In vitro safety evaluation method for substances using immortalized human myeloid cells

Abstract

[Problem] To discover a method having greater stability, reproducibility, economicalness, and ease of operation by using human immortalized myeloid cells in an in vitro method for evaluating the safety of a substance. [Solution] A method for evaluating the skin sensitization capacity and / or pyrogenicity of a test substance, a method for detecting a skin sensitizing substance and / or pyrogenic substance in a sample, and a method for evaluating an effect on a function of immune cells in a sample using human immortalized myeloid cells, wherein the method includes measuring the amount of IL-6 and / or IL-8 produced in the culture broth of human immortalized myeloid cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international application claims priority based on Japanese Patent Application No. 2019-208711, filed with the Japan Patent Office on November 19, 2019, the entire contents of which are incorporated herein by reference. [Technical field]

[0002] The present invention relates to a method for evaluating the skin sensitization and / or pyrogenicity of a substance in vitro using human immortalized myeloid cells, a method for detecting a skin sensitizing substance and / or a pyrogenic substance in a sample, and a method for evaluating the effect of a sample on the function of immune cells. [Background technology]

[0003] When providing products or substances to be consumed, ingested, or applied to humans, it is necessary to know in advance whether the substances contained in the product are safe and will not cause allergic reactions.

[0004] For example, a method for evaluating whether a substance induces allergies involves administering or applying the substance to laboratory animals such as mice and guinea pigs and observing the inflammatory reaction in the skin of the laboratory animals. In addition, a pyrogenic test using rabbits is used to check whether foods, cosmetics, etc. are contaminated by viruses, bacteria, etc. However, from the viewpoint of animal protection, there is a noticeable trend to reduce the pain of animals used and the number of animals used, and there is a demand for a shift to alternative methods.

[0005] To date, methods for evaluating skin sensitization using laboratory animals have been established, including the Guinea Pig Maximization Test (GPMT), Adjuvant and Patch Test (A&P), Buehler Test (BT), and Local Lymph Node Assay (LLNA).

[0006] For example, in the GPMT, A&P, and BT tests, guinea pigs are used as experimental animals and sensitized by administering a test solution containing a dissolved substance subcutaneously or by applying an occlusive patch several times. The test solution is then applied again by applying an occlusive patch, and after the test solution is removed, the skin reaction is observed and erythema and edema are scored to evaluate the presence or absence of skin sensitization.

[0007] In the LLNA test, mice are used as experimental animals. After the test solution is applied to the mouse's ears several times, a radioactively labeled substance (e.g., 3H-thymidine) is administered intravenously. The amount of the labeled substance taken up by the lymph nodes in the ear is measured, and the presence or absence of skin sensitization is evaluated using the activation of lymphocyte proliferation as an indicator.

[0008] As mentioned above, a test using rabbits has been established as an evaluation method for pyrogen testing. After measuring the rectal temperature with a temperature sensor in advance, the test liquid is administered to the rabbit's ear vein, etc., and the rise in body temperature is measured several hours later, and the degree of rise is judged based on a standard to evaluate whether or not a pyrogen is present.

[0009] In recent years, mainly in Europe, the 3Rs of animal welfare (Replacement, Reduction, and Refinement) have been recommended. Furthermore, animal testing of cosmetics and their ingredients has been completely banned in Europe since 2009, and laws have been enacted that prohibit the sale of products that have undergone animal testing. As a result, evaluation methods to replace animal testing have been developed and reported in recent years.

[0010] For example, skin sensitization is a reaction that occurs when a causative substance comes into contact with and penetrates the skin. The mechanism is as follows: 1) the substance comes into contact with the skin, 2) the substance is absorbed through the skin, 3) it binds to proteins and is phagocytosed by dendritic cells, 4) the phagocytosed dendritic cells become activated and migrate to the regional lymph nodes, and 5) T cells in the regional lymph nodes receive antigen presentation from dendritic cells, become activated, and proliferate. When these steps occur, immune induction occurs, and when the same substance comes into contact again, inflammatory cytokines are released from the proliferated T cells, causing an allergic inflammatory reaction (erythema and edema).

[0011] Based on this mechanism, alternative tests for in vitro evaluation have been developed. For example, the Direct Peptide Reactivity Assay (DPRA) is a method that uses liquid chromatography to measure the ease with which a protein binds to a substance. In addition, the human Cell Line Activation Test (h-CLAT) is a method that uses THP-1 cells to measure the expression of activation markers with a flow cytometer during the process of dendritic cell activation. These are methods that do not use animals.

[0012] h-CLAT is an alternative method for skin sensitization testing that was included in the Organisation for Economic Co-operation and Development (OECD) guidelines in 2016. Shiseido Co., Ltd. and others found that the amount of CD86 or CD54 and MHC Class II cell surface proteins on Langerhans cells, which are antigen-presenting cells in the skin, is increased by skin sensitizing substances, and an alternative method was developed that utilizes this phenomenon. However, since Langerhans cells are only found in the epidermis, and it is difficult to stably supply dendritic cells that exhibit Langerhans cell-like properties, an alternative method, h-CLAT, using human mononuclear cells (THP-1), was established.

[0013] As an alternative to experimental animals, the h-CLAT is highly useful because, compared to, for example, the LLNA evaluation, it does not use animals (mice) for the evaluation and the test period is short. However, as an in vitro evaluation method, it is more complicated to operate and has many restrictions than other established methods, such as DPRA. For example, it is necessary to prepare THP-1 cells and understand the concentration of the added agent in advance through preliminary tests, and a flow cytometer is used to measure surface markers. Furthermore, there are issues such as the need for continuous measurement of positive and vehicle controls and low reproducibility (approximately 80%) (Non-Patent Document 1).

[0014] In addition, THP-1 cells do not have sufficient amounts of the oxidoreductase cytochrome P450, which becomes activated as they differentiate from monocytes, and therefore have the problem that they cannot detect substances that exhibit skin sensitization through oxidation / reduction (Non-Patent Document 2).

[0015] Therefore, a method that can evaluate the skin sensitization potential of substances in vitro more easily and stably than the h-CLAT was required.

[0016] Alternative methods to experimental animals have also been developed for pyrogen testing. One method uses the blood of horseshoe crabs, which are not mammals, making it possible to determine contamination with gram-negative bacteria, and another measurement method uses Mono-Mac-6 cells derived from human acute monocytic leukemia and human peripheral blood, and utilizes the production of cytokines such as IL-6 by stimulating the cells with a specimen that may be contaminated by bacteria.

[0017] However, only gram-negative bacteria can be detected in horseshoe crab blood. Also, Mono-Mac-6 has a lower sensitivity than human blood. Furthermore, although human peripheral blood can be used to evaluate human conditions in vitro, it is difficult to provide a stable supply of blood obtained through blood donations, and individual differences may occur.

[0018] Therefore, a method for detecting skin sensitizers and / or pyrogens that uses tissues and cells similar to human dendritic cells and can be supplied in large quantities in a stable manner was required.

[0019] In addition, the function of immune cells is exerted by the cytokines they release, but until now there has been no system capable of testing the direct effects of drugs, foods, etc. on immune cells.

[0020] For example, one method of evaluating the effect of food ingredients on the function of immune cells is to use human peripheral blood, but this requires using blood obtained through donations, etc., which is difficult to provide on a stable basis and can result in individual differences.

[0021] Therefore, there was a demand for a method that could be easily and stably supplied and that could evaluate functionality in vitro. [Prior art documents] [Non-patent literature]

[0022] [Non-Patent Document 1] Skin Sensitization Test Evaluation Report JaCVAM Skin Sensitization Test Data Compilation Committee [Non-Patent Document 2] Takao Ashikaga et al.,ATLA 38,275-284,2010 Summary of the Invention

[0023] In other words, the present invention provides an in vitro method for evaluating the skin sensitization and / or pyrogenicity of a substance, a method for detecting skin sensitizing substances and / or pyrogenic substances in a sample, and a method for evaluating the effect of a sample on the function of immune cells, which aim to find methods that are more stable, reproducible, economical, and easy to operate. [Means for solving the problem]

[0024] The present inventors conducted studies using monocytes and dendritic cells prepared by various methods in order to seek a method for providing cells suitable for in vitro skin sensitization and / or pyrogenicity evaluation, detection of skin sensitizers and / or pyrogens, and evaluation of effects on immune cell functions, which are suitable for in vitro skin sensitization and / or pyrogenicity evaluation, detection of skin sensitizers and / or pyrogens, and evaluation of effects on immune cell functions. As a result, they found that by using proliferative human monocytes obtained by introducing genes into CD14-positive cells derived from human umbilical cord blood, ES cells, iPS cells, bone marrow cells, or peripheral blood, and cells (e.g., dendritic cells) having phagocytic ability obtained by inducing differentiation of the monocytes, it is possible to evaluate skin sensitization and / or pyrogenicity, detect skin sensitizers and / or pyrogens, and evaluate effects on immune cell functions, and thus completed the present invention. In particular, by using these cells, 1) it is possible to stably provide cells, 2) it is possible to provide cells with a uniform state (passage number, differentiation state), and 3) it is possible to detect reactions closer to those in vivo. In assessing skin sensitization and / or pyrogenicity, and evaluating the effect on immune cell function, the inventors focused on the fact that the addition of a substance results in the production of cytokines (IL-6, IL-8, etc.), and have now provided a novel method for testing skin sensitization and / or pyrogenicity, and for evaluating the effect on immune cell function.

