KR20180007256A - Culture medium composition for promoting human stem cell proliferaton comprising egg cell medium - Google Patents

Abstract

The present invention relates to a medium composition for culturing human stem cells containing an egg-derived stem cell culture solution as an active ingredient. To this end, the egg-derived stem cell culture solution enables effective proliferation of human stem cells while keeping the same under undifferentiated state, and brings skin cell proliferation efficacy, thereby being used as cosmetic compositions for relieving skin wound, pharmaceutical compositions for treating skin wound, and medium compositions for culturing human stem cells using the egg-derived stem cell culture solution.

Description

[0001] The present invention relates to a culture medium composition for promoting human stem cell proliferation,

The present invention relates to a culture medium for cell proliferation, a composition for skin cell proliferation, and a composition for treating wounds using the culture medium, while keeping human stem cells containing an egg-derived stem cell culture fluid of birds as an active ingredient in an undifferentiated state will be.

Stem cells are collectively referred to as undifferentiated cells at the stage before differentiation into each cell constituting the tissue, and specific differentiation stimulation (environment) differentiates into specific cells. Stem cells can proliferate (expand) because they can self-renew the same cells as themselves by cell division, unlike the differentiated cells where cell division is stopped. Further, when the differentiation stimulus is applied, It is characterized by its plasticity in differentiation because it can be differentiated into other cells by different environment or differentiation stimulus. There are two ways to obtain such stem cells. The first is to obtain embryos from embryos (embryonic stem cells), and the second is to collect stem cells (adult stem cells) that are retained in various parts of the adult body. Although there are differences in function, embryonic stem cells and adult stem cells all have differentiation characteristics into different types of cells.

Embryonic stem cells are derived from fertilized eggs (embryos) produced by fertilization of sperm, the male reproductive cells, and the reproductive cells of the female reproductive cells. When the embryo grows from the mother's stomach to the baby, about 2 trillion cells are produced. After the fertilization of the oocyte and the sperm, the cells up to 8 years of age have all-around properties that can be generated as a complete entity. Transplantation into the uterus can occur as a complete entity. Embryonic stem cells have the ability to occur as these various types of cells.

Adult stem cells are stem cells extracted from the growing body tissues and are primitive cells just before they are differentiated into specific organs. Adult stem cells are difficult to proliferate and tend to differentiate easily, but they can differentiate into tissue-specific progenitor cells inherent in the human body. The adult stem cells may be, but are not limited to, bone marrow, umbilical cord blood, blood, liver, skin, gastrointestinal tract, placenta, nerve, adrenal, epithelial and uterine stem cells.

Human embryonic stem cells have a pluripotency that is capable of producing all the cells of our body, and self-renewal ability to make infinite numbers of self-similar cells. These two properties together are called "stemness". Embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells), and induced pluripotent stem cells (iPSCs) . Induced pluripotent stem cells have self-renewal ability and universality, which means that they can differentiate into all types of cells including three germ layers.

Specifically, cultivation of embryonic stem cells is usually divided into culturing including feeder cells and culture not containing them. First, a method currently used for culturing stem cells, particularly human embryonic stem cells, is a method using mouse embryonic fibroblast or human foreskin fibroblast as a feeder cell, It is co-culture. This is the way feeder cells supply factors necessary for culturing human embryonic stem cells while maintaining stem cell characteristics. Feeder cells are treated with Mitomycin C or irradiated with radioactivity to stop the growth of feeder cells and then supply factors necessary for growth to human embryonic stem cells while maintaining stem cell characteristics. The culture medium used for co-cultivation is a medium supplemented with 20% knockout serum replacer (Gibco) in 80% Knockout DMEM medium (Gibco). In addition to the medium, glutamine, mercaptoethanol, A basic fibroblast growth factor (bFGF) may be added. Until now, it is not known in detail which factors provided in the feeder cells are important for stem cell maintenance of human embryonic stem cells.

Human embryonic stem cells cultured by co-culture with mouse embryonic fibroblasts have many difficulties in clinical use. This is because there is a possibility of transmission by xenogen. Therefore, attempts have been made to cultivate human embryonic stem cells in the absence of feeder cells. However, until now, there has not been established a method for culturing human embryonic stem cells for a long period of time by using a feeder-free culture medium and an extracellular matrix of a culture container in which animal-derived substances are completely removed Respectively.

On the other hand, a method of culturing embryonic stem cells without containing feeder cells has been proposed, in which mTeSR medium or the like is used to coat the surface of the plate with matrigel at a low temperature. However, when matrigel is used, a complicated problem has arisen since the surface of the plate is kept at a low temperature during the subculture of embryonic stem cells and a reagent treatment process for removing the matrigel is added. In addition, mTeSR culture medium (feeder-free media, Stem cell technologie) requires expensive protein coating (matrigel, BD science). The above method also requires changing the medium with mTsSR meida every day, and the cost of the medium itself is expensive.

