KR102092604B1 - Method for producing monocyte derived from bone marrow - Google Patents

Abstract

The present invention relates to a method for producing bone marrow-derived monocytes. According to the manufacturing method of the present invention, in the process of differentiating the bone marrow-derived hematopoietic stem cells into monocytes, it induces differentiation and proliferation of hematopoietic stem cells together. When using such a manufacturing method, the yield of monocytes is improved. In addition, not only the differentiation ability to immature dendritic cells is improved, but also the mature dendritic cells that mature the immature dendritic cells have an improved immune function. Therefore, the manufacturing method of the present invention can be usefully used to prepare dendritic cells for the treatment of anti-cancer immune cells.

Description

METHOD FOR PRODUCING MONOCYTE DERIVED FROM BONE MARROW}

The present invention relates to a method for producing bone marrow-derived monocytes.

Dendritic cells (DCs) are essential elements of induction of all immune responses, including innate immunity and adaptive immunity. Dendritic cells differentiate from hematopoietic stem cells and exist in various subtypes in vivo. Dendritic cells can react specifically to antigens to induce an immune response, but they also participate in reactions that induce immune tolerance to antigens beneficial to the body.

Recently, studies on anticancer immune cell therapeutics using dendritic cells have been actively conducted. Specifically, dendritic cells present antigens to CD8 + T cells and CD4 + T cells to activate these cells. Activated CD8 + T cells and CD4 + T cells participate in the adaptive immune system and exhibit anticancer activity. Moreover, activated CD8 + T cells and CD4 + T cells are known to have excellent anti-cancer effects and no side effects.

Dendritic cells for clinical use are obtained by isolating hematopoietic stem cells or monocytes from the patient's bone marrow or peripheral blood to differentiate into dendritic cells, adding antigen and activating them. The dendritic cells thus obtained are injected back into the patient and used as a therapeutic agent for immune cells. Dendritic cells injected into the body can induce antigen-specific immune responses by delivering specific antigens to T cells.

In addition, the subtype of dendritic cells is an important factor in determining the efficacy of a therapeutic agent. Recently, CD141 + Clec9a + subtype dendritic cells have been found to play a leading role in anti-cancer immune function in vivo .

However, the number of dendritic cells that can be obtained directly from blood and lymphoid tissues is extremely small and difficult to separate. In addition, in the case of differentiation from monocytes isolated from blood, the number of cells decreases during differentiation. In addition, since the number of cells that can be obtained from the bone marrow is greatly reduced with age, this is a major obstacle to the development of immune cell therapeutics using dendritic cells.

Meanwhile, as the age increases, the amount of fat in the bone marrow increases significantly. As a method for separating monocytes using it, a method of separating monocytes from bone marrow adipose tissue (KR 10-1387302 B1) has been reported. However, no method for efficiently obtaining CD141 + Clec9a + subtype dendritic cells from the monocytes thus obtained has been reported.

KR 10-1387302 B1

Accordingly, the present inventors completed the present invention by developing a production method for inducing differentiation and proliferation from hematopoietic stem cells to monocytes together, in order to increase the yield of monocytes, which are progenitor cells of dendritic cells.

The present invention comprises the steps of i) culturing bone marrow-derived hematopoietic stem cells in the presence of a conditioned medium; And ii) obtaining monocyte cells.

In addition, the present invention provides monocytes produced by the method for producing monocytes derived from bone marrow.

Furthermore, the present invention comprises the steps of i) culturing the bone marrow-derived hematopoietic stem cells in the presence of a conditioned medium; ii) obtaining monocyte cells; And iii) differentiating the obtained monocytes into immature dendritic cells in the presence of GM-CSF and IL-4.

In addition, the present invention provides immature dendritic cells that differentiate the monocytes.

Furthermore, the present invention provides a medium for culturing hematopoietic stem cells derived from bone marrow containing a conditioned medium.

The method of manufacturing monocytes of the present invention induces differentiation and proliferation of hematopoietic stem cells together in the process of differentiation of bone marrow-derived hematopoietic stem cells into monocytes. When using such a manufacturing method, the yield of monocytes is improved. In addition, not only the differentiation ability to immature dendritic cells is improved, but also the mature dendritic cells matured in the immature dendritic cells have an improved immune function. Therefore, the manufacturing method of the present invention can be usefully used to prepare dendritic cells for the treatment of anti-cancer immune cells.