[0025] Furthermore, the present invention solves these problems by using immortalized CD14-positive cells derived from human umbilical cord blood, ES cells, iPS cells, bone marrow cells, or peripheral blood. That is, by adopting a method for immortalizing CD14-positive cells, it is possible to easily obtain a population of only myeloid cells in a stable manner. Therefore, compared with conventional cells, it is possible to stably provide myeloid cells that are closer to living organisms, and these cells are considered to be suitable for safety evaluation. Thus, the present invention provides safety evaluation such as skin sensitization and / or pyrogenicity evaluation and detection of skin sensitizing substances and / or pyrogenic substances using immortalized myeloid cells. Furthermore, the present invention provides evaluation of the effect of a sample on the function of immune cells using immortalized myeloid cells.

[0026] Specifically, the present invention relates to the following inventions: (1) A method for evaluating the skin sensitization and / or pyrogenicity of a test substance, comprising measuring the amount of IL-6 and / or IL-8 produced in a culture medium obtained by culturing human immortalized myeloid cells in the presence of the test substance. (2) The method according to (1), wherein the human immortalized myeloid cells are human immortalized monocytes or dendritic cells prepared from the human immortalized monocytes. (3) The method according to (1) or (2), wherein the concentration of the test substance during the culture of the human immortalized myeloid cells includes at least three different concentrations between greater than 0 μg / mL and less than or equal to 1000 μg / mL. (4) The method according to any one of (1) to (3), wherein the culture time is selected from at least one period between 3 hours and 48 hours. (5) The method according to any one of (1) to (4), wherein the amount produced is measured by an immunological assay. (6) The method according to (5), wherein the immunological assay is ELISA. (7) The method according to any one of (1) to (6), further comprising determining that the test substance has skin sensitization and / or pyrogenicity when the production amount is 1.5 times or more compared to the production amount of a negative control. (8) A method for detecting a skin sensitizer and / or a pyrogen in a sample, comprising measuring the amount of IL-6 and / or IL-8 produced in a culture medium obtained by culturing human immortalized myeloid cells in the presence of the sample. (9) The method according to (8), wherein the human immortalized myeloid cells are human immortalized monocytes or dendritic cells prepared from the human immortalized monocytes. (10) The method according to (8) or (9), wherein the concentration of the sample during the culture of the human immortalized myeloid cells includes at least three different concentrations. (11) The method according to any one of (8) to (10), wherein the culture time is selected from at least one period between 3 hours and 48 hours. (12) The method according to any one of (8) to (11), wherein the amount produced is measured by an immunological assay. (13) The method according to (12), wherein the immunological assay is ELISA. (14) The method according to any one of (8) to (13), further comprising determining that the sample contains a skin sensitizer and / or a pyrogen if the amount of production is increased relative to the amount of production in a negative control. (15) The method according to any one of (8) to (14), wherein the skin sensitizer and / or pyrogen is lipopolysaccharide (LPS) and / or Staphylococcus aureus Cowan 1 (SAC). (16) A method for evaluating the effect of a sample on the function of immune cells, comprising measuring the amount of cytokine produced in a culture medium obtained by culturing human immortalized myeloid cells in the presence of the sample. (17) The method according to (16), wherein the human immortalized myeloid cells are human immortalized monocytes or dendritic cells prepared from the human immortalized monocytes. (18) The cytokine is IL-1a, IL-1b, IL-1ra, IL-2, IL-2Ra, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-12(p70), IL-12(p40), IL-13, IL-15, IL-16, IL-17A, IL-18, CTACK, Eotaxin, FGF basic, G-CSF, GM-CSF, GRO-α, HGF, IFN-α2, IFN-γ, IP-10, LIF, MCP-1(MCAF), MCP-3, M-CSF, MIF, MIG, MIP-1α, MIP-1 The method according to (16) or (17), which is selected from the group consisting of β, β-NGF, PDGF-BB, RANTES, SCF, SCGF-β, SDF-1a, TNF-α, TNF-β, TRAIL, and VEGF-A. (19) The method according to any one of (16) to (18), wherein the effect on the function of the immune cells is selected from the group consisting of an effect of increasing the amount of cytokine production from immune cells, an effect of decreasing the amount of cytokine production from immune cells, an inflammation-inducing effect by immune cells, an inflammation suppression-inducing effect by immune cells, an immune activation-inducing effect by immune cells, an immune suppression-inducing effect by immune cells, an effect of stimulating mast cells by immune cells, an effect of enhancing IgE production by immune cells, an effect of suppressing IgE production by immune cells, an effect of enhancing IgA production by immune cells, an effect of proliferating and activating lymphocytes by immune cells, an effect of increasing eosinophil production by immune cells, an effect of promoting leukocyte migration by immune cells, an effect of inducing apoptosis by immune cells, an effect of suppressing apoptosis by immune cells, an effect of increasing virus resistance by immune cells, an effect of promoting cell proliferation by immune cells, an effect of inducing angiogenesis by immune cells, a wound healing effect by immune cells, an effect of promoting hematopoietic cell proliferation by immune cells, an effect of differentiating precursor cells by immune cells, and an effect of inhibiting cell differentiation by immune cells. (20) The method according to any one of (16) to (19), wherein the culture time is 3 to 48 hours. (21) The method according to any one of (16) to (20), further comprising determining that the sample is functional if the amount of cytokine produced in the culture medium obtained by culturing the human immortalized myeloid cell in the presence of the sample is at least twice as much as the amount of cytokine produced in the culture medium obtained by culturing the human immortalized myeloid cell in the presence of a negative control. (22) The method according to any one of (16) to (21), wherein the sample is a functional beverage. (23) A kit for use in the method according to any one of (16) to (22), comprising human immortalized monocytes or dendritic cells prepared from the human immortalized monocytes. Effect of the Invention

[0027] The present invention enables research and evaluation of safety tests related to the detection of skin sensitization and pyrogenicity of substances. For example, the use of the present invention enables precise and sensitive evaluation of safety research and evaluation, which has been difficult to carry out because of the difficulty in stably evaluating blood cell materials derived from human blood donations, and makes it possible to carry out safety research and evaluation. In addition, the method of the present invention enables safety evaluation of drugs with less error. Furthermore, due to the above-mentioned characteristics, the method of the present invention can also be used as a screening method for safety evaluation. In addition, due to the above-mentioned characteristics, the method of the present invention can also be used as a method for evaluating the effect of a sample on the function of immune cells and a method for screening substances that have an effect on the function of immune cells. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows the results of skin sensitization and / or pyrogenicity evaluation of each test substance and the results of a conventional skin sensitization evaluation method. [Diagram 2] 2A and 2B are graphs showing the amounts of IL-6 and IL-8 produced in the culture supernatant obtained by culturing iPS-ML2 for 6 and 24 hours in the presence of various concentrations of LPS, as measured by ELISA. The vertical axis of the graph shows the measured values ​​by ELISA, and the horizontal axis of the graph shows the concentration of LPS. FIG. 2C and 2D are graphs showing the amounts of IL-6 and IL-8 produced in the culture supernatant obtained by culturing iPS-ML2 for 6 and 24 hours in the presence of various concentrations of SAC, as measured by ELISA. The vertical axis of the graph shows the measured values ​​by ELISA, and the horizontal axis of the graph shows the dilution ratio of SAC. [Diagram 3] The amount of IL-6 produced in the culture supernatant obtained by culturing iPS-ML2 for 24 hours in the presence of each concentration of functional beverage was measured by ELISA, and the graph shows the production ratio (Stim. Index), which is the value obtained by dividing the measured value of the cell supernatant after culturing in the presence of each functional beverage by the measured value of the negative control. The vertical axis of the graph shows the production ratio, and the horizontal axis of the graph shows the concentration (%) of the functional beverage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] <Terminology> As used herein, the term "skin sensitization" refers to a delayed hypersensitivity reaction, a phenomenon in which inflammation of the skin occurs due to an excessive immune response caused by a substance.

[0030] In this specification, the term "substance" may be a monatomic molecule or a compound.

[0031] As used herein, the term "pyrogenic substance" refers to a substance that, when taken into the body, causes an increase in body temperature, such as endotoxin, peptidoglycan, exotoxins from gram-positive bacteria, viruses, pathogenic bacteria, pathogenic fungi, lipopolysaccharide (LPS), Staphylococcus aureus Cowan 1 (SAC), etc. Such an increase in body temperature may be caused by an immune response of a sample inoculated with the substance.

[0032] In the present specification, a "myeloid cell" is defined as a cell expressing a CD11b molecule or a CD33 molecule, and the origin is not particularly limited, but examples thereof include myeloid cells derived from pluripotent stem cells, or myeloid cells collected from a living body (e.g., peripheral blood). Specific examples of myeloid cells include monocytes, dendritic cells, macrophages, etc.

[0033] In this specification, the term "test substance" is not particularly limited as long as it is a substance to be evaluated for skin sensitization and / or pyrogenicity.

