Cell population and method of obtaining it

Abstract

To provide cell populations, and methods for obtaining the same.SOLUTION: The cell population of the present invention can be obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a medium containing serum and up to four factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor.SELECTED DRAWING: None

Description

[Technical Field]

[0001]

[0001] The present invention relates to a cell population and a method for obtaining the same. The cell population of the present invention is obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum. The present invention also relates to a therapeutic composition for ischemic diseases, inflammatory diseases, or intractable wounds, comprising a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum. [Background technology]

[0002]

[0002] In recent years, cell transplantation therapy using bone marrow mononuclear cell transplantation and endothelial progenitor cells (EPCs) obtained from peripheral blood stem cell collection has become common for ischemic diseases. Therefore, there has been a particular need for technology to culture EPCs in large quantities. A method for in vitro amplification of endothelial progenitor cells from CD34 and / or CD133-positive cells has made it possible to provide an efficient EPC culture technology (Patent Document 1). Furthermore, a method for analyzing the differentiation dynamics of endothelial cells has revealed the existence of cells that form large endothelial cell-like colonies (differentiated EPC colonies) and cells that form small endothelial cell-like colonies (undifferentiated EPC colonies), making it possible to predict and understand the therapeutic effect of cell transplantation (Patent Document 2). In addition, a method for efficiently amplifying CD34 and / or CD133-positive cells from bone marrow mononuclear cells has been shown (Patent Document 3).

[0003]

[0003] As described in International Publication WO2014 / 0561154, a method for amplifying a cell population enriched with vascular endothelial progenitor cells or anti-inflammatory / immune tolerance-inducing cells from mononuclear cell fractions derived from bone marrow, umbilical cord blood, or peripheral blood (hereinafter referred to as the "QQ-MNC method" in this specification) is described. This method involves culturing mononuclear cells in a serum-free medium containing five factors: (1) stem cell factor (SCF), (2) interleukin-6 (IL-6), (3) FMS-like tyrosine kinase 3 ligand (FL), (4) thrombopoietin (TPO), and (5) vascular endothelial growth factor (VEGF), thereby amplifying a cell population containing EPCs in vitro. [Prior art documents] [Patent Documents]

[0004]

[0004] [Patent Document 1] International release WO2006 / 090882 [Patent Document 2] International release WO2006 / 090886 [Patent Document 3] International release WO2006 / 093172 [Patent Document 4] International release WO2014 / 0561154 [Overview of the project] [Problems that the invention aims to solve]

[0005]

[0005] As a result of diligent research, the present inventors have succeeded in obtaining a new cell population that has vascular regeneration and wound healing capabilities and is different from the cell population obtained by the QQ-MNC method, by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum, and have conceived the present invention.

[0006]

[0006] The present invention aims to provide a cell population. The cell population of the present invention is obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum.

[0007]

[0007] The present invention also aims to provide a method for obtaining a cell population. The method of the present invention comprises culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum.

[0008]

[0008] The present invention further aims to provide a composition for the treatment of ischemic diseases, inflammatory diseases, or intractable wounds. The composition of the present invention also comprises a group of cells obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum. [Means for solving the problem]

[0009]

[0009] The present invention includes, but is not limited to, the following embodiments. [Aspect 1] A group of cells obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum. [Aspect 2] The cell population according to aspect 1, wherein the culture medium contains three or four factors selected from stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor. [Aspect 3] The cell population according to aspect 1 or 2, wherein the serum is bovine or human serum. [Aspect 4] A group of cells according to any one of aspects 1-3, wherein serum is contained in the culture medium at a concentration of 0.5% to 10% by volume. [Aspect 5] A cell group according to any one of aspects 1-4, wherein the total percentage of cells that are CD206(+), CD34(+), or CD3(+) is 60% or more, and the percentage of cells that are CCR2(-) is 95% or more. [Aspect 6] The cell population according to aspect 5, wherein 50% or more of the cells in the CD206(+) population are CXCR4(+). [Aspect 7] The cell population according to aspect 5 or 6, wherein 80% or more of the cells in the CD206(+) population are CXCR4(+). [Aspect 8] A method for obtaining a cell population according to any one of aspects 1 to 7, comprising culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum. [Aspect 9] The method according to aspect 8, wherein in the cell population, the total percentage of cells that are CD206(+), CD34(+), or CD3(+) is 60% or more, and the percentage of cells that are CCR2(-) is 95% or more. [Aspect 10] The method according to aspect 8 or 9, wherein in the cell population, 50% or more of the cells are CXCR4(+) among the CD206(+) cells. [Aspect 11] The method according to any one of Aspects 8 - 10, wherein the medium contains three or four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor. [Aspect 12] The method according to any one of Aspects 8 - 11, wherein the serum is fetal bovine serum. [Aspect 13] The method according to any one of Aspects 8 - 12, wherein the serum is contained in the medium at a concentration of 0.5% by volume or more and 10% by volume or less. [Aspect 14] A therapeutic composition for treating an ischemic disease, an inflammatory disease, or a refractory wound, comprising the cell group according to any one of Aspects 1 - 7. [Aspect 15] The therapeutic composition according to Aspect 14, wherein the ischemic disease is limb ischemia. [Aspect 16] The therapeutic composition according to Aspect 14 or 15, wherein the ischemic disease is limb ischemia accompanied by ulcers. [Aspect 17] The therapeutic composition according to any one of Aspects 14 - 16, which promotes angiogenesis and / or wound healing.