1 is a view showing the proliferation rate of cultured bone marrow-derived monocytes according to the presence or absence of a culture medium.
2 is a view showing the yield of immature dendritic cells differentiated using cultured bone marrow-derived monocytes according to the presence or absence of a conditioned medium.
3 is a diagram showing the yield of bone marrow-derived monocytes obtained by culturing hematopoietic stem cells according to the presence or absence of a conditioned medium and immature dendritic cells differentiated using the same.
4 is a diagram confirming the expression level of phenotypic markers of immature dendritic cells differentiated from bone marrow-derived monocytes.
5 is a diagram confirming that immature dendritic cells differentiated from bone marrow-derived monocytes express CD141 and Clec9a, which are the next-generation dendritic cell-specific phenotypic markers.
6 is a view showing the secretion of cytokines secreted by immature dendritic cells differentiated from bone marrow-derived monocytes.
7 is a view showing the amount of IFN-r secreted by immature or mature dendritic cells differentiated from bone marrow-derived monocytes depending on the presence or absence of a conditioned medium.
8 is a view showing the amount of IL-12p70 secreted by immature or mature dendritic cells differentiated from bone marrow-derived monocytes depending on the presence or absence of a conditioned medium.
9 is a view showing the amount of secretion of IP-10 secreted from immature or mature dendritic cells differentiated from bone marrow-derived monocytes depending on the presence or absence of a conditioned medium.
10A is a diagram confirming induction of proliferation of T cells by immature dendritic cells differentiated from bone marrow-derived monocytes.
10B is a diagram confirming induction of proliferation of T cells by mature dendritic cells differentiated from bone marrow-derived monocytes.
11 is a view confirming a tumor antigen-specific immune response using mature dendritic cells differentiated from bone marrow-derived monocytes.

Hereinafter, the present invention will be described in detail.

In one aspect of the present invention provides a method for producing bone marrow-derived monocytes. Specifically, i) culturing the bone marrow-derived hematopoietic stem cells in the presence of a conditioned medium; And ii) obtaining monocytes.

The adjusted medium is at least selected from the group consisting of Angiogenin, Chitinase 3-like 1, Dkk-1, IGFBP-3, IL-6, Pentraxin 3, Serpin E1, SDF-1, TIMP-1, Thrombospondin-1 and VEGF. It may contain one protein.

The term "Angiogenin" used in the present invention, also called ribonuclease 5, refers to a protein that functions as a growth factor that increases the number of cells by inducing proliferation of hematopoietic progenitor cells in addition to the formation of new blood vessels. The Angiogenin may be derived from humans, or may be a protein having the amino acid sequence of GenBank: AAA51678.1.

The term "Chitinase-3-like protein 1" used in the present invention, also called YKL-40, refers to a secreted glycoprotein of about 40 kDa size encoded by the CHI3L1 gene. The Chitinase-3-like protein 1 may be derived from humans, or may be a protein having the amino acid sequence of GenBank: AAH39132.1.

The term "Dkk-1" used in the present invention is an abbreviation for Dickkopf-related protein 1 and refers to a protein known to control hematopoietic stem cell regeneration. The Dkk-1 may be derived from humans, or may be a protein having an amino acid sequence of NCBI Reference Sequence: NP_036374.1.

The term "IGFBP-3" used in the present invention is an abbreviation of insulin growth factor binding protein 3 and refers to a hormone that induces hematopoietic stem cell proliferation. The IGFBP-3 may be human-derived and may be a protein having the amino acid sequence of GenBank: CAA46087.1.

The term "IL-6" used in the present invention means a cytokine that stimulates proliferation of hematopoietic stem cells and maintains long-term repopulation function. The IL-6 may be human-derived, and may be a protein having the amino acid sequence of GenBank: AAH15511.1.

The term "Pentraxin 3" used in the present invention, also called pentraxin-related protein PTX3 or TNF-inducible gene 14 protein (TSG-14), refers to a cytokine that promotes pathogen recognition of dendritic cells and macrophages. it means. The Pentraxin 3 may be human-derived, and may be a protein having the amino acid sequence of UniProtKB / Swiss-Prot: P26022.3.

The term "Serpin E1" used in the present invention, also called Plasminogen activator inhibitor-1, refers to a protein encoded by the SERPINE1 gene. The Serpin E1 may be human-derived, or may be a protein having the amino acid sequence of GenBank: AAA60003.1.

The term "SDF-1" used in the present invention, also called CXCL12, means a chemokine known to be expressed only from bone marrow among hematopoietic stem cells. The SDF-1 plays an essential role in maintaining a quiescent pool of hematopoietic stem cells. The SDF-1 may be of human origin, and may be a protein having the amino acid sequence of UniProtKB / Swiss-Prot: P48061.1.

The term "TIMP-1" used in the present invention is an abbreviation of TIMP metallopeptidase inhibitor 1 and refers to a protein that inhibits MMPs to increase cell proliferation and performs anti-apoptotic function. The TIMP-1 may be derived from a human, and may be a protein having the amino acid sequence of UniProtKB / Swiss-Prot: P01033.1.

The term "Thrombospondin-1" used in the present invention means a protein that inhibits neovascularization and tumorigenesis. The Thrombospondin-1 may be derived from humans, or may be a protein having the amino acid sequence of GenBank: AAI36471.1.

