WO2019216450A1 - Method for culturing stem cells secreting anti-inflammatory components, and anti-inflammatory composition containing said stem cell culture medium - Google Patents

Abstract

An embodiment of the present invention provides: a method for culturing umbilical cord blood-derived pluripotent stem cells that secrete anti-inflammatory components for alleviating and treating inflammatory diseases; and an anti-inflammatory composition containing the stem cell culture medium. Specifically, the anti-inflammatory components can inhibit the amount of inflammatory substances such as nitric oxide (NO) and prostaglandin E2 (PGE2) produced, and more specifically, can inhibit the production of inflammatory substances by inhibiting the expression of iNOS and COX2 genes and proteins associated with inflammatory substances.

Description

Stem cell culture method for secreting anti-inflammatory components and anti-inflammatory composition comprising the stem cell culture solution

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.

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. A wide variety of tissues including amniotic membrane, placenta, bone marrow, nerves, muscles, adipose tissue, skin tissue (skin, liver or gastric mucosa, penis foreskin), hair follicle, pancreas, bladder, testes, cornea, teeth (teeth, wisdom teeth), menstrual blood Can be derived from. 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.

Currently, it is easy to separate and culture stem cells from bone marrow, but it is not easy to obtain bone marrow, and it is known that it is practically difficult to solve the immune rejection reaction when transplanting stem cells of another person. In contrast, 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. There is an advantage. In other words, 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.

Regarding inflammatory diseases, 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.

SUMMARY OF THE INVENTION 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.

The technical problem to be achieved by the present invention is not limited to the technical problem mentioned above, and other technical problems not mentioned above may be clearly understood by those skilled in the art from the following description. There will be.

In order to achieve the above technical problem, the cord blood-derived multipotent stem cell culture method for secreting an anti-inflammatory component according to an aspect of the present invention,

Separating monocytes from umbilical cord blood;

Culturing the monocytes to obtain umbilical cord blood-derived multipotent stem cells;

Stimulating said cord blood-derived multipotent stem cells in a stem cell anti-inflammatory media;

Washing the stimulated cord blood-derived multipotent stem cells; And

Culturing the washed umbilical cord blood-derived multipotent stem cells in a culture medium;

It may include.

In an embodiment of the present invention, the stem cell anti-inflammatory media,

αMEM or IMDM; And

And at least one component selected from the group consisting of TGF-β, TNF-α, IL-3, IL-6, and MEM vitamins.

In an embodiment of the present invention, the stem cell anti-inflammatory media,

αMEM or IMDM is 10 to 30 parts by weight; And

At least one component selected from the group consisting of TGF-β, TNF-α, IL-3, IL-6, and MEM vitamins may include 1 × 10 ⁻⁸ to 17 × 10 ⁻⁶ parts by weight;

It may include.

In an embodiment of the present invention, the stem cell anti-inflammatory media,

IMDM is 10 to 30 parts by weight; And

TGF-β is 1 x10 ^-8 to 1x10 ^-7 parts by weight, TNF-α 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, and MEM vitamin 8 x10 ^-6 to 17x10 ^-6 parts by weight;

It may include.

In an embodiment of the invention, the stimulating step may be characterized in that made for 20 to 28 hours.

In an embodiment of the present invention, the stimulating step may be characterized in that at 35 to 38 ℃.

In an embodiment of the present invention, the cord blood-derived multipotent stem cell culture medium,

one culture selected from the group consisting of αMEM, IMDM, DMEM / F12, and Medium199; And

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.

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 αMEM, IMDM, DMEM / F12 and Medium199 is 10 to 30 parts by weight; And

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.

In an embodiment of the present invention, the cord blood-derived multipotent stem cell culture medium,

Medium199 is 10 to 30 parts by weight; And

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;

It may include.

In an embodiment of the present invention, the anti-inflammatory component may be characterized by inhibiting NO (Nitric oxide) production.

In an embodiment of the present invention, the NO production may be suppressed by suppressing the expression level of i-NOS (inducible Nitric Oxide Synthase).

In an embodiment of the present invention, the anti-inflammatory component may be characterized by inhibiting the production of PGE ₂ (Prostaglandin E ₂ ).

In an embodiment of the present invention, the PGE ₂ production may be suppressed by suppressing the expression level of COX2 (Cyclooxygenase 2).

Anti-inflammatory composition according to another aspect of the present invention, may include a culture medium secreted by umbilical cord blood-derived multipotent stem cells cultured by the method according to the present invention.

