Method for producing cord blood-derived myeloid-derived suppressor cells from human cord blood CD34+ cells, cord blood-derived myeloid-derived suppressor cells produced by same production method, and pharmaceutical use thereof

Abstract

The present invention relates to: a method for producing cord blood-derived myeloid-derived suppressor cells from human cord blood CD34+ cells; cord blood-derived myeloid-derived suppressor cells produced by the production method; and pharmaceutical use thereof. More specifically, according to the present invention, a large amount of cord blood-derived myeloid-derived suppressor cells is cultured using a small amount of single cord blood through CD34+ proliferation stabilization, re-addition of a culture supernatant for culture, and the like, and thus the existing application of cord blood, which is stored in cord blood banks and limited only to transplantation use, can be expanded to use for the production of personalized cord blood-derived myeloid-derived suppressor cells, and it is possible to secure raw cells capable of commercial production of allogeneic cord blood-derived myeloid-derived suppressor cells using a small amount of raw materials, thus increasing availability.

Description

Method for producing cord blood-derived myeloid suppressor cells from human cord blood CD34+ cells, cord blood-derived myeloid suppressor cells produced by the method, and pharmaceutical use thereof

[0001] The present invention relates to a method for producing cord blood-derived myeloid suppressor cells from human cord blood CD34+ cells, cord blood-derived myeloid suppressor cells produced by the method, and pharmaceutical uses thereof.

[0002] Myeloid-derived suppressor cells (MDSCs) are a group of immature myeloid cells derived from the bone marrow that suppress the function of immune cells. They were first reported to suppress immune responses in solid tumors (Murdoch C et al. Nat Rev Cancer., vol.8(8), pp.618-31(2008)). MDSCs are immature myeloid cells that exist in an immature state due to failure to fully differentiate into granulocytes in tumors, autoimmune diseases, and infections. These cells are absent in healthy individuals, but accumulate in peripheral blood, lymphoid organs, spleen, and cancer tissues in pathological conditions such as infection, inflammatory response, cancer, and autoimmunity. MDSCs are CD11b-positive in mice. + Gr1 + In cells, Lin in humans - HLA-DR - CD11b + CD33 +are defined as. These cells are a highly heterogeneous population of myeloid cells (consisting of several different types) and are one of the hematopoietic stem cell precursors that develop into macrophages, dendritic cells, and granulocytes at various stages of hematopoietic differentiation. In particular, these cells are classified into two groups: monocytic and granulocytic. These two subtypes are distinguished by the expression of CD14 in humans and Ly6C and Ly6G in mice.

[0003] MDSCs are known to effectively suppress activated T cells, and the mechanism by which MDSCs regulate T cells is known to suppress T cell activity by maximizing the metabolism of L-arginine, an essential amino acid, through nitric oxide synthase, reactive oxygen species (ROS), and an enzyme called arginase. That is, by expressing arginase-1, an enzyme that consumes arginine, at high levels, they effectively lower the concentration of arginine in the surrounding area. When arginine is present at low levels, immune cells, T cells, cannot function properly, and not only does the arginine-dependent enzyme iNOS no longer produce nitric oxide (NO), an important mediator of immune function, but also nitric oxide (NO2), a powerful toxic oxidant that suppresses immunity, is generated, causing oxidative damage to cell membrane phospholipids in macrophages and other cell lines. Therefore, it is known to inhibit the proliferation of T lymphocytes through arginase and NOS activation and to inhibit the function of T lymphocytes through the formation of ROS.

[0004] In addition, recent studies have reported that MDSCs suppress the function of CD8 T cells, which are important for immune function in killing cancer cells or virus-infected cells, and that MDSCs increase in inflammatory responses and sepsis diseases and exhibit immunosuppressive functions.

[0005] However, cord blood MDSCs (hereinafter referred to as "cord blood-derived myeloid suppressor cells" or "CBMS") still face many limitations, such as the difficulty in obtaining them in large quantities. Furthermore, it is difficult to always obtain cells of a uniform morphology when isolating and culturing CBMS, and research is being conducted to address these issues.

[0006] The purpose of the present invention is to provide a method for mass producing CBMS having excellent immunosuppressive activity from a single unit of CD34+ cells isolated from umbilical cord blood.

[0007] Another object of the present invention is to provide CBMS having excellent immunosuppressive properties and its pharmaceutical use.

[0008] In order to achieve the above purpose, the present invention comprises a first step of producing a CD34+ starting cell population by culturing a single unit of CD34+ cells isolated from umbilical cord blood in a cell culture medium supplemented with growth factors, cytokines, StemRegenin I, and cyclosporin A for 6 to 8 days;

[0009] A second step of culturing the CD34+ starting cell population in a cell culture medium supplemented with GM-CSF and SCF for 13 to 15 days; and

[0010] A method for mass producing cord blood derived myeloid suppressor cells (CBMS) from CD34+ cells is provided, comprising a third step of culturing the culture obtained from the second step in a cell culture medium supplemented with GM-CSF and SCF for 6 to 8 days, and then further culturing the culture for 6 to 8 days in a cell culture medium supplemented with GM-CSF, SCF, and dexamethasone.

[0011] In addition, the present invention provides cord blood derived myeloid suppressor cells (CBMS) manufactured according to a mass production method of the cord blood derived myeloid suppressor cells (CBMS), exhibiting the following phenotypic characteristics and having immunosuppressive properties:

[0012] - Shows negative immunological characteristics for CD (cluster of differentiation) 3, CD19, and CD56

[0013] - Shows positive immunological characteristics for CD33, CD11b, and CD14

[0014] The present invention also provides a composition for preventing or treating an autoimmune disease or allergic disease, comprising the cord blood derived myeloid suppressor cell (CBMS).

[0015] The present invention also provides a method for treating an autoimmune disease or an allergic disease, comprising administering a therapeutically effective amount of cord blood derived myeloid suppressor cells (CBMS) to a subject in need thereof.

[0016] The present invention cultivates a large quantity of cord blood-derived myeloid suppressor cells using a small quantity of a single umbilical cord blood through CD34+ proliferation stabilization and culture with culture supernatant re-addition, etc., and thereby enables expansion of the use of umbilical cord blood stored in existing cord blood banks and used only for transplantation to the manufacture of customized myeloid suppressor cells. In addition, it is possible to secure raw material cells capable of commercially producing allogeneic cord blood-derived myeloid suppressor cells using a small quantity of raw materials, thereby increasing the utility value.

[0017] Figure 1 is a diagram illustrating the proliferation / differentiation process of CD34+ cells into cord blood-derived myeloid suppressor cells (CBMS) derived from the same type of cord blood according to the existing culture method (standard product) (A) and the novel culture method of the present invention (B).

[0018] Figure 2A is a schematic diagram of a conventional CD34+ cell separation method and a CD34+ cell separation method of the present invention. Figure 2B shows the results of CD34+ cell proliferation according to the CD34+ cell separation method of the present invention.

