Novel human immortalized myeloid cells

Abstract

The purpose of the present invention is to provide human immortalized myeloid cells (for example, human immortalized monocytes) that can be used in a method for quickly testing a pyrogen remaining in pharmaceutical products, vaccines, medical devices, etc. and has a cytokine (for example, TNF-α)-producing ability without the need for pretreatment. The problem is solved by providing a method for producing human immortalized myeloid cells, the method comprising: (A) a step for forcibly expressing (i) a cMYC gene and (ii) at least one gene selected from the group consisting of a BMI1 gene, an EZH2 gene, an MDM2 gene, an MDM4 gene, and an HIF1A gene in human myeloid blood cells; and (B) a step for freezing, in the presence of a magnetic field, the human immortalized myeloid cells obtained in the step (A).

Description

Novel immortalized human myeloid cells

[0001] The present invention relates to a method for producing immortalized human myeloid cells capable of producing cytokines without requiring pretreatment.

[0002] Currently, various blood cell lines established and developed for research purposes are used in disease analysis and drug development in the fields of immunology and infectious diseases. Human-derived white blood cells are often used, particularly in drug development for immune and infectious diseases in humans. For example, test substances are added to blood cells, and the cytokines and chemokines produced by the cells, as well as changes in cell surface proteins, are measured and evaluated. Among these, cytokine production, one of the roles of immune cells, has received considerable research attention. Numerous research reports, from basic to clinical studies, have examined the production of important inflammatory cytokines such as IL-1β, IL-6, and TNF-α, as well as the relationship between the production of anti-inflammatory cytokines and disease. Therefore, rapid assessment of cytokine production in response to stimulation by test substances is often required. However, prior preparation is generally required, such as culturing cells in a stable state and pre-treating them with stimulants to enhance cytokine production. For example, when evaluating the production of TNF-α, an important inflammatory cytokine, in THP-1 cells, which are used in research as a monocytic cell line, pretreatment with PMA or other methods is required (Non-Patent Document 1).

[0003] Pyrogen testing is currently used as one of the methods for testing for microbial contamination in pharmaceuticals, vaccines, and medical devices. This test is specified in the Japanese Pharmacopoeia and includes rabbit pyrogen testing and endotoxin testing using horseshoe crab blood. In recent years, with the development of pharmaceuticals with various modalities, production methods using cells, such as gene therapy, have become more common, moving away from traditional industrial production methods for small molecule drugs produced in chemical plants. As a result, small batch production and manual labor have become more common, increasing and diversifying the risk of microbial contamination compared to traditional standardized industrial chemical manufacturing. In particular, many viruses infect humans only and are species-specific, making it impossible to evaluate them using non-human species such as rabbits and horseshoe crabs. In addition, there is a global movement to reduce animal testing from an ESG perspective. Against this backdrop, there is a desire to replace traditional animal-based pyrogen testing methods with alternative testing methods using human cells. In Europe, the European Pharmacopoeia has been including a pyrogen testing method using human blood and human monocyte cells in its 2.6.30 format since 2010 (Non-Patent Document 2). India has included this method since 2018, and there are also moves toward including it in its pharmacopoeias in Japan and the United States.

[0004] The Monocyte Activation Test (MAT) is a pyrogen test that uses human blood and human monocyte cells. Monocytes, which are white blood cells, have various receptors that recognize microorganisms. When contaminated with microorganisms, chemokines and cytokines are produced via these receptors. This immune response is called innate immunity or primary immunity. The representative receptors that recognize these microorganisms are called Toll-like receptors (TLRs), and 10 types of TLRs are currently known in humans. These TLRs specifically recognize various pathogenic microorganisms and their parts, producing cytokines and other substances as part of the immune response.

[0005] For example, let us explain the innate immunity when a virus enters the body using the West Nile virus, a (+)-strand RNA virus. When the West Nile virus infects a cell, viral replication occurs within the cell. During this process, a large amount of double-stranded RNA (dsRNA) is produced as a viral genome replication intermediate. When the infected cell undergoes apoptosis or other such events, the dsRNA within the cell is released. When this released dsRNA is taken up into the cell by phagocytes such as macrophages or dendritic cells, it is recognized (bound) and activated by TLR3, a Toll-like receptor (TLR) found in the endoplasmic reticulum and endosomes. The activated receptor promotes the production of inflammatory cytokines and other proteins via intracellular signaling pathways. TLRs respond to a variety of microorganisms. For example, RNA viruses are recognized by TLR3, TLR7, and TLR8, DNA viruses by TLR9, not only viruses but also Gram-positive bacteria are recognized by TLR2, and Gram-negative bacteria by TLR4. Binding activates the cell's defense function. Not only bacteria and viruses, but also mold (fungi) and yeast are recognized by TLRs. As a result, chemokines and cytokines are produced (Non-Patent Document 3).

[0006] The monocyte activation test (MAT) utilizes this phenomenon to evaluate the presence of microbial contamination that could cause adverse events such as fever when drugs or medical devices are introduced into the human body before shipping. Specifically, the test substance, such as a drug, is added to human cells before shipping, and the production of cytokines and other substances resulting from the immune response is measured and evaluated. This test is listed in the European Pharmacopoeia (10th edition, 2.6.30), and reagent companies have offered two main types of MAT products to date. One type uses peripheral blood mononuclear cells (PBMCs), which are fractionated blood or the white blood cell component of blood, as the cell source. The other type uses human monocyte-type cells such as Mono-Mac-6 and THP-1 cells as the cell source. Cytokines produced by the cells, such as IL-6 and IL-1β, are also measured and evaluated.

[0007] The actual MAT testing method is described below. The test substance, such as a pharmaceutical product, is added to cells and cultured. When the test substance is contaminated with microorganisms, the cells are activated via Toll-like receptors and produce inflammatory cytokines. The inflammatory cytokines produced by the cells are quantitatively evaluated after approximately 20 hours of culture. In most test methods, the culture supernatant is removed and the amount of cytokines in the supernatant is measured using ELISA or other methods to measure the inflammatory cytokines produced by the cells. Therefore, performing a series of MAT tests requires "culture of cells and test substance" (hereinafter referred to as culture time) and "cytokine measurement using ELISA or other methods" (hereinafter referred to as measurement time). For example, on the first day, the cells and test substance are mixed and cultured until the next day (culture time: approximately 20 hours), and the culture supernatant is collected on the next day and the amount of cytokines is measured by ELISA (measurement time: approximately 2-5 hours). This requires two days from the start of culture to obtaining the measurement results (hereinafter referred to as total test time).

[0008] Conventional MAT requires a total test time of about two days from the start of incubation to the acquisition of measurement results, and requires a lot of manual work during the incubation process and ELISA measurement. As a result, there are issues with throughput when used for pre-shipment testing of pharmaceuticals, etc., and no fully satisfactory MAT products have been provided.

[0009] On the other hand, a simple endotoxin test (LAL test) is available as a pyrogen test. This endotoxin test (LAL test) uses the mechanism by which horseshoe crab blood components coagulate when they come into contact with lipopolysaccharides (endotoxins), which are the cell walls of gram-negative bacteria. The evaluation time is simple, about a few hours. Although the LAL test only evaluates gram-negative bacteria and has issues such as a narrow range of microorganisms that can be evaluated, it is used as a standard pre-shipment test. Although MAT has advantages such as the ability to comprehensively evaluate many microorganisms, its replacement with the LAL test has not progressed due to the difference in work time and simplicity compared to the LAL test.