The term "VEGF" used in the present invention refers to a glycoprotein that is involved in angiogenesis as a vascular endothelial cell growth factor and is an essential element for the survival and reproliferation of hematopoietic stem cells. The VEGF may be of human origin, and may be a protein having the amino acid sequence of GenBank: AAK95847.1.

Preferably, the adjusted medium is all of the Angiogenin, Chitinase 3-like 1, Dkk-1, IGFBP-3, IL-6, Pentraxin 3, Serpin E1, SDF-1, TIMP-1, Thrombospondin-1, VEGF It can contain.

In one embodiment, the adjusted medium may be a supernatant of the medium in which the mesenchymal stem cells are cultured. The mesenchymal stem cells may be isolated from umbilical cord blood, bone marrow or mononuclear cells present in peripheral blood. Specifically, the mesenchymal stem cells may be isolated from bone marrow-derived mononuclear cells.

The term "monocyte" used in the present invention means connective tissue, lymphoid tissue, and mononuclear mesenchymal cells present in blood. The mesenchymal cells are undifferentiated cells constituting the mesenchymal differentiated from mesoderm. Therefore, the mononuclear cells can be differentiated into cells that make hematopoietic tissue, bone tissue, connective tissue, or cartilage tissue.

The supernatant may be collected while culturing mesenchymal stem cells. Specifically, the supernatant may be separated by collecting the culture medium 1 to 20 times while culturing the mesenchymal stem cells. More specifically, the supernatant may be separated by collecting the culture medium 1 to 15 times while culturing the mesenchymal stem cells. More specifically, the supernatant may be separated by collecting the culture medium 1 to 10 times while culturing the mesenchymal stem cells. In one embodiment of the present invention, the culture medium was collected 5 times while culturing the mesenchymal stem cells. The supernatant may be collected every 4 to 7 days during the culture period.

The adjusted medium may include a supernatant separated from at least one culture medium of each culture medium collected while culturing mesenchymal stem cells. For example, the conditioned medium may include a supernatant separated from the first collected culture medium among the culture medium collected five times while culturing mesenchymal stem cells. In addition, the adjusted medium may include a supernatant separated from the second collected culture medium of the culture medium collected five times while culturing mesenchymal stem cells. Furthermore, the conditioned medium may include a supernatant separated from the third collected culture medium among the culture medium collected five times while culturing mesenchymal stem cells. In addition, the culture medium may include a supernatant separated from the fourth collected culture medium of the culture medium collected five times while culturing mesenchymal stem cells. Furthermore, the adjusted medium may include a supernatant separated from the fifth collected culture medium among the culture medium collected five times while culturing mesenchymal stem cells.

In addition, the culture medium may be a collection of each supernatant collected while culturing mesenchymal stem cells, or a supernatant of each of the collected supernatant. In one embodiment of the present invention, the control medium was used to collect the supernatant of each culture medium collected while culturing the mesenchymal stem cells.

Cultivation of the mesenchymal stem cells may be performed for 1 to 90 days. Specifically, the culture of the mesenchymal stem cells may be performed for 14 to 80 days. More specifically, the culture of the mesenchymal stem cells may be performed for 21 to 70 days. More specifically, the culture of the mesenchymal stem cells may be performed for 35 to 56 days. In one embodiment of the present invention, the period of culturing mesenchymal stem cells was performed for 35 to 56 days.

The culture temperature of the mesenchymal stem cells may be 25 ℃ to 40 ℃. Specifically, the culture temperature of the mesenchymal stem cells may be 37 ℃.

The mesenchymal stem cells can be cultured at a CO ₂ concentration of 5% to 30%. Specifically, the mesenchymal stem cells can be cultured at a concentration of 5% CO ₂ .

The medium used for culturing the mesenchymal stem cells may be a serum-free medium, and may further include serum and antibiotics. Specifically, it may be DMEM low glucose (1 g / L) to which 10% (v / v) FBS and 10 mg / ml concentration of gentamicin are added.

The term "serum-free medium" used in the present invention means a culture medium and a culture medium that does not contain serum for culturing animal tissues or cells. Specifically, the serum-free medium is Dulbecco's Modified Eagle's Medium (DMEM), Endothelial differentiation medium (EDM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, a-MEM It may be one selected from the group consisting of (a-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and Iscove's Modified Dulbecco's Medium. Specifically, the serum-free medium may be DMEM low glucose (1 g / L) serum-free medium.

The term "serum" as used in the present invention means a clear supernatant released from blood after the blood is completely coagulated. In addition, for the cultivation of animal cells, serum must be added to the synthetic medium, and bovine serum is the most common for using bovine, horse, and human serum. Depending on the blood collection time, fetal bovine serum (FBS), cow's newborn bovine serum, calf serum, and bovine serum can be distinguished. Specifically, it may be one selected from the group consisting of fetal bovine serume (FBS) and bovine calf serume (BCS). Preferably, the FBS may be treated at a concentration of 10% (v / v).

