WO2018226051A2 - Serum-free medium composition for culturing cells including exosome derived from human stem cell - Google Patents

Abstract

The present invention provides a novel serum-free medium composition for culturing cells, wherein an exosome that induces cell adhesion and proliferation is separated from a human stem cell, and the exosome is contained in a basic medium. A serum-free medium composition for culturing cells according to the present invention contains genes, proteins, and growth factors related to cell growth, and thus can assist cell adhesion and growth even without other additives such as cell activators or growth factors, and can induce and promote cell proliferation. The present invention can minimize the disadvantages of serum by using a medium in which an exosome is added instead of animal serum, or by minimizing the amount of serum used, and can ultimately be used in the culturing of stem cells for cell therapy for clinical application.

Description

Serum-free medium composition for cell culture comprising human stem cell-derived exosomes

This application claims priority based on Korean Patent Application No. 10-2017-0070592 filed on June 7, 2017, and all the contents disclosed in the specification and drawings of the application are incorporated in this application.

The present invention relates to a serum-free medium composition for culturing cells containing exosomes derived from human stem cells as an active ingredient, more specifically, containing exosomes derived from human stem cells, it is possible to minimize cell damage And a new serum-free medium composition for cell culture that can aid in cell adhesion and growth and induce and promote cell proliferation.

The present invention also relates to an animal serum replacement agent comprising an exosome derived from human stem cells, a serum-free medium composition for cell culture containing the same, and a method of culturing cells using the same.

In addition, the present invention can overcome the shortcomings of the serum by using a medium in which exosomes are added instead of animal serum, and can be applied to a serum-free medium composition for stem cell culture, thereby culturing stem cells without animal serum. And finally to a method for culturing stem cells for cell therapy for clinical applications.

Serum contains a variety of hormones in the insulin or polypeptide family that promote cell growth and function, growth factors that regulate cell division and function, and adhesion and diffusion factors such as fibronectin, transferrin as a binding protein, lipids and minerals. Many trace elements are included. Serum is also a buffer that regulates the osmotic pressure and pH of the culture, and has a viscous action that protects cells from proteolytic inhibition and physical shock. Cells can be cultured using serum containing these various functions.

Stem cells have the property of continuously producing the same cells as themselves for a certain period of time in an undifferentiated state, and differentiate into specific cells under suitable conditions. Adult stem cells can be obtained from bone marrow, blood, brain, skin, and fat, and are known to be able to differentiate into various tissue cells. In particular, adult stem cells such as adipose derived stem cells, bone marrow derived stem cells, umbilical cord or umbilical-cord blood derived stem cells Research into the development of autologous or other cell therapy is also actively underway.

However, in the case of using a medium containing animal serum to culture such human stem cells, even if washed with a solution without animal serum before clinical application, a significant amount of animal serum remains in the cell acting as a strong heterologous antigen, animal pathogenicity Or other factors are very likely. In addition, animal serum may have a change in composition depending on the time and place of collection, it is difficult to administer to the patient stem cells cultured in a serum-containing medium.

To overcome this, various stem cell cultures have been developed. Conventional cell culture media have attempted to use human serum instead of animal serum, but human serum is not used for stem cell culture for clinical applications because its source is limited and the risk of viruses and other infections cannot be excluded. Only very limited research is used for specific purposes. Recently, various methods for culturing stem cells in serum-free medium have been tried. Among them, the serum halving method is a method of gradually lowering the concentration of serum contained in the medium and finally culturing the cells in serum-free. However, this method has difficulty in proliferating cells, is likely to progress to apoptosis, and takes a long time, thus preparing a stem cell for administration to a patient.

Currently, the method of culturing stem cells by adding hormones, growth factors or purified proteins instead of serum is most commonly used. Although it is added to the basic medium by combining components necessary for adhesion and growth of stem cells such as amino acids, vitamins, insulin-like factors, epidermal growth factor, fibroblast growth factor, protease inhibitors, proteins and selenium, And growth factors are sold at a very high price, it is difficult to use a combination of various components.

On the other hand, it is known that various cells existing in multicellular organisms, including humans, secrete nanosized vesicles called exosomes. Exosomes are vesicles with the same membrane structure as cell membranes and are known to deliver membrane components, proteins, RNA, and the like to other cells and tissues. In particular, exosomes secreted from stem cells contain various growth factors and cytokines secreted by stem cells, and are known to regulate cell adhesion, growth, and differentiation. In addition, since exosomes are removed impurities such as cell waste, antibiotics, serum in the cell culture in the separation process, it can be used safely and equivalent to the effect of the cell culture.

