KR101849631B1 - Culture medium composition for promoting human stem cell proliferaton comprising egg cell medium - Google Patents

Abstract

The present invention relates to a culture medium for culturing human stem cells comprising an egg-derived stem cell culture liquid as an active ingredient, wherein the egg stem cell culture liquid maintains human stem cells in an undifferentiated state and effectively proliferates, The composition can be used as a culture medium for culture of human stem cells, a pharmaceutical composition for treating skin wound, and a cosmetic composition for skin wound healing.

Description

Culture medium composition for promoting human stem cell proliferaton comprising egg cell medium}

The present invention relates to a medium composition for cell proliferation, a composition for proliferation of skin cells, and a composition for wound treatment using the culture medium while maintaining human stem cells containing a stem cell culture solution derived from algae as an active ingredient in undifferentiated state. will be.

Stem cells collectively refer to undifferentiated cells in a stage before differentiation into each cell constituting a tissue, and differentiation proceeds to specific cells by a specific differentiation stimulation (environment). Stem cells have the property of proliferation (expansion) because they can produce cells identical to themselves (self-renewal) by cell division, unlike differentiated cells in which cell division is stopped. It is differentiated into cells, but it is characterized by having plasticity in differentiation because it can be differentiated into other cells by different environments or different differentiation stimulation. There are two main ways to obtain these stem cells. The first is to obtain stem cells (embryonic stem cells) from embryos generated from fertilized eggs, and the second is to recover stem cells (adult stem cells) that are kept in each part of the adult body. Although there are differences in function, both embryonic stem cells and adult stem cells have the characteristics of being able to differentiate into various types of cells.

Embryonic stem cells are derived from fertilized eggs (embryos) produced by fertilization of sperm, which is a male reproductive cell, and egg, which is a female reproductive cell. When a fertilized egg grows into a baby in the mother's stomach, about 2 trillion cells are produced, and the cells up to the 8-cell stage after fertilization of the egg and sperm have a universal property that can develop into one complete individual. When implanted in the uterus, it can develop into a single, complete individual. Embryonic stem cells have the ability to develop as these various types of cells.

Adult stem cells are stem cells extracted from grown body tissues, and are primitive cells just before differentiation into cells of specific organs. Adult stem cells are difficult to proliferate and have a strong tendency to differentiate easily, but can differentiate into tissue-specific progenitor cells inherent in the human body. The adult stem cells may be bone marrow-derived, cord blood-derived, blood-derived, liver-derived, skin-derived, gastrointestinal-derived, placenta-derived, nerve-derived, adrenal-derived, epithelial-derived, and uterine-derived stem cells, but are not limited thereto.

Human embryonic stem cells have pluripotency, which is capable of producing all cells in our body, and self-renewal, capable of producing cells that resemble themselves indefinitely. The combination of these two properties is called "stemness." Pluripotent stem cells include embryonic germ cells, embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells), and induced pluripotent stem cells (iPSCs). Includes. Induced pluripotent stem cells have self-renewal ability and pluripotency, which means that they can differentiate into all types of cells including three germ layers.

Specifically, the culture of embryonic stem cells is usually divided into a culture containing feeder cells and a culture not containing them. First, the method currently commonly used to cultivate stem cells, especially human embryonic stem cells, is a ball using mouse embryonic fibroblasts or human foreskin fibroblasts as feeder cells. It is a co-culture. This is a method in which the feeder cells supply the factors necessary for cultivation while maintaining the stem cellularity of human embryonic stem cells. The feeder cells are treated with mitomycine C or irradation to stop the growth of the feeder cells, and then supply the factors necessary for growth while maintaining stem cell properties to human embryonic stem cells. The medium used for co-culture is mainly 80% Knockout DMEM media (Gibco) with 20% knockout serum replacer (Gibco) added.Additionally to this medium, glutamine, mercaptoethanol, non-essential amino acids, basic fiber Basic fibroblast growth factor (bFGF) can be added. Until now, it is not known in detail what factors provided by feeder cells are important for maintaining stem cell properties of human embryonic stem cells.

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

Meanwhile, as a method of culturing embryonic stem cells without including feeder cells, a method of coating the surface of a plate with a matrigel at a low temperature using mTeSR medium or the like has been proposed. However, in the case of using Matrigel, there was a complicated problem because a reagent treatment process for maintaining the plate surface at a low temperature and removing Matrigel was added during the passage of embryonic stem cells. In addition, in the case of mTeSR culture (feeder-free media, Stem cell technologie), an expensive protein coating (matrigel, BD science) is required. The above method also had to change the badge to mTsSR meida every day, and there was a problem that the price of the badge itself was expensive.

