WO2019216450A1 - Procédé de culture de cellules souches sécrétant des composants anti-inflammatoires, et composition anti-inflammatoire contenant ledit milieu de culture de cellules souches - Google Patents

Procédé de culture de cellules souches sécrétant des composants anti-inflammatoires, et composition anti-inflammatoire contenant ledit milieu de culture de cellules souches Download PDF

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WO2019216450A1
WO2019216450A1 PCT/KR2018/005317 KR2018005317W WO2019216450A1 WO 2019216450 A1 WO2019216450 A1 WO 2019216450A1 KR 2018005317 W KR2018005317 W KR 2018005317W WO 2019216450 A1 WO2019216450 A1 WO 2019216450A1
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inflammatory
cord blood
umbilical cord
multipotent stem
stem cell
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김준홍
윤성지
쵄쇼웬
최정혜
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주식회사 휴코드
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Definitions

  • the present invention relates to a method for culturing umbilical cord blood-derived multipotent stem cells secreting an anti-inflammatory component, and an anti-inflammatory composition comprising the stem cell culture. More specifically, a method of culturing umbilical cord blood-derived multipotent stem cells and a stem cell secreting a component having an effect of inhibiting the production of NO (Nitric Oxide) and PGE 2 (Prostaglandin E 2 ) by inhibiting iNOS and COX 2 It relates to an anti-inflammatory composition comprising a culture solution.
  • NO Nitric Oxide
  • PGE 2 Prostaglandin E 2
  • Stem cells are pluripotent cells capable of differentiating into all the cells constituting our body and have self-renewal ability, and cells capable of regeneration when there is damage to cells and tissues. These stem cells are largely divided into embryonic stem cells and adult stem cells. Embryonic stem cells may be derived from pre-implantation embryos, and adult stem cells are pluripotent cells capable of differentiating into specific cell types.
  • Stem cells are also classified into autologous and allogenic stem cells because one of the biggest side effects of transplanting stem cells into the patient's body is the rejection of the transplant. . Therefore, it is often divided into autologous or other stem cells. In the case of adult stem cells, many clinical trials are currently being conducted. In particular, adult stem cell transplantation may determine whether the stem cells are autologous or other stem cells.
  • cord blood is easier to acquire than bone marrow, and when a lot of cord blood is obtained, cord blood stem cells that match or are closest to the patient's histocompatibility gene can be used to solve the immune rejection reaction.
  • umbilical cord blood is commercially advantageous over embryonic stem cells that cannot be used realistically as a source of stem cells, and commercially available because it does not require the harvesting of bone marrow, fat, etc. in the adult body like adult stem cells. It is easier to say.
  • allergic contact dermatitis, psoriasis, and atopic dermatitis which are typical skin diseases, are inflammatory diseases mediated by T cells, which are immune cells, and steroidal anti-inflammatory drugs are used to treat these inflammatory diseases. It is shown. Therefore, it is necessary to develop a new material having low toxicity and no side effects, and the present inventors have developed a material having an anti-inflammatory effect using cord blood-derived multipotent stem cells.
  • the present invention has been made in an effort to provide an anti-inflammatory composition comprising a cord blood-derived multipotent stem cell culture method and a stem cell culture medium that secrete a substance that replaces a conventional anti-inflammatory agent having many side effects.
  • cord blood-derived multipotent stem cell culture method for secreting an anti-inflammatory component according to an aspect of the present invention
  • the stem cell anti-inflammatory media in an embodiment of the present invention, the stem cell anti-inflammatory media
  • the stem cell anti-inflammatory media in an embodiment of the present invention, the stem cell anti-inflammatory media
  • ⁇ MEM or IMDM is 10 to 30 parts by weight
  • At least one component selected from the group consisting of TGF- ⁇ , TNF- ⁇ , IL-3, IL-6, and MEM vitamins may include 1 ⁇ 10 ⁇ 8 to 17 ⁇ 10 ⁇ 6 parts by weight;
  • the stem cell anti-inflammatory media in an embodiment of the present invention, the stem cell anti-inflammatory media
  • IMDM is 10 to 30 parts by weight
  • TGF- ⁇ is 1 x10 -8 to 1x10 -7 parts by weight
  • TNF- ⁇ is 1 x10 -8 to 5x10 -8 parts by weight
  • IL-3 is 1x10 -8 to 5x10 -8 parts by weight
  • IL-6 is 1x10 - 8 to 5x10 -8 parts by weight
  • the stimulating step may be characterized in that made for 20 to 28 hours.