Brief Description of the Drawings

[0010]

[0010] [Figure 1] FIG. 1 shows the results of fractionation by flow cytometry (FACS) of the constituent cell components of RE-01 prepared from the peripheral blood of a healthy subject. As a comparative example, the FACS results of peripheral blood mononuclear cells before culture are also shown. [Figure 2] FIG. 2 shows the analysis results of flow cytometry (FACS) of the constituent cell components of RE-01 prepared from the peripheral blood of a healthy subject different from that in FIG. 1. [Figure 3] FIG. 3 shows the analysis results of flow cytometry (FACS) of the constituent cell components of RE-01 prepared from the peripheral blood of a 53-year-old diabetic female patient. As a comparative example, a cell group (MNC-QQ) obtained by culturing using a serum-free medium containing five factors was used. [Figure 4]Figure 4 shows the results of investigating the angiogenic ability of RE-01 using human umbilical vein endothelial cells (HUVECs). For comparative examples, the results were also investigated when MNC-QQ was used instead of RE-01, and when HUVECs were used alone. Figure 4A shows the change in the number of formed lumens over time from the start of culture, and Figure 4B shows the number of formed lumens 2.75 hours after the start of culture. Figure 4C shows the number of fluorescently labeled test cells incorporated into the lumen structure 2.75 hours after the start of culture. [Figure 5] Figure 5 shows the results of flow cytometry (FACS) fractionation of the constituent cellular components of RE-01 prepared from peripheral blood of patients with severe lower limb ischemia. [Modes for carrying out the invention]

[0011]

[0011] 1.Cell group This invention relates to a group of cells.

[0012] The cell population of the present invention is obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum.

[0012]

[0013] The above cell population is obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood. The "mononuclear cells" used to obtain the above cell population are a general term for cells with a round nucleus contained in peripheral blood, bone marrow, or umbilical cord blood, and include lymphocytes, monocytes, macrophages, vascular endothelial progenitor cells, hematopoietic stem cells, etc. For example, mononuclear cells can be obtained by collecting bone marrow, umbilical cord blood, or peripheral blood from an animal and extracting the fraction using, for example, a blood collection tube for mononuclear cell separation, or by density gradient centrifugation. The density gradient centrifugation method is not particularly limited as long as a mononuclear cell fraction is formed. For example, Histopaque-1077 (Sigma-Aldrich) may be used.

[0013]

[0014] The animal species from which bone marrow, umbilical cord blood, or peripheral blood is derived is not particularly limited. Animal species include mammals in general, including humans, to which cell transplantation therapy is applied for diseases such as ischemic diseases, inflammatory diseases, or intractable wounds. From the perspective of clinical application, humans are preferred.

[0014]

[0015] The subjects from which the bone marrow, umbilical cord blood, or peripheral blood is derived are not particularly limited. In one embodiment, for example, they may be healthy individuals, diabetic patients, or patients with severe lower limb ischemia.

[0016] Stem cell factor (SCF) is a glycoprotein with a molecular weight of approximately 30,000, consisting of 248 amino acids. While soluble and membrane-bound forms exist due to alternative splicing, any type of SCF is acceptable for obtaining the above-mentioned cell population, as long as it is useful for culturing mononuclear cells. The soluble form is preferred. The origin of the SCF is not particularly limited. Non-limitingly, recombinants with a stable supply are preferred, and human recombinants are particularly preferred. Commercially available SCFs are known. The concentration of SCF in the culture medium varies depending on the type of SCF used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant SCF, non-limitingly, for example, 10-1000 ng / mL, preferably 50-500 ng / mL, and more preferably about 100 ng / mL.

[0015]

[0017] Interleukin-6 (IL-6) is a glycoprotein with a molecular weight of 210,000 that was isolated as a factor that induces the terminal differentiation of B cells into antibody-producing cells. IL-6 is generally known to be involved in immune responses, proliferation and differentiation of hematopoietic and nervous system cells, and acute phase reactions. The IL-6 used to obtain the above cell population is not particularly limited and can be selected as appropriate. When used for culturing human mononuclear cells, human IL-6 is preferred, and recombinant IL-6 that is expected to have a stable supply is particularly preferred. Commercially available IL-6 is known. The concentration of IL-6 in the culture medium varies depending on the type of IL-6 used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant IL-6, it is non-limited to, for example, 1 to 500 ng / mL, preferably 5 to 100 ng / mL, and more preferably about 20 ng / mL.

[0016]

[0018] FMS-like tyrosine kinase 3 ligand (FL) is known as a ligand for receptor tyrosine kinases that play an important role in the early regulation of hematopoiesis. Several alternative splicing products are known, and some have been reported to stimulate the proliferation of hematopoietic stem cells. Any type of FL may be used to obtain the above cell population, as long as it is useful for culturing mononuclear cells. Commercially available types are known. The concentration of FL in the culture medium varies depending on the type of FL used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant Flt-3 ligand, it is non-limiting, for example, 10 to 1000 ng / mL, preferably 50 to 500 ng / mL, and more preferably about 100 ng / mL.

[0017]

[0019] Thrombopoietin (TPO) is a type of hematopoietic cytokine that specifically acts on the process by which megakaryocytes are produced from hematopoietic stem cells, and is known to promote megakaryocyte production. The origin of the TPO used to obtain the above cell population is not particularly limited. Recombinant TPO that can be expected to have a stable supply is preferred, and human recombinant TPO is particularly preferred. Commercially available TPO is known. The concentration of TPO in the culture medium varies depending on the type of TPO used and is not particularly limited as long as it is useful for culturing mononuclear cells, but in the case of human recombinant TPO, it is not limited to, for example, 1 to 500 ng / mL, preferably 5 to 100 ng / mL, and more preferably about 20 ng / mL.