In addition, the antibiotic may be one selected from the group consisting of penicillin, streptomycin and gentamicin. As an example, gentamicin may be treated at a concentration of 10 mg / ml.

The term "hematopoietic stem cell" used in the present invention means a cell that is present in a small number in peripheral blood, but mainly produces blood cells such as white blood cells, red blood cells or platelets through autogenous replication and differentiation in the bone marrow. Hematopoietic stem cells collected from human bone marrow express a CD34 phenotype marker.

The term "monocyte" as used in the present invention means a cell in a specific differentiation stage of macrophage-based cells. Specifically, the bone marrow differentiates and matures into hematopoietic stem cells, monoblasts, and shear cells into monocytes and then flows into the tissues after it flows into the blood and then differentiates into macrophages, tissue cells, Langerhans cells, dendritic cells, and the like. Human monocytes express the CD14 phenotype marker.

The term "CD14" used in the present invention is called a differentiation cluster 14 and mainly refers to a receptor protein that is expressed on the surface of macrophages and functions to recognize bacterial lipid polysaccharides. Monocytes expressing the CD14 on the cell surface may be differentiated into immature dendritic cells by treating IL-4 and GM-CSF.

The hematopoietic stem cells may be cultured for 10 to 15 days, but are not limited thereto. In one embodiment of the present invention, the hematopoietic stem cells were cultured for 11 days.

The culture temperature of the hematopoietic stem cells may be 25 ℃ to 40 ℃. Specifically, the culture temperature of the hematopoietic stem cells may be 37 ℃.

The hematopoietic stem cells can be cultured at a CO ₂ concentration of 5% to 30%. Specifically, the hematopoietic stem cells may be cultured at a CO ₂ concentration of 5%.

In addition, the culture medium may be present at a concentration of 5 to 20% (v / v), 7 to 15% (v / v) or 9 to 12% (v / v) of the total culture medium during the period of culturing hematopoietic stem cells. . Specifically, the adjusted medium may be present at a concentration of 10% (v / v) of the total culture medium during the period of culturing hematopoietic stem cells.

In the step of culturing the hematopoietic stem cells, GM-CSF, Flt3L, and SCF may be further treated to be cultured. In the step of culturing the hematopoietic stem cells, GM-CSF may be further treated at 1 to 3,000, 100 to 2000, 500 to 1,500 International Units / ml. In addition, in the step of culturing the hematopoietic stem cells, SCF may be further treated with 1 to 1,000, 100 to 900, and 300 to 700 IU / ml. In addition, in the step of culturing the hematopoietic stem cells, Flt3L may be further treated with 1 to 1,000, 100 to 900, and 300 to 700 IU / ml. Specifically, in the step of culturing the hematopoietic stem cells, GM-CSF may be further cultured by further treating at concentrations of 1,000 IU (International Unit) / ml, SCF 500 IU / ml and Flt3L 500 IU / ml.

The term “GM-CSF” used in the present invention is a granulocyte macrophage colony-stimulating factor, secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells and fibroblasts. Refers to the protein being. The GM-CSF functions as a growth factor for white blood cells and stimulates stem cells to differentiate into granulocytes and monocytes. Monocytes migrate to tissues and mature into macrophages and dendritic cells. The GM-CSF may be human-derived, and may be a protein having the amino acid sequence of GenBank: AAA52578.1.

The term “FLT3L” used in the present invention is an abbreviation of FMS-like tyrosine kinase 3 ligand, and induces the proliferation of hematopoietic progenitor cells in addition to the formation of new blood vessels to activate hematopoietic progenitor cells to activate immunity. It means a cytokine that functions as a growth factor that increases the number of cells. The FLT3L may be human-derived, and may be a protein having the amino acid sequence of GenBank: AAA19825.1.

The term “SCF” used in the present invention is also called hematopoietic cell growth factor, kit ligand, KL ligand, KL, and SF factor, and 248 amino acids. It means a membrane-bound protein having a molecular weight of about 30,000. The SCF may be human-derived, or may be a protein having the amino acid sequence of GenBank: AAA85450.1.

In another aspect, the present invention provides a monocyte prepared by the method of manufacturing a monocyte of the present invention.

The present inventors confirmed that the mononuclear cells prepared by the method for preparing the monocytes of the present invention have a higher proliferation rate than the mononuclear cells cultured by the conventional culture method (FIGS. 1 and 3). In addition, it was confirmed that the monocytes have better differentiation ability into immature dendritic cells than monocytes cultured by conventional culture methods (FIGS. 2 and 3).

Therefore, the monocytes produced by the method for preparing monocytes of the present invention can be usefully used for dendritic cell production.