  • the stimulating step may be characterized in that at 35 to 38 °C.
  • the cord blood-derived multipotent stem cell culture medium in an embodiment of the present invention, the cord blood-derived multipotent stem cell culture medium
  • one culture selected from the group consisting of ⁇ MEM, IMDM, DMEM / F12, and Medium199;
  • It may be characterized by including; at least one component selected from the group consisting of insulin, transferrin, sodium selenite, MEM vitamin and human albumin.
  • the cord blood-derived multipotent stem cell culture medium in an embodiment of the present invention, the cord blood-derived multipotent stem cell culture medium
  • One culture medium selected from the group consisting of ⁇ MEM, IMDM, DMEM / F12 and Medium199 is 10 to 30 parts by weight;
  • At least one component selected from the group consisting of insulin, transferrin, sodium selenite, MEM vitamins, and human albumin may be 1 x 10 -6 to 5 x 10 -2 parts by weight.
  • the cord blood-derived multipotent stem cell culture medium in an embodiment of the present invention, the cord blood-derived multipotent stem cell culture medium
  • Medium199 is 10 to 30 parts by weight
  • Insulin 1 x10 -3 to 5x10 -2 part by weight of transferrin is 1 x10 -3 to 5x10 -2 part by weight of sodium selenite is 1 x10 -6 to 5x10 -5 parts by weight
  • MEM vitamins 8 x10 -6 to 17 ⁇ 10 ⁇ 6 parts by weight and human albumin are 5 ⁇ 10 ⁇ 3 to 15 ⁇ 10 ⁇ 3 parts by weight;
  • the anti-inflammatory component may be characterized by inhibiting NO (Nitric oxide) production.
  • the NO production may be suppressed by suppressing the expression level of i-NOS (inducible Nitric Oxide Synthase).
  • i-NOS inducible Nitric Oxide Synthase
  • the anti-inflammatory component may be characterized by inhibiting the production of PGE 2 (Prostaglandin E 2 ).
  • the PGE 2 production may be suppressed by suppressing the expression level of COX2 (Cyclooxygenase 2).
  • Anti-inflammatory composition may include a culture medium secreted by umbilical cord blood-derived multipotent stem cells cultured by the method according to the present invention.
  • the anti-inflammatory composition may be characterized in that it comprises at least one selected from the group consisting of IL-8, IL-10, IL-18, GM-CSF and MIP-1 ⁇ .
  • the anti-inflammatory composition is 40 to 50 parts by weight of IL-8, 0.05 to 0.07 parts by weight of IL-10, 1 to 2 parts by weight of IL-18, 0.4 to 0.6 of GM-CSF
  • the parts by weight and MIP-1 ⁇ may be characterized by including 20 to 30 parts by weight.
  • Anti-inflammatory composition comprising a culture medium secreted by umbilical cord blood-derived multipotent stem cells according to an embodiment of the present invention can be used in medicine or functional cosmetics for the improvement or treatment of inflammatory diseases.
  • HSCM anti-inflammatory components
  • Figure 2 is a result of measuring the amount of nitric oxide (NO; Nitric Oxide) production according to the concentration of anti-inflammatory component (HSCM).
  • NO nitric oxide
  • HSCM anti-inflammatory component
  • Figure 3 is a result of measuring the production amount of prostaglandin E 2 (PGE 2 ; Prostaglandin E 2 ) according to the concentration of anti-inflammatory component (HSCM).
  • Figure 4 is a result of measuring the gene expression of inflammatory factors iNOS, COX2, IL-1 ⁇ , IL-6 and TNF- ⁇ according to the concentration of anti-inflammatory component (HSCM).
