordingly, each embodiment has a general scope.

  In the examples, percentages are in terms of mass unless otherwise specified, temperature is in degrees Celsius unless otherwise specified, and pressure is atmospheric pressure unless otherwise specified.

Example 1 Method for Separating Monocytes from Peripheral Circulating Blood Venous blood is collected from one or more human donors, preferably in a vacuum blood collection system or bag containing a normal anticoagulant product such as lithium heparin. Circulating blood was obtained.

Preferably, monocytes are separated from circulating blood by the following method.
1. The blood is centrifuged using a medium for lymphocyte separation, and mononuclear cells are collected.
-Label with an antibody cocktail such as anti-CD3, anti-CD7, anti-CD16, anti-CD19, anti-CD56, anti-CD123 antibody or anti-glycophorin A bound to magnetic beads. Pass through a magnetized column and collect only the unlabeled monocytes that elute.
Or labeled with an antibody specific for monocytes bound to magnetic beads, for example with an anti-CD14 antibody. Through the magnetic column, only labeled monocytes are retained in the column. After elution from the column, the labeled monocytes are recovered.
-Or labeled with an antibody specific for monocytes, eg anti-CD16 antibody, conjugated to a fluorescent dye such as phycoerythrin. After sorting the cells by flux cytometry, collect only the labeled monocytes.
2. These monocytes are separated using any physical separation method well known to those skilled in the art, particularly using sedimentation or centrifugation, and used directly in subsequent cultures.
3. From a 100 mL sample of blood, up to about 150 million (± 20 million) mononuclear cells are extracted and up to 40 million monocytes are purified.

Example 2 Method for Freezing Monocytes Isolated from Peripheral Circulating Blood Monocytes obtained in Example 1 were suspended in a nutrient medium, such as RPMI medium supplemented with serum and a cryoprotectant such as DMSO (dimethyl sulfoxide). Cloudy and then frozen.
Cell mortality upon monocyte thawing is less than 30%.

From the peripheral circulating blood of a 100 mL sample, up to 80 × 10 ⁶ monocytes are frozen and up to 76 × 10 ⁶ monocytes are recovered after thawing.

Example 3: Co-cultured keratinocytes and LC multi-cell monolayer model Obtaining monocytes: see Example 1 or Example 1 in culture dishes, for example in 6-well culture dishes, 1-2 x 10 ^Six human keratinocytes and 1-2 × 10 ⁶ human monocytes (obtained in Example 1 or 2) are cultured in a nutrient medium, for example in a K-SFM type medium. Co-culture is continued for 6 days in nutrient medium, for example K-SFM type medium, without adding exogenous cytokines.

The cells are recovered by enzymatic methods well known to those skilled in the art, in particular by trypsin treatment, and cells composed of 2 × 10 ⁵ keratinocytes and monocytes in a mixed cell lysate together with monoclonal anti-langerin antibodies are collected. Incubate and analyze by flux cytometry. Up to 40% Langerin + LC is observed.

Example 4: Co-cultured fibroblasts, DDC and multi-cell monolayer model Obtaining monocytes: see Example 1 or Example 2 in a culture dish, for example in a 6-well culture dish, 1-2 × 10 ⁶ human keratinocytes and 1 to 2 × 10 ⁶ human monocytes (obtained in Example 1 or 2) are cultured in a nutrient medium, for example, an FBM type medium. Co-culture is continued for 6 days in nutrient medium, eg FBM type medium, without the addition of exogenous cytokines.

The cells are collected by an enzymatic method well known to those skilled in the art, particularly by trypsin treatment, and cells composed of 2 × 10 ⁵ keratinocytes and monocytes in a mixed cell lysate are treated with monoclonal anti-DC-SIGN. Culture with antibody and analyze by flux cytometry. Up to 60% DC-SIGN + DCC is observed.

Example 5: Use of the single-layer multicellular model described in Examples 3 and 4 to examine the distribution of cytokines secreted by the effect of the active ingredient Evaluation of stimulation, sensitization and allergic activity of the active ingredient for human skin And for predicting inflammatory or anti-inflammatory activity, cytokines secreted into the culture supernatant, eg IL-1 and IL-6, IL-8, IL-10, TNF- α and INF-γ are quantified.
This model is made by the method described in Example 3 or 4.
-Add retinol to the culture medium to a final concentration of 0.05% and let stand for 3 days.
-The culture supernatant is then collected and analyzed.

  It can be seen that retinol causes stimulation of inflammatory cytokine secretion.