In an embodiment of 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-1α.

In an embodiment of the present invention, 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-1α 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.

The effects of the present invention are not limited to the above-described effects, but should be understood to include all the effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a result of cytotoxicity (MTT Assay) of anti-inflammatory components (HSCM) secreted by cord blood-derived multipotent stem cells according to the present invention.

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).

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-1β, IL-6 and TNF-α according to the concentration of anti-inflammatory component (HSCM).

5 is a result of measuring the protein expression of inflammatory factors iNOS and COX2 according to the concentration of anti-inflammatory component (HSCM).

Hereinafter, the preparation method of the umbilical cord blood-derived multipotent stem cells secreting an anti-inflammatory component (HSCM) and the effects thereof according to the present invention will be described in detail. However, these Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these.

Example 1 Screening of Umbilical Cord Blood

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 ℃ was selected.

Example 2 Isolation and Culture of Multipotent Stem Cells from Umbilical Cord Blood

Umbilical cord blood that was stored frozen at minus 196 ° C. was thawed immediately in a 37 ° C. water bath, and cord blood transferred within 24 hours from the mother was used as it was. To separate monocytes from umbilical cord blood, umbilical cord blood was diluted in double doses with αMEM (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 αMEM, 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 αMEM 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) with 10 to fetal bovine serum (FBS; fetal bovine serum, Jeil Biotech Services) ml), G-CSF (granulocyte colonystimulating factor; 10 ng / ml), IL-3 (interleukin-3; 10 ng / ml) and IL-6 (interleukin-6; 10 ng / ml) It was suspended at a concentration of 1 × 10 ⁶ / cm 2. After multiplying the selected cells for 5 days, multipotent stem cells were obtained.

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-β (Prosepc, Israel) 10-100pg / ml, TNF-α (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 ℃ incubator for 24 hours. After the stem cell stimulation, 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.

Test Example 1 Cytotoxicity Test (MTT Assay)

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 ℃, 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. After incubation, 10 μl of 5 mg / ml MTT reagent was dispensed into the medium containing the test solution, and then incubated in 37 ° C., 5% CO ₂ incubator for 4 hours by blocking the light of a 96 well plate. When the culture was completed, the medium containing the MTT reagent was removed, and 100ul of DMSO (dimethyl sulfoxide) was added to dissolve the MTT-formazan crystal, and the cell viability was confirmed by measuring the absorbance at 570 nm.

The results are shown in FIG. 1, which shows that even if the concentration of anti-inflammatory component (HSCM) is increased, it does not significantly affect cell survival.

Test Example 2 Determination of Nitric Oxide Production in Cells Cultured in Medium Containing Anti-Inflammatory Component (HSCM)

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 로 10 ⁵ cells / well in a 48 well plate was incubated for 24 hours in 37 ℃, 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. Then, all of the culture solution was taken, transferred to a 1.5ml tube, and centrifuged at 14,000 rpm for 20 minutes to obtain a supernatant. 100 μl of the obtained supernatant was transferred to a 96 well plate, 100 μl of Griess reagent (cayman, USA) was added and reacted at room temperature for 20 minutes, and the absorbance was measured at 540 nm with an ELISA reader. A standard curve was prepared using NaNO ₂ standard, and NO production was quantified by comparison.

The results are shown in Figure 2, and looking at this, it can be seen that the concentration of Nitric Oxide decreases as the concentration of HSCM increases.

Test Example 3 Determination of Prostaglandin E2 Production in Cells Cultured in Medium containing Anti-Inflammatory Component (HSCM)

In order to measure PGE ₂ (Prostaglandin E ₂ ), 250 μl 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. 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 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. Then, 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). Add 50ul of standard and sample (5 times dilution using EIA buffer) to 96 well plates coated with Goat anti-mouse, and add 50ul of Tracer solution and monoclonal antibody solution to the wells for 18 hours at 4 ℃. Reacted. After the reaction was washed 5 times with Washing Buffer and reacted with 200ul Ellman reagent 60 ~ 90 minutes was measured by absorbance at 405 ~ 420nm with ELISA reader. Standard curves were prepared and compared to quantify PGE ₂ production.

The results are shown in FIG. 3, and it can be seen that as the concentration of anti-inflammatory component (HSCM) increases, the concentration of PGE ₂ decreases.

