Method for preparing dendritic cells by cluster control culture

Abstract

To provide a preparation method of a dendritic cell by using IL-4 in which the quality of a DC to be obtained is improved.SOLUTION: A method for preparing a dendritic cell in which a cytotoxic T-cell with high antigen-presenting function is induced from a monocyte includes: differentiating and inducing a monocyte separated from peripheral blood into a dendritic cell by culturing using culture medium containing IL-4 and GM-CSF; and maturating an obtained immature dendritic cell in the presence of prostaglandin E2 and OK432 in the state where spheroid is formed in an immature dendritic cell by cluster controlled culture.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for preparing dendritic cells from monocytes.

Background Art

[0002] Dendritic cells (DC) are powerful antigen-presenting cells in vivo and are known to induce an immune response by presenting an antigen to T cells. In addition, it is known that DC acts directly not only on T cells but also on B cells, NK cells, NKT cells, etc., and plays a central role in the immune reaction. Immature DC acquires high T cell-stimulating ability with an increase in the expression of CD40, CD80, CD86, etc. by receiving antigen stimulation, migrates to peripheral lymphoid tissues, and induces an immune response by activating T cells specific to the captured antigen.

[0003] Generally, several kinds of cytokines are known as substances that can induce the differentiation of dendritic cells from hematopoietic progenitor cells. For example, there are many reports on the induction of DC differentiation by the combined use of GM-CSF and IL-4 (Non-Patent Document 1). In addition, substances that can induce DC differentiation alone or in combination with other cytokines have also been reported (Non-Patent Document 2). For example, TNF-α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-13, IL-15, HGF (Hepatocyte growth factor), CD40 ligand, M-CSF, Flt3 ligand, c-kit ligand, TGF-β, etc. have been reported.

[0004] In the method of inducing DC differentiation by combining GM-CSF and IL-4, it is carried out by an adherent culture method. Mononuclear cells (monocytes and lymphocytes) are seeded on a culture dish, the lymphocytes are washed, and the adhered monocytes are used for culture. Culturing is performed for 5 to 7 days in the presence of GM-CSF / IL-4, the cells are collected by washing with the medium and scraping (physically peeling off), and the medium is replaced with a medium (Fresh medium) containing an adjuvant (immunopotentiator) OK432 to prepare mature DC.

[0005] In addition, a method for preparing dendritic cells using G-CSF (Patent Document 1) and a method for preparing dendritic cells by non-adherent culture using IFN (Patent Document 2) have been reported.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] In the method for preparing dendritic cells using pre-evaluated IL-4, the cells were cultured in a low-adhesion dish, and the viable cell rate and yield of the processed specific cell product (WT1-IL-4-dendritic cell vaccine) were good. However, the quality variation of IL-4-dendritic cells (IL-4-DC), which are dendritic cells prepared using IL-4, was a major problem, and there was a major issue that maturation was inhibited.

[0009] The present invention aims to provide a method for preparing IL-4-DC prepared using IL-4, which improves the quality of the obtained DC.

Means for Solving the Problem

[0010] In view of the problem of large variations in quality in the method for preparing dendritic cells using IL-4, the present inventors have now considered the optimization of a method for producing IL-4-DC with uniform quality. As a result, (1) by performing cluster control culture (spheroid culture) using a cluster control culture dish to form clusters of uniform size, maturation was promoted, IFN-γ production was significantly increased, and furthermore, a mature type with a higher degree of maturation of surface traits than conventional dendritic cells using IL-4 was obtained. Furthermore, in IL-4-DC prepared by cluster control culture, it was found that the expression of the BCL2A1 gene was amplified and the lifespan of dendritic cells was extended, leading to the completion of the present invention.

[0011] That is, the present invention is as follows. [1] A method for preparing dendritic cells with high antigen-presenting function capable of inducing cytotoxic T cells from monocytes, comprising differentiating and inducing monocytes separated from peripheral blood into dendritic cells by culturing them in a medium containing IL-4 and GM-CSF, and maturing the obtained immature dendritic cells in a state where spheroids are formed in the immature dendritic cells by cluster control culture in the presence of prostaglandin E2 and OK432. [2] The method according to [1], wherein the cluster control culture is performed using a spheroid formation culture container. [3] The method according to [1] or [2], wherein the immature dendritic cells are matured in a state where spheroids are formed in the immature dendritic cells by cluster control culture at a density of 1.8×10 6 cells / ml to 2.2×10 6 cells / ml. [4] The method according to any one of [1] to [3], comprising culturing in a serum-free medium containing IL-4 and GM-CSF by non-adherent culture for 3 to 8 days, adding prostaglandin E2 and OK432, and maturing in a state where spheroids are formed in the immature dendritic cells by cluster control culture for 10 to 36 hours. [5] Culturing using IL-4 at 1 ng / mL to 1,000 ng / mL, GM-CSF at 10 ng / mL to 1,000 ng / mL, prostaglandin E2 at 5 ng / mL to 50 ng / mL, and OK432 at 5 μg / mL to 50 μg / mL, by any one of the methods of [1] to [4]. [6] Adding a cancer-specific antigen during the maturation of dendritic cells, by any one of the methods of [1] to [5]. [7] The method of any one of [1] to [6], wherein the viable cell rate of the obtained dendritic cells is 90% or more, and the yield, which is the ratio of the number of obtained dendritic cells to the number of monocytes during culture, is 15% or more. [8] The method of any one of [1] to [7], wherein the obtained dendritic cells have an increased positive rate of CD80 and CD40, and further an increased intensity of expression (ΔMFI) of CD80, CD86, and CD40, compared to dendritic cells prepared without performing cluster control culture. [9] The method of any one of [1] to [8], wherein the obtained dendritic cells have an increased production of IFN-γ, compared to DCs prepared without performing cluster control culture.

[10] The method of any one of [1] to [9], wherein the obtained dendritic cells have an increased expression level of the BCL2A1 gene, compared to DCs prepared without performing cluster control culture.

[11] The method of preparing dendritic cells from any one of [1] to

[10] monocytes, wherein the obtained dendritic cells have a longer lifespan under 4°C and physiological saline suspension conditions, compared to DCs prepared without performing cluster control culture.

[12] Dendritic cells obtained by the method of preparing dendritic cells from any one of [1] to

[11] monocytes.

[13] A pharmaceutical composition containing the dendritic cells of

[12] .

[14] The pharmaceutical composition of

[13] , which has anti-cancer immune activity and can be used for cancer treatment.

[15] A method for predicting the lifespan of dendritic cells using the gene expression level of BCL2A1 in dendritic cells as an index, wherein the higher the gene expression level of BCL2A1, the longer the predicted lifespan. [Effect of the Invention]

[0012] By the method for preparing dendritic cells (DCs) of the present invention, DCs with high antigen-presenting function capable of inducing cytotoxic T cells can be obtained in a high yield in a short period of time. Further, the DCs obtained by the method for preparing dendritic cells (DCs) of the present invention have a high degree of maturity and a long lifespan, and thus can be suitably used for cancer immunotherapy.