[0034] As used herein, the term "sample" refers to bodily fluids (e.g., blood, tears, saliva, or urine) or tissues derived from humans or animals, plants and their extracts, foods such as fruits and vegetables and their extracts, foods such as processed foods and beverages, soaps, shampoos, rinses and treatments, detergents, dyes, fibers, cloth, cosmetics, pharmaceuticals, compounds, mixtures (e.g., foods, food extracts, plant extracts), trace substances in the air, exhaust gases, waste liquids, industrial waste, cell culture fluid, cultured cells, cell culture media, cell culture additives, cell preservation fluids, health functional foods, supplements, etc.

[0035] In this specification, "health functional foods" refers to a group of foods such as processed foods and beverages that are consumed for the purpose of maintaining or improving health, including foods for specified health uses, foods with nutrient functions, and foods with functional claims. In this specification, "functionality" refers to properties that are effective in maintaining and improving health (e.g., bioregulation, improving immunity, preventing diseases, etc.). In this specification, "functional beverages" refers to beverages that contain functional ingredients (e.g., lactic acid bacteria).

[0036] In this specification, the term "about" refers to a range of ±10%.

[0037] <Method for preparing immortalized monocytes> In the present invention, the starting monocytes can be obtained by, for example, collecting them from peripheral blood, inducing their differentiation from pluripotent stem cells, or inducing their differentiation from other somatic cells such as fibroblasts.

[0038] When monocytes are collected from peripheral blood, a method for separating and preparing cells expressing CD14 molecules in human peripheral blood is known. For example, human peripheral blood is gently diluted with an equal volume of physiological saline, phosphate-buffered physiological saline, or Hank's buffered saline. The diluted blood is gently layered on Ficoll (registered trademark) (GE Healthcare) in a centrifuge tube and centrifuged at 15 to 30°C and 500 to 1000 x G for 20 minutes. A white band-like layer between the yellowish plasma and the transparent Ficoll is collected as a peripheral blood mononuclear cell (PBMC) fraction consisting of lymphocytes and monocytes. The collected peripheral blood mononuclear cell fraction may be further washed as necessary before use. Monocytes can be obtained by further collecting cells expressing CD14 molecules from the collected PBMC fraction. For example, monocytes can be separated and collected by contacting PBMC with a solid phase bound to an anti-CD14 antibody to bind the monocytes to the solid phase, and washing and removing unbound cells. For example, a method using magnetic beads as such a solid phase is known (Dynabeads (registered trademark) CD14 (Thermo Fisher Scientific Inc.), etc.). Furthermore, monocytes can be obtained by directly contacting peripheral blood with a solid phase to which an anti-CD14 antibody is bound, without isolating PBMCs from peripheral blood.

[0039] When monocytes are obtained by differentiating pluripotent cells, methods for inducing monocytes from pluripotent cells are known. As used herein, "pluripotent stem cells" refer to cells having pluripotency and self-renewal ability. As used herein, "pluripotency" is synonymous with multipotency and refers to the state of cells that can be differentiated into cells of multiple lineages by differentiation. As used herein, pluripotency includes a state in which a cell can be differentiated into all types of cells that constitute a living body (totipotency), a state in which a cell can be differentiated into all types of cells except for extraembryonic tissues (pluripotency), a state in which a cell can be differentiated into cells belonging to some cell lineages (multipotency), and a state in which a cell can be differentiated into one type of cell (unipotency). Thus, the term "pluripotent stem cells" in this specification includes stem cells, embryonic stem (ES) cells, cloned embryo-derived embryonic stem cells ("ntES cells") obtained by nuclear transfer, germline stem cells ("GS cells"), embryonic germ cells ("EG cells"), induced pluripotent stem (iPS) cells, and hematopoietic stem cells. Whether a certain cell is a pluripotent stem cell can be determined, for example, when the test cell forms an embryoid body in an in vitro culture system, or when the test cell differentiates into a desired cell after being cultured (differentiation treatment) under differentiation-inducing conditions. Methods for obtaining pluripotent stem cells are known, and for example, it has been reported that iPS cells can be obtained by introducing three or more types of reprogramming genes (Oct3 / 4, Sox2, Klf4, c-Myc, L-Myc, Nanog, Lin28, Esrrb, Glis1, E-cadherin, shp53, UTX, and H1foo, etc.) into somatic cells.

[0040] The differentiation of pluripotent stem cells into monocytes can be induced, for example, by contacting the pluripotent stem cells with macrophage colony-stimulating factor (M-CSF) and interleukin 3 (IL-3) (see Fernadondo O. Martinez et al., Experimental Hematology (2008); 36: 1167-1175), or with IFN-γ or PMA (Annabelle Grolleau et al., J Immunol 1999; 162: 3491-3497). From the monocytes induced to differentiate, only cells expressing CD14 may be collected and purified by the above-mentioned method, if necessary.

[0041] Methods for inducing differentiation of monocytes from other somatic cells such as fibroblasts include the method described by Takahiro Suzuki et al., “Reconstruction of Monocyte Transcriptional Regulatory Network Accompanies Monocytic Functions in Human Fibroblasts,” PLoS One (2012) doi:10.1371 / journal.pone.0033474.

[0042] The obtained monocytes can be given proliferation ability while maintaining the function of the monocytes by introducing at least one gene selected from the BMI1 gene, EZH2 gene, MDM2 gene, MDM4 gene, HIF1A gene, BCL2 gene, and LYL1 gene and the cMYC gene. Preferably, at least one gene selected from the BMI1 gene, EZH2 gene, MDM2 gene, MDM4 gene, and HIF1A gene is combined with the cMYC gene, or the BMI1 gene and the BCL2 gene or the LYL1 gene and the cMYC gene. The introduction of these genes into monocytes can be performed with reference to the descriptions in WO2012 / 043651 and JP2017-131136A. For example, the genes can be introduced by infecting monocytes with a lentiviral vector carrying these genes.

[0043] <Preparation of dendritic cells> Dendritic cells can be induced from immortalized monocytes as previously reported (Francoise Chapuis et al., Eur J Immunol. (1997) 27(2):431-41; Marc Dauer et al., J Immunol (2003) 170(8):4069-4076; Figdor CG et al., Nat Med. (2004) 10(5):475-80; Helmut Jonuleit et al., Eur J Immunol. (1997) 27(12):3135-42), for example, by culturing monocytes in the presence of 200 IU / ml GM-CSF and 200 IU / ml IL-4.

[0044] <Evaluation of skin sensitization and / or pyrogenicity of substances using immortalized monocytes or dendritic cells> Evaluation of skin sensitization and / or pyrogenicity of a substance using immortalized monocytes or dendritic cells is carried out by the following steps.

[0045] As the first step, cells for evaluation are prepared. The cells used for evaluation are the immortalized monocytes or dendritic cells described above. Examples of media used for evaluation and / or for culturing before and after evaluation include IMDM solution, RPMI solution, α-MEM solution, MEM medium, and DMEM solution. In addition, additives may include human or bovine plasma protein fractions, fetal bovine serum or human serum, glucose, sugars such as D-mannitol, amino acids, nucleic acid bases such as adenine, sodium hydrogen phosphate, α-tocopherol, linoleic acid, cholesterol, sodium selenite, human holotransferrin, human insulin, ethanolamine, 2-mercaptoethanol, G-CSF, GM-CSF, sodium bicarbonate, methylcellulose, and the like. In addition, antibiotics such as gentamicin, ampicillin, penicillin, and strepromycin; inorganic salts; and buffers such as HEPES, phosphate buffer, and acetate buffer may be added as necessary. Specific examples include α-MEM (10% fetal bovine serum) medium supplemented with non-essential amino acids, a culture medium prepared by adding 2 or 5% HPL (human platelet lysate, a supplement derived from human platelets) to α-MEM, or a medium prepared by adding 10% fetal bovine serum, sodium bicarbonate, 100 units / mL penicillin, and 100 mg / mL streptomycin to RPMI1640 medium (SIGMA) containing hypoxanthine and HEPES (SIGMA).

[0046] Immortalized monocytes or dendritic cells are seeded in the above-mentioned medium, and the cells are passaged a predetermined number of times. The number of passages may be, for example, once, twice, three times, four times, or five or more times. The cells after passage are then washed a predetermined number of times (for example, twice, three times, or four or more times), and the cells are adjusted to a predetermined concentration using the medium used for the evaluation, and then the evaluation is performed using the cell concentration. The cell concentration can be set according to the protocol of the immunological measurement method described below.

[0047] In the second step, the immortalized monocytes or dendritic cells are cultured in the presence of a test substance. The test substance is dissolved in a dissolving solution and used for evaluation. Ethanol, DMSO, water, PBS, etc. may be used as the dissolving solution for dissolving the test substance. The test substance is diluted so as to contact the cells at a predetermined final concentration and used for evaluation. The predetermined final concentration is one or more types, preferably at least three types, between 0 μg / mL and 1000 μg / mL or less. The concentrations to be adjusted may be three types, four types, five types, or six or more types. The test substance may be diluted using the above-mentioned medium.

[0048] The culture time of the immortalized monocytes or dendritic cells may be set to one type of time or two types of time. Also, three or more types of time may be set. For example, the culture time may be set to any time selected from 3 to 48 hours, preferably 6 to 24 hours. Also, the temperature during culture may be about 37°C, and the carbon dioxide concentration during culture may be about 5%.