[0018]

[0020] Vascular endothelial growth factor (VEGF) is a growth factor that specifically acts on vascular endothelial progenitor cells (EPCs) and is known to be mainly produced in perivascular cells. Several types of VEGF proteins of different sizes are produced by alternative splicing, but any type of VEGF may be used to obtain the above-mentioned cell population, as long as it enables EPC colony formation. VEGF165 is preferred. The origin of the VEGF is not particularly limited. Recombinant VEGFs that are expected to have a stable supply are preferred, and human recombinant VEGFs are particularly preferred. Commercially available recombinant VEGFs are known. The concentration of VEGF in the culture medium varies depending on the type of VEGF used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant VEGF165, for example, it is not limited to about 5 to 500 ng / mL, preferably about 20 to 100 ng / mL, and more preferably about 50 ng / mL.

[0019]

[0021] The various factors added to the culture medium used for culturing mononuclear cells may, without limitation, be unified by factors derived from the same animal species as the animal from which the mononuclear cells originate. By unifying the origin of the mononuclear cells and various factors in this way, cell cultures suitable for allogeneic transplantation, such as allogeneic transplantation, can be obtained. Furthermore, by using mononuclear cells derived from the individual from which the cell transplant is intended, it is also possible to obtain cell cultures suitable for allogeneic transplantation.

[0020]

[0022] The above-mentioned components can be dissolved to a predetermined concentration in a culture medium, or a concentrated solution (stock solution) of each component can be prepared in advance and diluted to a predetermined concentration in the culture medium to prepare a culture medium for culturing mononuclear cells. For example, the necessary components can be dissolved in a commercially available culture medium to a predetermined concentration, and then sterilized by filtration sterilization, or a sterilized stock solution can be aseptically added to a commercially available culture medium and diluted to prepare a culture medium. Filtration sterilization can be carried out in accordance with methods commonly used in this field, for example, using a 0.22 μm or 0.45 μm Millipore filter.

[0021]

[0023] The "culture medium" used in this invention can be any medium commonly used in the field, for example, a culture medium known as a culture medium for hematopoietic stem cell proliferation can be used. Examples of basal media that can be used as culture media include Stemline II, DMEM, MEM, IMDM, RPMI, SCGM, and EBM.

[0022]

[0024] To obtain the above cell population, the culture medium for mononuclear cells contains serum, in addition to four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0023]

[0025] The type of serum is not particularly limited. Non-limitingly, in one embodiment, the serum is bovine or human serum. In one embodiment, the serum is fetal bovine serum and / or human serum albumin. The concentration of serum in the culture medium is also not particularly limited. In one embodiment, the serum is contained in the culture medium at concentrations of 0.1% by volume or more, 0.3% by volume or more, or 0.5% by volume or more. There is no particular upper limit to the concentration of serum in the culture medium. In one embodiment, it is within 30% by volume, within 20% by volume, within 10% by volume or within 5% by volume. Non-limitingly, the serum is contained in the culture medium at concentrations of 0.1% by volume or more and 20% by volume, or at concentrations of 0.5% by volume or more and 10% by volume.

[0024]

[0026] Mononuclear cell culture is performed by adding a cell suspension containing mononuclear cells to a culture medium containing the factors and serum mentioned above. Alternatively, body fluids containing mononuclear cells themselves (e.g., bone marrow fluid, umbilical cord blood, peripheral blood) can be used as the cell suspension. The culture conditions for mononuclear cells are not particularly limited and can be those commonly used in this field. Non-limited conditions include, for example, culturing at approximately 37°C under a 5% CO2 atmosphere. The culture period is non-limited to, for example, 3 days or more, and can be within 5 days, 6 days, 7 days, or 10 days. For example, 3-10 days or 3-6 days. The concentration of mononuclear cells in the culture medium is not particularly limited as long as it allows for mononuclear cell culture, but for example, approximately 0.1 to 10 × 10⁻⁶. 6 Cells / ml, more preferably about 0.5-5 × 10⁶ 6 The value is cells / ml.

[0025]

[0027] In this specification, "cell population" refers to the collective term for cells obtained by culturing mononuclear cells in a culture medium containing the factors and serum described above.

[0028] The culture medium used for culturing mononuclear cells contains four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0026]

[0029] In one embodiment, the culture medium contains two or more factors selected from stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, or three or more factors. In one embodiment, the culture medium contains two to four factors selected from the above five factors. In one embodiment, the culture medium contains three or four factors selected from stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0027]

[0030] Non-limitingly, the culture medium used for culturing mononuclear cells, and therefore the culture medium used in the culture method of the present invention, includes, for example, i) a combination of FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, or ii) a combination of stem cell factor, FMS-like tyrosine kinase 3 ligand, and vascular endothelial growth factor. The medium more preferably contains a combination of about 80-120 ng / ml of FMS-like tyrosine kinase 3 ligand, about 15-25 ng / ml of thrombopoietin, and about 40-60 ng / ml of vascular endothelial growth factor.

[0028]

[0031] In one embodiment, a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum, contains more CD206(+), CD34(+), or CD3(+) cells than the mononuclear cell population before culture. Non-limitingly, the proportion of CD206(+), CD34(+), or CD3(+) cells is amplified by 1.5 times or more, 2 times or more, or 3 times or more by culture. Non-limitingly, the total number of CD206(+), CD34(+), or CD3(+) cells in the cell population is 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total cell population. Furthermore, the cell population contains more CCR2(-) cells than the mononuclear cell population before culture. Non-limitingly, the proportion of CCR2(-) cells is amplified by culture by 1.5 times or more, 2 times or more, or 3 times or more. Non-limitingly, in a cell population, CCR2(-) cells constitute 60% or more, 80% or more, 90% or more, or 95% or more of the total cell population. Non-limitingly, in one embodiment, 60% or more of the cells are CD206(+), CD34(+), and CD3(+), and 95% or more of the cells are CCR2(-).

[0029]

[0032] By culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum, a cell population containing a higher proportion of CD206(+), CD34(+), and CD3(+) can be stably obtained. Furthermore, the proportion of CXCR4-positive cells among the CD206-positive cells, including anti-inflammatory macrophages M2, is significantly increased compared to the cell population obtained by the QQ-MNC method. Non-limitingly, the inventors have found that the following effects can be observed with the above culture method.