  • HSCM anti-inflammatory component
  • HSCM anti-inflammatory component
  • HSCM anti-inflammatory component
  • Umbilical cord blood was transferred within 24 hours from the mother who received the consent, or only the nucleated cell layer was recovered, and cryopreserved cord blood stored in a cryogenic freezer at minus 196 °C was selected.
  • umbilical cord blood was diluted in double doses with ⁇ MEM (alpha-minimum essential medium, Jeil Biotech Services, Korea) or DMEM (Dulbecco's modified Eagle's medium) and centrifuged at 300 xg for 10 minutes at room temperature. .
  • the separated buffy coat layer was harvested and diluted again with twice the capacity of ⁇ MEM, then superimposed on Ficoll-Hypaque, and centrifuged at 300xg for 30 minutes at room temperature.
  • Ficoll-Hypaque a polymer of Ficoll (a polymer of sucrose) and Hypaque (sodium ditrizoate), is mainly used to separate monocytes from blood.
  • Ficoll-Hypaque has a specific gravity of 1.077g / ml, and monocytes are lighter than this, but red blood cells are heavier than that, so the specific gravity can be separated. In other words, when the blood is centrifuged on Ficoll-Hypaque, monocytes are collected on Ficoll-Hypaque.
  • Monocytes obtained by such a density gradient centrifugation method were washed twice with ⁇ MEM for washing with no additives.
  • the resulting monocytes contained antibiotics (1000 U / ml penicillin G, 1000 ug / ml streptomycin sulfate, Gibco-BRL), antifungal agents (0.25 ug / ml amphotericin B), and 2 mM glutamine (Sigma).
  • Stem Cell Factor 50 ng / ml
  • GM-CSF granulocyte-macrophage colony-stimulating factor; 10 ng / ml
  • FBS fetal bovine serum
  • G-CSF granulocyte colonystimulating factor
  • 10 ng / ml fetal bovine serum
  • IL-3 interleukin-3; 10 ng / ml
  • IL-6 interleukin-6
  • Umbilical cord blood-derived multipotent stem cells were obtained and washed three times with PBS and then stem cell anti-inflammatory media (IMDM 500ml, TGF- ⁇ (Prosepc, Israel) 10-100pg / ml, TNF- ⁇ (Prospec, Istrael) 10-50pg / ml, IL-3 (Prospec, Istrael) 10-50pg / ml, IL-6 (Prospec, Istrael) 10-50pg / ml and MEM Vitamin (Gibco, USA) 1-2% (8,000-17,000pg / ml) Stimulated by incubating in a 37 °C incubator for 24 hours.
  • the cells were washed three times with PBS and cultured medium (500 ml of medium 199, 1-50 ug / ml of insulin (Sigma, USA), 1-50 ug / ml of transferrin (Sigma, USA), sodium selenite (Sigma, USA) ) 0.001 ⁇ 0.05ug / ml, MEM vitamin (Gibco, USA) 1 ⁇ 2% (0.008 ⁇ 0.017ug / ml), human albumin (Sigma, USA) 0.5 ⁇ 1.5% (5 ⁇ 15ug / ml)
  • the cells were incubated in an incubator. The cultures were collected by replacing the medium once every two days, and the collected cultures were used after each filter (Top Filter system, Corning) and then refrigerated and frozen.
  • MTT assay was performed to confirm the cytotoxicity of HSCM, an anti-inflammatory substance secreted by cord blood-derived multipotent stem cells according to the present invention.
  • Raw 264.7 cells were incubated for 24 hours in 37 °C, 5% CO 2 incubator with 100ul each of 3X10 3 cells / well in a 96 well plate with DMEM containing 10% FBS. After incubation, the medium was removed, and experimental groups (HSCM 0, 10, 20, 30, 40, 50%) were prepared, treated with triplicate for each group, and then incubated in 37 ° C. and 5% CO 2 incubator for 24 hours.
