KR101334243B1 - Method for manufacturing culture solution of fetus-derived mesenchymal stem cells from amniotic fluids using shear stress - Google Patents

Abstract

The present invention provides a method for producing a culture of stem cells comprising the step of applying a wound stimulation to stem cells, the culture of the stem cells prepared by the method, a cosmetic composition for improving the skin condition comprising the culture as an active ingredient, pharmaceutical The present invention relates to a composition and a method for mass production of a protein related to wound treatment, by using a culture of stem cells prepared by applying wound stimulation according to the present invention, a composition having better cell growth and skin regeneration ability can be provided.

Description

Method for manufacturing culture solution of fetus-derived mesenchymal stem cells from amniotic fluids using shear stress}

The present invention provides a method for producing a culture of stem cells comprising the step of applying a wound stimulation to stem cells, the culture of the stem cells prepared by the method, a cosmetic composition for improving the skin condition comprising the culture as an active ingredient, pharmaceutical A method of mass production of proteins associated with compositions and wound healing.

Stem cells are cells that are capable of self-proliferation and have the ability to differentiate into other cells through suitable environment and stimulation. Stem cells have the potential to develop into cells with characteristic phenomena and specialized functions.

As far as is known, adult stem cells are known to have the ability to differentiate into various cells. Adult stem cells are bone marrow ( Science 276, 71-74, 1997; Science 284, 143-147, 1999; Science 287, 1442-1446, 2000), skeletal muscle ( Proc . Natl Acad . Sci . USA 96, 14482-14486, 1999; Nature 401, 390-394, 1999) and Fat tissue ( Tissue Eng 7, 211-228, 2001; J. Cell . Physiol . 206, 229-237, 2006), each of which can differentiate into similar lines.

Mesenchymal stem cells derived from bone marrow, a type of adult stem cell, have been used for a long time and have been proven to be effective. However, isolation and the acquisition of large amounts of cells are difficult, so there is an urgent need for alternative sources. As a result of research showing that the cells isolated from adipose tissue as well as other tissues have similar characteristics to human bone marrow-derived mesenchymal stem cells, a new hypothesis has been made that stem cells exist in the amniotic fluid protecting fetuses. .

After fertilization takes place on the wall of the uterus after fertilization, the embryo is surrounded by amniotic sac filled with amniotic fluid. Since amniotic fluid contains a mixture of substances from the fetus's body, the amniotic fluid can be analyzed to determine whether the fetus's chromosomes are abnormal or not infected with bacteria. Amniotic fluid not only keeps the fetus free to move, but also protects the fetus from external shocks and stimuli, prevents bacterial infections, and helps to regulate the body's body temperature.

Before the fetus is born, the amniotic fluid tests various information on the health of the fetus. At this point, the amniotic fluid can be extracted without harming the mother from the beginning of pregnancy until after the birth. The cells used in the test are discarded after the test. If the patient's consent is obtained, the cells can be used for research purposes. Thus, amniotic cells have the advantage of easily obtaining a large amount of cells compared to other adult stem cells that have been studied previously. Amniotic fluid cells have an advantage of superior multipotency compared to other adult stem cells.

On the other hand, bFGF (basic fibroblast growth factor), a cytokine necessary for culturing stem cells, is expensive, and thus there is a problem of costly to obtain mesenchymal stem cells. Therefore, in obtaining mesenchymal stem cells and preparing a culture solution of the mesenchymal stem cells, it is required in the art to develop a method for producing more efficiently.

Thus, the present inventors cultured the cells in a medium containing selenium having an antioxidant effect instead of expensive bFGF in order to obtain mesenchymal stem cells that can be easily cultured in vitro while having a multipotency from human amniotic fluid that can be easily separated. Confirmed that the growth of mesenchymal stem cells is promoted, and when wound stimulation is applied during the preparation of the conditioned medium culture of the mesenchymal stem cells prepared by the above method, it is confirmed that the wound healing and skin regeneration ability is excellent. By this, the present invention was completed.

An object of the present invention is to provide a method for producing a culture of stem cells comprising the step of applying wound stimulation to stem cells.

Another object of the present invention is to provide a culture of stem cells prepared by the above method.

Still another object of the present invention is to provide a cosmetic composition and a pharmaceutical composition for improving skin condition comprising the culture as an active ingredient.