Stem cell exosomes contain complex components such as protein, insulin-like growth factor, epithelial cell growth factor, fibroblast growth factor, vascular epithelial cell growth factor, various fatty acids and antioxidants to help stem cell adhesion and growth.

Therefore, the present invention has developed a serum-free culture medium containing stem cell-derived exosomes that can minimize cell damage of stem cells and help adhesion and growth.

An object of the present invention is to isolate the exosomes that induce cell attachment and proliferation from human stem cells, and to provide a new serum-free medium composition for culturing cells containing exosomes in the basal medium. It is an object of the present invention to provide a serum-free medium composition for stem cell culture comprising extracting an exosome having the efficacy of stem cell proliferation and culture from human adipose stem cells and a mixture thereof.

Specifically, an object of the present invention contains exosomes derived from human stem cells, which can minimize cell damage, aid in cell adhesion and growth, and induce and promote cell proliferation. Serum medium composition is provided.

It is also an object of the present invention to provide an animal serum replacement agent comprising an exosome derived from human stem cells, a serum-free medium composition for cell culture containing the same, and a method of culturing cells using the same.

In addition, an object of the present invention can overcome the shortcomings of serum by using a medium in which exosomes are added instead of animal serum, and can be applied to serum-free medium composition for stem cell culture to culture stem cells without animal serum. And finally, to provide a method for culturing stem cells for cell therapy for clinical applications.

However, the problems of the present invention as described above are exemplary, and the scope of the present invention is not limited thereby.

In order to achieve the above object, the present invention is to isolate the exosomes to induce cell adhesion and proliferation from human stem cells, animal serum replacement agent comprising the isolated exosomes, cell culture containing exosomes in the basal medium Serum-free medium composition, and a method for culturing cells using the same.

As used herein, the term “exosome” refers to a membrane vesicle that is secreted from various cell types, and serves various roles such as delivering membrane components, proteins, and RNA to other cells and tissues. It is known.

In one embodiment of the present invention, the stem cells derived from exosomes may be adult stem cells, more specifically, may be selected from the group consisting of adipose derived stem cells, bone marrow stem cells and umbilical cord blood stem cells, Preferably, it may be selected from the group consisting of human adipose derived stem cells, human bone marrow stem cells, and human umbilical cord blood stem cells, and more preferably human adipose derived stem cells, but is not limited thereto.

The animal serum replacement agent and / or serum-free medium composition for cell culture of one embodiment of the present invention is preferably used for culturing animal cells, more preferably for culturing stem cells.

The serum-free medium composition for cell culture of one embodiment of the present invention is characterized in that it contains exosomes derived from human stem cells, preferably human fat-derived stem cells, as an active ingredient, and does not include animal serum. Exosomes derived from human stem cells, preferably human adipose-derived stem cells are used by mixing in a basic medium at a constant rate to maximize the proliferation effect of animal cells, preferably stem cells.

In one embodiment of the present invention, human stem cell-derived exosomes isolated from serum-free, antibiotic-free medium contain growth factors effective for promoting growth and maintenance of various proliferation-related cells, such as cell activators or growth factors. It can be effectively applied as a serum-free medium composition for culturing animal cells, preferably stem cells, without additives. Therefore, the animal serum replacement agent and / or serum-free medium composition for cell culture of one embodiment of the present invention can culture animal cells, preferably stem cells, without animal serum.

As used herein, the term 'basal medium' refers to a culture medium containing essential ingredients necessary for growth and proliferation of cells in vitro. The basal medium may be artificially synthesized or commercially prepared medium.

Commercially prepared media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), RPMI 1640, F-10, F-12, α-Mineral Essential Medium: Gibco, Invitrogen, Newyork , Glasgow's Mineral Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium (IMDM).

Specifically, the exosomes may be included in the medium at a concentration of 0.01 to 1,000 μg / mL.

On the other hand, the exosomes can be prepared using a conventional exosome separation method, for example

1) culturing the stem cells in the culture medium and then passage in serum-free and antibiotic-free medium;

2) recovering the cell culture supernatant;

3) centrifuging the recovered cell culture supernatant; And

4) may be prepared by the step of separating and purifying the exosomes, but is not limited thereto.