Therefore, the present inventors have made efforts to develop a culture medium capable of effectively culturing human stem cells without feeder cells, and as a result, it has been confirmed that human stem cells proliferate actively in a conditioned medium containing a culture medium of avian stem cells derived from avians , And that the egg stem cell culture solution exhibits a significant skin cell proliferation effect and can be used as a culture medium for human stem cell culture or a wound healing composition.

Conventionally, when a feeder cell is used in a method of culturing a stem cell, there is a risk of heterologous infection. When a medium in which a feeder cell such as mTeSR medium is not used is used, a complicated process and an expensive price . Accordingly, the present invention has been devised to solve such a conventional problem, and thus, a culture solution capable of effectively culturing human stem cells without feeder cells has been developed.

That is, an object of the present invention is to provide a culture medium for culturing human stem cells, a pharmaceutical composition for treating skin wound, and a cosmetic composition for skin wound healing, which comprises a stem cell culture fluid derived from Egg of algae as an active ingredient.

In order to achieve the above object,

The present invention provides a culture medium for culturing human stem cells, which comprises an egg-derived stem cell culture fluid of algae as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for treating skin wound, which comprises a stem cell culture fluid derived from avian egg as an active ingredient.

Further, the present invention provides a cosmetic composition for skin wound healing, which contains a stem cell culture fluid derived from avian egg as an active ingredient.

In addition,

1) adding avian egg-stem cells to a serum-free medium containing a microcarrier coated with gelatin or fibronectin; And

2) treating the serum-free medium of step 1) with at least one inhibitor selected from the group consisting of a ROCK inhibitor, a MEK inhibitor, an ERK inhibitor, and a GSK-3b inhibitor and then culturing the cells in an egg-derived stem cell culture solution And a manufacturing method thereof.

The culture medium for human stem cell culture, which contains the stem cell culture fluid derived from the egg of the present invention as an effective ingredient, can effectively proliferate human stem cells while maintaining undifferentiated state without feeder cells, It can be used as an inexpensive and effective medium for human stem cell culture, a pharmaceutical composition for treating skin wound, and a cosmetic composition for improving skin condition.

Figure 1 A shows a photograph of an epiblast in which egg totipotent cells are present. In particular, the inside of the circles represents the area pellucida, and there are fully differentiated cells in this area. FIG. 1B shows the fifth stage of the occurrence of the egg, showing that the egg-differentiated cells are differentiated into neural stem cells.
Figure 2 shows each of the differentiated cells obtained from the egg cells in four different ways. A is a fully differentiated cell which was isolated and cultured from an epiblast in the first stage of development. B is a fully differentiated cell that is isolated and cultured from the blood of primordial embryo. C is a fully differentiated cell that is isolated and cultured from primordial germ cells. D is a stellate differentiated cell.
Figure 3 shows the difference in the differentiation of erythrocyte differentiation between the experimental group treated with inhibitor (red curve) and the control group (black curve) not treated with inhibitor as a graph of erythrocyte differentiation cell proliferation. It was confirmed that the progenitor cells proliferated only when the inhibitor was treated.
FIG. 4 is a photograph of human pre-differentiated cells cultured in egg cell culture. Morphologically, it was confirmed that human stem cells were grown in an undifferentiated state.
FIG. 5A shows the results of confirming the expression of undifferentiated stem cell markers in human embryonic stem cells through cell immuno staining. In the photograph, the red part represents the part of the OCT4 gene that is a precursor of the differentiated stem cell marker, and the part B of FIG. 5 represents the supporting cells and the stem cells indicated by arrows as a result of nuclear staining.
FIG. 6 shows PCR results showing that human embryonic stem cells express genes important for maintaining undifferentiated states such as OCT4, SOX2 and NANOG. As a result, NANOG, SOX2, and OCT4 genes were expressed in human stem cell culture medium. Among them, SOX2 showed an increased expression pattern than Line 2 using a typical stem cell undifferentiated growth medium. This indicates that human stem cells remain undifferentiated in Egg cell culture medium, and it is more effective than the case of using a typical stem cell undifferentiated growth medium.
FIG. 7 shows the results of culturing human embryonic stem cells in culture medium for 1, 2, 3, 4, and 5 days in 20% KO conditioned medium and Egg suspension conditioned medium, The results of measurement of proliferation are shown. As a result, it was confirmed that human embryonic stem cells proliferated more effectively when cultured in egg suspension medium than 20% KO conditioned medium.
8 shows the results of culturing HatCaT cells, which are skin cells, on a medium containing DMEME medium, DMEM + EGF (100 ng / mL) medium, egg cell culture medium 0.1%, 0.5% The results are compared. It was confirmed that HatCaT cells proliferated effectively when cultured on a medium containing 0.5% to 1% of egg cell culture medium, and that the growth promoting effect was excellent when cultured skin cells in egg cell culture medium than other condition medium.

Hereinafter, the present invention will be described in more detail.

The present invention provides a culture medium for culturing human stem cells, which comprises an egg-derived stem cell culture fluid of algae as an active ingredient.

