KR102153637B1 - Serum-free medium composition for stem cell culture and methods for culturing stem cell - Google Patents

Abstract

The present invention relates to a serum-free medium composition for culturing stem cells and a method for culturing stem cells. In the present invention, by proliferating and culturing stem cells in a serum-free medium containing Taxifolin, the stem cells can be stably proliferated and cultivated. In addition, the stem cell culture solution cultured by the culture method according to the present invention is expected to promote skin regeneration and improve skin elasticity and wrinkles, and thus can be used in external preparations for skin or cosmetics.

Description

Serum-free medium composition for stem cell culture and methods for culturing stem cell}

The present invention relates to a serum-free medium composition for culturing stem cells for culturing skin stem cells, and a method of culturing stem cells in the serum-free medium composition for culturing stem cells.

Skin stem cells play an important role in maintaining skin health and physiological and biochemical homeostasis. Stem cells present in the skin also have abnormal functions due to aging, and accordingly, various problems occur as the homeostasis of the skin is broken. Therefore, reactivating aged stem cells will have the same effect as reversing the age of the skin.

The skin consists of three layers from the outside: the epidermis, the dermis and the subcutaneous fat layer. The epidermis is the thinnest among the three layers, and the thickness varies depending on the area. The epidermis is mainly composed of keratinocytes, and is composed of melanocytes that produce melanin pigments and Langerhans cells that perform immunological functions. The dermis is mainly composed of connective tissue and matrix composed of collagen and elastic fibers, and contains nerves, blood vessels, lymphatic vessels, muscles, hair follicles, sebaceous glands and sweat glands.

It is known that all components of human skin are derived from ectoderm or mesoderm. The epidermis, hair follicles, sebaceous glands, and sweat glands originate in the ectoderm, and melanocytes, nerves, and special sensory receptors are derived from the neuroectoderm. Melanocytes derived from neuroectoderm reach the skin 8 weeks after development, and at 4 to 5 months of development, dendritic processes develop and move melanosomes to surrounding keratinocytes. In this way, embryonic stem cells undergo differentiation according to the embryonic period, and each has characteristics appropriate to its function. Even after adulthood, a certain number of stem cells remain in the tissues, but stem cells mainly exist in two places in the skin. The first is in the hair follicle. These cells are known to play an important role in the regeneration of the epidermis as cells before cell differentiation occur, and play an important role in hair regeneration and growth. The second is the basal cell layer of epidermis called the interfollicular epidermis between the hair follicle and the hair follicle. It was found that the stem cells located here play an important role in protecting skin health by managing fibroblasts in the dermis as well as the epidermis. Stem cells here are widely used in skin stem cell research because they have the advantage of being large in quantity and easy to obtain. The skin is continuously renewed, and stem cells present in the epithelium of the skin, that is, epithelial stem cells of the skin, are involved in the repair of the epithelium after a wound (see Non-Patent Document 1). The epithelial stem cells of the skin are also referred to as skin stem cells, and when the skin stem cells are activated, skin wounds such as trauma can be treated or wound healing can be promoted, and wrinkle relief of the skin can also be expected. have. Integrin β1 and integrin α6 are used as indicators of skin stem cells, and it is reported that the maintenance of skin stem cells required for epithelial morphogenesis and differentiation is regulated by the p63 protein.

Meanwhile, in order to develop a pharmaceutical composition or a cosmetic composition using human skin stem cells, it is necessary to stably proliferate and cultivate the skin stem cells while minimizing the possibility of contamination. A conventionally known method of culturing human skin stem cells includes the use of 5 to 20% of Fetal bovine Serum, that is, a serum-containing medium. The fetal bovine serum contains high molecular proteins such as albumin and nutrients, and contains components necessary for cell culture such as hormones and adhesion factors. Fetal bovine serum also has growth factors such as insulin or EGF, and plays a role in neutralizing toxic components of the medium. However, there are unidentified trace components in fetal bovine serum, and these trace components can have a significant effect on the growth of cells. Trace components in fetal bovine serum are not only very difficult to analyze, but also their components may vary depending on the time or place of production, causing considerable difficulty in establishing a reproducible test and production process. In addition, since fetal bovine serum is collected from animals, there is a risk of contamination from other factors such as animal diseases such as mad cow disease or viruses. Furthermore, when using a product through cell culture, it is difficult to purify the serum components and the process is often complicated.

