KR102296986B1 - Development of a chemically defined stem cell culture media to overcome the harmfulness of fetal bovine serum - Google Patents

Abstract

The present invention relates to a serum-free medium composition for culturing stem cells and a method for culturing stem cells using the same. The serum-free medium composition of the present invention is safe as the composition does not use animal-derived serum, and despite the non-use of the animal-derived serum, has effects of exhibiting a relatively excellent cell differentiation rate and maintaining stem cell characteristics.

Description

Development of a chemically defined stem cell culture media to overcome the harmfulness of fetal bovine serum

The present invention relates to the development of a chemically defined stem cell culture medium for overcoming the harmfulness of fetal bovine serum components.

In the case of cells cultured using fetal bovine serum, there may be tissue variability, and the price is high, so it is rare in terms of time. In addition, using this culture medium can become a source of contamination of mycoplasmas, bacteriophages and viruses, and while it contains toxins, it can cause cell surface modification by adsorption and binding of serum proteins. Separation of cell products may be a source of disturbance, and excessive growth of fibroblasts in the initial culture may be encouraged. In addition, although it reduces the cytotoxic action of immunoglobulins during heat inactivation, even unstable components are destroyed, so the results are not always satisfactory compared to serum that is not heat-treated.

The addition of an energy source (serum) to the culture medium contains various harmful components, and in the United States, more than 500,000 cows are sacrificed to make this serum a year, so NGO groups are taking action against production.

In addition, many problems are reported when FBS is added to cosmetics because it is derived from cattle (animals), and shock death may occur when stem cells are cultured and injected into humans, so there is a high demand for the development of a serum-free medium. .

Therefore, as a means to replace this, much effort is being made to make a culture solution without the addition of serum. However, it is known that the stem cell development rate is lowered when compared to the culture medium with and without serum.

Numerous references are referenced throughout this specification and citations thereof are indicated. The disclosures of the cited documents are incorporated herein by reference in their entirety to more clearly describe the content of the present invention and the level of the art to which the present invention pertains.

Republic of Korea Patent Publication No. 10-2020-0120956

In humans, except for special cells, various types of cells are used for culture by adding fetal bovine serum (FBS) as an energy source to a culture medium (DMEM, Ham F-10, etc.) that is almost similar.

Among the special cells, human germ cells grow only in a culture medium that is completely different from that described above. World-vitro fertilization cases of domestic Mary Hospital and the best treatment lot (Journal of Fertility and Sterility. Vol . 75, No. 4, pp 833, 2000.) Follicular person in a culture medium developed by itself in liquid (A thesis for Thousands of babies have already been born using the degree of Ph.D. By San Hyun Yoon, Korea university.

The present invention succeeded in developing a culture medium for culturing stem cells for the first time in the world by modifying the culture medium. In addition, in the state in which follicular fluid (FF) was added instead of fetal bovine serum, better results were obtained compared to the existing stem cell culture medium. For various reasons, such as moral issues, the use was restricted.

Therefore, naturally, stem cells were also looking for their substitutes. Most of the alternative substances (human serum albumin, plasmatain, etc.) are expensive and difficult to use, and a more economically and effectively available synthetic material was found in the components of the oocyte, and chemically defined optimally by adjusting the content. By developing a stem cell culture medium, the present invention was completed.

Accordingly, an object of the present invention is to provide a medium composition for stem cell culture that does not contain animal-derived serum.

Another object of the present invention is to provide a method for culturing stem cells without using animal serum, characterized in that the medium composition for stem cell culture is used.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

One aspect of the present invention is (i) a basal medium containing taurine and a chelating agent, and (ii) as an additive, a complex protein of HSA and globulin (Synthetic Protein; SP), characterized in that it comprises a stem cell culture medium to provide a composition.

The term "basic medium" used in the present invention means a medium that can support stem cell growth and survival in vitro, and a conventional medium used in the art suitable for culturing cells. include all Depending on the type of cell, the medium and culture conditions can be selected.

As the basal medium, for example, MR-IVF ^TM , DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, aMEM (a Minimal essential Medium), There are GMEM (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, etc., but preferably MR-IVF ^TM culture medium can be used.

MR-IVF ^TM culture medium contains taurine and a chelating agent, which is known to be involved in energy production by acting on mitochondria. In addition, it is an amine that reacts well with hypochlorite to form N-chlorotaurine, which is relatively stable when reacted, and is then reduced to chlorine ions and taurine. It is reported that this process shows an antioxidant effect. In addition, taurine stabilizes cell membranes and is effective in delaying and preventing carcinogenesis caused by lipid peroxidation. Due to this antioxidant effect, it reduces lipid peroxidation of cells and prevents deterioration of motility.