Brief Description of the Drawings

[0013]

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Modes for Carrying Out the Invention

[0014] The present invention will be described in detail below. In this specification, "A to B" (where A and B are numerical values) shall represent "A or more and B or less" unless otherwise specified. In this specification, "%" shall represent "v / v%" unless otherwise specified.

[0015] The present invention is a method for preparing dendritic cells (DC) from monocytes. Mononuclear cells are white blood cells, and mononuclear cells are divided into monocytes and lymphocytes. Mononuclear cells include peripheral blood-derived mononuclear cells (PBMC), bone marrow-derived mononuclear cells, spleen cell-derived mononuclear cells, and umbilical cord blood-derived mononuclear cells. Among these, peripheral blood-derived mononuclear cells are preferred. Mononuclear cells can also be separated using an apheresis device. Fresh unfrozen mononuclear cells or frozen mononuclear cells may be used. Even when frozen mononuclear cells are used, the antigen-presenting ability and phagocytic and degradative abilities of the finally obtained dendritic cells do not decrease.

[0016] 1. Separation of monocytes from mononuclear cells Monocytes include monocytes derived from peripheral blood, bone marrow, spleen cells, and umbilical cord blood, among which monocytes derived from peripheral blood are preferred. Monocytes are characterized by CD14 positivity. When collecting monocytes from a living body, they can be separated using FACS (Fluorescent activated cell sorter), a flow cytometer, a magnetic separation device, etc. using the presence of CD14 as an indicator. They can also be separated using a component collection (apheresis) device. Furthermore, they can be separated by density gradient centrifugation using Ficoll (registered trademark), etc. The animal species from which monocytes are derived is not limited, and mammals such as mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans can be used. As FACS and flow cytometers, for example, FACS vantage (manufactured by Becton Dickinson), FACS Calibur (manufactured by Becton Dickinson), etc. can be used. As a magnetic separation device, for example, autoMACS (registered trademark) (Miltenyi Biotec), etc. can be used. For example, from peripheral blood mononuclear cells (PBMC), using CD14 microbeads conjugated with CD14 as an indicator, they can be isolated using AutoMACS (registered trademark) and CliniMACS (registered trademark) technologies.

[0017] In addition, since monocytes have the property of easily adhering to a culture vessel, mononuclear cells can be seeded on an adherent culture dish, cultured, and monocytes can be adhered to the culture dish to separate them from lymphocytes. At this time, as the culture medium, either a serum-supplemented medium or a serum-free medium can be used. An albumin-supplemented medium may also be used. The medium is not limited, and any medium that can be used for culturing human lymphoid cells can be used. For example, AIM-V (registered trademark, Thermo Fisher Scientific), X-VIVO5 (registered trademark), HL-1 (trademark, Lonza), BIOTARGET (trademark)-1 SFM (Cosmo Bio Co., Ltd.), DCO-K (Nissui Pharmaceutical Co., Ltd.), DMEM, MEM, RPMI1640, IMDM, etc. can be used.

[0018] Since monocytes have the property of strongly adhering to a container, monocytes can be cultured by adherent culture. Monocytes are adhered to a culture container such as a culture dish, petri dish, plate, or flask, and non-adherent cells are removed to separate and recover them. A culture container for adherent cells to which cells can adhere may be used. As the culture container for adherent cells, commercially available ones can be widely used. As the culture container for adherent cells, a low-adhesion culture container or a high-adhesion culture container may be used.

[0019] The pH during culture for the separation of monocytes is preferably about 6 to 8. The culture is usually carried out at about 30 to 40 °C for 15 minutes to 48 hours, more preferably 15 minutes to 36 hours, more preferably 15 minutes to 24 hours, more preferably 10 hours to 24 hours, more preferably 12 hours to 36 hours, more preferably 12 hours to 24 hours, more preferably 16 hours to 24 hours, and particularly preferably 18 hours to 24 hours. During the culture, medium replacement, aeration, and stirring may be added as necessary. For example, carbon dioxide gas may be added, and the carbon dioxide gas may be added at 2.5 to 10%, preferably 2.5 to 7.5%, and more preferably 5%.

[0020] After adherent culture, non-adherent cells are removed by washing, and monocytes can be separated by adherent culture. At this time, the washing is performed 1 to 5 times, preferably 2 times.

[0021] 2. Method for preparing dendritic cells (DC) from monocytes Dendritic cells can be prepared using the monocytes separated by the method for separating monocytes from the above-mentioned mononuclear cells.

[0022] The separated monocytes are first induced to differentiate into DC. Immature DC can be obtained by inducing differentiation into DC. Next, the immature DC are cultured and matured in the presence of a specific cytokine to obtain DC with a high antigen-presenting function and capable of inducing cytotoxic T cells.

[0023] To induce differentiation into DCs, isolated monocytes may be cultured in the presence of cytokines such as GM-CSF (granulocyte-macrophage colony-stimulating factor), which has DC-inducing activity, and IL-4. At this time, other cytokines having DC-inducing activity may also be used. Examples of other cytokines having DC-inducing activity include cytokines such as IL-2 (interleukin 2), IL-3 (interleukin 3), IL-6 (interleukin 6), IL-7 (interleukin 7), IL-12 (interleukin 12), IL-13 (interleukin 13), IL-15 (interleukin 15), TNF (tumor necrosis factor)-α, HGF (hepatocyte growth factor), TGF (transforming growth factor)-β, CD40 ligand, c-kit ligand, IFNα (interferon α), flt3 ligand, etc.

[0024] The concentration of IL-4 or other cytokines having DC-inducing activity used for culturing to induce differentiation of DCs is 1 ng / mL to 1,000 ng / mL, preferably 10 ng / mL to 100 ng / mL.

[0025] The concentration of GM-CSF used for culturing to induce differentiation is 100 U / ml to 10,000 U / mL, preferably 500 U / mL to 2,000 U / mL, more preferably 800 U / ml to 1,200 U / mL, and particularly preferably 1,000 U / mL. Alternatively, it is 10 ng / mL to 1,000 ng / mL, preferably 20 ng / mL to 200 ng / mL, more preferably 20 ng / mL to 100 ng / mL.

[0026] By examining the expression of surface antigens of monocytes or DCs using FACS or the like, the concentration at which cells of the desired degree of differentiation can be obtained can be appropriately determined.

[0027] The culture for inducing differentiation in the presence of GM-CSF and IL-4 is carried out for 2 to 10 days, preferably 3 to 8 days, more preferably 4 to 6 days, and particularly preferably 5 days. Immature DCs can be obtained by culturing in the presence of GM-CSF and IL-4.