Example 6: Use of the active ingredient of the multi-cell monolayer model described in Examples 3 to 4 for the evaluation of immunostimulatory or immunosuppressive activity Generates an immune response and / or tolerance to active and inactive ingredients In order to evaluate the ability of LC and DCC to induce a response, the type of phenotype was examined by flux cytometry in the following manner.
-Make a model by the method described in Example 3 or Example 4.
-The active ingredient is then added in various concentrations to the culture medium and left for 3 days.
-The cells are then harvested by trypsinization.
Mixed cell lysates containing 2 × 10 ⁵ cells consisting of keratinocytes and LC, or fibroblasts and DCC, were prepared from various antibodies (anti-CCR7, anti-HLA-DR, anti-CD80, anti-CD83, anti-CD86, Culture in a solution containing anti-DC-LAMP).

  By determining the cell phenotype, it is possible to determine the immune activity (induction and / or increase in labeled molecule expression) or immunosuppression status (inhibition and / or suppression of labeled molecule expression) of the tested active ingredient.

Example 7: Use of the multi-cell monolayer model described in Examples 3-4 for measuring the immunomodulatory activity of an active ingredient The immunomodulatory effect of the active ingredient after stimulation, sensitization, or allergic stress,
Check by the following method.
-This model is made by the method described in Example 3 or Example 4.
-300 μl of TNP (2,4,6-trinitrobenzenesulfonic acid) is then added at a concentration of 5 mM and left at 37 ° C. for 30 minutes.
-After this stimulation, the active ingredient is added to the culture medium in various concentrations and left for 2 days.
-The medium containing the cells is collected for examination of the secretion of immunocytokines such as IL-12. The collected cells are cultured with anti-langerin antibody or anti-DC-SIGN antibody, and then LC or DCC is separated by flux cytometry, respectively.
-Seed separated LCs or DCCs in Boyden-type transfer chambers (5-8 μm membrane pores with or without MATRIGEL ^™ coating) and incubate at 37 ° C. for up to 72 hours.
-Determine the number of LCs or DCCs moved, for example by counting with an optical microscope.

  As a result of migration tests and / or IL-12 synthesis and secretion tests, the immunomodulatory properties of the active ingredient under test are revealed.

Example 8: Three-dimensional multicellular regenerative dermis model with LC Obtaining monocytes: See Example 1 or Example 2.

This model was produced by the following method.
-0.5 to 1 × 10 ⁶ normal human skin keratinocytes are seeded in a Boyden chamber type (porous membrane with a pore size of 0.4 μm) insert and in a nutrient medium, for example, preferably a calf having a final concentration of 1 mM Serum and ascorbic acid, preferably EGF (epidermal growth factor) with a final concentration of 10 ng / mL, preferably hydrocortisone with a final concentration of 0.4 μg / mL, preferably a concentration with a final concentration of 0.12 IU / mL Umulin, preferably isplel with a final concentration of 0.4 μg / mL, and preferably triiodothyronine with a final concentration of 2.10 ⁻⁹ M, preferably adenine with a final concentration of 24.3 μg / mL, preferably final The cells are cultured for 2 days in a DMEM-glutamax type medium supplemented with normosin at a concentration of 100 μg / mL.

1-5 × 10 ⁴ monocytes are then seeded on the surface corresponding to the epithelium, which is further cultured for 2 days.
-This culture is further cultured for 10 days at the gas-liquid interface in the same medium used for immersion culture (but not calf serum, hydrocortisone, umulin, isprel and triiodothyronine).
-The culture is placed in an amorphous resin such as Tissue Tech (R) and frozen directly in liquid nitrogen.
Perform immunohistochemical examination of histological sections and identify cell types using specific antibodies.
-As a result of labeling, the presence of LC (Langerin + cells) is confirmed.

Conclusion As shown by the observation of lectin, ie Langerin, monocytes added in the regenerated epidermis model differentiate into LC-type DCs.

Example 9: Three-dimensional multicellular staining and / or neuronal regeneration epidermis model with LC Obtaining monocytes: see Example 1 or Example 2 It is prepared by simultaneously seeding 1 × 10 ⁵ normal human skin-derived melanocytes and / or Merkel cells and keratinocytes.

  In addition to the labeling described in Example 8, a melanocyte (HMB45) labeling and DOPA reaction is performed to detect melanin, and an anti-keratin 20 antibody is labeled to identify Merkel cells.

CONCLUSIONS As shown by the observation of lectins, ie Langerin, monocytes incorporated into colored and / or neural regenerating epidermis models differentiate into LC-type DCs.