  • FIG. 1 shows that even if the concentration of anti-inflammatory component (HSCM) is increased, it does not significantly affect cell survival.
  • HSCM anti-inflammatory component
  • Nitric oxide (NO) production experiments were performed using the Griess method using raw 264.7 macrophage cells.
  • DMEM containing 10% FBS and 250ul of 1 ⁇ 10 5 cells / well in a 48 well plate was incubated for 24 hours in 37 °C, 5% CO 2 incubator. After incubation, the medium was removed, and the experimental group (HSCM 0, 10, 30, 50%) was placed in a DMEM medium containing 10% FBS, and 1ug / ml LPS (lipopolysaccharide), which is a source of inflammation, was added thereto at 37 ° C and 5% CO 2. Incubated for 24 hours in an incubator.
  • PGE 2 Prostaglandin E 2
  • 250 ⁇ l of 1X10 5 cells / well was added to 48 well plates with DMEM containing 10% FBS and incubated in 37 ° C. and 5% CO 2 incubator for 24 hours.
  • the medium was removed, and the experimental group (HSCM 0, 10, 30, 50%) was placed in a DMEM medium containing 10% FBS, and 1 ug / ml of lipopoliysaccharide (LPS), a source of inflammation, was added thereto at 37 ° C and 5% CO 2.
  • the incubator was incubated for 24 hours.
  • the culture solution was collected, transferred to a 1.5ml tube, and centrifuged at 14,000 rpm for 20 minutes to obtain a supernatant.
  • the obtained supernatant was measured using a PGE 2 EIA kit (cayman, USA).
  • Test Example 4 Analysis of anti-inflammatory gene expression in cells cultured in medium containing anti-inflammatory component (HSCM) (RT-PCR method)
  • RT-PCR was performed to investigate the effect of HSCM on gene expression of inflammatory factors iNOS, COX2, IL-1 ⁇ IL-6 and TNF- ⁇ .
  • RNAzol B reagent for gene expression analysis. After 1 ml of RNAzol B reagent was added to dissolve the cells, the tissues were denatured, transferred to 1.5 ml tubes, and 200 ul of chloroform was added and vortexed for 20 seconds to ensure complete mixing. After reaction at room temperature for 15 minutes, the supernatant was obtained by centrifugation at 14,000 rpm for 20 minutes, and inverted with the same amount of isopropyl alcohol, and left to stand at room temperature for 10 minutes. The sample was centrifuged at 14,000 rpm for 15 minutes to obtain RNA pellet.
  • RNA concentration and purity were measured at OD 260/280 nm.
  • Single-strand cDNA synthesis was performed by mixing 1 ug of oligo-d (T) primer (100 pmol) with 1 ug of total RNA extracted and reacting at 65 ° C. for 10 minutes, followed by rapid cooling.
  • T oligo-d
  • To this template add 2m each of 10mM dNTP (TaKaRa Bio Inc., Japan), 0.1M DTT and 5x RT buffer (10mM Tris-Cl, 50mM KCl, 2.5mM MgCl2) and add 100 units of M-MLV RTase (BioNeer, Korea) After the addition, the total amount was corrected to 20 ul using distilled water treated with DEPC. Samples were terminated by inactivating reverse transcriptase through a reaction at 25 ° C. for 5 minutes and at 42 ° C. for 1 hour, followed by reaction at 72 ° C. for 15 minutes.
  • RT-PCR reaction was performed using the synthesized cDNA and primers of each gene. Reaction conditions were template 1ul and primer 0.5ul (10pmol), 2.5mM dNTP 0.5ul, 10X PCR buffer [10mM Tris-Cl (pH8.3), 50mM KCl, 2.5mM MgCl2] 2.5ul, Taq polymerase 2 unit / ul After the addition was carried out using a sterile distilled water to correct the total amount to 25ul to perform a PCR reaction. The amplification products obtained from the PCR reactions were subjected to electrophoresis using 1.5% agarose gel, and the detection intensity of each PCR product was analyzed using an image analysis system (Kodak EDAS290). Relative quantification of band detection intensity was corrected based on the housekeeping genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and actin.