[0010] M. E. Lund et. al. , J. Immunol. Methods., 430 (2016), p64-70. Ph. Eur. 11-5 Edition, 2.6.30. M. Carty et. al. , Clin Exp Immunol. , 2010 Sep, 161(3), p397-406.

[0011] The present invention has been made in view of the above-mentioned problems of the prior art, and aims to provide immortalized human myeloid cells (e.g., immortalized human monocytes) capable of producing cytokines (e.g., TNF-α) without requiring pretreatment, which can be used in methods for rapidly testing for pyrogens remaining in pharmaceuticals, vaccines, medical devices, etc.

[0012] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have succeeded in producing immortalized human peripheral blood-derived monocytic cells (hereinafter also referred to as "aMylc-Z") capable of producing TNF-α without the need for pretreatment by cryopreserving immortalized human peripheral blood-derived monocytic cells (produced by introducing an immortalizing gene into monocytic cells) in the presence of a magnetic field. Furthermore, it was confirmed that when cultured with LPS (0.1 EU / mL), these cells produce 150 pg / mL or more of TNF-α within 3.5 hours and more than 1 hour after the start of culture. Furthermore, aMylc-Z was shown to have superior cell numbers and cell viability upon thawing, as well as to have higher responsiveness to pyrogens (e.g., LPS), compared to conventional human peripheral blood-derived immortalized monocytic cells (aMylc) frozen by conventional freezing methods. In addition, the present inventors discovered for the first time that by using these cells in combination with a quantitative measurement method using the inflammatory cytokine TNF-α as an indicator, it is possible to evaluate the immune response of minute amounts of microorganisms and components in an extremely short period of time when the microorganisms are sensitized, thereby completing the present invention.

[0013] That is, the present invention relates to the following: [1] A method for producing immortalized human myeloid cells, comprising: (A) forcibly expressing in human myeloid blood cells: (i) the cMYC gene, and (ii) 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 (B) freezing the immortalized human myeloid cells obtained in step (A) in the presence of a magnetic field. [2] The method according to [1], wherein the gene is forcibly expressed in the human myeloid blood cells by introducing the gene into the human myeloid blood cells. [3] The method according to [1] or [2], wherein the human myeloid blood cells are human peripheral blood-derived monocytes. [4] The method according to [1] or [2], wherein the human myeloid blood cells are derived from pluripotent stem cells. [5] The method of any of [1] to [4], wherein M-CSF and / or GM-CSF are further forcibly expressed in the human myeloid blood cells of step (A) or the human immortalized myeloid cells obtained in step (A). [6] The method of any of [1] to [5], wherein the human immortalized myeloid cells obtained in step (B) are cells that produce 150 pg / mL or more of TNF-α within more than 1 hour but 3.5 hours from the start of culture when cultured with the addition of LPS (0.1 EU / mL). [7] Human immortalized myeloid cells obtained by the method of any of [1] to [6]. [8] Human immortalized myeloid cells that produce 150 pg / mL or more of TNF-α within more than 1 hour but 3.5 hours from the start of culture when cultured with the addition of LPS (0.1 EU / mL).

[0014] The human immortalized myeloid cells (e.g., aMylc-Z) of the present invention do not require pretreatment, allowing for convenient testing for pyrogens. Furthermore, the human immortalized myeloid cells (e.g., aMylc-Z) of the present invention are excellent in cell number and cell viability upon thawing, and furthermore, are highly responsive to pyrogens (e.g., LPS), making them suitable for use in pyrogen testing and other applications. Furthermore, the rapid pyrogen testing method of the present invention using human immortalized myeloid cells (e.g., aMylc-Z) shortens the pyrogen testing time, which conventional methods require approximately two days, to within a few hours (e.g., the total test time from the start of culture to the completion of measurement is within three hours). Additionally, the human immortalized myeloid cells (e.g., aMylc-Z) of the present invention can also be used for screening antiviral drugs and drugs targeting immune diseases.

[0015] 1 is a diagram showing the flow of a conventional MAT test method. 2 is a diagram showing the results of a comparison of MAT tests between aMylc-Z and conventional aMylc. 3 is a diagram showing the results of a rapid MAT test (sample transfer method) for aMylc-Z. 4 is a diagram showing the results of a rapid MAT test (direct method) for aMylc-Z. 5 is a diagram showing the results of a MAT test using TNF-α and IL-6 as indicators when non-endotoxin is added to aMylc-Z, and the results of a conventional MAT test. 6 is a diagram showing the test results of a MAT (continuous evaluation method) for aMylc-Z. 7 is a diagram showing the results of a comparison of TNF-α production activity between aMylc-Z and conventional aMylc.

[0016] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited to this.

[0017] (Conventional Monocyte Activation Test (MAT)) The Monocyte Activation Test (MAT) is listed in the European Pharmacopoeia in sections 2.6.30 and 2.6.40. Therefore, reagent companies and others provide products for evaluating the MAT (e.g., PyroMAT: Merck, CTLMAT: MAT Biotech) as evaluation kits. The MAT evaluation kit consists of cells (supplied as frozen vials) and a cytokine detection evaluation kit, which can also be purchased separately. The cells (PBMCs or monocyte-type cells) are thawed from frozen vials, mixed with the test substance according to the protocol, and then cultured. These products require an incubation time of approximately 18 to 22 hours after mixing to measure cytokines such as IL-6 or IL-1β produced by the cells in response to stimulation with the test substance. After incubation, the amount of cytokine produced is evaluated using a detection kit. Detection kits typically use an enzyme reaction and a cytokine measurement method (ELISA) using an absorbance meter. A typical detection and evaluation kit requires approximately 3 to 5 hours for measurement after culture, while a simple detection and evaluation kit incorporating a sandwich antibody requires approximately 2 hours. Therefore, the process involves mixing the cells and the test substance on the first day to start culture, and then recovering the culture supernatant on the second day to measure the cytokines produced. When performing MAT, the "total test time" from the start of culture to obtaining test results is generally approximately 2 days.

[0018] (Conventional Rapid Measurement Method for Monocyte Activation Test (MAT)) As mentioned above, most current monocyte activation test products use ELISA to measure inflammatory cytokines (IL-6), which requires a "total test time" of approximately two days for the MAT. There are also methods for rapid MAT evaluation that use cells pre-loaded with mechanisms to detect gene changes such as NF-κB, or evaluation kits with simplified detection reagents. However, because it is necessary to wait for receptor signal activation and cytokine production due to stimulation by the test substance, it has been difficult to shorten the "total test time" from the start of culture to the acquisition of test results (for example, within three hours).

[0019] (Rapid Measurement Method for Monocyte Activation Assay (MAT) of the Present Invention) Based on these findings, the present inventors conducted extensive research and successfully produced immortalized human peripheral blood-derived monocytes (aMylc-Z) capable of producing TNF-α without the need for pretreatment. These immortalized human peripheral blood-derived monocytes were prepared by introducing an immortalizing gene into human monocytes extracted from human peripheral blood. These immortalized human myeloid cells were then cryopreserved in the presence of a magnetic field. These immortalized human myeloid cells produce the inflammatory cytokine TNF-α in a short period of time upon stimulation with a pyrogen or the like, without pretreatment. Therefore, by using this cytokine as an indicator and further combining it with a luminescent reagent, the present inventors have successfully completed a rapid measurement method for the monocyte activation assay (MAT), which enables MAT evaluation (cultivation and measurement) to be performed in a short period of time (e.g., within 3 hours of the total test time from the start of culture to the completion of measurement). As used herein, "pretreatment" refers to a treatment in which cells are contacted with a specific substance in advance to promote cytokine production. For example, vitamin D (10 ng / mL) or PMA (8-200 nM) may be added to cells 1 to 2 days before the start of MAT to promote cytokine production (see Chanput, W. et al., Food Funct., 2010, 1(3), 254-26).