Furthermore, the present invention comprises the steps of i) culturing the bone marrow-derived hematopoietic stem cells in the presence of a conditioned medium; ii) obtaining monocytes; And iii) differentiating the obtained monocytes into immature dendritic cells in the presence of GM-CSF and IL-4.

The culture temperature of steps i) to ii) may be 25 ° C to 40 ° C. Specifically, the culture temperature of steps i) to ii) may be 37 ° C.

The conditions of the CO ₂ concentration in steps i) to ii) may be 5% to 30%. Specifically, the CO ₂ concentration conditions in steps i) to ii) may be 5% CO ₂ .

In addition, it provides an immature dendritic cell produced by the method for producing immature dendritic cells of the present invention.

It was confirmed that the immature dendritic cells differentiated from the monocytes prepared by the above-described monocyte production method simultaneously express CD141 + and Clec9a +, which are specific phenotypic markers of next-generation dendritic cells having excellent anticancer immune function (FIGS. 4 and 5).

In addition, it was confirmed that the expression of IL-12p70 in which the immature dendritic cells exhibited anti-cancer immune activity increased. In addition, it was confirmed that the immature dendritic cells express well IFN-r showing anti-cancer immune action. Furthermore, when the immature dendritic cells were differentiated into mature dendritic cells, it was confirmed that IL-12p70, IFN-r, and the expression of IP-10 cytokines recognized as markers of next-generation dendritic cells increased (FIGS. 6 and 9). ).

Therefore, immature dendritic cells differentiated from monocytes produced by the method for preparing monocytes of the present invention can be usefully used for the development of cell therapy products using dendritic cells.

In addition, the present invention provides a medium for culturing bone marrow-derived hematopoietic stem cells comprising a conditioned medium.

The adjusted medium is at least selected from the group consisting of Angiogenin, Chitinase 3-like 1, Dkk-1, IGFBP-3, IL-6, Pentraxin 3, Serpin E1, SDF-1, TIMP-1, Thrombospondin-1 and VEGF. It may contain one protein. Preferably, the adjusted medium is all of the Angiogenin, Chitinase 3-like 1, Dkk-1, IGFBP-3, IL-6, Pentraxin 3, Serpin E1, SDF-1, TIMP-1, Thrombospondin-1, VEGF It can contain. The Angiogenin, Chitinase 3-like 1, Dkk-1, IGFBP-3, IL-6, Pentraxin 3, Serpin E1, SDF-1, TIMP-1, Thrombospondin-1 and VEGF are as described above in the bone marrow-derived monocyte manufacturing method. same.

The adjusted medium may be a supernatant of the medium in which the mesenchymal stem cells are cultured. The mesenchymal stem cells may be isolated from umbilical cord blood, bone marrow or mononuclear cells present in peripheral blood. Specifically, the mesenchymal stem cells may be isolated from bone marrow-derived mononuclear cells.

The supernatant may be collected while culturing mesenchymal stem cells. Specifically, the supernatant may be separated by collecting the culture medium 1 to 20 times while culturing the mesenchymal stem cells. More specifically, the supernatant may be separated by collecting the culture medium 1 to 15 times while culturing the mesenchymal stem cells. More specifically, the supernatant may be separated by collecting the culture medium 1 to 10 times while culturing the mesenchymal stem cells. In one embodiment of the present invention, the culture medium was collected 5 times while culturing the mesenchymal stem cells. The supernatant may be collected every 4 to 7 days during the culture period.

The adjusted medium may include a supernatant separated from at least one culture medium of each culture medium collected while culturing mesenchymal stem cells. For example, the conditioned medium may include a supernatant separated from the first collected culture medium among the culture medium collected five times while culturing mesenchymal stem cells. In addition, the adjusted medium may include a supernatant separated from the second collected culture medium of the culture medium collected five times while culturing mesenchymal stem cells. Furthermore, the conditioned medium may include a supernatant separated from the third collected culture medium among the culture medium collected five times while culturing mesenchymal stem cells. In addition, the culture medium may include a supernatant separated from the fourth collected culture medium of the culture medium collected five times while culturing mesenchymal stem cells. Furthermore, the adjusted medium may include a supernatant separated from the fifth collected culture medium among the culture medium collected five times while culturing mesenchymal stem cells.

In addition, the culture medium may be a collection of each supernatant collected while culturing mesenchymal stem cells, or a supernatant of each of the collected supernatant. In one embodiment of the present invention, the control medium was used to collect the supernatant of each culture medium collected while culturing the mesenchymal stem cells.

Cultivation of the mesenchymal stem cells may be performed for 1 to 90 days. Specifically, the culture of the mesenchymal stem cells may be performed for 14 to 80 days. More specifically, the culture of the mesenchymal stem cells may be performed for 21 to 70 days. More specifically, the culture of the mesenchymal stem cells may be performed for 35 to 56 days. In one embodiment of the present invention, the period of culturing mesenchymal stem cells was performed for 35 to 56 days.