[0030]

[0033] CD206 positivity increases, including in anti-inflammatory macrophages M2. CD34-positive cells, including vascular endothelial progenitor cells (EPCs), and CD133-positive cells increase; T cells, especially CD3-positive cells such as Helper T cells and Angiogenic T cells, increase.

[0031]

[0034] On the other hand, a decrease in inflammatory monocytes / macrophages, specifically CCR2-positive cells, B cells (CD19-positive cells), and NK cells (CD56-positive cells), was observed.

[0035] Non-limitingly, in one embodiment, a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum, shows a higher recovery rate than the cell population obtained by the QQ-MNC method. Furthermore, it exhibits a higher viability and superior stability after recovery. These characteristics lead to higher therapeutic efficacy.

[0032]

[0036] In one embodiment, a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum, contains a greater proportion of CD206(+) cells with CXCR4(+) than the mononuclear cell population before culture. Non-limitingly, the proportion of CD206(+) cells with CXCR4(+) is amplified by 1.5 times or more, 2 times or more, or 3 times or more by culture. Non-limitingly, the proportion of CD206(+) cells with CXCR4(+) is 50% or more, 60% or more, 70% or more, or 80% or more. In one embodiment, the proportion of CD206(+) cells with CXCR4(+) is 50% or more. In one embodiment, the proportion of CD206(+) cells with CXCR4(+) is 80% or more.

[0033]

[0037] The cell population of the present invention contains a particularly large number of CXCR4(+) cells among the CD206(+) compared to the cell population obtained by the QQ-MNC method.

[0038] 2. Method for obtaining cell populations The present invention also relates to a method for obtaining a population of cells.

[0034]

[0039] The method of the present invention comprises culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum.

[0035]

[0040] The "cell population," "mononuclear cells," "stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor," "serum," "culture medium," etc., are as described in "1. Cell Population" above.

[0036]

[0041] The proportions of CD206(+), CD34(+), and CD3(+) cells, the proportion of CCR2(-) cells, and the proportion of CXCR4(+) cells among the CD206(+) cells in the cell population are as described in "1. Cell Population" above.

[0037]

[0042] In one embodiment, in the cell population obtained by the above method, more than 80% of the cells are CD206(+), CD34(+), and CD3(+), and more than 95% of the cells are CCR2(-).

[0038]

[0043] In one embodiment, in the cell population obtained by the above method, more than 50% of the cells are CXCR4(+) among the CD206(+) cells.

[0044] In one embodiment, the culture medium in the above method comprises three or four factors selected from stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0039]

[0045] In one embodiment, the serum in the above method is bovine or human serum. In one embodiment, the serum is fetal bovine serum. In one embodiment, the serum is contained in the culture medium at a concentration of 0.5% to 10% by volume.

[0040]

[0046] 3. Therapeutic composition The present invention further relates to a therapeutic composition for ischemic diseases, inflammatory diseases, or intractable wounds. The therapeutic composition of the present invention includes the cell group described in "1. Cell Group". The mononuclear cells from which the cell group is derived may be derived from the subject to which the therapeutic composition is used (autologous) or from a subject other than the subject to which the therapeutic composition is used (allogeneic).

[0041]

[0047] "Ischemic diseases" are conditions caused by a decrease in blood volume, leading to reduced blood flow within tissues and resulting in tissue damage such as cell degeneration, atrophy, and fibrosis. Ischemia is broadly classified into obstructive ischemia, compressive ischemia, spastic ischemia, and compensatory ischemia depending on its cause. If ischemia persists, cell degeneration, atrophy, and fibrosis occur. "Ischemic diseases" include limb ischemia, ischemic ulcers, ischemic heart disease, and cerebral infarction. "Cerebral infarction" (or cerebral softening) refers to cerebral ischemia caused by the occlusion or narrowing of arteries that nourish the brain, resulting in necrosis or near-necrosis of brain tissue due to lack of oxygen or nutrients. It is classified into cerebral thrombosis and cerebral embolism. "Limb ischemia" is a disease in which the arteries that supply blood to the limbs are narrowed or blocked. If this obstructive arteriosclerosis becomes severe, it can lead to severe limb ischemia, causing symptoms such as pain and intractable ulcers, and in the worst case, amputation may be necessary. In a non-limiting manner, the limbs are, in one embodiment, the lower limbs.

[0042]

[0048] In a non-limiting manner, in one aspect, ischemic disease is ischemia of the limbs. Ischemic disease is ischemia of the limbs accompanied by ulcers.

[0049] "Inflammatory diseases" are a general term for diseases that cause symptoms due to abnormalities such as tissue damage caused by some reason. Inflammatory diseases include Crohn's disease, cirrhosis, hepatitis, ulcerative colitis, and inflammatory bowel disease.

[0043]

[0050] The above-mentioned therapeutic composition promotes angiogenesis and / or wound healing. Therefore, it can be applied to diseases that can be treated by angiogenesis and / or wound healing. Examples of "diseases that can be treated by angiogenesis and / or wound healing" include ischemic diseases (e.g., myocardial infarction, ischemic heart diseases such as angina pectoris, lower limb ischemia such as lower limb ischemic arteriosclerosis, Buerger's disease), and vascular injuries. It can also be used to heal wounds such as skin ulcers or to create artificial blood vessels. The effects of applying the therapeutic composition can be confirmed by methods that are already known. For example, if the disease that can be treated by angiogenesis is a lower limb ischemic disease, the therapeutic effect after transplantation can be evaluated by examining, for example, the lower limb blood flow and the rate of necrosis improvement. The increase in blood flow can be measured by measuring the value of laser Doppler imaging analysis. The rate of necrosis improvement can be measured visually as a limb salvage score.