Therefore, there is a need in the art to develop a method capable of stably proliferating and culturing human-derived skin stem cells in a serum-free medium without heterogeneous serum such as fetal bovine serum.

1.Epidermal Stem Cells of the Skin, Cedric Blanpain, Elaine Fuchs, Annual Review of Cell and Developmental Biology, November 2006, Vol. 22, Pages 339-373

Accordingly, the present inventors studied a compound capable of proliferating and culturing stem cells, specifically, skin stem cells, in a serum-free medium condition for various compounds derived from natural products. As a result, it was confirmed that stem cells were effectively proliferated and cultured in a serum-free medium containing Taxifolin, and the present invention was completed.

Accordingly, an object of the present invention is to provide a serum-free medium composition for culturing stem cells containing Taxifolin and a method of culturing stem cells in a serum-free medium containing Taxifolin.

As a means for solving the above problems, the present invention provides a serum-free medium composition for culturing stem cells comprising a compound represented by the following Formula 1:

[Formula 1]

As another means for solving the above problems, the present invention provides a method of culturing stem cells comprising culturing the stem cells in a serum-free medium containing the compound represented by Formula 1 above.

As another means for solving the above problems, the present invention provides a skin regeneration or wrinkle improvement external preparation for skin comprising a culture medium cultured by the stem cell culture method described above.

As another means for solving the above problems, the present invention provides a cosmetic composition for skin regeneration or wrinkle improvement comprising a culture medium cultured by the stem cell culture method described above.

In the present invention, by proliferating and culturing stem cells in a serum-free medium containing Taxifolin, the stem cells can be stably proliferated and cultivated.

In addition, the stem cell culture solution cultured by the culture method according to the present invention is expected to promote skin regeneration and improve skin elasticity and wrinkles, and thus can be used in skin external preparations or cosmetics.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention is the use of a compound represented by the following formula 1 (hereinafter referred to as a compound of formula 1) for culturing stem cells, a serum-free medium composition for culturing stem cells containing the compound of formula 1, and the formula 1 It provides a method of culturing stem cells comprising culturing the stem cells in a serum-free medium containing the compound of.

[Formula 1]

The compound of Formula 1 may include all possible isomers, and, for example, may include the compound of Formula 2 below.

[Formula 2]

The compound of Formula 2 is Taxifolin.

The compound of Formula 1 and the compound of Formula 2 may be synthesized and used, or a commercially available compound may be used.

In the present invention, the term'stem cell' is a cell that can divide cells by itself and has the ability to differentiate into a wide variety of specific cell types. The type of stem cells is not particularly limited, and in one embodiment, the stem cells may be skin stem cells. The “skin stem cells” refer to stem cells that can differentiate into cells that make up the skin (epidermis, dermis and subcutaneous fat layer). The cells constituting the skin include keratinocytes, melanocytes, and fibroblasts (mainly responsible for the biosynthesis of collagen and elastin) present in the epidermis.

The type of skin stem cells is not particularly limited. The skin stem cells used in the present invention can be used regardless of where they originate from. For example, skin stem cells can be obtained from a known source of skin stem cells, such as hair follicles or the basal layer of the epidermis, and the animal to be collected may be a mammal. In one embodiment, the mammal may include, but is not limited to, a human, mouse, rat, guinea pig, rabbit, monkey, pig, horse, cow, sheep, antelope, dog, or cat. Preferably, the mammal may be a human. Such a method of obtaining skin stem cells from a source of skin stem cells is well known in the art.