It is another object of the present invention to provide a method for mass production of proteins associated with wound healing.

As one embodiment for achieving the above object, the present invention comprises the steps of (a) applying a wound stimulation to stem cells; And (b) culturing the stem cells in a serum-free medium for 1 day to 10 days to provide a conditioned medium.

In the present invention, step (a) is a step of applying wound stimulation to stem cells.

As used herein, the term “stem cell” refers to a cell capable of self-proliferation and having the ability to differentiate into another cell through a suitable environment and stimulation, and having a potential to develop into a cell having characteristic phenomena and specialized functions. to be. The stem cells are preferably mesenchymal stem cells, more preferably may be fetal derived mesenchymal stem cells in the amniotic fluid.

In the present invention, the term "mesenchymal stem cells (MSCs)" is a cell that assists in making cartilage, bone, fat, myeloid epilepsy, muscle, nerves, etc., in adults generally stay in the bone marrow but the cord blood, It exists in peripheral blood, other tissues, etc., and means the cell which can be obtained from these. For the purposes of the present invention, the mesenchymal stem cells of the present invention may preferably be fetal-derived mesenchymal stem cells in the amniotic fluid.

Amniotic fluid from the mother contains many chemicals from the fetus's body that can produce most of the cells in the body and are easy to harvest. In addition, heterologous (heterogenous) cells exist in the amniotic fluid, and the present inventors have confirmed that there are homogenous mesenchymal stem cells having the same shape as fibroblasts characteristic of mesenchymal stem cells.

In the present invention, the amniotic fluid-derived mesenchymal stem cells are preferably obtained by subcultured amniotic fluid-derived cells in a medium containing FBS and selenium, more preferably obtained by subcultured in a medium further containing bFGF. can do.

The amniotic fluid-derived cells may be obtained by culturing amniotic fluid obtained from the mother, removing the remaining cells except the cells adhered to the bottom of the culture vessel, and separating the cells adhered to the bottom using trypsin and centrifugation.

As used herein, the term "culture media" refers to a medium that enables the growth and survival of fetal-derived cells in amniotic fluid in vitro and is suitable for the culture of fetal-derived cells in amniotic fluid. It includes all the usual medium used in the field, it is possible to select the medium and culture conditions according to the type of cells. The medium used for the culturing is preferably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source and a trace element component. Such cell culture minimal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI1640, F-10, F-12, α Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (GMEM) and Iscove's Modified Dulbecco's Medium (IMEM), but are not limited thereto. In addition, the medium may include an antibiotic such as penicillin, streptomycin, or gentamicin.

The medium of the present invention is characterized by containing selenium together with FBS. In one embodiment of the present invention, by confirming that long-term cell growth is possible upon addition of bFGF and selenium to the medium, the production of fetal mesenchymal stem cells in the amniotic fluid can be cultured in a medium to which selenium is added instead of bFGF. It was confirmed. The content of the FBS in the medium of the present invention may be preferably from 2 to 15%, more preferably from 5 to 10%, even more preferably from 10%, the content of the selenium is preferably Preferably from 1 to 20 ng / ml, more preferably from 2.5 to 10 ng / ml, even more preferably from 5 ng / ml.

The medium may further contain bFGF together with FBS and selenium. Through the co-addition of selenium and bFGF in the medium can exhibit a more excellent effect in the production of fetal mesenchymal stem cells in the amniotic fluid. The content of the bFGF may be preferably 1 to 20ng / ml, more preferably 2 to 10ng / ml, and even more preferably 4ng / ml. In one embodiment of the present invention it was confirmed that the best cell growth in the medium containing 10% FBS, 4ng / ml bFGF and 5ng / ml selenium (Fig. 3).

In addition, the medium may preferably use low glucose DMEM medium, and may further include 1% L-glutamine and 1% penicillin-streptomycin.

In the present invention, the term "shear stress" is an artificial stimulus through a wound similar to a wound applied to the human body applied to stem cells, and through the wound stimulation, it is possible to derive growth factors or effective ingredients related to wound healing. have. In one embodiment of the present invention, by applying the wound stimulation to the stem cells, it was observed that the cell growth and wound healing ability is improved compared to the group without the wound stimulation (Figs. 7 and 9).