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-1βIL-6 and TNF-α. Dispense the raw 264.7 macrophage cells into DMEM containing 10% FBS at a concentration of 1Ⅹ10 ⁵ cells / well in a 24 well plate and incubate in 37 ° C and 5% CO ₂ incubator. Then remove the culture medium and test group ( HSCM 0, 10, 30, 50%) with 10% FBS was placed in a containing DMEM culture solution was added to inflammation caused LPS of 1ug / ml here 37 ℃, 5% CO in the _second condition 24 Incubated for hours. Then, the medium was removed, washed once with PBS, and total RNA was extracted from the cultured cells using 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. The sample was centrifuged at 14,000 rpm for 10 minutes with 70% ethanol for RNA, and dried for 5 minutes using an aspirator. The dried RNA sample was added with 25ul of distilled water treated with 0.1% DEPC (diethyl pyrocarbonate) and reacted at 55 ° C for 10 minutes to dissolve the pellet and used as a sample for cDNA synthesis. RNA concentration and purity were measured at OD 260/280 nm.

(1) cDNA synthesis

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. 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.

(2) RT-PCR

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.

The results are shown in Figure 4, and comparing the bands, it can be seen that as the concentration of HSCM increases, the gene expression of iNOS, COX2, IL-1βIL-6 and TNF-α related to the inflammatory agent is gradually suppressed. .

Test Example 5: Analysis of anti-inflammatory gene expression in cells cultured in a medium containing anti-inflammatory components (HSCM) (Western blot method)

This study was conducted to investigate the anti-inflammatory effects of HSCM by checking the protein expression levels of iNOS and COX2 that are directly involved in NO and PGE ₂ produced during inflammatory reactions. The experimental group ( HSCM 0, 10, 30, 50%) was treated with 1 μg / ml LPS for 24 hours in Raw 264.7 macrophage cells, and then cultured medium was removed and collected by scraper by adding 1 ml of PBS. Washed by centrifugation at 14,000 rpm for 20 minutes. After centrifugation, the supernatant was removed and 500ul lysis buffer (150mM NaCl, 50mM Tris-HCl pH7.5, 1% NP40, 0.1% SDS, 1mM PMSF) was added and lysed by an ultrasonic grinder, and centrifuged at 14,000 rpm for 10 minutes. 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. TBST solution containing 5% skim milk with rabbit polyclonal anti iNOS (Millipore) as an antibody of iNOS, rabbit polyclonal anti COX2 (Millipore) as an antibody of COX2, goat polyclonal anti actin (Santa-Curz) as an antibody of Actin It was diluted to and reacted at room temperature for 1 hour 30 minutes. After washing three times with TBST solution, as a secondary antibody, anti-goat and anti-rabbit IgG conjugated with HRP (Horse Radish peroxidase) was diluted in TBST solution containing 5% skim milk and reacted at room temperature for 1 hour. I was. Then, after washing the membrane three times with TBST immobilon western kit. (Millipore Cat. No. WBKLS0100) was used for 1 minute to confirm the intensity of the band developed by the LAS-3000 analyzer.

The results are shown in FIG. 5, and it can be seen that as the concentration of HSCM increases, expression of iNOS protein and COX2 protein related to NO and PGE ₂ which cause inflammation is gradually suppressed.

Comparative Example 1 Comparison of anti-inflammatory components secreted from umbilical cord blood-derived multipotent stem cells cultured in a medium containing anti-inflammatory components (HSCM) and anti-inflammatory components secreted from umbilical cord blood-derived multipotent stem cells cultured in a medium containing soy protein hydrolysates

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-1α 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.

(Unit: pg)

Anti-inflammatory	Umbilical Cord Blood-derived Multipotent Stem Cells Cultivated in Soy Hydrolysate Containing Medium of Domestic Publication (10-2009-0090850 or 10-2013-0104924)	Umbilical cord blood-derived multipotent stem cells stimulated and cultured according to the present invention
IL-8	4790.71 ± 1267.38	42458.46 ± 4279.86
IL-10	1.18 ± 0.02	60.12 ± 1.06
IL-18	0	1135.79 ± 22.01
GM-CSF	20.15 ± 8.58	484.37 ± 11.14
MIP-1a	8.78 ± 0.66	25704.68 ± 742.22

Referring to the results 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-1α. IL-8 is 8.8 times, IL-10 is 50 times, GM-CSF is 24 times and MIP-1α 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. In addition, 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.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is represented by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention.