Accordingly, the present inventors have made efforts to develop a culture medium that can effectively cultivate human stem cells without feeder cells, and as a result, it has been confirmed that human stem cells actively proliferate without feeder cells in a conditioned medium containing a stem cell culture solution derived from algae eggs. In addition, the present invention was completed by revealing that the egg-derived stem cell culture solution exhibits a significant skin cell proliferation effect and can be used as a culture medium composition for human stem cells or a composition for wound treatment.

In the conventional method of culturing stem cells, when feeder cells are used, there is a risk of infection between different species, and when a medium without feeder cells such as mTeSR medium is used, there is a problem in terms of complicated process and expensive price. There was. Accordingly, the present invention has been devised to solve such a conventional problem, and has developed a culture solution capable of effectively culturing human stem cells without feeder cells.

That is, an object of the present invention is to provide a medium composition for human stem cell culture, a pharmaceutical composition for treating skin wounds, and a cosmetic composition for improving skin wounds, comprising a stem cell culture solution derived from algae as an active ingredient.

To achieve the above object,

The present invention provides a medium composition for culturing human stem cells comprising a stem cell culture solution derived from algae as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for treating skin wounds containing a stem cell culture solution derived from algae as an active ingredient.

In addition, the present invention provides a cosmetic composition for improving skin wounds containing a stem cell culture solution derived from algae as an active ingredient.

In addition, the present invention

1) adding egg-derived stem cells of algae to a serum-free medium containing microcarriers 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, followed by culturing; Provides a manufacturing method.

The human stem cell culture medium composition containing the egg-derived stem cell culture medium of the present invention as an active ingredient can effectively proliferate human stem cells while maintaining an undifferentiated state without feeder cells. Since it has a proliferative effect, it can be used as an inexpensive and effective human stem cell culture medium, a pharmaceutical composition for treating skin wounds, and a cosmetic composition for improving skin condition.

1A shows a picture of an egg epiblast in which egg totipotent cells are present. In particular, the inside of the marked circle is an area pellucida, and there are predifferentiated cells in this area. FIG. 1B shows the five stages of egg development and shows the state in which egg predifferentiated cells are differentiated into neural stem cells.
2 shows each of the predifferentiated cells obtained from egg cells by four different methods. A is a predifferentiated cell isolated and cultured from an egg epiblast in the first stage of development. B is a predifferentiated cell isolated and cultured from primitive embryonic blood. C is a predifferentiated cell isolated and cultured from primitive germ cells. D is a dedifferentiated egg predifferentiated cell.
3 is a graph of proliferation of egg predifferentiated cells, showing the difference in growth of egg predifferentiated cells between the experimental group treated with the inhibitor (red curve) and the control group without the inhibitor (black curve). It was confirmed that egg predifferentiated cells proliferated only when the inhibitor was treated.
4 is a photograph of human predifferentiated cells cultured in egg cell culture, and it was confirmed that human stem cells morphologically maintained an undifferentiated state and grown.
5A is a result of confirming the expression of undifferentiated stem cell markers in human embryonic stem cells through cell immunostaining. In the photo, the red part is the part in which the OCT4 gene, which is a marker for predifferentiated stem cells, is expressed, and B in FIG. 5 shows the support cells and stem cells indicated by arrows as a result of nuclear staining.
6 is a result of confirming through PCR that human embryonic stem cells express genes important for maintaining undifferentiated states such as OCT4, SOX2, and NANOG. As a result of using egg cell culture medium as a human stem cell medium, all of the NANOG, SOX2, and OCT4 genes were expressed. Among them, SOX2 showed an increased expression pattern than that of Line 2 using a typical stem cell undifferentiated growth medium. This indicates that human stem cells remain undifferentiated in the egg cell culture medium, and it is confirmed that it is more effective than the case of using a typical stem cell undifferentiated growth medium.
7 is a cell in which human embryonic stem cells were cultured for 1, 2, 3, 4, 5 days on 20% KO conditioned media and Egg suspension conditioned media, and then the number of hESCs was quantified. The measurement results of proliferation are shown. As a result, it was confirmed that human embryonic stem cells proliferated effectively when cultured in Egg suspension cinditioned media rather than 20% KO conditioned media.
Figure 8 shows the growth rate after culturing the skin cells, HatCaT cells, on a medium containing DMEME, a medium containing DMEM + EGF (100 ng/mL), and a medium containing 0.1%, 0.5%, and 1% egg cell culture solutions for 3 days, respectively. This is the result of comparison. When cultured on a medium containing 0.5% to 1% of egg cell culture medium, HatCaT cells proliferated effectively, and it was confirmed that the proliferation promoting effect was superior when culturing skin cells on the egg cell culture medium than other conditions.

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

The present invention provides a medium composition for culturing human stem cells comprising a stem cell culture solution derived from algae as an active ingredient.

The egg of the present invention is an egg of a bird, and the bird is a chicken, turkey, duck, geese, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary ) Taxonomically, including, but not limited to, all species, subspecies, or rass of the Aves class. Particularly preferred is Gallus gallus or a kind of chicken, such as white leg horn, brown leg horn, Barred-Rock, Sussex, California gray, Italian oartidge-color (Italian oartidge- colored).