[0049] As a third step, IL-6 and / or IL-8 in the culture medium of the immortalized monocytes or dendritic cells after the culture is measured. The culture medium may be, for example, a culture supernatant. The measurement may be performed using a known immunological measurement method. The immunological measurement method may be, for example, ELISA (Enzyme-Linked Immuno Sorbent Assay), immunochromatography, radioimmunoassay, or Western blot. Furthermore, these immunological measurements may be performed using commercially available kits.

[0050] Whether or not a test substance has skin sensitization and / or pyrogenicity is judged based on the measured amount of IL-6 and / or IL-8 produced. Specifically, when the amount of IL-6 and / or IL-8 produced in a culture solution of immortalized monocytes or dendritic cells obtained by culturing for an arbitrary time in the presence of a test substance is increased compared to a negative control, the test substance is judged to have skin sensitization and / or pyrogenicity. For example, the increase may be set to a certain increase rate relative to the negative control as a threshold, and when the amount of production exceeds the increase rate, the test substance may be judged to have skin sensitization and / or pyrogenicity. Such an increase rate may be a value between 1.2 and 2.0 times, for example, 1.5 times. In addition, when evaluation is performed using a test substance at multiple concentrations, when the amount of IL-6 and / or IL-8 produced in a culture solution of the cells obtained by culturing in the presence of at least one of the multiple concentrations prepared for the test substance is increased compared to a negative control, the test substance is judged to have skin sensitization and / or pyrogenicity. In addition, when evaluation is performed by setting multiple incubation times in the presence of the test substance, if the amount of IL-6 and / or IL-8 produced in the culture medium of the cells obtained by culturing the cells in the presence of a given concentration of the test substance is increased compared to the negative control during at least one of the multiple incubation times set, the test substance is determined to have skin sensitization and / or pyrogenicity.

[0051] As a negative control, a dissolving solution for dissolving the test substance may be used, or a medium used for culturing immortalized monocytes or dendritic cells may be used.

[0052] <Detection of skin sensitizers and / or pyrogens in samples using immortalized monocytes or dendritic cells> Detection of skin sensitizers and / or pyrogens in a sample using immortalized monocytes or dendritic cells is carried out by measuring the amount of IL-6 and / or IL-8 produced in a culture medium obtained by culturing the immortalized monocytes or dendritic cells in the presence of the sample.

[0053] The method for detecting skin sensitizers and / or pyrogens in a sample is basically the same as that described above in <Evaluation of skin sensitization and / or pyrogenicity of substances using immortalized monocytes and dendritic cells>, so this section will only detail the differences.

[0054] The skin sensitizer and / or pyrogen (e.g., LPS and / or SAC) is dissolved in a dissolving solution and used for the evaluation. Such a dissolving solution may be ethanol, DMSO, water, PBS, etc. Furthermore, the skin sensitizer and / or pyrogen is diluted so as to contact with cells at a predetermined final concentration and used for the evaluation.

[0055] The predetermined final concentration is, for example, between 0.0001 pg / mL and 1,000,000 pg / mL in the case of LPS, and a dilution ratio of 10 in the case of SAC. 1 Times to 10 10 One or more, preferably at least three, concentrations are prepared between 100 and 200 fold. The number of concentrations to be prepared may be three, four, five, or six or more. The skin sensitizer and / or pyrogen may be diluted using the medium described in the section "Evaluation of skin sensitization and / or pyrogenity of a substance using immortalized monocytes and dendritic cells." Such skin sensitizer and / or pyrogen may be used as a positive control in the detection of skin sensitizer and / or pyrogen in a sample.

[0056] The sample used for the evaluation may be diluted at an arbitrary dilution ratio. The arbitrary dilution ratio may be selected from a range of 1.2 to 100. The dilution ratios selected may be two, three, or four or more. The sample may be diluted with water, physiological saline, a medium used for culturing immortalized monocytes or dendritic cells, or the like.

[0057] The detection of a skin sensitizer and / or a pyrogen in a sample is determined based on the measured amount of IL-6 and / or IL-8 produced. Specifically, when the amount of IL-6 and / or IL-8 produced in a culture solution of immortalized monocytes or dendritic cells cultured for an arbitrary time in the presence of a sample is increased compared to a negative control, the sample is determined to contain a skin sensitizer and / or a pyrogen. In addition, when evaluation is performed after multiple culture times in the presence of a sample are set, when the amount of IL-6 and / or IL-8 produced in the culture solution of the cells is increased compared to a negative control for at least one of the multiple culture times set, the sample is determined to contain a skin sensitizer and / or a pyrogen.

[0058] Furthermore, when the sample is diluted at multiple dilution factors and evaluated, if the amount of IL-6 and / or IL-8 produced in the culture medium of the cells obtained by culturing the cells in the presence of at least one of the samples diluted at multiple dilution factors is increased compared to a negative control, the sample is determined to contain a skin sensitizer and / or a pyrogen.

[0059] As a negative control, a dissolving solution for dissolving a skin sensitizer and / or a pyrogen, physiological saline, a medium used for culturing immortalized monocytes or dendritic cells, or the like may be used.

[0060] <Evaluation of the effect of samples using immortalized monocytes or dendritic cells on immune cell function> The function of immune cells can be measured by measuring various cytokines produced by immortalized monocytes or dendritic cells. Thus, the present invention makes it possible to evaluate the effect of a sample on the function of immune cells by measuring various cytokines produced by such immortalized monocytes or dendritic cells. Examples of cytokines produced by immortalized monocytes or dendritic cells include interleukins, chemokines, tumor necrosis factor superfamily, interferons, growth factors, hematopoietic factors, colony-stimulating factors, etc.

[0061] Interleukins include IL-1α, IL-1β, IL-1Ra, IL-2, IL-2Ra, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, I Examples include L-9, IL-10, IL-11, IL-12 (p70), IL-12 (p40), IL-13, IL-15, IL-16, IL-17A, IL-18, and the like.

[0062] Among these interleukins, for example, IL-1α, IL-1β, IL-1Ra, IL-6, IL-8, IL-12, IL-17A, and IL-18 are known as inflammatory cytokines and are involved in skin inflammation and body temperature rise. On the other hand, IL-4 and IL-10 are known as anti-inflammatory cytokines. Thus, in one example, the function of immune cells may be an inflammation-inducing effect such as skin inflammation or body temperature rise, or an inflammation-suppressing effect.

[0063] Interleukins are also known to have various physiological activities related to health. IL-4 and IL-13 are known to induce IgE production, and IL-9 is known to stimulate mast cell proliferation. On the other hand, IL-12 is known to have an effect of suppressing IgE production. Thus, in one example, the function of immune cells may be a mast cell stimulating effect, an effect of enhancing IgE production, or an effect of suppressing IgE production.

[0064] In addition, IL-5 and IL-6 are known to enhance IgA production (Jpn J. Lactic Acid Bact, 2007, 18(2), 54-57). Thus, in one example, the function of the immune cells may be an enhancement of IgA production.

[0065] IL-2 and IL-2Ra are known to have the effects of proliferation and activation of T cells, B cells, and natural killer (NK) cells. IL-7 is known to be involved in the survival, differentiation, etc. of B cells, T cells, and NK cells. IL-15 is known to have the effects of proliferation and activation of T cells and B cells. Thus, in one example, the function of immune cells may be the proliferation and activation of lymphocytes (i.e., T cells, B cells, and NK cells).

[0066] IL-5 is known to have the effect of increasing eosinophil production, and therefore, in one example, the function of the immune cells may be the effect of increasing eosinophil production.

[0067] It is known that IL-17A induces the production of inflammatory cytokines, IL-18 cooperates with IL-2 to induce the production of interferon-γ, and IL-18 alone induces the production of IL-4. Thus, in one example, the function of immune cells may be an action to increase the production of cytokines.

[0068] Chemokines include IL-8, CTACK, Eotaxin, GRO-α, IP-10, MCP-1, MCP-3, MIG, MIP-1α, MIP-1β, RANTES, SDF-1α, etc. These chemokines induce migration of leukocytes from blood vessels into inflamed tissues.

[0069] For example, IL-8 is thought to have a defensive effect against infectious diseases (pathogen elimination effect) through activation of neutrophils, etc. (The Japanese Journal of Antibiotics, 2013, 66-6, 305-310). In addition, CTACK is a skin-specific chemokine and is known to be involved in increased production of eosinophils. Eotaxin is known to have strong eosinophil chemotactic activity. GRO-α is known to have a cell division function and a neutrophil activator. IP-10 is known to induce migration of monocytes, macrophages, etc. MCP-1 (MCAF) and MCP-3 are known to induce migration of monocytes and macrophages. MIG is known to induce migration of T cells and some macrophages to inflammatory sites. MIP-1α is known to induce migration of monocytes and basophils. MIP-1β is known to induce migration of monocytes and eosinophils. RANTES is known to induce the migration of monocytes, eosinophils, and basophils. SDF-1α is a potent chemotactic factor for lymphocytes, and is known to recruit lymphocytes to newly formed blood vessels and to be involved in angiogenesis in vivo.

[0070] Among the above-mentioned interleukins, IL-16 is also known to have the effect of inducing migration of CD4-positive T cells.

[0071] Thus, in one example, the function of an immune cell may be the promotion of migration of white blood cells (i.e., neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages), or the defense against infectious diseases.