The culture temperature of the mesenchymal stem cells may be 25 ℃ to 40 ℃. Specifically, the culture temperature of the mesenchymal stem cells may be 37 ℃.

The mesenchymal stem cells can be cultured at a CO ₂ concentration of 5% to 30%. Specifically, the mesenchymal stem cells can be cultured at a concentration of 5% CO ₂ .

The medium used for culturing the mesenchymal stem cells may be a serum-free medium, and may further include serum and antibiotics. Specifically, it may be DMEM low glucose (1 g / L) to which 10% (v / v) FBS and 10 mg / ml concentration of gentamicin are added.

The term "serum-free medium" used in the present invention means a culture medium and a culture medium that does not contain serum for culturing animal tissues or cells. Specifically, the serum-free medium is Dulbecco's Modified Eagle's Medium (DMEM), Endothelial differentiation medium (EDM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, a-MEM It may be one selected from the group consisting of (a-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and Iscove's Modified Dulbecco's Medium. Specifically, the serum-free medium may be DMEM low glucose (1 g / L) serum-free medium.

The term "serum" as used in the present invention means a clear supernatant released from blood after the blood is completely coagulated. In addition, for the cultivation of animal cells, serum must be added to the synthetic medium, and bovine serum is the most common for using bovine, horse, and human serum. Depending on the blood collection time, fetal bovine serum (FBS), cow's newborn bovine se-rum, calf serum, and bovine serum can be distinguished. Specifically, it may be one selected from the group consisting of fetal bovine serume (FBS) and bovine calf serume (BCS). Preferably, the FBS may be treated at a concentration of 10% (v / v).

In addition, the antibiotic may be one selected from the group consisting of penicillin, streptomycin and gentamicin. As an example, gentamicin may be treated at a concentration of 10 mg / ml.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples.

Example  One. Media  Produce

In order to prepare the conditioned medium, human bone marrow was collected and subjected to a ficoll process, thereby obtaining mononuclear cells. Thereafter, mesenchymal stem cells were isolated from bone marrow-derived mononuclear cells. The supernatant of the culture medium collected during the period of culturing the separated mesenchymal stem cells was collected and mixed. After that, it was manufactured through inspection and filter filtration.

Specifically, after collecting human bone marrow, the bone marrow was mixed 1: 1 with 1xDPBS. Thereafter, the bone marrow collected to have a 1: 1 volume ratio with Piccol was poured over Piccol. After centrifugation, only the white layer was collected and washed 3 times with 1xDPBS. After that, only the petritte was left and transferred to a T flask for incubation.

The mesenchymal cells were cultured in DMEM low glucose (1 g / L) medium mixed with FBS 10% (v / v) and gentamicin sol at a concentration of 10 mg / ml by mononuclear cells obtained through the Ficoll process. Only stem cells were isolated. In order to proliferate purely isolated mesenchymal stem cells, the culture was performed for 35 to 56 days. During the cultivation, 25% (v / v) medium of the total culture medium was added to the stem cell culture medium every 3 to 5 days, and cultured at 37 ° C. under a condition of 5% CO ₂ .

The culture medium used was collected while replacing with a new medium every 4-7 days during the culture period, and then centrifuged to collect the supernatant. During the period of culturing monocytes, the culture solution was recovered 5 times. The collected supernatant was examined through Mycoplasma, sterile and virus tests, and filtered through a 0.2 μm filter.

Experimental example  One. Media  Ingredient confirmation

Through the proteomics technique, the composition of the control medium was confirmed. Samples were prepared by subdividing a certain amount of the supernatant collected in Example 1. Thereafter, the small sample was taken out and used as needed while being stored at a temperature of -20 ° C or -70 ° C. The sample was performed according to the manufacturer's manual using the Proteome Profiler Human XL Cytokine Array Kit and Human angiogenesis Array Kit (R & D systems, ARY022B).

As a result, Angiogenin, Chitinase 3-like 1, Dkk-1, IGFBP-3, IL-6, Pentraxin 3, Serpin E1, SDF-1, TIMP-1, Thrombospondin not found in culture medium containing FBS in conditioned medium It was confirmed that proteins such as -1 and VEGF were present.

Example  2. Marrow-derived monocyte production

Example  2.1. CD34 + hematopoietic stem cells from bone marrow preparation

After only mononuclear cells were isolated from the bone marrow using Ficoll, CD34 + hematopoietic stem cells were isolated using magnetic beads (MACS, Milteney Biotech) with CD34 antibody attached.

Example  2.2. Differentiation into CD14 + monocytes

CD34 + hematopoietic stem cells obtained in Example 2.1. Were mixed with FMEM 10% (v / v), gentamicin at a concentration of 10 mg / mL and DMEM low glucose (1 g / L) medium mixed with the control medium prepared in Example 1. Incubation was performed for 10 to 17 days. During the cultivation, the culture medium was cultured under conditions of 37 ° C. and CO ₂ 5% while maintaining the concentration of the adjusted medium at 10% (v / v) of the total culture. As a result, monocytes were obtained.