The exosomes are not only loaded with genetic information, proteins, and growth factors of stem cells, but the exosomes themselves may serve as carriers. Exosomes consisting of lipids of about 50-150 nm in size are biocompatible because they are cell-derived materials, and the uptake of cells is also very good.

Problems of animal serum because human stem cell-derived exosomes contained in the animal serum replacement agent and / or serum-free medium composition for cell culture of one embodiment of the present invention are purified components that do not contain antibiotics, serum, or harmful factors of the culture solution. It is possible to overcome this, and because it is a cell-derived lipid transporter, the cell penetration and effective factor delivery efficiency is very excellent. A serum-free medium composition for cell culture containing such exosomes as an active ingredient promotes the attachment and proliferation of animal cells, preferably stem cells.

Therefore, the serum-free medium composition for cell culture of one embodiment of the present invention can be used as a composition for stem cell culture for clinical application.

Another aspect of the invention is the step of separating the exosomes from human stem cells; Preparing a serum-free medium composition for cell culture comprising the isolated exosomes; And it provides a cell culture method comprising culturing the cells in the serum-free medium composition for cell culture.

In the cell culture method of an embodiment of the present invention, the human stem cells may be selected from the group consisting of human adipose derived stem cells, human bone marrow stem cells and human umbilical cord blood stem cells, more preferably human adipose derived stem cells It may be, but is not limited thereto.

In the cell culture method of one embodiment of the present invention, the serum-free medium composition for cell culture is preferably used for the culturing of animal cells, more preferably for the culturing of stem cells derived from exosomes.

In the cell culture method of an embodiment of the present invention, the cell culture-free serum medium composition is characterized in that it does not contain animal serum.

In addition, in one embodiment of the present invention, the cell culture-free serum medium composition is characterized in that it contains substantially no animal serum.

The term “substantially free of” may be completely devoid of the component, or contain little of the component to the extent that the effect may be the same as if the component was completely lacking. In other words, a composition that is "substantially free" of components or elements may still actually contain these items unless there is a measurable effect thereof. The term “substantially free of” refers to up to 5 wt%, up to 4 wt%, up to 3 wt% or up to 2 wt%, preferably up to 1 wt%, more preferably 0.1, based on the total content of the composition unless otherwise indicated. It may mean up to weight percent, even more preferably up to 0.01 weight percent.

Animal serum substitutes comprising exosomes derived from human stem cells of the present invention, and serum-free medium composition for cell culture containing the same contains genes, proteins, growth factors, etc., which are related to cell growth, and thus cell activators or growth factors. Without other additives, it can help cells attach and grow and induce cell proliferation. Human stem cell-derived exosomes used in the present invention can overcome the problems of animal serum because it is a purified component that does not contain antibiotics, serum, or harmful eyeballs of the culture medium, cell penetration and effective factors because of cell-derived lipid transporter The transmission efficiency is very good.

Therefore, the present invention can overcome the shortcomings of the serum by using a medium in which exosomes are added instead of animal serum, and can be applied to a serum-free medium composition for stem cell culture, thereby culturing stem cells without animal serum. Finally, it can be used for culturing stem cells for cell therapy for clinical applications.

On the other hand, the scope of the present invention is not limited by the effects as described above.

Figure 1 (Fig. 1a to 1c) shows the results for the characterization of exosomes produced from human adipose derived stem cells,

Figure 1a is the concentration of stem cell exosomes confirmed using a nanoparticle tracking analysis (Nanoparticle Tracking Analysis, NTA),

Figure 1b is the form of stem cell exosomes confirmed using a transmission electron microscope (Transparent Electron Microscope, TEM), and

1c is a diagram of surface marker analysis of exosomes using flow cytometry (n = 3). CD9, CD63, and CD81, which are specific markers of exosomes, were found to express 94.67%, 98.22%, and 89.64%, respectively (n = 5). The scale bar of the image is 100 nm (black) and 20 nm (white), respectively.