The wound stimulus may be used without limitation, including physical stimuli capable of applying a wound similar to a wound applied to the human body. However, the wound stimulus may be a multi pipette, a micro pipette, A scraper or the like may be used, more preferably, multiple pipettes may be used, and even more preferably, it may be added by scratching with the multiple pipettes. The wound stimulation may be preferably performed 1 to 7 times, more preferably 3 to 5 times.

In an embodiment of the present invention, when wound stimulation was applied using a multi-pipette inserted with seven tips, it was confirmed that excellent cell growth occurred when the wound stimulation was not applied, and four wound stimuli were applied. It was confirmed that the best cell growth occurred (Fig. 6).

In the present invention, step (b) is a step of preparing a conditioned medium by culturing the stem cells in serum-free medium for 1 to 10 days.

In the present invention, the term "conditioned medium" is a liquid suspension culture when the cell reaches the apoptotic growth phase of the cell division, the dividing cells are removed by centrifugation or filtration, and only the culture medium is collected and mixed with the culture substrate. Say one badge. This uses an unknown growth factor that is extracted in the medium from the dividing cells, and is widely used for low density cell plating or protoplast culture.

For the purposes of the present invention, the conditioned medium composition of the present invention is a composition comprising a solution in which amniotic fluid-derived mesenchymal stem cells are removed from a medium in which amniotic fluid-derived mesenchymal stem cells are cultured. It refers to a composition containing abundant substances such as growth factors derived from stem cells, preferably Apo-1 / Fas, epidermal growth factor (EGF), IP-10 (Interferon-γ inducible protein-10), Leptin, MIP4, MMP3, Rantes, Interferon-gamma (IFNγ), human transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNFα), tumor necrosis factor receptor I ( TNFRI), tumor necrosis factor receptor II (TNFRII), cell adhesion factor-1 (ICAM-1), vascular cell adhesion factor-1 (VCAM-1), vascular endothelial growth factor (VEGF), interleukin-1 beta (IL -1β), interleukin-1 receptor alpha (IL-1Rα), IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-12 And IL-15 Can. In addition, genes related to wound treatment such as collagen type III, MMP1, fibronectin, vitronectin, syndecan 2, syndecan 4, SPP1, and elastin may be expressed in large amounts.

In the present invention, in order to obtain a conditioned medium of fetal-derived mesenchymal stem cells, the mesenchymal stem cells obtained from amniotic fluid containing Ham's F-12 nutrient mixture containing amino acids or their analogues and vitamins or their analogs It is preferable to use a serum culture medium. Serum-free medium containing Ham's F-12 nutrient mixture according to the present invention is based on DMEM without a pH indicator such as phenol red and Ham's F-12 nutrient mixture is added at a ratio of approximately 1: 0.5-2. . At this time, it is possible to add oxidants such as L-glutamine, energy metabolites such as sodium pyruvate, and carbon regulators such as sodium bicarbonate. This mixture is used to secrete various minerals and amino acids involved in cell growth and homeostasis and to enhance cell stability and maintenance in subculture after initial culture of mesenchymal stem cells. Vitamin nutrients and other factors, which can promote higher production of growth factors, are formed in a proportion.

The present invention may include the step of collecting the conditioned medium culture solution, thereby finally preparing a culture solution of fetal-derived mesenchymal stem cells in the amniotic fluid. Collection of the conditioned medium culture may be performed by a method known in the art, for example, may be collected by centrifugation or filtration.

As another aspect, the present invention provides a culture of stem cells prepared by the above method.

The culture medium may be prepared as described above, in one embodiment of the present invention confirmed that the conditioned medium culture prepared by applying the wound stimulation has excellent cell growth and wound healing effect, and additionally in the amniotic fluid-derived medium In the production of leaf stem cells, when cultured in a medium containing selenium and bFGF, it was confirmed that there is more excellent cell growth and wound healing effect (Figs. 7, 9 and 11).

As another aspect, the present invention provides a cosmetic composition for improving skin conditions comprising the culture solution as an active ingredient. Specifically, the skin condition may be whitening, wrinkles, skin damage by ultraviolet rays, skin elasticity, pore expansion, skin wounds or skin scars.