The chelating agent is an organic compound that forms two or more bonds between a central atom and two or more bound ligands, EDTA (ethylenediamine tetra-acetic acid), EDTAC (EDTA plus Cetavlon), EGTA ((ethylene glycol-bis ( β-aminoethyl ether)-N,N,N',N'-teraacetic cid) and tetracycline-hydrochloride (tetracycline-hydrochloride), but are not limited thereto.

Chelating agents such as EDTA chelate heavy metal substances in the culture medium outside the cell membrane to facilitate the movement of other substances important for cell development, thereby increasing cell development and increasing the absorption of amino acids to induce protein synthesis. It helps to develop stably.

In one embodiment, the taurine in the stem cell culture medium composition of the present invention may be added at a concentration of 10 to 500 mM, and the concentration of the chelating agent at a concentration of 1 to 1000 mM.

On the other hand, when cosmetics are made with a raw material solution cultured with stem cells cultured with animal-derived serum, side effects such as skin rash, erythema, psoriasis, necrosis, pigmentation, and dermatitis may appear. It is advantageous to use a material extracted from a serum-free culture medium as a cosmetic raw material to prevent even such a minor side effect in advance.

Accordingly, in a preferred embodiment, the present invention provides a serum-free medium composition, specifically, a serum-free culture medium composition for culturing stem cells that does not contain animal-derived serum while including a cell activator in the basal medium.

The SP (Synthetic Protein) protein added to the medium composition of the present invention is a substance that can replace animal-derived serum and follicular fluid (FF), and contains 80±1% by volume of HSA (Human serum albumin) and 20± It is a complex protein consisting of 1% by volume of pure globulin. The SP (Synthetic Protein) protein exhibits the most efficient stem cell differentiation when the albumin:globulin complex ratio is 8:2 (v:v).

In one embodiment, the amount of the SP protein added to the stem cell culture medium composition of the present invention may be 5 to 20% by weight based on the total weight of the composition.

The medium composition of the present invention may further include one or more growth factors selected from the group consisting of Epidermal Growth Factor (EGF), Insulin-like Growth Factor (IGF), and Fibroblast Growth Factor (FGF).

The FGF may preferably be a basic fibroblast growth factor (bFGF), which, like other FGF relatives, has a wide range of somatic cell viability and includes embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, It plays a role in various biological processes such as invasion. It is also synthesized and secreted by human adipocytes, and the concentration of FGF2 correlates with BMI in blood samples. In addition, it was found to act on the proliferative form of the metaphyseal bone marrow cells after binding to the fibrous compound growth factor receptor 1 and activating phosphate 3-kinase. FGF2 is an important component of human embryonic stem cell culture media. Growth factors are necessary for cells to remain in an undifferentiated state, although the mechanism by which cells play this role has not been defined. This has been demonstrated to induce greemlin expression, which is known to inhibit the induction of differentiation by osteogenic proteins. FGF2 promotes the differentiation of stem cells together with BMP4. After differentiation, cells treated with BMP4 and FGF2 are generally known to promote osteoblast and chondrocyte differentiation in higher amounts than untreated stem cells.

In one embodiment, the addition amount of the growth factor in the medium composition for culturing stem cells of the present invention may be 1 to 1000 ng/ml.

In addition, the medium composition of the present invention may further include one or more additives selected from the group consisting of ascorbic acid or a salt thereof, hyaluronic acid or a salt thereof, and mercaptoethanol.

The ascorbic acid or its salts refer to several vitamin C and vitamin C activity in animals including oxidized form molecules such as dihydroascorbic acid. Since ascorbate and ascorbic acid interconvert their forms depending on pH, one of them is both naturally conserved in the body if one of them is inserted into the cell. Vitamin C is a cofactor in at least eight enzymatic reactions, including several reactions that cause the most severe symptoms of scurvy when collagen synthesis reactions are disrupted. In animals, these responses are particularly important for wound healing and prevention of bleeding in capillaries. Ascorbate also acts as an antioxidant, protecting against oxidizing substances. Ascorbic acid salts (e.g., the anion of ascorbic acid) require a variety of essential metabolic requirements in all animals and plants. It is made internally in almost all organisms.