[0072] The tumor necrosis factor superfamily includes tumor necrosis factor (TNF)-α, TNF-β, and TNF related apoptosis-inducing ligand (TRAIL). TNF-α and TNF-β are known to be involved in infection defense and antitumor effects by increasing the expression of cell adhesion molecules, inducing apoptosis, and enhancing inflammatory mediators (IL-1, IL-6, prostaglandin E2, etc.). TRAIL is also known to induce apoptosis in cancer cells.

[0073] Thus, in one example, the function of an immune cell may be the induction of apoptosis.

[0074] Interferons include IFN-α (1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, 21), IFN-β, and IFN-γ, etc. Interferons are known to increase the virus resistance of cells by suppressing viral replication.

[0075] Thus, in one example, the function of the immune cells may be the increase in viral resistance.

[0076] Examples of growth factors include basic fibroblast growth factor (FGF basic), hepatocyte growth factor (HGF), nerve growth factor (NGF), platelet derived growth factor (PDGF), hematopoietic stem cell growth factor (SCGF) α and β, vascular endothelial growth factor (VEGF) AE, and epidermal growth factor (EGF).

[0077] FGF basic is known to play an important role in promoting cell division, angiogenesis, and wound healing. HGF is known to play an important role in promoting cell division, angiogenesis, and tissue repair. NGF is known to promote the extension of nerve axons and the synthesis of neurotransmitters, maintain nerve cells, and repair cells when damaged. PDGF is known to be mainly involved in regulating the migration and proliferation of mesenchymal cells, and is also involved in angiogenesis and wound healing. SCGFα and β are known to be mainly involved in the proliferation of hematopoietic cells. VEGF AE is known to be involved in the proliferation of vascular endothelial cells, angiogenesis, and the like. EGF is also known to be involved in the proliferation of epithelial cells and wound healing.

[0078] Thus, in one example, the function of the immune cells may be a cell proliferation promoting effect, an angiogenesis inducing effect, or a wound healing effect.

[0079] Examples of hematopoietic factors include stem cell factor (SCF), etc. SCF is a hematopoietic growth factor that functions through signal transduction via the c-kit receptor, and is known to be an important factor for the survival, proliferation, and differentiation of hematopoietic cells.

[0080] Among the above-mentioned interleukins, IL-3 is also known to act as a hematopoietic factor that supports the proliferation of immature blood cells.

[0081] Thus, in one example, the function of the immune cells may be the promotion of hematopoietic cell proliferation.

[0082] Colony-stimulating factors include granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte colony-stimulating factor (M-CSF). GM-CSF acts on granulocyte / macrophage precursor cells to differentiate into granulocyte precursor cells and macrophage precursor cells, G-CSF acts on granulocyte precursor cells to differentiate into neutrophils, and M-CSF acts on macrophage precursor cells to differentiate into macrophages, and they are known to exhibit various physiological actions (Nihonnai Kai Zasshi 1996, 85, 844-849).

[0083] Thus, in one example, the function of an immune cell may be the differentiation of a progenitor cell.

[0084] Another cytokine is Leukemia Inhibitory Factor (LIF), which is a cytokine of the IL-6 family that inhibits cell differentiation and affects cell growth. Thus, in one example, the function of immune cells may be a cell differentiation inhibitory effect.

[0085] In addition, macrophage migration inhibitory factor (MIF) induces cytokines such as TNF-α and IL-1β to aggravate inflammatory symptoms such as allergies, while it is known to have an effect of controlling the expression induction of the cancer suppressor gene p53 and to suppress apoptosis induced by p53 (Carcinogenesis. 2009, 30, 1597-605). Thus, in one example, the function of immune cells may be an effect of controlling the expression induction of p53 or an effect of suppressing apoptosis.

[0086] The functions of the immune cells are not limited to those described above, and may be, for example, an effect of reducing the amount of cytokine production from immune cells, an effect of inducing immune activation by immune cells, or an effect of inducing immune suppression by immune cells.

[0087] The evaluation method of the present invention can be carried out by measuring the amount of cytokine produced in a culture medium obtained by culturing immortalized monocytes or dendritic cells in the presence of a sample. In this method, the effect on immune cell function can be evaluated using the amount of cytokine produced as an indicator. Specific evaluation methods are described in detail below.

[0088] The effect of the sample on the function of immune cells can be evaluated in accordance with the above-mentioned <Evaluation of skin sensitization and / or pyrogenicity of substances using immortalized monocytes and dendritic cells>.

[0089] In the evaluation method of the present invention, the cytokines whose production amounts are measured are preferably IL-6, IL-8, IL-12 (p40), MIP-1α, MIP-1β, GRO-α, TNF-α, MCP-1 (MCAF), IFN-γ, and G-CSF.

[0090] The sample can be used for evaluation after being dissolved, suspended or diluted with a dissolving solution, suspension or diluting solution, or as it is. Examples of such dissolving solutions include organic solvents (ethanol, DMSO, etc.), water, culture media (such as the culture media described in the section "Evaluation of skin sensitization and / or pyrogenicity of substances using immortalized monocytes and dendritic cells"), and buffer solutions (such as PBS). Commercially available products (such as functional drinks or medicines) may also be used as samples.

[0091] The sample may be dissolved or diluted so as to contact with cells at a predetermined final concentration or dilution ratio, and used for evaluation. One or more types of final concentrations, preferably at least three types, may be prepared between 0% and 50%. The types of final concentrations or dilution ratios may be three, four, five, or six or more.

[0092] The amount of cytokine produced in the culture solution of the immortalized monocytes or dendritic cells after the culture is measured. The culture solution to be measured is generally a culture supernatant, but a cell lysate may be used as necessary. The measurement can be performed using a known immunological measurement method. The immunological measurement method may be, for example, ELISA (Enzyme-Linked Immuno Sorbent Assay), immunochromatography, radioimmunoassay, Western blot, or multiplex assay. Furthermore, these immunological measurements may be performed using a commercially available kit.

[0093] Whether or not a sample has an effect on immune cell function is determined based on the measured amount of cytokine production. Specifically, when the amount of cytokine production in a culture solution of immortalized monocytes or dendritic cells obtained by culturing for an arbitrary time in the presence of a sample is increased compared to a negative control, the sample is determined to have an effect on immune cell function. In this case, for example, the increase may be set to a certain increase rate relative to the negative control as a threshold, and when the amount of production exceeds the increase rate, the sample may be determined to have functionality. Such an increase rate may be 1.2 times or more, specifically, it may be 2 times, 3 times, 5 times, or 10 times or more.

[0094] In addition, when evaluation is performed using samples of multiple concentrations, the sample may be determined to have an effect on immune cell function if the amount of cytokine produced in the culture medium of the cells obtained by culturing in the presence of at least one sample of the multiple concentrations prepared is increased compared to a negative control. In addition, when evaluation is performed after multiple culture times in the presence of a sample are set, the sample may be determined to have an effect on immune cell function if the amount of cytokine produced in the culture medium of the cells obtained by culturing in the presence of a sample of any concentration is increased compared to a negative control for at least one of the multiple culture times set.

[0095] As a negative control, for example, a medium used for culturing immortalized monocytes or dendritic cells may be used.

[0096] <Evaluation kit> The present invention relates to a kit for evaluating the skin sensitization and / or pyrogenicity of a substance, detecting a pyrogen in a sample, and / or evaluating an effect on immune cell function, comprising human immortalized monocytes or dendritic cells prepared from the human immortalized monocytes. The kit may contain, in addition to the above-mentioned monocytes or dendritic cells, a package and an instruction manual, and may also contain a culture medium, culture additives, laboratory equipment, an anti-IL-6 antibody and / or an anti-IL-8 antibody, any anti-cytokine antibody, and any standard substance. As the culture medium, the medium described in the section "Evaluation of the skin sensitization and / or pyrogenicity of a substance using immortalized monocytes or dendritic cells" can be used. EXAMPLES

[0097] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. All documents cited throughout this application are incorporated by reference in their entirety.

[0098] Example 1: Preparation of immortalized monocytes derived from human peripheral blood and preparation of dendritic cells (Creation of immortalized monocytes derived from human peripheral blood) Human peripheral blood-derived immortalized monocytes were produced with reference to previous reports (WO2012 / 043651 and JP2017-131136). In detail, CD14 positive fractions were extracted from human peripheral blood, and genes (c-MYC, BMI1, and BCL-2) reported for the production of human immortalized monocytes were introduced according to previous reports. The immortalized monocyte line was cultured in α-MEM medium containing 20% ​​FBS (Cytiva Inc., Cat #: SH30088.03), 50 ng / mL M-CSF (Peprotech Inc., Cat #: AF-300-25), and 50 ng / mL GM-CSF (Peprotech Inc., Cat #: 300-03). After 3 to 5 weeks of culture, the cells were obtained as proliferative cells and stored under liquid nitrogen using a commercially available cell preservation solution, such as the serum-free cell preservation solution Bambanker (GCLTEC Inc., Cat #: CS-02-001).

[0099] (Verification of prepared cells) The surface markers of the prepared cells were measured, and morphological observations were performed using Giemsa staining and the like. Surface markers were measured using CD11 and CD14 antibodies (both Biolegend) using a Sysmex flow cytometer, CyFlowspace. The expression of CD11 and CD14 was confirmed by the measurement, confirming that the prepared cells expressed monocyte-like markers. For staining, Giemsa staining or May-Giemsa staining was performed based on known staining methods using a commercially available staining solution (Sigma), and measurements were performed using a high-magnification microscope (400 to 1000 times).

