KR20220104321A - Method for enhancing generation or increasing productivity of exosomes and/or extracellular vesicles - Google Patents

Abstract

The present invention provides a method for promoting the production of exosomes and/or extracellular vesicles, or a method for improving productivity thereof, wherein the method comprises the step for culturing animal-derived cells in a medium containing at least one selected from the group consisting of insulin, transferrin, and selenious acid or a salt thereof. The method for promoting the production of exosomes and/or extracellular vesicles, or the method for improving productivity thereof of the present invention can economically and efficiently produce, at high yield, exosomes and/or extracellular vesicles that can be effectively used commercially and/or clinically and, in particular, has the advantage of being able to produce exosomes and/or extracellular vesicles in larger quantities when compared with conventional methods.

Description

Exosomes and/or extracellular vesicles promoting production or productivity enhancement method {Method for enhancing generation or increasing productivity of exosomes and/or extracellular vesicles}

The present invention relates to a method for promoting production of exosomes and/or extracellular vesicles or improving productivity.

In addition, the present invention relates to a medium for promoting production of exosomes and/or extracellular vesicles or improving productivity.

Recently, it has been reported that cell secretome contains various bioactive factors that regulate cell behavior. In particular, cell secretion contains 'exosome' or Because it contains 'extracellular vesicle', research on its components and functions is being actively conducted.

Cells release various membrane types of ERs to the extracellular environment, and these ERs are commonly referred to as extracellular vesicles (EVs). The extracellular vesicles are also called cell membrane-derived ERs, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, they are used separately from exosomes.

Exosomes are endoplasmic reticulum with a size of several tens to hundreds of nanometers made of a double phospholipid membrane identical to the structure of the cell membrane, and the inside contains proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known Exosomes contain specific genetic material and bioactive factors according to the nature and state of the cell from which they are derived. In the case of proliferating stem cell-derived exosomes, it regulates cell behaviors such as cell migration, proliferation and differentiation, and reflects the characteristics of stem cells related to tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

That is, exosomes called avatars of cells contain bioactive factors such as growth factors similar to cells, and serve as a carrier for transporting bioactive factors between cells, that is, communication between cells. Exosomes are known not only to be released from animal cells such as stem cells, immune cells, fibroblasts and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae. .

Conventional techniques for separating such exosomes and/or extracellular vesicles include ultracentrifugation, density gradient centrifugation, ultrafiltration, and tangential flow filtration (TFF). ), size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total There is an exosome extraction kit (total exosome isolation kit), or a polymer based precipitation method.

However, as described above, although various methods for separating exosomes and/or extracellular vesicles have been proposed, continuous improvement can be made in the technical field related to the production of exosomes and/or extracellular vesicles as in other technical fields. there will be For example, it is necessary to provide a new exosome and/or extracellular vesicle production technology capable of improving the productivity of exosomes and/or extracellular vesicles secreted or released per cell (or cell culture medium).

On the other hand, it should be understood that the matters described as the above background art are only for improving understanding of the background of the present invention, and are not cited as approval that they can be used as "prior art" of the present invention.

Republic of Korea Patent Publication No. 10-1948237 (2019.02.14)

It is an object of the present invention to provide a method for promoting or improving productivity of exosomes and/or extracellular vesicles.

Another object of the present invention is to provide a medium for promoting the production of exosomes and/or extracellular vesicles or for improving productivity.

However, the problems of the present invention as described above are exemplary, and the scope of the present invention is not limited thereto. Further, other objects and advantages of the present invention will become more apparent by the following detailed description of the invention, claims and drawings.

The present inventors have been intensively researching on a method for producing exosomes and/or extracellular vesicles that can improve the productivity of exosomes and/or extracellular vesicles, insulin, transferrin, and acetylenic acid or salts thereof When the cells are cultured in a medium containing at least one selected from the group consisting of, it was confirmed that the productivity of exosomes and/or extracellular vesicles secreted or released per cell (or cell culture medium) is improved, thereby completing the present invention.

The present invention relates to a method for promoting the production of exosomes and/or extracellular vesicles, comprising culturing an animal-derived cell in a medium containing at least one selected from the group consisting of insulin, transferrin, and acetylenic acid or a salt thereof. Or it provides a productivity improvement method.