Example 10: Three-dimensional multicellular epithelial model of gingival and vaginal regenerating mucosa with LC Obtaining monocytes: see Example 1 or Example 2 This model is made by the method described in Example 8. However, some changes are made as follows.

Replace normal human keratinocytes with normal human gingival and vaginal mucosal epithelial cells.
The concentration of serum in the culture medium is 1%.
-Cultivation is carried out by dipping in the medium.
-The labeling result indicates the presence of LC (Langerin + cells).
Conclusion As shown by the observation of lectin, ie Langerin, monocytes incorporated into epithelial models of gingival and vaginal regenerating mucosa differentiate into DC or LC type.

Example 11: Three-dimensional multi-cell regenerative skin model This model is obtained by further culturing a regenerated epidermis on a regenerated dermal culture.

This regenerated dermis model is produced by the following method.
-0.5 to 1 × 10 ⁶ normal human skin fibroblasts are seeded onto a collagen matrix substrate cross-linked with diphenylphosphoryl azide and preferably in nutrient medium, for example with 10% calf serum Is cultured for 14 days in DMEM-glutamax supplemented with ascorbic acid at a final concentration of 1 mM, preferably EGF (epidermal growth factor) at a final concentration of 10 ng / mL, and preferably at a final concentration of 100 μg / mL.

This regenerated skin model is obtained by the following method.
-0.5 to 1 x 10 ⁶ normal human keratinocytes are seeded on the skin equivalent and in nutrient medium, for example calf serum, preferably with a final concentration of 1 mM ascorbic acid, and also preferably with a final concentration Is 10 ng / mL EGF (epidermal growth factor), preferably a final concentration of 0.4 μg / mL hydrocortisone, preferably a final concentration of 0.12 IU / mL umulin, and preferably a final concentration of 0.4 μg / Ml isprel, preferably a final concentration of 2 × 10 ⁻⁹ M triiodothyronine, preferably a final concentration of 24.3 μg / mL adenine, and preferably a final concentration of 100 μg / mL normosin Cultured in DMEM-glutarnax / ham F-12 (ratio = 3/1 V / V). Incubation is continued for 7 days under immersion conditions.

  The culture is placed at the gas-liquid interface and further immersed in the same medium for 14 days. However, calf serum and hydrocortisone, isprel, triiodothyronine and umulin are excluded.

Conclusion Both cell types of skin, epidermis and dermis can be regenerated in vitro.

Example 12: Three-dimensional multicellular regenerative dermis model containing DCC, macrophages and endothelial cells Obtaining monocytes: See Example 1 or Example 2 Regenerated dermis is cultured according to Example 11.

This model is obtained by the following method.
−1 to 5 × 10 ⁴ monocytes are seeded on the surface of the skin equivalent and further cultured for 7 days.
-The culture is contained in an amorphous resin such as Tissue Tech® and directly frozen in liquid nitrogen.
Perform immunohistochemical examination of histological sections and identify cell types using specific antibodies.
-As a result of labeling, the coexistence of DDC (DC-SIGN + cells), macrophages (CD68 + cells) and endothelial cells (CD31 + cells and CD36 + cells) can be seen.

Conclusion These monocytes that have been incorporated into the regenerated dermis model and cultured can differentiate into DDC-type DC, and can differentiate into macrophages and endothelial cells.

Example 13: Three-dimensional multicellular regenerative chorionic model containing IDC, macrophages and endothelial cells Obtaining monocytes: see Example 1 or Example 2 This model was made by the method described in Example 12. The differences are as follows.
The fibroblasts used are those derived from normal human gingiva and vaginal mucosa.
As a result of labeling, coexistence of IDC (DC-SIGN + cells), macrophages (CD68 + cells), and endothelial cells (CD31 + cells and CD36 + cells) can be seen.

Conclusion Monocytes that have been incorporated into and cultured in the gingival and vaginal-type regenerating chorion model can differentiate into DCs of IDC type and can differentiate into macrophages and endothelial cells.

Example 14: Three-dimensional multicellular regenerative skin model containing LC Obtaining monocytes: see Example 1 or Example 2 A regenerated skin model is cultured according to Example 11.
This model is obtained in the following way.
−1 to 5 × 10 ⁴ monocytes are seeded with keratinocytes on the skin equivalent.
-As a result of labeling, the presence of LC (Langerin + cells and Barbeck + granules) is observed in the epithelial part of the culture.

Conclusion Monocytes cultured with keratinocytes in this regenerated skin model differentiate into LC type DCs.