The above-mentioned hyaluronic acid is a hydrophilic material, and as a biosynthetic natural substance that has a moisturizing action on the skin of animals, etc., the salt of hyaluronic acid is not particularly limited as long as it is a salt that is acceptable as a medicine. For example, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, succinic acid, citric acid, tartaric acid, adipic acid, gluconic acid, glucoheptoic acid, gluconic acid Lonic acid, terephthalic acid, methanesulfonic acid, lactic acid, hemanic acid, 1,2-ethanedisulfonic acid, isethionic acid, lactobionic acid, oleic acid, pamoic acid, polygalacturonic acid, stearic acid, tannic acid, trifluoromethanesulfonic acid, benzene salts with organic acids such as sulfonic acid, p-toluenesulfonic acid, lauryl sulfate ester, methyl sulfate, naphthalenesulfonic acid, and sulfosalicylic acid; quaternary ammonium salts such as methyl bromide and methyl iodide; salt with halogen ions, such as a bromine ion, a chlorine ion, and an iodine ion; salts with alkali metals such as lithium, sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; metal salts with iron, zinc, etc.; salt with ammonia; Triethylenediamine, 2-aminoethanol, 2,2-iminobis(ethanol), 1-deoxy-1-(methylamino)-2-D-sorbitol, 2-amino-2-(hydroxymethyl)-1 and salts with organic amines such as ,3-propanediol, procaine, and N,N-bis(phenylmethyl)-1,2-ethanediamine.

The 　 hyaluronic acid 　 salt added to the composition of the present invention may preferably be sodium hyaluronate.

The mercaptoethanol may be preferably beta-mercaptoethanol (β-mercaptoethanol; β-ME). It is used to reduce disulfide bonds and is known to act as a biological antioxidant by scavenging hydroxyl radicals. It is widely used because the hydroxyl group gives solubility in water and reduces volatility.

Because of their ability to disrupt the structure of proteins, they have been used in protein analysis, for example, to ensure that protein solutions contain monomeric protein molecules instead of disulfide-linked dimers or higher-order oligomers. Inhibits the oxidation of reducing agent residues, and is therefore used in various enzyme assays as a buffer component by being included in the enzymatic reaction to maintain protein activity.

In one embodiment, the addition amount of the ascorbic acid or its salt in the stem cell culture medium composition of the present invention is 1 to 100 μg / ml, the addition amount of the hyaluronic acid or its salt is 10 to 500 μg / ml, the mercaptoethanol The addition amount may be 1 or more and less than 50 μg/ml.

In addition, the medium composition of the present invention may further include one or more additives selected from the group consisting of growth hormone (GH), cholesterol and insulin. Cholesterol forms the basis of hormones, and as an essential substance for life, it is used to form cell membranes in all organs and tissues of the body. It is used as a cell membrane component to make the stem cell membrane safe, and is also used as a component to promote differentiation into adipocytes.

In one embodiment, the addition amount of the growth hormone (GH) in the medium composition for stem cell culture of the present invention is 40 to 500 ng / ml, the addition amount of the cholesterol is 1 to 100 μg / ml, the addition amount of the insulin is 10 to 3000 ng/ml.

In addition, the stem cell culture medium composition of the present invention may further include one or more adhesion factors selected from fibronectin and gelatin, and the amount of the adhesion factors added may be 1 to 100 ng/ml.

In one embodiment, the medium composition for culturing stem cells of the present invention does not contain the cytokines IL-1b and TNF-α.

In the present inventors' experiments, the addition of a substance did not promote the development of cells, so the addition of one substance did not promote the development of the cells, and it was found that the synergistic effect appeared as the development of the cells was improved by adding one substance at a time. It is estimated. However, on the contrary, not all cell growth factors enhance cell growth, but substances such as cytokines IL-1b and TNF-α rather inhibit cell development in the present inventors' experiments.

In addition, the stem cell culture medium composition of the present invention may further include transferrin, selenium, L-alanine-L-glutamine, etc. as trace elements.

As the transferrin (Transferrin) and selenium (Selenium), Gibco Insulin-Transferrin-Selenium (ITS-G) may be commercially purchased and used, and as the L-alanine-L-glutamine, Glutamax may be commercially purchased. can also be used.

As described above, when the medium composition for culturing stem cells of the present invention is used, stem cells can be cultured at a high development rate without using animal serum.

In a preferred embodiment, the serum-free medium composition of the present invention comprising SP as a serum substitute simultaneously contains hyaluronic acid or a salt thereof, ascorbic acid or a salt thereof, cholesterol and beta-mercaptoethanol, regardless of the content of growth factors. 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 100% or more, most preferably 110% or more of the stem cell development rate obtained when fetal bovine serum (FBS) is included It can represent more than the stem cell development rate. The development rate refers to a value calculated as a percentage based on the number of cells obtained after the initial cell count and the number of cells obtained after culturing when the cells are seeded.