[0020] Hereinafter, one embodiment of human immortalized myeloid cells capable of producing cytokines without requiring pretreatment and a rapid test method for pyrogens using the same will be described. For convenience of explanation, the following will be described using specific cytokines, specific cells, specific pyrogens, test reagents, etc., but the present invention is not limited to these embodiments.

[0021] (Method for producing immortalized human myeloid cells)

[0022] In one embodiment of the present invention, there is provided a method for producing immortalized human myeloid cells (hereinafter also referred to as "the present production method"), comprising: (A) forcibly expressing in human myeloid blood cells (i) the cMYC gene, and (ii) 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 (B) freezing the immortalized human myeloid cells obtained in step (A) in the presence of a magnetic field.

[0023] As used herein, "myeloid blood cells" are defined as cells that express CD11b molecules or CD33 molecules, and their origin is not particularly limited, but examples include myeloid blood cells derived from pluripotent stem cells, or myeloid blood cells collected from a living organism (e.g., peripheral blood). Specific examples of myeloid blood cells include monocytes, dendritic cells, and macrophages. The myeloid blood cells are preferably human myeloid blood cells.

[0024] As used herein, the term "pluripotent stem cells" refers to cells that have the ability to proliferate under artificially constructed conditions (in vitro), such as in a test tube, and can differentiate into all cells that constitute a living organism. In the present invention, embryonic stem cells or induced pluripotent stem cells (induced pluripotent stem cells, iPS cells) are preferably used as pluripotent stem cells, and induced pluripotent stem cells are more preferably used. Examples of embryonic stem cells and induced pluripotent stem cells used in the present invention include those described in WO 2012 / 043651. Furthermore, a method for inducing differentiation of pluripotent stem cells into myeloid blood cells can also be performed, for example, by the method described in WO 2012 / 043651.

[0025] The present invention will be explained below using "aMylc-Z" as an example of "human immortalized myeloid cells" produced by this production method. "aMylc-Z" is an immortalized monocyte cell derived from human peripheral blood that has the ability to produce TNF-α without requiring pretreatment.

[0026] Monocytes can be prepared by a method of fractionating and collecting monocytes from peripheral blood or umbilical cord blood, or by a method of proliferating hematopoietic cells separated from peripheral blood or umbilical cord blood and then inducing their differentiation.

[0027] When monocytes are collected from peripheral blood or umbilical cord blood, they can be obtained as cells expressing CD14 molecules in human blood by known separation and preparation methods. For example, human peripheral blood is gently diluted with an equal volume of physiological saline, phosphate-buffered saline, or Hank's buffer solution, gently layered on Ficoll (registered trademark) (GE Healthcare) in a centrifuge tube, and centrifuged at 15-30°C and 500-1000 xg for approximately 20 minutes. The white band-like layer between the yellowish plasma and the transparent Ficoll can be 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 PBMCs with a solid phase bound to an anti-CD14 antibody to allow the monocytes to bind to the solid phase, and then washing to remove unbound cells. For example, a method using magnetic beads as such a solid phase is known (e.g., Dynabeads (registered trademark) CD14 (Thermo Fisher Scientific Inc.)). Alternatively, monocytes can be obtained by directly contacting peripheral blood with a solid phase bound to an anti-CD14 antibody, without separating PBMCs from the peripheral blood.

[0028] Methods for obtaining monocytes by culturing and differentiating tissue stem cells, such as hematopoietic cells obtained from peripheral blood or umbilical cord blood, are known. As used herein, "tissue stem cells" refer to cells present in bone marrow, liver, etc., which have the ability to differentiate into various cells and have the ability to self-replicate. Examples of tissue stem cells include hematopoietic cells. Furthermore, as used herein, "tissue" is synonymous with tissues that constitute a living body, such as the liver, heart, and blood.

[0029] For example, the following method has been reported for inducing differentiation of hematopoietic cells obtained from peripheral blood or umbilical cord blood into monocytes. Specifically, hematopoietic cells are first collected from peripheral blood or umbilical cord blood using magnetic beads or the like, using their characteristic surface marker, CD34, as an indicator. The collected CD34-positive cells are expanded and cultured in a culture medium containing growth factors such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), and FMS-like tyrosine kinase 3 ligand (FLT-3L). The differentiation of proliferated CD34-positive cells into monocytes can be induced by culturing them in a culture medium containing, for example, SCF, macrophage colony-stimulating factor (M-CSF), IL-3, and FLT-3L (see Malgorzata Stec et al., Journal of Leukocyte Biology, (2007); 82; 594-602). If necessary, monocytes may be purified by collecting only cells expressing CD14 using the method described above.

[0030] Monocyte immortalization can be achieved by forcibly expressing at least one gene selected from the group consisting of the BMI1 gene, EZH2 gene, MDM2 gene, MDM4 gene, HIF1A gene, BCL2 gene, and LYL1 gene, as well as the cMYC gene, in the monocytes obtained as described above, thereby imparting proliferation ability while maintaining monocyte function. Forced expression is preferably achieved by introducing the above genes into monocytes. Of the above genes, a combination of the BMI1 gene, the BCL2 gene, and the cMYC gene is preferably used, and one or more of the EZH2 gene, the MDM2 gene, the MDM4 gene, the HIF1A gene, and the LYL1 gene may also be introduced. Introduction of these genes into monocytes can be performed by referring to the descriptions in International Publication No. 2012 / 043651 and Japanese Patent Application Laid-Open No. 2017-131136. Gene introduction can be achieved using the gene introduction method described below.

[0031] The proliferation ability of human immortalized monocytic cells can be maintained by further introducing M-CSF and GM-CSF. Thus, the method of this embodiment may further include introducing M-CSF and GM-CSF into human immortalized monocytic cells. Alternatively, cells expressing M-CSF and GM-CSF may be used as the human immortalized monocytic cells. Introduction of these genes into immortalized myeloid cells or human immortalized monocytic cells can be performed by referring to the description in Japanese Patent Application Laid-Open No. 2018-171005.

[0032] The human immortalized monocytes obtained by the above method can be grown in a low-adhesion cell culture plate in a culture medium containing a basal medium such as MEMα, serum, or serum substitute, and additives such as M-CSF and GM-CSF. A sufficient amount of cells grown by cell growth is then frozen under magnetic field control of water molecule orientation (i.e., step (B)). For example, a frozen cell solution containing the human immortalized monocytes is sealed in a cryovial and frozen for 30 minutes or more in a magnetic field-activated freezer (Riho Freeze, Proton Freezer) at approximately -35°C under a uniform magnetic flux of 100-300 Gauss. This allows for the freezing of cells with smaller intracellular ice particles than conventional freezing. The inventors believe that this reduces the decrease in cytokine production due to cell damage during thawing, and enables the use of human immortalized monocytes with high cell viability and maintained cytokine production, even when subjected to MAT testing immediately after thawing. For example, thawed human immortalized monocytes can be rapidly evaluated without pretreatment using the production of cytokines such as TNF-α in response to stimulation with a test substance as an indicator. These are so-called immortalized human monocytes capable of producing TNF-α without the need for pretreatment.

[0033] In one embodiment of the present invention, there is provided an immortalized human myeloid cell produced by the production method.