Example 15: Three-dimensional multi-cell coloration and / or neural regeneration epidermis model containing LC Obtaining monocytes: see Example 1 or Example 2 This regeneration skin model containing LC was cultured according to Example 14. To do.

This model is obtained in the following way.
−1 to 5 × 10 ⁴ normal human skin-derived melanocytes and / or Merkel cells, along with keratinocytes and monocytes, are seeded in the regenerating dermis.
In addition to the label described in Example 14,
Labeling of melanocytes (HMB45) and DOPA reaction is performed for detection of melanin, and labeling using anti-keratin 20 antibody is performed for identification of Merkel cells.

Conclusion Monocytes cultured with keratinocytes and melanocytes and / or Merkel cells in this regenerated skin model differentiate into LC-type DCs.

Example 16: Three-dimensional multicellular regenerative mucosal model with LC Obtaining monocytes: see Example 1 or Example 2 This model is obtained according to the method described in Example 14. The changes are as follows.
-Keratinocytes are replaced with normal human gingival and vaginal mucosal epithelial cells, and fibroblasts are normal human gingival and vaginal mucosal fibroblasts.
-The concentration of calf serum in the culture medium at the epithelialization stage is 1%.
-All cultures are carried out by immersion in the medium.
-As a result of the labeling, the presence of LC (Langerin + cells and Barbeck + granules) is observed in the epithelial part of the culture.

Conclusion Monocytes cultured with gingival or vaginal epithelial cells in this regenerating mucosa model differentiate into LC type DCs.

Example 17: Three-dimensional multicellular regenerative skin model containing DCC, macrophages and endothelial cells Obtaining monocytes: See Example 1 or Example 2 This regenerated skin model is cultured according to Example 11.

This model is obtained in the following way.
−1 to 5 × 10 ⁴ monocytes are seeded on the surface of the skin equivalent and further cultured for 7 days.
As a result of the labeling, the coexistence of DCC (DC-SIGN + cells), macrophages (CD68 + cells) and endothelial cells (CD31 + cells and CD36 + cells) is confirmed.
Conclusion Monocytes that have been incorporated into and cultured in the skin part of the regenerated skin model differentiate into DDC-type DCs, and at the same time differentiate into macrophages and endothelial cells.

Example 18: Three-dimensional multicellular staining and / or neural regenerated epidermis model comprising DCC, macrophages and endothelial cells This model is prepared according to the method described in Example 17 with 1-5 × 10 ⁴ normal human skin. It is obtained by simultaneously seeding melanocytes and / or Merkel cells derived from keratinocytes.

  In addition to the labeling described in Example 17, melanocyte (HMB45) labeling and DOPA reaction are performed for melanin detection, and anti-keratin 20 antibody labeling is performed for Merkel cell identification.

Conclusion Monocytes that have been incorporated into the skin of a colored and / or neurally regenerated epidermis model and cultured are differentiated into DDC-type DCs, and simultaneously differentiated into macrophages and endothelial cells.

Example 19: Three-dimensional multicellular model of regenerating mucosa containing IDC, macrophages and endothelial cells Obtaining monocytes: see Example 1 or Example 2 This model is obtained by the method described in Example 17. However, the changes are as follows.
-Change keratinocytes to normal human gingiva and vaginal mucosal epithelial cells and change fibroblasts to normal human gingiva and vaginal mucosa derived fibroblasts.
-The concentration of calf serum in the culture medium at the epithelialization stage is 1%.
-All cultures are carried out by immersion in the medium.
As a result of labeling, coexistence of IDC (DC-SIGN + cells), macrophages (CD68 + cells) and endothelial cells (CD31 + cells and CD36 + cells) is observed.

Conclusion Monocytes that have been incorporated into and cultured in the gingival and vaginal-type regenerative mucosa model junctions differentiate into IDC-type DCs and simultaneously differentiate into macrophages and endothelial cells.

Example 20: Three-dimensional multicellular regenerative skin model containing LC, DCC, macrophages and endothelial cells Obtaining monocytes: See Example 1 or Example 2 This model is obtained in the following manner.
-This regenerated dermis model containing DCC, macrophages and endothelial cells is made by the method described in Example 12.
A regenerated skin model containing -LC is obtained according to Example 14.
-As a result of labeling, LC (Langerin + cells and -Barbeck + granules) was observed in the epidermis part of the culture, DCC (DC-SIGN + cells), macrophages (CD68 + cells) and endothelial cells (CD31 + cells) in the skin part of the culture. And CD36 + cells).

Conclusion Monocytes cultured in the skin part in the regenerated skin model and then cultured with keratinocytes differentiate into LC-type DCs in the epidermis part, and simultaneously differentiate into macrophages and endothelial cells in the skin part.