[0044]

[0051] The manner in which the above therapeutic composition is used is not particularly limited. It may be the cell population itself, which is the active ingredient, or the cell population suspended in a liquid medium. The liquid medium may be any liquid that can be injected into a human, for example, isotonic electrolyte solutions, phosphate buffer, physiological saline, or serum-free medium such as DMEM can be used. The liquid medium may also contain compounds that are favorable for cell survival, such as albumin. Preferably, serum derived from the patient is used as a compound containing albumin. If cryopreservation is required, the cells may be suspended in a cryopreservation solution or similar.

[0045]

[0052] The therapeutic compositions described above are not particularly limited to any subject requiring treatment for ischemic disease, inflammatory disease, or intractable wounds. Non-limited applications include humans, monkeys, chimpanzees, dogs, cats, cattle, horses, mice, guinea pigs, and the like.

[0046]

[0053] 4. Kit The present invention also relates to a kit for culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood and obtaining a cell population.

[0047]

[0054] The above kit includes a culture medium containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum. Each factor and serum may be pre-added to the culture medium, or they may be stored in separate containers and added to the culture medium at the time of use.

[0048]

[0055] The "cell population," "mononuclear cells," "stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor," "serum," "culture medium," etc., are as described in "1. Cell Population" above.

[0049]

[0056] 5.Treatment method etc. The present invention also relates to a method for treating ischemic diseases, inflammatory diseases, or intractable wounds, comprising applying the cell population described in "1. Cell Population" to the target of need.

[0050]

[0057] The present invention further relates to the use of the cell population described in "1. Cell Population" in a method for treating ischemic diseases, inflammatory diseases, or intractable wounds, or in a composition for treating ischemic diseases, inflammatory diseases, or intractable wounds, including applying the cell population to the target area where necessary.

[0051]

[0058] Regarding "ischemic diseases," "inflammatory diseases," or "intractable wounds," "application methods of cell groups," and "target populations," etc., please refer to the information provided in "3. Therapeutic Compositions." [Examples]

[0052]

[0059] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and change the present invention based on the description herein, and such modifications are within the technical scope of the present invention.

[0053]

[0060] Example 1: Culture of RE-01 cell population In this example, mononuclear cells were isolated from peripheral blood and cultured.

[0061] (1) Isolation of mononuclear cells 100 mL of peripheral blood was collected from healthy volunteers or diabetic patients using BD Vacutainer® CPT® mononuclear cell isolation tubes (manufactured by BD). After blood collection, the tubes were centrifuged and then stored refrigerated for transport until culture was started upon arrival. The portion of the CPT® tube containing mononuclear cells and plasma above the gel barrier was collected into a centrifuge tube, the gel barrier and other parts of the tube were rinsed with a small amount of EDTA-PBS, and the contents were collected into the same centrifuge tube. The centrifuge tube containing the collected cells was made up with EDTA-PBS, then centrifuged (300xg, room temperature, 15 minutes), and the cells were collected as sediment. The collected cells were incubated in ACK hemolysis buffer (15 mL / tube) (Gibco, Thermo-Fisher) at room temperature for 5 minutes to remove any contaminating red blood cells. The ACK hemolysis buffer was composed of NH4Cl 8,290 mg / l; KHCO3 1,000 mg / l; and EDTA.Na2·2H2O 37 mg / l. Subsequently, the solution was made up with EDTA-PBS, and the cells were collected by centrifugation (200xg, room temperature, 10 minutes) or (100xg, room temperature, 15 minutes), and this procedure was repeated twice.

[0054]

[0062] (2) Culture in serum medium (1) The mononuclear cells collected in step (1) were placed in 1 mL of growth medium (50 ng / mL VEGF) 165 Cell counting was performed using trypan blue in stemline® II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich, Cat No. S0192) supplemented with 20 ng / mL TPO, 100 ng / mL Flt-3 ligand, 100 units / mL penicillin, 100 μg / mL streptomycin, and 0.5% FBS. A portion of this suspension was used to count cells. In the resulting cell proliferation medium, the cell concentration was increased to 1 x 10⁻⁶. 6The solution was prepared to 1 / mL and 2 mL was seeded into each well of a 6-well culture plate. The cells were cultured for 5 days under normal culture conditions (37°C, 5% CO2). After 5 days of culture, the cells were collected in a centrifuge tube and centrifuged three times at 250xg (~1000 rpm) for 7-10 minutes. After centrifugation, samples were removed for cell counting, viability measurement, and QC (confirmation) testing. The remaining cells were suspended in PlasmaLyte A and 2.5% human albumin serum and 8x10⁻¹⁴ 5 The concentration was adjusted to the desired number of cells / mL. Hereafter, the resulting cell population may be referred to as "RE-01".

[0055]

[0063] Example 2: Characterization of the RE-01 cell population

[0064] (1) Flow cytometry analysis In this example, in Example 1, flow cytometry analysis was performed on a cell population (referred to as "RE-01") obtained by culturing peripheral blood from healthy individuals using a culture medium containing three types of factors and serum, in order to further clarify the characteristics of the cell population.

[0056]

[0065] Cells (1.5 × 10⁶) suspended in FACS buffer (composition: 2 mM EDTA-PBS with 2% FBS added) 6 10 μL of FC blocking reagent (Milteny) was added to 300 μL of cells in FACS buffer and incubated at 4°C for 30 minutes. The incubated cell population was dispensed in equal volumes into staining reaction tubes (100 μL / tube x 3 tubes). 2 μL of each primary antibody was added to each aliquot and cultured at 4°C for 20 minutes. The cells were then washed twice with 1 mL of FACS buffer and the stained cells were suspended in FACS buffer (5 x 10⁶). 5 Cells were mixed in 200-300 μL of FACS buffer. Flow cytometry was performed using a BD FACSAria® III cell sorter (BD).