[0019] Figure 3A shows the results of comparing the phenotypes of the raw material cells after CD34+ cell proliferation according to the existing culture method (standard product) and the new culture method of the present invention. Figure 3B shows the results of comparing the distributions of CD3+, CD19+, CD34+, and CD56+ cells after isolating CD34+ cells according to the existing culture method (standard product) and the new culture method of the present invention, thereby demonstrating the high purity of the raw material CD34+ cells in the new culture method.

[0020] Figure 4A is a graph showing the proliferation and differentiation process from CD34+ cells into CBMS according to the existing culture method (standard product) and the novel culture method of the present invention. Figure 4B is a graph showing the change in CD34+ expression rate during the culture process from starting cells to CBMS according to the existing culture method (standard product) and the novel culture method of the present invention. Figure 4C shows the percentage of CD34+ cells in the final product according to the density of the starting cells in the third step. Figure 4D shows the difference in the proliferation rate of CBMS according to the existing culture method (standard product) and the novel culture method of the present invention.

[0021] Figure 5 shows the distribution of CBMS phenotypes according to the existing culture method (standard product) and the new culture method of the present invention.

[0022] Figure 6 compares the immunosuppressive capacity of CBMS according to the existing culture method (standard product) and the novel culture method of the present invention.

[0023] Figure 7 shows the cell viability by solvent used for washing and cryopreservation of CBMS according to the existing culture method (standard product) and the new culture method of the present invention.

[0024] Figure 8 shows the proliferation rate (A) and the effect on promoting differentiation into CBMS (B) depending on the presence or absence of cyclosporin A during the proliferation stabilization process (first step) of CD34+ cells according to the novel culture method of the present invention.

[0025] Figure 9 shows the proliferation rate (A) at each time point of adding dexamethasone (3rd stage; 0.32 μM) after stabilization of proliferation of CD34+ cells according to the novel culture method of the present invention, and the survival rate (B) at each concentration of dexamethasone (0.04 μM, 0.32 μM).

[0026] Figure 10 shows the results of an efficacy test of CBMS according to the existing culture method (standard product) and the novel culture method of the present invention in a humanized GVHD mouse model. A shows the survival rate of the GVHD mouse model after CBMS administration, B shows the Th1, Th2, and Th17 ratios of T cells collected from the spleen of the GVHD mouse model, and C shows the Th2 ratio (IL-4, IL-5) of the lymph nodes of the GVHD mouse model.

[0027] Figure 11A shows a schematic diagram of the signal transduction pathway related to the distinction of Signal 1 (Growth Factor), Signal 2 (Upstream Activation), and Signal 3 (Signal Factor) of CBMS according to the existing culture method (standard product) and the new culture method of the present invention, and the immunosuppressive activity. Figure 11B shows a comparison of the expression level of STAT3 corresponding to Signal 3 between the existing culture method CBMS and the culture method CBMS of the present invention. Figure 11C shows a list of CD markers and Signaling Factors indicating the difference in the expression level of CBMS according to the existing culture method (standard product) and the new culture method of the present invention.

[0028] Hereinafter, the configuration of the present invention will be described in detail.

[0029] The present invention comprises a first step of producing a CD34+ starting cell population by culturing a single unit of CD34+ cells isolated from umbilical cord blood in a cell culture medium supplemented with growth factors, cytokines, StemRegenin I, and cyclosporin A for 6 to 8 days;

[0030] A second step of culturing the CD34+ starting cell population in a cell culture medium supplemented with GM-CSF and SCF for 13 to 15 days; and

[0031] A method for mass producing cord blood derived myeloid suppressor cells (CBMS) from CD34+ cells, comprising a third step of culturing the culture obtained from the second step in a cell culture medium supplemented with GM-CSF and SCF for 6 to 8 days, and then further culturing the culture for 6 to 8 days in a cell culture medium supplemented with GM-CSF, SCF, and dexamethasone.

[0032] In order to solve the problem of difficulty in obtaining CBMS in large quantities and difficulty in always obtaining cells of uniform shape in isolating and culturing CBMS, the inventors of the present invention used only one unit of umbilical cord blood CD34+ cells as raw material cells instead of the existing manufacturing method of mixing 10 units of umbilical cord blood, and used the existing (approximately 3x10 6 We have established culture conditions that allow stable proliferation while maintaining existing characteristics using more than 10 times less than the conventional umbilical cord blood (CBMS) cells. Therefore, in addition to its use as a homologous cell, it can be used to manufacture customized therapeutics, dramatically expanding the uses of umbilical cord blood stored in existing umbilical cord blood banks. CD34+ cells stabilized for proliferation through this method can be used in CBMS culture because they maintain their original phenotype and hematopoietic capacity.

[0033] In addition, the "culture suspension re-addition culture method" was used for mass production of CBMS. That is, it is a culture method that utilizes the characteristics of CBMS that differentiate through cell-cell interactions by leaving attached cells in the semi-adherent state of CBMS and centrifuging the suspended cells and re-adding fresh medium at a certain ratio. This method reduces the loss of suspended cells caused by medium replacement and uses less growth factors, and since there is no need to add cytokines, it is not only cheaper than other methods, but also affects the increase of immunosuppressive function by maintaining appropriate interaction between adherent cells and suspended cells, and can effectively obtain a large quantity of CBMS that are uniformly differentiated using CD34+ cells of higher purity without worrying about mixing other cells, so it has outstanding superiority over other obtaining methods.

[0034] In addition, the culture method of the present invention overcomes the phenomenon of low cell proliferation and differentiation rates and survival rates in large-capacity culture vessels compared to existing culture methods through a process of increasing cell proliferation rate, activity, and survival rate by optimizing the number of additions and method of adding dexamethasone in the final harvesting stage, and by optimizing the washing solution and cryopreservative for CBMS, the cell yield and survival rate after thawing of CBMS can be stably maintained even after thawing, so that it can be usefully used for stabilizing mass production of CBMS and enhancing immunosuppressive activity, and can be dramatically improved to a level that allows commercialization.

[0035] In this specification, the term "existing culture method" refers to a culture method referring to the method for producing cord blood immunosuppressive cells disclosed in Korean Patent Publication No. 10-2022-0167528, which uses 10 units of cord blood derived from different individuals as a raw material sample by mixing (pooling). Specifically, 10 units of cord blood derived from different individuals are mixed (pooled), CD34+ cells are separated therefrom, and then 1x10 per well are cultured in a 48-well plate using IMDM medium with a cytokine combination of GM-CSF (100 ng / mL) / SCF (50 ng / mL). 5 / mL for 3 weeks, 5x10 per well 5 / mL and cultured for an additional 3 weeks, and CBMS expressing a specific cell phenotype were selected.