Figure 2 shows the results of the cell proliferation evaluation of human adipose derived stem cells according to the exosome concentration. Cell proliferation was evaluated by incubating human adipose derived stem cells (ASC, 1 × 10 ⁴ cell / well) for 3 days in a 48-well plate, and the degree of cell proliferation was determined by CCK-8 reagent (Dojindo, cat no. DJB4000X) was used for absorbance analysis. Exo-proliferation efficiency than some containing the cultured cells in serum-free medium resulting positive control (growth medium) is natatjiman, it was confirmed that the growth rate is improved as compared to the negative control (serum-free medium, SFM) in all experimental groups (P <0.001). In particular, stem cells cultured in a medium containing 50 μg / mL of exosomes were confirmed to improve cell growth efficiency more than two times compared to the negative control group.

Figure 3 shows the results of evaluating the cell proliferation induction of exosomes in culture medium containing a low concentration of serum (Serum 1%, 3%, 5%). Culture medium was prepared by adding 50 μg / mL of exosomes to all culture conditions except for positive control (Serum 10%). Human adipose derived stem cells (ASC, 1 × 10 ⁴ cell / well) were evaluated by incubating for 3 days in 48-well plates (n = 5), respectively. (a) is a graph showing the cell proliferation rate in each culture medium of different composition, (b) is a table quantifying the values of the (a) graph. In all experimental groups except the positive control group, it was confirmed that the stem cells cultured in the exosome-treated culture medium improved the proliferation rate by 1.5 times or more ( P <0.001).

Figure 4 shows the results of the differentiation effect of stem cells cultured in a serum-free medium containing exosomes to adipocytes. Evaluation of differentiation efficacy of stem cells was carried out in the growth of human adipose derived stem cells (ASC, 1 × 10 ⁴ cells / well) in 24-well plates (DMEM + FBS 10% + P / S 1%) and exo Cell density was increased by culturing in serum-free medium (DMEM + 50 μg / mL exosomes) for 3 days to increase cell density, and then changed into an adipocyte differentiation-inducing medium for differentiation oil for 2 weeks (n = 3). (a) is an optical micrograph of human adipose derived stem cells cultured in a normal culture medium and a serum-free medium containing exosomes, (b) is a fat cell induced differentiation induced through differentiation induction medium in (a) It is an optical microscope photograph evaluated for differentiation ability by red O staining. Scale bars are 200 μm (white) and 50 μm (black) respectively. Evaluation of differentiation ability into adipocytes confirmed that the differentiation function of stem cells cultured in serum-free medium containing exosomes was well maintained.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples merely illustrate the contents of the present invention and do not limit or limit the scope of the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can be easily inferred to be within the scope of the present invention. References cited in the present invention are incorporated herein by reference.

Example 1 Isosome Isolation from Human Adipose Stem Cells

Human adipose stem cell-derived exosomes were extracted during the process of culturing human adipose stem cells.

Specifically, human adipose stem cells (Company: CellBio, Cat #: CB-ADMSC-001) are DMEM (Dulbecco) containing 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin (penicillin / streptomycin). Modified Eagle Medium high glucose; Gibco, Cat #: 11995065). 24 hours before extracting the exosomes, the cells were replaced with serum-free, antibiotic-free, phenol-free medium (Company: Gibco, Cat #: 31053028) and cultured for 24 hours, and then cell culture supernatants were recovered. After the supernatant was recovered, normal culture medium was added to the stem cells and cultured. This process was repeated until passage 7.

The recovered cell culture supernatant was centrifuged at 2,000 × g, 4 ° C. for 5 minutes, primary filtering step using a cell strainer (pore size; 40 μm), and a bottle top filter (pore size). ; 0.22 μm) to remove cell debris and wastes by a second filtering step. After the first purification step, tangential flow filtration (TFF) was used for exosome enrichment and further purification. At this time, the filter specification used was MWCO 300 kDa (Molecular Weight Cut-Off).

The exosomes extracted in the process of proliferation were used as the stem cell culture additive composition.

Example 2 Characterization of Human Adipose-Derived Stem Cell Exosomes

Exosomes extracted from human adipose derived stem cells of Example 1 were exosomes using a nanoparticle tracking analysis (NTA), a transmission electron microscope (TEM), and a flow cytometry. The concentration and purity of, size, morphology and CD surface markers were confirmed (Figure 1).

In order to evaluate the concentration of exosomes, exosomes are measured by performing sample dilution (dilution solution; 1X phosphate buffer saline (PBS) solution) so that the number of nanoparticles identified on the screen per frame can be within the range of 30 to 40 (FIG. 1A). The extracted exosomes concentration was 1.0 × 10 ¹⁰ particles / mL on average, it was confirmed that the average size is about 164 nm (n = 3).

