Patent Application
20050008623
The present invention relates essentially to a process for the in vitro culture of CD14+ monocytes, to a culture medium and to the use of the process in a method for the assessment of immunotoxicity/immunotolerance, in a method for the study and selection of active principles, in a method for the physio-pathological study of skin and mucous membranes and in a method of cell and/or tissue engineering and therapy.
Dendritic cells (DC) are antigen-presenting cells which are considered to be guardians of the immune system. They are in fact located almost everywhere, namely in the thymus, the systemic circulation and the secondary lymphoid organs and also in the peripheral tissues such as the skin and mucous membranes, whether they can be monostratal or of the malpighian type, i.e. comprising a multistratal epithelium, namely those of the vagina, the outer cervix, the vulva, the perianal region, the esophagus and the mouth. Although in very small numbers in the organism, DC are at the center of the triggering of specific immune responses, exerting control over the specificity, intensity and nature of the immune response, and are located at the interface of innate and acquired immunity. Apart from their function of “switching on” the immune response, DC also have a role to play in the induction of peripheral tolerance.
DC precursors are derived from the differentiation of CD34+ hemopoietic precursors in the same way as numerous populations of the immune system and blood cells. They are transported by the blood to the skin and mucous membranes, where they differentiate and reside in the form of immature DC. Two types of DC can be described according to their in vivo location:
Following the capture of an antigen, LC and/or IDC migrate towards the lymph nodes. This migration correlates with an activation of the LC and/or IDC, with a modification of the expression of chemokine receptors (loss of expression of the CCR6 receptor and acquisition of expression of CCR7) and adhesion molecules, and with a modification of their phenotypic and functional characteristics. For example, in the case of LC, the Birbeck's granules become disorganized and their morphology is perturbed. In the lymphatic ganglia, the interaction between the CD40 receptor of the DC and its ligand CD40-L situated on the T lymphocytes induces a maturation of the DC into “interdigitated DC”, which are characterized by the membranous expression of the antigen CD83 and the co-stimulation markers CD80 and CD86, and by a massive membranous translocation of the class II molecules of the major histocompatibility complex, such as HLA-DR. These activated mature DC thus become producers of TNFα and IL-12.
A valuable use of LC, especially in combination with epithelial cells derived either from skin or from human mucous membranes, consists in integrating them into a system or model of “reconstructed skin” or “reconstructed mucous membrane” (cf. publication by Régnier, JID 1997; patent EP 0 789 074 to L'OREAL; Sivard P. Peaux et muqueuses reconstruites (Reconstructed skin and mucous membranes), Nouv. Dermatol., 2001, 20, 520-523). In particular, this could serve as a biological basis for methods said to be alternatives to animal experimentation, which should be increasingly used for in vitro evaluation of the tolerance and/or efficacy of products, such as pharmaceutical and cosmetic products.
In fact, these uses are currently limited, or even non-existent, due to the absence of a reasonably exploitable process for obtaining LC reliably on the industrial scale, and due to the imperfection of the models described.
Patent EP 0 789 074 to L'OREAL is concerned with a skin model or equivalent and the use of CD34+ precursors derived from umbilical cord blood. The skin equivalent is in fact only an epidermis equivalent since the cells are deposited on a matrix which is a de-epidermized dermis, i.e. a dead dermis containing no living cells.
Whatever the case may be, IDC are never obtained (nor are macrophages or endothelial cells) because the dermis is not “living”.
Furthermore, the number of CD34+ cells is limited since they are obtained from umbilical cord blood.
A publication by Geissmann (F. Geissmann, C. Prost, J-P. Monnet, M. Diy, N. Bruce and O. Hermine; 1998; J. Exp. Med., vol. 187, number 6, 961-966) describes the use of CD14+ monocytes obtained from circulating blood, as well as their culture in suspension for 6 days (in the presence of GM-CSF, TGFβ1 and IL-4) to give LC.
According to the protocol described in said publication, the cells are cultivated in suspension and not on a three-dimensional model. Also, the presence of neither IDC nor other cells (macrophages, endothelial cells) is described.
One main object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the in vitro generation, from a single cellular precursor, of the two living populations of dendritic cells of the skin and the mucous membranes, namely Langerhans cells (or LC) and interstitial dendritic cells (or IDC).
Another main object of the present invention is to solve the novel technical problem consisting in the provision of a single precursor which is easily obtainable because it is present in the circulating blood and particularly in the peripheral circulating blood of a human or animal individual.
Another main object of the present invention is to solve the novel technical problem consisting in the provision of a single precursor which is present in sufficient quantity to allow the in vitro generation of cells in numbers such that they can be used on the industrial scale.
Another main object of the present invention is to solve the novel technical problem consisting in the provision of a single precursor which allows the in vitro generation of cells in a perfectly reproducible manner, particularly without variability as a function of the donor.
Another main object of the present invention is to solve the novel technical problem consisting in the provision of a single precursor which allows the rapid in vitro generation of cells (7 to 8 days of culture are required to obtain LC).
Another main object of the present invention is to solve the novel technical problem consisting in the provision of a single precursor which allows the in vitro generation of cells having the same phenotype and the same functions as those present in vivo.
Another main object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the in vitro generation of dendritic cells, namely Langerhans cells and/or interstitial dendritic cells, at different, targeted steps of differentiation/maturation, i.e. at a step of preconditioned and undifferentiated cells, or at a step of differentiated and immature cells, or at a step of mature cells, or at a step of interdigitated cells.
Another main object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the in vitro generation, from a single cellular precursor, of either predominantly Langerhans cells (or LC), or predominantly interstitial dendritic cells (or IDC), or a dual population of Langerhans cells and interstitial dendritic cells (or LC/IDC).
Another main object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the in vitro generation, from a single cellular precursor of dendritic cells, namely Langerhans cells (or LC) and interstitial dendritic cells (or IDC), including the in vitro generation of subpopulations of these LC and/or CDI, these subpopulations being different ones from the others by their phenotypes and/or their functional properties.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the use of these cells in therapy.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the in vitro generation of dendritic cells, namely Langerhans cells and/or interstitial dendritic cells, for medical or biomedical applications such as anticancer cell therapy, for example an injection of DC capable of stimulating the immune response; cell therapy in cases of autoimmune disease through the creation of an immunotolerance situation, for example by producing anergic T cells; gene therapy for diseases affecting the immune system; and the development and production of vaccines.