The egg of the present invention is an egg of an alga and the bird is a chicken, turkey, duck, geese, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary ), Including, but not limited to, all species, subspecies, or rass of the Aves class. Particularly preferred are gallus gallus or chicken varieties such as White Leghorn, Brown Leghorn, Barred-Rock, Sussex, California Gray, Italian oartridge- colored.

The stem-derived stem cells of the present invention are pluripotent stem cells obtained from the blastoderm of embryonic egg embryos corresponding to stage-X or XI at the development stage of Eyal-Giladi and Kochav (EG & K, 1976) ), Egg embryos were cultured in the incubator for 3-5 days at 38 ° C and the blood was collected from embryos that reached the development stage of H & H (Hamburger and Hamilton, 1951) stage 14-17. Embryonic germ cells (EGCs) isolated from the gonads formed in H & H (Hamburger and Hamilton, 1951) stage 28 after incubation for 5-6 days at 38 ° C were incubated at 38 ° C for 11 days. Induced pluripotent stem cells obtained by treatment with Yamanaka transcription factor (OCT4, Sox2, KIF-4, c-Myc) and over-differentiation of embryonic fibroblasts (EFs) cells, iPS Cs). ≪ / RTI >

However, the present invention is not limited to this. 1) First, egg-derived stem cells obtained by the above method are coated with gelatin or fibronectin 2) the various kinds of cell signal circuit inhibitors, preferably the inhibitors, in the serum-free medium are added to a serum-free medium containing a GSK inhibitor, a GSK-3b inhibitor, a MEK inhibitor, a TGF -beta inhibitor, a ROCK inhibitor, a BMP inhibitor, and an ERK inhibitor, and more preferably at least one inhibitor selected from the group consisting of a ROCK inhibitor, a MEK inhibitor, an ERK inhibitor, and a GSK-3b inhibitor Step.

The reason for using a microcarrier coated with gelatin or fibronectin is as follows. Generally, in order to proliferate stem cells in vitro, stem cells are cultured on a flat surface pre-coated with extracellular matrix (ECM) protein or nutrient cells in a flask. Flat culture, however, often fails to support the long-term growth of stem cells because of its limited surface area, which often requires subculturing. As a solution to this, microcarrier-based methods can be used for stem cell culture, and microcarriers have a high surface-area-to-volume ratio and thus overcome the surface area limitation of stem cell growth on flat surfaces . In addition, by using a microcarrier coated with a polymer such as gelatin or fibronectin in the present invention, algae precursor differentiated stem cells are effectively attached to microcarriers and exhibit high efficiency in the subsequent culturing step, scale up and mass production stage .

On the other hand, serum-free medium (SFM) is used as a medium for stem cell growth of algae, which means a medium containing no serum that enables cell survival and cell growth. Adding serum to the culture medium is essential for cell culture to provide universal nutrients for growth of all cell lines, but serum has many limitations. Animal sera are deviating from each other in unit of production, are expensive, are potential causes of contamination such as disease-inducing viruses and mycoplasma, and also have a large amount of components present in the serum, resulting in separation of recombinant proteins secreted in the medium and high- Is very difficult. Because of this problem, when culturing stem cells, it is preferable to use serum-free medium containing no serum in the medium. This medium need not be a chemically purified medium, but may also include hydrolysates derived from animals and plants. The serum-free medium of the present invention is preferably limited to a non-animal origin that does not contain animal or human origin components, and natural serum proteins are replaced by recombinant proteins. However, the serum-free medium of the present invention does not include proteins (PF medium: protein-free medium) and / or chemical purification (CDM medium: chemically purified medium).

Examples of commercially available SFM media are as follows. (Cambrex ref 12-766Q, catalog 2003), HyQ (Cambrex ref 12-766Q, Catalog 2003), Pro 293 S-CDM (Cambrex ref 12765Q, Catalog 2003), LC17 (Cambrex Ref BESP302Q), Pro CHO 5-CDM (Hyclone Ref SH30515-02), HyQ SFM4 CHO-Utility (Hyclone Ref SH30516.02), HyQ PF293 (Hyclone ref SH30356.02), HyQ PF Vero (Hyclone Ref SH30352.02) (JRH Biosciences ref 14560-1000M), Ex cell 302 serum-free medium (JRH Biosciences ref 14312-1000M), Ex cell 65319 (JRH Biosciences), Ex cell 65421 (JRH Biosciences), Ex Ex-cell 65627 (JRH Bioscience), Ex cell 65627 (JRH Bioscience), Ex cell 65627 (JRH Bioscience), Ex cell 65628 (JRH Bioscience), Ex cell 65629 SIGMA-Aldrich, ref G-9916).

The concentration of each of the inhibitors treated with serum-free medium for the preparation of the precursor stem cells is not limited, but preferably the inhibitor comprises about 0.3 [mu] M to about 30 [mu] M GSK-3b inhibitor, about 10 nM to about 10 [ A MEK inhibitor, about 5 μM of a ROCK inhibitor at about 50 nM, and most preferably the inhibitor treats about 3 μM of a GSK-3b inhibitor, about 1 μM of a MEK inhibitor, and about 0.5 μM of a ROCK inhibitor .