[0028] In the present invention, when immature DCs are matured after being induced to differentiate from monocytes, monocytes are cultured by cluster control culture. Here, "cluster control culture" refers to a culture performed in a state where spheroids are formed in cells during culture. A spheroid refers to a spherical cell aggregate (cluster) in which cells aggregate and agglomerate to form a 3D three-dimensional structure. "Cluster control culture" is also referred to as "spheroid culture". "Cluster control culture" may be performed under cell culture conditions that enable the formation of three-dimensional cell spheroids even when the cell population is in a floating state. Cluster control culture is sometimes referred to as culture using a cluster control culture vessel (culture dish), or simply as cluster control even when the culture is controlled.

[0029] Cluster control culture (spheroid culture) can be performed, for example, by the following method described in Koshiro Kusamori et al., Drug Delivery System 28-1, 2013, PP.45-53. (1) Culture using a culture plate with a non-adhesive bottom When using such a plate, spheroids are formed by the adhesion of cells floating in the culture medium to each other. (2) Hanging drop cell culture Cells are suspended in the culture medium and a small amount is dropped inside the lid of the culture plate, and the droplet is cultured upside down. As a result, the cells gather at the bottom of the droplet and adhere to each other to form spheroids. (3) Culture using a microwell fabricated using micromolding technology By culturing in U-shaped or V-shaped wells, cells adhere to each other in the microwell to form spheroids. A container having such a microwell is called a spheroid formation culture vessel (culture dish) or a cluster control culture vessel (culture dish). (4) Rotary cell culture method Spheroids are formed by rotating a cell culture vessel containing the culture medium.

[0030] In the present invention, any cluster control culture including the above method can be employed, but preferably it is carried out using a spheroid formation culture vessel (culture dish) or a cluster control culture vessel (culture dish).

[0031] Examples of the spheroid formation culture vessel (culture dish) or the cluster control culture vessel (culture dish) include EZSPHERE (registered trademark) microplate / plate / dish (AGC Techno Glass Co., Ltd.), PAMCELL (3D Cell Spheroid Culture Plate) (Funakoshi Co., Ltd.), Corning (registered trademark) spheroid microplate (Corning), SPHERICALPLAE 5D (Mito Kogyo Co., Ltd.), Cell-able (registered trademark) plate (Toyobo Co., Ltd.), NanoCultute Plase / Dish (Medical Biology Institute Co., Ltd.), etc. Among these, a culture vessel (culture dish) having a well with a pore diameter of 1,000 μm or less and a depth of 500 μm or less, having no flat portion in the well, and having a protein low-adhesion coat on the inner surface of the well is preferred. As such a culture vessel (culture dish), EZSPHERE (registered trademark) microplate 24well (AGC Techno Glass Co., Ltd.) can be mentioned. The EZSPHERE (registered trademark) microplate 24well has about 470 wells (spheroid wells) with a diameter of about 400 to 500 μm and a depth of 150 to 200 μm uniformly processed up to the wall surface without gaps on the culture surface in 24 wells. Also, the surface is coated with a protein low-adhesion coat, and the seeded cells form spheroids (clusters) uniformly in the well. Since the well shape is a mortar type structure, the cell sphericity is uniform and cell recovery is easy. Since there is no flat portion, all the seeded cells fall into the well and form spheroids (clusters) without losing cells.

[0032] The maturation of immature DCs is achieved by culturing the immature DCs in a maturation medium. As the maturation medium, a serum-free medium containing at least prostaglandin E2 (PGE2) and OK432 is used. The maturation medium may further contain IL-4. As the serum-free medium, the serum-free medium described in the method for separating monocytes from the above-mentioned monocytes can be used.

[0033] The density of immature DCs during maturation is 1.1×10 6 cells / ml to 1.0×10 7 cells / ml, preferably 1.5×10 6 cells / ml to 5.0×10 6 cells / ml, more preferably 1.5×10 6 cells / ml to 2.5×10 6 cells / ml, more preferably 1.8×10 6 cells / ml to 2.2×10 6 cells / ml, particularly preferably 2.0×10 6 cells / ml.

[0034] The concentration of PG2E used for culturing is 1 ng / mL to 100 ng / mL, preferably 5 ng / mL to 50 ng / mL, more preferably 5 ng / mL to 20 ng / mL. The concentration of OK432 is 1 μg / mL to 100 μg / mL, preferably 5 μg / mL to 50 μg / mL, more preferably 5 μg / mL to 20 μg / mL. When adding IL-4, the concentration of IL-4 is 1 ng / mL to 1000 ng / mL, preferably 10 ng / mL to 100 ng / mL.

[0035] By examining the expression of the surface antigen of DCs by FACS or the like, the concentration at which cells of the desired degree of maturity can be obtained can be appropriately determined.

[0036] Culturing with the maturation medium for 10 to 48 hours, preferably 10 to 36 hours, more preferably 10 to 24 hours, particularly preferably 18 to 24 hours, can obtain DCs with high antigen-presenting function and capable of inducing cytotoxic T cells.

[0037] The total culture period for separating monocytes from mononuclear cells and further maturing them is 3 to 10 days, preferably 3 to 7 days, more preferably 5 to 8 days, still more preferably 5 to 7 days, and most preferably 4 days.

[0038] By cluster control culture, the spheroids formed in the wells contain about 3,000 monocytes or less, and the diameter of the spheroids is about 10 to 1,000 μm.

[0039] 3. Characteristics of the obtained DCs (1) Viable cell ratio and yield In the method of the present invention, the viable cell ratio of the obtained mature DCs is high, and the yield is also high. The viable cell ratio of the obtained DCs is 70% or more, preferably 80% or more, more preferably 90% or more, according to the criteria of the NIH (National Institutes of Health). Also, the recovery rate of DCs (the ratio of the number of obtained DC viable cells to the number of seeded monocytes) is 5% or more, preferably 10% or more, more preferably 15% or more. Furthermore, the purity of DCs is 90% or more, preferably 95% or more.

[0040] (2) Surface antigens In DCs prepared by cluster control culture, CD80, CD86, CD83, CD40, CCR7, HLA-ABC, HLA-DR, CD14, and CD11c are positive. CD14 is a marker for monocytes, CD197 (CCR7) is a molecule that promotes migration to lymph nodes, and CD11c is a dendritic cell marker. Also, CD80 and CD40 are co-stimulatory molecules involved in the antigen-presenting ability to T cells, CD83 is a maturation marker for dendritic cells, and HLA-DR is a molecule involved in antigen presentation.