[0036] In addition, the term "standard" refers to CD11b+, CD33+, CD14+, CD15- and HLA-DR selected from CBMS differentiated according to the conventional culture method mentioned above. LOW Refers to CBMS expressing a cell phenotype including .

[0037] As used herein, the term "CD34+ cell" refers to a cell capable of self-renewal and differentiation into all types of blood cells, including erythroid, myeloid, and lymphoid lineages. CD34+ cells are used in the clinic as a cell therapy to treat various hematopoietic malignancies, including leukemia. Currently, three sources of HSCs are used for transplantation: umbilical cord blood (UCB), mobilized peripheral blood (mPB), and bone marrow (BM). Due to the limited availability of these cells for clinical transplantation, many attempts have been made in the past to expand them ex vivo to obtain sufficient numbers of transplantable CD34+ cells. Most strategies for expanding CD34+ cells focus on regulating the renewal and survival of CD34+ cells, which are mediated by intrinsic factors (e.g., transcription factors and signaling molecules) or environmental signals (e.g., cytokines, chemokines, stromal cells, and adhesion molecules).

[0038] The mass production method of the CBMS of the present invention is explained step by step as follows.

[0039] Step 1 is to manufacture a CD34+ starting cell population to resolve the shortage of starting cell numbers in culture by proliferating CD34+ cells, which are raw material cells isolated from a single unit.

[0040] Specifically, step 1 is

[0041] A step of isolating a single unit of CD34+ cells from umbilical cord blood; and

[0042] 3 5x10 5 A step of preparing a CD34+ starting cell population by mixing the isolated CD34+ cells with cell culture medium supplemented with growth factors, cytokines, stemlegenin I and cyclosporin A and culturing them in a round-bottomed vessel containing a gas-permeable culture surface for 6 to 8 days.

[0043] In the past, CD34+ cells were isolated from pooled 10 units, but the present invention is characterized by isolating CD34+ cells from a single unit of cell sample from cord blood and using them for differentiation into CBMS. Therefore, since the isolation of CD34+ cells from a single unit (1 unit) is less than that of the existing method, it is not appropriate to apply the existing separation method. Therefore, the present invention reduces costs by using a tube instead of a column by using CliniMACS CD34 microbeads in the Easysep magnet cell separation method, and can isolate CD34+ cells with high purity even from a small unit of cord blood MNC. In addition, by performing proliferation stabilization of CD34+ cells, the number of cells required for initial culture can be obtained (when using a column, the efficiency of separation of a small number of cells is greatly reduced). CliniMACS CD34 beads, clinical beads from Miltenyi, are used to label CD34+ cells, and cell separation is performed using a positive separation method using the Easysep magnet from Stemcell. The development of this separation method has replaced the existing animal-derived raw material CD34 microbeads with limited commercial use, and has also improved the method of collecting cells using simple magnetic force on the MACS stand, which used to collect cells by applying pressure to the cells in a column format, thereby greatly increasing the survival rate of the collected cells.

[0044] Next, the isolated CD34+ cells are mixed with cell culture medium supplemented with growth factors, cytokines, stemlegenin I, and cyclosporin A and cultured for 6 to 8 days in a round-bottomed vessel containing a gas-permeable culture surface to prepare a CD34+ starting cell population.

[0045] The above cell culture medium may contain growth factors, cytokines, stemlegenin I, and cyclosporin A as supplementary components. For example, the cell culture medium may be SFEM (Serum-Free Expansion Medium), SFEMII (Serum-Free Expansion Medium II), AOF (Animal Origin-Free), XF, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (αMinimal essential Medium), GMEM (Glasgow's Minimal essential Medium), or IMDM (Iscove's Modified Dulbecco's Medium).

[0046] Growth factors may be used alone or in combination with one or more of TPO (thrombopoietin), FLT3 (fms-like tyrosine kinase 3), Flt3L (fms-like tyrosine kinase 3 ligand), SCF (Stem cell factor), or LDL (Low-density lipoprotein).

[0047] Cytokines may be used alone or in combination with one or more of IL-2, IL-3, IL-6, IL-7, IL-9, IL-11, IL-15, or IL-21.

[0048] StemRegenin I (SRI; 4-(2-(2-(benzo[b]thiphen-3-yl)-9-isopropyl-9H-purin-6-yl)amino)ethyl)phenol hydrochloride, CH-223191 (1-Methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide) is an aryl hydrocarbon receptor inhibitor that can be used for the initiation of differentiation, cell expansion (increase in cell number), etc., and the concentration 0.5μM to 1μM can be used, but is not limited thereto.

[0049] Cyclosporin A is an immunosuppressant that suppresses cytokines secreted when T lymphocytes in our body cause a rejection reaction against a transplanted organ, and is mainly used to suppress interleukin-2, gamma interferon, etc. According to one specific example of the present invention, the group to which cyclosporin A was added showed an effect of increasing the proliferation rate by about twice compared to the control group during the initial 7-day proliferation process of CD34+ cells, and at the same time, the group to which cyclosporin A was added showed an increase in the content of CMP (Common myeloid progenitor) and GMP (granulocyte / monocyte progenitor) compared to the control group, which promoted differentiation into CBMS, so the effect of shortening the culture period can be expected. Although the effect on the increase in the proliferation rate and differentiation of CD34+ cells was unexpected, it is thought that this phenomenon will have a positive effect during the culture process.

[0050] Cyclosporin A can be added to the medium at a concentration of 160 to 640 ng / mL, and within this range, the proliferation rate of CD34+ cells can be improved and differentiation into CBMS can be promoted.

[0051] The circular-bottomed vessel including the above-mentioned gas-permeable culture surface refers to a vessel used for amplifying a population of immune cells, and may be a gas-permeable vessel through which gas is permeated, or a vessel through which gas is supplied including a gas supply system. Preferably, the vessel itself is designed to be gas-permeable, so that it may be a gas-permeable vessel. For example, it may be a flask or culture bag such as a Grex 10, Grex 10M, Grex 10M-CS, Grex 100, Grex 100M, Grex 100M-CS, Grex 500, Grex 500M, or Grex 500M-CS flask, but is not limited thereto.

[0052] A Grex device comprising a Grex vessel is a cell culture flask having an air-permeable membrane at the base that supports a large medium volume without compromising gas exchange (Bajgain et al., 2014, Molecular Therapy-Methods & Clinical Development, 14015), and the air-permeable device may be a bioreactor. The bioreactor may be a WAVE bioreactor. The bioreactor may be a stirred tank bioreactor.

[0053] The present invention makes culturing CBMS easier in terms of time and method than using a conventional 48-well plate by using a Grex container.

[0054] According to one specific example of the present invention, separated CD34+ cells are mixed with SFEMII (Serum-Free Expansion Medium II) supplemented with CD34+ Expansion Supplement (containing Flt3L, SCF, IL-3, IL-6, and TPO) and cultured in a Grex-10M cell culture vessel for 6 to 8 days to prepare a CD34+ starting cell population, and when the proliferation and stabilization of CD34+ cells are achieved, the total number of cells shows a proliferation rate of about 50 to 100 times that of the existing culture method, and on average, shows a proliferation rate of 73.3 times.