Hereinafter, with reference to the accompanying drawings will be described the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and like reference numerals designate like parts throughout the specification.

Throughout the specification, when a part is said to be "connected (connected, contacted, coupled)" with another part, it is not only "directly connected" but also "indirectly connected" with another member in between. "Includes the case. In addition, when a part is said to "include" a certain component, this means that it may further include other components, without excluding the other components unless otherwise stated.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. As used herein, the terms "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Umbilical cord blood-derived multipotent stem cell culture method for secreting an anti-inflammatory component according to an aspect of the present invention 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. For example, as 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. According to an embodiment of the present invention, 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). Specifically, 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.

According to an embodiment of the present invention, 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-1α. 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.

According to an embodiment of the invention, the stem cell anti-inflammatory media is αMEM or IMDM; And at least one component selected from the group consisting of TGF-β, TNF-α, IL-3, IL-6, and MEM vitamins.

According to an embodiment of the present invention, the stem cell anti-inflammatory customized medium is αMEM or IMDM 10 to 30 parts by weight; And at least one component selected from the group consisting of TGF-β, TNF-α, IL-3, IL-6, and MEM vitamins, may include ¹ × 10 ⁻⁸ to 17 × 10 ⁻⁷ .

According to an embodiment of the present invention, the MEM vitamin may be composed of Choline chloride, D-Calcium pantothenate, Folic Acid, Nicotinamide, Pyridoxal hydrochloride, Riboflavin, Thiamine hydrochloride and i-Inositol. Preferably, 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.

According to an embodiment of the present invention, the stem cell anti-inflammatory medium, αMEM or IMDM is 10 to 30 parts by weight; And TGF-β is 1x10 ^-8 to 17x10 ^-7 parts by weight, TNF-α 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.

According to a preferred embodiment of the present invention, the stem cell anti-inflammatory media, 15 to 25 parts by weight of IMDM; And TGF-β is 1x10 ^-8 to 1x10 ^-7 parts by weight, TNF-α is 1x10 ^-8 to 5x10 ^-8 parts by weight, IL-3 is 1x10 ^-8 to 5x10 ^-8 parts by weight, and IL-6 is 1x10 ^-8 to 5x10 ^-8 parts by weight, and MEM vitamins may be an 8x10 ^-6 to 17x10 ^-6 wt.

According to another embodiment of the invention, αMEM or IMDM is 400 to 600ml; And at least one component selected from the group consisting of TGF-β, TNF-α, IL-3, IL-6, and MEM vitamins may include 1 to 100 pg / ml.

According to another embodiment of the present invention, the IMDM is 450 to 550 ml; And 1 to 100 pg / ml for TGF-β, 1 to 100 pg / ml for TNF-α, 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. More preferably, the TGF-β is 10 to 100 pg / ml, the TNF-α is 10 to 50 pg / ml, the IL-3 is 10 to 50 pg / ml, the IL-6 is 10 to 50 pg / ml and the The MEM vitamin can be between 8000 and 17000 pg / ml.

According to an embodiment of the present invention, the stimulating step may be performed for 20 to 28 hours. Preferably from 23 to 25 hours. When stimulated for less than 20 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.

According to an embodiment of the present invention, the stimulating step may be performed at 35 to 38 ° C. Preferably it may be made at 36 to 37.5 ℃. 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.

According to an embodiment of the present invention, the anti-inflammatory component may inhibit NO (Nitric oxide) production. 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. The NOS can be broadly divided into nNOS (neuronal NOS), eNOS (endothelial NOS), and i-NOS (inducible NOS). In particular, i-NOS is independent of intracellular calcium concentration and stimulated by LPS, IFN-γ, IL-1 and TNF-α 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.

Referring to Figure 2, it can be seen that 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.

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.

According to another embodiment of the present invention, the anti-inflammatory component may inhibit the production of PGE ₂ (Prostaglandin E2). The PGE ₂ is one of inflammatory mediators such as NO, and may mediate vasodilation, edema, fever, and pain. In addition, it is possible to induce the production of matrix metalloproteinases (MMPs) in inflammatory diseases involved in tissue damage, a large amount of PGE ₂ is produced in macrophages obtained from rheumatoid arthritis patients, the resulting PGE ₂ inflammatory response in rheumatoid arthritis And may play an important role in the mechanism of tissue destruction. The synthesis of PGE ₂ begins with the production of arachidonic acid from membrane phospholipids by enzymatic action of phospholipase A2. Arachidonic acid is enzymatically converted to prostaglandin G2 and then to the unstable metabolite prostaglandin H2, both of which are promoted by cyclooxygenase (COX). COX exists in two or more types of isoenzymes. Among them, COX1 is continuously expressed, which is responsible for physiological functions such as platelet aggregation, gastric mucosa protection, and renal function regulation. COX2 is expressed by stimulation such as inflammation, and thus prostaglandin produced by COX2. It may be involved in inflammatory reactions and cell proliferation.