[0041] Whether these surface antigens are positive or negative can be determined by microscopic observation or the like as to whether cells are stained with an antibody against these antigens labeled with a chromogenic enzyme, a fluorescent compound, or the like. For example, cells may be immunostained with these antibodies to determine the presence or absence of surface antigens. It can also be determined using magnetic beads conjugated with the antibody. Further, it can also be determined whether there is a surface antigen using FACS or a flow cytometer. That the surface antigen is negative means that when analyzed using FACS as described above, it is not sorted as a positive cell, and when the expression is examined by immunostaining, no expression is observed. Even if it is expressed to such an extent that it cannot be detected by these methods, it is judged to be negative.

[0042] In the DCs prepared by cluster control culture, the positive rates of CD80 and CD40 increase compared to the DCs prepared without cluster control culture, and furthermore, the expression intensities (ΔMFI) of CD80, CD86 and CD40 increase. The positive rate of CD80 is 70% or less in the DCs prepared without cluster control culture, while it is 75% or more in the DCs prepared by cluster control culture. The ratio of the positive rate of CD80 in the DCs prepared by cluster control culture to the positive rate of CD80 in the DCs prepared without cluster control culture is 1.05 or more, preferably 1.1 or more, more preferably 1.15 or more. Regarding the expression intensity, the ratio of the expression intensity of CD80 in the DCs prepared by cluster control culture to the expression intensity of CD80 in the DCs prepared without cluster control culture is 1.1 or more, preferably 1.15 or more. The ratio of the expression intensity of CD86 in the DCs prepared by cluster control culture to the expression intensity of CD86 in the DCs prepared without cluster control culture is 1.2 or more, preferably 1.3 or more, more preferably 1.4 or more. The ratio of the expression intensity of CD40 in the DCs prepared by cluster control culture to the expression intensity of CD40 in the DCs prepared without cluster control culture is 1.05 or more. Here, the DCs prepared without cluster control culture are also the DCs prepared by inducing the differentiation of DCs by combining conventional GM-CSF and IL-4, and are also the DCs prepared by the method described in Akagawa K.S. et al., Blood, Vol.88, No.10 (November 15), 1996: pp.4029-4039. The phenotype indicates that the maturity of the DCs prepared by cluster control culture is improved compared to the DCs prepared without cluster control culture.

[0043] (3) Cytokine production ability The production amounts of the following cytokines are as follows when DCs are at 2×10 6It is the value measured by Bio-plex assay kit (Bio-Rad Labs) for cytokines in the collected culture supernatant after suspending in AIM-V medium to a cell density of cells / ml, seeding in a culture dish, culturing at 37°C and 5% CO2 for 18 to 24 hours, and then collecting the culture supernatant.

[0044] In DCs prepared by cluster control culture, the production of IFN-γ, which is one of the cytokines that enhance the induction of cytotoxic T cells, is significantly increased compared to DCs prepared without cluster control culture. The ratio of the IFN-γ production level in DCs prepared by cluster control culture to that in DCs prepared without cluster control culture is 1.1 or more, preferably 1.2 or more, more preferably 1.25 or more.

[0045] (4) Gene expression In DCs prepared by cluster control culture, the expression of BCL2A1, which is one of the anti-apoptosis genes, is increased compared to DCs prepared without cluster control culture. When compared by the fold change relative to the control, in DCs prepared by cluster control culture, the expression level of BCL2A1, which is one of the anti-apoptosis genes, increases by 1.2-fold or more, preferably 1.3-fold or more, compared to DCs prepared without cluster control culture. Also, the mRNA expression quotient of BCL2A1 increases by 1.5-fold or more, preferably 1.7-fold or more, more preferably 1.8-fold or more, even more preferably 1.9-fold or more.

[0046] (5) Survival rate of the obtained DCs In the case of DCs prepared by cluster control culture, the rate of decrease in viable cell ratio over time is lower under storage conditions (4°C, suspended in physiological saline) compared to DCs prepared without cluster control culture. For example, when DCs prepared without cluster control culture are stored under the above conditions for 6 hours, the viable cell ratio is less than 70%, whereas for DCs prepared by cluster control culture, the viable cell ratio is 70% or more. The ratio of the viable cell ratio of DCs prepared by cluster control culture stored under the above conditions for 6 hours to the viable cell ratio of DCs prepared without cluster control culture stored under the above conditions for 6 hours is 1.1 or more. When DCs prepared without cluster control culture are stored under the above conditions for 12 hours, the viable cell ratio is less than 53%, whereas for DCs prepared by cluster control culture, the viable cell ratio is 55% or more. The ratio of the viable cell ratio of DCs prepared by cluster control culture stored under the above conditions for 12 hours to the viable cell ratio of DCs prepared without cluster control culture stored under the above conditions for 12 hours is 1.1 or more. When DCs prepared without cluster control culture are stored under the above conditions for 24 hours, the viable cell ratio is less than 35%, whereas for DCs prepared by cluster control culture, the viable cell ratio is 36% or more. The ratio of the viable cell ratio of DCs prepared by cluster control culture stored under the above conditions for 24 hours to the viable cell ratio of DCs prepared without cluster control culture stored under the above conditions for 24 hours is 1.1 or more. When DCs prepared without cluster control culture are stored under the above conditions for 48 hours, the viable cell ratio is less than 19%, whereas for DCs prepared by cluster control culture, the viable cell ratio is 19% or more. The ratio of the viable cell ratio of DCs prepared by cluster control culture stored under the above conditions for 48 hours to the viable cell ratio of DCs prepared without cluster control culture stored under the above conditions for 48 hours is 1.1 or more.

[0047] This means that the obtained DCs have a long lifespan. Therefore, the lifespan of the obtained DCs is extended. This means that the performance of the DCs after preparation is less likely to decline, leading to an improvement in the quality of DC vaccines.

[0048] Note that the viable cell rate can be predicted to be improved by the anti-apoptotic effect due to the increased gene expression of BCL2A1. Therefore, the gene expression level of BCL2A1 can be a biomarker for ensuring the quality of IL-4-DC prepared by the method of the present invention. The present invention can measure the gene expression level of BCL2A1 in the prepared DC, and using the expression level as an index, it is possible to examine the resistance to the decrease in the viable cell rate of DC, that is, the ability to maintain viable DC cells. When the viable DC cells are maintained, it indicates that the quality of DC during storage or transportation is maintained and is less likely to decrease. The present invention also includes a method for predicting whether the quality of DC is maintained.