[0100] (Preparation of dendritic cells from immortalized monocytes) The stored immortalized monocyte line was maintained and prepared by culturing in α-MEM medium (containing 20% ​​FBS, 50 ng / mL M-CSF, and 50 ng / mL GM-CSF) in a 37°C, 5% CO2 incubator with medium replacement every 3 days. Dendritic cells were prepared by culturing the immortalized monocytes in the presence of 50 ng / mL M-CSF, 50 ng / mL GM-CSF, and 100 ng / mL IL-4 for 3 days to differentiate them into dendritic cells.

[0101] Example 2 (Preparation of immortalized monocytes derived from human induced pluripotent stem cells) Immortalized monocytes derived from human induced pluripotent stem (iPS) cells were prepared with reference to previous reports (WO2012 / 043651, JP 2017-131136 and JP 2018-171005). In detail, undifferentiated iPS cells (provided by the Center for iPS Cell Research and Application, Kyoto University) were seeded on a 10 cm diameter dish (AGC Technoglass Co., Ltd., Cat #: 1012-100) or a 6-well plate (Corning Inc., Cat #: 3516) that had been coated with laminin 511 (iMatrix-511-E8, Nippi Co., Ltd., Cat #: 892-012) as a basement membrane in advance, and differentiation induction culture was started using a culture medium (hereinafter referred to as α-MEM medium (containing 20% ​​FBS)) containing 20% ​​FBS (Cytiva Inc., Cat #: SH30088.03) in α-MEM medium (Fujifilm Wako Pure Chemical Co., Ltd., Cat #: 137-17215). After that, the culture was continued while changing the α-MEM medium (containing 20% ​​FBS) once every 3 days. 14 to 21 days after the start of differentiation induction, TrypLE TMEqual amounts of Select (containing 1 mM ethylenediaminetetraacetic acid (EDTA)) (GIBCO Inc., Cat#: A12859-01) and 0.5 mM EDTA / PBS (Nacalai Tesque, Inc., Cat#: 13567-84) were mixed, and the cells were treated (37°C, 60 minutes) with the prepared solution (final concentration 0.75 mM EDTA) to dissociate and recover, and a cell suspension was prepared by pipetting. Next, the cell suspension collected from one 10 cm diameter dish or one well of a 6-well plate was suspended in 10 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd., Cat#: 044-29765) (containing 10% FBS (Cytiva Inc., Cat#: SH30088.03)), and seeded on one or two 10 cm diameter dishes without feeder cells or laminin coating, and left to stand in a 37°C, 5% CO2 incubator. After about 16 hours, cells that did not adhere to the dish were collected and passed through a 100 micrometer mesh (BD Falcon Cell Strainer) to obtain a cell suspension from which aggregated cell clumps had been removed.

[0102] The cell suspension obtained above, from which the aggregated cell masses had been removed, was seeded in α-MEM medium (containing 20% ​​FBS, 100 ng / mL M-CSF (Peprotech Inc., Cat #: AF-300-25), and 100 ng / mL GM-CSF (Peprotech Inc., Cat #: 300-03)) and cultured in a T25 flask (CellSeed, HydroCell, Cat #: CSF025, or Thermo Fisher Scientific Inc., Cat #: 169900). After 3 to 9 days, floating or weakly adherent cells appeared, and the number of cells gradually increased. The floating cells (iPS-MCs) were collected and the expression of the leukocyte marker CD45 and myeloid cell markers CD11b and CD33 was confirmed using a flow cytometer (not shown).

[0103] Using a third-generation lentiviral vector (SignaGen Inc.) that is a proliferation-deficient strain of the human immunodeficiency virus type 1 (HIV-I) and has an EF1a protein expression promoter, genes reported for the generation of mouse immortalized hematopoietic stem cells (Myc, Myb, Hob8, TLX1, E2A-pbx1, MLL, Ljx2, RARA, Hoxa9, Notch1, v-raf / v-myc, MYST3-NCOA2, Evi1, HOXB6, HOXB4, β-catenin, Id1) or genes reported for the generation of human immortalized monocytes (c-MYC and at least one of BMI1, EZH2, MDM2, MDM4, and HIFIA) were introduced into the suspension cells (iPS-MCs) obtained above, and immortalized monocytes derived from human induced pluripotent stem cells were generated. This immortalized monocyte line was cultured in α-MEM medium containing 20% ​​FBS, 50ng or 50-100ng / mL M-CSF, and 50ng or 50-100ng / mL GM-CSF in a 24-well plate, and obtained as proliferative cells 3-5 weeks after the start of culture. Human induced pluripotent stem cell-derived immortalized monocyte cells were stored under liquid nitrogen using a commercially available cell preservation solution, such as the serum-free cell preservation solution Bambanker (GCLTEC Inc., Cat♯:CS-02-001).

[0104] Example 3: Preparation of immortalized monocytes derived from human induced pluripotent stem cells expressing M-CSF and GM-CSF Human induced pluripotent stem cell-derived immortalized monocytes expressing M-CSF and GM-CSF were prepared with reference to a previous report (JP Patent Publication 2018-171005). In detail, expression vectors for human M-CSF gene and human GM-CSF gene were simultaneously introduced into the human induced pluripotent stem cell-derived immortalized monocytes obtained in Example 2 using a third-generation lentivirus vector (SignaGen Inc.) that is a Human Immunodeficiency Virus Type 1 (HIV-I) proliferation-deficient strain and has an EF1a protein expression promoter. From 3 days after gene introduction, the cells were cultured in α-MEM (containing 20% ​​FBS) medium that does not contain either human M-CSF or GM-CSF, and 3 to 5 weeks after the start of culture, the cells were obtained as proliferative cells and stored under liquid nitrogen using a commercially available cell preservation solution. Hereinafter, the human immortalized monocytes prepared in Example 3 will be referred to as iPS-ML2.

[0105] Example 4: Evaluation test of skin sensitization and / or pyrogenicity of substances using immortalized monocyte line The skin sensitization and / or pyrogenicity evaluation test was performed according to the following procedure. iPS-ML2 was used in the test. iPS-ML2 cells that had been frozen were thawed at room temperature, washed, and then seeded in a T-25 (Thermo Fisher Scientific Inc.) flask containing 10 mL of medium (α-MEM (GIBCO) containing 10% FCS), and then passaged three or more times before being used in the test. 1. The iPS-ML2 floating in the T-25 flask was transferred to a 15 mL tube, which was then centrifuged at room temperature and 300×G for 5 minutes, and the supernatant was discarded. 2. 10 mL of PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the iPS-ML2 pellet, and the mixture was centrifuged at room temperature and 300×G for 5 minutes, and the supernatant was discarded. This procedure was repeated once more. 3. After washing, the iPS-ML2 was suspended in culture medium (α-MEM (GIBCO) containing 10% FCS). 4. The number of cells was counted using trypan blue. 5. 1×10 per well in a 96-well flat plate (Coring) 4iPS-ML2 cells were seeded at a cell density of 10 cells / 100 μL. 6. As test substances, the compounds shown in Table 1 below were dissolved in a dissolving solution, and this solution was adjusted to a concentration of 400 μg / mL in medium (α-MEM (GIBCO) containing 10% FCS). The test substance at 7.400 μg / mL was diluted two-fold with medium (α-MEM (GIBCO) containing 10% FCS) to prepare a total of 11 concentrations. 8. 100 μL of each concentration of the test substance was added to each well of the plate prepared in item 5. This resulted in a 2-fold dilution of the test substance, with the highest final concentration of the substance being 200 μg / mL. 9. Six hours after the start of iPS-ML2 culture, the culture supernatant was collected from each well. The collected supernatant was stored at -80°C until use in ELISA. 10. The amount of each cytokine in the collected culture supernatant was measured using the IL-6 ELISA KIT (BIOLEGEND, ELISA MAX TM DELUXE SETHUMAN IL-6) and IL-8 ELISA KIT(BIOLEGEND, ELISA MAX TM Measurements were performed using a plate reader (TECAN, Infinite 200PRO M nano) according to the protocol of the DELUXE SET HUMAN IL-8.

[0106] [Table 1] (result) As a result of the test, for N7, no production of IL-6 or IL-8 was observed 6 hours after addition to iPS-ML2.

[0107] On the other hand, P1, P3, and P4 produced 1.5 times or more IL-6 and IL-8 6 hours after addition to iPS-ML2 compared to the negative control of medium without the test substance. Also, P2, P5, and P6 produced 1.5 times or more IL-8 6 hours after addition to iPS-ML2 compared to the negative control of medium without the test substance. In Figure 1, cells with a production level of 1.5 times or more compared to the negative control of medium without the test substance are indicated as having skin sensitization and / or pyrogenicity (P), and cells with a production level of less than 1.5 times are indicated as not having skin sensitization and / or pyrogenicity (N).