Another main object of the invention is to solve the novel technical problem consisting in the provision of a solution for the in vitro generation of dendritic cells, namely Langerhans cells and/or interstitial dendritc cells, and for their integration into models, including models of skin tissues or mucous membranes.
Another main object of the present invention is to solve the novel technical problem consisting in the- provision of a solution for the in vitro generation of preconditioned cells which, when integrated into a complete skin or mucous membrane model, i.e. a model comprising both an epithelium and a connective matrix, are capable, by virtue of the cellular environment, preferably fibroblasts and epithelial cells, and the matricial environment, of locating in the epithelium in order to differentiate into Langerhans cells, and in the connective matrix in order to differentiate into interstitial dendritic cells, macrophages and endothelial cells, and of acquiring a functionality comparable to that of Langerhans cells, interstitial dendritic cells, macrophages and endothelial cells in vivo.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the study and/or selection of substances, such as active principles.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the in vitro generation of endothelial cells and macrophages.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for obtaining an equivalent of immunocompetent skin or mucous membrane.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a model/tool for studying the physio-pathology of the different types of cells and tissues to which the invention relates, a model/tool for pharmacotoxicological study, for example with the aim of performing in vitro tests for predicting the immunotoxicity or allergenicity of external agents, and a model/tool for studying substances with immunomodulating properties.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the use of these various models in therapy.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the use of a model especially for the purpose of studying the immunostimulant or immunosuppressant activity of an active principle or evaluating or inducing an immunotolerance by said active principle.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the use of a model for studying the physiopathology of epithelial barriers; irritation of skin or mucous membranes; aggressions of a biological nature, for example viruses, retroviruses such as HIV, bacteria, molds, microorganisms and particulate antigens; photo-toxicity; photoprotection; the effect of an active principle, particularly a cosmetic or pharmaceutical active principle; and the effect of finished products, particularly cosmetic or pharmaceutical products; and for studying the mechanisms of infection by a pathogenic agent.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the use of a model for detecting the presence of a pathogenic agent, for example viruses, retroviruses such as HIV, bacteria, molds, microorganisms and particulate antigens.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the use of a model for a medical, biomedical or cosmetic application, in particular for modulating the immune or tolerance response, in vitro or in vivo, following an environmental aggression, particularly of the physical type, such as UV irradiation, or of the chemical or biological type, particularly for the purpose of preventive or curative therapy.
Another object of the present invention is to solve the novel technical problem consisting in the provision of a solution for the use of a model for tissue and cell engineering applications; medical or biomedical applications such as anticancer cell therapy, for example an injection of DC capable of stimulating the immune response; cell therapy in cases of autoimmune disease through the creation of an immunotolerance situation, for example by producing anergic T cells; gene therapy for diseases affecting the immune system; and the development and production of vaccines.
The present invention makes it possible for the first time to solve each of the above-mentioned technical problems in a safe, reliable and reproducible manner which can be used on the industrial and commercial scale and especially on the cosmetic and/or pharmaceutical and/or medical industrial scale.
The invention consists mainly in the in vitro generation, from a living single precursor, i.e. the CD14+ monocyte present in the peripheral circulating blood, of at least the two populations of dendritic cells of the skin and mucous membranes, namely Langerhans cells and interstitial dendritic cells.
Within the framework of the invention, the term “cells” is always to be understood as meaning “living cells”, unless indicated otherwise.
According to the invention, the term “peripheral circulating blood” is to be understood as meaning blood taken from any living being having a blood system in which the blood flows in a circuit, especially at the periphery, and particularly animals and mammals, preferably humans.
According to the invention, the term “fresh blood”, is blood from which the extraction of CD14+ monocytes is initiated and performed preferably not later than 24 hours after the taking of blood on an individual.
Thus, according to a first feature, the present invention relates to the use of CD14+ monocytes isolated from peripheral circulating blood for obtaining, by differentiation, at least one mixed population of Langerhans cells and of interstitial dendritic cells, both Langerhans cells and interstitial dendritic cells being preconditioned and undifferentiated, and/or differentiated and immature, and/or mature, and/or interdigitated.
According to one advantageous characteristic of the use of CD14+ monocytes, the extraction of CD14+ monocytes is performed from fresh blood i.e. initiated and performed preferably not later than 24 hours after taking of blood on an individual, preferably not later than 18 hours, preferably not later than 12 hours, preferably not later than 6 hours and still preferably the extraction is immediately initiated just after the taking of blood and performed not later than 5 hours.
According to one advantageous characteristic of the use of CD14+ monocytes, the differentiation results in the presence of different subpopulations of LC and/or IDC.
According to one advantageous characteristic of the use of CD14+ monocytes, the differentiation results in the presence of at least one additional subpopulation of preconditioned undifferentiated cells, and/or differentiated cells, such as cells of the macrophage type and/or cells of the endothelial type.
According to one advantageous characteristic of the use of CD14+ monocytes, the differentiation is effected by culture of these CD14+ monocytes in a culture medium containing at least the two cytokines GM-CSF and TGFβ, preferably TGFβ1.
According to one advantageous characteristic of the use of these CD14+ monocytes, the distribution between the populations of LC and IDC depends on the presence of a third cytokine at a given concentration and for a given period of time during said culture, said cytokine preferably being the cytokine IL-13.
In another advantageous variant, the culture is carried out in the presence of the cytokine IL-13 for at most about two days so as to favor differentiation into LC, i.e. favor the predominant formation of LC.
In another advantageous variant, the culture is carried out in the presence of the cytokine IL-13 for about 6 days in order to favor the formation of IDC.
In another advantageous variant, the culture is carried out in the presence of the cytokine IL-13 for about 4 days in order to favor the formation of a dual population of LC/IDC.
According to another advantageous characteristic, an additional degree of differentiation of LC and IDC can be obtained by carrying out said culture in the presence of the cytokine TNFα.
The culture can advantageously be carried out in the presence of TNFα at a given concentration and for a given period of time, the latter being less than about 18 hours, in order to obtain immature Langerhans cells and immature interstitial dendritic cells while at the same time avoiding a maturation of these cells into mature activated dendritic cells.
According to another characteristic of the invention, the culture in the presence of TNFα is carried out at a given concentration and for a given period of time, the latter being more than about 20 hours, in order to obtain a maturation into mature activated dendritic cells.