As used herein, the term "extracellular vesicles (EVs)" generally encompasses membrane vesicles, ectosomes, shedding vesicles, microparticles, or equivalents thereof. used in the sense that Depending on the isolation environment, conditions and methods, etc., the extracellular vesicle may have the same meaning as an exosome, and may have the same or similar size as the exosome, but may also have a meaning including nanovesicles that do not have the composition of the exosome. On the other hand, in the present specification, the term exosome used in relation to production promotion or productivity is used to encompass the extracellular vesicles.

As used herein, the term "exosomes" refers to endoplasmic reticulum with a size of several tens to hundreds of nanometers (preferably about 30 to 200 nm) consisting of a double phospholipid membrane identical to the structure of the cell membrane (provided that the separation target is The particle size of exosomes may vary depending on the type of cell to be used, separation method and measurement method) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015)) DOI 10.1007/s00216-015-8535-3). Exosomes contain proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known

On the other hand, the term "exosome" used herein has a nano-sized vesicle structure secreted from animal-derived cells and released into the extracellular space and has a composition similar to that of exosomes (eg, exosomes). -It means to include all similar vesicles).

In the present invention, the type of animal-derived cells from which exosomes and/or extracellular vesicles are derived is not limited, but may be stem cells or immune cells as an example not limiting the present invention. The stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, embryonic stem cell-derived mesenchymal stem cells, or induced pluripotent stem cell-derived mesenchymal stem cells. The immune cells may be T cells, B cells, NK cells, cytotoxic T cells, dendritic cells or macrophages.

As an example not limiting the present invention, the adult stem cells include one or more adult stem cells selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and pluripotent stem cells. It may be a stem cell. The mesenchymal stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton's jelly and placenta. Preferably, the adult stem cells may be mesenchymal stem cells, for example, stem cells derived from fat, bone marrow, umbilical cord or umbilical cord blood, and more preferably, adipose-derived stem cells. The type of the stem cell or the immune cell is not limited as long as there is no risk of infection by a pathogen and does not cause immune rejection, but preferably a human stem cell or human-derived immune cell.

However, as long as it does not cause adverse effects on the human body, it is of course possible to use various animal cells that are used in the art or that can be used in the future. For example, it is also possible to use HEK 293 cells or HEK 293T cells. Therefore, it should be understood that the adipose-derived stem cells used in the examples to be described below are examples of animal cells that can be used in the present invention, and the present invention is not limited thereto.

As used herein, the term "pre-treatment" refers to culturing animal-derived cells in a medium containing at least one selected from the group consisting of insulin, transferrin, and acetylenic acid or a salt thereof, and "pre-treated exosomes and/or The term "extracellular vesicles" refers to exosomes and/or extracellular vesicles obtained by culturing animal-derived cells in a medium containing at least one selected from the group consisting of insulin, transferrin, and acetylenic acid or a salt thereof. Pre-treatment may be made in the cell proliferation process and / and may be made in the cell culture process for exosome production (so-called, conditioning process).

As used herein, the term "untreated" refers to culturing animal-derived cells in a medium that does not contain at least one selected from the group consisting of insulin, transferrin, and acetylenic acid or a salt thereof, and "untreated exosomes" And / or extracellular vesicles or untreated control "exosomes obtained without treatment with at least one selected from the group consisting of insulin, transferrin, and selenic acid or a salt thereof in any stage of the cell culture step and the exosome separation step and/or extracellular vesicles.

As used herein, the term "serum-free medium" or "SFM" refers to, for example, a culture medium in which serum such as Fetal Bovine Serum (FBS) is not added to a basal medium (optionally including antibiotics and/or antifungals) means In addition, as used herein, the term "conditioning culture media" refers to a culture medium (optionally antibiotic and / or including antifungal agents). As an example not to limit the present invention, the basal medium is α-MEM, DMEM, DMEM F12, 199 medium, RPMI-1640, L-15, Hum F-10, Hum F-12, DM-160, DM-170, etc. can

The present invention promotes the production of exosomes and/or extracellular vesicles, comprising the step of culturing an animal-derived cell in a medium containing at least one selected from the group consisting of insulin, transferrin, and acetylenic acid or a salt thereof. It provides a way to improve productivity.