The composition of the present invention can be used for improving the skin condition. In one embodiment of the present invention confirmed that the wound healing effect of the conditioned medium obtained from the amniotic fluid-derived mesenchymal stem cells prepared by culturing in a medium containing selenium and bFGF, was prepared by applying wound stimulation It was confirmed that the medium (CM2) showed a superior effect than the control and no wound stimulation (Figs. 9 and 11).

The cosmetic composition of the present invention may include, in addition to the culture medium, components commonly used in cosmetic compositions such as antioxidants, stabilizers, solubilizers, conventional auxiliaries such as vitamins, pigments and flavors, and carriers, and the effect thereof. It may further comprise a skin absorption promoting material to enhance.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsion may be used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol Fatty acid esters of 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan may be used.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

As another aspect, the present invention provides a pharmaceutical composition for the treatment of skin damage, wrinkles, skin wounds or skin scars by ultraviolet rays comprising the culture medium as an active ingredient.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, which is commonly used in the preparation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, and know. Nate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils However, it is not limited thereto. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes such as oral or parenteral routes such as oral, rectal or intravenous, muscular, subcutaneous, intra-uterine, Can be administered by injection. Preferably, it is applied by transdermal administration during parenteral administration, more preferably by topical application by application.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The dose of the pharmaceutical composition of the present invention can be administered in an amount of 0.1-100 mg / kg on an adult basis once or several times a day in the case of an oral formulation, and in the case of an external preparation, It is preferable to apply it once to 5 times a day in an amount of 3.0 ml and continue for 1 month or longer. However, the dosage does not limit the scope of the present invention.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation can be used in any form suitable for pharmaceutical preparations, including powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations such as aerosols, external preparations such as ointments, creams, suppositories, and sterile injectable solutions. It may further comprise a dispersant or stabilizer.

In another aspect, the present invention provides a method for mass production of a protein associated with wound healing. Specifically, (a) subcultured amniotic fluid-derived cells in medium containing FBS and selenium to obtain fetal mesenchymal stem cells in the amniotic fluid; (b) applying wound stimulation to the obtained amniotic fluid-derived mesenchymal stem cells; And (c) culturing the stem cells in a serum-free medium for 1 to 10 days to prepare a conditioned medium, which is collagen type III, MMP1, fibronectin, vitronectin, syndecan from fetal mesenchymal stem cells in the amniotic fluid. 2, syndecan 4, SPP1 and elastin provides a method for producing a large amount of one or more proteins selected from the group consisting of.

In the present invention, step (a) is a step of obtaining cultured amniotic stem-derived mesenchymal stem cells by subcultured with amniotic fluid-derived cells in a medium containing FBS and selenium, which is as described above, wherein the medium is added to bFGF It may contain.

In the present invention, step (b) is a step of applying a wound stimulus to the obtained amniotic fluid-derived mesenchymal stem cells, the wound stimulation is as described above.

In one embodiment of the present invention, compared to the condition medium (CM1) prepared by applying the wound stimulation, compared to the condition medium (CM1) prepared without the DMEM / F12 serum-free medium and wound irritation, not the conditioned medium, Expression of collagen type III, vitronectin, syndecan 2, syndecan 4, fibronectin, MMP1, SPP1, elastin genes related to the treatment was confirmed to be high (Fig. 10).

Using the culture of stem cells prepared by applying a wound stimulation according to the present invention can provide a composition having better cell growth and skin regeneration ability.