[0034] In another embodiment of the present invention, there is provided an immortalized human myeloid cell comprising exogenous (i) a cMYC gene and (ii) at least one gene selected from the group consisting of a BMI1 gene, an EZH2 gene, an MDM2 gene, an MDM4 gene, and an HIF1A gene, and which produces cytokines upon contact with a pyrogen without pretreatment.

[0035] (Immortalized human myeloid cells) In one embodiment of the present invention, there is provided immortalized human myeloid cells produced by the production method of the present invention.

[0036] The "immortalized human myeloid cells" produced by this production method are cells that, when cultured with the addition of LPS (0.1 EU / mL), produce 150 pg / mL or more of TNF-α within more than 1 hour but within 3.5 hours from the start of culture. Thus, one embodiment of the present invention provides immortalized human myeloid cells that, when cultured with the addition of LPS (0.1 EU / mL), produce 150 pg / mL or more of TNF-α within more than 1 hour but within 3.5 hours from the start of culture.

[0037] The "human immortalized myeloid cells" produced by this production method may be cells that produce 150 pg / mL or more of TNF-α within more than 1 hour to 3.5 hours from the start of culture, for example, cells that produce 150 pg / mL or more of TNF-α 1.5 hours, 2 hours, 2.5 hours, 3 hours, or 3.5 hours from the start of culture. Furthermore, in one embodiment of the present invention, the "human immortalized myeloid cells" produced by this production method may be cells that produce 150 pg / mL or more of TNF-α, 160 pg / mL or more, 170 pg / mL or more, 180 pg / mL or more, 190 pg / mL or more, 200 pg / mL or more, 210 pg / mL or more, 220 pg / mL or more, 230 pg / mL or more, 240 pg / mL or more, or 250 pg / mL or more from more than 1 hour to 3.5 hours from the start of culture. The higher the amount of TNF-α produced within 3.5 hours and more than 1 hour from the start of the culture, the better, and there is no particular upper limit, but it is, for example, 2000 pg / mL or less.

[0038] In one embodiment of the present invention, the "immortalized human myeloid cells" produced by this production method are cells that produce 150 pg / mL or more of TNF-α 3.5 hours after the start of culture. Whether or not the cells produce 150 pg / mL or more of TNF-α within 3.5 hours, more than 1 hour after the start of culture when cultured in the presence of LPS (0.1 EU / mL), can be confirmed by the method described in the Examples.

[0039] In one embodiment of the present invention, after step (B), the method may comprise a step of selecting immortalized human myeloid cells that produce 150 pg / mL or more of TNF-α within more than 1 hour and within 3.5 hours from the start of culture when cultured in the presence of LPS (0.1 EU / mL). Therefore, in one embodiment of the present invention, there is provided the following method: (A) a step of forcibly expressing in human myeloid blood cells: (i) the cMYC gene, and (ii) 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; (B) a step of freezing the human immortalized myeloid cells obtained in step (A) in the presence of a magnetic field; and (C) a step of selecting, from the human immortalized myeloid cells obtained in step (B), cells that produce 150 pg / mL or more of TNF-α within more than 1 hour and within 3.5 hours from the start of culture when cultured with the addition of LPS (0.1 EU / mL).

[0040] (Sample transfer method: rapid testing method for pyrogens using human immortalized myeloid cells) In one embodiment of the present invention, there is provided a method for detecting pyrogens in a sample, comprising the steps of culturing human immortalized myeloid cells in the presence of the sample (also referred to as the "culturing step") and measuring the amount of cytokines produced in the culture medium obtained in the culturing step (also referred to as the "measuring step"), wherein the human immortalized myeloid cells have been frozen and stored in the presence of a magnetic field (hereinafter also referred to as "the present detection method").

[0041] In another embodiment of the present invention, there is provided a method for evaluating the thermogenicity of a test substance, comprising the step of culturing human immortalized myeloid cells in the presence of the test substance (also referred to as the "culture step"), and the step of measuring the amount of cytokine produced in the culture medium obtained in the culture step (also referred to as the "measurement step"), wherein the human immortalized myeloid cells have been frozen and stored in the presence of a magnetic field (hereinafter also referred to as the "present evaluation method").

[0042] 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, soap, shampoo, rinse and treatment, detergent, dye, fiber, cloth, cosmetics, pharmaceuticals, medical devices, compounds, mixtures (e.g., foods, food extracts, plant extracts), trace substances in the air, exhaust gases, waste liquids, industrial waste, cell culture medium, cultured cells, cell culture media, cell culture additives, cell preservation solutions, health-promoting foods, supplements, and the like.

[0043] As used herein, the term "pyrogenic substance" refers to a substance that, when taken into the body, causes an increase in body temperature, and examples thereof include endotoxin, peptidoglycan, exotoxin from gram-positive bacteria, viruses, pathogenic bacteria, pathogenic fungi, yeast, 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. In one embodiment of the present invention, the pyrogenic substance detected by this detection method is preferably lipopolysaccharide (LPS).

[0044] As used herein, "freezing in the presence of a magnetic field" means freezing cells under control of the orientation of water molecules by a magnetic field. Specific aspects of freezing in the presence of a magnetic field are described above (Method for producing immortalized human myeloid cells). Furthermore, "frozen in the presence of a magnetic field and stored" means that the cells were frozen under control of the orientation of water molecules by a magnetic field and stored in a frozen state until use.

[0045] As used herein, the term "cytokine" refers to a substance produced by cells in response to sensitization with a pyrogen, etc. Examples of cytokines include, but are not limited to, 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, and FGF. Examples of cytokines include 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. In one embodiment of the present invention, the fact that conventional MAT requires two days to complete is one of the issues that must be resolved before a universal test can be implemented. Therefore, it is preferable that the cytokine be produced within five hours after contact with a pyrogen. Using such cytokines as indicators allows rapid detection of pyrogens in a sample. Examples of such cytokines include TNF-α, IL-6, and IL-1β. In one embodiment of the present invention, the cytokine is preferably TNF-α.

[0046] In this specification, the term "test substance" is not particularly limited as long as it is a substance whose pyrogenicity is to be evaluated.

[0047] The immortalized human myeloid cells are preferably immortalized human monocytes or dendritic cells prepared from the immortalized human monocytes. The immortalized human monocytes are produced, for example, by the method described above (Method for producing immortalized human myeloid cells). Furthermore, dendritic cells can be induced from human immortalized monocytes according to previous reports (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.

[0048] The immortalized human myeloid cells may be derived from peripheral blood or from pluripotent stem cells, preferably from human peripheral blood.

[0049] In the following description, "TNF-α" will be mainly used as the "cytokine" and "immortalized human monocyte cells" will be mainly used as the "immortalized human myeloid cells".

[0050] A rapid test method for pyrogens using human immortalized monocytes capable of producing TNF-α, which does not require pretreatment, can be carried out by carrying out a step of producing cytokines by contacting the human immortalized monocytes with a sample or a test substance (culture step), and a step of measuring the produced cytokines (measurement step).