According to another advantageous characteristic, the concentration of cytokine GM-CSF is between 0.1 and 4000 IU/ml, advantageously between 1 and 2000 IU/ml and more precisely about 400 IU/ml; the concentration of cytokine TGFβ, preferably TGFβ1, is between 0.01 and 400 ng/ml, advantageously between 1 and 100 ng/ml and more precisely about 10 ng/ml; the concentration of cytokine IL-13, if this cytokine is present in the medium, is between 0.01 and 400 ng/ml, advantageously between 1 and 100 ng/ml and more precisely about 10 ng/ml; and the concentration of cytokine TNFα, if this cytokine is present in the medium, is between 0.1 and 4000 IU/ml, advantageously between 1 and 1000 IU/ml and more precisely about 200 IU/ml.
According to another advantageous characteristic of the use of the CD14+ monocytes, the LC and IDC obtained have functional phenotypes identical to those found in vivo.
According to another advantageous characteristic, the culture of said LC and IDC is carried out in a three-dimensional culture environment comprising, in particular, at least epithelial and stromal cells.
Advantageously, according to one characteristic of this additional differentiation, when the epithelial and stromal cells are distinctly separated, the LC are located mainly in the region of the epithelial cells and the IDC are located mainly in the region of the stromal cells.
Advantageously, according to one characteristic of the use of these CD14+ monocytes, endothelial cells and macrophages are obtained by differentiation from certain cells derived from the culture, particularly when they are placed in a three-dimensional environment.
Advantageously, according to one characteristic of the use, cells, preferably preconditioned cells, are obtained which, when integrated into a complete skin or mucous membrane model, i.e. a model comprising both an epithelium and a connective matrix, are capable, by virtue of the cellular environment, preferably fibroblasts and epithelial cells, and the matricial environment, of locating in the epithelium in order to differentiate into Langerhans cells, and in the connective matrix in order to differentiate into interstitial dendritic cells, macrophages and endothelial cells, and of acquiring a functionality comparable to that of Langerhans cells, interstitial dendritic cells, macrophages and endothelial cells in vivo.
According to a second feature, the present invention further relates to a process for the in vitro culture of CD14+ monocytes which comprises:
According to one advantageous characteristic, in this process for the in vitro culture of CD14+ monocytes, the culture takes place in the presence of at least the cytokines GM-CSF and TGFβ, preferably TGFβ1 .
According to another advantageous characteristic of the present invention, in the process for the in vitro culture of CD14+ monocytes, the culture takes place in the presence of a third cytokine at a given concentration and for a given period of time during said culture, said cytokine preferably being the cytokine IL-13.
In one variant of this advantageous characteristic, the culture is carried out in the presence of the cytokine IL-13 for at most about two days so as to favor differentiation into LC.
In another variant of this advantageous characteristic, the culture is carried out in the presence of the cytokine IL-13 for about six days in order to favor the formation of IDC.
In another advantageous variant of this characteristic, the culture is carried out in the presence of the cytokine IL-13 for about 4 days in order to favor the formation of a mixed population of LC/IDC.
According to one advantageous characteristic of the present invention, in the process for the in vitro culture of CD14+ monocytes, the culture takes place in the presence of the cytokine TNFα.
In one variant of this advantageous characteristic, the culture in the presence of TNFα is carried out at a given concentration and for a given period of time, the latter being less than about 18 hours, in order to obtain differentiation of the cells into still immature Langerhans cells and interstitial dendritic cells while at the same time avoiding a maturation into activated mature dendritic cells.
According to another advantageous characteristic, the culture in the presence of TNFα is carried out at a given concentration and for a given period of time, the latter being more than about 20 hours, in order to obtain a maturation into activated mature dendritic cells.
According to another advantageous characteristic of present invention, the extraction of CD14+ monocytes is performed from fresh blood i.e initiated and performed preferably not later than 24 hours after taking of blood on an individual, preferably not later than 18 hours, preferably not later than 12 hours, preferably not later than 6 hours and still preferably the extraction is immediately initiated just after the taking of blood and performed not later than 5 hours.
According to another advantageous characteristic of the present invention, in the process for the in vitro culture of CD14+ monocytes, the culture takes place in a three-dimensional culture environment, particularly in the presence of at least epithelial cells and stromal cells.
According to another advantageous characteristic of the present invention, an additional degree of differentiation is obtained by carrying out the culture of said Langerhans cells and interstitial dendritic cells in a three-dimensional culture environment comprising, in particular, at least distinctly separated epithelial and stromal cells.
According to another advantageous characteristic of the present invention, after culture with the cytokines in the process for the in vitro culture of CD14+ monocytes, a complementary stimulation of maturation is effected in particular by interaction of the dendritic cells with CD40-ligand, or by addition of the cytokine TNFα or lipopolysaccharide, for a sufficient period of time to obtain a phenotypic and functional maturation of said cells.
According to another advantageous characteristic of the present invention, the process for the in vitro culture of CD14+ monocytes comprises integration of a dual population of LC and IDC, in variable proportions, into a three-dimensional culture model.
In another variant of this last advantageous characteristic, the three-dimensional culture model includes skin models, mucous membrane models, dermis models, chorion models, epidermis models and epithelium models.
In another variant of this last advantageous characteristic, the three-dimensional culture model comprises a matricial support (of dermis or chorion) preferably selected from:
In another variant of this last advantageous characteristic, the three-dimensional culture model used consists of the above-mentioned model onto whose surface epithelial cells, particularly keratinocytes, have been deposited.
In one variant of this last advantageous characteristic, the three-dimensional culture model used consists of a model into which has been incorporated at least one complementary cell type, for example nerve cells and/or endothelial cells (EC) and/or melanocytes and/or lymphocytes and/or adipocytes and/or appendages of skin, such as scalp hair, other body hair and sebaceous glands.
In another variant, certain cells derived from the culture differentiate into endothelial cells and macrophages, particularly when they are placed in a three-dimensional environment comprising at least epithelial and stromal cells.
The invention relates in general terms to a culture process comprising the use of CD14+ monocytes in a manner described above or in a manner resulting from the following description, including the Examples, taken in its entirety.
According to a third feature, the present invention relates to a medium for the in vitro culture of CD14+ monocytes which comprises a basic culture medium combined with at least two cytokines, namely the cytokine GM-CSF and the cytokine TGFβ, preferably TGFβ1.
Advantageously, the culture medium combined with said two cytokines is also combined with the cytokine IL-13, which is preferably physically separated so that it can be introduced into the culture medium at a given moment during culture.
According to one advantageous characteristic of this third feature, the culture medium combined with said two cytokines is also combined with the cytokine TNFα, which is preferably physically separated so that it can be introduced into the culture medium at a given moment during culture.