[0049] 4. Dendritic cell therapy The DC prepared by the method of the present invention can be used for dendritic cell therapy. Examples of dendritic cell therapy include cancer immunotherapy known as dendritic cell vaccine therapy. For example, dendritic cells can be prepared from the monocytes of a subject by the method of the present invention, and the obtained dendritic cells can be returned to the subject for use in cancer treatment or prevention, etc. At this time, the prepared dendritic cells act non-specifically for cancer types and can exhibit a cancer treatment effect. Further, when culturing dendritic cells, it is possible to obtain dendritic cells having cancer-specific anti-cancer immune activity by adding a cancer-specific antigen specific to a specific cancer such as WT1 peptide and culturing. It is preferable to add a cancer-specific antigen specific to a specific cancer such as WT1 peptide during the maturation of dendritic cells. The dendritic cells can induce cancer type-specific cytotoxic T cells. It can also be used for the treatment of bacterial and viral infections. In the treatment of infections, DC prepared by culturing monocytes by non-adherent culture in the presence of IL-4, GM-CSF, PGE2 and OK432 by the method of the present invention is useful. The prepared dendritic cells may be administered to the subject by intradermal administration, subcutaneous administration, intravenous administration or intranodal administration, etc. The dosage and administration time can be appropriately determined according to the type of disease of the subject, the severity of the disease and the state of the subject.

Examples

[0050] The present invention will be specifically described by the following examples, but the present invention is not limited by these examples.

[0051] [Example 1] Comparison of IL-4DCs Prepared Using Adherent Culture Dishes or Low-Adhesion Culture Dishes During the Maturation Process This example was conducted as preliminary test (1). In this example, the comparison of cell morphology and phenotype of IL-4DCs prepared by the method of using adherent culture dishes (adherent method) or the method of using low-adhesion culture dishes (low-adhesion method) during the maturation process was verified.

[0052] Method Protocol (Adherent Method) Peripheral blood mononuclear cells (PBMCs) derived from patients collected by apheresis were seeded on an adherent culture dish (Corning Primaria™ 100mm Standard Cell Culture Dish) using AIM-V medium. By culturing for 18 to 24 hours under the conditions of 37°C and 5% CO2, the cells were adhered to the bottom surface of the culture dish to select monocytes and lymphocytes.

[0053] Subsequently, differentiation induction into IL-4-DC was performed using AIM-V medium supplemented with 50 ng / ml recombinant human (rh) IL-4 and 100 ng / ml GM-CSF for the adherent cells. Five days after the start of differentiation, the cells were collected and cultured for 18 to 24 hours using AIM-V medium supplemented with a maturation medium in which various reagents (5 ng / ml rhIL-4, 10 μg / ml OK432, 10 ng / ml PGE2) were mixed on an adherent culture dish (Corning Primaria™ 100mm Standard Cell Culture Dish) to mature IL-4-DC, and then collected and the cell morphology and phenotype were evaluated. The evaluation of the phenotype was performed by detecting cell surface antigens (CD11c, CD14, CD40, CD80, CD83, CD86, HLA-ABC, HLA-DR, CCR7, PD-L1, and PD-L2) expressed on DCs by flow cytometry using labeled antibodies.

[0054] Protocol (low adhesion method) Peripheral blood mononuclear cells (PBMCs) derived from a patient, collected by apheresis, were seeded onto an adherent culture dish (Corning Primaria (trademark) 100 mm Standard Cell Culture Dish) using AIM-V medium. By culturing for 24 hours under the conditions of 37 °C and 5% CO2, the cells were adhered to the bottom surface of the culture dish, and monocytes and lymphocytes were sorted.

[0055] Subsequently, induction of differentiation into IL-4-DCs was performed using AIM-V medium supplemented with 50 μg / ml recombinant human IL-4 and 100 ng / ml GM-CSF for the adherent cells. Five days after the start of differentiation, the cells were collected and cultured for 18 - 24 hours using a maturation medium in which various reagents (10 μg / ml OK432, 10 ng / ml PGE2) were mixed and an AIM-V medium supplemented with 20 μg / ml tumor antigen peptide (WT-1: Wilms tumor1) in a low adhesion culture dish (Prime Surface (registered trademark) Petri dish 90, Sumitomo Bakelite) to mature the IL-4-DCs, and then the cells were collected and the cell morphology and phenotype were evaluated.

[0056] Results Comparison of cell morphology Recovery of IL-4-DCs was easier in the culture using a low adhesion culture dish that did not require scraping than in the culture using an adherent culture dish.

[0057] Figure 1 shows the cell morphologies of IL-4-DCs prepared by the adhesion method and IL-4-DCs prepared by the low adhesion method. Images #1 to #3 are of IL-4-DCs prepared by the adhesion method (IL-4-DC / adhesion method), and images #4 - #6 are of IL-4-DCs prepared by the low adhesion method (IL-4-DC / low adhesion method). In the IL-4-DC / adhesion method, adherent cells were observed in the culture dish. In the IL-4-DC / low adhesion method, prominent cluster formation in suspension was observed, and no adherent cells were observed.

[0058] Phenotypic comparison Figure 2-1, Figure 2-2, and Figure 2-3 show the positive rates (%) of CD11c, CD14, CD40, and CD80 (Figure 2-1), CD83, CD86, HLA-ABC, and HLA-DR (Figure 2-2), and CCR7, PD-L1, and PD-L2 (Figure 2-3), respectively. IL-4-DCs prepared by the adherent method had N = 26, and those prepared by the low-adherent method had N = 11. Similar to the IL-4-DCs prepared by the adherent method, the IL-4-DCs prepared by the low-adherent method met the minimum quality standard (CD86+ / HLA-DR+), but showed low maturity (increase in CD14 positive rate and decrease in CD83, CCR7, and PD-L2 positive rates).

[0059] Summary From the results of Example 1, the significant cluster formation observed in the low-adherent method may affect the maturation of dendritic cells.

[0060] [Example 2] Preparation of IL-4-DC by varying cell seeding density during the maturation process This example was conducted as a preliminary test (2) to verify the maturation of dendritic cells under conditions where the seeding cell density and the number of cells were reduced to suppress cluster formation during maturation.

[0061] Method First, in the process of preparing dendritic cell vaccines, focusing on the cluster formation when using low-adherent culture dishes during the maturation process, the effect of cluster formation due to cell seeding density on the phenotype was evaluated. The protocol is shown in Figure 3.

[0062] Protocol Peripheral blood mononuclear cells (PBMCs) from patients collected by apheresis were seeded onto adherent culture dishes using AIM-V medium. By culturing for 24 hours under the conditions of 37°C and 5% CO2, the cells were adhered to the bottom of the culture dish, and monocytes and lymphocytes were sorted.

[0063] Subsequently, differentiation induction into IL-4-DCs was performed using AIM-V medium supplemented with 50 μg / ml recombinant human IL-4 and 100 ng / ml GM-CSF for adherent cells. Five days after the start of differentiation, the cells were collected and cultured for 18 - 24 hours using AIM-V medium supplemented with mature medium mixed with various reagents (10 μg / ml OK432, 10 ng / ml PGE2) and 20 mixed mature tumor antigen peptides (WT-1: Wilms tumor1) at low and high cell seeding densities in a low-adhesion culture dish to mature the IL-4-DCs. The low density was 1×10 6 cells / ml and the medium volume was 6 ml. Also, the high density was 2×10 6 cells / ml and the medium volume was 10 ml.