Referring to Figure 2, it can be seen that 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.

According to an embodiment of the present invention, the anti-inflammatory component may inhibit the PGE ₂ by inhibiting the expression level of 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.

According to an embodiment of the present invention, the culture medium, one medium selected from the group consisting of αMEM, 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.

According to an embodiment of the present invention, the culture medium is one selected from the group consisting of αMEM, 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.

According to another embodiment of the present invention, the culture medium, Medium199 is 10 to 30 parts by weight; And 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.

According to a preferred embodiment of the present invention, the culture medium, Medium199 is 15 to 25 parts by weight; And 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.

According to another embodiment of the present invention, the culture medium, one medium selected from the group consisting of αMEM, 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.

According to a preferred embodiment of the present invention, 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.

According to an embodiment of the present invention, 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-1α. Preferably, the anti-inflammatory composition, 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 and MIP-1α may comprise 20 to 30 parts by weight.

According to another embodiment of the present invention, 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-1α may comprise 20 to 30 ng / ml.

Claims (11)

1. Separating monocytes from umbilical cord blood;

   Culturing the monocytes to obtain umbilical cord blood-derived multipotent stem cells;

   Stimulating the obtained cord blood-derived multipotent stem cells in a stem cell anti-inflammatory medium;

   Washing the stimulated cord blood-derived multipotent stem cells; And

   Culturing the washed umbilical cord blood-derived multipotent stem cells in a culture medium;

   Including, umbilical cord blood-derived multipotent stem cell culture method for secreting anti-inflammatory components.
2. The method of claim 1,

   The stem cell anti-inflammatory customized medium,

   αMEM or IMDM; And

   At least one component selected from the group consisting of TGF-β, TNF-α, IL-3, IL-6 and MEM vitamins;

   Umbilical cord blood-derived multipotent stem cell culture method for secreting an anti-inflammatory component, comprising a.
3. The method of claim 1,

   The stimulating step is characterized in that made for 20 to 28 hours, umbilical cord blood-derived multipotent stem cell culture method for secreting anti-inflammatory components.
4. The method of claim 1,

   The stimulating step is characterized in that made at 35 to 38 ℃, umbilical cord blood-derived multipotent stem cell culture method for secreting anti-inflammatory components.
5. The method of claim 1,

   The culture medium,

   one culture selected from the group consisting of αMEM, IMDM, DMEM / F12, and Medium199; And

   At least one component selected from the group consisting of insulin, transferrin, sodium selenite, MEM vitamins and human albumin;

   Umbilical cord blood-derived multipotent stem cell culture method for secreting an anti-inflammatory component, comprising a.
6. The method of claim 1,

   The anti-inflammatory component is characterized in that to inhibit the production of NO (Nitric oxide), umbilical cord blood-derived multipotent stem cell culture method that secretes anti-inflammatory components.
7. The method of claim 6,

   Inhibiting the production of NO is characterized in that the expression level of i-NOS (inducible Nitric oxide synthase) is suppressed, umbilical cord blood-derived multipotent stem cell culture method that secretes anti-inflammatory components.
8. The method of claim 1,

   The anti-inflammatory component is characterized in that to inhibit the production of PGE ₂ (Prostaglandin E2), umbilical cord blood-derived multipotent stem cell culture method for secreting an anti-inflammatory component.
9. The method of claim 8,

   Inhibiting the production of PGE ₂ is characterized in that the expression level of COX2 (Cyclooxygenase 2) is suppressed, umbilical cord blood-derived multipotent stem cell culture method that secretes anti-inflammatory components.
10. Anti-inflammatory composition comprising a culture medium secreted by umbilical cord blood-derived multipotent stem cells cultured by the method according to claim 1.
11. The method of claim 10,

    The anti-inflammatory composition is characterized in that it comprises at least one selected from the group consisting of IL-8, IL-10, IL-18, GM-CSF and MIP-1α, anti-inflammatory composition.

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