[0057]

[0066] Specifically, the expression of each cell surface marker was examined using antibodies against each cell surface marker in the obtained cell population. The antibodies used for each cell surface marker were all commercially available products as listed below.

[0058]

[0067] Anti-CD206 antibody: PE / Cy7-labeled anti-human CD206 (MMR) antibody (manufactured by BioLegend); Anti-CD34 antibody: PE-labeled anti-human CD34 antibody (manufactured by Bio Legend); Anti-CD3 antibody: Alexa Fluor700-labeled anti-CD3 antibody (manufactured by Bio Legend); Anti-CXCR4 antibody: APC-labeled anti-human CD184 (CXCR4) antibody (manufactured by BD Corporation); Anti-CCR2 antibody: PerCP / Cy5.5 labeled human CD192 (CCR2) antibody (manufactured by BioLegend).

[0059]

[0068] The results are shown in Figure 1. As shown in Figure 1, RE-01 can be broadly divided into three live cell populations based on the size of the constituent cellular components (horizontal axis: FSC) and the density of cellular components such as granules contained within the cells (vertical axis: SSC) in FACS scatter analysis (referred to as "region A," "region B," and "region C" in this specification). In peripheral blood mononuclear cells before culture, only regions A and B are present, and region C is not observed. Region A mainly contains lymphocytes, and region B contains monocytes. In RE-01 obtained by culture using growth medium (serum medium (2) in Example 1), vascular endothelial progenitor cells (CD34-positive cells) migrate from region A to region B, and region C, which contains many M2 macrophages (CD206-positive cells), appears.

[0060]

[0069] In addition, the results of flow cytometry analysis of RE-01 obtained by culturing monocytes collected from another healthy individual using the medium containing the three types of factors and serum described in Example 1 are shown in FIG. 2. Similarly, in the case of healthy individuals of different individuals, three regions A, B, and C appeared. Also, the total of cells that are CD206(+), CD34(+), or CD3(+) was 86.57%, and since CCR2(+) was 0.33%, the cells that are CCR2(-) were 99.67%. Furthermore, the cells that are CXCR4(+) among CD206(+) were 92.97%.

[0061]

[0070] Example 3 Comparison between RE-01 cell group and MNC-QQ cell group

[0071] (1) Culturing of RE-01 and MNC-QQ Monocytes collected from healthy individuals and diabetic patients were cultured using the medium containing three types of factors and serum in the same manner as in Example 1 to obtain RE-01.

[0062]

[0072] As a comparative example, after isolating monocytes in the same manner as RE-01 cells, stemline (registered trademark) II Hematopoietic Stem Cell Expansion Medium (manufactured by Sigma-Aldrich, Cat No. S0192) was used. Serum was not added, and 100 units / mL penicillin, 100 μg / mL streptomycin, and 50 ng / mL VEGF 165 , 20 ng / mL TPO, 100 ng / mL Flt-3 ligand, 100 ng / mL SCF, and 20 ng / mL IL-6 were aseptically added to prepare five types of each factor. The obtained cells were adjusted to 1x10 6 / mL in a growth medium, seeded 2 mL each into wells of a 6-well culture plate, and cultured for 7 days under normal culture conditions (37 °C, 5% CO2). After 7 days of culture, the cells were collected into a centrifuge tube, and centrifugal washing was performed 3 times at 250 xg (~1000 rpm) for 7 - 10 minutes. After centrifugation, except for samples for cell counting, viability measurement, and QC (confirmation) test, the remaining cells were suspended in PlasmaLyte

[0073] A, 2.5% human albumin serum to 8x10 5The solution was prepared to a concentration of cells / mL. Hereafter, the resulting cell population may be referred to as "MNC-QQ".

[0063]

[0073] (2) Flow cytometry analysis In Example 1, flow cytometry was performed on cell populations obtained by culturing peripheral blood from diabetic patients using a medium containing three factors and serum, and cell populations obtained by culturing using serum-free medium containing the five factors described in (1) above (MNC-QQ), and the two were compared. Specifically, the expression of each cell surface marker was examined using antibodies against each cell surface marker in the obtained cell populations.

[0064]

[0074] The results are shown in Figure 3. RE-01 showed higher recovery and survival rates after culture compared to MNC-QQ. Furthermore, it had a higher proportion of C-region cells, which are newly formed cell groups after culture, and in particular, a higher proportion of CD206+ cells, one of the cell types responsible for the effectiveness of the cell group. Surprisingly, the proportion of CXCR4+ cells within the CD206+ cells was significantly higher, indicating that the cell composition of the RE-01 and MNC-QQ cell groups differed.

[0065]

[0075] (3) acLDL uptake capability In Example 1, we examined the acLDL (acetylated low-density lipoprotein) uptake ability of a cell population (RE-01) obtained by culturing cells prepared from peripheral blood mononuclear cells of diabetic patients using a medium containing three factors and serum, and, as a comparative example, a cell population (MNC-QQ) obtained by culturing cells using a serum-free medium containing five factors.

[0066]

[0076] RE-01 or MNC-QQ (1x10) collected in a 1.5 mL tube 5The cells were suspended in 500 μL of basal medium (Stemline II), 5 μL of acLDL labeled with Alexa Fluor488 was added, and the tubes were incubated at 37°C for 60 minutes. After incubation, 1 mL of FACS buffer was added to the tubes and mixed by inversion. Then, the tubes were centrifuged for 5 minutes (250 x g, 4°C) and the cells were collected as sediment. The collected cells (RE-01) were suspended in 300 μL of FACS buffer and stored on ice until measurement. Immediately before measurement, DAPI (2 μL) was added, and the percentage of cells that incorporated acLDL and DAPI was determined by flow cytometry.

[0067]

[0077] The results are shown in Table 1 below.