[0064] Results During the process of generating IL-4-DCs, the formation of clusters 24 hours after seeding at each cell density (high density: high and low density: low) at maturation was observed by phase-contrast microscopy. The results are shown in Figure 4. Marked cluster formation was observed when the cell seeding density was high.

[0065] Furthermore, the cells were collected and their phenotypes were compared. The results are shown in Figures 5-1 and 5-2. Figure 5-1 shows the positive rates of CD80, CD86, CD83, CD40, CCR7, HLA-ABC, HLA-DR, CD14, and CD11c, and Figure 5-2 shows the expression intensity (ΔMFI) of each surface antigen. "High" and "low" indicate the cell seeding density at maturation. CD80, CD83, and CD40 were increased in expression at low density compared to high density in terms of cell seeding density (N = 4). In terms of the expression intensity (ΔMFI), CD80, CD86, CD83, CD40, CCR7, HLA-ABC, and HLA-DR were increased.

[0066] Summary When compared with IL-4-DCs prepared by culturing using a culture dish, IL-4-DCs prepared by culturing using a low-adhesion culture dish were more likely to form clusters, met the minimum quality standards (CD86+, HLA-DR+) as a DC vaccine in terms of phenotype, but showed a tendency of low maturity (from Example 1).

[0067] When using a low-adhesion culture dish during the maturation process of IL-4-DC, formation of large clusters was observed when seeding cells at a high density (N = 4).

[0068] During the maturation process, an increase in the expression of dendritic cell maturation markers (CD40, CD80, CD83) was observed in IL-4-DCs prepared by seeding at a low density (N = 4). Furthermore, in terms of the intensity of expression (ΔMFI), CD80, CD86, CD83, CD40, CCR7, HLA-ABC, and HLA-DR increased.

[0069] Therefore, it was found that cluster formation during the maturation process affects the quality of dendritic cell vaccines.

[0070] [Example 3] Preparation of dendritic cells by cluster-controlled culture During the maturation process when preparing IL-4-DC, the quality improvement of IL-4DC and the validity of the preparation method were examined when performing cluster control by a culture dish and culturing with an optimal cell density.

[0071] Example 3 was conducted as this test to examine the effects of cluster control using culture dishes on the viable cell rate, yield, and phenotype of IL-4-DCs. That is, a group using a low-adhesion culture dish (PrimeSurface (registered trademark) dish plate 24F, Sumitomo Bakelite) in which cluster formation due to fluctuations in cell seeding density affects the quality of dendritic cells (IL-4-DC) was used as a control, and IL-4-DCs (cluster-controlled IL-4-DCs) prepared using a cluster control culture dish (EZSPHERE (registered trademark) microplate 24 well, AGC Techno Glass Co., Ltd.) to strictly and uniformly control clusters were compared (N = 10). The EZSPHERE (registered trademark) microplate 24 well used had approximately 470 wells with a diameter of about 400 to 500 μm and a depth of 150 to 200 μm (spheroid wells) uniformly processed on the culture surface up to the wall surface without gaps in 24 wells. In addition, the surface was coated with a protein low-adhesion coating, and the seeded cells formed spheroids (clusters) uniformly within the wells. Since the well shape is a mortar-like structure, the cell spheroids are uniform and cell recovery is easy. Since there is no flat part, all seeded cells fall into the wells and form spheroids (clusters) without losing cells.

[0072] The evaluation items were as follows. (i) Phase-contrast microscopy findings (observation of cell morphology) (Example 3) (ii) Comparison of viable cell rate and yield between IL-4-DC and cluster-controlled IL-4-DC (Example 3) (iii) Comparison of 3D images of clusters by phase-contrast microscopy (Example 3) (iv) Phenotype comparison by flow cytometry (FCM) (Example 3) (v) Comparison of cytokine production ability (Example 4) (vi) Comprehensive gene expression analysis by microarray (Example 5) (vii) mRNA expression analysis of apoptosis and anti-apoptosis related genes (Example 5) (viii) Comparison of viable cell ratios over time under DC vaccine storage conditions (4°C, suspended in physiological saline) (Example 6)

[0073] Method Protocol Peripheral blood mononuclear cells (PBMC) derived from patients collected by apheresis were seeded onto an adherent culture dish (Corning Primaria™ 60mm Standard Cell Culture Dish) using AIM-V medium. The cells were cultured for 18 - 24 hours under the conditions of 37°C and 5% CO2 to adhere the cells to the bottom of the culture dish and to select monocytes and lymphocytes.

[0074] Subsequently, IL-4-DC differentiation induction was performed using AIM-V medium supplemented with 50 ng / ml rhIL-4 and 100 ng / ml GM-CSF for the adherent cells. Five days after the start of differentiation, the cells were collected and various reagents (10 μg / ml OK432, 10 ng / ml PGE2) were mixed with the maturation medium and AIM-V medium supplemented with 20 μg / ml tumor antigen peptide (WT1) were used in a cluster control culture dish (EZSPHERE® microplate 24 well) or a low-adhesion culture dish (PrimeSurface® dish plate 24F, Sumitomo Bakelite). IL-4-DC was matured by culturing at a cell density of 2×10 6 cells / ml for 18 - 24 hours. Figure 6A shows the protocol using a low-adhesion culture dish (PrimeSurface® dish plate 24F, Sumitomo Bakelite), and Figure 6B shows the protocol using a cluster control culture dish (EZSPHERE® microplate 24 well).

[0075] Subsequently, the viable cell rate and yield were measured, and further, observation of cell morphology was performed using a phase-contrast microscope. In addition, the comparison of phenotypes by IL-4-DCs prepared by performing cluster control by culturing using an IL-4-DC and a cluster control culture dish (EZSPHERE (registered trademark) microplate 24 well) was evaluated by flow cytometry (FACS).

[0076] Note that the IL-4-DC prepared by performing cluster control by culturing using a cluster control culture dish (EZSPHERE (registered trademark) microplate 24 well) is referred to as "cluster control IL-4-DC".

[0077] Results (1) Comparison of viable cells and yield Figure 7 shows microscopic images of cells in wells of cultures using a low-adhesion culture dish (PrimeSurface (registered trademark) dish) and cultures using a cluster control culture dish (EZSPHERE (registered trademark)). Figure 8 shows the area of cell clusters (μm 2 )(A), viable cell rate (%) (B), and yield (%) (C). The yield was calculated by the formula "number of seeded mononuclear cells ÷ viable cell rate".

[0078] The area of cell clusters was analyzed from the microscopic images of each group. As shown in Figure 8A, in the culture using the cluster control culture dish, a uniform cell area was shown compared to the culture using the low-adhesion culture dish (N = 3).