[0055] In the method of the present invention, the second step is a step of proliferation using a culture supernatant re-addition culture method while differentiating the CD34+ starting cell population into CBMS using differentiation factors of GM-CSF and SCF.

[0056] Specifically, the second stage is 0.1 to 1x10 8 A step of mixing a CD34+ starting cell population of cell / L with a cell culture medium supplemented with GM-CSF and SCF and culturing the cells for 6 to 8 days in a round-bottomed vessel containing a gas-permeable culture surface; and

[0057] The method may include mixing the culture obtained from the above step and the cell culture medium supplemented with fresh GM-CSF and SCF at a certain ratio, dispensing the mixture into a circular-bottomed vessel including a plurality of gas-permeable culture surfaces, and culturing the mixture for 6 to 8 days.

[0058] The GM-CSF may be added to the cell culture medium at a concentration of 50 ng / mL to 200 ng / mL. The SCF may be added to the cell culture medium at a concentration of 10 ng / mL to 100 ng / mL. Within the above range, CD34 +　Cell proliferation can be relatively increased. Preferably, the GM-CSF and SCF can be added to the cell culture medium at a concentration ratio of 1:0.8 to 0.3.

[0059] The above cell culture medium may be SFEM (Serum-Free Expansion Medium), SFEMII (Serum-Free Expansion Medium II), AOF (Animal Origin-Free), XF, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (αMinimal essential Medium), GMEM (Glasgow's Minimal essential Medium), or IMDM (Iscove's Modified Dulbecco's Medium). Specifically, IMDM medium may be used, but is not limited thereto.

[0060] The circular-bottomed vessel including the above-mentioned gas-permeable culture surface may be a Grex vessel, and specifically, Grex-100M may be used, but is not limited thereto.

[0061] The step of mixing the culture to which the above culture supernatant re-addition method is applied and the cell culture medium supplemented with fresh GM-CSF and SCF is preferably performed by mixing the culture and the cell culture medium supplemented with GM-CSF and SCF in a volume ratio of 1:1 to 9, dispensing the mixture into a circular-bottomed vessel including a plurality of gas-permeable culture surfaces, and culturing the mixture, thereby increasing the cell number.

[0062] In the method of the present invention, the third step is a step of further culturing the culture obtained from the second step in a cell culture medium supplemented with GM-CSF, SCF, and dexamethasone to complete proliferation and differentiation into CBMS.

[0063] Specifically, the third step is

[0064] 2.5 to 5x10 from step 2 8 A step of mixing the cell / L culture with a cell culture medium supplemented with GM-CSF and SCF and culturing it for 6 to 8 days in a vessel with a round bottom containing a gas-permeable culture surface; and

[0065] It may include a step of mixing the culture obtained from the above step and a cell culture medium supplemented with GM-CSF, SCF and dexamethasone at a certain ratio and culturing for 6 to 8 days in a vessel with a circular bottom including a gas-permeable culture surface.

[0066] The third stage, in which the differentiation of the final product, CBMS, can be completed, is performed by increasing the number of starting cells compared to the second stage of culture, and finally, when CD34+ cells reach a purity of 1% or less, the culture is terminated. It is recommended that the number of starting cells in the third stage be cultured at a higher density than that of the starting cells in the second stage, and preferably 2.5 to 5x10 per Grex-100M (1L). 8 cell / L. According to one specific example of the present invention, the minimum cell density at which CD34+ cells, etc. reach a purity of 1% or less is about 2.5x10 8 cell / L, and the maximum cell density is about 5x10 8 It is about cell / L.

[0067] Dexamethasone can be supplemented at a concentration of 0.04 μM to 0.32 μM. Within this range, cell proliferation and cell viability can be increased.

[0068] The above cell culture medium may be SFEM (Serum-Free Expansion Medium), SFEMII (Serum-Free Expansion Medium II), AOF (Animal Origin-Free), XF, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (αMinimal essential Medium), GMEM (Glasgow's Minimal essential Medium), or IMDM (Iscove's Modified Dulbecco's Medium). Specifically, IMDM medium may be used, but is not limited thereto.

[0069] The circular-bottomed vessel including the above-mentioned gas-permeable culture surface may be a Grex vessel, and specifically, Grex-100M may be used, but is not limited thereto.

[0070] A circular-bottomed vessel including the above-described gas-permeable culture surface can be effective in enabling CD34+ cells to reach a purity of 1% or less when differentiating into CBMS. That is, in the case of a square-bottomed culture vessel, residual space remains at the rim due to the structure, and the cells do not completely fill the bottom of the culture vessel, so that complete differentiation of CD34+ cells into CBMS does not occur (the purity of CD34+ cells exceeds 1%), whereas in the case of a Grex series culture vessel with a circular bottom, the cells completely fill the bottom of the culture vessel, so that rapid proliferation of the cells can be maintained.

[0071] In addition, the step of mixing the culture to which the above culture supernatant re-addition culture method is applied and the cell culture medium supplemented with fresh GM-CSF, SCF and dexamethasone is preferably performed by mixing the culture and the cell culture medium supplemented with GM-CSF, SCF and dexamethasone in a volume ratio of 1:1 to 9 and culturing in a vessel with a circular bottom including a gas-permeable culture surface, thereby increasing the cell number. It is preferable to culture by mixing the culture and the cell culture medium in a volume ratio of 1:1.

[0072] The method of the present invention may further include a step of washing CBMS differentiated from CD34+ cells using Hartmann Solution (HS) as a washing solution after the third step culture.

[0073] According to the existing method, sterile saline solution used as a cell washing solution has the disadvantage of lowering the cell viability during the washing process, but if cells are washed with Hartmann's solution, which has a pH and composition similar to plasma, the cell viability can be increased by acting as a buffer for the cells.

[0074] The method of the present invention may further include a step of freezing CBMS differentiated from CD34+ cells using CryoStor10 (CS10) as a cryopreservative after the third stage culture or using a solution in which CS10 and Hartmann solution (HS) are mixed in a volume ratio of 9 to 1:1.

[0075] Compared to the combination of saline solution + albumin, which is a conventional cell cryopreservative, the combination of CryoStor10 (CS10) and Hartmann solution (HS) increases cell viability, and specifically, when mixed in a volume ratio of 9 to 1:1, it exhibits cell viability equivalent to that of CryoStor10 (CS10). Therefore, by mixing and using the inexpensive Hartmann solution instead of the expensive CS10, there is an effect of reducing the production cost.