For example, the method of promoting or enhancing the production of exosomes and/or extracellular vesicles of one embodiment of the present invention comprises the steps of (a) culturing the animal-derived cells in a growth medium to proliferate the cells, (b) the Animal-derived cells are cultured in a serum-free medium containing at least one selected from the group consisting of insulin, transferrin, and acetylenic acid or a salt thereof, and a conditioned medium containing exosomes secreted or released from the animal-derived cells. media) and then separating the exosomes and/or extracellular vesicles from the collected culture medium.

In addition, the method for promoting or enhancing the production of exosomes and/or extracellular vesicles of one embodiment of the present invention is (a) at least one selected from the group consisting of insulin, transferrin, and selenic acid or a salt thereof in animal-derived cells. Proliferating the cells by culturing them in a medium containing the; Culturing in a medium, collecting a conditioned media containing the exosomes secreted or released from the animal-derived cells, and then separating the exosomes and/or extracellular vesicles from the collected culture medium.

In the method for promoting or improving productivity of exosomes and/or extracellular vesicles of one embodiment of the present invention, the concentration of insulin is about 0.01 μg/mL to 100 μg/mL, and the concentration of transferrin is about 0.01 μg/mL to about 0.01 μg/mL to 100 μg/mL, and the concentration of acetylenic acid or a salt thereof may be about 0.01 ng/mL to 100 ng/mL. For example, preferably, the concentration of insulin is about 0.03 μg/mL to 50 μg/mL, the concentration of transferrin is about 0.03 μg/mL to 50 μg/mL, and the concentration of acetylenic acid or a salt thereof is about 0.03 ng. /mL to 50 ng/mL. More preferably, the concentration of insulin is about 0.08 μg/mL to 2 μg/mL, the concentration of transferrin is about 0.044 μg/mL to 1.1 μg/mL, and the concentration of acetylenic acid or a salt thereof is about 0.0536 ng/mL to about 0.0536 ng/mL 1.34 ng/mL.

In the method for promoting or improving productivity of exosomes and/or extracellular vesicles of one embodiment of the present invention, at least one selected from the group consisting of insulin, transferrin, and acetylenic acid or a salt thereof is insulin, transferrin, or sub selenic acid or a salt thereof, respectively, or a combination of insulin, transferrin, and selenic acid or a salt thereof.

In one embodiment of the present invention promoting or enhancing the production of exosomes and/or extracellular vesicles, the salt of selenium acid may be, for example, sodium selenite.

In one embodiment of the present invention, in the method for promoting or enhancing the production of exosomes and/or extracellular vesicles, the amount of exosomes and/or extracellular vesicles produced per cell (or cell culture medium) is exosomes and/or extracellular vesicles. content (tetraspanin content, eg CD81 content), or the number of exosomes and/or extracellular vesicles particles per cell (or cell culture medium).

The present invention provides a medium for promoting production of exosomes and/or extracellular vesicles derived from animal cells or enhancing productivity, comprising at least one selected from the group consisting of insulin, transferrin, and acetylenic acid or a salt thereof.

In one embodiment of the present invention, in the medium for promoting or enhancing the production of animal cell-derived exosomes and/or extracellular vesicles, the concentration of insulin is about 0.01 μg/mL to 100 μg/mL, and the concentration of transferrin is about 0.01 μg/mL to 100 μg/mL, and the concentration of acetylenic acid or a salt thereof may be about 0.01 ng/mL to 100 ng/mL. For example, preferably, the concentration of insulin is about 0.03 μg/mL to 50 μg/mL, the concentration of transferrin is about 0.03 μg/mL to 50 μg/mL, and the concentration of acetylenic acid or a salt thereof is about 0.03 ng. /mL to 50 ng/mL. More preferably, the concentration of insulin is about 0.08 μg/mL to 2 μg/mL, the concentration of transferrin is about 0.044 μg/mL to 1.1 μg/mL, and the concentration of acetylenic acid or a salt thereof is about 0.0536 ng/mL to about 0.0536 ng/mL 1.34 ng/mL.