At this time, the proliferation rate (3,500 + 600%) of monocytes cultured in the culture medium containing the conditioned medium was higher than the proliferation rate (2,300 + 800%) of monocytes cultured in the existing culture medium (FIG. 1).

Example  3. Immature Into dendritic cells  differentiation

In order to differentiate the bone marrow-derived monocytes obtained in Example 2 into immature dendritic cells, GM-CSF at a concentration of 1000 IU / ml and IL-4 at a concentration of 1000 IU / ml were treated with 37 ° C. and 5% CO for 5 to 6 days. Cultured under ₂ conditions.

As a result, after proliferation and cultivation of CD34 + hematopoietic stem cells (1x10 ⁵ cells) with monocytes in a culture medium containing a conditioned medium, the yield (3,780,000 + 600%) of the immature dendritic cells differentiated from the cultured monocytes was added to the existing culture medium. It was higher than the yield (1,300 + 100%) of immature dendritic cells differentiated from cultured monocytes (FIGS. 2 and 3).

Experimental example  3. Immature Dendritic  Characteristics check

Experimental example  3.1. Immaturity In dendritic cells  Phenotype Marker  Confirm

Anti-HLA-ABC antibody, anti-CD80 antibody, anti-CD86 antibody, anti-CCR7 antibody, anti-CD141 labeled with FITC, APC (Allophycocyanin) and PE (Phycoerythrin) on the immature dendritic cells obtained in Example 3. After staining by treatment with antibody and anti-Clec9a antibody (eBioscience), fluorescence intensity was measured through a flow cytometer (CytoFLEX Flow Cytometry Analyzer, Beckman, USA).

As a result, the expression of HLA-ABC, CD80 / 86, and CCR7, which are functional markers of dendritic cells, increased in the immature dendritic cells. Simultaneous expression of CD141 + and Clec9a +, which are specific phenotypic markers of next-generation dendritic cells with excellent anticancer immune function. It was confirmed that (Fig. 4 and Fig. 5).

Experimental example  3.2. Immaturity In dendritic cells  Confirmation of cytokine secretion

The amount of IL-12p70, IFN-r and IP-10 secreted by the immature dendritic cells obtained in Example 3 into the culture medium was confirmed by an ELISA method. The ELISA method was performed using an ELISA kit, and was performed according to the ELISA kit manual.

As a result, the expression level of IL-12p70, which is known to contribute to the anti-cancer immune function, increased in the immature dendritic cells. In addition, it was confirmed that IFN-r was well expressed in immature dendritic cells. Furthermore, it was confirmed that despite the state of the immature dendritic cells, IP-10 recognized as a marker of the next generation dendritic cells was expressed (FIGS. 6 to 9).

Experimental example  4. Mature Dendritic  Characteristics check

Experimental example  4.1. Maturity Into dendritic cells  differentiation

To the immature dendritic cell culture solution (1x10 ⁶ cells) obtained in Example 3, 10 to 50 µg / ml Rg3 was added and cultured for 18 to 48 hours to mature. The Rg3 was purchased from Vitrosis.

Experimental example  4.2. Maturity In dendritic cells  Confirmation of cytokine secretion

The amount of IL-12p70, IFN-r and IP-10 secreted into the culture medium by the mature dendritic cells obtained in Experimental Example 4.1. Was confirmed by an ELISA method. The ELISA method was performed using an ELISA kit, and was performed according to the ELISA kit manual.

As a result, the expression level of IP-10 cytokines recognized as markers of IL-12p70, IFN-r and next-generation dendritic cells in the mature dendritic cells increased (FIGS. 7 to 9).

Experimental example  4.3. Maturity On dendritic cells  by T cell  Proliferation confirmation

The immature or mature dendritic cells obtained in Example 3. and Experimental Example 4.1 were treated with mytomicin-C to block the proliferative function. After separating lymphocytes from human peripheral blood using picol, the immature or mature dendritic cells and lymphocytes in a 1:10 ratio of FBS 10% (v / v), 10 mg / ml concentration of gentamicin sol , 1 ㎍ / ㎖ concentration of PHA (Phytohemagglutin) in DMEM low glucose (1 g / L) medium was co-cultured for 4 days at 37 ℃, 5% CO ₂ conditions. After 4 days, the number of living cells was quantified through CCK8 analysis to confirm the T cell proliferation rate.

As a result, the T cell proliferation rate of the experimental group co-cultured with immature dendritic cells and lymphocytes increased by 298.62 + 76.79% compared to the control group with only lymphocytes. In addition, the T cell proliferation rate of the experimental group in which the mature dendritic cells and lymphocytes were co-cultured increased by 440.2 + 30.41% compared to the control group in which only lymphocytes were present (FIGS. 10A and 10B).