[0079] When comparing the viable cell rate and yield of IL-4-DCs prepared using a low-adhesion culture dish and cluster control IL-4-DCs, as shown in Figures 8B and C, no significant difference in the median value was observed (N = 10).

[0080] (2) Observation of cell morphology After culturing for 24 hours using a low-adhesion culture dish and a cluster control culture dish at maturity, 3D cell observation was performed on the prepared IL-4-DCs using an all-in-one fluorescence microscope (BZ-X800). The cell observation images are shown in Fig. 9. Fig. 9A shows the prepared IL-4-DC (IL-4-DC) using a low-adhesion culture dish, and Fig. 9B shows the IL-4-DC prepared using a cluster control culture dish (cluster control IL-4-DC). Fig. 10 shows the thickness in the XZ direction (A) and the thickness in the YZ direction (B) of the cluster. As shown in Fig. 10, it was confirmed that the thickness of the cluster was thinner in the cluster control culture dish.

[0081] (3) Phenotype The phenotypes of IL-4-DCs prepared using a low-adhesion culture dish and a cluster control culture dish during the maturation process were evaluated by FACS (N = 10). The results are shown in Figs. 11-1 and 11-2. Fig. 11-1 shows the positive rates of CD80, CD86, CD83, CD40, CCR7, HLA-ABC, HLA-DR, CD14, and CD11c, and Fig. 11-2 shows the intensity of expression (ΔMFI) of each surface antigen. "-" indicates the results of IL-4-DCs prepared using a low-adhesion culture dish, that is, IL-4-DCs prepared without cluster control, and "+" indicates the results of IL-4-DCs prepared using a cluster control culture dish (cluster control IL-4-DC), that is, IL-4-DCs prepared with cluster control. As shown in Figs. 11-1 and 11-2, the expressions of CD40 and CD80 were significantly increased in cluster control IL-4-DCs (N = 10). The intensities of the expressions of CD40, CD80, and CD86 were significantly increased in cluster control IL-4-DCs (N = 10)

[0082] Summary When cluster formation was strictly controlled during the maturation process using a cluster control culture dish, a significant increase in the number of positive cells for CD80 and CD40, which are dendritic cell maturation markers, was observed (N = 10).

[0083] When compared with the low-adhesion culture dish, when the cluster was strictly controlled during the maturation process using the cluster-controlled culture dish, the expression levels of CD80, CD86, and CD40, which are dendritic cell maturation markers, were significantly increased (N = 10).

[0084] Therefore, it was shown that strict cluster control by the culture dish significantly improved the maturity of dendritic cells during the maturation process.

[0085] [Example 4] Cytokine production performance comparison Example 4 was conducted as this test. Cytokines (IL-10, IFN-γ, TNF-α, IL-12, IL-6) involved in the induction of cytotoxic T cells secreted from cluster-controlled IL-4-DC were measured using a Bio-plex assay kit (Bio-Rad Labs) (N = 9).

[0086] Method The experimental protocol is shown in Fig. 12. Fig. 12A shows the protocol for evaluating the cytokine levels of IL-4-DC prepared using a low-adhesion culture dish, and Fig. 12B shows the protocol for evaluating the cytokine levels of IL-4-DC prepared using a cluster-controlled culture dish. The cytokines evaluated were IFN-γ, IL-12 (p70), IL-10, IL-6, and TNF-α.

[0087] Results The results of the cytokine evaluation are shown in Fig. 13. "-" indicates the results of IL-4-DC prepared using a low-adhesion culture dish, that is, IL-4-DC prepared without cluster control, and "+" indicates the results of IL-4-DC prepared using a cluster-controlled culture dish (cluster-controlled IL-4-DC), that is, IL-4-DC prepared with cluster control.

[0088] When the cluster was strictly controlled during the maturation process using the cluster-controlled culture dish, the production of IFN-γ, which is one of the cytokines that enhance the induction of cytotoxic T cells, was significantly increased (N = 9).

[0089] Summary During the maturation process, strict cluster control using culture dishes was found to amplify the cytokine-producing ability of dendritic cells, particularly IFN-γ.

[0090] [Example 5] Influence of Cluster Control by Culture Dishes on Gene Expression of IL-4-DC Example 5 was conducted as this test. In Example 5, by performing cluster control, it was evaluated by comprehensive gene expression analysis using a microarray whether the genes related above were fluctuating.

[0091] From the results of Examples 3 and 4, it was found that cluster control during the maturation process affects the phenotype and cytokine-producing ability of IL-4-DC. Next, in the preparation of IL-4-DC, the changes in gene expression caused by cluster control were comprehensively analyzed using a microarray. Also, focusing on apoptosis-promoting and anti-apoptosis-related genes, the mRNA expression was evaluated by real-time PCR.

[0092] Method IL-4-DC was prepared from PBMC collected from three patients using a low-adhesion culture dish, and cluster-controlled IL-4-DC was prepared using a cluster control culture dish, and comprehensive gene expression analysis was performed. Also, from the results of the comprehensive gene expression analysis, apoptosis-promoting and anti-apoptosis-related genes were focused on, and the mRNA expression was evaluated by real-time PCR (N = 4).

[0093] Figure 14 shows the experimental protocol. Figure 14A shows the experimental protocol using IL-4-DC prepared using a low-adhesion culture dish, and Figure 14B shows the experimental protocol using IL-4-DC prepared using a cluster control culture dish.

[0094] Results Figure 15 shows the results of a comprehensive gene expression analysis of IL-4-DCs. It shows the expression of survival-promoting genes (BCL2, BCL2L1, BCL2L2, MCL1, and BCL2A1) and apoptosis-promoting genes (BAX, BOK, BAK1). "-" indicates the results of IL-4-DCs prepared using low-adhesion culture dishes, i.e., IL-4-DCs prepared without cluster control, and "+" indicates the results of IL-4-DCs prepared using cluster control culture dishes (cluster control IL-4-DCs), i.e., IL-4-DCs prepared with cluster control.

[0095] Compared with IL-4-DCs prepared using low-adhesion culture dishes, an increase in the gene expression of BCL2A1, which is one of the anti-apoptosis genes, was detected in IL-4-DCs prepared using cluster control culture dishes (cluster control IL-4-DCs) (N = 3). Since the expression of BCL2A1, which is one of the anti-apoptosis genes, increased commonly in three patients, it was suggested that anti-apoptosis genes vary to increase by cluster control.

[0096] Figure 16 shows the results of the evaluation of anti-apoptosis gene mRNA expression of IL-4-DCs. The vertical axis shows the fold change in mRNA expression relative to the control. Figure 16 shows the results of BCL2, BAX, BCL2 / BAX, and BCL2A1. "-" indicates the results of IL-4-DCs prepared using low-adhesion culture dishes, i.e., IL-4-DCs prepared without cluster control, and "+" indicates the results of IL-4-DCs prepared using cluster control culture dishes (cluster control IL-4-DCs), i.e., IL-4-DCs prepared with cluster control.