  • Test Example 5 Analysis of anti-inflammatory gene expression in cells cultured in a medium containing anti-inflammatory components (HSCM) (Western blot method)
  • lysis buffer 150mM NaCl, 50mM Tris-HCl pH7.5, 1% NP40, 0.1% SDS, 1mM PMSF
  • 500ul lysis buffer 150mM NaCl, 50mM Tris-HCl pH7.5, 1% NP40, 0.1% SDS, 1mM PMSF
  • 500ul lysis buffer 150mM NaCl, 50mM Tris-HCl pH7.5, 1% NP40, 0.1% SDS, 1mM PMSF
  • Separation and cell membrane components were removed. Protein samples obtained from the cultured cells were quantified using the BCA method, 20ug of lysate was electrophoretically separated by 12% SDS-PAGE, and the protein was transferred to the PVDF membrane at 90V for 50 minutes using a transfer solution.
  • the blocking of the membrane was carried out for 1 hour at room temperature in TBST (50mM Tris-HCl pH7.6, 150mM NaCl, 0.2% Tween 20) solution containing 5% skim milk.
  • IL-8 which is a major component related to anti-inflammatory, among the components secreted by umbilical cord blood-derived multipotent stem cells cultured in soy hydrolyzate-containing medium disclosed in Korean Patent Publication No. 10-2009-0090850 or Korean Patent Publication No. 10-2013-0104924
  • the contents of IL-10, IL-18, GM-CSF and MIP-1 ⁇ were compared with the contents of the components secreted from umbilical cord blood-derived multipotent stem cells stimulated and cultured according to the present invention and the results are shown in Table 1.
  • umbilical cord blood-derived multipotent stem cells stimulated and cultured according to the present invention are the major anti-inflammatory components IL-8, IL-10, IL-18, GM-CSF and MIP-1 ⁇ .
  • IL-8 is 8.8 times
  • IL-10 is 50 times
  • GM-CSF is 24 times
  • MIP-1 ⁇ is 2927 times higher than cord blood-derived multipotent stem cells cultured by the culture techniques described in the published patents. You can see more secretion.
  • IL-18 was not secreted from the cord blood multipotent stem cells according to the patents, but was secreted from the cord blood multipotent stem cells according to the present invention as shown in Table 1 above.
  • Umbilical cord blood-derived multipotent stem cell culture method for secreting an anti-inflammatory component comprises the steps of: 1) separating monocytes from umbilical cord blood; 2) culturing the monocytes to obtain umbilical cord blood-derived multipotent stem cells; 3) stimulating the obtained cord blood-derived multipotent stem cells in a stem cell anti-inflammatory customized medium; 4) washing the stimulated cord blood-derived multipotent stem cells; And 5) culturing the washed umbilical cord blood-derived multipotent stem cells in a culture medium.
  • the umbilical cord blood refers to umbilical cord blood coming out of the umbilical cord at birth, and may contain hematopoietic stem cells (stematopoietic stem cells) that make leukocytes, red blood cells, and platelets, and mesenchymal stem cells that make cartilage, bone, muscle, and nerves. (Mesenchymal stem cells) may also be included.
  • the multipotent stem cell refers to a stem cell having a differentiation capacity capable of differentiating only into cells specific to a certain tissue and organs.
  • a cell having a multipotency an adult stem such as HSC, MSC, and NSC Cells may be included.
  • Adult pluripotent stem cells having such multipotency may be involved in the growth and development of prenatal, neonatal, and adult tissues and organs, as well as in maintaining homeostasis of adult tissues and inducing regeneration upon tissue damage.
  • the multipotent stem cells can secrete several components and these components can act on damaged cells or tissues in vivo to improve damaged sites.
  • the multipotent stem cells may directly repair damaged cells or tissues, but protect the damaged cells from apoptosis, create new blood vessels, and break down hardened proteins to improve the function of tissues. Not only can it be revived, but it can also release various physiological factors (called the paracrine effect).