According to another advantageous characteristic of this third feature, the concentration of cytokine GM-CSF in the culture medium is between 0.1 and 4000 IU/ml, advantageously between 1 and 2000 IU/ml and more precisely about 400 IU/ml; the concentration of cytokine TGFβ, preferably TGFβ1, is between 0.01 and 400 ng/ml, advantageously between 1 and 100 ng/ml and more precisely about 10 ng/ml; the concentration of cytokine IL-13, if this cytokine is present in the medium, is between 0.01 and 400 ng/ml, advantageously between 1 and 100 ng/ml and more precisely about 10 ng/ml; and the concentration of cytokine TNFα, if this cytokine is present in the medium, is between 0.1 and 4000 IU/ml, advantageously between 1 and 1000 IU/ml and more precisely about 200 IU/ml.
According to a fourth feature, the invention relates to a cell population comprising at least one mixed population of Langerhans cells and interstitial dendritic cells—both Langerhans cells and interstitial dendritic cells being preconditioned and undifferentiated, and/or differentiated and immature, and/or mature, and/or interdigitated—which are obtainable from CD14+ monocytes and especially by the use as defined above, or by the culture process according to the above description, or by the use of the culture medium as described above.
According to a fifth feature, the invention relates to the use of the mixed population of LC and IDC obtained from the above-mentioned use of CD14+ monocytes, or by the above-mentioned culture process, or the use of the above-mentioned culture medium for the in vitro generation of dendritic cells, namely Langerhans cells and/or interstitial dendritic cells, for medical or biomedical applications such as anticancer cell therapy, for example an injection of DC capable of stimulating the immune response; cell therapy in cases of autoimmune disease through the creation of an immunotolerance situation, for example by producing anergic T cells; gene therapy for diseases affecting the immune system; and the development and production of vaccines.
Again, according to a sixth feature, the present invention relates to the use of the mixed population of LC and IDC obtained from the above-mentioned use of CD14+ monocytes, or by the above-mentioned culture process, or the use of the above-mentioned culture medium, or as described above, for the manufacture of a suspension, monolayer or three-dimensional, monocellular or multicellular study model.
According to one advantageous characteristic of this fifth feature, the study model is selected from:
According to one advantageous characteristic, this model comprises mainly either LC, or IDC, or a mixture of LC/IDC, or a mixture of LC/IDC/endothelial cells/macrophages, or a mixture of IDC/endothelial cells/macrophages.
The tissue model is defined as being able to be an epidermis model consisting mainly of keratinocytes, a connective matrix model, called a dermis in the case of skin and chorion in the case of a mucous membrane, containing mainly stromal cells, an epithelium model consisting mainly of epithelial cells, a skin model consisting of an epidermis and a dermis, or a mucous membrane model consisting of an epithelium and a chorion.
Normal healthy cells, pathological cells or cells derived from lines can be used in these models; these cells can be of human or animal origin.
Epithelial cells, pigmentary cells, nerve cells etc. can be introduced into the epithelial part in addition to the cells generated according to the invention.
Stromal cells (particularly fibroblasts), T lymphocytes, adipocytes and appendages of skin (scalp hair, other body hair, sebaceous glands) can be introduced into the connective matrix in addition to the cells generated according to the invention.
According to a seventh feature, the present invention relates to a complete model of reconstructed skin or reconstructed mucous membrane, or a model of reconstructed dermis or reconstructed chorion, or a model of reconstructed epithelium, particularly an epidermis model, or any other suspension, monolayer or three-dimensional, monocellular or multicellular model comprising at least one mixed population of LC/IDC as obtained above from CD14+ monocytes.
According to one advantageous characteristic, this model of reconstructed tissue, or other model, is selected from:
According to one advantageous characteristic, this model comprises mainly either LC, or IDC, or a mixture of LC/IDC, or a mixture of LC/IDC/endothelial cells/macrophages, or a mixture of IDC/endothelial cells/macrophages.
Advantageously, according to one characteristic of this model, the LC are located in the epithelial part and the IDC, macrophages and endothelial cells, when present, are located in the connective matrix.
Advantageously, the invention relates to a model as described above wherein cells are present which provide architecture, especially stromal cells, particularly fibroblasts, and/or epithelial cells, particularly keratinocytes, and/or other cell types, especially T lymphocytes, and/or nerve cells, and/or pigmentary cells, particularly melanocytes, and cells which provide immune defense, especially LC, IDC and/or macrophages, and cells which provide vascularization, especially endothelial cells, as well as adipocytes.
According to an eight feature, the present invention relates to the use of at least one of said mixed populations of LC and IDC as a model for the study and/or selection of active principles.
The term “active principle” is to be understood as meaning any substance, product or composition which is potentially capable of exhibiting an activity of value in industry, particularly in the cosmetic industry, pharmaceutical industry, dermopharmaceutical industry, food industry, agrifoodstuffs industry, etc.
An ninth feature of the invention relates to the use of an above-mentioned model especially for the purpose of studying the immunostimulant or immunosuppressant activity of an active principle or evaluating or inducing an immunotolerance by said active principle.
According to a tenth feature, the invention relates to the use of an above-mentioned model for studying the physiopathology of epithelial barriers; irritation of the skin or mucous membranes; aggressions of a biological nature, for example viruses, retroviruses such as HIV, bacteria, molds, microorganisms and particulate antigens; phototoxicity; photoprotection; the effect of active principles, particularly cosmetic or pharmaceutical active principles; and the effect of finished products, particularly cosmetic or pharmaceutical products; and for studying the mechanisms of infection by a pathogenic agent. In particular, the invention makes it possible to use the models for studying the mechanisms involved in the phenomena of infection, replication and transmission of viruses, including retroviruses such as HIV, and to research and develop therapeutic methods (including vaccines, drugs etc.).
According to a eleventh feature, the present invention relates to the use of an above-mentioned model for detecting the presence of a pathogenic agent, for example viruses, retroviruses such as HIV, bacteria, molds, microorganisms and particulate antigens.
According to an twelfth feature, the present invention relates to the use of an above-mentioned study model for a cosmetic, medical or biomedical application, in particular for modulating the immune or tolerance response, in vitro or in vivo, following an environmental aggression, particularly of the physical type, especially UV irradiation, or of the chemical or biological type, including the immunological type, particularly for the purpose of preventive or curative therapy.