To confirm the shape of the extracted exosomes by transmission electron microscopy, the exosome sample was treated with 1% phosphotungstic acid solution for 1 minute and subjected to negative staining. As a result, spherical nanoparticle morphology was observed, and the average particle size was about 100 nm (FIG. 1B).

In addition, the expression level of CD9, CD63, and CD81, known as exosome membrane surface markers, were confirmed by flow rate analyzer. After attaching the reagent (SBI, CD9 / 63 / 81-EXO FLOW Capture kit) and the fluorescent marker to which the surface marker specific antibody is bound to the magnetic bead on the surface of the exosome, the expression of the surface marker of the exosome was measured using a flow rate analyzer. It was confirmed (FIG. 1C).

Example 3 Evaluation of Stem Cell Proliferation Induction in Serum-Free Medium Containing Exosomes

Serum-free culture medium containing exosomes was prepared by mixing as shown in Table 1 below.

 Table 1. Preparation of exosome serum-free medium composition

group	GM	SFM		EXO 10	EXO 20	EXO 30	EXO 40	EXO 50
Badge (DMEM)	10 mL
Fetal Bovine Serum (FBS)	10%	-	-	-	-	-	×
Antibiotics1%	100 μL
Exosomes	-	-	10 μg / mL	20 μg / mL	30 μg / mL	40 μg / mL	50 μg / mL

^* GM: Growth medium, positive control ^* SFM: Serum free medium, negative control

^* EXO 10 ~ 50: Serum free medium with exosomes, sample

* Antibiotics: Penicillin / streptomycin

Evaluation of proliferation induction of stem cells was evaluated after incubation of human adipose derived stem cells (ASC, 1 × 10 ⁴ cells / well) in a 48-well plate for 3 days. Cell proliferation was measured by using the CCK-8 kit (manufactured by Dojindo, Cat #: DJB4000X) (n = 5). The CCK-8 reagent reacts with live intracellular dehydrogenase to change the pigment of the reagent, which allows quantitative assessment of cell proliferation rate. Stem cell proliferation medium (GM group; DMEM, fetal bovine serum 10%, penicillin / streptomycin 1%) for positive control group and serum-free medium (SFM group; DMEM, penicillin / streptomycin 1%) for negative control group It was.

As a result of culturing stem cells in a serum-free medium containing exosomes, it was confirmed through a significant evaluation that proliferation was induced in all experimental groups as compared to the negative control group ( P <0.001), especially 50 ㎍ / mL exosomes It was confirmed that the culture medium is well induced more than two times compared to the negative control group (Fig. 2).

< Example  4> In cultures containing different serum concentrations Exosomes  Evaluation of Stem Cell Proliferation by Addition

Increasing the induction of cell proliferation of human adipose derived stem cells by adding 50 μg / mL of the exosomes isolated in Example 1 to a culture medium containing low serum, specifically 1%, 3%, and 5% serum Evaluation was performed (Table 2, FIG. 3). The evaluation was performed by culturing human adipose derived stem cells (ASC, 1 × 10 ⁴ cells / well) for 3 days in a 48-well plate, and the positive control group was a stem cell proliferation medium (GM group; DMEM, right) Fetal serum 10%, penicillin / streptomycin 1%) and serum-free medium (SFM group; DMEM, penicillin / streptomycin 1%) were used as negative controls (n = 5).

Table 2. Preparation of Medium Composition by Serum Concentration

group

Serum 1%

Serum 3%

Serum 5%

Serum 10%

Badge (DMEM)

10 mL

Fetal Bovine Serum (FBS)

One%

One%

3%

3%

5%

5%

10%

Antibiotics1%

100 μL

Exosome (50 μg / mL)

-

+

-

+

-

+

-

^* SFM: Serum free medium As a result, it was confirmed that in all experimental groups except the positive control group (serum 10%), the growth rate of the stem cells cultured in the exosome-treated culture conditions improved on average by 1.5 times or more ( P <0.001).

< Example  5> Exosomes  Contained Serum free  Stem Cells Cultured in Medium Eruption  evaluation

To confirm the functionality of stem cells cultured in serum-free medium containing exosomes, differentiation efficacy evaluation was performed.