[0076] The present invention also relates to cord blood derived myeloid suppressor cells (CBMS) produced according to the above method, exhibiting the following phenotypic characteristics, and having immunosuppressive properties:

[0077] - Shows negative immunological characteristics for CD (cluster of differentiation) 3, CD19, and CD56

[0078] - Shows positive immunological characteristics for CD33, CD11b, and CD14

[0079] CD34 isolated from the above umbilical cord blood + The CBMS of the present invention, which is induced to differentiate from cells, is CD11b + CD33 + CD14 + They may be monocytic myeloid suppressor cells expressing the cellular phenotype.

[0080] The functions associated with the additives of the above CBMS and the functional classification of their signaling factors are shown in the table below. Growth factors such as GM-CSF and S-CSF are involved in proliferation and differentiation upstream. Cytokines influence activation, and the additionally added stemlegenin I, cyclosporin A, and dexamethasone enhance proliferation and immunosuppression functions. As a result, changes in downstream signaling factors may occur, resulting in functional differences in the culture method of the present invention.

[0081] In addition, when cultured with the additive substance of the above CBMS, it is possible to find differences in the expression levels of surface CD markers other than CD33, CD11b, and CD14, which are known MDSC markers, compared to the existing culture method CBMS, and this can be linked to changes in signaling factors to explain the enhanced immunosuppressive capacity. The list of CD markers is as follows.

[0082]

[0083] The present invention also relates to a composition for preventing or treating an autoimmune disease or allergic disease, comprising the cord blood derived myeloid suppressor cell (CBMS).

[0084] When the CBMS of the present invention is administered to a humanized GVHD mouse model, it shows a high survival rate, and compared to CBMS manufactured by a conventional culture method, it shows an excellent survival rate with at least 1 / 10 the number of administrations. In addition, when the ratio of Th1, Th2, and Th17 in T cells collected from the spleen of a humanized GVHD mouse model administered CBMS was confirmed, the expression of each marker cytokine (IL-4 and IL-5) was decreased. When the blood IgE concentration was confirmed, the disease was improved in atopic mice.

[0085] Therefore, the CBMS of the present invention can be used for the prevention or treatment of autoimmune diseases or allergic diseases, such as atopic dermatitis, psoriasis, Behcet's disease, polymyositis / dermatomyositis, Sjogren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, or scleroderma.

[0086] The above pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. For oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. may be used. For injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used. For topical administration, bases, excipients, lubricants, preservatives, etc. may be used.

[0087] The pharmaceutical composition may be prepared in various forms by mixing it with the pharmaceutically acceptable carrier described above. For example, for oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it may be prepared in the form of unit dose ampoules or multiple doses.

[0088] Additionally, the pharmaceutical composition may include a surfactant capable of improving membrane permeability. Such surfactants may be, but are not limited to, those derived from steroids, cationic lipids such as N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), or various compounds such as cholesterol hemisuccinate and phosphatidyl glycerol.

[0089] The pharmaceutical composition may be administered together with or sequentially with the aforementioned pharmacological or physiological components, or may be administered in combination with additional conventional therapeutic agents, either sequentially or simultaneously. Such administration may be single or multiple administrations. Taking all of the above factors into account, it is important to administer an amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination readily available to those skilled in the art.

[0090] The term "administration" used in the present invention means providing the pharmaceutical composition of the present invention to a subject by any suitable method. The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount, which is an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human, as considered by a researcher, veterinarian, doctor, or other clinician, i.e., an amount that induces alleviation of symptoms of a disease or disorder to be treated. It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration for the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and can be adjusted according to various factors, including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the treatment period, and concurrently used drugs. The pharmaceutical composition of the present invention may be administered in an amount of 1x10 6 3x10 inland 7 It can be administered in an amount of cells / kg / day, and can be administered once a day or divided into several doses.

[0091] The term "subject" as used herein means a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed or treated by administration of the CBMS, and preferably means a human.

[0092] The present invention also provides a method for treating an autoimmune disease or an allergic disease, comprising administering a therapeutically effective amount of cord blood derived myeloid suppressor cells (CBMS) to a subject in need thereof.

[0093] The above CBMS can be administered in a pharmaceutically effective amount to treat autoimmune or allergic diseases. The dosage may vary depending on various factors, including the type of disease, the patient's age and weight, the nature and severity of symptoms, the type of current treatment, the number of treatments, the form and route of administration, and can be readily determined by experts in the field.

[0094] The above target is the same as the target for administration of the pharmaceutical composition of the present invention.

[0095] Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

[0096] <Example 1> Production of homogeneous CBMS from CD34+ cells using existing CBMS culture method and new culture method

[0097] A novel proliferation / differentiation method was established by changing the CD34+ isolation method and the CD34+ proliferation stabilization step in the existing CBMS proliferation / differentiation method. As illustrated in Fig. 1, the novel culture method attached CliniMACS CD34 microbeads to MNC isolated from one umbilical cord blood cell, and then positively separated using an EasySep Magnet. After applying a 7-day CD34+ cell proliferation stabilization period, the cells were cultured using a Grex culture vessel (Wilson Wolf Manufacturing, LLC).

[0098] (Week -1) (CD34+ isolation and preparation of CD34+ starting cell population) Frozen cord blood-derived MNCs were thawed, washed with IMDM medium, and passed through a 40 ㎛ strainer to remove clumped cells. After washing the cells with MACS rinsing solution (buffer) containing 2% FBS, 1x10 cells were seeded. 7 8 ㎕ of CD34 CliniMACS sorting beads were added to each bead and stained for 30 minutes. The beads were washed with MACS rinsing solution containing 2% FBS, suspended in 1 mL, transferred to a 5 mL tube mounted on an EasySep magnet, and an additional 2 mL was added to make a total of 3 mL of buffer, and left to stand for 15 minutes. While the tube was mounted on the magnet, the buffer was tilted to collect CD34- cells, and 3 mL of buffer was added and left to stand for 15 minutes (repeated twice more). The buffer was poured into the CD34- cell tube, and the tube was removed from the magnet. CD34+ cells were collected with 1 mL of buffer. The separated CD34+ cells were mixed with SFEMII (Serum-Free Expansion Medium II) containing CD34+ Expansion Supplement and incubated at 1 x 10 4 After culturing for 7 days under / mL conditions, cells were collected again and the total cell number was measured.

[0099] At least 3x10 when starting culture 5More than 1x10 10+ CD34+ cells were cultured in SFEMII (Serum-Free Expansion Medium II: Iscove's MDM, Bovine serum albumin, Recombinant human insulin, Human transferrin (iron-saturated), 2-Mercaptoethanol) supplemented with CD34+ Expansion Supplement (containing Flt3L, SCF, IL-3, IL-6, and TPO) and 320 ng / mL cyclosporin A. 4 / mL and cultured in Grex-10M for 7 days.

[0100] (Week 0) CD34+ cells (0.1-1x10) proliferated in Grex-10M after 7 days 5 Cells (100 cells / mL) were collected, washed with IMDM culture medium, transferred to Grex-100M, and then IMDM culture medium containing 100 ng / mL of hGM-CSF and 50 ng / mL of hSCF was added up to 1000 mL, and proliferation culture was performed for an additional 7 days.