[0051] The medium used in the culture step may be selected based on the culture conditions used for culturing blood cells (e.g., An in vitro monocytic culture method and establishment of a human monocytic cell, Katsuyuki Kadoi, Veterinaria Italiana, 2011, 47(2), 139-146). In one embodiment, when culturing human immortalized monocytes capable of producing TNF-α, which do not require pretreatment, in contact with a sample or a test substance, the evaluation conditions used with human immortalized monocytes are used as a reference (e.g., MEMα medium conditions containing 10% bovine serum and growth factors (Haruta M, Hum Immunol. 2013 Oct;74(10):1400-8)). For example, if the evaluation conditions are MEMα (Shimadzu Diagnostics or Gibco) or RPMI 1640 medium (SIGMA) containing a human platelet-derived lysate, or a bovine serum substitute such as KSR (KnockOut Serum Replacement, Gibco), or bovine serum, the evaluation conditions are used as is. However, since MAT is an alternative to animal testing, it is desirable not to use animal-derived reagents or components, or to use only the minimum amount of them. The time required for the culture step (culture time) can be set appropriately depending on the cytokines to be evaluated for the test substance and cells, but when TNF-α production is used as an indicator, it can be, for example, 30 minutes to 3.5 hours, preferably 1 to 3 hours, more preferably 1.5 to 2.5 hours, and even more preferably 2 hours. Other culture conditions can be determined in accordance with general culture conditions for human cells, for example, 37°C, 5% CO ２ The procedure can be carried out under conditions of 5% CO ２ is not essential and may be under atmospheric composition.

[0052] The time required for the measurement step (measurement time) may vary depending on the cytokine measurement method. For example, when measuring cytokines using a luminescent reagent, the measurement time mainly includes the following three steps: first, adding a luminescent substrate to bind to the cytokine, then adding a luminescent reagent to cause the substrate to emit light, and finally, measuring with a measuring device. The time required for this series of steps can be, for example, 20 minutes to 1.5 hours, preferably 30 minutes to 1.2 hours, and more preferably 40 minutes to 1.0 hour.

[0053] In this embodiment, the total time for the culture step and the measurement step (i.e., the total test time from the start of culture of the human immortalized monocyte cells to the completion of measurement) is, for example, 50 minutes to 4.5 hours, preferably 1 to 4 hours, more preferably 1.5 to 3.5 hours, and even more preferably 2 to 3 hours.

[0054] The test evaluation is completed by measuring the amount of cytokine produced in the culture supernatant after the incubation period. The amount of cytokine produced in the culture supernatant is preferably measured using a luminescent reagent. For example, quantitative evaluation can be performed using a cytokine detection kit that uses a commercially available luminescent reagent and a luminometer. The luminescent reagent is not particularly limited, but examples include Lumit™ TNF-α (Human) (Promega).

[0055] In one embodiment of the present invention, the method further comprises the step of measuring the amount of production of a second cytokine, different from the cytokine (e.g., TNF-α), in the culture medium obtained in the culturing step. The time at which cytokines are produced when blood cells are sensitized to a pyrogen varies depending on the cytokine. For example, Jansky et al. reported that when peripheral blood mononuclear cells (PBMCs) are sensitized to the pyrogen LPS, the time at which cytokines such as TNF-α and IL-6 are produced varies (L. Jansky et al., Physiol. Res., 2003, 52, 593-598). Therefore, by further measuring the amount of production of a second cytokine, different from TNF-α, in the culture medium, it is possible to more accurately evaluate the pyrogen in the sample or to evaluate the pyrogenicity of the test substance.

[0056] In one embodiment of the present invention, the method includes measuring the amount of production of a cytokine (for convenience, referred to as a "first cytokine") in a first culture medium obtained in the culturing step, and measuring the amount of production of a second cytokine different from the first cytokine in a second culture medium obtained in the culturing step, wherein the first culture medium and the second culture medium are culture mediums obtained at different culture times in the culturing step. For example, a culture supernatant is obtained from a culture medium obtained by culturing human immortalized monocytes in the presence of a sample for 3 hours, and the amount of production of a first cytokine (e.g., TNF-α) in the culture supernatant (first culture medium) is measured. Subsequently, a culture supernatant is obtained from a culture medium obtained by culturing the cells for 20 hours, and the amount of production of a second cytokine (e.g., IL-6) different from the first cytokine in the culture supernatant (second culture medium) is measured.

[0057] The first cytokine and the second cytokine are not particularly limited as long as they are substances produced by sensitization with a pyrogen or the like and are different from each other. Examples of the first cytokine and the second cytokine include 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, and FGF. Examples of cytokines that can be used include 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. In one embodiment of the present invention, for example, the first cytokine may be a cytokine produced within 5 hours after contact with a pyrogen (e.g., TNF-α, IL-6, IL-1β, etc.), and the second cytokine may be a cytokine other than the first cytokine from the cytokines exemplified above.

[0058] The present detection method and the present evaluation method may comprise, prior to the culturing step, a step of thawing human immortalized myeloid cells that have been frozen and stored in the presence of a magnetic field. The present detection method and the present evaluation method may comprise, prior to the culturing step, a step of thawing step (B) of the present production method and the human immortalized myeloid cells that have been frozen in step (B). The present detection method and the present evaluation method may comprise, prior to the culturing step, a step of thawing human immortalized myeloid cells that have been frozen in step (A), step (B), and step (B) of the present production method.

[0059] Therefore, in one embodiment of the present invention, there is provided a method for detecting a pyrogen in a sample, comprising the steps of thawing immortalized human myeloid cells that have been frozen and stored in the presence of a magnetic field, culturing the immortalized human myeloid cells in the presence of a sample, and measuring the amount of cytokine produced in the culture medium obtained in the culturing step.

[0060] In addition, in one embodiment of the present invention, there is provided a method for detecting a pyrogen in a sample, comprising the steps of: (B) freezing human immortalized myeloid cells in the presence of a magnetic field; thawing the human immortalized myeloid cells frozen in step (B); culturing the human immortalized myeloid cells in the presence of a sample; and measuring the amount of cytokine produced in the culture medium obtained in the culturing step.

[0061] Furthermore, in one embodiment of the present invention, there is provided a method for detecting a pyrogen in a sample, comprising: (A) forcibly expressing in human myeloid blood cells (i) the cMYC gene, and (ii) 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; (B) freezing human immortalized myeloid cells in the presence of a magnetic field, thawing the human immortalized myeloid cells frozen in step (B), culturing the human immortalized myeloid cells in the presence of a sample, and measuring the amount of cytokine produced in the culture medium obtained in the culturing step.

[0062] In another embodiment of the present invention, there is provided a method for evaluating the pyrogenicity of a test substance, comprising the steps of thawing immortalized human myeloid cells that have been frozen and stored in the presence of a magnetic field, culturing the immortalized human myeloid cells in the presence of a test substance, and measuring the amount of cytokine produced in the culture medium obtained in the culturing step.

[0063] In another embodiment of the present invention, there is provided a method for evaluating the pyrogenicity of a test substance, comprising: (B) a step of freezing human immortalized myeloid cells in the presence of a magnetic field; a step of thawing the human immortalized myeloid cells frozen in step (B); a step of culturing the human immortalized myeloid cells in the presence of a test substance; and a step of measuring the amount of cytokine produced in the culture medium obtained in the culturing step.

[0064] Furthermore, in another embodiment of the present invention, there is provided a method for evaluating the pyrogenicity of a test substance, comprising: (A) forcibly expressing in human myeloid blood cells (i) the cMYC gene, and (ii) 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; (B) freezing human immortalized myeloid cells in the presence of a magnetic field, thawing the human immortalized myeloid cells frozen in step (B), culturing the human immortalized myeloid cells in the presence of the test substance, and measuring the amount of cytokine produced in the culture medium obtained in the culturing step.

[0065] (Direct method for rapid detection of pyrogens using human immortalized myeloid cells) In one embodiment of the present invention, a method for detecting pyrogens in a sample is provided, which comprises measuring the amount of cytokines produced in a culture medium while culturing human immortalized myeloid cells in the presence of the sample, wherein the human immortalized myeloid cells have been frozen and stored in the presence of a magnetic field.