The medium composition for culturing stem cells containing the compound of Formula 1 of the present invention is a serum-free medium. The serum-free medium refers to any culture medium that does not contain more than a certain amount of serum derived from animals including humans (animal-derived serum). For example, the serum-free medium may contain animal-derived serum in an amount of less than 0.1% by weight or less than 0.01% by weight based on the total composition content, and specifically may not contain animal-derived serum.

General media for culturing stem cells are known in the art. These media contain salts, vitamins, buffers, energy sources, amino acids and other substances, and contain about 5 to 20% of animal-derived serum when culturing stem cells to provide universal nutrients for the growth of cells to be cultured. Is done. However, since animal-derived serum contains unidentified trace components, it affects the growth of cells, is difficult to analyze, and has difficulty in establishing a reproducible test and production process.

The present invention provides a serum-free medium composition containing the compound of Formula 1 instead of animal-derived serum required for proliferation and cultivation of stem cells. Accordingly, the present invention can stably proliferate and cultivate stem cells so as to replace animal-derived serum, and establish a reproducible test and production process.

The concentration of the compound of Formula 1 in the serum-free medium composition for stem cell culture is not particularly limited, and may be adjusted in the range of 1 to 500 μM.

The present invention also provides a method for culturing stem cells comprising culturing the stem cells in a serum-free medium containing the compound of Formula 1.

In the present invention, the type of stem cells is not particularly limited, and may be skin stem cells, and the serum-free medium is as described above.

The concentration of the compound of Formula 1 is not particularly limited, and may be adjusted in the range of 1 to 500 μM in the medium.

In one embodiment, the method for culturing stem cells according to the present invention,

(1) culturing skin stem cells in a medium containing animal-derived serum; And

(2) culturing the skin stem cells cultured in step (1) in a serum-free medium containing the compound of Formula 1 may be included.

The medium for early stage cell culture including serum is preferably a medium suitable for maintaining and storing cell types such as skin stem cells. In the present invention, CNT-57, DMEM (Dulbecco's Modified Eagle's Medium), and aMEM (alpha Minimal Essential Medium), which are generally used for cell culture, are used, and include serum commonly used for cell culture.

It is preferable to add 0.1 to 20% of fetal bovine serum (FBS) to the serum, and a compound having a component similar to that of animal-derived serum, for example, bovine pituitary extract (BPE) or the like may be used.

It is also advisable to add antibiotics, antifungal agents and reagents that prevent the growth of mycoplasma, which causes contamination.

As antibiotics, antibiotics commonly used for cell culture, such as penicillin, streptomycin, or fungizone, may be used. It is preferable to use amphotericin B as an antifungal agent and tylosin as a mycoplasma inhibitor, and gentamicin, ciprofloxacin, azithromycin, etc. can prevent mycoplasma contamination.

If necessary, oxidizing nutrients such as glutamine and energy metabolites such as sodium pyruvate may be further added.

General culture conditions for initial culture are cultured in a humidity 90 to 95%, temperature 25 to 40°C, 5 to 10% CO ₂ incubator by applying the most suitable conditions for cell culture, and final concentration in case of 5 to 10% CO ₂ culture A carbon control source such as sodium bicarbonate may be added to 0.17 to 0.22% by weight.

The cumulative doubling time is maintained until 70 to 80% confluence of the cells in culture in the flask, and the cells are harvested and subcultured preferably at the time of 75% confluence.

The skin stem cells cultured in the above step may be cultured for 1 to 3 days at 25 to 40° C. in a serum-free medium to which the compound of Formula 1 is added.

The serum-free medium may be used without limitation as long as it is a medium suitable for maintaining and storing a cell type as a medium for early stage cell culture that does not contain serum.

The serum-free medium may further include growth factors or cytokines in the medium.

As the growth factor, IGF, bFGF, TGF, HGF, EGF, VEGF, or PDGF may be used alone or in two or more, but is not particularly limited thereto.