In one embodiment of the present invention, the medium for promoting or improving productivity of animal cell-derived exosomes and/or extracellular vesicles is insulin, transferrin, or selenic acid or a salt thereof, respectively, or insulin, transferrin, and acetylenic acid. or a combination of salts thereof.

In an embodiment of the present invention, in the medium for promoting production of exosomes derived from animal cells and/or extracellular vesicles or improving productivity, the salt of selenium acid may be, for example, sodium selenite.

According to the present invention, it is possible to improve the productivity of exosomes and/or extracellular vesicles secreted or released per cell (or cell culture medium). Therefore, the method of promoting or enhancing the production of exosomes and/or extracellular vesicles of the present invention is economically and efficiently with high yields of exosomes and/or extracellular vesicles that can be utilized commercially and/or clinically. It can be produced, in particular, there is an advantage that can produce a large amount of exosomes and / or extracellular vesicles compared to the conventional method.

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

1a is a particle size distribution obtained by performing NTA (Nanoparticle Tracking Analysis) on exosomes and/or extracellular vesicles obtained by culturing stem cells in an untreated medium not containing insulin, transferrin or sodium selenite; It is a graph showing the number of particles.
Figure 1b is a graph showing the particle size distribution and the number of particles obtained by performing NTA on the exosomes and / or extracellular vesicles obtained by culturing stem cells in a medium containing insulin.
1c is a graph showing the particle size distribution and the number of particles obtained by performing NTA on exosomes and/or extracellular vesicles obtained by culturing stem cells in a medium containing transferrin.
1d is a graph showing the particle size distribution and the number of particles obtained by performing NTA on exosomes and/or extracellular vesicles obtained by culturing stem cells in a medium containing sodium selenite.
1e is a graph showing the particle size distribution and the number of particles obtained by performing NTA on exosomes and/or extracellular vesicles obtained by culturing stem cells in a medium containing insulin, transferrin and sodium selenite.
2 is an exo per unit mL when stem cells are cultured in a medium containing insulin, transferrin, or sodium selenite alone or a combination thereof (hereinafter referred to as "productivity enhancing material") according to an embodiment of the present invention; It is a comparative graph showing that the number of small particles (ie, exosome productivity) is significantly increased compared to the untreated control not treated with the productivity enhancing material.
Figure 3 shows that when stem cells are cultured in a medium containing insulin, transferrin or sodium selenite alone or a combination thereof according to an embodiment of the present invention, the exosome content (CD81 content) per unit mL is a productivity enhancing material. It is a graph showing a marked increase compared to the untreated control group that was not treated.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or limit the scope of the present invention. What can be easily inferred by those skilled in the art from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention. The references cited herein are incorporated herein by reference.

Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Example

Example 1: Cultivation of cells

Dulbecco's Modified Eagle Medium (DMEM) culture medium (hereinafter referred to as "growth medium" or "GM ") was prepared. In addition, serum-free addition of 2 mM L-glutamin, 1 mM sodium pyruvate, 100 Units/mL penicillin and 100 μg/mL streptomycin to phenol-free DMEM. A medium (hereinafter referred to as "SFM") was prepared.

After suspending human adipose-derived stem cells in a growth medium, inoculated at a density of 6000 cells/cm ² in a 25T flask and cultured at 5% CO ₂ , 37° C. until the cell confluency reached 80% or more. . On the other hand, in the case of pretreatment of human adipose-derived stem cells with insulin, transferrin, or sodium selenite alone or a combination thereof (hereinafter referred to as “productivity enhancing substance”) in the cell proliferation stage, 0.08 to 2 μg/mL of insulin, Cultivate human adipose-derived stem cells in a growth medium supplemented with 0.044 to 1.1 μg/mL of transferrin or 0.0536 to 1.34 ng/mL of sodium selenite, respectively, or a combination thereof.