[0066] The rapid pyrogen testing method using human immortalized monocytes capable of producing TNF-α, which does not require pretreatment, can also provide evaluation results more rapidly by measuring the amount of TNF-α produced during culture of the human immortalized monocytes (e.g., by adding a mixture of antibodies to be evaluated and then adding a luminescent reagent). For convenience, this method is referred to as the "direct method." Hereinafter, only matters not explained in the above (rapid pyrogen testing method using human immortalized myeloid cells: sample transfer method) will be explained. For the rest, the description in the above (rapid pyrogen testing method using human immortalized myeloid cells: sample transfer method) is incorporated by reference.

[0067] In one embodiment, the "direct method" can be performed by adding a TNF-α antibody mixture to a well, for example, immediately after the start of culture to 6 hours later, preferably 30 minutes to 3 hours later, more preferably 45 minutes to 2 hours later, and even more preferably 1 hour later. After the addition of the TNF-α antibody mixture, the wells are further cultured, for example, for 30 minutes to 3 hours, preferably 45 minutes to 2 hours, and more preferably 1 hour, and then a luminescent reagent is added for quantitative evaluation. The direct method can be performed by simultaneously performing the culture step and the step of adding the TNF-α antibody mixture as a luminescent substrate for the measurement step and allowing it to bind to TNF-α produced in the culture medium. Subsequent addition of a luminescent reagent for quantitative evaluation enables even simpler and more rapid evaluation.

[0068] The time required for the process in this embodiment (i.e., the total test time from the start of culturing the human immortalized monocyte cells to the completion of the measurement) is, for example, 30 minutes to 3 hours, preferably 1 to 2.5 hours, and more preferably 2 hours.

[0069] In one embodiment of the present invention, there is provided a method for detecting a pyrogen in a sample, comprising the steps of thawing immortalized human myeloid cells that have been frozen and stored in the presence of a magnetic field, and measuring the amount of cytokine produced in the culture medium while culturing the immortalized human myeloid cells in the presence of the sample.

[0070] In addition, in one embodiment of the present invention, there is provided a method for detecting a pyrogen in a sample, comprising: (B) a step of freezing human immortalized myeloid cells in the presence of a magnetic field; a step of thawing the human immortalized myeloid cells frozen in step (B); and a step of measuring the amount of cytokine produced in the culture medium while culturing the human immortalized myeloid cells in the presence of the sample.

[0071] Furthermore, in one embodiment of the present invention, there is provided a method for detecting a pyrogen in a sample, comprising: (A) forcibly expressing in human myeloid blood cells (i) the cMYC gene, and (ii) 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; (B) freezing the human immortalized myeloid cells in the presence of a magnetic field; thawing the human immortalized myeloid cells frozen in step (B); and measuring the amount of cytokine produced in the culture medium while culturing the human immortalized myeloid cells in the presence of the sample.

[0072] The present invention will be described in more detail below using examples, but these are not intended to limit the scope of the present invention. Note that all references cited throughout this specification are incorporated herein by reference in their entirety.

[0073] Example 1: Preparation of human peripheral blood-derived immortalized monocytes (aMylc-Z) capable of producing TNF-α without requiring pretreatment (Preparation of human peripheral blood-derived immortalized monocytes) Human peripheral blood-derived immortalized monocytes were prepared with reference to previous reports (WO 2012 / 043651 and JP 2017-131136 A). Specifically, a CD14-positive fraction was extracted from human peripheral blood, and c-Myc, BMI-1, and genes described in the human immortalized monocyte cell report were introduced into a lentiviral vector, which was then used to introduce the vector into CD14-positive cells to produce the immortalized monocytes. Human peripheral blood-derived immortalized monocytic cells were cultured in DCO-K (Shimadzu Diagnostics) medium containing 2.5% human platelet-derived lysate or 10% KSR (Knockout Serum Replacement, Gibco), 50 ng / ml M-CSF, and 50 ng / ml GM-CSF, and were harvested as proliferative cells 2 to 5 weeks after the initiation of culture.

[0074] (Confirmation of the prepared cells) To confirm that the prepared cells were monocyte-like cells, morphological observation was performed by evaluating the color of the concentrated cells and measuring surface markers. The color of the concentrated cells was evaluated by visually observing the color of the cells when they were collected in a centrifuge tube. Surface marker measurements were performed using a CD14 antibody (Biolegend) and a BD flow cytometer, Accuri.

[0075] (Preparation of immortalized monocytes (aMylc-Z) using a magnetic field cryopreservation method and conventional method (aMylc)) The cells were expanded using cell culture plates (CellSeed) with an ultra-low adhesion coating. Cells that had proliferated more than 100 times their pre-expansion size were frozen at 8 x 10 using a commercially available cell freezing solution. ５The cells were packaged in 0.5 mL cells / vial and frozen using two methods. 1. Immortalized monocyte cells using a magnetic field cryopreservation method (aMylc-Z): They were rapidly frozen at -35°C using a magnetic field-based freezer (Proton Freezer, Ryoho Freeze Systems). After one hour, the frozen cell vials were stored in an ultra-low temperature freezer at -80°C or below. 2. Conventional method (aMylc): They were placed in a freezing container (BICEL, Nippon Freezer Co., Ltd.) pre-cooled to 4°C and placed in an ultra-low temperature freezer at -80°C. The next day, they were removed from the container and stored in an ultra-low temperature freezer at -80°C or below.

[0076] (MAT test comparison of immortalized monocyte cells (aMylc-Z) cryopreserved using a magnetic field) A MAT test using endotoxin was performed using frozen aMylc-Z and aMylc cells. Human peripheral blood-derived immortalized monocyte cells (aMylc-Z) cryopreserved using a magnetic field and human peripheral blood-derived immortalized monocyte cells (aMylc) frozen using a conventional freezing method were thawed from cryovials in a 37°C water bath and incubated at 2.5 x 10 in MEMα culture medium containing human platelet lysate (2.5%) or artificial serum (10%). ４ cells / mL. Separately, endotoxin (Merck, Endotoxin Standard) was prepared according to the method attached to the reagent to a concentration of 2000 EU / mL, and then diluted with saline at a dilution ratio of 2 to give a concentration of 0.8 to 0.0625 EU / mL in the evaluation wells. 100 μL of the diluted endotoxin solution and saline were added to a 96-well plate, 4 wells per group, and then 100 μL of the culture medium containing the human peripheral blood-derived immortalized monocyte cells (aMylc-Z or aMylc) was added to each well. The mixed 96-well plate was then incubated in an incubator (37°C, 5% CO ２ ) and cultured at 4°C for 20 hours. 20 hours after the start of culture, 50 μL of the culture supernatant was collected from each well. The collected culture supernatant was measured with an absorbance meter using an ELISA kit (LegendMAX™ IL-6 (Human), Biolegend) that uses IL-6 as an indicator, according to the attached measurement method.

[0077] The results are shown in Figure 2. Human peripheral blood-derived immortalized monocyte cells (aMylc-Z) cryopreserved using a magnetic field and human peripheral blood-derived immortalized monocyte cells (aMylc) frozen using a conventional freezing method were used to compare the cell count upon thawing, cell viability, and reactivity to a pyrogen (LPS) when each type of cell was used. The results showed that aMylc-Z exhibited better values ​​than aMylc in terms of cell count, cell viability, and cytokine production upon sensitization to a pyrogen. For example, in Lot. A, the cell count and cell viability were 80 x 10 ４ When the frozen vial of cells was thawed, the number of cells in aMylc was 55.8 × 10 ４ cells, cell viability was 88%, while aMylc-Z was 69.8 × 10 ４ The cell viability was 98%. It was also revealed that the amount of IL-6 produced in response to LPS stimulation showed a significant difference from 0.1 EU / mL.