Figure 1 shows a variety of shapes (heterogenous population: left) in culturing amniotic fluid-derived cell lines in medium supplemented with bFGF and / or selenium, but the same shape (three sub-cultures) homogenous enrichment (right: 4 passages), showing mesenchymal stem cells.
FIG. 2 shows karyotype of fetal-derived cells from amniotic fluid isolated from mothers, showing that sex chromosomes come from XY and are derived from non-natal amniotic fetuses; bFGF alone, selenium alone, or bFGF Normal karyotype analysis occurred in both the and selenium co-added groups.
Figure 3 is an experimental result for determining the appropriate concentration of selenium, it shows that the cell growth of the best results at 5ng / ml. The figure below shows normal cell growth in long-term culture by 3T3 assay, and normal cell growth occurs in both passages of selenium alone and co-addition in 26 passages, and beta-gal staining at 40 passages. Cell growth is stopped (+/-: bFGF addition / selenium addition, − / +: bFGF no addition / selenium addition, + / +: bFGF addition / selenium addition, the same below).
Figure 4 shows the analysis of the immunological phenotype of the cell through a flow cytometry, the results show that all three groups have similar characteristics as mesenchymal stem cells.
Figure 5 shows that the fetal-derived cells in the amniotic fluid has a differentiation capacity similar to that of bone marrow-derived mesenchymal stem cells with a multipotency, through differentiation into fat (A), bone (B), cartilage (C) The differentiation markers indicate differentiation at the mRNA level.
FIG. 6 shows cell growth of conditioned amniotic fluid-derived cell lines with conditioned medium (CM1) prepared using cells cultured in medium supplemented with bFGF and / or selenium and conditioned medium (CM2) prepared by additional wound stimulation. The results of the comparative experiment (a), the cell division number determination experiment (b) for producing CM2, and the experiment (c) for confirming the optimal condition of the number of wound stimulation are shown.
Figure 7 shows the result of comparing the effect of a large amount of growth factors present in the conditioned medium prepared by applying the wound stimulation on the growth of fibroblasts and the group without the wound stimulation and the control group.
Figure 8 shows the results confirmed by the real-time PCR analysis of the genes of cyclin A and E related to cell cycle analysis and cell proliferation using BrdU assay.
9 shows the results of comparing the effects of wound healing of skin fibroblasts on in vitro of conditioned medium (CM1 and CM2) prepared using fetal-derived mesenchymal stem cells in amniotic fluid.
10 shows the results of quantitative analysis of genes related to wound healing in vitro of conditioned medium (CM1 and CM2) prepared using fetal-derived mesenchymal stem cells in amniotic fluid.
FIG. 11 shows that there is a difference in wound regeneration ability after applying the control, CM1 and CM2 conditioned medium to the skin after wounding the mouse skin.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. However, the following examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

Example  1: Identification of Allogeneic Mesenchymal Stem Cells in Cultured Amniotic Fetal-derived Cell Lines

There are many floats in the amniotic fluid obtained from the mother. To separate it, the amniotic fluid was placed in a T-flask and incubated at 37 ° C. The next day, the rest were removed except for the cells on the floor. Cells adhered to the bottom were separated on the bottom using trypsin and collected by centrifugation, and then in low glucose DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS (fetal bovine serum), 1% L-glutamine, 1% penicillin- Cells were seeded in 100 mm cell culture dishes by resuspension in basal medium consisting of streptomycin, and 5 ng / ml selenium or 4 ng / ml bFGF. After 12 to 24 hours, the new medium was replaced. Amniotic fetal-derived cell lines had a variety of shapes (heterogenous population), but after three or four passages, only one fibroblast (homogenous population) was confirmed (FIG. 1). The medium was changed every 2 to 3 days and subcultured at a cell density of 80-90%.

Example  2: determination of fetal stem cells in amniotic fluid by karyotyping

In order to determine whether the cells cultured in Example 1 are fetal-derived cells in the amniotic fluid, karyotyping was performed.

The number, size, and shape of chromosomes are called karyotypes, and chromosomal mutations and sex can be determined by examining the fetus's chromosomes through karyotyping. In order to perform karyotyping, chromosomes were analyzed by stopping cell division with colcemid for 1 to 2 hours and observing by G-banding staining. As a result of the karyotype analysis, it was confirmed that the fetal-derived cells in the amniotic fluid have normal chromosomes and the sex chromosomes appear as XY in the cell culture through the medium containing bFGF and selenium. In addition, bFGF alone, selenium alone, and cell culture through co-addition of bFGF and selenium all showed normal karyotype results (FIG. 2).