As the vessel used for producing the egg-stem cell culture of the avian embryo of the present invention, a tank bioreactor, a WaveTM bioreactor, a Bello (TM) bioreactor, a spinner flask, a flask and a cell factory may be used. Most preferably, it is preferred to use a continuous stirred tank bioreactor with controlled temperature, air circulation, pH and other conditions.

In general, the cell number scale-up step increases the scale using a master or working cell bank vial through T-flasks or roller bottles of various sizes, and preferably increases the scale finally in the bioreactor. A seed production bioreactor (typically 20-30 L volume) is then used for further incubation of the cell suspension produced. The volume ratio of the secondary bioreactor to the seed bioreactor depends on the number of cells proliferated in the primary bioreactor, but is preferably in the range of 3: 1 to 10: 1, i.e., 6 to 8: 1.

The avian stem cell culture solution produced by the above method has the characteristic of maintaining and multiplying human stem cells in an undifferentiated state. In order to confirm this, human stem cells were cultured at 35-37 ° C for 2-5 days in a medium containing avian stem cell culture fluid as an active ingredient, and the morphology of human stem cells was observed under a microscope (see FIG. 4) In order to confirm the expression pattern of the OCT4 gene, which is a nuclear stain and a totally differentiated stem cell marker, immunofluorescence staining using an antibody was performed (see FIG. 5). Also, by using the Nanog, Sox-2 and OCT4 gene PCR, Human embryonic stem cells were cultured at 35-37 ° C for 2-5 days in the stem cell culture fluid of avian egg. In order to confirm the retention of cell undifferentiation, total RNA was isolated, and RT-PCR was performed using primers specific for each gene (See Fig. 6). In order to confirm the cell proliferative ability of the stem cell culture fluid derived from avian egg, human stem cells were cultured in a culture medium of avian stem cell derived from avian The cells were cultured under the same culture conditions, and the number of cells was measured. The proliferation cell potency was compared with the number of cells in the positive control group (see FIG. 7).

In a specific example of the present invention, the present inventors have shown a method for obtaining a meiotic cell from avian Egg cells and a method for producing a stem cell culture liquid derived from an avian egg. In the experimental example, (See FIG. 4), OCT-4 staining (see FIG. 5), and NANOG, SOX2 and OCT4 gene expression PCR tests (see FIG. 6) were performed to confirm whether the undifferentiated state was maintained during the culture, It was confirmed that human stem cells and skin cells were effectively proliferated on an egg-derived stem cell culture medium (see FIGS. 7 and 8)

As a result, the egg-derived stem cell culture solution of the present invention can be used as a culture medium for human stem cells because it can effectively proliferate human stem cells without maintaining the feeder cells in an undifferentiated state.

In addition, the present invention provides a pharmaceutical composition for treating skin wound, which comprises a stem cell culture fluid derived from avian egg as an active ingredient.

The wound of the present invention refers to a state in which a living body is damaged, and may include damage to any part of the body, such as a tissue constituting an inner or outer surface of a living body.

The stem cell culture fluid derived from the avian egg may promote skin cell proliferation and have activity for wound healing and skin regeneration but is not limited thereto and the active ingredient of the present invention may be used in an amount of 0.1-100 wt% % ≪ / RTI >

In the specific examples and experimental examples of the present invention, HaCaT (keratinocyte, Thermo) skin cells were cultured on an egg stem cell culture medium for a predetermined period of time in order to confirm the skin cell proliferating ability in a stem cell culture fluid of avian egg, Effective skin cell proliferating ability was confirmed (see Fig. 8).

As a result, it was confirmed that the skin cell proliferation effect was excellent in the skin cell culture on the egg-derived stem cell culture solution of the bird of the present invention. As a result, the stem cell culture solution of the avian egg- .

The pharmaceutical composition comprising the human stem cell culture liquid of the present invention may further contain an appropriate carrier, excipient and diluent commonly used in addition to the egg stem cell culture fluid as an effective ingredient. In addition, solid pharmaceutical preparations or liquid pharmaceutical preparations can be used for the preparation of pharmaceutical compositions. The preparation additive may be either organic or inorganic.

Examples of the excipient include lactose, sucrose, saccharin, glucose, corn starch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate and silicon dioxide. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethylcellulose, methylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylmethylcellulose, calcium citrate, Dextrin, pectin, and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any coloring agent may be used as long as it is usually allowed to be added to pharmaceuticals. These tablets and granules can be suitably coated with sugar (sugar coating), gelatin coating, and others as required. If necessary, preservatives, antioxidants and the like may be added.

The pharmaceutical composition of the present invention can be prepared into any formulation conventionally produced in the art (for example, Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), the form of the formulation is not particularly limited, It may preferably be an external preparation. The external preparation of the present invention may be in the form of a sheet, a liquid coating agent, a spray agent, a lotion agent, a cream agent, a papermaking agent, a powder, a penetration pad agent, a spray agent, a gel agent, a pasta agent, a liniment agent, an ointment agent, Absorbents and the like may be included. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a formulation generally known in all pharmaceutical chemistries.