The serum-free culture medium composition of the present invention is not only safe because it does not use animal-derived serum, but also has the effect of exhibiting a relatively excellent cell differentiation rate and maintaining stem cell characteristics despite the non-use of animal-derived serum. Therefore, the medium composition of the present invention can be usefully used for culturing stem cells used for medical and cosmetic purposes. It shows superiority in growth of human adipose-derived stem cells in serum-added medium without adding serum, contains the components required by cells, and can produce components beneficial to humans at high cost.

1 is a graph comparing the cell development rate when FBS is added to DMEM basal medium and when FF is added to MR basal medium.
2 shows the results of observing the number of ^{stem cells after seeding 10x10 5} stem cells using SP combining albumin and globulin in various ratios and culturing for 7 days.
Figure 3 shows the cell development rate according to the concentration of additives FBS, SP, SM, low-concentration growth factor and low-concentration cytokines in the MR culture medium.
Figure 4 shows the cell development rate according to the concentration of the additive material FBS, SP, SM, high-concentration growth factor and high-concentration cytokine in the MR culture medium.
Figure 5 shows the cell development rate according to the additives FBS, SP, SM, ascorbic acid, beta-mercaptoethanol and cholesterol in the MR culture medium.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples.

Example

Materials and Methods

DMEM was mainly used as the culture medium used for the existing stem cell culture, but in this patent, the YS medium developed for embryo culture is modified and a basic medium similar to the experiment (in the MR-IVF ^TM phase experiment, stem cell growth is shown like DMEM. ), and substances such as EGF, FGF, GH, glutamax, hyaluronic acid, Insulin, selenium, and transferrin that promote stem cell development were added.

However, in the comparative experiment DMEM, Minimal non-essential medium (MNEM) and antibiotics were not added, so 1% of each was added.

The culture medium preparation for the present invention was finally adjusted to pH 7.4 to 7.6 and osmotic pressure to 280 ± 5 mOsm/kg and used to culture adipose-derived stem cells.

Table 1 below shows the composition of the culture medium (MR-IVF ^TM ) used as the basal medium, Table 2 shows the additives used in the experiment, and Table 3 shows the components and contents of follicular eggs of postmenopausal women.

Group component Concentration mineral NaCl 58.44 mM KCl 74.55 mM CaCl 1.8 mM NaHCO3 84.01 mM KHCO3 100.1 mM MgSO4.7HO 246.50 mM organic matter Glucose 5.56 mM Glutamine 146.10 mM Lactate 112.10 mM Pyruvate 110.00 mM Taurine 125.10 mM amino acid Minimum essential medium 1% (v:v) Minimum non-essential medium 1% (v:v) vitamin MEM vitamin solution 0.25% (v:v) chelate compound EDTA 372.2 mM Antibiotic penicillin
Streptomycin 0.07 g/l
0.05 g/l

additional substances trace elements Selenium, Hyaluronic acid, Glutamax,
β-mercaptoethanol, Trasferrin hormone Insulin, Growth hormone, Cholesterol synthetic protein Synthetic Protein (SP) growth factor EGF, b-FGF, IGF cytokine IL-1b, TNF-α vitamin Ascorbic acid

As the Synthetic Protein (SP), a complex protein prepared by combining albumin (80% by volume) and globulin (20% by volume) that promotes cell growth was used. Specifically, pure albumin powder and pure globulin powder purchased from Sigma were used by dissolving in 10 ml of culture medium at an albumin 8:globulin 2 ratio (v:v).

Composition Concentration Nonessential amino acid (g/dL) Alanine 305.5 Asparagine 98.8 Aspartic acid 8.1 Glutamic acid 79.5 Glycine 179.7 Proline 125.2 Serine 69.7 Taurine 23.0 Essential amino acid (g/dL) Arginine 65.9 Cystine 12.3 Glutamine 347.0 Histidine 79.1 Isoleucine 29.8 Leucine 53.4 Lysine 123.7 Methionine 15.5 Phenylalanine 34.5 Hydroxyproline 16.0 Threonine 114.8 Tryptophan 34.5 Tyrosine 29.3 Valine 135.9 Albumin & globulin (g/dL) albumin 4.56 Globulin α1-globulin 0.37 α2-globulin 0.43 β-globulin 0.87 γ-globulin 1.33 Hormones (U/dL) Estrogen 100 Progesterone 1.6 FSH (mIU) 524 LH (mIU) 76 Insulin (mIU) 0.3 Prolactine (ug) 114.2 Growth hormone (ng) 422 Growth factors & cytokines (U/dL) IL-1 (ng) 2.33 TNFα (ng) 0.74 IGF-I (ug) 10.57 IGF-II (ng) 278

A thesis for the degree of Ph.D. By San Hyun Yoon, Korea university. 2005.

Fat separation, washing and cell seeding for cell extraction

01. Adipose tissue was collected, placed in a 50 ml tube, and operated at 1,000 rpm for 1-2 minutes using a homogenizer to crush the fat.