[0078] Example 2 Rapid Measurement and Evaluation of Endotoxin Using Human Peripheral Blood-Derived Immortalized Monocyte Cells (aMylc-Z) Capable of Producing TNF-α Without Pretreatment (Sample Transfer Method) Human peripheral blood-derived immortalized monocyte cells (aMylc-Z) were thawed from a frozen vial in a 37°C water bath and incubated at 2 x 10 in MEMα culture medium containing human platelet lysate (2.5%) or artificial serum (10%). ５ cells / mL. Separately, LPS (Merck, Endotoxin standard) was prepared at 2000 EU / mL according to the method attached to the reagent, and diluted with saline to give 0.4, 0.025, and 0.0625 EU / mL in the evaluation wells. The endotoxin diluted solution and saline were added to a 96-well plate at 100 μL / well, 2 wells per group, and then 100 μL of the culture medium containing the human peripheral blood-derived immortalized monocyte cells (aMylc-Z) was added to each well. The mixed 96-well plate was then placed in an incubator (37°C, 5% CO ２) were cultured. 10 μL of the culture supernatant was collected from each well 1 to 3 hours after the start of culture (culture time: 1 to 3 hours). Next, the measurement step was carried out. The collected culture supernatant was used for measurement using a luminoassay kit (Lumit™ TNF-α (Human), Promega) with TNF-α as an indicator. Specifically, the collected culture supernatant (10 μL) was transferred to a 96-well plate (white, Corning). Next, a mixture of two TNF-α antibodies (2x, 10 μL) prepared according to the kit's instructions was added and allowed to react for 45 minutes at room temperature. After 45 minutes, Lumit™ substrate solution (5x, 5 μL) was added to each well, and the plate was shaken for 1 minute, followed by measurement using a luminometer (Glomax, Promega). Therefore, the incubation time was 1 to 3 hours, and the measurement time was within 1 hour, with the total test time being 2 to 4 hours.

[0079] The results are shown in Figure 3. Human peripheral blood-derived immortalized monocytic cells (aMylc-Z) were used, and TNF-α production in the culture supernatant of each well was measured using a luminometer 1 to 3 hours after LPS stimulation. The X axis represents the LPS concentration (EU / mL), and the Y axis represents the luminometer readings (RLU / sec). After 1 hour of incubation, no significant increase in the readings was observed. However, after 2 hours of incubation, the average reading was 895 RLU / sec with 0.025 EU / mL of LPS stimulation, which was higher than the blank reading (580 RLU / sec). Even after 3 hours of incubation, the average reading was 1933 RLU / sec with 0.025 EU / mL of LPS stimulation (compared to the blank reading of 675 RLU / sec after 3 hours). Using human peripheral blood-derived immortalized monocytic cells (aMylc-Z) and TNF-α as an indicator, the measured values ​​of 0.025 EU / mL after 2 and 3 hours were both found to be greater than the blank value, demonstrating that MAT can be formed within a culture time of 3 hours.

[0080] Example 3 Rapid Measurement and Evaluation of Endotoxin Using Human Peripheral Blood-Derived Immortalized Monocyte Cells (aMylc-Z) Capable of Producing TNF-α Without Pretreatment (Direct Method) Human peripheral blood-derived immortalized monocyte cells (aMylc-Z) capable of producing TNF-α without pretreatment were thawed from a frozen vial in a 37°C water bath and incubated at 2 x 10 in MEMα culture medium containing human platelet lysate (2.5%) or artificial serum (10%). ５ cells / mL. Separately, LPS (Merck, Endotoxin standard) was prepared at 2000 EU / mL according to the method attached to the reagent, and diluted with saline to give 0.4, 0.025, and 0.0625 EU / mL in the evaluation wells. The endotoxin diluted solution and saline were added to a 96-well plate at 100 μL / well, 2 wells per group, and then 100 μL of the culture medium containing the human peripheral blood-derived immortalized monocyte cells (aMylc-Z) was added to each well. The mixed 96-well plate was then placed in an incubator (37°C, 5% CO ２ ). One hour after the start of incubation, a mixture of two TNF-α antibodies (50x, 10 μL) prepared according to the method of a luminoassay kit (Lumit™ TNF-α (Human), Promega) using TNF-α as an indicator was added to each well, and incubation continued for another 1 to 2 hours. This combined part of the measurement process into the incubation process. After incubation, 20 μL of the culture supernatant from each well was collected and transferred to a 96-well plate (white, Corning). Next, Lumit™ substrate solution (20x, 5 μL) was added to each well, and the plate was shaken for 1 minute, followed by measurement with a luminometer. Therefore, the "total test time" using this method was 2 to 3 hours.

[0081] The results are shown in Figure 4. The X-axis represents the LPS concentration (EU / mL), and the Y-axis represents the luminometer readings (RLU / sec). After 1 hour of incubation, the antibody was added, and pretreatment for measurement was performed in the same plate. After a total of 2 hours of incubation and pretreatment, the average value was 555 RLU / sec with 0.025 EU / mL LPS stimulation, indicating an increase in the measured value compared to the blank (276 RLU / sec). After an additional 1 hour of incubation, the average value was 1465 RLU / sec with 0.025 EU / mL LPS stimulation, indicating an increase in the measured value even with the blank (1002 RLU / sec), although an increase in the blank value was also observed.

[0082] (Example 4) Rapid measurement and evaluation of non-endotoxins using human peripheral blood-derived immortalized monocyte cells (aMylc-Z) capable of producing TNF-α without requiring pretreatment. We investigated whether human peripheral blood-derived immortalized monocyte cells (aMylc-Z) can be used for MAT even in the absence of endotoxins, using their TNF-α production ability as an indicator. Human peripheral blood-derived immortalized monocyte cells (aMylc-Z) were thawed from a frozen vial in a 37°C water bath and incubated at 2 x 10 in MEMα culture medium containing human platelet lysate (2.5%) or artificial serum (10%). ５ The non-endotoxin diluted solution and saline were added to a 96-well plate at 100 μL / well, 4 wells / group, and then 100 μL of the culture medium containing the human peripheral blood-derived immortalized monocyte cells (aMylc-Z) was added to each well. The mixed 96-well plate was then placed in an incubator (37°C, 5% CO ２ ) were cultured. Three hours after the start of culture, 50 μL of the culture supernatant was collected from each well (culture time: 3 hours). The collected culture supernatant was measured using an ELISA kit (LegendMAX™ TNF-α (Human), Biolegend) using TNF-α as an indicator. For comparison, the same non-endotoxin culture was cultured for 20 hours according to the conventional MAT test method, and the amount of IL-6 produced was measured by ELISA.

[0083] The results are shown in Figure 5. The X axis represents the LPS concentration (EU / mL), and the Y axis represents the measured value (OD value) measured by an absorbance meter. When non-endotoxins such as Flagellin, Pam3CSK4, Poly(I:C), and HKSA were added to human peripheral blood-derived immortalized monocytic cells (aMylc-Z), it was confirmed that the amount of TNF-α produced in the culture supernatant increased after 3 hours in all samples. Furthermore, a comparison with a conventional MAT evaluation using IL-6 production as an index after 20 hours of culture revealed that both TNF-α and IL-6 were correlated with each other for all non-endotoxin test substances.