As the cytokines, IL-1, IL-4, IL-6, IFN-γ, IL-10, or IL-17 may be used alone or two or more, but are not particularly limited thereto.

The skin stem cell culture medium cultured in a serum-free medium to which the compound of Formula 1 is added may have a high concentration of active ingredients, for example, Decorin, MCP-1, DKK-3, TIMP-2, Angiogenin, Follistatin, Osteoprotegerin, betaIG -H3, MMP-10, MMP-7, LYVE-1, XEDAR, IL-22, RANK, IGFBP-6, Axl, FLRG, NRG1-beta1, PAI-1, Ferritin, Galectin-7, Resistin, Bate2 M, TSLP, VEGF-C, CD40, sTNT R1, IL-6, TRAIL R2, Osteopontin, TIMP-1, Growth Hormon, Thromboppoietin, CXCL-16, DPPIV, HVEM, IL-17B, IL-3, bFGF, IFN-γ , TGF, HGF or PDGF may be included.

Korean Patent Registration No. 10-1026070 suggests that skin stem cells cultured in a serum-free medium are effective in improving wrinkles by promoting collagen expression in skin fibroblasts. Through this, it can be predicted that the skin stem cell culture medium cultured in a serum-free medium containing the compound of Formula 1 of the present invention will also be effective in skin regeneration or wrinkle improvement.

Accordingly, the skin stem cell culture solution obtained according to the stem cell culture method of the present invention can be used as an active ingredient such as skin external preparations or cosmetics for skin regeneration or wrinkle improvement through the above-described useful ingredients. The culture solution may be used as a fraction.

When the culture solution is used for external skin, additionally fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, Commonly used for external skin preparations such as ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators or lipid vesicles. It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients used. In addition, the above ingredients may be introduced in amounts generally used in the field of dermatology.

When the culture solution is provided as an external preparation for skin, it may be a formulation such as an ointment, patch, gel, cream, or spray, but is not limited thereto.

When the culture solution is used as a cosmetic product, the cosmetic product prepared by containing the culture solution as an active ingredient may be prepared in the form of a general emulsified formulation or a solubilized formulation. For example, a lotion such as a flexible lotion or a nutritional lotion; Emulsions such as facial lotion or body lotion; Creams such as nourishing cream, moisture cream, or eye cream; essence; Cosmetic ointment; spray; Gel; pack; Sunscreen; Makeup base; Foundations such as liquid type, solid type or spray type; powder; Makeup removers such as cleansing cream, cleansing lotion, and cleansing oil; It may have a formulation such as a cleaning agent such as a cleansing foam, soap, and body wash.

In addition, the cosmetics are fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers in addition to the culture solution. , Nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators, or adjuvants commonly used in the cosmetic field, such as lipid vesicles. It may contain.

The culture solution may contain a relatively high concentration of the culture solution in the case of a wash-off type cosmetics such as a makeup remover or detergent in which the active ingredient stays on the skin within a short period of time when it is commercialized as a cosmetic. On the other hand, in the case of leave-on type cosmetics such as lotions, emulsions, creams or essences in which the active ingredient stays on the skin for a long period of time, the compound of Formula 1 is contained in a lower concentration than the wash-off type cosmetics. It would be fine. Although not limited thereto, in one embodiment of the present invention, the composition comprises 0.000001% by weight to 10% by weight (preferably 0.001% by weight to 10% by weight, more preferably 0.1 to 10% by weight) based on the total weight of the culture solution. 10% by weight). When the composition of the present invention contains less than 0.000001% by weight of the culture medium, sufficient skin regeneration or wrinkle improvement effect cannot be expected, and when it contains more than 10% by weight, unwanted reactions such as allergies may occur or There may be problems, so this is to prevent it.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

< Reference example 1> Taxipoline ( Taxifolin ) Of substance information

[Formula 2]