According to a thirteenth feature of the present invention, the reconstructed tissue, reconstructed skin, reconstructed mucous membrane or study model can be used for tissue and cell engineering applications; medical or biomedical applications such as anticancer cell therapy, for example an injection of DC capable of stimulating the immune response; cell therapy in cases of autoimmune disease through the creation of an immunotolerance situation, for example by producing anergic T cells; gene therapy of diseases affecting the immune system; and the development and production of vaccines.
According to yet another feature, the present invention also covers any potentially active substance whose activity has been demonstrated through the use of at least the mixed population of cells obtained from CD14+ monocytes, especially by putting into effect any one of the foregoing features capable, in particular, of utilizing a study model.
By virtue of the invention, an easily accessible source of circulating monocytes is used through the possibility of using selectable donor blood bags. The number of CD14+ precursors present in circulating blood is high and makes it possible to produce a large number of LC and IDC in vitro with a high degree of reproducibility and feasibility.
In addition, the culture of CD14+ monocytes makes it possible to produce both LC and IDC, thereby providing a culture model suitable for the high-speed screening of substances intended in particular for applications to the skin or mucous membranes. This culture model therefore constitutes a satisfactory and complete tool because it utilizes at least LC and/or IDC at the same time; consequently, it constitutes an alternative method to animal experimentation and makes it possible especially to observe the ethical conventions in force according to the legislation of the cosmetic industry.
The invention also makes it possible to use the culture model in association with the models of reconstructed skin or reconstructed mucous membrane, affording the in vitro generation of a single model of “endothelialized immunocompetent reconstructed skin” or “endothelialized immunocompetent reconstructed mucous membrane” which is physiologically very similar to normal human skin or normal human mucous membrane. This model may be used for studying the physiopathology of epithelial barriers, irritation of the skin or mucous membranes, aggressions of a biological nature (for example viruses, retroviruses such as HIV, bacteria, molds, particulate antigens), phototoxicity, photoprotection, and the effect of active principles, particularly pharmaceutical and cosmetic active principles, and of finished products, particularly cosmetic and pharmaceutical products.
The invention makes it possible to generate different populations of DC whose different functionalities enable all the phenomena involved in the organism's infection/defense processes to be taken into account.
In addition, remarkably and unexpectedly, once integrated into a model of reconstructed skin or reconstructed mucous membrane, the cells generated in vitro from CD14+ monocytes, themselves isolated from peripheral circulating blood, are capable of:
It is seen that the invention affords major technical improvements allowing reliable and reproducible use on the industrial and commercial scale, particularly in the cosmetic and/or pharmaceutical industry, and that it can have major clinical implications.
A summary of the operating protocol used will give a better understanding of the different orientations of the CD14+ monocyte culture process.
Generation of Cells on the Basis of the Following Protocols, After Extraction of CD14+ Monocytes from Peripheral Circulating Blood:
Protocol 1:
CD14+ cultivated in suspension for 2 days in the presence of GM-CSF, TGFβ1 and IL-13, then for an additional 4 days in the presence of GM-CSF and TGFβ1→to D6: pre-LC (undifferentiated and immature)
Addition of TNFα (<18 h) in suspension→predominance of LC (differentiated and immature)
Protocol 2:
CD14+ cultivated in suspension for 6 days in the presence of GM-CSF, TGFβ1 and IL-13→to D6: pre-IDC (undifferentiated and immature)
Addition of TNFα (<18 h) in suspension→predominance of IDC (differentiated and immature)
Protocol 3:
CD14+ cultivated in suspension for 4 days in the presence of GM-CSF, TGFβ1 and IL-13, then for an additional 2 days in the presence of GM-CSF and TGFβ1→to D6: pre-LC and pre-IDC (undifferentiated and immature)
Addition of TNFα (<18 h) in suspension→homogeneous mixed population of LC and IDC (differentiated and immature)
Protocol 4:
CD14+ cultivated in suspension for 6 days in the presence of GM-CSF, TGFβ1 and IL-13, for either 2 days, 4 days or 6 days→to D6: pre-LC and pre-IDC (undifferentiated and immature)
Addition of TNFα (>20 h) in suspension→activated cells (differentiated and mature and no longer either LC or IDC)
If the cells obtained according to protocol 1, 2 or 3 are integrated into three-dimensional culture models (preferably at the undifferentiated cell stage=pre-LC and/or pre-IDC), it is observed that:
Other advantageous objects and characteristics of the invention will become clearly apparent to those skilled in the art from the following description referring to several Examples, which are given by way of illustration and cannot therefore in any way limit the scope of the invention.
In the Examples, the temperature is in degrees Celsius or is room temperature and the pressure is atmospheric pressure, unless indicated otherwise.
Process for the Separation of CD14+ Monocytes from Peripheral Circulating Blood
Peripheral circulating blood is harvested by drawing venous blood from one or more human donors into vacutainers or plastic bags containing conventional anticoagulant products such as heparin-lithium or citrate phosphate dextran.
Advantageously, the CD14+ monocytes can be separated from this circulating blood according to the protocol described by Geissmann et al. in J. EXP. MED. vol. 187, no. 6, 16 March 1998, pages 961-966, published by The Rockefeller University Press, in the following manner:
The CD14+ monocytes are recovered from the eluate by any physical separation process well known to those skilled in the art, especially by sedimentation or centrifugation, and are eluted as such for the subsequent cultures.
Per 100 milliliters of blood withdrawn, about 150 million (±20 million) mononuclear cells are extracted and up to 40 million CD14+ monocytes are purified. Depending on the culture conditions used (cf. the Examples below), from 12 to 16 million Langerhans cells and/or interstitial dendritic cells are generated.
Culture of Isolated CD14+ Monocytes to Give Undifferentiated and Immature Dendritic Cells
CD14+ monocytes, as obtained in Example 1, are cultivated at a rate of about 1 million per milliliter in RPMI 1640 culture medium supplemented with 10% of decomplemented fetal calf serum and initially containing two cytokines, namely the cytokine GM-CSF at a rate of 400 International Units/milliliter (or IU/ml) and the cytokine TGFβ1 at a rate of 10 nanograms/milliliter.
The culture is carried out at 37° C. in a humid atmosphere containing 5% of CO2.