In the external preparation of the present invention, pharmaceutically acceptable carriers include hydrocarbons such as vaseline, liquid paraffin, gelling hydrocarbons (also known as plastid bases) and the like; Vegetable oils such as medium-chain fatty acid triglyceride, lard, hard fat, cacao butter and the like; Higher fatty alcohols such as cetanol, stearyl alcohol, stearic acid and isopropyl palmitate; fatty acids and esters thereof; Water-soluble bases such as macrogol (polyethylene glycol), 1,3-butylene glycol, glycerol, gelatin, white sugar, sugar alcohol and the like; Emulsifiers such as glycerin fatty acid esters, polyoxyl stearate, and polyoxyethylene hardened castor oil; Acrylic acid ester, and sodium alginate; Jet agents such as liquefied petroleum gas and carbon dioxide; And preservatives such as p-hydroxybenzoic acid esters. The external preparation of the present invention can be prepared by using these methods according to a conventional method. In addition, a stabilizer, a flavoring agent, a coloring agent, a pH adjuster, a diluent, a surfactant, a preservative, an antioxidant and the like may be blended as needed. The external preparation of the present invention can be applied on top of the local window by a conventional method. In addition, the external preparation according to the present invention can be used by being adhered to a solid support such as a wound dressing cover of a conventional band-aid. As an aspect of the present invention, the solid support is first coated with an adhesive layer to improve the adhesion of the culture solution to the solid support. Examples of the pressure sensitive adhesive may include polyacrylate and cyanoacrylate.

The pharmaceutically effective amount of the present invention may vary depending on the type of wound of the patient, the site of application, the number of treatment, the treatment time, the formulation, the condition of the patient, the kind of the adjuvant and the like. Although the amount to be used is not particularly limited, a daily effective amount of the pharmaceutical composition of the present invention is usually 1 to 50 μl / cm ² Preferably from 5 to 20 [mu] l / cm < ² > Lt; / RTI > The above-mentioned daily dose may be administered once a day or two or three times a day at appropriate intervals, or intermittently administered at intervals of several days. However, the above-mentioned amount of the pharmaceutical composition of the present invention may be varied depending on the administration route, age, sex, weight, severity of the patient, wound type, application site, treatment time, treatment time, The effective amount does not limit the scope of the present invention in any aspect.

The present invention also provides a cosmetic composition for skin regeneration or skin wound healing which comprises an egg stem cell culture fluid as an active ingredient.

The egg-derived stem cell culture solution of the present invention can effectively proliferate skin cells and can be used as a composition for skin cell proliferation or a cosmetic composition for skin regeneration or skin wound healing using the composition as an active ingredient.

The skin condition improving cosmetic composition comprising the human stem cell culture liquid of the present invention may contain an acceptable carrier in a cosmetic preparation. Herein, "an acceptable carrier for a cosmetic preparation" is a known or used compound or composition that can be contained in a cosmetic preparation, or a compound or composition to be developed in the future, which is toxic, instable or irritating It says nothing. The carrier may be included in the cosmetic composition of the present invention in an amount of about 1% by weight to about 99.99% by weight, preferably about 90% by weight to about 99.99% by weight of the composition, based on the total weight thereof. However, since the ratio varies depending on the formulations described below in which the cosmetic composition of the present invention is prepared, and on the specific application site (face, neck, etc.) of the cosmetic composition and its preferable application amount, But is not limited to.

Examples of the carrier include an alcohol, an oil, a surfactant, a fatty acid, a silicone oil, a wetting agent, a moisturizer, a viscous modifier, an emulsifier, a stabilizer, an ultraviolet scattering agent, an ultraviolet absorber, a coloring agent and a perfume. The compounds / compositions which can be used as the above-mentioned alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscous modifier, emulsifier, stabilizer, ultraviolet scattering agent, ultraviolet absorber, The person skilled in the art can select and use the appropriate substance / composition. As an embodiment of the present invention, the cosmetic composition according to the present invention may contain glycerin, butylene glycol, propylene glycol, polyoxyethylene hydrogenated castor oil, ethanol, triethanolamine, etc., besides the stem cell culture derived from the egg, Antiseptic, antiseptic, coloring agent, purified water and the like can be contained as needed. The cosmetic composition according to the present invention can be prepared in various forms such as lotion, essence, gel, emulsion, lotion, cream (water-in-oil type, water-in-water type, multiphase), solution, suspension (Soft capsules, hard capsules) with a coating such as anhydrous products (oil and glycol), gel, mask, pack, powder, or gelatin. The skin of the present invention includes not only a face but also a scalp and a whole body. The cosmetic composition applicable to such scalp is a shampoo, a rinse, a treatment, a hair removal agent, etc., and a body cleanser And can be manufactured in various forms as an application of the composition. The method for preparing a cosmetic composition for skin regeneration or skin wound healing according to the present invention is not limited to the above-mentioned manufacturing method, The cosmetic composition according to the present invention can also be prepared by a method in which the production method is partially modified.