Example  3: Establishment of proper concentration of selenium, in cell culture bFGF Replaceability with selenium and bFGF Confirmation of synergy effect through joint addition of selenium

In order to determine the proper concentration of selenium, under 2% and 10% FBS serum conditions, the culture conditions were changed depending on whether 4ng / ml bFGF was added and the concentration of added selenium was 0ng / ml, 2.5ng / ml, 5ng. Cell growth was compared by dividing into / ml and 10ng / ml. As a result, serum showed higher cell growth when 10% FBS was added than when 2% FBS was added, and selenium promoted cell growth regardless of whether bFGF was added. Was the highest. In addition, the cell growth effect of the cell culture in the medium added with bFGF and selenium was much higher than the addition of bFGF or selenium alone, and the cell growth was the most active when 4ng / ml bFGF and 5ng / ml selenium were added. (FIG. 3). From this, selenium can be used to replace the existing bFGF in culturing amniotic fluid-derived fetal cells, and when bFGF and selenium were co-added, it was confirmed that excellent cell growth was possible.

In addition, when adding selenium instead of bFGF, or co-addition of bFGF and selenium, the results of long-term cell culture were confirmed to confirm the possibility of alternative addition and co-addition even in long-term cell culture. Amniotic fetal stem cells obtained from mothers were initially added to low glucose DMEM with 10% FBS, 1% L-glutamine, 1% penicillin-streptomycin, 4ng / ml bFGF alone, 5ng / ml selenium alone and The culture method of 3T3 in the basal medium consisting of co-addition confirmed that cell growth was possible without difficulty even in long term cell growth. As a result, normal cell growth was possible even up to 40 passages by subculture every 2 or 3 days, showing similar results in the addition of selenium and bFGF alone, confirming the feasibility, and co-addition of bFGF and selenium. Showed the best cell growth ability (FIG. 3).

Example  4: Amniotic fluid of fetal derived cells With mesenchymal stem cells  Similarity check

We confirmed whether fetal-derived cells in amniotic fluid have characteristics of multipotent mesenchymal stem cells.

First, immunological phenotypes were analyzed by flow cytometry. When fetal-derived amniotic cells were compared with human bone marrow-derived mesenchymal stem cells, CD13, CD29, and CD44 were expressed similarly to bone marrow-derived mesenchymal stem cells (FIG. 4). Although the expression of these surfaces is a specific marker for mesenchymal stem cells, different characteristics may be present in each cell, so we examined the differentiation ability to bone, fat, and chondrocytes, which are representative characteristics of mesenchymal stem cells.

Amniotic fetal-derived cells are differentiated like human bone marrow-derived mesenchymal stem cells, and bones are osteopontin and osteocalcin, genes that are specifically expressed as bone forms, and fat is fat. The gene involved in the formation of lipoprotein lipase (LPL), fatty acid binding protein (aP2) and peroxysome proliferator-activated receptor gamma (PPAR) It was confirmed by reverse transcription chain reaction (RT-PCR) that cartilage was formed and differentiated into representative gene type 2 collagen (Type II collagen), type 1 collagen (Type I collagen) and aggrecan (aggrecan). Alizarin red S staining to confirm the formation of calcium salts when differentiating into bones. Lipid differentiation results in lipid droplets and oil red O staining to stain them. In order to stain the cartilage and the cartilage, the cartilage-specific secretion substrate was identified by the specific staining method for each differentiation using Alcian blue staining (Alcian blue staining) (FIG. 5). As a result, it was confirmed that the fetal-derived cells in the amniotic fluid of the present invention are cells having similar characteristics to the bone marrow-derived mesenchymal stem cells.

Example  5: Amniotic fluid-derived cell lines Conditioned  Badge manufacturing

Conditioned medium was prepared using an amniotic fluid-derived cell line. Production conditions for conditioned media were established from amniotic fetal-derived mesenchymal stem cells prepared by culturing in bFGF alone, selenium alone, or media co-added with bFGF and selenium.

First, mesenchymal stem cells established by three conditions were separated from the bottom by trypsin treatment in a 100 mm cell culture dish. Collect the separated cells in a 15ml tube and centrifuged to loosen the collected cells in 5 ~ 10ml medium and mix well. The cells were added to low glucose DMEM in 10% FBS, 1% L-glutamine, 1% penicillin-streptomycin, and basal medium in three conditions consisting of 4ng / ml bFGF alone, 5ng / ml alone and co-addition. It was suspended and seeded in a 100 mm cell culture dish. After 12 hours, the cells were changed to conditioned medium DMEM / F12 serum-free medium and incubated for 72 hours. After 72 hours, the culture medium in each condition was transferred to a tube, followed by centrifugation, and a 0.20 μm syringe filter to produce a conditioned medium.