[0108] The results of this evaluation were compared with those of each test substance in conventional evaluation methods (LLNA, h-CLAT) (see Figure 1). The LLNA evaluation results (LLNA potency) were cited from ATLA 38, 275-284, 2010. If there is no skin sensitization, it is described as Non-sensitiser, if there is weak skin sensitization, it is described as weak, and as the strength of skin sensitization increases, it is described as moderate, strong, and extreme. The results of the h-CLAT (h-CLAT CD86 and CD54) were cited from ATLA 38, 275-284, 2010, and a positive (P) was determined when the expression increased by 150% or more (CD86) or 200% or more (CD54) compared to the negative control, and a negative (N) was determined when it decreased below that level. In this case, if either CD86 or CD54 was positive, it was determined to have skin sensitization.

[0109] P1 to P6 have been confirmed to have skin sensitization in the LLNA, and in the h-CLAT, an increase in the expression levels of CD86 and / or CD54 was observed in cells to which P1 to P6 had been added. Therefore, the results of this evaluation correlated with those of the h-CLAT.

[0110] Therefore, it is believed that by adding each test substance to iPS-ML2 prepared by the method described in this Example and culturing for 6 hours, and measuring the amount of IL-6 and / or IL-8 produced in the culture supernatant obtained, the skin sensitization and / or pyrogenicity of the test substance can be evaluated.

[0111] In addition, when similar evaluations of skin sensitization and / or pyrogenicity were performed on different cell lots, no differences in the results of skin sensitization and / or pyrogenicity were observed between the different lots, confirming that this evaluation method has high reproducibility.

[0112] In addition, since this evaluation can be performed by ELISA, it can be performed by a simpler method than conventional methods (e.g., h-CLAT). Therefore, this evaluation can be used, for example, as a screening test before performing a test by a conventional method.

[0113] (Example 5) Test for detection of skin sensitizers and / or pyrogens in samples using immortalized human monocytes The test to detect skin sensitizers and / or pyrogens in the samples was performed according to the following procedure. iPS-ML2 was used for the test. iPS-ML2 cells were frozen and thawed at room temperature, washed, and then seeded in a T-25 flask (Thermo Fisher Scientific Inc.) containing 10 mL of medium (α-MEM (GIBCO) containing 10% FCS), and then passaged three or more times before being used for the test. 1. The iPS-ML2 floating in the T-25 flask was transferred to a 15 mL tube, which was then centrifuged at room temperature and 300×G for 5 minutes, and the supernatant was discarded. 2. 10 mL of PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the iPS-ML2 pellet, and the mixture was centrifuged at room temperature and 300×G for 5 minutes, and the supernatant was discarded. This procedure was repeated once more. 3. After washing, the iPS-ML2 was suspended in culture medium (α-MEM (GIBCO) containing 10% FCS). 4. The number of cells was counted using trypan blue. 5. 1×10 per well in a 96-well flat plate (Coring) 4 iPS-ML2 cells were seeded at a cell density of 10 cells / 100 μL. 6. As samples containing skin sensitizers and / or pyrogens, LPS and SAC were dissolved and suspended in a dissolving solution. In the case of LPS, this dissolving solution was prepared in medium (α-MEM (GIBCO) containing 10% FCS) so that the concentration was 2,000,000 pg / mL. In the case of SAC, it was diluted 5-fold with medium (α-MEM (GIBCO) containing 10% FCS). 7. 2,000,000 pg / mL LPS and 5-fold diluted SAC were diluted 10-fold with medium (α-MEM (GIBCO) containing 10% FCS) to prepare a total of 10 concentrations. 8. 100 μL of samples containing skin sensitizers and / or pyrogens of various concentrations were added to each well of the plate prepared in item 5. As a result, the concentrations of skin sensitizers and / or pyrogens were diluted 2-fold, the maximum final concentration of LPS was 1,000,000 pg / mL, and the minimum dilution ratio of SAC was 10-fold. 9. 6 hours and 24 hours after the start of iPS-ML2 culture, the culture supernatant was collected from each well. The collected supernatant was stored at -80°C until use in ELISA. 10. The amount of each cytokine in the collected culture supernatant was measured using the IL-6 ELISA KIT (BIOLEGEND, ELISA MAX TM DELUXE SETHUMAN IL-6) and IL-8 ELISA KIT(BIOLEGEND, ELISA MAX TM Measurements were performed using a plate reader (TECAN, Infinite 200PRO M nano) according to the protocol of the DELUXE SET HUMAN IL-8.

[0114] (result) LPS or SAC was added to iPS-ML2 at various concentrations, and the amounts of IL-6 and IL-8 produced in the culture supernatant of iPS-ML2 after culturing for 6 and 24 hours are shown in FIG.

[0115] For LPS, the production of IL-6 and IL-8 in the culture supernatant increased in a concentration-dependent manner compared to the negative control of medium alone both after 6 and 24 hours of culture (see Figures 2A and 2B). Note that the negative controls are shown on the vertical axis of the graphs in Figures 2A and 2B as circle points (negative control after 6 hours of culture) and square points (negative control after 24 hours of culture).

[0116] For SAC, the lower the dilution ratio, i.e., the higher the concentration, the greater the amount of IL-6 and IL-8 produced compared to the negative control of medium alone, both after 6 and 24 hours of culture (see Figures 2C and 2D). As in the case of LPS described above, the negative controls are shown on the right side of the graphs in Figures 2C and 2D as circle points (negative control after 6 hours of culture) and square points (negative control after 24 hours of culture).

[0117] Therefore, it is believed that skin sensitizers and / or pyrogens can be detected by adding a sample containing a skin sensitizer and / or pyrogen to iPS-ML2 prepared by the method described in this Example, culturing for 6 hours and / or 24 hours, and measuring the amount of IL-6 and / or IL-8 produced in the culture supernatant obtained.

[0118] The detection of skin sensitizers and / or pyrogens by this detection method was confirmed to have the same or better detection performance as the detection limit of conventional tests (e.g., PBMC tests). Furthermore, when similar detection tests were performed on different cell lots, no difference was observed in the results between the lots. In other words, this detection method has been shown to have high reproducibility.

[0119] (Example 6) Evaluation test of cytokine production by functional beverage using immortalized monocyte line The effect of the functional beverage on cytokine production from the immortalized monocyte line was evaluated. For the test, iPS-ML2 prepared in Example 3 was used. The following functional beverages were used as test samples.

[0120] [Table 2] The specific test was carried out according to the following procedure. 1. Frozen iPS-ML2 was thawed at room temperature, 10 mL of PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the pellet, and the mixture was centrifuged at room temperature at 300×G for 5 minutes, and the supernatant was discarded. This operation was repeated once more. 2. After washing, the iPS-ML2 were suspended in 10 mL of culture medium containing 10% FBS (Cytiva Inc., Cat#: SH30088.03) or α-MEM (Gibco BRL) supplemented with 2 or 5% HPL (Human platelet lysate, human platelet-derived supplement (AdvantaCell, Cat#: HPCHXCGL50)). 3. The number of cells was counted using trypan blue. 4. 1×10 per well in a 96-well flat plate (Corning) 4 iPS-ML2 cells were seeded at a cell density of 10 cells / 100 μL. 5. As samples, various commercially available functional beverages shown in Table 2 were diluted two-fold with culture medium (α-MEM (GIBCO) containing 10% FCS) to prepare a total of 11 concentrations ranging from the original solution (100%) to 0.05%. 6. 100 μL of each concentration of functional beverage was added to each well of the plate prepared in item 5. This resulted in a two-fold dilution of the functional beverage, with the maximum final concentration being 50% and the minimum final concentration being 0.025%. 7. iPS-ML2 was cultured in a 37°C, 5% CO2 incubator, and the culture supernatant was collected from each well 24 hours after the start of culture. The collected supernatant was stored at -80°C until use in ELISA. 8. The amount of each cytokine in the collected culture supernatant was measured using the IL-6 ELISA KIT (BIOLEGEND, ELISA MAX TM Measurements were performed using a plate reader (TECAN, Infinite 200PRO M nano) according to the protocol for IL-6 (DELUXE SETHUMAN IL-6, Cat♯:430504). 9. The amount of IL-6 produced was expressed as the stimulation index, which was calculated using the following formula. Note that the medium was used as a negative control.

[0121] Stimulation Index = (measured amount of IL-6 produced by cell supernatants to which various functional beverages were added) / (measured amount of IL-6 produced by negative control) (result) Figure 3 shows the ratio of IL-6 production in the culture supernatant of iPS-ML2 after adding various concentrations of various functional beverages to iPS-ML2 and culturing for 24 hours. The horizontal axis of the graph shows the concentration of the sample (%), and the vertical axis shows the ratio of production to the production amount of the negative control (=1). Note that the negative control is shown as a square dot on the vertical axis of the graph in Figure 3.

[0122] For example, when iMUSE (registered trademark) was used as a sample, the IL-6 production ratio was about 17 times when added at a concentration of 6.25%. From these results, it was found that there was a difference in the maximum IL-6 production amount between various samples, and that the IL-6 production amount changed depending on the dilution concentration of the sample. It was also found that under conditions of high sample concentration, for example when the final concentration of the sample was around 50%, sufficient evaluation results could not be observed in many groups due to changes in osmotic pressure.

[0123] Therefore, this example demonstrated that the produced iPS-ML2 cells can be used to measure the amount of cytokines such as IL-6 produced by immune cells and to use this as an indicator for the degree of inflammation induction and for screening evaluation.