Example 21: Three-dimensional multicellular staining and / or neural regeneration epidermis model containing LC, DCC, macrophages and endothelial cells Obtaining monocytes: See Example 1 or Example 2 This model is obtained in the following manner .
-A regenerated dermis model containing DCC, macrophages and endothelial cells is obtained according to Example 12.
A colored and / or nerve regeneration epidermis model containing -LC is prepared according to Example 15.
-As a result of labeling, LC (Langerin + cells and Barbeck granules) in the epidermis portion of the culture, DCC (DC-SIGN + cells), macrophages (CD68 + cells) and endothelial cells (CD31 + cells) in the skin portion of the culture CD36 + cells) coexistence is observed.

Conclusion Monocytes obtained by culturing in the skin part and then culturing with keratinocytes, melanocytes and / or Merkel cells in a colored and / or neuronal regeneration epidermis model differentiate into LC type DC in the epidermis part, and in the skin part Differentiates into DDC type DC and simultaneously differentiates into macrophages and endothelial cells.

Example 22: Three-dimensional multicellular regenerative mucosal model containing LC, IDC, macrophages and endothelial cells Obtaining monocytes: See Example 1 or Example 2 This model is obtained in the following manner.
-This regenerated chorionic model containing IDC, macrophages and endothelial cells is prepared according to Example 13.
A regenerated mucosa model containing -LC is prepared according to Example 16.
-As a result of labeling, LC (Langerin + cells and Verbeck + granules) in the epithelial part, IDC (DC-SIGN + cells), macrophages (CD68 + cells), and endothelial cells (CD31 + cells and CD36 + cells) in the connection part of the culture Coexistence is recognized.

Conclusions Monocytes cultured in the connected part of the gingival and vaginal type regenerative mucosa model and then cultured with epithelial cells differentiated into LC type DC in the epithelial part, differentiated into IDC type DC in the connected part, and at the same time macrophages And differentiate into endothelial cells.

Example 23: Use of the three-dimensional multicellular regenerative skin model described in Example 20 for testing the effects of solar UV irradiation To examine the effects of various environmental factors, particularly the effects of solar UV irradiation, The migration of LC and DCC and their phenotype in the regenerated skin model by immunohistochemical examination were evaluated by the following method.
-This model is obtained as described in Example 20.
- 42 days in culture, for example, corresponds to the UVA and 0.5 J / cm ² of UVB 2J / cm ^2, the single dose is in solar energy 526kJ / cm ^2, irradiating the reconstructed skin (sun Light irradiator Suntest CPS +, Atlas). Incubation is continued for another 2 days.
A series of antibodies (anti-CD1a, anti-CD80, anti-CD83, anti-CD86, anti-CCR7, anti-DC-LAMP, anti-DC-SIGN, to observe LC and DCC migration and then identify their phenotypic conditions Immunohistochemical tests are performed using anti-langerin, anti-HLA-DR).

  After sunlight UV irradiation, LC migration is observed in the skin part of the culture, and acquisition of CCR7 receptor expression is observed on the LC and DCC. Moreover, only LC which moved to the skin part expresses aging label DC-LAMP.

Conclusion This multicellular model can therefore predict UV stress by targeting LCs and DCCs with migration, activation and ripening capabilities.

Example 24: Use of the three-dimensional multicellular regenerative skin model described in Example 20 to evaluate the effectiveness of the active ingredient In order to evaluate the anti-inflammatory action of the active ingredient for human skin, it was applied to a culture supernatant. The secretion of inflammatory cytokines such as IL-1, IL-6, IL-8, TNF-α, and INF-γ is measured by the following method.
This model is obtained by the method described in Example 20.
- 42 days in culture, for example, corresponds to the UVA and 0.5 J / cm ² of UVB 2J / cm ^2, the single dose is in solar energy 526kJ / cm ^2, irradiating the reconstructed skin (sun Light irradiator Suntest CPSs +, Atlas).
-Then 8 μl of 3% antioxidant, for example with or without flavagram (R) (Hesperitin laurate, Engelhard) and additionally Flavenger (R) (quercitin caprylate, Engelhard) A cosmetic preparation is applied to the regenerated skin and left for 10 days.
-After completion of the treatment, the regenerated skin is further cultured in the immersion medium for 48 hours, and then the effectiveness of the antioxidant treatment is evaluated as follows. 1. Cell viability test (methylthiazole tetrazolium-MTT test),

2. Analysis of cytokine secretion in culture supernatant by flux cytometry (CBA-cytometric bead array)

  By the method of the present invention, it can be seen that sunlight UV stress results in decreased cell viability and increased production of inflammatory interleukins. Therefore, it is desirable to suppress the increase in the synthesis of this inflammatory molecule and cell mortality using appropriately selected active ingredients. Of the active ingredients tested, two compounds, flavagrams and flavangers, showed a tendency to maintain these two variables at their original values, indicating efficacy.