Within the framework of the invention, the culture medium is initially supplemented with a third cytokine, namely the cytokine IL-13 at a rate of 10 nanograms/milliliter. On day 4 of culture, the same culture medium devoid of IL-13 is added and the culture is continued for a further two days. On day 6 of culture, undifferentiated and immature dendritic cells are generated which are capable of orientating themselves towards the pathways of differentiation into Langerhans cells and interstitial dendritic cells:
Culture of Isolated CD14+ Monocytes to Give Undifferentiated and Immature Dendritic Cells Capable of Orientating Themselves Preferentially Towards the Pathway of Differentiation into Interstitial Dendritic Cells (IDC)
CD14+ monocytes, as obtained in Example 1, are cultivated at a rate of about 1 million per milliliter in RPMI 1640 culture medium supplemented with 10% of decomplemented fetal calf serum and initially containing two cytokines, namely the cytokine GM-CSF at a rate of 400 IU/ml and the cytokine TGFβ1 at a rate of 10 ng/ml.
The culture is carried out at 37° C. in a humid atmosphere containing 5% of CO2.
Within the framework of the invention, the culture medium is initially supplemented with a third cytokine, namely the cytokine IL-13 at a rate of 10 ng/ml. After 6 days of culture, undifferentiated and immature dendritic cells are generated which are capable of orientating themselves preferentially towards the IDC differentiation pathway:
Culture of Isolated CD14+ Monocytes to Give Undifferentiated and Immature Dendritic Cells Capable of Orientating Themselves Preferentially Towards the Pathway of Differentiation into Langerhans Cells (LC)
CD14+ monocytes, as obtained in Example 1, are cultivated at a rate of about 1 million per milliliter in RPMI 1640 culture medium supplemented with 10% of decomplemented fetal calf serum and initially containing two cytokines, namely the cytokine GM-CSF at a rate of 400 IU/ml and the cytokine TGFβ1 at a rate of 10 ng/ml.
The culture is carried out at 37° C. in a humid atmosphere containing 5% of CO2.
The culture medium is initially supplemented with a third cytokine, namely the cytokine IL-13 at a rate of 10 ng/ml. Before 2 days of culture at the most, the same culture medium devoid of IL-13 is added up to day 6 of culture. On day 6, undifferentiated and immature dendritic cells are generated which are capable of orientating themselves preferentially towards the pathway of differentiation into Langerhans cells:
Culture of Isolated CD14+ Monocytes to Give Mainly Interstitial Dendritic Cells
CD14+ monocytes, as obtained in Example 1, are cultivated at a rate of about 1 million per milliliter in RPMI 1640 culture medium supplemented with 10% of decomplemented fetal calf serum and initially containing two cytokines, namely the cytokine GM-CSF at a rate of 400 IU/ml and the cytokine TGFβ1 at a rate of 10 ng/ml.
The culture is carried out at 37° C. in a humid atmosphere containing 5% of CO2.
Within the framework of the invention, the culture medium is initially supplemented with a third cytokine, namely the cytokine IL-13 at a rate of 10 ng/ml. After 6 days of culture, the cytokine TNFα is added at a rate of 200 IU/ml over less than 18 hours to give mainly interstitial dendritic cells:
Culture of Isolated CD14+ Monocytes to Give Mainly Langerhans Cells
CD14+ monocytes, as obtained in Example 1, are cultivated at a rate of about 1 million per milliliter in RPMI 1640 culture medium supplemented with 10% of decomplemented fetal calf serum and initially containing two cytokines, namely the cytokine GM-CSF at a rate of 400 IU/ml and the cytokine TGFβ1 at a rate of 10 ng/ml.
The culture is carried out at 37° C. in a humid atmosphere containing 5% of CO2.
Within the framework of the invention, the culture medium is initially supplemented with a third cytokine, namely the cytokine IL-13 at a rate of 10 ng/ml. Before 2 days of culture at the most, the same culture medium devoid of IL-13 is added up to day 6 of culture. On day 6, the cytokine TNFα is added at a rate of 200 IU/ml over at most 18 hours to give mainly Langerhans cells:
Culture of Isolated CD14+ Monocytes to Give a Homogeneous Dual Population of Langerhans Cells and Interstitial Dendritic Cells
CD14+ monocytes, as obtained in Example 1, are cultivated at a rate of about 1 million per milliliter in RPMI 1640 culture medium supplemented with 10% of decomplemented fetal calf serum and initially containing two cytokines, namely the cytokine GM-CSF at a rate of 400 IU/ml and the cytokine TGFβ, at a rate of 10 ng/ml.
The culture is carried out at 37° C. in a humid atmosphere containing 5% of CO2
Within the framework of the invention, the culture medium is initially supplemented with a third cytokine, namely the cytokine IL-13 at a rate of 10 ng/ml. After 4 days of culture, the same culture medium-devoid of IL-13 is added for a further 2 days. On day 6, the cytokine TNFα is added at a rate of 200 IU/ml over at most 18 hours, making it possible to generate a dual population of Langerhans cells and interstitial dendritic cells:
Culture of Isolated CD14+ Monocytes to Give Mainly Activated Mature Dendritic Cells
CD14+ monocytes, as obtained in Example 1, are cultivated at a rate of about 1 million per milliliter in RPMI 1640 culture medium supplemented with 10% of decomplemented fetal calf serum and initially containing two cytokines, namely the cytokine GM-CSF at a rate of 400 IU/ml and the cytokine TGFβ1 at a rate of 10 ng/ml.
The culture is carried out at 37° C. in a humid atmosphere containing 5% of CO2.
Within the framework of the invention, the culture medium is initially supplemented with a third cytokine, namely the cytokine IL-13 at a rate of 10 ng/ml. The culture is carried out up to day 6, irrespective of the incubation time of the cytokine IL-13. On day 6, the cytokine TNFα is added at a rate of 200 IU/ml over more than 20 hours to generate activated mature dendritic cells:
Use of the Population of Mainly Langerhans Cells in a Suspension Monocellular Model of Migration
Generation of the cells: cf. Example 6.
To evaluate the migratory capacity of Langerhans cells generated in vitro towards any kind of aggression, for example an aggression of a biological nature, such as a microorganism, for example a microorganism of the bacterial type, migration chambers are used which have two compartments separated by a membrane with a porosity of 8 to 5 micrometers, which may or may not be covered with a matrix imitating a basal membrane (Matrigel™ type), or Boyden chamber, according to the following protocol:
After simulation with mannan, the migration indices are between 1.6 and 1.9, i.e. the Langerhans cells generated in vitro and stimulated with mannan migrate 1.6 to 1.9 times more than the untreated Langerhans cells.
The Langerhans cells generated in vitro are capable of migrating under the effect of a stimulant, indicating that they are functional and that this test can be used as a study model for evaluating the effect of potentially aggressive/allergizing agents.