[0124] (Example 7) Comprehensive evaluation test of cytokines produced by functional beverages using immortalized monocyte lines A comprehensive evaluation test of the cytokines produced by the samples was performed according to the following procedure. iPS-ML2 was used for the test. iPS-ML2 cells were frozen and thawed at room temperature, washed, and then seeded in a T-25 flask (Thermo Fisher Scientific Inc.) containing 10 mL of medium (α-MEM (GIBCO) containing 10% FCS). The cells were then passaged three or more times and used for the test. 1. The iPS-ML2 floating in the T-25 flask was transferred to a 15 mL tube, which was then centrifuged at room temperature at 300×G for 5 minutes, and the supernatant was discarded. 2. 10 mL of PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the iPS-ML2 pellet, and the mixture was centrifuged at room temperature and 300×G for 5 minutes, after which the supernatant was discarded. This procedure was repeated once more. 3. After washing, the iPS-ML2 were suspended in 10 mL of medium (α-MEM (GIBCO) containing 10% FCS) or in culture medium prepared by adding 2 or 5% HPL (Human platelet lysate, human platelet-derived supplement (AdvantaCell, Cat♯:HPCHXCGL50)) to α-MEM (Gibco BRL). 4. The number of cells was counted using trypan blue. 5. 1×10 per well in a 96-well flat plate (Corning) 4 iPS-ML2 cells were seeded at a cell density of 10 cells / 100 μL. 6. The functional drink (iMUSE (registered trademark)) was diluted with culture medium (α-MEM (GIBCO) containing 10% FCS) to a final concentration of 10%, and 100 μL was added per well of the plate prepared in step 5. 7. iPS-ML2 was cultured at 37°C in a 5% CO2 incubator, and the culture supernatant was collected from each well 6 hours and 24 hours after the start of culture. The collected supernatant was stored at -80°C until use in ELISA. 8. The amount of each cytokine in the collected culture supernatant was analyzed using Bio-Plex Pro TMMeasurements were performed using a measuring device (product name: Bio-Plex 200, Bio-RAD) according to the protocol for the Human Cytokine Screening 48-Plex Panel (Bio-RAD, Cat#: 12007283). 9. The production amount of each cytokine is expressed as the production ratio (stimulation index), and the production ratio is calculated using the following formula. As a negative control, the medium was used.

[0125] Stimulation Index = (measured cytokine production in the supernatant of cells with iMUSE® added) / (measured cytokine production in the negative control) (result) A comparison of cytokine production amounts by addition of iMUSE (registered trademark) is shown in Table 3. In Table 3, + indicates that the production amount ratio is 2.5 to 5 times, ++ indicates that the production amount ratio is 5 to 10 times, and +++ indicates that the production amount ratio is 10 times or more.

[0126] [Table 3] For example, in the case of IL-8, the amount produced after 24 hours was 10 times higher (+++). As a result, it was revealed that cytokines such as IL-8 were also produced in addition to IL-6 shown in Example 6.

[0127] Therefore, it was demonstrated that the iPS-ML2 cells produced can be used to evaluate various cytokine production by immune cells, and that this cytokine evaluation can be used to evaluate the cytokine production performance of various substances.

Claims

1. A method for producing a human myeloid cell derived from peripheral blood or pluripotent stem cells, comprising: (A) the cMYC gene, and (B) at least one gene selected from the group consisting of the BMI1 gene, the EZH2 gene, the MDM2 gene, the MDM4 gene, and the HIF1A gene. and measuring the amount of IL-6 and / or IL-8 produced in a culture medium obtained by culturing human immortalized myeloid cells into which the above-mentioned compound has been introduced in the presence of the test substance.

2. The method according to claim 1, wherein the human immortalized myeloid cells are human immortalized monocytes or dendritic cells prepared from the human immortalized monocytes.

3. The method according to claim 1 or 2, wherein the concentration of the test substance during the culture of the human immortalized myeloid cells includes at least three different concentrations between greater than 0 μg / mL and less than or equal to 1000 μg / mL.

4. The method according to any one of claims 1 to 3, wherein the culture time is selected from at least one period between 3 hours and 48 hours.

5. The method according to any one of claims 1 to 4, wherein the amount of production is measured by an immunological assay.

6. The method of claim 5 , wherein the immunoassay is an ELISA.

7. The method according to any one of claims 1 to 6, further comprising determining that the test substance has skin sensitization and / or pyrogenicity when the production amount is 1.5 times or more relative to the production amount of a negative control.

8. A method for the treatment of peripheral blood or pluripotent stem cell-derived human myeloid cells, comprising: (A) the cMYC gene, and (B) at least one gene selected from the group consisting of the BMI1 gene, the EZH2 gene, the MDM2 gene, the MDM4 gene, and the HIF1A gene. A method for detecting a skin sensitizer and / or a pyrogen in a sample, comprising culturing a human immortalized myeloid cell into which a compound has been introduced in the presence of the sample, and measuring the amount of IL-6 and / or IL-8 produced in a culture medium obtained by the culture in the presence of the sample.

9. The method according to claim 8, wherein the human immortalized myeloid cells are human immortalized monocytes or dendritic cells prepared from the human immortalized monocytes.

10. The method according to claim 8 or 9, wherein the concentration of the sample during the culture of the human immortalized myeloid cells includes at least three different concentrations.

11. The method according to any one of claims 8 to 10, wherein the culture time is selected from at least one period between 3 hours and 48 hours.

12. The method according to any one of claims 8 to 11, wherein the production amount is measured by an immunological assay.

13. The method of claim 12, wherein the immunoassay is an ELISA.

14. The method according to any one of claims 8 to 13, further comprising determining that the sample contains the skin sensitizer and / or pyrogen if the amount of production is increased relative to the amount of production of a negative control.

15. 15. The method of any one of claims 8 to 14, wherein the skin sensitizer and / or pyrogen is lipopolysaccharide (LPS) and / or Staphylococcus aureus Cowan 1 (SAC).

16. A method for producing a human myeloid cell derived from peripheral blood or a pluripotent stem cell, comprising: (A) the cMYC gene, and (B) at least one gene selected from the group consisting of the BMI1 gene, the EZH2 gene, the MDM2 gene, the MDM4 gene, and the HIF1A gene. A method for evaluating the effect of a sample on the function of immune cells, comprising culturing human immortalized myeloid cells into which the above-mentioned has been introduced in the presence of the sample, and measuring the amount of cytokines produced in a culture medium obtained by the above-mentioned method.

17. The method according to claim 16, wherein the human immortalized myeloid cells are human immortalized monocytes or dendritic cells prepared from the human immortalized monocytes.

18. The cytokine is IL-1a, IL-1b, IL-1ra, IL-2, IL-2Ra, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-12 (p40), IL-13, IL-15, IL-16, IL-17A, IL-18, CTACK, Eotaxin, FGF The method of claim 16 or 17, wherein the inhibitor is selected from the group consisting of basic, G-CSF, GM-CSF, GRO-α, HGF, IFN-α2, IFN-γ, IP-10, LIF, MCP-1 (MCAF), MCP-3, M-CSF, MIF, MIG, MIP-1α, MIP-1β, β-NGF, PDGF-BB, RANTES, SCF, SCGF-β, SDF-1a, TNF-α, TNF-β, TRAIL, and VEGF-A.

19. 19. The method according to any one of claims 16 to 18, wherein the effect on the function of the immune cells is a function selected from the group consisting of an effect of increasing an amount of cytokine production from immune cells, an effect of decreasing an amount of cytokine production from immune cells, an inflammation-inducing effect by immune cells, an inflammation suppression-inducing effect by immune cells, an immune activation-inducing effect by immune cells, an immune suppression-inducing effect by immune cells, an effect of stimulating mast cells by immune cells, an effect of enhancing IgE production by immune cells, an effect of suppressing IgE production by immune cells, an effect of enhancing IgA production by immune cells, an effect of proliferating and activating lymphocytes by immune cells, an effect of increasing eosinophil production by immune cells, an effect of promoting leukocyte migration by immune cells, an effect of inducing apoptosis by immune cells, an effect of suppressing apoptosis by immune cells, an effect of increasing virus resistance by immune cells, an effect of promoting cell proliferation by immune cells, an effect of inducing angiogenesis by immune cells, a wound healing effect by immune cells, an effect of promoting hematopoietic cell proliferation by immune cells, an effect of differentiating precursor cells by immune cells, and an effect of inhibiting cell differentiation by immune cells.

20. The method according to any one of claims 16 to 19, wherein the culture time is from 3 to 48 hours.

21. 21. The method of any one of claims 16 to 20, further comprising determining that the sample is functional if the amount of cytokine produced in the culture medium obtained by culturing the human immortalized myeloid-lineage cells in the presence of the sample is at least twice as much as the amount of cytokine produced in the culture medium obtained by culturing the human immortalized myeloid-lineage cells in the presence of a negative control.

22. 22. The method of any one of claims 16 to 21, wherein the sample is a functional beverage.

23. A method for treating a human monocyte derived from peripheral blood or pluripotent stem cells, comprising: (A) the cMYC gene, and (B) at least one gene selected from the group consisting of the BMI1 gene, the EZH2 gene, the MDM2 gene, the MDM4 gene, and the HIF1A gene.

23. A kit for use in the method according to any one of claims 1 to 22, comprising human immortalized monocytes transduced with or dendritic cells prepared from said human immortalized monocytes.

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