When the density of the cultured cells is 80% or more, the control group untreated with a productivity enhancing material is washed with Phosphate Buffered Saline (PBS), replaced with SFM, and cultured for 24 to 72 hours, and then the supernatant (hereinafter , culture) was recovered (incubation for exosome production and culture medium recovery step). In addition, when the density of the cultured cells is 80% or more, the productivity-enhancing substance-treated group is washed with phosphate buffer, followed by 0.08 to 2 μg/mL of insulin, 0.044 to 1.1 μg/mL of transferrin, or 0.0536 to 1.34 ng/mL. Replace with SFM (or alternatively, SFM is possible if human adipose-derived stem cells have been pretreated in the cell proliferation stage) containing mL of sodium selenate each or a combination thereof and incubate for 24 to 72 hours, and then the supernatant ( Hereinafter, the culture medium) was recovered (culture and culture medium recovery step for exosome production).

Example 2: Isolation and purification of exosomes

Ultrafiltration was used for separation and concentration of exosomes from the culture medium recovered in Example 1. A centrifugal filter was used as a filter for the ultrafiltration method, and this filter may be selected by various molecular weight cut off (MWCO). Exosomes were selectively isolated and concentrated using the selected MWCO, and particles smaller than the MWCO, proteins, lipids, nucleic acids, and low molecular weight compounds were removed.

A centrifugal filter of MWCO 100,000 Da (Dalton) was used to separate and concentrate the exosomes. While the culture medium was concentrated to a volume of about 1/10 using ultrafiltration, substances smaller than MWCO were removed to separate exosomes.

On the other hand, as a method of isolating the exosomes from an animal-derived cell culture medium including stem cells, various methods known in the art may be used in addition to the separation method as described above. For example, for the separation of exosomes, ultracentrifugation, density gradient centrifugation, tangential flow filtration (TFF), size exclusion chromatography (Size Exclusion Chromatography), ions Ion Exchange Chromatography, Immunoaffinity Capture, Microfluidics Based Isolation, Exosome Precipitation, Total Exosome Isolation Kit or Polymer Based Precipitation A known separation method such as Polymer Based Precipitation can be used. However, the separation method of the exosomes is not limited to the methods described above, and of course, various separation methods that are used in the art or that can be used in the future are acceptable.

Example 3: Evaluation of exosome productivity based on exosome characterization and particle number

Nanoparticle Tracking Analysis (NTA) was used to measure the size and concentration of the exosomes isolated according to Example 2. NTA result graphs showing the particle size and distribution of the exosomes isolated according to Example 2 are shown in FIGS. 1a to 1e.

In addition, for accurate exosome productivity comparison analysis, the experimental group was classified as follows according to whether insulin, transferrin, or sodium selenite were included in the medium composition:

(1) untreated control group (Untreated): an experimental group in which exosomes were obtained according to Example 2 after culturing stem cells in a medium not containing insulin, transferrin and sodium acetate;

(2) Insulin treated group (Insulin treated, I): an experimental group in which exosomes were obtained according to Example 2 after culturing stem cells in a medium containing insulin;

(3) Transferrin treated group (Transferrin treated, T): an experimental group in which exosomes were obtained according to Example 2 after culturing stem cells in a medium containing transferrin;

(4) Sodium selenite alone treated group (Sodium Selenite treated, S): an experimental group in which exosomes were obtained according to Example 2 after stem cells were cultured in a medium containing sodium selenite;

(5) Treatment group containing a combination of insulin, transferrin and sodium selenite (ITS treated, ITS): After culturing stem cells in a medium containing insulin, transferrin and sodium selenite according to Example 2 Experimental group obtained exosomes.

Insulin (indicated as "Insulin treated" in FIG. 1B), transferrin (indicated as "Transferrin treated" in FIG. 1C) or sodium selenite (indicated as "Sodium Selenite treated" in FIG. 1D) according to one embodiment of the present invention Human adipose-derived stem cells were cultured using conditioned culture media containing either or both (indicated as "ITS treated" in FIG. 1E). As described above, after culturing human adipose-derived stem cells using a conditioned culture medium, exosomes obtained using ultrafiltration were performed in an untreated medium (indicated as “Untreated” in FIG. 1A ) that did not contain a productivity enhancing material. After culturing adipose-derived stem cells, it was found that the particle size distribution was uniform compared to the exosomes obtained using ultrafiltration.