In particular, the cosmetic composition for skin regeneration or the cosmetic composition for skin wound healing can be produced in the form of a general emulsified formulation and a solubilized formulation, by using a conventionally known manufacturing method, in addition to the manufacturing method specifically disclosed in the present invention. When emulsified formulations and solubilized form cosmetic compositions are prepared, cosmetics of emulsified form include nutritional lotion, cream, essence and the like, and solubilized form cosmetics have softening longevity. It can also be prepared in the form of adjuvants which can be applied topically or systemically, which are conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base. In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing an oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) May be provided in the form of a follicular dispersing agent of the type, cream, skin, lotion, powder, ointment, spray or conical stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

The cosmetic composition for skin regeneration or the skin regeneration cosmetic composition according to the present invention may further comprise at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, , A perfume, a surfactant, water, an ionic or nonionic emulsifier, a filler, a sequestering and chelating agent, a preservative, a vitamin, a barrier, a wetting agent, a essential oil, a dye, a pigment, a hydrophilic or lipophilic active agent, Or any other ingredient conventionally used in cosmetics, or adjuvants commonly used in the cosmetics or dermatology fields. And the above ingredients may be introduced in amounts commonly used in the dermatology field. Such a cosmetic composition according to the present invention includes forms of functional cosmetics such as skin astringency, anti-inflammation, wound, wound healing, and anti-aging.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> Of birds From Egg cells Pre-differentiable cells ( pluripotent  cell separation

<1-1> Isolation of exocrine cells (PSCs) from the blastoderm of Egg embryos

Eggs of white leghorn species, a kind of chicken, were used.

The developmental stage according to Eyal-Giladi and Kochav (EG & K, 1976), that is, the blastoderm of stage-X or XI stage embryo was clipped using surgical scissors. The vitelline and egg yolk of the scraped blastodule were removed from the PBS buffer, and blastodermal cells with differentiation ability were obtained. BFGF (10 ng / ml), IGF-1 (20 ng / ml) were incubated with STO cells (ATCC collection) in which growth was inhibited by treating mitochondria C (Mitomycin C) (1%), IL-6 (1%), IL-11 (1%), LIF (1%) and FBS (10%). As a result of co-cultivation, pre-differentiated cells formed communities, and specific culture methods were referred to Pain B et al. (1996). The embryonic stem cells (ESCs), which are pluripotent cells, were obtained by isolating and culturing the Egg blastoderm (FIG. 1A) in which pluripotent cells were present (FIG. 2A) .

<1-2> Isolation of pre-differentiable cells (PSCs) from blood produced from embryos

Egg embryos were incubated for 5 days at 38 ° C in H & H (Hamburger and Hamilton, 1951) stage 14-17, and the blood of this period contained promodial germ cells (PGC). Blood from this stage was collected from the heart and arteries and treated with mitomycin C to inhibit the growth of STO cells and DMEM medium containing SCF (6 ng / ml), bFGF (4 ng / ml) and 8% FBS Lt; / RTI &gt; Embryonic stem cells (ESCs) were obtained by differentiating and differentiating cells from the differentiated cells after 2 weeks of co-cultivation. A detailed culture method was described by van de lavoir et al. (2006) (Fig. 2B).

<1-3> Isolation of total differentiable cells (PSCs) from gonads

Embryonic germ cells (EGCs) develop in the gonads when the embryos are incubated for 6 days at 38 ° C. EGCs were isolated from the gonad and the cells were treated with bFGF (10 ng / ml), IGF-1 (10 μg / ml) together with STO cells (ATCC collection) in which growth was inhibited by treatment with mitomycin C (1%), IL-6 (1%), IL-11 (1%), LIF (1%) and FBS EGCs with differentiating ability were obtained (Fig. 2C).

<1-4> Isolation of ESCs from pre-differentiation-differentiating cells

Embryonic fibroblasts (EFs) were obtained by culturing embryos in 10% DMEM medium. A vector capable of overexpressing the Yamanaka factor (Oc4, c-Myc, Sox2, KIF-4) was introduced into the fibroblast using an electrophoresis (electrophoresis) Egg embryo cells were co-cultured for 4 weeks in DMEM medium containing 10% FBS together with STO cells inhibiting cell growth with mitomycin C. As a result of co-culture, a population of induced pluripotent stem cells (iPSCs) having a total differentiation ability was obtained (FIG. 2D).

Example 2: Preparation of stem cell culture liquid derived from avian egg

<2-1> Co-culture of egg-stem cells and feeder cells

(10 ng / ml), IGF-1 (20 ng / ml) and SCF (10 ng / ml) were added to the medium containing fetal bovine serum (FBS) to maintain the growth and undifferentiation of the stem- , Serum-free medium (SFM (10 ng / ml)), and IL-6 (20 ng / ml) ) To prepare an egg-derived stem cell culture solution.