[0101] (Week 1) After removing the supernatant from Grex-100M, leaving 500 mL, the cells were suspended in the remaining supernatant using a pipet aid, dispensed into new Grex-100M (100 mL each), and added to 1000 mL of IMDM culture medium supplemented with 100 ng / mL of hGM-CSF and 50 ng / mL of hSCF, and cultured for proliferation and differentiation for an additional 7 days.

[0102] (Week 2) Cells were collected from each Grex-100M container, washed with culture medium, and counted. 2.5x10 8 The cells were placed in new Grex-100M containers and cultured for an additional 7 days by adding IMDM culture medium containing 100 ng / mL of hGM-CSF and 50 ng / mL of hSCF up to 1000 mL.

[0103] (Week 3) 500 mL of the supernatant was removed, and IMDM culture medium containing 100 ng / mL hGM-CSF, 50 ng / mL hSCF, and 0.32 μM dexadethasone was added up to 1000 mL, and the cells were collected after an additional 7 days of culture.

[0104] <Experimental Example 1> Comparison of the high-purity separation and proliferation stabilization processes of CD34+ cells.

[0105] Figure 2A is a description of the raw material preparation step of the -1 week of the new culture method.

[0106] In addition, as shown in Fig. 2B, after the stabilization of proliferation of CD34+ cells, the total cell number showed a proliferation rate of approximately 50 to 100 times, and the average proliferation rate was 73.3 times.

[0107] <Experimental Example 2> Comparison of phenotypes of CD34+ cells isolated using the existing culture method (standard product) and the new culture method.

[0108] The purity of the CD34+ cells isolated in Example 1 immediately after isolation and the CD34 expression rate after 7 days of culture were compared by flow cytometry analysis using anti-CD34 antibodies. The purity of the source cells isolated from the existing and new culture methods was confirmed, and the presence of cells other than CD34+ cells was confirmed by staining with anti-CD3, anti-CD19, and anti-CD56.

[0109] Figure 3 shows the purity of CD34+ cells isolated at week -1 of the new culture method and the proliferated CD34+ cells at week 0. The purity of CD34+ cells isolated in the existing culture method was confirmed to be 53.1%. The percentage of CD34+ cells isolated in the new culture method was 89.2%, and 83.7% after 7 days of proliferation stabilization. The results of the new culture method showed a higher CD34+ cell expression rate than the existing culture method even after proliferation stabilization. Although it was confirmed that CD34+ cells in the new culture method decreased by 5.5% during the 7-day proliferation culture process, it is considered a sufficient advantage considering the total number of proliferated cells, and it was confirmed that CBMS culture can be started with an average of 73.3 times more starting cells with high purity compared to the existing culture method.

[0110] Among the cells excluding CD34+ cells, CD3+, CD19+, and CD56+ cells were significantly less distributed in the culture method of the present invention compared to the existing culture method. Therefore, in the culture method of the present invention, the starting population cells, CD34+ cells, had a high level of purity (85-95%), and compared to the existing culture method, there were fewer mixed cells at the beginning, and it was confirmed that the culture started with approximately 25% more starting cells compared to the existing culture method that started with the purity of CD34+ cells (50-70%).

[0111] <Experimental Example 3> Comparison of CBMS proliferation rates according to existing and new culture methods.

[0112] To analyze the proliferation rate and CD34+ expression rate of cells cultured using the existing and new culture methods, total cell counts and flow cytometry were performed every 7 days to confirm the proliferation rate and CD34+ expression rate.

[0113] The existing culture method measured the proliferation rate of cells every 7 days, and the new culture method measured the proliferation rate of cells at -1, 0, 2, and 4 weeks. As shown in Figure 4A, the graph confirmed that the existing culture method went through a phase of rapid cell proliferation from week 0 to week 3, and then the proliferation rate decreased and differentiated from week 4 to week 6. In the new culture method, the graph confirmed that the cells went through a phase of rapid cell proliferation from week 0 to week 2, and then continued to proliferate and differentiate from week 3 to week 4.

[0114] In the culture method of the present invention, the purity of CD34+ cells, which are separated with a high level of purity (85-95%) and are the starting population cells in the existing culture method, starts at 50-70%, so the culture starts with about 25-35% more starting cells (Fig. 4B).

[0115] The density condition required for cell differentiation and proliferation stage in the second week is 2.5x10 8 5.0x10 8 Proliferation and differentiation were compared with the conventional culture method by setting it to cell / Grex-100M (1L).

[0116] As shown in Figure 4C, the minimum cell density of the stage 3 starting cells that can complete differentiation of the final product is 2.5x10 8 cells / L, and it is expected that final differentiation will be completed at a density higher than this, which can completely fill the cell culture vessel, and in this experiment, 5.0x10 8cells / L. In order for the differentiation of CBMS to be complete, the bottom of a circular culture vessel such as Grex-100M must be completely filled with cells, and if there is residual space at the bottom of the culture vessel, it appears that the terminal differentiation conditions are not reached (i.e., maintenance of rapid cell proliferation, high CD34+ expression, decreased CD14+ expression, etc.). This result was also confirmed through the phenomenon that terminal differentiation is not reached in square-bottomed vessels such as T75 and T175 when the cells do not completely fill the bottom of the culture vessel due to the structural residual space remaining at the edge.

[0117] It was confirmed that CD34+ cells cultured using the new culture method differentiated into CBMS, and the characteristics were the same as those shown in the existing culture method. It was also confirmed that the proliferation rate was maintained to some extent even in the third stage where the density was changed compared to the existing culture method.

[0118] In order to set the completion point of proliferation and differentiation, the expression rate of CD34+ of CBMS according to the existing and new culture methods was confirmed by flow cytometry analysis at each week, and the completion point of culture was set to week 4, when the expression rate of CD34+ was less than 1%.

[0119] As shown in Figure 4D, the final proliferation rate was confirmed to be approximately 600 times for the existing culture method and approximately 20,000 times for the new culture method, and a dramatically improved proliferation rate was confirmed in the new culture method compared to the existing culture method.

[0120] <Experimental Example 4> Comparison of CBMS phenotypes and the presence of other immune cells according to existing and new culture methods.

[0121] To analyze the phenotype of cells cultured using the existing and new culture methods, flow cytometry analysis was performed on cultured CBMS to confirm the expression rates of CD33+CD11b+CD14+ and CD3+, CD19+, CD34+, and CD56+, respectively.

[0122] As shown in Fig. 5, the cell phenotype was measured as CD33+CD11b+CD14+, and it was confirmed that both the existing and new culture methods showed similar phenotypes at the time of culture completion, confirming that the same cells were cultured by both methods. CD3, CD19, CD34, and CD56, which are representative phenotypes of T cells, B cells, stem cells, and NK cells, were measured, and values ​​less than 1% were confirmed for all items, confirming that no other immune cells other than CBMS remained.