The egg-derived stem cell of the present invention is a pluripotent stem cell obtained from the blastoderm of an egg fertilized egg of an embryo corresponding to stage-X or XI in the development stage of Eyal-Giladi and Kochav (EG &K, 1976). ), egg fertilized eggs were cultured in an incubator at 38°C for 3-5 days, and blood was collected from embryos that reached H&H (Hamburger and Hamilton, 1951) stage 14-17. Muscle obtained by culturing at 38°C for 5-6 days and culturing embryonic germ cells (EGCs) and egg fertilized eggs isolated from gonads formed at H&H (Hamburger and Hamilton, 1951) stage 28 at 38°C for 11 days. Dedifferentiated stem cells (induced pluripotent stem cells) obtained by treating embryonic fibroblasts (EFs) obtained from tissues with Yamanaka transcription factors (OCT4, Sox2, KIF-4, c-Myc) or by overexpressing miRNA-302 clusters. cells, iPSCs).

Meanwhile, it is preferable to cultivate the algae egg-derived stem cell culture solution as follows, but is not limited thereto: 1) First, the egg-derived stem cells obtained through the above method are coated with gelatin or fibronectin. After addition to the serum-free medium containing the microcarrier (Microcarrier), 2) various kinds of cell signal circuit inhibitors in the serum-free medium, preferably the inhibitor is a GSK inhibitor, GSK-3b inhibitor, MEK inhibitor, TGF -β inhibitors, ROCK inhibitors, BMP inhibitors, ERK inhibitors, and the like, and more preferably treating and culturing any one or more inhibitors selected from the group consisting of ROCK inhibitors, MEK inhibitors, ERK inhibitors and GSK-3b inhibitors. Go through the steps.

The reasons for using a microcarrier coated with gelatin or fibronectin are as follows. In general, in order to proliferate stem cells in vitro, stem cells are cultured in a flask on a flat surface pre-coated with extracellular matrix (ECM) proteins or feeder cells. However, planar culture cannot support long-term growth of stem cells due to its limited surface area, so subculturing is often required. As a solution to this, a microcarrier-based method can be used for stem cell cultivation, and since the microcarrier has a high surface-area-to-volume ratio, it can overcome the surface area limitation of stem cell growth on a flat surface. I can. In addition, by using a microcarrier coated with a polymer such as gelatin or fibronectin in the present invention, the egg starchy stem cells of algae are effectively attached to the microcarrier, and show high efficiency in the subsequent cultivation step, scale-up and mass production step. .

On the other hand, a serum-free medium (SFM) is used as a medium for growth of stem cells derived from algae eggs, which means a medium to which serum is not added to enable cell survival and cell growth. In order to provide universal nutrients for the growth of all cell lines, adding serum to the culture medium is essential for cell culture, but serum has many limitations. Animal serum varies greatly from manufacturing unit to manufacturing unit, is expensive, and can be a potential contaminant such as disease-causing virus and mycoplasma. Also, separation and high purity purification of recombinant proteins secreted in the medium due to many components present in serum The disadvantage is that it is very difficult. Because of this problem, when culturing stem cells, it is preferable to use a serum-free medium that does not contain serum in the medium. This medium does not need to be a chemically purified medium and may contain hydrolysates derived from animals or plants. The serum-free medium of the present invention is preferably limited to non-animal origin that does not contain components of animal or human origin, and natural serum proteins are replaced with recombinant proteins. However, the serum-free medium of the present invention does not contain proteins (PF medium: protein-free medium) and/or chemically purified (CDM medium: chemically purified medium).

Examples of commercially available SFM media are as follows. Episerf (InVitrogen Ref 10732-022, Catalog 2003), Pro 293 S-CDM (Cambrex ref 12765Q, Catalog 2003), LC17 (Cambrex Ref BESP302Q), Pro CHO 5-CDM (Cambrex ref 12-766Q, Catalog 2003), HyQ SFM4CHO (Hyclone Ref SH30515-02), HyQ SFM4CHO-Utility (Hyclone Ref SH30516.02), HyQ PF293 (Hyclone ref SH30356.02), HyQ PF Vero (Hyclone Ref SH30352.02), Ex Cell 293 Medium (JRH Biosciences ref) 14570-1000M), Ex cells VPRO medium (JRH Biosciences ref 14560-1000M), Ex cells 302 serum-free medium (JRH Biosciences ref 14312-1000M), Ex cells 65319 (JRH Biosciences), Ex cells 65421 (JRH Biosciences), Ex Cell 6625 (JRH Bioscience), Ex cell 65626 (JRH Bioscience), Ex cell 65627 (JRH Bioscience), Ex cell 65628 (JRH Bioscience), Ex cell 65629 (JRH Bioscience), gene therapy medium 3 (animal component) ( SIGMA-Aldrich, ref G-9916).