Use of a Population of Mainly Interstitial Dendritic Cells in a Suspension Monocellular Model of Cytokine Secretion
Generation of the cells: cf. Example 5.
To quantify the protein secretion of cytokines secreted by interstitial dendritic cells generated in vitro, for example interleukin 12 or IL-12, towards any kind of aggression, for example an aggression of a chemical nature, particularly an allergen such as TNP or 2,4,6-trinitrobenzenesulfonic acid, we can use assays of the ELISA (Enzyme Linked Immuno-Sorbent Assay) type according to the following protocol:
The interstitial dendritic cells generated in vitro and stimulated with TNP secrete IL-12p75 at concentrations of between 2.1 and 2.7 nanograms IL-12p75/1 million cells/milliliter, whereas the untreated interstitial dendritic cells secrete IL-12p75 at concentrations of less than 0.1 nanogram/1 million cells/milliliter.
The interstitial dendritic cells generated in vitro increase their secretion of immunoactivating cytokine under the effect of a stimulant, indicating that they are functional and that this test can be used as a study model for evaluating the effect of potentially aggressive/allergizing agents.
Use of a Dual Population of LC and IDC in a Suspension Bicellular Model of Antigen Internalization
Generation of the cells: cf. Example 7.
The advantage of a substantially homogeneous dual population of LC and IDC is the possibility of interacting with both cell types as in the in vivo situation. To study the internalization pathways of Langerhans cells and interstitial dendritic cells generated in vitro, i.e. their capacity to capture the antigen, we used dextran and the flow cytometry technique according to the following protocol:
Dendritic cells generated in vitro are capable of internalizing antigens, indicating that they are functional and that this test can be used as a model for studying the internalization of antigens.
Use of a Dual Population of LC and IDC in a Suspension Bicellular Model to Study Maturation Pathways of Both LC and IDC
Generation of the cells: cf. Example 7
On day 6, the cytokine TNFα is added at a rate of 200 IU/ml for 48 h.
The advantage of a substantially homogeneous dual population of LC and IDC is the possibility of interacting with both cell types as in the in vivo situation. To study the maturation pathways of LC and IDC generated in vitro, we analyzed the intracytoplasmic expression of maturation marker DC-LAMP on both LC and IDC. To this end, the experiments were performed according to the following protocol:
Use of a Population of Activated Mature Dendritic Cells in a Suspension Monocellular Model of Allogenic Stimulation of Naive T Lymphocytes
Generation of the cells: cf. Example 8.
To study whether mature dendritic cells generated in vitro are capable of acquiring the functionality of interdigitated dendritic cells, i.e. capable of stimulating the proliferation of allogenic naive T lymphocytes, we performed mixed lymphocyte reactions according to the following protocol:
After interaction with CD40-ligand, the activated dendritic cells generated in vitro strongly stimulate the proliferation of naive T lymphocytes (between 12.103 and 16.103 cpm) compared with activated dendritic cells, which induce a low proliferation of naive T lymphocytes (between 3.103 and 6.103 cpm).
Dendritic cells generated in vitro are capable of acquiring the functionality of interdigitated dendritic cells, i.e. capable of acquiring high allostimulant capacities, indicating that they are functional -and that this test can be used as a model for studying allostimulation.
Monolayer Multicellular Model of Keratinocytes and LC in Co-Culture
Generation of the cells: cf. Example 4 or 6.
1.105 keratinocytes are inoculated into culture dishes of the 6-well plate type in a Clonetics medium (reference: KGM-2) for a period of immersion culture up to confluence of the keratinocytes. At the point of confluence, 1 to 3.105 dendritic cells generated in vitro according to Example 4 or 6 are added. The culture is maintained for a further 3 to 4 days in RPMI 1640 culture medium supplemented with 10% of decomplemented fetal calf serum and initially containing two cytokines, namely the cytokine GM-CSF at a rate of 400 IU/ml and the cytokine TGFβ1 at a rate of 10 nanograms/milliliter.
Monolayer Multicellular Model of Fibroblasts and Interstitial Dendritic Cells in Co-Culture
Generation of the cells: cf. Examples 3 and 5.
1.105 fibroblasts are inoculated into culture dishes of the 6-well plate type in DMEM-Glutamax medium supplemented with 10% of Hyclone II calf serum, penicillin at a concentration of 100 IU/milliliter and gentamicin at a final concentration of 20 micrograms/milliliter for a period of immersion culture up to confluence of the fibroblasts. At the point of confluence, 1 to 3.105 dendritic cells generated in vitro according to Example 3 or 5 are added. The culture is maintained for a further 3 to 4 days.
Three-Dimensional Multicellular Model of Reconstructed Epidermis or Reconstructed Epithelium of Gingival Mucous Membrane Containing Epithelial Cells and Langerhans Cells
The model is prepared according to the following protocol:
Three-Dimensional Multicellular Models of Reconstructed Dermis or Reconstructed Gingival Chorion Containing Populations of Interstitial Dendritic Cells, Macrophages and Endothelial Cells
Generation of the cells: cf. Example 3, 4, 5, 6 or 7.
The model is prepared according to the following protocol:
The markers used reveal the presence of interstitial dendritic cells (DC-SIGN+), macrophages (macrophage marker from Novocastra: clone 3A5-monoclonal antibody NCL-MACRO) and endothelial cells (V-CAM+).
Three-Dimensional Multicellular Model of Reconstructed Skin Containing Populations of Langerhans Cells, Interstitial Dendritic Cells, Macrophaaes and Endothelial Cells
Generation of the cells: cf. Example 4 or 6.
The model is prepared according to the following protocol:
Three-Dimensional Multicellular Model of Pigmented Reconstructed Skin Containing Populations of Langerhans Cells, Interstitial Dendritic Cells, Macrophages and Endothelial Cells
The model is prepared according to the protocol described in Example 18, 10,000 melanocytes being co-inoculated with the keratinocytes and the dendritic cells generated in vitro.
In addition to the markers described in Example 18, the melanocytes are immunolabeled (MELAN-A) and an immunohistochemical study is carried out (DOPA reaction).
Three-Dimensional Multicellular Model of Reconstructed Skin Containing Populations of Interstitial Dendritic Cells, Macrophages and Endothelial Cells
Generation of the cells: cf. Example 3, 5 or 7.
The model is prepared by following the protocol described in Example 18.
The markers used reveal the presence of interstitial dendritic cells (DC-SIGN+), macrophages (macrophage marker from Novocastra: clone 3A5-monoclonal antibody NCL-MACRO) and endothelial cells (V-CAM+) in the dermis.