02. Centrifuged at 1,200 rpm for 5 minutes.

03. The oil in the upper layer was removed.

04. Add 25 ml of saline and 25 ml of the fat layer, and shake well.

05. Centrifuged at 1,200 rpm for 3 minutes.

06. 20 ml of adipose tissue and collagen AIDS were mixed in a 1:1 ratio.

07. It was left in a shaking incubator at 37°C at 200 speed for 60 minutes.

08. 50 ml each was dispensed and centrifugation was performed at 1,200 rpm for 5 minutes.

09. After removing the supernatant, leaving only about 5 ml in the lower layer, 20 ml of saline was added and mixed.

10. The cell suspension was filtered through a 100 μm filter and added 1:1 with saline.

11. Centrifuged at 1,200 rpm for 5 minutes.

12. After removing the supernatant, 1-2 ml of saline was added to the remaining pellet.

13. 10-20 μl was collected, and the number of cells was measured after staining with 0.4% trypan blue and 1:1.

14. Cells were seeded in a 30Ψ culture dish (3-10 X 10 ³ cells/cm ² ).

15. 37° C., 5% CO ₂ Incubated for 3-4 days in an incubator.

subculture

01. After removing the culture medium, cells were washed with PBS.

02. After applying PBS/0.05% trypsin-EDTA to the culture dish, it was left in an incubator at 37°C for 5 minutes.

03. After shaking gently to float the cells by pipetting, it was diluted 1:1 with the culture solution.

04. Transferred to a 50 ml tube, centrifuged at 1,000 rpm for 5 minutes, and washed with a culture solution.

05. 10-20 ul was collected and the number of cells was measured after 1: 1 staining with 0.4% trypan blue.

06. Incubated under freezing or 37°C, 5% CO _{2 conditions.}

cell freezing

01. After culturing, the stem cells were mixed well with the freezing culture solution and put into a 1.8 ml freezing tube and frozen.

02. Using a cell freezer, -1°C drop per minute from 4°C to -4°C, -20°C decrease per minute from -4°C to -45°C, +10°C increase per minute from -45°C to -10°C, - From 10°C to -20°C, the temperature drops at -0.5°C per minute, and at -20°C to -80°C, drops by -1°C per minute. When the freezing process is complete, it is immediately immersed in a liquid nitrogen tank and stored until use.

cell lysis

01. The lower part of the freezing tube was immersed in a 37°C water bath and shaken for 1-2 minutes.

02. Put 9 ml of the culture solution in a 15 ml centrifuge tube, and mix with the thawed cell suspension.

03. Centrifuged at 1,000 rpm for 5 minutes.

04. Steps 2 and 3 were repeated twice.

05. 10-20 ul was collected and mixed with 0.4% trypan blue 1:1 to measure the number of cells after staining.

06. Incubated at 37° C., 5% CO ₂ conditions.

Experiments for the development of serum-free medium

1. Experiment with Fetal Bovine Serum (FBS) and follicular fluid (FF)

As shown in Table 4 below, FBS was added to DMEM and FF was added to MR-IVF (MR) to test the possibility of stem cell culture.

division Basic Badge (V:V) Additives (V:V) DMEM DMEM (90%) FBS (10%) MR MR (90%) FF (10%)

After culturing the group in which 10% of FBS was added to the DMEM and 10% of FF in the MR culture medium was cultured for 1 week, the number of cells was measured. counted based on the number), suggesting the possibility of adipose-derived stem cell culture medium (FIG. 1).

However, since FF is a human-derived material, it is currently impossible to use it, so an experiment was conducted to find a substitute for it.

2. Experiment with HSA (human serum albumin)

As shown in Table 5 below, an experiment was conducted with HSA, which is currently widely used in human embryo culture.

division Basic Badge (V:V) Additives (V:V) MF DMEM (90%) FBS (10%) MRF MR (90%) FBS (10%) MRS MR (90%) SP (10%) MRH MR (95%) HSA (5%) MRHS MR (92.5%) HSA (2.5%) + SP (5%)

As a result of measuring the number of cells after culturing the stem cells for one week, it was confirmed that there was no significant difference in stem cell growth between the two groups as the cells continued to differentiate at 6.1-fold and 6.3-fold, respectively, in the MF and MRF groups to which FBS was added. However, it was slightly lower in other groups.

Many cells have a property of growing by living on the bottom of a culture dish. So, the group to which FBS was added attached well to the culture dish, but the group to which SP and HSA were added did not stick to the culture dish, so fibronectin and gelatin, which are known to be adherent substances, were added. was confirmed, and the highest adhesion rate was confirmed at the concentration of 50 ug/ml. However, due to the difference in price between the two adhesives, gelatin was used in the same ratio in all experiments from now on in consideration of the economical aspect.