The concentration of each inhibitor treated in a serum-free medium for the production of egg predifferentiated stem cells is not limited, but preferably, the inhibitor is from about 0.3 μM to about 30 μM of GSK-3b inhibitor, from about 10 nM to about 10 μM. MEK inhibitors, may comprise from about 50 nM to about 5 μM ROCK inhibitor, most preferably the inhibitor is treated with about 3 μM GSK-3b inhibitor, about 1 μM MEK inhibitor and about 0.5 μM ROCK inhibitor. This is desirable.

Containers used to prepare the algal egg-derived stem cell culture solution of the present invention may include a tank bioreactor, a WaveTM bioreactor, a BelloTM bioreactor, a spinner flask, a flask, and a cell factory that are continuously stirred, but are not limited thereto. Most preferably, it is preferable to use a continuously stirred tank bioreactor in which temperature, air circulation, pH and other conditions are controlled.

In general, in the step of scaling up the number of cells, the scale is increased using master or working cell bank vials through T-flasks or roller bottles of various sizes, preferably finally increasing the scale in the bioreactor. Then, a seed-producing bioreactor (typically 20-30 L volume) is used for further cultivation of the prepared cell suspension. The volume ratio of the secondary bioreactor to the seed bioreactor depends on the range of the number of cells grown in the primary bioreactor, but is preferably in the range of 3:1 to 10:1, that is, 6 to 8:1.

The algal egg-derived stem cell culture medium prepared by the above method maintains and proliferates human stem cells in an undifferentiated state. To confirm this, human stem cells were cultured at 35-37°C for 2-5 days in a medium containing a stem cell culture solution derived from algae as an active ingredient, and then the morphology of human stem cells was observed under a microscope (see FIG. 4). , Nuclear staining and immunofluorescence staining using an antibody was performed to confirm the expression pattern of the OCT4 gene, which is a predifferentiated stem cell marker. In addition, Nanog, Sox-2, OCT4 gene PCR test was used to confirm the human stem cell. In order to confirm the maintenance of cell undifferentiation, human embryonic stem cells were cultured at 35-37°C for 2-5 days in a stem cell culture medium derived from algae, and then total RNA was isolated and RT using a primer specific for each gene. -PCR was performed. (Refer to Fig. 6) In order to confirm the cell proliferation ability of the stem cell culture solution derived from algae, human stem cells were cultured in an appropriate culture condition on the stem cell culture solution derived from algae, and the number of cells was measured. Compared with the number of cells in the positive control group, the proliferative cell ability was confirmed (see Fig. 7).

In a specific embodiment of the present invention, the present inventors have shown a method of obtaining pluripotent cells from an algal egg cell and a method of preparing a stem cell culture solution derived from an algae, and in the experimental example, human stem cells on an egg-derived stem cell culture solution In order to check whether the undifferentiated state was maintained during cultivation, a morphological analysis method (see FIG. 4), OCT-4 staining (see FIG. 5), and a PCR test for expression of NANOG, SOX2, and OCT4 genes were performed (see FIG. 6). When human stem cells and skin cells were cultured on an egg-derived stem cell culture medium, it was confirmed that they proliferated effectively (see FIGS. 7 and 8).

In conclusion, the stem cell culture medium derived from algae of the present invention can effectively proliferate and maintain human stem cells in an undifferentiated state without feeder cells, and thus can be used as a human stem cell culture medium.

In addition, the present invention provides a pharmaceutical composition for treating skin wounds containing a stem cell culture solution derived from algae 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 an individual, such as a tissue constituting an internal or external surface of the living body.

The algae egg-derived stem cell culture medium promotes skin cell proliferation and may be active in wound treatment and skin regeneration, but is not limited thereto, and the active ingredient of the present invention is 0.1-100 weight based on the total weight of the composition. It can contain in %.

In specific examples and experimental examples of the present invention, in order to confirm the ability of skin cell proliferation on the stem cell culture medium derived from algae, HaCaT (keratinocyte, Thermo) skin cells were cultured on the stem cell culture medium derived from the egg for a certain period of time, and then the growth rate was compared. Effective skin cell proliferation ability was confirmed (see Fig. 8).

In conclusion, it was confirmed that the skin cell proliferation effect was excellent when the skin cells were cultured on the stem cell culture solution derived from the algae of the present invention, and the stem cell culture solution derived from the algae was an active ingredient of the pharmaceutical composition for the treatment of skin wounds. Can be used as

The pharmaceutical composition comprising the human stem cell culture solution of the present invention may further include a suitable carrier, excipient, and diluent that are commonly used in addition to the egg-derived stem cell culture solution as an active ingredient. In addition, solid or liquid additives for preparation may be used in the preparation of pharmaceutical compositions. The additive for formulation may be either organic or inorganic.