[0097] When clusters were strictly controlled during the maturation process using cluster control culture dishes, an increase in the mRNA expression of BCL2A1, which is one of the anti-apoptosis-related genes, was observed.

[0098] Summary During the maturation process, strict cluster control using culture dishes showed an increase in the mRNA expression of BCL2A1, which may be involved in the lifespan of dendritic cells.

[0099] [Example 6] Influence of Cluster Control by Culture Dishes on the Survival Rate of IL-4-DC Example 6 was conducted as this test. IL-4-DC was prepared, and in the state of the same conditions as the DC vaccine delivery state (4°C, suspended in physiological saline), the viable cell rate over time (0 hr, 6 hr, 12 hr, 24 hr, 48 hr) was evaluated by trypan blue staining. The protocol is shown in Fig. 17.

[0100] Results Fig. 18 shows the influence of cluster control by culture dishes on the viable cell rate over time in IL-4-DC. Figs. 18A, B, and C show each of the three results. In the figure, "IL-4-DC" indicates IL-4-DC prepared using a low-adhesion culture dish, that is, IL-4-DC prepared without cluster control. In IL-4-DC prepared using a cluster control dish compared with IL-4-DC prepared using a low-adhesion culture dish, the decay of the viable cell rate over time was maintained without alternation (N = 2).

[0101] Summary When strict cluster control was performed during the maturation process using a cluster control culture dish, maintenance and improvement of the viable cell rate were observed in the preservation state under vaccine delivery conditions (4°C, suspended in physiological saline).

[0102] Therefore, during the maturation process, an increase in the mRNA expression of BCL2A1, which is said to be involved in the lifespan of dendritic cells, was observed along with the maintenance of viable cells in vitro when strict cluster control was performed using culture dishes.

[0103] Fig. 19 shows a summary of the quantitative results in Example 2 (preliminary test) (Fig. 19A) and Example 3 (this test) (Figs. 19B and 19C).

[0104] As shown in Fig. 19A, from the results of Example 2, a decrease in the maturation markers of dendritic cells was observed when the cell seeding density was high during the maturation process (N = 4).

[0105] Also, from the results of Example 3, as shown in Fig. 19B, no difference was observed in the cluster-controlled IL-4-DC viable cell rate and recovery rate (N = 10). Furthermore, as shown in Fig. 19C, an increase in the maturation markers was observed in the IL-4-DC prepared by performing cluster control using a culture dish during the maturation process (N = 10).

[0106] Fig. 20 shows a summary of the quantitative results of Example 4 (Fig. 20A) and Example 5 (Fig. 20B). As shown in Fig. 20A, the production of IFN-γ involved in cytotoxic T cell induction significantly increased in the IL-4-DC prepared by performing cluster control using a culture dish during the maturation process (N = 9). Also, as shown in Fig. 20B, an increase in the expression of BCL2A1, which is one of the anti-apoptosis-related genes and may be involved in the lifespan of dendritic cells, was observed in the IL-4-DC prepared by performing cluster control using a culture dish during the maturation process (N = 3 and N = 4).

[0107] Fig. 21 shows a summary of the quantitative results of Example 6. In the IL-4-DC prepared by performing cluster control using a culture dish during the maturation process, maintenance of the attenuation of the viable cell rate over time was observed (N = 3).

[0108] From the results of this example, it was found that in the process of preparing IL-4-DC using a low-adhesion culture dish, the cell seeding density at maturity affects the cluster formation and maturity of dendritic cells.

[0109] In the production process of IL-4-DC, controlling the cluster at the time of maturation was found to not only promote the maturation of IL-4-DC, but also improve the viable cell rate due to increased gene expression of BCL2A1, leading to an improvement in the quality as a DC vaccine. In addition, it was found that the gene expression level of BCL2A1 becomes a biomarker that can guarantee the quality of IL-4-DC.

Industrial Applicability

[0110] The dendritic cells (DCs) prepared by the method of the present invention can be used in dendritic cell therapy.

Claims

1. A method for preparing dendritic cells with high antigen-presenting function and capable of inducing cytotoxic T cells from monocytes, comprising differentiating and inducing monocytes separated from peripheral blood into dendritic cells by culturing them using a medium containing IL-4 and GM-CSF, and maturing the obtained immature dendritic cells in a state where spheroids are formed by cluster control culture performed using a spheroid formation culture vessel in the presence of prostaglandin E2 and OK432.

2. The method according to claim 1, wherein the spheroid formation culture vessel is a culture vessel having a well with a recess having a pore diameter of 1,000 μm or less and a depth of 500 μm or less, having no flat portion in the well, and having a protein low-adhesion coat on the inner surface of the well.

3. The method according to claim 2, wherein the spheroid formation culture vessel is an EZSPHERE (registered trademark) microplate 24 well.

4. Immature dendritic cells are at a density of 1.8×10 6 cells / ml to 2.2×10 6 cells / ml, and mature in a state where spheroids are formed in the immature dendritic cells by cluster control culture, the method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 4, comprising culturing for 3 to 8 days by non-adherent culture using a serum medium containing IL-4 and GM-CSF, adding prostaglandin E2 and OK432, and maturing in a state where spheroids are formed in immature dendritic cells by cluster control culture for 10 to 36 hours.

6. The method according to any one of claims 1 to 5, wherein the culture is performed using 1 ng / mL to 1,000 ng / mL of IL-4, 10 ng / mL to 1,000 ng / mL of GM-CSF, 5 ng / mL to 50 ng / mL of prostaglandin E2, and 5 μg / mL to 50 μg / mL of OK432.

7. The method according to any one of claims 1 to 6, wherein a cancer-specific antigen is added during the maturation of dendritic cells.

8. The method according to any one of claims 1 to 7, wherein the viable cell rate of the obtained dendritic cells is 90% or more, and the yield, which is the ratio of the number of the obtained dendritic cells to the number of monocytes during culture, is 15% or more.

9. The method according to any one of claims 1 to 8, wherein the obtained dendritic cells have an increased positive rate of CD80 and CD40 and an increased intensity of expression (ΔMFI) of CD80, CD86, and CD40 compared to dendritic cells prepared without performing cluster control culture.

10. The method according to any one of claims 1 to 9, wherein the obtained dendritic cells have an increased production of IFN-γ as compared to DCs prepared without performing cluster control culture.

11. The method according to any one of claims 1 to 10, wherein the obtained dendritic cells have an increased expression level of the BCL2A1 gene as compared to DCs prepared without performing cluster control culture.

12. The method for preparing dendritic cells from monocytes according to any one of claims 1 to 11, wherein the obtained dendritic cells have a longer lifespan under physiological saline suspension conditions at 4°C as compared to DCs prepared without performing cluster control culture.

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