<2-2> Inhibitor treatment on SFM medium

(10 ng / ml), IGF-1 (20 ng / ml) and SCF (10 ng / ml) were added to the SFM medium of Example <2-1> to establish culture conditions of single- ), IL-6 (10 ng / ml), LIF (1000 U) with ROCK inhibitor (Y27632, 0.5 μM), MEK inhibitor (PD98059, 1 μM), GSK-3b inhibitor (CHIR-98014 CHIR- ) Were cultured in KO-DMEM / F12 medium for 5 days. At this time, it was confirmed that the cells proliferated while keeping the stem cell state without co-culturing with feeder cells such as STO cells.

In order to confirm that the inhibitor treatment is essential, the differentiation of the cells was observed after incubation for 5 days on the SFM medium of Example <2-1> in which no inhibitor was added for 5 days. As a result, as shown in Fig. 3, it was confirmed that the egg-precursor cells cultured in the medium without the inhibitor did not proliferate or die, and that the egg-precursor cells cultured in the medium supplemented with the inhibitor actively proliferated. Therefore, it was confirmed that when the inhibitor was essentially added in the serum-free suspension culture condition, the progenitor cells proliferated (Fig. 3).

&Lt; 2-3 > Stem Cell Suspension Culture Using Microcarriers

Egg-derived stem cells were suspended and cultured using a microcarrier coated with gelatin or Fibronectin as an adherend to increase the efficiency of stem cell culture derived from avian eggs. To coat the microcarriers, microcarriers were immersed in gelatin (0.1%) or fibronectin (0.1%) at 37 ° C for 2 hours and then dried on a clean bench. BFGF (10 ng / ml), IGF- 1 (20 ng / ml), SCF (10 ng / ml), LIF (1000 U) and MEK inhibitor (PD98059, 1 uM), and GSK-3b inhibitor (CHIR-98014 CHIR-98014 CHIR-98014, In the KO-DMEM / F12 medium containing the embryonic stem cells, the embryonic stem cells were suspended and cultured for 3 days. After cultivation, the cells were cultured in a medium containing no GSK-3b inhibitor and PD98059, a MEK inhibitor, for 2 to 3 days. As a result, the resulting culture was used as a culture medium for growing human stem cells while maintaining their undifferentiated state. When compared with conventional microcarriers, it was confirmed that when the microcarrier coated with gelatin or fibronectin protein was used, the stem-derived stem cells adhered well to the microcarriers and the cells were scaled up efficiently in the bioreactor .

<Experimental Example 1> Morphological confirmation of undifferentiation maintenance in human stem cell culture

Human embryonic stem cells were cultured at 37 占 폚 for 3 days in a medium containing an avian embryonic stem cell culture solution prepared by the method of Example <2-3> as an active ingredient. After that, the morphology of human stem cells was observed with a microscope. As a result, it was confirmed that morphological development such as tissue and organ development was not progressed and maintained in a cellular state as shown in FIG. 4 (FIG. 4).

< Experimental Example  2> OCT4  Confirmation of whether undifferentiated state is maintained when human stem cells are cultured using staining

OCT4 immunofluorescence staining was performed to confirm whether human stem cells cultured in avian stem cell culture fluid maintained undifferentiated state.

In order to examine the expression pattern of the OCT4 gene, which is a precursor for the differentiation of stem cells, human embryonic stem cells were cultured in 20% SR DMEM / F12 medium except for various growth factors. Immunofluorescence staining was performed using anti-mouse IgG, which was labeled with Cy-3 as a secondary antibody against mOct4 (mouse monoclonal OCT4, Santa Cruz) as a secondary antibody. Nuclear staining was performed using DAPI.

As a result, as shown in FIG. 5A, it was confirmed that OCT4 gene was expressed in the red-stained region and the stem cells were undifferentiated, and nuclear differentiation and supporting cells were confirmed through nuclear staining (FIG. 5B).

< Experimental Example 4> Nanog , Sox-2, OCT4  gene PCR  Identification of undifferentiated maintenance of culture of human stem cells using the test method

In order to determine whether human embryonic stem cells were maintained in an undifferentiated state after incubating human embryonic stem cells for 2 days at 37 ° C. in the avian stem cell culture fluid prepared by the method of Example 2, Nanog, Sox-2, OCT4 And the expression of genes important for maintaining the undifferentiated state as shown in Fig.

Specifically, MEF medium and hESCs_20% SR DMEM / F-12 were isolated from human embryonic stem cells harvested by culturing on bFGF-containing medium and egg stem cell culture medium. Then, PCR was carried out using primers specific for Nanog, Sox-2, OCT4 and a comparative gene, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene. The primers used for RT-PCR are as follows. NANOG (F: 5'-GCTGAGATGCCTCACACGGAG-3` SEQ ID NO: 1, R: 5`-TCTGTTTCTTGACTGGGACCTTGTC-3` SEQ ID NO: 2), SOX2 (F: 5`-GTCATTTGCTGTGGGTGATG-3` SEQ ID NO: 5, AAGAACATGTGTAAGCTGCGGC-3 &apos; SEQ ID NO: 6)