Examples of excipients include lactose, sucrose, white sugar, glucose, corn starch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. As a binder, for example, polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, arabic rubber, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, Dextrin, pectin, and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any colorant that is permitted to be added to pharmaceuticals can be used. These tablets and granules can be appropriately coated with sugar, gelatin coating, or other necessary. In addition, preservatives, antioxidants, and the like may be added as necessary.

The pharmaceutical composition of the present invention may be prepared in any formulation conventionally prepared in the art (e.g., Remington's Pharmaceutical Science, the latest edition; Mack Publishing Company, Easton PA), and the form of the formulation is not particularly limited, Preferably, it may be an external preparation. The external preparations of the present invention include sheet agents, liquid coating agents, sprays, lotions, creams, pads, powders, penetration pads, sprays, gels, pasta agents, liniment agents, ointments, aerosols, powders, suspensions, transdermal agents. It may be included in the form of a conventional external preparation such as an absorbent. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a formula generally known for all pharmaceutical chemistry.

In the external preparation of the present invention, the pharmaceutically acceptable carrier may vary depending on its formulation, but may include hydrocarbons such as petrolatum, liquid paraffin, and gelled hydrocarbons (also known as plastibase); Animal and vegetable oils such as medium-chain fatty acid triglyceride, pork fat, hard fat, and cacao oil; Higher fatty acid alcohols such as cetanol, stearyl alcohol, stearic acid and isopropyl palmitate, and fatty acids and esters thereof; Water-soluble bases such as macrogol (polyethylene glycol), 1,3-butylene glycol, glycerol, gelatin, sucrose, and sugar alcohol; Emulsifiers such as glycerin fatty acid ester, polyoxyl stearate, and polyoxyethylene hardened castor oil; Adhesives such as acrylic acid ester and sodium alginate; Propellants such as liquefied petroleum gas and carbon dioxide; And preservatives such as paraoxybenzoic acid esters, and the like, and the external preparation of the present invention can be prepared by using them according to a conventional method. Further, in addition to these, stabilizers, flavors, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants, and the like may be blended as necessary. The use of the external preparation of the present invention can be applied to the local wound by a conventional method. In addition, the external preparation according to the present invention may be used by being adhered to a solid support such as a wound peeling 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 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 wound type of the patient, the application site, the number of treatments, the treatment time, the formulation, the condition of the patient, the type of adjuvant and the like. The amount used is not particularly limited, but the daily effective amount of the pharmaceutical composition of the present invention is 1 to 50 μl/cm ^{2 when the cell culture solution is applied to the wound.} May be, preferably 5 to 20 µl/cm ² Can be The daily dose may be administered once a day, divided into 2-3 times a day at appropriate intervals, or intermittently administered at intervals of several days. However, the amount of use of the pharmaceutical composition of the present invention is various such as administration route, patient's age, sex, weight, patient's severity, type of wound, application site, number of treatments, treatment time, formulation, patient's condition, type of adjuvant, etc. Since it is determined in light of related factors, the effective amount does not limit the scope of the present invention in any aspect.

In addition, the present invention provides a cosmetic composition for skin regeneration or skin wound improvement comprising an egg-derived stem cell culture solution as an active ingredient.

The egg (egg)-derived stem cell culture solution of the present invention can effectively proliferate skin cells, and thus may be used as a composition for proliferating skin cells or a cosmetic composition for skin regeneration or skin wound improvement containing the same as an active ingredient.

The cosmetic composition for improving skin condition comprising the human stem cell culture solution of the present invention may contain an acceptable carrier in a cosmetic preparation. Here, the term "acceptable carrier in cosmetic formulations" is a compound or composition that is already known and used that can be included in a cosmetic formulation, or is a compound or composition to be developed in the future, and has more toxicity, instability or irritation than the human body can adapt when in contact with the skin. Say nothing. The carrier may be included in the cosmetic composition of the present invention in about 1% by weight to about 99.99% by weight, preferably about 90% by weight to about 99.99% by weight, based on the total weight of the cosmetic composition. However, since the ratio varies depending on the formulation as described below in which the cosmetic composition of the present invention is prepared and its specific application site (face, neck, etc.) or its preferred application amount, the ratio is in any aspect of the present invention. It does not limit the scope of.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizing agents, viscosity modifiers, emulsions, stabilizers, ultraviolet scattering agents, ultraviolet absorbers, color developing agents, fragrances, and the like. Compounds/compositions that can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizing agents, viscosity modifiers, emulsions, stabilizers, ultraviolet scattering agents, ultraviolet absorbers, coloring agents, fragrances, etc. are already known in the art. Therefore, those skilled in the art can select and use an appropriate corresponding material/composition. As an embodiment of the present invention, the cosmetic composition according to the present invention may include glycerin, butylene glycol, propylene glycol, polyoxyethylene hydrogenated castor oil, ethanol, triethanol amine, etc., in addition to the egg-derived stem cell culture solution, Preservatives, perfumes, coloring agents, purified water, etc. may be included in trace amounts as needed. The cosmetic composition according to the present invention can be prepared in various forms, such as lotions, essences, gels, emulsions, lotions, creams (oil-in-water type, water-in-oil type, multi-phase), solutions, suspensions (anhydrous and aqueous). , Anhydrous products (oil and glycol-based), gel, mask, pack, powder, or capsules (soft capsules, hard capsules) with a film such as gelatin may be prepared in the form of a formulation. Skin in the present invention is a concept that includes not only the face, but also the scalp and the whole body, and as cosmetic compositions that can be applied to such scalp, there are shampoos, conditioners, treatments, hair growth agents, etc., and body cleansers that can be applied to the whole body, etc. It can be manufactured in various forms for the use of. The manufacturing method of the cosmetic composition for skin regeneration or skin wound improvement containing the egg-derived stem cell culture solution according to the present invention is not limited to the above-described manufacturing method, and those of ordinary skill in the art to which the present invention pertains, the above The cosmetic composition according to the present invention can be prepared by a method in which the manufacturing method is partially modified.