Example  6: Amniotic fetal cell line derived from mother Conditioned  Determination of the Appropriate Number of Cells and the Number of Wound Stimuli in Media Preparation

In the preparation of the conditioned medium of Example 1, a wound-like wound applied to the human body to the fetus-derived mesenchymal stem cells in the amniotic fluid may be artificially stimulated to derive growth factors or effective ingredients related to wound regeneration or wound healing. Check if there is. For three groups of amniotic fetal stem-derived mesenchymal stem cells prepared by placing 250,000 stem cells in a 100 mm culture dish each and then culturing in a medium supplemented with bFGF alone, selenium alone or bFGF and selenium The results of cell growth were observed in the conditioned medium prepared by stimulation and wound stimulation. The wound stimulus was applied by scratching using a multi-pipette fitted with seven tips. As a result, compared to the group without the wound stimulation (CM1) in the wound stimulation group (CM2), it was confirmed that the results of excellent cell growth independently of the addition of bFGF or selenium (Fig. 6 a).

In addition, to establish the optimal conditions for cell division, 250,000, 500,000, 750,000, 1 million and 1.25 million stem cells were placed in a culture dish in 100 mm culture dishes, respectively, and 10% FBS, 1 in low glucose DMEM. Incubate for 12 to 18 hours with the addition of a medium consisting of co-addition of% L-glutamine, 1% penicillin-streptomycin, 4 ng / ml bFGF, and 5 ng / ml selenium, using a multi-pipette with 7 tips Artificial scratches were applied through scratches. After washing three times with PBS again and incubated in DMEM / F12 serum-free medium culture for 3 days, and harvested only the culture medium, it was centrifuged and filtered to 0.22um to produce a conditioned medium. As a result, when seeding 750,000, 1 million and 1.25 million stem cells (seeding) similarly showed an excellent cell growth effect (Fig. 6b).

In order to establish the optimal condition for the number of wound stimulation, two, four and six wounds, respectively, were used for the seeding of 750,000 stem cells having a relatively small cell division number in the group showing the effect of excellent cell growth in the experiment. Cell growth in the stimulus was compared. As a result, it was confirmed that the results of the best cell growth in the group subjected to four wound stimulation with multiple pipettes (Fig. 6c).

Example  7: Amniotic fetal origin In mesenchymal stem cells Conditioned  Competitive Comparison of Cell Growth by Media Preparation

The wound stimulation was not performed using the conditioned medium prepared by applying wound stimulation to the amniotic fluid-derived mesenchymal stem cells prepared by three methods of bFGF alone, selenium alone, or co-addition prepared in Example 1. The growth rate of fibroblast cells (BJ cells) and the serum-free DMEM / F12 culture medium added antibiotics alone were compared between the group and the control group. 30,000 fibroblasts were dispensed in a 24-well culture dish and subjected to a comparative culture experiment for 3 days, and cell growth was compared through crystal violet staining. As a result, it was confirmed that the cell growth was significantly more active than the control group in the wound stimulation group, the cell growth was more active than the group without the wound stimulation (Fig. 7).

Example  8: Stem Cell Culture Solution by Wound Stimulation BrdU assay Wow real - time PCR Efficacy analysis through

When fibroblast growth was compared with the conditioned medium prepared using mesenchymal stem cells cultured in a medium supplemented with bFGF and selenium, and the conditioned medium prepared by applying additional wound stimulation, It was confirmed that the cell growth was more active, BrdU assay was performed on the conditioned medium prepared by applying wound stimulation. As a result, it was confirmed through the cell cycle analysis using the BrdU assay that the cell culture occurs actively during the addition of selenium alone and co-addition of bFGF and selenium (FIG. 8). In addition, the results of cyclin A and E genes related to cell proliferation were confirmed by real-time PCR analysis. Also, the expression of proteins related to cell proliferation was increased during the addition of selenium alone and the co-addition of bFGF and selenium. (FIG. 8).