SP was used in the experiment with human albumin and globulin based on HSA. In order to examine the effect of the albumin:globulin ratio on stem cell differentiation, 10x10 ⁵ stem cells were seeded using SP combined in various ratios and cultured for 7 days, then the number of cells was observed, and the result is shown in FIG. 2 shown in As a result of the experiment, it was surprisingly confirmed that the albumin:globulin (8:2) ratio resulted in the most efficient stem cell differentiation ( FIG. 2 ). Therefore, it was used in the experiment with a combination in the same ratio thereafter.

3. Effect on the growth of stem cells between the FBS-added group and the various addition groups

In order to evaluate the effect on the growth of stem cells between the FBS-added group and the various addition groups, the medium was prepared and tested as shown in Table 6 below.

division Basic Badge (V:V) Additives (V:V) MF DMEM (90%) FBS (10%) MRF MR (90%) FBS (10%) MRS MR (90%) SP (10%) MRH MR (95%) HSA (5%) MRHS MR (92.5%) HSA (2.5%) + SP (5%) MRF5F MR (85%) FBS (10%) + FF (5%) MRF10F MR (80%) FBS (10%) + FF (10%)

As a result of the experiment, it was confirmed that the cell division rate increased by 28% and 39%, respectively, in the group in which 5% and 10% of FF was added to FBS. However, HSA and FF were expensive and difficult to obtain, so SP was decided as an additive material.

4. Cell development experiment when growth factors and cytokines are added

As shown in Table 7 below, cell development experiments were performed by adding growth factors and cytokines to DMEM and MR cultures.

division Basic Badge (V:V) Additives (V:V) MF DMEM (90%) FBS (10%) MRF MR (90%) FBS (10%) MRSM MR (98%) SM (ITS 1% + glutamax 1% + galatin 50ug + EGF 1ng + IGF 1ng + bFGF 1ng + GH 1ng + IL-1b 1ng) MRSSM MR (88%) SP (10%) + SM (ITS 1% + glutamax 1% + galatin 50ug + EGF 1ng + IGF 1ng + bFGF 1ng + GH 1ng + IL-1b 1ng)

As a result of the experiment, it was confirmed that there was little difference in cell division as a result of culturing with growth factors and cytokines added to the DMEM and MR cultures.

Therefore, when the growth factor was at a low concentration (1 ng/ml) and the cytokine at a low concentration (1 ng/ml), the cell development rate did not increase. After this, the experiment was performed by increasing each concentration.

5. Cell development experiment according to the concentration of HSA, SM (supplemental material), growth factors and cytokines in MR culture medium

As shown in Table 8 below, HSA, SM, growth factors and cytokines were added to the MR culture medium to prepare a medium.

division Basic medium (V:V) Additives (V:V) MF DMEM (90%) FBS (10%) MRF MR (90%) FBS (10%) MRSM MR (98%) SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 1 ng + IGF 1 ng + bFGF 1 ng + GH 1 ng + IL-1b 1 ng + TNF-a 1 ng) MRHSM MR (88%) HSA (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 1 ng + IGF 1 ng + bFGF 1 ng + GH 1 ng + IL-1b 1 ng + TNF-a 1 ng) MRHSMEGF MR (88%) HSA (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 5 ng + IGF 1 ng + bFGF 1 ng + GH 1 ng + IL-1b 1 ng + TNF-a 1 ng) MRHSMIGF MR (88%) HSA (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 1 ng + IGF 5 ng + bFGF 1 ng + GH 1 ng + IL-1b 1 ng + TNF-a 1 ng) MRHSbFGF MR (88%) HSA (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50ug + EGF 1ng + IGF 1ng + bFGF 5ng + GH 1ng + IL-1b 1ng + TNF-a 1ng) MRHSMGH MR (88%) HSA (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 1 ng + IGF 1 ng + bFGF 1 ng + GH 5 ng + IL-1b 1 ng + TNF-a 1 ng) MRHSM
-IL,TN-a MR (88%) HSA (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50ug + EGF 1ng + IGF 1ng + bFGF 1ng + GH 1ng + IL-1b 5ng + TNF-a 1ng) MRHSM
-IL, TNF-a MR (88%) HSA (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 1 ng + IGF 1 ng + bFGF 1 ng + GH 1 ng + IL-1b 1 ng + TNF-a 5 ng)

A comparative experiment between FBS and HSA was conducted, and the effect of HSA was observed on the development of stem cells by fixing the concentration and changing the concentration of growth factors and cytokines, and the results are shown in FIG. 3 .