  • the multipotent stem cells may secrete substances or anti-cancer substances that promote cell growth factors that help the growth and regeneration of the skin and antioxidants that protect the skin from harmful free radicals and reduce inflammation, and the like. Can be.
  • the step of stimulating the obtained cord blood-derived multipotent stem cells in a stem cell anti-inflammatory tailored medium can activate the anti-inflammatory signaling mechanism of stem cells, as a result IL-8 Can induce secretion of anti-inflammatory components such as IL-10, IL-18, GM-CSF, MCP-1 ⁇ . That is, among the various signaling mechanisms of the cord blood-derived multipotent stem cells, the anti-inflammatory signaling mechanism may be specifically activated more than the general state, and as a result, the state of the stem cells to be suitable for secreting anti-inflammatory components. Can change.
  • the stem cell anti-inflammatory media is ⁇ MEM or IMDM; And at least one component selected from the group consisting of TGF- ⁇ , TNF- ⁇ , IL-3, IL-6, and MEM vitamins.
  • the stem cell anti-inflammatory customized medium is ⁇ MEM or IMDM 10 to 30 parts by weight; And at least one component selected from the group consisting of TGF- ⁇ , TNF- ⁇ , IL-3, IL-6, and MEM vitamins, may include 1 ⁇ 10 ⁇ 8 to 17 ⁇ 10 ⁇ 7 .
  • the MEM vitamin may be composed of Choline chloride, D-Calcium pantothenate, Folic Acid, Nicotinamide, Pyridoxal hydrochloride, Riboflavin, Thiamine hydrochloride and i-Inositol.
  • the MEM vitamin is 80 to 120 ug Choline chloride, 80 to 120 ug D-Calcium pantothenate, 80 to 120 ug Folic Acid, 80 to 120 ug Nicotinamide, 80 to 120 ug Pyridoxal hydrochloride, 8 to 12 ug Riboflavin, 80-120 ug Thiamine hydrochloride and 180-220 ug i-Inositol.
  • the stem cell anti-inflammatory medium, ⁇ MEM or IMDM is 10 to 30 parts by weight;
  • TGF- ⁇ is 1x10 -8 to 17x10 -7 parts by weight, TNF- ⁇ was 1 x10 -8 to 17x10 -7 parts by weight,
  • IL-3 is 1x10 -8 to 17x10 -7 parts by weight,
  • IL-6 is 1x10 - 8 to 17 ⁇ 10 ⁇ 7 parts by weight and the MEM vitamin may be 1 ⁇ 10 ⁇ 8 to 17 ⁇ 10 ⁇ 7 parts by weight.
  • the stem cell anti-inflammatory media 15 to 25 parts by weight of IMDM;
  • TGF- ⁇ is 1x10 -8 to 1x10 -7 parts by weight
  • TNF- ⁇ is 1x10 -8 to 5x10 -8 parts by weight
  • IL-3 is 1x10 -8 to 5x10 -8 parts by weight
  • IL-6 is 1x10 -8 to 5x10 -8 parts by weight
  • MEM vitamins may be an 8x10 -6 to 17x10 -6 wt.
  • ⁇ MEM or IMDM is 400 to 600ml; And at least one component selected from the group consisting of TGF- ⁇ , TNF- ⁇ , IL-3, IL-6, and MEM vitamins may include 1 to 100 pg / ml.
  • the IMDM is 450 to 550 ml; And 1 to 100 pg / ml for TGF- ⁇ , 1 to 100 pg / ml for TNF- ⁇ , 1 to 100 pg / ml for IL-3, 1 to 100 pg / ml for IL-6, and 1 to 100 pg / ml for MEM vitamins; It may include.
  • the TGF- ⁇ is 10 to 100 pg / ml
  • the TNF- ⁇ is 10 to 50 pg / ml
  • the IL-3 is 10 to 50 pg / ml
  • the IL-6 is 10 to 50 pg / ml
  • the The MEM vitamin can be between 8000 and 17000 pg / ml.
  • the stimulating step may be performed for 20 to 28 hours. Preferably from 23 to 25 hours.