Example 25: Use of the three-dimensional multicellular regenerative skin model described in Examples 14, 17 and 20 for the evaluation of the immunostimulatory or immunosuppressive activity of an active ingredient. In order to assess whether it induces a response that produces immunity and / or tolerance, the effect of these cells on stimulating the proliferation of allogeneic untreated T lymphocytes is assessed in the following manner: .
This model is obtained by the method described in Examples 14, 17 and 20.
42 days after cultivation, the active ingredient is applied directly with a cotton swab (topical application) or added to the culture medium (systemic application) and treated for 10 days.
-After treatment, cultures are treated with trypsin (1 mg / mL) for 2 hours at 37 ° C and then with collagenase (1 mg / mL) to recover LC and / or DCC.
-The LC and / or DCC are cultured for 3 days with allogeneic untreated T lymphocytes in RPMI medium supplemented with 10% human AB serum. LCs and / or DCs with cell numbers ranging from 125 to 8,000 are obtained and cultured with 10 ⁵ untreated T lymphocytes. On the third day of culture of the mixed lymphocytes, 20 μl of 5 mCi tritinium thymidine is added and treated for 18 hours.
-This graph is a graph plotting the number of LC and / or DCC on the horizontal axis and the amount cpm of tritinium thymidine introduced into allogeneic untreated T lymphocytes (counts per minute) on the vertical axis. Show.

Compared to untreated LC and / or DCC that grows only a little (1 × 10 ³ to 4 × 10 ³ cpm) of untreated T lymphocytes, after treatment with active ingredient X, LC and / or DCC Strongly stimulates lymphocyte proliferation (5 × 10 ^{4 to} 7 × 10 ⁴ cpm).

Example 26: Use of three-dimensional multicellular model for screening of active ingredient capable of regulating allergic reaction The immunoregulatory effect of an active ingredient after stimulating, sensitizing and causing allergic stress is determined by the following method. Investigate.
This model is obtained according to the method described in Example 12.
After culturing for 21 days, preferably a final concentration of 2% stimulating substance (sodium dodecyl sulfate) and preferably a final concentration of 0.25% sensitizer DNCB (1,4-chloro-dinitrobenzene) in the culture medium And process for 24 hours.
-The culture medium thus obtained is then treated for 3 days with 100 μl of a cosmetic preparation with or without a final concentration of 3% stabilizer.
-After treatment, the culture supernatant is collected and measured for example IL-10 and IL-12;
Immunohistochemical testing is performed using a series of antibodies, anti-CD80, anti-CD83, anti-CD86, anti-CCR7, anti-DC-LAMP, anti-HLA-DR.

  The immunoregulatory ability of the active ingredient to be tested is determined from the results of IL-10 and IL-12 secretion and the DCC phenotype test by immunohistochemistry.

CONCLUSION The multicellular model of this patent thus provides a test tool by which the potential immunomodulatory effects of active ingredients can be assessed by screening DCCs with the ability to secrete, activate and mature cytokines.

Claims (29)