As a result of adding HSA as a preliminary test for SP, the group with the addition showed a higher development rate than the group without it.

High development was confirmed in the group with 5 ng/ml of EGF, FGF, and GH added with the growth factor, and in particular, the group with the addition of the growth factor bFGF showed rather good results.

In the case of SM, the performance of FBS was not achieved, and the doses of growth factors and cytokines were adjusted, and the following experiment was performed.

It was confirmed that cell development was accelerated when growth factors were added to the MR culture medium. However, we have seen that the synergistic effect of good ingredients increases cell development when several are added rather than one. Accordingly, the following experiment was performed by adding several concentrations.

6. Experiment with cell development according to the concentration of SP, SM, growth factors and cytokines in MR culture medium.

As shown in Table 9 below, SP, SM, growth factors and cytokines were added to the MR culture medium to prepare a medium.

division Basic medium (V:V) Additives (V:V) MRF MR (90%) FBS (10%) MRSSMG5 MR (88%) SP(10%) + SM(ITS 1% + Glutamax 1% + Galatin 50ug + EGF 1ng + IGF 1ng + bFGF 1ng + GH 5ng ) MRSSMG10 MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 5 ng + IGF 5 ng + bFGF 5 ng + GH 10 ng ) MRHSMG20 MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 20 ng + IGF 20 ng + bFGF 20 ng + GH 20 ng ) MRHSMG50 MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 50 ng + IGF 50 ng + bFGF 50 ng + GH 50 ng ) MRHSMG5
-IL, TNF-a MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 5 ng + IGF 5 ng + bFGF 5 ng + GH 5 ng + IL-1b 5 ng + TNF-a 5 ng) MRHSMG20
-IL, TNF-a MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 20 ng + IGF 20 ng + bFGF 20 ng + GH 20 ng + IL-1b 10 ng + TNF-a 10 ng) MRHSMG50
-IL, TNF-a MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Galatin 50 ug + EGF 50 ng + IGF 50 ng + bFGF 50 ng + GH 50 ng + IL-1b 20 ng + TNF-a 20 ng)

In the preliminary experiment, it was confirmed that stem cell growth was different according to the concentration of various growth hormones, and the concentration was increased to prove the relationship with the concentration of growth factors.

Thus, by adjusting the concentration of growth hormone to 5, 10, 20, and 50, growth factor to 1, 5. 20 and 50, and cytokine to 5, 10, and 20, an experiment at which concentration promotes cell development was performed and the results are shown in FIG. 4 .

As a result of the experiment, as a preliminary experiment for the development of SP materials, HSA was used as a preliminary experiment to combine growth factors and cytokines. As a result, there was no significant difference in the SP-added group, but it was confirmed that stem cell development was promoted by about 5%. In particular, the best results were obtained for growth factor and cytokine 50.

The next experiment was conducted on whether the best concentration and several additional substances lead to the development of stem cells.

7. Cell division experiment by increasing the concentration of SP, SM, growth factors and cytokines in MR culture medium

As shown in Table 10 below, SP, SM, growth factors and cytokines were added to the MR culture to prepare a medium.

division Basic medium (V:V) Additives (V:V) MRF MR (90%) FBS (10%) MRSSMG50 MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Gelatin 50 ug + EGF 100 ng + IGF 100 ng + bFGF 100 ng + GH 50 ng + Hyaluronate 200 ug + A + C + M) MRSSMG100 MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Gelatin 50 ug + EGF 100 ng + IGF 100 ng + bFGF 100 ng + GH 100 ng + Hyaluronate 200 ug + A + C + M) MRSSMG200 MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Gelatin 50 ug + EGF 200 ng + IGF 200 ng + bFGF 200 ng + GH 200 ng + Hyaluronate 200 ug + A + C + M) MRSSMG100
-IL, TNF-a MR (88%) SP(10%) + SM(ITS 1% + Glutamax 1% + Gelatin 50ug + EGF 100ng + bFGF 100ng + GH 100ng + Hyaluronate 200ug + IL-1b 2ng + TNF-a 1ng + A + C + M) MRSSMG200
-IL, TNF-a MR (88%) SP (10%) + SM (ITS 1% + Glutamax 1% + Gelatin 50 ug + EGF 200 ng + bFGF 200 ng + GH 200 ng + Hyaluronate 200 ug + IL-1b 2 ng + TNF-a 1 ng + A + C + M)

* A: Ascorbic acid 25 μg/mL, C: Cholesterol 30 μg/mL, M: Beta-mercaptoethanol In the preliminary experiment, stem cell growth was confirmed to be different depending on the concentration of various growth hormones, and the concentration was increased. The purpose of this study was to prove the relationship between growth factor concentrations. In addition, the best results were obtained at 25 μg/mL (A) for ascorbic acid, 30 μg/mL for cholesterol (C), and 5 μg/mL (M) for beta-mercaptoethanol. Therefore, these concentrations were added to each group to see the results. In particular, from 50 μg/mL or more of mercaptoethanol, it had a rather adverse effect, and cell death of adipose-derived stem cells was observed.