In particular, the cosmetic composition for skin regeneration or the cosmetic composition for improving skin wounds may be prepared in the form of a general emulsified formulation and a solubilized formulation using a conventionally known manufacturing method in addition to the manufacturing method specifically disclosed in the present invention. When prepared as a cosmetic composition of an emulsified formulation and a solubilized formulation, cosmetic products of an emulsified formulation include nutrient lotion, cream, essence, etc., and a cosmetic of a solubilized formulation includes a softening lotion. In addition, by containing a dermatologically acceptable medium or base, it may be prepared in the form of an adjuvant that can be applied topically or systemically used in the field of dermatology. In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or paste dry products, emulsions obtained by dispersing the oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), nonionic It can be provided in the form of a vesicle dispersant in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick. In addition, it may be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the cosmetic composition for skin regeneration or the cosmetic composition for improving skin wounds of the present invention may further include fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents. , Fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles Or it may contain adjuvants commonly used in the field of cosmetics or dermatology, such as any other ingredients commonly used in cosmetics. And the above ingredients may be introduced in an amount generally used in the field of dermatology. The cosmetic composition according to the present invention includes forms of functional cosmetics such as skin astringent, anti-inflammatory, wound, wound healing, and anti-aging.

Hereinafter, the present invention will be described in detail by examples and experimental examples.

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

< Example 1> From algae egg cells Isolation of pluripotent cells

<1-1> Isolation of pluripotent cells (PSCs) from egg-fertilized blastocysts

One type of chicken, White Leghorn, was used.

The blastoderm of the embryonic stage according to Eyal-Giladi and Kochav (EG &K, 1976), that is, stage-X or XI stage, was cut out using surgical scissors. The viteline and egg yolk of the cut-out blastocyst were removed in PBS buffer, and blastocyst cells having pluripotency were obtained. The resulting blastocyst cells were treated with mitomycine C (10 ug/ml) for 2 hours to inhibit growth with STO cells (ATCC collection) together with bFGF (10 ng/ml) and IGF-1 (20 ng/ml), mSCF (1%), IL-6 (1%), IL-11 (1%), LIF (1%), and FBS (10%). As a result of co-culture, predifferentiated cells formed a colony, and the specific culture method was referred to Pain B et al. (1996). Thereafter, the egg blastoderm (Fig. 1A) containing pluripotent cells was isolated and cultured to obtain embryonic stem cells (ESCs), which are pluripotent cells (Fig. 2A). .

<1-2> Isolation of pluripotent cells (PSCs) from blood produced in fertilized eggs

If egg fertilized eggs are cultured at 38°C for 5 days in an incubator, H&H (Hamburger and Hamilton, 1951) stage 14-17 is reached, and promodial germ cells (PGC) are contained in the blood of this period. DMEM medium containing STO cells, SCF (6 ng/ml), bFGF (4 ng/ml), and 8% FBS, which inhibited the growth of blood from the heart and arteries at this stage and treated with mitomycin C It was co-cultured on the bed. Two weeks after co-culture, a colony of predifferentiated cells was formed, from which predifferentiated cells were separated and cultured to obtain embryonic stem cells (ESCs). For detailed culture method, refer to van de lavoir et al. (2006) (Fig. 2B).

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

If egg fertilized eggs are cultured at 38℃ for 6 days, H&H stage 28 is reached and embryonic germ cells (EGCs) develop in the gonad. EGCs were isolated from the gonads, and the cells were treated with mitomycin C (10 ug/ml) for 2 hours to inhibit growth, together with STO cells (ATCC collection), bFGF (10 ng/ml), IGF-1 ( 20 ng/ml), mSCF (1%), IL-6 (1%), IL-11 (1%), LIF (1%), FBS (10%) EGCs with differentiation ability were obtained (Fig. 2C).