[0123] <Experimental Example 5> Comparison of the immunosuppressive activity of CBMS according to the existing and new culture methods.

[0124] To analyze the established immunosuppressive capacity, PBMCs were labeled with CFSE (Carboxyfluorescein succinimidyl ester) and co-cultured with Dynabeads, magnetic beads that stimulate T cells, at a 1:1 (PBMC:CBMS) ratio for 6 days. Then, the cell surface was stained with anti-CD3, CD4, and CD8 antibodies, and the T cell proliferation capacity was confirmed.

[0125] As shown in Figure 6, the conventional culture method demonstrated a 50-60% suppressive effect on CD4+ T cells and CD8+ T cells. CBMS cultured using the novel culture method demonstrated a 90-100% suppressive effect on CD4+ T cells and CD8+ T cells. Compared to the conventional culture method, this enhanced T cell suppressive effect was confirmed, demonstrating functional improvements.

[0126] <Experimental Example 6> Comparison of cell viability according to the isolation conditions of CD34+ cells from single-cell cord blood and changes in washing solution and cryopreservation solution during CBMS culture

[0127] The CD34+ cell isolation method, which is an intermediate step between the existing and new culture methods, the cryopreservative, cell washing solution, and culture vessel were changed to increase the cell proliferation rate and culture efficiency. Hartmann's solution was used as the cell washing solution. The cryopreservative was prepared by using the existing cryopreservative (albumin 40%, sterile saline 50%, DMSO 10%) and the new cryopreservative (CS10, Hartmann's solution, different ratio conditions) and used for cell freezing, and frozen under the same conditions in a Controlled Rate Freezer (CRF). The frozen cells were thawed after at least 7 days and the cell viability and cell recovery rate were measured.

[0128] As shown in Fig. 7, this is a description of the final CBMS production stage in the 4th week of the new culture method. In the case of sterile saline used as a cell washing solution, there was a problem of reducing cell viability during the washing process. In the process of improving cell viability, it was confirmed that the pH of saline was 5.7, which is low compared to the body environment of 7.4, so Hartmann's solution was selected as a new cell washing solution because it has a buffering effect and is inexpensive. Hartmann's solution is a prescription drug known to have a pH and composition similar to plasma, so it has a buffering effect on cells and a higher cell viability was confirmed compared to sterile saline during washing.

[0129] The cell cryopreservative was also changed from the existing saline + albumin combination to 50% CS10 + 50% Hartmann's solution. When cells were frozen with 100% CS10, increased cell viability was observed both during freezing and after thawing. However, due to the high unit price of CS10, optimized conditions for the cryopreservative, including the much less expensive Hartmann's solution, were established, thereby improving its industrial applicability.

[0130] <Experimental Example 7> Comparison of the effects of adding cyclosporin A during the proliferation stabilization process of CD34+ cells.

[0131] Cyclosporin A is also used to suppress GvHD during hematopoietic stem cell (CD34+) transplantation, and is known to have no effect on hematopoietic stem cells (CD34+ cells). Therefore, cyclosporin A is an immunosuppressant, and it was added after CD34+ cell separation to further control the presence of small amounts of NK or T cells (NK cells can rapidly proliferate even when mixed in small amounts).

[0132] To this end, CD34+ cells sorted from umbilical cord blood MNC were cultured in IMDM culture medium supplemented with 100 ng / mL of hGM-CSF and 50 ng / mL of hSCF, and cyclosporin A was added at a concentration of 320 ng / mL to confirm whether the proliferation rate was improved and cell differentiation was promoted.

[0133] As shown in Fig. 8, when cyclosporin A was added in the -1 week of the new culture method, the proliferation rate measured after 7 days of stabilization of CD34+ cell proliferation in the group to which cyclosporin A was added was confirmed to be 73-fold, which is a 1.7-fold increase compared to the 43-fold proliferation rate in the group to which cyclosporin A was not added. In addition, the proportion of Granulocyte-macrophage progenitor (GMP, CD34+CD38+CD45RA+) in the group to which cyclosporin A was added was 30.7%, which is a 14% increase compared to 16.7% in the group to which cyclosporin A was not added, which is thought to improve the entry into the differentiation stage into CBMS.

[0134] In conclusion, the group with cyclosporine A added showed an increase in the proliferation rate compared to the control group during the initial 7-day proliferation process of CD34+ cells. At the same time, the group with cyclosporine A added showed an increase in the content of CMP (Common myeloid progenitor) and GMP (granulocyte / monocyte progenitor) compared to the control group, which promoted differentiation into CBMS. Therefore, a shortening of the culture period can be expected. Although the increase in the proliferation rate and the effect on differentiation of CD34+ cells were unexpected phenomena, it is thought that this phenomenon will have a positive effect during the culture process.

[0135] <Experimental Example 8> Comparison of proliferation rate, survival rate, and differentiation according to the addition of dexamethasone after stabilization of CD34+ cell proliferation.

[0136] Dexamethasone was added to the culture medium at two concentrations, 0.04 μM and 0.32 μM, 7 days prior to cell collection, and its effects on CBMS proliferation rate, survival rate, and differentiation promotion were investigated. After dexamethasone treatment in the 3rd and 4th weeks, the survival rate was checked 7 days after addition to determine the appropriate addition time.

[0137] As shown in Fig. 9, when dexamethasone (0.32 μM) was treated in the 3rd and 4th weeks of the new culture method, cell proliferation was confirmed in the group treated with dexamethasone in the 3rd week of the new culture method, 7 days later, in the 4th week. In contrast, in the group treated with dexamethasone in the 4th week of the new culture method, the proliferation rate was confirmed to decrease by 0.67 times in the 5th week, 7 days later. Based on the results, the appropriate dexamethasone treatment time in the new culture method was confirmed to be in the 3rd week.

[0138] In groups treated with both hGM-CSF / hSCF and dexamethasone, an increase in cell proliferation rate was observed compared to the group treated with only hGM-CSF / hSCF. Furthermore, cell viability was found to increase proportionally with the dexamethasone treatment concentration (0.04 μM, 0.32 μM). It is believed that future treatment with cyclosporin A can maximize culture efficiency.

[0139] <Experimental Example 9> Comparison of the therapeutic effects of CBMS obtained through a new and existing culture method in a humanized GVHD mouse model.

[0140] After establishing a humanized GVHD model in immunodeficient mice, the efficacy of CBMS on GVHD was confirmed by administering CBMS produced by the method described in Fig. 1. Humanized NSG mice were irradiated with 200 cGY x-rays, and the next day, 1x10 human PBMCs were injected 6 After cell administration, body weight, engraftment rate, and GVHD score were recorded to confirm the creation of a GVHD model. Five weeks after human PBMC administration, cultured CBMS were administered to determine survival rates and cytokine secretion levels. The existing culture conditions served as a control, and CBMS cultured using the novel culture method were administered at low doses at 3-4-day intervals for a total of two and four administrations, respectively, to confirm their efficacy.