Three-Dimensional Multicellular Model of Reconstructed Vaginal Mucous Membrane Containing Populations of Langerhans Cells, Interstitial Dendritic Cells, Macrophacies and Endothelial Cells
Generation of the cells: cf. Example 4 or 6.
The model is prepared according to the protocol described in Example 18, with the following modifications: the keratinocytes are replaced with vaginal epithelial cells, the fibroblasts are derived from vaginal mucous membrane and the culture is carried out totally as an immersion culture in the culture medium.
The epithelial cell cultures are then maintained as immersion cultures for 12 to 18 days in the same culture medium, except that the percentage of calf serum is reduced from 10 to 1%.
The markers used reveal the presence of Langerhans cells (Langerin+) in the epithelium and interstitial dendritic cells (DC-SIGN+), macrophages (macrophage marker from Novocastra: clone 3A5-monoclonal antibody NCL-MACRO) and endothelial cells (V-CAM+) in the chorion.
Three-Dimensional Multicellular Model of Reconstructed Vaginal Mucous Membrane Containing Populations of Interstitial Dendritic Cells, Macrophages and Endothelial Cells
Generation of the cells: cf. Example 3, 5 or 7.
The model is prepared according to the protocol described in Example 18, with the following modifications: the keratinocytes are replaced with vaginal epithelial cells, the fibroblasts are derived from vaginal mucous membrane and the culture is carried out totally as an immersion culture in the culture medium. The epithelial cell cultures are then maintained as immersion cultures for 12 to 18 days in the same culture medium as that used for the immersion culture, except that the percentage of calf serum is reduced from 10% to 1%.
The markers used reveal the presence of interstitial dendritic cells (DC-SIGN+), macrophages (macrophage marker from Novocastra: clone 3A5-monoclonal antibody NCL-MACRO) and endothelial cells (V-CAM+) in the chorion.
Use of Any One of the Models Described in Example 16, 18, 19 or 20 for Studying LC/Epithelial Environment Interactions
After preparation of the model, the E-cadherin is labeled.
Expression of the adhesion molecule E-cadherin is found on the Langerhans cells and the epithelial cells, representing possible interactions of the heterophilic type via this protein between the Langerhans cells and the neighboring epithelial cells.
Use of the Model of Reconstructed Epidermis Described in Example 16 for Studying the Influence of UVB Radiation
To study the influence of various environmental factors, particularly UV radiation and more precisely UVB, which mainly penetrates the epidermis, we evaluated the migration and the phenotypic profile of Langerhans cells in a model of reconstructed epidermis after UVB irradiation by means of immunohistochemical studies according to the following protocol:
After UVB irradiation, an estimated decrease of more than 50% is observed in the number of Langerhans cells in the epidermal compartment, together with a decrease e.g. in the labeling intensity of the co-stimulation molecule CD86 on the Langerhans cells remaining in the epidermis.
Use of the Model of Reconstructed Skin Described in Example 20 for Studying the Influence of UVA Radiation
To study the influence of various environmental factors affecting the dermis of the skin, particularly UV radiation and more precisely UVA, we evaluated the phenotypic profile of interstitial dendritic cells in a model of reconstructed skin after UVA irradiation by means of immunohistochemical studies according to the following protocol:
After UVA irradiation, a decrease is observed for example in the labeling intensity of the molecules HLA-DR and CD86 on the interstitial dendritic cells present in the dermis.
Use of the Model of Reconstructed Skin Described in Any One of Examples 18, 19 and 20 for Studying the Profile of Cytokines Secreted Under the Effect of an Active Principle
To evaluate the potentially sensitizing or allergizing power and assess a possible pro- or anti-inflammatory activity of an active principle intended for the human skin, we quantified the secretion of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, IL-12, TNFα, INFγ etc. and immunosuppressant cytokines such as IL-2, IL-10 etc. by ELISA according to the following protocol:
It is observed that the retinol 10S causes a stimulation of the pro-inflammatory cytokines.
Use of the Model of Reconstructed Epidermis Described in Example 16 for Screening Active Principles Capable of Modulating Allergic Reactions
The immunomodulating effect of an active principle after the induction of an allergizing stress is studied according to the following protocol:
The combined results of the migration test and the IL-12 synthesis make it possible to establish the immunomodulating profile of the active principles tested.
Use of a Model of Reconstructed Skin or Reconstructed Mucous Membrane Obtained According to Any One of Examples 18, 19 and 21 for Studying the Immunostimulant or Immunosuppressant Activity of an Active Principle or Evaluating and/or Inducing an Immunotolerance
To evaluate the capacity of Langerhans cells and/or interstitial dendritic cells to induce or not to induce immune and/or tolerogenic responses towards an active principle, we studied the phenotypic profile of Langerhans cells and/or interstitial dendritic cells by immunohistochemistry in the three-dimensional culture models according to the following protocol:
Use of a Model Containing Mainly Interstitial Dendritic Cells in Suspension, Obtained According to Example 10 for Studying the Immunostimulant or Immunosuppressant Activity of an Active Principle or Evaluating and/or Inducing an Immunotolerance
The study is carried out according to the following protocol:
The phenotypic profile of the cells makes it possible to define the immuno-modulating effect of the active principles tested.
Use of Any One of the Models of Reconstructed Mucous Membrane Described in Example 21 or 22 for Studying Infection by HIV
The study is carried out according to the following protocol:
Infections are produced by the direct injection or deposition of the viral suspension (monocytotrophic strain HIV-1BaL at a concentration of 55 nanograms p24/106) in reconstructed mucous membranes after 35 days of culture using a needle. Incubation proceeds overnight at 37° C. and is followed by 4 washes with culture medium. The cultures are continued for one week and the following analyses are performed:
Preparation of Suspensions of Dendritic Cells Using a Serum-Free Culture Medium—Therapeutic Applications
The CD14+ culture protocol is identical to Examples 2, 3, 4, 5, 6, 7 and 8. However, the RPMI 1640 medium supplemented with 10% of fetal calf serum is replaced with a specific serum-free medium from STEMBIO with the reference StembioA: SB A 100.
The dendritic cells can then serve as targets for sensitization and as therapeutic tools (antigen-presenting cells) in cell immunotherapy.
| Number | Date | Country | Kind |
|---|---|---|---|
| 01/15942 | Dec 2001 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP02/14874 | 12/10/2002 | WO |