  A method for producing Langerhans cells (LC) or interstitial / dermal dendritic cells (IDC), or LC and IDC, from CD14 + monocytes derived from living organisms, particularly human peripheral circulating blood, comprising: A method characterized in that CD14 + monocytes are differentiated into LC or IDC, or into LC and IDC, in the presence of a cellular environment comprising epithelial cells such as keratinocytes and / or mesenchymal cells such as dermal fibroblasts.   The CD14 + monocytes are placed in the presence of a cellular environment comprising epithelial cells such as keratinocytes and / or mesenchymal cells such as dermal fibroblasts, and CD14 + monocytes are introduced into IDCs and macrophages and / or endothelial cells, or The method according to claim 1, wherein the method is differentiated into LC, IDC, macrophage and / or endothelial cell.   The method according to claim 1 or 2, wherein the differentiation of CD14 + monocytes is performed without adding a significant amount of exogenous cytokines.   The method according to any one of claims 1 to 3, wherein the monocytes are put in the presence of a cellular environment containing epithelial cells such as keratinocytes, and the monocytes are differentiated into immature functional LC.   The method according to any one of claims 1 to 4, wherein monocytes are placed in the presence of a cellular environment containing mesenchymal cells such as dermal fibroblasts, and the monocytes are differentiated into immature functional IDCs.   Monocytes are placed in the presence of a cellular environment containing epithelial cells such as keratinocytes and mesenchymal cells such as dermal fibroblasts, and mesenchymal cells such as dermal fibroblasts, and monocytes are subjected to typical LC and IDC. The method according to any one of claims 1 to 5, which is differentiated.   A method of culturing CD14 + monocytes comprising a fusion of CD14 + monocytes derived from the peripheral circulating blood of an organism, particularly a human, within a cell or tissue model, wherein the cell or tissue model is an epithelial cell such as keratinocytes, and / or Epithelial cells, such as keratinocytes, that contain mesenchymal cells such as dermal fibroblasts and to differentiate CD14 + monocytes into LC, or into IDC, or into LC and IDC in the model And / or culturing in the presence of mesenchymal cells such as dermal fibroblasts.   The method according to claim 7, wherein the culture of CD14 + monocytes is performed without adding a significant amount of exogenous cytokines.   The method according to claim 7 or 8, wherein the cell or tissue model is selected from the group consisting of an epidermis model, an epithelial model, a dermis model, a chorionic model, a skin model, and a mucosal model. The cell or tissue culture model has the following materials:
-Collagen-based or fibrin-based gels or films containing mesenchymal cells, in particular fibroblasts,
-A collagenous pore that may contain one or more glycosaminoglycans and / or chitosan as needed and may or may not contain fused mesenchymal cells, especially fibroblasts Quality matrix,
The following groups, which may or may not contain mesenchymal cells, in particular fibroblasts:
Semipermeable synthetic membranes, especially semipermeable nitrocellulose membranes, semipermeable nylon membranes, polytetrafluoroethylene (Teflon (R), PTFE) membranes or sponges, semipermeable polycarbonates or polyethylene terephthalate (PET) Membrane, inorganic membrane having a capillary porous structure of aluminum oxide (semipermeable anopore membrane), cellulose acetate or ester (HATF) membrane, semipermeable biopore CM membrane, semipermeable polyester membrane,
And an inert support treated for polycarbonate-based or polystyrene-based culture, which may or may not contain mesenchymal cells, in particular fibroblasts,
10. A method according to any one of claims 7 to 9, comprising a skin or chorionic matrix support selected from the group consisting of.   11. The method according to claim 10, wherein the cell or tissue model used comprises the skin or chorion support having epithelial cells, in particular keratinocytes, on the surface.   The cell or tissue model is at least one other cell type, such as neurons and / or Merkel cells and / or endothelial cells and / or macrophages, and / or melanocytes and / or lymphocytes and / or adipocytes and / or The method according to claim 10 or 11, comprising skin appendages such as bristles, hair and sebaceous glands.   The method according to any one of claims 7 to 12, wherein CD14 + monocytes are cultured in a cell or tissue model containing at least mesenchymal cells to differentiate some monocytes into endothelial cells and / or macrophages. .   8. The cell or tissue model includes an epithelial portion and a connecting matrix, LC is substantially localized in the epithelial portion, and IDC, macrophages and / or endothelial cells are substantially localized in the connecting matrix. The method as described in any one of -12. A method for differentiating CD14 + monocytes into LC and / or IDC comprising the following steps:
-Collecting CD14 + monocytes from circulating blood;
-Maintaining CD14 + monocytes under conditions that do not promote differentiation into DC; and-CD14 + monocytes under conditions that promote differentiation into LC, or IDC, or LC and IDC, keratinocytes, etc. Placing in the presence of a cellular environment comprising mesenchymal cells such as epithelial cells and / or fibroblasts,