[0068] [Table 1]

[0069]

[0078] As shown in Table 1, high acLDL uptake was observed overall, particularly in the C region. This supports the idea that the compositional differences shown in Example 2 are linked to functional differences between the two cell groups (RE-01 and MNC-QQ).

[0070]

[0079] (4)Angiogenic ability In this example, mononuclear cells collected from the blood of healthy individuals were cultured in serum medium using the same method as in Example 1 (RE-01), and their angiogenic ability was examined. As a comparative example, a cell population (MNC-QQ) obtained by culturing in serum-free medium containing five factors was used. Human umbilical vein endothelial cells (HUVEC) with lumen-forming ability (obtained from Lonza, Basel, Switzerland) were used in passages of 8-10, and each cell group was co-cultured with the HUVEC. 50 μl / well of basement membrane matrix (Corning® Matrigel® (Corning, USA, NY)) was dispensed into a 96-well plate and coated at 37°C for 30 minutes. Meanwhile, each cell population was suspended in 500 μl IMDM + 5 μl DiI-Ac-LDL to label with DiI-Ac-LDL and incubated at 37°C for 1 hour.

[0071]

[0080] After labeling, each cell group was divided into 1x10 3 / 20μl, and HUVEC 5x10 3 Each cell was resuspended in PBS to a volume of 20 μl. The cell-count-adjusted suspensions were mixed with the test cell group and HUVEC in a 1:1 ratio, and 50 μl of each was added to Matrigel. The cells were cultured for more than 10 hours at 37°C under 5% CO2 in a time-lapse fluorescence microscope (Olympus, Tokyo, Japan), while taking photographs. After the culture period, the number of formed lumens and the number of fluorescently labeled test cells incorporated into the lumens were visually counted at 100x magnification.

[0072]

[0081] The results are shown in Figure 4. Figure 4A shows the change in the number of formed lumens over time from the start of culture, and Figure 4B shows the number of formed lumens 2.75 hours after the start of culture. Figure 4C shows the number of fluorescently labeled test cells incorporated into the lumen structure 2.75 hours after the start of culture. The lumen formation ability when HUVEC and RE-01 were co-cultured for a certain period of time was improved compared to culturing HUVEC alone or co-culturing HUVEC with MNC-QQ. Furthermore, more RE-01 cells were incorporated into the formed lumen structure. In other words, it was revealed that RE-01 had higher in vitro angiogenesis ability than MNC-QQ cultured for a longer period (7 hours) in serum-free medium in vitro. This supports the idea that the difference in composition shown in Example 3 is linked to the difference in function between the two cell groups (RE-01 and MNC-QQ).

[0073]

[0082] (5) Effects using a mouse lower limb ischemia model The therapeutic effect of mononuclear cells cultured using the same method as in Example 1 on lower limb ischemia was investigated using a mouse model of lower limb ischemia. As a comparative example, a cell population (MNC-QQ) obtained by culturing in serum-free medium containing five factors was used.

[0074]

[0083] (6) Stability Test The stability of mononuclear cells collected from the blood of healthy individuals and cultured in the same manner as in Example 1 was examined up to 72 hours after harvesting by measuring cell number and viability. As a comparative example, a cell population (MNC-QQ) obtained by culturing in serum-free medium containing five factors was used.

[0075]

[0084] Example 4: Culture and characterization of RE-01 cell population In this example, mononuclear cells were isolated and cultured from the peripheral blood of patients with severe lower limb ischemia. Furthermore, the characteristics of the resulting cell population were evaluated.

[0076]

[0085] (1) Isolation of mononuclear cells Mononuclear cells were isolated from the peripheral blood of patients with severe lower limb ischemia. Isolation was performed in the same manner as in Example 1.

[0086] Specifically, 100 mL of peripheral blood was collected from patients with severe lower limb ischemia using a BD Vacutainer® CPT® mononuclear cell isolation tube (manufactured by BD). After blood collection, the tube was centrifuged and then stored refrigerated for transport until culture was started upon arrival. The portion of the CPT® tube containing mononuclear cells and plasma above the gel barrier was collected into a centrifuge tube, the gel barrier and other parts of the tube were rinsed with a small amount of EDTA-PBS, and the contents were collected into the same centrifuge tube. The centrifuge tube containing the collected cells was made up with EDTA-PBS, then centrifuged (300xg, room temperature, 15 minutes), and the cells were collected as sediment. The collected cells were incubated in ACK hemolysis buffer (15 mL / tube) (Gibco, Thermo-Fisher) at room temperature for 5 minutes to remove any contaminating red blood cells. The ACK hemolysis buffer was composed of NH4Cl 8,290 mg / l; KHCO3 1,000 mg / l; and EDTA.Na2·2H2O 37 mg / l. Subsequently, the solution was made up with EDTA-PBS, and the cells were collected by centrifugation (200xg, room temperature, 10 minutes) or (100xg, room temperature, 15 minutes), and this procedure was repeated twice.

[0077]

[0087] (2) Culture in serum medium The isolated mononuclear cells were cultured in serum medium in the same manner as in Example 1. The differences from Example 1 are that Iscove's Modified Dulbecco's Medium (IMDM) was used as the basic medium instead of stemline®, and human albumin serum was added in addition to FBS as the serum.

[0078]

[0088] Specifically, the mononuclear cells collected in (1) are placed in 1 mL of growth medium (50 ng / mL VEGF 165The cells were suspended in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20 ng / mL TPO, 100 ng / mL Flt-3 ligand, 100 units / mL penicillin, 100 μg / mL streptomycin, 0.5% FBS, and 0.5% human albumin serum, and a portion of this was used for cell counting using trypan blue. In the resulting cell growth medium, the cell concentration was increased to 1 x 10⁻⁶. 6 The solution was prepared to 1 / mL and 2 mL was seeded into each well of a 6-well culture plate. The cells were cultured for 5 days under normal culture conditions (37°C, 5% CO2). After 5 days of culture, the cells were collected in a centrifuge tube and centrifuged three times at 250xg (~1000 rpm) for 7-10 minutes. After centrifugation, samples were removed for cell counting, viability measurement, and QC (confirmation) testing. The remaining cells were suspended in PlasmaLyte A and 2.5% human albumin serum and 8x10⁻¹⁴ 5 The concentration was adjusted to cells / mL.