  • the stem cells according to an embodiment of the present invention is not sufficiently stimulated to secrete anti-inflammatory components, and if stimulated for more than 28 hours, the stem cells excessively stimulated In addition to the anti-inflammatory ingredients in addition to the other components come out can lower the anti-inflammatory purity.
  • the stimulating step may be performed at 35 to 38 ° C. Preferably it may be made at 36 to 37.5 °C. Stimulation of stem cells at temperatures below 35 ° C may reduce the overall activity of the cells and may not result in sufficient stimulation. Stimulation of stem cells at temperatures above 38 ° C may inhibit the activity of intracellular enzymes and the like. As a result, the cells may die and secretion of the anti-inflammatory substances according to the embodiment of the present invention may not occur properly.
  • Washing the stimulated umbilical cord blood-derived multipotent stem cells may include stimulating the stem cell anti-inflammatory media after the stimulation in the stem cell anti-inflammatory media and before culturing in the culture medium. During this step, the components secreted from the cord blood-derived multipotent stem cells are also removed. In the stimulating step, the components secreted from the umbilical cord blood-derived multipotent stem cells are not components corresponding to the anti-inflammatory action, which is the object of the present invention, and thus should be removed through a washing process.
  • the culturing the washed umbilical cord blood-derived multipotent stem cells in a culture medium is a step of culturing to produce and secrete anti-inflammatory components from the umbilical cord blood-derived stem cells that have undergone washing after the stimulation is completed. That is, the components secreted from the cord blood-derived multipotent stem cells cultured in the culturing may include components capable of anti-inflammatory action.
  • the anti-inflammatory component may inhibit NO (Nitric oxide) production.
  • NO Nitric oxide
  • the NO is known to play a role in blood pressure regulation, neurotransmission, platelet aggregation inhibitory, immune function, and can be synthesized by nitric oxide synthase (NOS) from L-arginine in various tissues and cells.
  • NOS nitric oxide synthase
  • the NOS can be broadly divided into nNOS (neuronal NOS), eNOS (endothelial NOS), and i-NOS (inducible NOS).
  • i-NOS is independent of intracellular calcium concentration and stimulated by LPS, IFN- ⁇ , IL-1 and TNF- ⁇ in various cells such as macrophages, vascular smooth muscle cells, endothelial cells, hepatocytes and cardiomyocytes. It can be activated and produce a large amount of NO for a long time. However, when NO is generated more than necessary, it may cause harmful effects on the living body by causing vasodilation due to shock, tissue damage caused by inflammatory response, and damage to nerve tissue.
  • the anti-inflammatory component (HSCM) secreted from stem cells can inhibit the NO production through the umbilical cord blood-derived multipotent stem cell culture method according to an embodiment of the present invention.
  • the anti-inflammatory component may inhibit NO production by inhibiting the expression level of i-NOS (inducible Nitric oxide synthase). 4 and 5 show that the anti-inflammatory component can inhibit not only gene expression of i-NOS but also protein expression.
  • the anti-inflammatory component may inhibit the production of PGE 2 (Prostaglandin E2).
  • PGE 2 is one of inflammatory mediators such as NO, and may mediate vasodilation, edema, fever, and pain.
  • MMPs matrix metalloproteinases
  • COX cyclooxygenase
  • the anti-inflammatory component (HSCM) secreted from stem cells through the umbilical cord blood-derived multipotent stem cell culture method according to an embodiment of the present invention can inhibit the amount of PGE 2 produced.
  • the anti-inflammatory component may inhibit the PGE 2 by inhibiting the expression level of COX2 (Cyclooxygenase 2).
  • COX2 Cyclooxygenase 2
  • 4 and 5 show that the anti-inflammatory component can inhibit not only the gene expression of COX2 but also protein expression.
  • the culture medium one medium selected from the group consisting of ⁇ MEM, IMDM, DMEM / F12 and Medium199; And at least one component selected from the group consisting of insulin, transferrin, sodium selenite, MEM vitamin, and human albumin.