CAS No.: 480-18-2

Where to buy: Sichuan Hongjie Import&Export CO., LTD

< Example 1> Serum-free In medium condition Culture of skin stem cells

Human epidermal stem cells purchased from Cellntec were added to a 48-well plate (6×10 ³ cells/well) and bovine pituitary extract (BPE) similar to fetal bovine serum was added. CNT-57 medium (Cellntec) was used for 24 hours in 5% CO ₂ and 37 ℃ conditions. Thereafter, the culture medium was removed through a suction tube, and then the medium component was removed using a PBS solution (GibcoBRL), and 20 ㎍/mL of the compound of Formula 2 was treated in CNT-57 medium containing no BPE, and 5% CO Incubated for 72 hours at ₂ and 37 ℃ conditions.

< Experimental example 1> CCK -8 Effect of promoting skin stem cell proliferation through evaluation method

The skin stem cells cultured in Example 1 were evaluated for CCK-8 (Cell Counting Kit-8). The CCK-8 evaluation is an analysis method that indirectly indicates the density of living cells by measuring the absorbance of formazan produced by decomposing Tetrazolium Salt by dehydrogenase in the intracellular electron transport system.

The cells were treated with a CCK-8 solution (1/10 of the medium volume was treated) at 37° C. for 2 hours, and then measured spectroscopically by measuring the absorbance at a wavelength of 450 nm. The effect of promoting the proliferation of skin stem cells of the compound of Chemical Formula 2 was obtained from the measured absorbance value compared to the control (CNT-57 medium added with BPE) according to Equation 1 below to obtain the proliferation rate (%), and the values are shown in Table 1 below. .

[Equation 1]

Proliferation rate (%) = (absorbance of sample treatment group/absorbance of control group) × 100

Skin stem cell proliferation promotion effect Added sample density Cell proliferation rate compared to control (%) Compound of formula 2 20 μg/mL 120% Control - 100%

As can be seen from the results in Table 1, skin stem cells in the medium condition treated with the compound of Formula 2 of the present invention exhibited excellent proliferation promoting effect.

< Experimental example 2> Stem cell properties of skin stem cells ( stemness ) Check maintenance effect

In order to confirm the effect of maintaining stem cell properties of the skin stem cells cultured in Example 1, the level of p63 expression was evaluated. p63 is an intracellular transcription factor and is well known as a maintenance marker for skin stem cells. After separating RNA from the cells, cDNA was synthesized, and then real-time-PCR was performed with Taqman stain solution to measure the expression level of p63. In this experiment, the concentration of RNA was normalized to S16 ribosomal RNA.

The experimental results are shown in Table 2 below. In Table 2 below, the value of the p63 increase fold refers to the fold compared to the control (CNT-57 medium with BPE added).

Effect of maintaining stemness (number of repetitions = 3) Added sample density p63 increase multiple (fold) Compound of formula 2 20 μg/mL 1.35 Control - 1.0

As can be seen from the results of Table 2, the medium condition treated with the compound of Formula 2 of the present invention promotes the expression of p63 in skin stem cells, thereby exhibiting excellent stem cell properties.

Claims (8)

Serum-free medium composition for culturing skin stem cells comprising a compound represented by the following Formula 1:
[Formula 1]

delete The method of claim 1,
The compound of Formula 1 is a serum-free medium composition for culturing skin stem cells, which is contained in a concentration of 1 to 500 μM.
A method of culturing skin stem cells comprising culturing the stem cells in a serum-free medium containing the compound represented by the following Formula 1:
[Formula 1]

delete The method of claim 4,
A method of culturing skin stem cells containing the compound of Formula 1 at a concentration of 1 to 500 μM.
An external preparation for skin for skin regeneration or wrinkle improvement comprising a culture medium cultured by the method of culturing skin stem cells according to claim 4.
A cosmetic composition for skin regeneration or wrinkle improvement comprising a culture solution cultured by the method of culturing skin stem cells according to claim 4.