As such, the experimental results of the cell development rate according to the additive materials FBS, SP, SM, ascorbic acid, beta-mercaptoethanol and cholesterol in the MR culture are shown in FIG. 5 .

As a result of the experiment, growth factors EGF, bFGF, and GH were added at 100 ng/ml, ascorbic acid at 25 μg/ml, cholesterol at 30 μg/ml, and beta-mercaptoethanol at 5 μg/ml in the group added with FBS. It was confirmed that the stem cell development rate was slightly improved.

Hyaluronic acid was found to grow best at a concentration of 200 ug/ml.

When comparing the group with and without the cytokines IL-1b and TNF-a, it was confirmed that the development rate of the group was lowered, which had an adverse effect on cell growth.

When analyzed comprehensively, it was found that the good components of the added substances work together to exert a synergistic effect, rather than increase the splitting rate by adding a certain substance.

Preferred embodiments obtained as a result of this experiment are exemplified in Table 11 below.

division matter substance concentration Basic medium (MR-IVF) EDTA
Taurine 372.2 mM
125.10 mM adhesion factor Gelatin 50 ug/ml growth factor EGF 100 ng/ml bFGF 100 ng/ml hormone growth hormone 50 ng/ml Choesterol 30 ug/ml Insulin 1000 ng/ml trace elements Transferrin 550 ng/ml selenium 500 pg/ml β-mercaptoethanol 5 ug/ml Proteins, vitamins and other additives Hyaluronate 200 ug/ml SP 5-20% HSA 2.5-10% FF 5-20% L-alanyl-L-glutamine 434.44 ug/ml Ascorbic acid 25 ug/ml

It is an object of the present invention to develop a culture solution composition capable of culturing adipose-derived stem cells without using animal-derived serum.

The serum-free medium composition of the present invention containing a cell activator is not only safe because it does not use animal-derived serum, but also has the effect of exhibiting a relatively excellent cell differentiation rate and maintaining stem cell characteristics because it uses a chemically purified material. .

Therefore, it contains very good ingredients required by cells, and blocks genetic mutations caused by protein denaturation in advance to eliminate unsafe factors that can come from culturing fetal bovine serum when used for human medical purposes. When made, side effects such as skin rash, erythema, and skin psoriasis can be avoided.

Claims (11)

(i) a basal medium containing taurine and a chelating agent, and (ii) a complex protein of human serum albumin (HSA) and globulin, ascorbic acid or a salt thereof, hyaluronic acid or a salt thereof, cholesterol and mercaptoethanol as additives. Characterized in that, a medium composition for culturing stem cells,
wherein the globulin is at least one selected from the group consisting of α1-globulin, α2-globulin, β-globulin and γ-globulin. The medium composition for culturing stem cells according to claim 1, wherein the composition does not contain animal-derived serum. The medium composition for stem cell culture according to claim 1, wherein the complex protein is composed of 80±1% by volume of HSA and 20±1% by volume of globulin. According to claim 1, wherein the chelating agent is characterized in that EDTA, stem cell culture medium composition. The method of claim 1, wherein the composition further comprises one or more growth factors selected from the group consisting of Epidermal Growth Factor (EGF), Insulin-like Growth Factor (IGF), and Fibroblast Growth Factor (FGF). A medium composition for stem cell culture. According to claim 1, wherein the composition is characterized in that it further comprises selenium (selenium), stem cell culture medium composition. According to claim 1, wherein the composition further comprises one or more additives selected from the group consisting of GH (Growth Hormone) and insulin, the stem cell culture medium composition. The medium composition for culturing stem cells according to claim 1, wherein the composition further comprises one or more adhesion factors selected from fibronectin and gelatin. The medium composition for culturing stem cells according to claim 1, wherein the composition does not contain the cytokines IL-1b and TNF-α. The method according to claim 1, wherein the amount of the ascorbic acid or its salt added is 1 to 100 μg/ml, the added amount of the hyaluronic acid or its salt is from 10 to 500 μg/ml, and the amount of the mercaptoethanol is 1 to less than 50 μg/ ml, characterized in that the medium composition for stem cell culture. A method of culturing stem cells without using animal-derived serum, characterized in that the medium composition for stem cell culture according to any one of claims 1 to 10 is used.

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