Experimental example  5. Maturity Dendritic Tumor antigen  Confirmation of specific immune response induction

HepG2 50-100 μg / ml of cell lysate was added to the mature dendritic cell culture obtained in Example 4.1., And cultured for 18-48 hours, followed by antigen training, followed by addition of 10-50 μg / ml Rg3 for 18-48 hours. And incubated to mature. Thereafter, the mature dendritic cells were treated with mitomycin to block the proliferative function. On the other hand, after separating lymphocytes from human peripheral blood using picol, mature dendritic cells trained with the HepG2 antigen and gentamicin (gentamicin) at concentrations of 10% (v / v) and 10 mg / mL IL-2, FBS sol) in a mixed DMEM medium at 37 ° C, 5% CO ₂ conditions for 14 days co-culture (co-culture). At this time, the ratio of mature dendritic cells and lymphocytes was simultaneously cultured at a ratio of 1:10.

A medium containing mature dendritic cells and 2% (20 μl / 1 ml) IL-2 antigen-trained every 2 to 3 days while cultured for 14 days was newly supplied. After 14 days, only living cells were separated and co-cultured with HepG2, H929 or PC3 tumor cell lines at a ratio of 1:10 at 37 ° C and 5% CO2 for 4 days. After 4 days, the number of living cells was quantified through CCK8 analysis to confirm cell viability. The HepG2, H929 or PC3 cell line was purchased from the American Cell Line Bank (ATCC).

As a result, gastric cancer cell line (HepG2) was killed by T cells induced by mature dendritic cells loaded with human gastric cancer cell line (HepG2) antigen, but did not affect the survival of prostate cancer (PC3) or plasmacytoma (H929) cell line. . Thus, it was confirmed that mature dendritic cells induce a killer immune response (CTL) specific to the tumor antigen (FIG. 11).

Claims (23)

i) culturing the bone marrow-derived hematopoietic stem cells in the presence of a conditioned medium; And
ii) a method for producing monocytes derived from bone marrow, comprising the step of obtaining monocytes,
The media is characterized in that it contains Angiogenin, Chitinase 3-like 1, Dkk-1, IGFBP-3, IL-6, Pentraxin 3, Serpin E1, SDF-1, TIMP-1, Thrombospondin-1 and VEGF proteins And
The conditioned medium is the supernatant of the culture medium collected during culturing the mesenchymal stem cells, and the mesenchymal stem cells are isolated from umbilical cord blood, bone marrow or mononuclear cells present in peripheral blood, and the supernatant is used during the culture period. It is characterized in that it is collected every 4 to 7 days, and the temperature for culturing the mesenchymal stem cells is 25 ° C to 40 ° C, and the mesenchymal stem cell culture is performed for 35 days to 56 days. Characterized in, the method for producing monocytes derived from bone marrow. delete delete delete delete delete delete delete According to claim 1,
The hematopoietic stem cell is characterized in that the culture at 25 ℃ to 40 ℃, bone marrow-derived monocyte production method. According to claim 1,
The hematopoietic stem cell is characterized by culturing in a CO ₂ concentration condition of 5% to 30%, bone marrow-derived monocyte manufacturing method. According to claim 1,
In the step of culturing the hematopoietic stem cells, characterized in that further cultured by GM-CSF, Flt3L and SCF, bone marrow-derived monocyte production method. According to claim 1,
The control medium is characterized in that it is present at a concentration of 5% (v / v) to 20% (v / v) of the total culture medium during the period of culturing hematopoietic stem cells, a method for producing monocyte-derived monocytes. A monocyte produced by the method of claim 1. i) culturing the bone marrow-derived hematopoietic stem cells in the presence of a conditioned medium;
ii) obtaining monocyte cells; And
iii) a method for producing immature dendritic cells, comprising the step of differentiating the obtained monocytes into immature dendritic cells in the presence of GM-CSF and IL-4,
The media is characterized in that it contains Angiogenin, Chitinase 3-like 1, Dkk-1, IGFBP-3, IL-6, Pentraxin 3, Serpin E1, SDF-1, TIMP-1, Thrombospondin-1 and VEGF proteins And
The conditioned medium is the supernatant of the culture medium collected during culturing the mesenchymal stem cells, and the mesenchymal stem cells are isolated from umbilical cord blood, bone marrow or mononuclear cells present in peripheral blood, and the supernatant is used during the culture period. It is characterized in that it is collected every 4 to 7 days, and the temperature for culturing the mesenchymal stem cells is 25 ° C to 40 ° C, and the mesenchymal stem cell culture is performed for 35 days to 56 days. Characterized by, immature dendritic cell manufacturing method. Immature dendritic cells prepared by the method of claim 14. delete delete delete delete delete delete delete delete

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