[0079]

[0089] (3) Flow cytometry analysis In this example, in (1) and (2), mononuclear cells isolated from the peripheral blood of patients with severe lower limb ischemia were cultured in a medium containing three types of factors and serum to obtain a cell population (referred to as "RE-01" as in Example 1). Flow cytometry analysis was performed to further clarify the characteristics of the cell population.

[0080]

[0090] Cells (1.5 × 10⁶) suspended in FACS buffer (composition: 2 mM EDTA-PBS with 2% FBS added) 6 10 μL of FC blocking reagent (Milteny) was added to 300 μL of cells in FACS buffer and incubated at 4°C for 30 minutes. The incubated cell population was dispensed in equal volumes into staining reaction tubes (100 μL / tube x 3 tubes). 2 μL of each primary antibody was added to each aliquot and cultured at 4°C for 20 minutes. The cells were then washed twice with 1 mL of FACS buffer and the stained cells were suspended in FACS buffer (5 x 10⁶). 5Cells were mixed in 200-300 μL of FACS buffer. Flow cytometry was performed using a BD FACSAria® III cell sorter (BD).

[0081]

[0091] Specifically, the expression of each cell surface marker was examined using antibodies against each cell surface marker in the obtained cell population. The antibodies used for each cell surface marker were all commercially available products as listed below.

[0082]

[0092] Anti-CD206 antibody: PE / Cy7-labeled anti-human CD206 (MMR) antibody (manufactured by BioLegend); Anti-CD34 antibody: PE-labeled anti-human CD34 antibody (manufactured by Bio Legend); Anti-CD3 antibody: Alexa Fluor700-labeled anti-CD3 antibody (manufactured by Bio Legend); Anti-CXCR4 antibody: APC-labeled anti-human CD184 (CXCR4) antibody (manufactured by BD Corporation); Anti-CCR2 antibody: PerCP / Cy5.5 labeled human CD192 (CCR2) antibody (manufactured by BioLegend).

[0083]

[0093] The results are shown in Figure 5. As shown in Figure 5, the same three regions, A, B, and C, appeared in the cell population cultured from mononuclear cells isolated from the peripheral blood of patients with severe lower limb ischemia, as in Example 2. Furthermore, 83.72% of the cells were CD206(+), CD34(+), or CD3(+), and 1.63% were CCR2(+), meaning that 98.37% of the cells were CCR2(-). In addition, 83.72% of the CD206(+) cells were CXCR4(+).

[0084]

[0094] In summary, it was revealed that, similar to the healthy individuals or diabetic patients in Example 1, RE-01 cell populations, particularly those in which more than 80% of cells are CD206(+), CD34(+), or CD3(+), more than 95% are CCR2(-), and more than 80% of the CD206(+) cells are CXCR4(+), were obtained from patients with severe lower limb ischemia by culturing them in a medium containing three types of factors and serum.

[0085] Industrial applicability

[0095] This invention provides a novel cell population with high vascular regeneration and wound healing capabilities. This cell population can be used, for example, as a therapeutic agent for patients with intractable limb ischemic diseases. This could lead to significant life innovations, such as improving patient quality of life (QOL) and reducing the burden of care, including the possibility of avoiding amputation, thus having great social significance. Furthermore, the provision of this novel cell population could lead to the development of new treatment strategies and provide insights into vascular lesions in general. Moreover, in addition to its high efficacy, this cell population remarkably achieves consistently high recovery and survival rates with shorter culture periods compared to existing QQ-MNC methods, and is also more stable after production, offering significant industrial advantages.

Claims

1. A kit for use in a method for obtaining a cell population from mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood, wherein the total percentage of cells is 60% or more that are CD206(+), CD34(+), or CD3(+), and the percentage of cells is 95% or more that are CCR2(-), A mixture containing four or fewer factors selected from the group consisting of stem cell factors, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, along with serum, in the same or separate containers, The method for obtaining the aforementioned cell population includes culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of 10-1000 ng / mL of stem cell factor, 1-500 ng / mL of interleukin-6, 10-1000 ng / mL of FMS-like tyrosine kinase 3 ligand, 1-500 ng / mL of thrombopoietin, and 5-500 ng / mL of vascular endothelial growth factor, and serum at a concentration of 0.1% to 20% by volume. In this context, the culture medium does not simultaneously contain both stem cell factors and interleukin-6 as factors. The aforementioned kit.

2. The kit according to claim 1, comprising three or four factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

3. The kit according to claim 1 or 2, wherein the serum is bovine or human serum.

4. The method for obtaining the aforementioned cell population includes culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a culture medium containing four or fewer factors selected from the group consisting of 50-500 ng / mL of stem cell factor, 5-100 ng / mL of interleukin-6, 50-500 ng / mL of FMS-like tyrosine kinase 3 ligand, 5-100 ng / mL of thrombopoietin, and 20-100 ng / mL of vascular endothelial growth factor, as well as serum at a concentration of 0.5% to 10% by volume. In this context, the culture medium does not simultaneously contain both stem cell factors and interleukin-6 as factors. The kit according to claim 1.

5. The kit according to any one of claims 1 to 4, wherein 50% or more of the cells in the CD206(+) group are CXCR4(+).

6. The kit according to any one of claims 1 to 4, wherein 80% or more of the cells in the CD206(+) are CXCR4(+).

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