The results of confirming the expression of these genes are shown in Fig. According to this line 1, MEF represents the expression level of undifferentiated stem cell marker genes when a growth medium containing only MEF is used as a mouse-derived supporting cell generally used for human stem cell culture. As a result, neither Nanog, Sox-2 nor OCT4 gene was expressed. In line 2, hESCs_20% SR DMEM F-12 is a typical stem cell undifferentiated growth medium, and Line 2 represents the expression level of human stem cell marker genes using a typical stem cell undifferentiated growth medium. As a result, both Nanog, Sox-2, and OCT4 genes were expressed, indicating that human stem cells maintained undifferentiated state. Line 3 represents the expression level of undifferentiated stem cell marker genes when used as human stem cell culture medium. As a result, both Nanog, Sox-2, and OCT4 genes were expressed. Among them, Sox-2 showed an expression pattern higher than that of Line 2. It was confirmed that human stem cells retained undifferentiated state in avian cell culture of avian algae, and it was confirmed to be more effective than the case of using a typical stem cell undifferentiated growth medium.

< Experimental Example  5> Origin of Egg Eggs Human stem cells of stem cell cultures Proliferative ability  Confirm

Human embryonic stem cells were cultured on 20% KO conditioned medium or Egg suspension conditioned media to confirm the human stem cell proliferative capacity of avian stem cell culture. Respectively.

Human embryonic stem cells were cultured on a 20% Serum Replacement (SR) -DMEM / F-12 conditioned media or bovine embryonic stem (FGF) medium supplemented with bFGF for 1 day, , 3, 4, and 5 days, and the number of cells was measured. As a result, it was confirmed that human embryonic stem cells proliferate more effectively when cultured on an agar cell culture medium than 20% KO conditioned medium (Fig. 7).

< Experimental Example  6> Egg Origin of Stem Cell Culture Skin cell Proliferative ability  Confirm

In order to confirm the skin cell proliferating ability of the avian stem cell culture fluid of the avian egg, HatCaT (keratinocyte, Thermo) cells were cultured in DMEM medium containing 10% FBS and cultured in DMEM without FBS for 1 day, Respectively. (Positive control) in which EGF (10 ng / mL) was added to DMEM (negative control) and DMEM medium not containing FBS (DMEM medium containing 0.1%, 0.5%, and 1% And grown for 3 days on each medium. As a result, it was confirmed that HaCaT cells proliferated effectively when cultured on a medium containing 0.5% and 1% of avian egg cell culture medium in the basic DMEM medium. In the DMEM medium containing EGF as the positive control group, It was confirmed that the DMEM medium containing the culture medium had excellent skin cell proliferation promoting effect (FIG. 8).

<110> Sonimedi <120> Culture medium composition for promoting human stem cell          proliferaton comprising egg cell medium <130> P2016-099 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NANOG F primer <400> 1 gctgagatgc ctcacacgga g 21 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NANOG R primer <400> 2 tctgtttctt gactgggacc ttgtc 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX2 F primer <400> 3 gtcatttgct gtgggtgatg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX2 R primer <400> 4 agaaaaacga gggaaatggg 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OCT4 F primer <400> 5 ggaaaggctt ccccctcagg gaa 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OCT4R primer <400> 6 aagaacatgt gtaagctgcg gc 22

Claims (11)

1. A culture medium for culturing human stem cells comprising an avian egg-derived stem cell culture fluid as an active ingredient.
The culture medium for culturing human stem cells according to claim 1, wherein the algae is chicken.
The culture medium for culturing human stem cells according to claim 1, wherein the egg stem cell culture medium maintains and proliferates human stem cells in an undifferentiated state.
The culture medium for human stem cell according to claim 1, wherein the egg-derived stem cells are cultured in a serum-free medium containing a microcarrier coated with gelatin or fibronectin Composition.
The culture medium for human stem cell culture according to claim 3, wherein the serum-free medium comprises at least one inhibitor selected from the group consisting of ROCK inhibitors, MEK inhibitors, ERK inhibitors and GSK-3b inhibitors.
The culture medium for culturing human stem cells according to claim 1, wherein the egg-derived stem cells are pluripotent stem cells (PSCs) extracted from egg cells.
The culture medium for stem cell culture according to claim 1, wherein the human stem cells are embryonic stem cells or adult stem cells.
The stem cell according to claim 7, wherein the adult stem cells are selected from the group consisting of bone marrow derived cells, cord blood derived cells, blood derived cells, liver derived cells, skin derived cells, gastrointestinal cells derived cells, placental cells, nerve cells, adrenal cells, epithelial cells, Wherein the culture medium for stem cell culture is a culture medium.
A pharmaceutical composition for treating skin wounds containing an egg-derived stem cell culture solution of avian influenza as an active ingredient.
A cosmetic composition for improving skin regeneration or skin wounds containing an egg-derived stem cell culture fluid of algae as an active ingredient.
1) adding egg-derived stem cells of algae to a serum-free medium containing a microcarrier coated with gelatin or fibronectin; And
2) treating the serum-free medium of step 1) with at least one inhibitor selected from the group consisting of a ROCK inhibitor, a MEK inhibitor, an ERK inhibitor, and a GSK-3b inhibitor and then culturing the cells in an egg-derived stem cell culture solution Gt;

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