<1-4> Isolation of ESCs from dedifferentiated pluripotent cells

The muscle tissue obtained by culturing egg fertilized eggs at 38° C. for 11 days was sectioned and cultured in 10% DMEM medium to obtain embryonic fibroblasts (EFs). In the fibroblast, a vector capable of overexpressing Yamanaka factors (Oc4, c-Myc, Sox2, KIF-4) was introduced using an electrophorator (invitrogene), and the vector was injected. Egg embryonic cells were co-cultured for 4 weeks in DMEM medium containing 10% FBS with STO cells inhibiting cell growth with mitomycin C. As a result of co-culture, a population of induced pluripotent stem cells (iPSCs) having pluripotency was obtained (FIG. 2D).

<Example 2> Preparation of stem cell culture solution derived from algae egg

<2-1> Egg-derived stem cells and feeder cells co-cultured

In order to maintain the growth and undifferentiation of egg-derived stem cells, growth factors bFGF (10 ng/ml), IGF-1 (20 ng/ml), SCF (10 ng/ml) in a medium containing fetal bovine serum (FBS) , IL-6 (20 ng/ml), IL-11 (20 ng/ml), and LIF (1000 units) were added, and a serum-free medium (SFM) was added using STO cells as feeder cells. ) To prepare an egg-derived stem cell culture solution.

<2-2> Inhibitor treatment on SFM medium

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

In order to confirm whether treatment with the inhibitor was essential, egg predifferentiated cells were cultured on the SFM medium of Example <2-1> to which no inhibitor was added, and then the proliferation difference was confirmed. As a result, as shown in FIG. 3, it was confirmed that egg predifferentiated cells cultured in the medium to which the inhibitor was not added did not proliferate or died, and egg predifferentiated cells cultured in the medium to which the inhibitor was added actively proliferated. Therefore, it was confirmed that egg predifferentiated cells proliferate only when an inhibitor is essentially added under serum-free suspension culture conditions (FIG. 3).

<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 attachment material in order to increase the cultivation efficiency of egg-derived stem cells of algae. To coat the microcarrier, the microcarrier was immersed in gelatin (Gelatin. 0.1%) or fibronectin (0.1%) for 2 hours at 37°C, and then dried in a clean bench, bFGF (10 ng/ml), IGF- 1 (20 ng/ml), SCF (10 ng/ml), LIF (1000 U) and MEK inhibitor (PD98059, 1 μM), GSK-3b inhibitor (CHIR-98014 CHIR-98014 CHIR-98014, 3 μM) Egg predifferentiated cells were suspended and cultured for 3 days in the contained KO-DMEM/F12 medium. After cultivation, it was cultured again for 2 to 3 days in a medium not containing the GSK-3b inhibitor and the MEK inhibitor PD98059. The resulting culture medium was used as a medium composition for proliferation while maintaining human stem cells undifferentiated. Compared with general microcarriers, when using a microcarrier coated with gelatin or fibronectin protein, it was confirmed that egg-derived stem cells adhere well to the microcarrier and the cells are scaled up efficiently in the bioreactor. .

< Experimental example 1> Morphological confirmation of undifferentiated maintenance during human stem cell culture

Human embryonic stem cells were cultured at 37° C. for 3 days in a medium containing the algal egg-derived stem cell culture solution prepared by the method of Example <2-3> as an active ingredient. Thereafter, the morphology of the human stem cells was observed under a microscope, and as a result, it was confirmed that the developmental processes such as tissue and organ development did not proceed morphologically as shown in FIG.

< Experimental Example 2> Confirmation of undifferentiated maintenance when culturing human stem cells using OCT4 staining

In order to confirm whether the human stem cells cultured in the algal egg-derived stem cell culture medium maintain an undifferentiated state, OCT4 immunofluorescence staining was performed.

At this time, egg predifferentiated stem cell culture medium was added to 20% SR DMEM/F12 medium excluding various growth factors, and used as a growth medium for human embryonic stem cells, and to confirm the expression pattern of the OCT4 gene, a predifferentiated stem cell marker 1 Immunofluorescence staining was performed using mOct4 (mouse monoclonal OCT4, Santa Cruz) as the secondary antibody and anti-mouse IgG tagged with Cy-3 as the secondary antibody. Nuclear staining was performed using DAPI.

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

< Experimental Example 4> Confirmation of undifferentiated maintenance during human stem cell culture using Nanog , Sox-2, OCT4 gene PCR test method

After culturing human embryonic stem cells at 37° C. for 2 days in the algal egg-derived stem cell culture solution prepared by the <Example 2> method, Nanog, Sox-2, OCT4 The expression of genes important for maintaining the undifferentiated state was confirmed through PCR.