  • the culture medium is one selected from the group consisting of ⁇ MEM, IMDM, DMEM / F12 and Medium199 10 to 30 parts by weight; And at least one component selected from the group consisting of insulin, transferrin, sodium selenite, MEM vitamin, and human albumin, may include 1 ⁇ 10 ⁇ 6 to 5 ⁇ 10 ⁇ 2 parts by weight.
  • the culture medium, Medium199 is 10 to 30 parts by weight;
  • Insulin 1x10 -6 to 5x10 -2 part by weight of transferrin is 1x10 -6 to 5x10 -2 part by weight of sodium selenite is 1x10 -6 to 5x10 -2 part by weight,
  • MEM vitamins 1x10 -6 to 5x10 -2 Parts by weight and human albumin may include 1 ⁇ 10 ⁇ 6 to 5 ⁇ 10 ⁇ 2 parts by weight.
  • the culture medium, Medium199 is 15 to 25 parts by weight;
  • Insulin 1x10 -3 to 5x10 -2 part by weight of transferrin is 1x10 -3 to 5x10 -2 part by weight of sodium selenite is 1x10 -6 to 5x10 -5 parts by weight,
  • MEM vitamins 8x10 -6 to 17x10 -6 Parts by weight and human albumin may include 5 ⁇ 10 ⁇ 3 to 15 ⁇ 10 ⁇ 3 parts by weight.
  • the culture medium one medium selected from the group consisting of ⁇ MEM, IMDM, DMEM / F12 and Medium199 is 400 to 600 ml; And at least one component selected from the group consisting of insulin, transferrin, sodium selenite, MEM vitamins, and human albumin may comprise 0.001 to 50 ug / ml.
  • Medium199 is 450 to 550 ml; And 1 to 50 ug / ml of insulin, 1 to 50 ug / ml of transferrin, 0.001 to 0.05 ug / ml of sodium selenite, 0.008 to 0.017 ug / ml of MEM vitamin and 5 to 15 ug / ml of human albumin; can do.
  • the anti-inflammatory composition according to another aspect of the present invention may include a culture medium secreted by the stem cells cultured by the cord blood-derived multipotent stem cell culture method.
  • the anti-inflammatory composition may comprise at least one selected from the group consisting of IL-8, IL-10, IL-18, GM-CSF and MIP-1 ⁇ .
  • IL-8 is 40 to 50 parts by weight
  • IL-10 is 0.05 to 0.07 parts by weight
  • IL-18 is 1 to 2 parts by weight
  • GM-CSF is 0.4 to 0.6 parts by weight
  • MIP-1 ⁇ may comprise 20 to 30 parts by weight.
  • the anti-inflammatory composition is 40 to 50ng / ml IL-8, 0.05 to 0.07ng / ml IL-10, 1 to 2ng / ml IL-18, 0.4 GM-CSF To 0.6 ng / ml and MIP-1 ⁇ may comprise 20 to 30 ng / ml.

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Abstract

L'invention concerne un mode de réalisation qui fournit : un procédé de culture de cellules souches pluripotentes dérivées du sang de cordon ombilical qui sécrètent des composants anti-inflammatoires pour soulager et traiter des maladies inflammatoires; et une composition anti-inflammatoire contenant le milieu de culture de cellules souches. En particulier, les composants anti-inflammatoires peuvent inhiber la quantité de substances inflammatoires telles que l'oxyde nitrique (NO) et la prostaglandine E2 (PGE2) produits, et plus spécifiquement, peut inhiber la production de substances inflammatoires par inhibition de l'expression de gènes et de protéines iNOS et COX2 associés à des substances inflammatoires.
PCT/KR2018/005317 2018-05-08 2018-05-09 Procédé de culture de cellules souches sécrétant des composants anti-inflammatoires, et composition anti-inflammatoire contenant ledit milieu de culture de cellules souches WO2019216450A1 (fr)

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KR102492888B1 (ko) * 2020-12-23 2023-01-30 에스씨엠생명과학 주식회사 줄기세포 프라이밍 조성물 및 프라이밍된 줄기세포

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