Including methods.   The cell model obtained by the method as described in any one of Claims 1-15 containing at least 1 sort (s) of said LC and / or IDC, and also a macrophage and / or endothelial cell as needed.   The epidermis model, epithelial model, dermis obtained by the method according to any one of claims 1 to 15, further comprising at least one of the LC and / or IDC, and, if necessary, macrophages and / or endothelial cells. A tissue model selected from the group consisting of a model, a chorionic model, a skin model, and a mucous membrane model.   An epithelial part comprising epithelial cells such as keratinocytes and a connected matrix comprising mesenchymal cells such as between skin or chorionic fibroblasts, wherein the LC is substantially localized in the epithelial part, IDC, macrophages and / or 18. The tissue model according to claim 17, wherein the endothelial cells are substantially localized in the connected matrix.   Frozen CD14 + monocytes isolated from the peripheral circulating blood of organisms, particularly humans.   At least one cell model as defined in claim 16 or at least one tissue model as defined in claim 17 or 18 as a test model in the cosmetic, dermatological, pharmaceutical field and / or active ingredient The method used for selection.   Especially for the assessment of the phenomena and activities occurring in the defense / infection process of an organism of at least one cell model as defined in claim 16 or at least one tissue model as defined in claim 17 or 18. In order to evaluate immunostimulation or immunosuppressive activity of a specific active ingredient, in order to evaluate and induce immune modulation by the active ingredient, conduct in vitro tests to predict allergic, stimulating and sensitizing activities of external reagents. A method used for conducting molecular or chemical tests, especially toxicity studies.   Physiological pathology of epithelial barrier of at least one cell model as defined in claim 16 or at least one tissue model as defined in claim 17 or 18; irritation of skin and mucous membranes; eg virus, HIV Biological attack, such as retroviruses, bacteria, filamentous fungi, microorganisms, particle antigens, etc .; phototoxicity; photoprotection; and the effects of active ingredients, especially cosmetic or pharmaceutical active ingredients; final products, especially cosmetic or pharmaceutical products The method used to test the mechanism of infection by pathogenic substances.   Testing of the mechanism of infection, replication, propagation of viruses, including retroviruses such as HIV, of at least one cell model as defined in claim 16 or at least one tissue model as defined in claim 17 or 18; Or a method used for exploring and developing new therapies, including the administration of vaccines and drugs.   At least one cell model as defined in claim 16, or at least one tissue model as defined in claim 17 or 18, of a pathogenic substance, for example a virus, a retrovirus such as HIV, a bacterium, a filamentous fungus, a microorganism, A method used for the detection of the presence of particle antigens and the like.   Method of using at least one cell model as defined in claim 16 or at least one tissue model as defined in claim 17 or 18 for medical, biomedical or cosmetic applications, in particular immunization in vitro or in vivo. Modulation of tolerance response, environmental attack, especially prevention of immune or tolerance response as a result of physical attack such as UV irradiation and chemical attack or biological attack such as irritation / allergy / sensitizer Or a method used for adjustment for therapeutic purposes.   Method, medical or biomedical application, for use in tissue engineering and cell engineering of at least one cell model as defined in claim 16 or at least one tissue model as defined in claim 17 or 18 Anti-cancer cell therapy by DC injection that can stimulate the immune response; methods used for cell therapy in the case of autoimmune diseases, for example, induction of immune tolerance stimulation by the production of anergic T cells; A method used for gene therapy of an invading disease, or a method used for the development and manufacture of a vaccine. The following steps:
Isolating CD14 + monocytes from the peripheral circulating blood of the organism,
-Seeding skin or mucosal cells on a support and culturing in nutrient medium to obtain regenerated tissue;
-Seeding the regenerated tissue with CD14 + monocytes simultaneously with or not simultaneously with skin or mucosal cells;
-Differentiating the regenerated tissue containing CD14 + monocytes and skin or mucosal cells into LC, a mixture of IDC and endothelial cells and / or macrophages, and a mixture of LC and IDC and endothelial cells and / or macrophages. (If the regenerated tissue is an epidermis model, the skin cells are epidermal keratinocytes, and if the regenerated tissue is a dermis model, the skin cells are skin fibroblasts, Is an epithelial model, the mucosal cells are epithelial mucosal cells, and if the regenerated tissue is a chorionic model, the mucosal cells are mucosal fibroblasts),
A method for manufacturing an organization model comprising: A method for producing regenerated skin or regenerated mucosa comprising keratinocytes or epithelial mucosal cells, and optionally epithelial parts comprising Merkel cells and / or melanocytes, and a connected matrix comprising dermis or chorionic fibroblasts, The following steps:
The surface of a dermis or chorionic model containing keratinocytes or epithelial mucosal cells in a nutrient medium, optionally in the presence of Merkel cells and / or melanocytes, and containing IDC and optionally endothelial cells and / or macrophages In the presence of the keratinocytes or epithelial mucosal cells (wherein the dermis or chorion model is obtained by the method for producing a dermis or chorion model as defined in claim 27), and A step of seeding and culturing CD14 + monocytes isolated from the peripheral circulating blood of an organism at the same time or not simultaneously with keratinocytes or epithelial mucosal cells under conditions where monocytes differentiate into LC;
A method consisting of:   The method according to claim 27 or 28, wherein the cell is a human cell.