Specifically, MEF medium, hESCs_20% SR DMEM/F-12 was cultured on a medium containing bFGF and an egg-derived stem cell culture medium to isolate total RNA from human embryonic stem cells harvested. Then, PCR was performed using primers specific for Nanog, Sox-2, OCT4, and the control 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:3, R:5 `-AGAAAAACGAGGGAAATGGG-3 SEQ ID NO:4), OCT4(F: 5`-GGAAAGGCTTCCCCCTCAGGGAA-3` SEQ ID NO:5, R: 5`- AAGAACATGTGTAAGCTGCGGC-3` SEQ ID NO:6)

The results of confirming the expression of these genes are shown in FIG. 6. According to this, Line 1 indicates the level of expression of undifferentiated stem cell marker genes when MEF is a mouse-derived support cell commonly used in human stem cell culture, and when a growth medium containing only MEF is used. As a result, neither Nanog, Sox-2 nor OCT4 genes were expressed. In Line 2, hESCs_20% SR DMEM F-12 is a typical stem cell undifferentiated growth medium, and Line 2 shows the level of expression of human stem cell marker genes when a typical stem cell undifferentiated growth medium is used. As a result, Nanog, Sox-2, and OCT4 genes were all expressed, indicating that human stem cells maintain undifferentiated. Line 3 shows the level of expression of undifferentiated stem cell marker genes when egg-derived stem cell culture medium is used as a human stem cell medium. As a result, Nanog, Sox-2, and OCT4 genes were all expressed. Among them, Sox-2 showed an increased expression pattern than that of Line 2. It was confirmed that human stem cells maintained undifferentiated in the egg cell culture medium of algae, and this was confirmed to be more effective than the case of using a typical stem cell undifferentiated growth medium.

< Experimental example 5> Human stem cells in stem cell culture solution derived from algae eggs Proliferative ability Confirm

In order to confirm the human stem cell proliferation ability of the algal egg-derived stem cell culture medium, the results were compared and analyzed after culturing human embryonic stem cells in 20% KO conditioned media or Egg suspension conditioned media. I did.

Human embryonic stem cells in 20% Serum Replacement (SR)-DMEM/F-12 condition medium with bFGF added or egg stem cells in suspension culture for 1 day without treatment with inhibitors 1, 2 , Incubated for 3, 4, and 5 days, and the number of cells was measured. As a result, it was confirmed that human embryonic stem cells proliferated effectively when cultured on an egg cell culture medium rather than a 20% KO condition medium (FIG. 7).

< Experimental example 6> Algae egg-derived stem cell culture solution Skin cells Proliferative ability Confirm

To check the skin cell proliferation ability of the algal egg-derived stem cell culture medium, the skin cells, HatCaT (keratinocyte, Thermo) cells, were cultured in 10% FBS DMEM medium and then cultured in DMEM without FBS for 1 day to grow cells. Was suppressed. In addition, DMEM without FBS (negative control), a medium containing EGF (10 ng/mL) added to DMEM medium (positive control), and 0.1%, 0.5%, and 1% of egg cell culture medium were included in DMEM medium. After culturing each of the medium for 3 days, the growth was compared. As a result, it was confirmed that HaCaT cells proliferated effectively when cultured on a medium containing 0.5% and 1% of algal egg cell culture medium in a basic DMEM medium. It was confirmed that the effect of promoting skin cell proliferation was excellent in the DMEM medium containing the culture medium (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> OCT4 R primer <400> 6 aagaacatgt gtaagctgcg gc 22

Claims (5)

The present invention relates to a stem cell-conditioned medium derived from avian (Egg)
The conditioned media
1) Conditioned medium in which the avian stem cells of avian eggs are cultured in a stem cell culture medium containing support cells and bFGF;
2) conditioned media in which avian egg-stem cells are cultured in a stem cell culture medium containing bFGF, ROCK inhibitor, MEK inhibitor and GSK 3b inhibitor; or
3) characterized that the avian stem cells of avian eggs are cultured in stem cell culture medium containing bFGF, MEK inhibitor and GSK 3b inhibitor and then conditioned medium additionally cultured in medium not containing MEK inhibitor and GSK 3b inhibitor To a culture medium for human embryonic stem cell culture.
The culture medium for culturing human embryonic stem cells according to claim 1, wherein the algae is chicken.
The culture medium for culturing human embryonic stem cell according to claim 1, wherein the egg-derived stem cell conditioned medium maintains and proliferates human embryonic stem cells in an undifferentiated state.
The method according to claim 1, wherein the egg-derived stem cells of the alga in 3) are cultured in a serum-free medium containing a microcarrier coated with gelatin or fibronectin. A medium composition for culturing embryonic stem cells.
The culture medium for culturing human embryonic stem cells according to claim 1, wherein the egg-derived stem cells are pluripotent stem cells (PSCs) extracted from egg cells.

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