Specifically, in order to evaluate the differentiation efficacy of stem cells, human adipose derived stem cells (ASC, 1 × 10 ⁴ cell / well) are radish containing proliferation medium (DMEM + FBS 10% + P / S 1%) and exosomes. Incubate in serum medium (DMEM + 50 μg / mL exosomes) for 3 days to increase cell density, and then to induce adipocyte differentiation medium (DMEM (Dulbecco Modified Eagle Medium high glucose; Gibco, Cat #: 11995065) , 5% FBS (fetal bovine serum), 0.5 mM IBMX (Isobutyl-1-methylxanthine; Sigma, Cat # I5879-1G), 10 μg / mL Insulin (Sigma, Cat # I16634-100), 1 μM Dexamethasone (Abcam, Cat # ab12074) and 100 μM Indomethacin (Sigma, Cat # I7378-5) were used for differentiation induction for 2 weeks, and oil red O staining was used to evaluate differentiation into adipocytes. Human adipose derived stem cells cultured in the serum-free medium containing it was confirmed that the oil-droplet was formed specifically observed in fat cells (Fig. 4). ).

Therefore, the exosomes produced from human adipose derived stem cells were found to be effective ingredients that help cell proliferation while maintaining the differentiation ability of human adipose derived stem cells.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. Those skilled in the art can make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

Claims (17)

1. A serum-free medium composition for cell culture containing human stem cell-derived exosomes as an active ingredient.
2. The method of claim 1,

   The human stem cell is serum-free medium composition for cell culture, characterized in that at least one selected from the group consisting of human bone marrow-derived stem cells and human adipose derived stem cells.
3. The method of claim 2,

   Serum-free medium composition for cell culture, characterized in that it contains exosomes derived from human adipose derived stem cells as an active ingredient and does not contain animal serum.
4. The method according to any one of claims 1 to 3,

   Serum-free medium composition for cell culture, characterized in that used for the culture of stem cells.
5. The method according to any one of claims 1 to 3,

   The exosomes are serum-free medium composition for cell culture contained in a concentration of 0.01 to 1,000 μg / mL in the basal medium.
6. The method of claim 5,

   The basal medium includes Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), RPMI 1640, F-10, F-12, a-Mineral Essential Medium (G-MEM), and Glasgow's Mineral Essential Medium (G-MEM). And, serum-free medium composition for cell culture, characterized in that at least one selected from the group consisting of IMDM (Iscove's Modified Dulbecco's Medium).
7. Animal serum substitutes, including human stem cell-derived exosomes.
8. The method of claim 7, wherein

   The human stem cells are animal bone substitutes, characterized in that at least one selected from the group consisting of human bone marrow-derived stem cells and human adipose derived stem cells.
9. The method according to claim 7 or 8,

   Animal serum substitute, characterized in that used in the culture of stem cells.
10. Separating exosomes from human stem cells;

    Preparing a serum-free medium composition for cell culture comprising the isolated exosomes; And

    Cell culture method comprising the step of culturing the cells in the serum-free medium composition for cell culture.
11. The method of claim 10,

    The human stem cell is a cell culture method, characterized in that at least one selected from the group consisting of human bone marrow-derived stem cells and human adipose derived stem cells.
12. The method of claim 10,

    The cell culture-free serum medium composition contains an exosome derived from human adipose derived stem cells as an active ingredient and does not contain animal serum.
13. The method according to any one of claims 10 to 12,

    Cell culture method, characterized in that used for the culture of stem cells.
14. The method according to any one of claims 10 to 12,

    The cell culture-free serum medium composition is a cell culture method containing the separated exosomes in a concentration of 0.01 to 1,000 μg / mL in the basal medium.
15. The method of claim 14,

    The basal medium is Dulbecco's Modified eagle's Medium (DMEM), Minimal Essential Medium (MEM), RPMI 1640, F-10, F-12, a-Mineral Essential Medium (G-MEM), and Glasgow's Mineral Essential Medium (G-MEM). And, and cell culture method characterized in that at least one selected from the group consisting of IMDM (Iscove's Modified Dulbecco's Medium).
16. The method of claim 1,

    The composition contains a exosome derived from human adipose derived stem cells as an active ingredient, serum-free medium composition for cell culture, characterized in that substantially no animal serum.
17. The method of claim 16,

    The serum-free medium composition for cell culture, characterized in that the composition substantially free of animal serum comprises animal serum in a concentration of 0 to 5% by weight in the total composition.

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