Example  9: in dermal fibroblasts Conditioned  Wound healing in the medium ( wound healing ) Check the effect

Using conditioned medium prepared from stem cells derived from amniotic fluid, it was confirmed whether the conditioned medium prepared by applying wound stimulation improved the wound healing effect. After artificial wounds were applied to skin-derived fibroblasts, a cultured medium of serum-free DMEM / F12 containing only antibiotics was used as a control group, and a conditioned medium of stem cells prepared through co-addition medium of bFGF and selenium (CM1). And conditioned medium (CM2) prepared by applying additional wound stimulation to the cell, and at 13 hours, the degree of migration from the cells treated with the conditioned medium prepared by applying the wound stimulus was almost completed. Showed the most complete wound healing effect, and the difference in the three groups was found to be the largest at 15 hours, wound healing of conditioned medium (CM2) prepared by applying wound stimulation from stem cells derived from amniotic fluid It was confirmed that the effect is the most excellent (Fig. 9).

Example  10: In dermal fibroblasts Conditioned  Quantitative Analysis of Genes Associated with Wound Healing Effects in Medium

At the time when migration rate was measured, expression of genes related to wound healing was confirmed by real-time PCR. Cell migration in conditioned medium (CM2) prepared by applying wound stimulation, as compared to DMEM / F12 serum-free medium and conditioned medium (CM1) prepared without applying wound stimulation, but not conditioned medium. Collagen type III, vitronectin, syndecan 2, syndecan 4, fibronectin, MMP1, SPP1, elastin, etc., genes that help wound healing in It was confirmed that the effect of wound healing was the highest in the conditioned medium prepared by applying wound stimulation (FIG. 10).

Example  11: mouse in vivo in Conditioned  Skin regeneration effect of badge

Animal experiments using mouse wounded skin were performed to determine the effect of conditioned medium on fetal-derived mesenchymal stem cells in amniotic fluid on skin regeneration. Artificial wounds were made using a punch of approximately 50 cm² (diameter 8 cm) into the skin of the mouse, followed by normal DMEM / F12 serum-free medium, conditioned medium (CM1) and wound stimulation prepared above. Conditioned medium (CM2) prepared by applying was applied to each of the wounds to measure the degree of wound closure (wound closure) of the wound. Differences between the three groups were best compared on day 9, while normal DMEM / F12 serum-free medium showed slower skin regeneration at the wound site, but CM1 showed higher wound closure than normal DMEM / F12 serum-free medium. Showed the most complete wound closure (FIG. 11).

Claims (11)

(a) subcultured amniotic fluid-derived cells in FBS (fetal bovine serum) and selenium-containing medium to obtain fetal mesenchymal stem cells in amniotic fluid;
(b) applying wound stimulation to said fetal mesenchymal stem cells in said amniotic fluid; And
(c) culturing the stem cells in a serum-free medium for 1 to 10 days to prepare a conditioned medium, a method for producing a culture of amniotic fluid-derived mesenchymal stem cells.
delete delete The method of claim 1, wherein the medium of step (a) further comprises bFGF.
The method of claim 1, wherein the wound stimulus is applied by scratching with a multi pipette.
The method of claim 5, wherein the scratch is added 3 to 5 times.
A culture of amniotic fluid-derived mesenchymal stem cells prepared by the method of any one of claims 1 and 4 to 6.
The cosmetic composition for improving the skin condition comprising a culture of the amniotic fluid-derived mesenchymal stem cells of claim 7 as an active ingredient, wherein the skin condition is wrinkles, skin damage by ultraviolet rays, skin elasticity, skin wounds or skin scars Cosmetic composition for improving skin condition.
A pharmaceutical composition for the treatment of skin damage, wrinkles, skin wounds or skin scars caused by ultraviolet rays comprising the culture medium of the amniotic fluid-derived mesenchymal stem cells of claim 7 as an active ingredient.
(a) passage of amniotic fluid-derived cells in medium containing FBS and selenium to obtain fetal mesenchymal stem cells in amniotic fluid;
(b) applying wound stimulation to the obtained amniotic fluid-derived mesenchymal stem cells; And
(C) collagen type III, MMP1, fibronectin, vitronectin, syndecan 2 from the amniotic fluid-derived mesenchymal stem cells comprising the step of culturing the stem cells in serum-free medium for 1 to 10 days to prepare a conditioned medium , mass production of one or more proteins selected from the group consisting of syndecan 4, SPP1 and elastin.
The method of claim 10, wherein the medium of step (a) further comprises bFGF.

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