[0141] In addition, the efficacy of CBMS on atopy was confirmed by administering CBMS produced by the method described in Fig. 1 to a mouse model of atopy induced by house dust mites. For this purpose, the back of NC / nga mice was shaved and hair was removed by applying depilatory cream. 100 mg of AD biostir (Df ointment) was applied immediately after the first shave, and then 4% SDS (in DW) was applied to the back, followed by Df ointment 4 hours later. Df ointment was applied twice a week, and after 4 weeks of application, Df was not applied during the 5th week.

[0142] As shown in Figure 10, the results of a nonclinical experiment conducted with CBMS produced by the new culture method showed an 80% survival rate for up to 63 days in mice administered CBMS by the existing culture method. The CBMS produced by the new culture method was produced under conditions of high dose 1x10 6 , low dose 1x10 5 It was administered under the condition, and although the number of administered cells was 1 / 10 less and the number of administrations was 4 times, which was the same as the conditions of the existing culture method, an improved survival rate of 90% was confirmed. As a result, it was confirmed in the mouse disease model that the CBMS produced by the new culture method was functionally improved while using a smaller number of cells than the existing culture method. In order to confirm the Th1, Th2, and Th17 ratios in T cells collected from the spleen of GVHD mice, each marker cytokine was confirmed by flow cytometry, and it was confirmed that the expression was statistically significantly reduced compared to the GHVD control group under the CBMS condition of the new culture method.

[0143] As a result of confirming the lymph node Th2 ratio of mice administered CBMS by the conventional culture method, it was confirmed that the expression of IL-4 and IL-5 was significantly reduced in the CBMS conditions produced by the novel culture method compared to the CBMS by the conventional culture method. To confirm the blood IgE concentration, the serum was separated and confirmed by ELISA as a result, it was confirmed that the concentration was significantly reduced in the mice administered CBMS produced by the novel culture method compared to the mice administered CBMS by the conventional culture method. It was confirmed that the CBMS by the novel culture method improved the disease in atopic mice compared to the conventional culture method.

[0144] <Experimental Example 10> Comparison of CBMS phenotype and signaling factor expression rate according to existing and new culture methods.

[0145] To analyze the phenotype of cells cultured using conventional and novel culture methods, flow cytometry analysis was performed on cultured CBMS to determine the expression rates of selected CD Markers and Signaling Factors, respectively. Unstained MDSC cultured cells were used as a control.

[0146] To confirm whether the signaling factor showing a difference in expression rate actually affects the increase in immunosuppressive ability, the immunosuppressive ability analysis described in Experimental Example 5 was performed after treating the blocker of the corresponding marker.

[0147] The present invention can be utilized for in vitro mass production of myeloid suppressor cells.

Claims

1. A first step of preparing a CD34+ starting cell population by culturing a single unit of CD34+ cells isolated from umbilical cord blood in a cell culture medium supplemented with growth factors, cytokines, StemRegenin I, and cyclosporin A for 6 to 8 days; A second step of culturing the CD34+ starting cell population in a cell culture medium supplemented with GM-CSF and SCF for 13 to 15 days; and A method for mass producing cord blood derived myeloid suppressor cells (CBMS) from CD34+ cells, comprising a third step of culturing the culture obtained from the second step in a cell culture medium supplemented with GM-CSF and SCF for 6 to 8 days, and then further culturing the culture for 6 to 8 days in a cell culture medium supplemented with GM-CSF, SCF, and dexamethasone.

2. In paragraph 1, A method wherein the growth factor is at least one selected from the group consisting of TPO, FLT3, Flt3L, SCF, and LDL (Low-density Lipoprotein).

3. In paragraph 1, A method wherein the cytokine is at least one selected from the group consisting of IL-2, IL-3, IL-6, IL-7, IL-9, IL-11, IL-15 and IL-21.

4. In paragraph 1, Cyclosporine A is supplemented at a concentration of 160 to 640 ng / mL.

5. In paragraph 1, Step 1 is 3 5x10 5 A method for preparing a CD34+ starting cell population, comprising mixing isolated CD34+ cells from a dog with a cell culture medium supplemented with growth factors, cytokines, stemlegenin I, and cyclosporin A and culturing the cells in a round-bottomed vessel containing a gas-permeable culture surface for 6 to 8 days.

6. In paragraph 5, A method wherein the circular-bottomed vessel comprising a gas-permeable culture surface is any one of Grex 10, Grex 10M, Grex 10M-CS, Grex 100, Grex 100M, Grex 100M-CS, Grex 500, Grex 500M or Grex 500M-CS.

7. In paragraph 1, the second step is 0.1 to 1x10 8 A step of mixing a CD34+ starting cell population of cell / L with a cell culture medium supplemented with GM-CSF and SCF and culturing the cells for 6 to 8 days in a round-bottomed vessel containing a gas-permeable culture surface; and A method comprising the step of mixing the culture obtained from the above step and the cell culture medium supplemented with fresh GM-CSF and SCF at a certain ratio, dispensing the mixture into a circular-bottomed vessel including a plurality of gas-permeable culture surfaces, and culturing the mixture for 6 to 8 days.

8. In paragraph 1, A method wherein dexamethasone is supplemented at a concentration of 0.04 μM to 0.32 μM.

9. In paragraph 1, the third step is 2.5 to 5x10 from step 2 8 A step of mixing the cell / L culture with a cell culture medium supplemented with GM-CSF and SCF and culturing it for 6 to 8 days in a vessel with a round bottom containing a gas-permeable culture surface; and A method comprising a step of mixing the culture obtained from the above step and a cell culture medium supplemented with GM-CSF, SCF and dexamethasone at a certain ratio and culturing for 6 to 8 days in a vessel with a circular bottom including a gas-permeable culture surface.

10. In paragraph 1, A method further comprising a step of washing cord blood-derived myeloid suppressor cells differentiated from CD34+ cells using Hartmann Solution (HS) as a washing solution after the third stage of culture.

11. In paragraph 1, A method further comprising a step of freezing cord blood-derived myeloid suppressor cells proliferated and differentiated from CD34+ cells using a solution in which CryoStor10 (CS10) and Hartmann solution (HS) are mixed in a volume ratio of 9 to 1:1 as a cryopreservative after the third stage of culture.

12. In paragraph 1, The cell culture medium is selected from the group consisting of SFEM (Serum-Free Expansion Medium), SFEMII (Serum-Free Expansion Medium II), AOF (Animal Origin-Free), XF, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (αMinimal essential Medium), GMEM (Glasgow's Minimal essential Medium), and IMDM (Iscove's Modified Dulbecco's Medium).

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