[0084] (Example 5) Continuous Cytokine Measurement and Evaluation of Test Substances Using Human Peripheral Blood-Derived Immortalized Monocyte Cells (aMylc-Z) Capable of Producing TNF-α Without Pretreatment (Continuous Evaluation Method) It was verified whether human peripheral blood-derived immortalized monocyte cells (aMylc-Z) could be used to continuously measure the cytokines of test substances and test them with two different cytokines (TNF-α and IL-6). Human peripheral blood-derived immortalized monocyte cells (aMylc-Z) were thawed from a frozen vial in a 37°C water bath and incubated at 2 x 10 in MEMα culture medium containing human platelet lysate (2.5%) or artificial serum (10%). ５ Separately, LPS (Merck, Endotoxin standard) as a test substance was prepared at 2000 EU / mL according to the method attached to the reagent, and a dilution series was prepared in the evaluation wells with physiological saline starting from 0.4 EU / mL at a common ratio of 2. The endotoxin dilution solution and physiological saline were added to a 96-well plate at 100 μL / well, 3 wells per group, and then 100 μL of the culture medium containing the human peripheral blood-derived immortalized monocyte cells (aMylc-Z) was added to each well. The mixed 96-well plate was then placed in an incubator (37°C, 5% CO ２) were cultured. Three hours after the start of culture, the 96-well plate was removed from the incubator, and 10 μL of the culture supernatant from each well was collected and placed back into the incubator for further culture. The collected culture supernatant was measured using a luminoassay kit (Lumit™ TNF-α (Human), Promega) using TNF-α as an indicator. Specifically, the collected culture supernatant (10 μL) was transferred to a 96-well plate (white, Corning). Next, a mixture of two TNF-α antibodies (2x, 10 μL) prepared according to the kit's instructions was added and allowed to react for 45 minutes at room temperature. After 45 minutes, Lumit™ substrate solution (5x, 5 μL) was added to each well, and the plate was shaken for 1 minute and then measured with a luminometer. Meanwhile, the collected 96-well plate was returned to the incubator and continued to be cultured for 20 hours from the start of culture. Twenty hours after the start of culture, the 96-well plate was removed from the incubator, and 10 μL of the culture supernatant was collected from each well. The collected culture supernatant was measured using a luminoassay kit (Lumit™ IL-6 (Human), Promega) using IL-6 as an indicator. Specifically, the collected culture supernatant (10 μL) was transferred to a 96-well plate (white, Corning). Next, a mixture of two IL-6 antibodies (2×, 10 μL) prepared according to the kit's instructions was added, and the reaction was allowed to proceed at room temperature for 45 minutes. After 45 minutes, Lumit™ substrate solution (5×, 5 μL) was added to each well, and the plate was shaken for 1 minute, after which measurements were made using a luminometer.

[0085] The results are shown in Figure 6. The X-axis represents the LPS concentration (EU / mL), and the Y-axis represents the luminometer readings (RLU / sec). When human peripheral blood-derived immortalized monocytic cells (aMylc-Z) were added with LPS, TNF-α production in the culture supernatant after 3 hours was confirmed to be concentration-dependent, with detectable LOD (limit of detection) values ​​as low as 0.0125 EU / mL. Furthermore, by continuing the culture, MAT evaluation using IL-6 production as an indicator under conventional 20-hour culture conditions was also confirmed to be detectable at an LOD of 0.0016 EU / mL. Thus, it was demonstrated that continuous cytokine production measurements were possible at 3 and 20 hours after the start of culture in the same well, and that TNF-α and IL-6 production could be detected in a similar manner.

[0086] Example 7 Evaluation of TNF-α production activity using human peripheral blood-derived immortalized monocyte cells (aMylc-Z) capable of producing TNF-α without requiring pretreatment and conventional human peripheral blood-derived immortalized monocyte cells (aMylc) A pyrogen test was carried out using human peripheral blood-derived immortalized monocyte cells (aMylc-Z) capable of producing TNF-α without requiring pretreatment and conventional human peripheral blood-derived immortalized monocyte cells, with TNF-α as the indicator. aMylc-Z and aMylc were thawed from frozen vials in a 37°C water bath and incubated at 1.0 x 10 ５ Separately, LPS (Merck, Endotoxin standard) as a test substance was prepared at 2000 EU / mL according to the method attached to the reagent, and a dilution series was prepared in the evaluation wells with physiological saline starting from 0.4 EU / mL at a common ratio of 2. The endotoxin dilution solution and physiological saline were added to a 96-well plate at 100 μL / well, 4 wells per group, and then 100 μL of the culture medium containing the human peripheral blood-derived immortalized monocyte cells (aMylc-Z or aMylc) was added to each well. The mixed 96-well plate was then placed in an incubator (37°C, 5% CO ２). 3.5 hours after the start of culturing, the 96-well plate was removed from the incubator, and 50 μL of the culture supernatant was collected from each well. The collected culture supernatant was measured using a luminoassay kit (Lumit™ TNF-α (Human), Promega) using TNF-α as an indicator. Specifically, the collected culture supernatant (50 μL) was transferred to a 96-well plate (white, Corning). Next, a mixture of two TNF-α antibodies (2×, 50 μL) prepared according to the kit's instructions was added, and the plate was incubated in an incubator (37°C, 5% CO) for 45 minutes. ２ After 45 minutes, Lumit™ substrate solution (5x, 25 μL) was added to each well, the plate was shaken for 10 seconds, and then measured in a luminometer after 5 minutes.

[0087] The results are shown in Figure 7. The X axis represents the LPS concentration (EU / mL), and the Y axis represents the luminometer measurement value (RLU / sec). It was confirmed that aMylc-Z could detect the amount of TNF-α produced in the culture supernatant after 3.5 hours of LPS addition in a concentration-dependent manner, with a limit of detection (LOD) value of 0.0125 EU / mL. In contrast, conventional aMylc cells produced a low amount of TNF-α, with an LOD value of 0.05 EU / mL.

[0088] The present invention can be widely used in various fields, for example, in rapid MAT evaluation of medicines, vaccines, medical devices, etc., cytokine research of test substances, etc. This application is based on Japanese Patent Application No. 2024-087456 (filing date: May 29, 2024), the contents of which are incorporated in full herein.

Claims

1. A method for producing immortalized human myeloid cells, comprising: (A) forcibly expressing in human myeloid blood cells: (i) the cMYC gene; and (ii) 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 (B) freezing the immortalized human myeloid cells obtained in step (A) in the presence of a magnetic field.

2. The method according to claim 1, wherein the gene is forcibly expressed in the human myeloid blood cells by introducing the gene into the human myeloid blood cells.

3. The method according to claim 1 or 2, wherein the human myeloid blood cells are human peripheral blood-derived monocytes.

4. The method of claim 1 or 2, wherein the human myeloid blood cells are derived from pluripotent stem cells.

5. The method according to claim 1 or 2, wherein M-CSF and / or GM-CSF are further forcibly expressed in the human myeloid blood cells of step (A) or the human immortalized myeloid cells obtained in step (A).

6. The method according to claim 1 or 2, wherein the human immortalized myeloid cells obtained in step (B) are cells that produce 150 pg / mL or more of TNF-α within more than 1 hour and within 3.5 hours from the start of culture when cultured with the addition of LPS (0.1 EU / mL).

7. Immortalized human myeloid cells obtained by the method of claim 1 or 2.

8. Human immortalized myeloid cells that, when cultured with the addition of LPS (0.1 EU / mL), produce 150 pg / mL or more of TNF-α within 3.5 hours and more than 1 hour from the start of culture.

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