In addition, as shown in FIG. 2 , as a result of comparing exosome productivity (the number of exosome particles) through NTA analysis according to an embodiment of the present invention, insulin, transferrin, or sodium selenite were each alone. Exosomes obtained by culturing human adipose-derived stem cells in a conditioned conditioned culture medium (indicated by "Insulin(I)", "Transferrin(T)" or "Sodium Selenite(S)" according to the productivity enhancing material in FIG. 2) It can be seen that the productivity of the exosomes (untreated exosomes) obtained by culturing human adipose-derived stem cells in a medium not containing a productivity-enhancing substance was significantly increased compared to the productivity of exosomes (untreated exosomes). On the other hand, the productivity of exosomes obtained by culturing human adipose-derived stem cells in a conditioned culture medium containing all of insulin, transferrin and sodium selenite (indicated by "ITS" in FIG. It was confirmed that the productivity of exosomes obtained by culturing human adipose-derived stem cells in a conditioned culture medium containing sodium selenate alone was significantly higher than that of exosomes (see FIG. 2).

From these results, the present invention can significantly improve the productivity of exosomes secreted or released per cell (or cell culture medium) by culturing the cells in a medium containing insulin, transferrin, or sodium selenite alone or a combination thereof. confirmed that.

Therefore, the present invention is to economically and efficiently produce exosomes and/or extracellular vesicles with uniform particle size distribution in high yield by culturing cells in a medium containing insulin, transferrin or sodium selenite alone or a combination thereof. can

Example 4: Evaluation of exosome productivity using exosome-specific markers

Since CD81 is a representative positive marker of exosomes, the CD81 content and the exosome content have linearity with each other, and an increase in the CD81 content means a proportional increase in the exosome content. Hereinafter, the content of exosomes was analyzed according to this principle. For this purpose, Exosome-Human CD81 Flow Detection Reagent was used. After mixing the exosome and exosome-human CD81 flow detection reagent overnight, it was reacted with PE Mouse anti-Human CD81 for 1 hour. After the reaction was completed, PE Mean Fluorescence Intensity (MFI) was measured using flow cytometry.

Meanwhile, a linear regression analysis was performed to calculate the CD81 content using the PE mean fluorescence intensity. That is, a linear regression analysis was performed using the concentration of the CD81 protein prepared by serial dilution and the MFI value according to the concentration. Using the standard quantitative analysis graph determined through this, the exosome content (CD81 content) of each of the experimental groups (1) to (5) classified in Example 3 was determined.

As a result, a conditioned culture medium containing insulin, transferrin, or sodium selenite alone, respectively (“Insulin (I)”, “Transferrin (T)” or “Sodium Selenite (S)” depending on the productivity enhancing material in FIG. 3 ) The content (CD81 content) of exosomes obtained by culturing human adipose-derived stem cells in “ ) was significantly increased compared to the content (CD81 content). On the other hand, the content of exosomes (CD81 content) obtained by culturing human adipose-derived stem cells in a conditioned culture medium (indicated as "ITS" in FIG. 3) containing all of insulin, transferrin, and sodium selenite was insulin, It was confirmed that the content of exosomes (CD81 content) obtained by culturing human adipose-derived stem cells in a conditioned culture medium containing transferrin or sodium selenate alone was increased.

From these results, the present invention can significantly improve the productivity of exosomes secreted or released per cell (or cell culture medium) by culturing the cells in a medium containing insulin, transferrin, or sodium selenite alone or a combination thereof. was confirmed again.

Accordingly, the present invention dramatically improves the productivity of exosomes and/or extracellular vesicles secreted or released per cell (or cell culture medium) by culturing the cells in a medium containing insulin, transferrin, or sodium selenite alone or a combination thereof. It can be increased to, and it is possible to economically and efficiently produce exosomes and / or extracellular vesicles that can be useful commercially and / or clinically in high yield, and in particular, compared to conventional methods, exosomes and / or It has the advantage of being able to mass-produce extracellular vesicles.

In the above, the present invention has been described with reference to the above examples, but 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 (8)

Insulin, transferrin, and a method of promoting or improving productivity of exosomes and/or extracellular vesicles, comprising the step of culturing in a medium comprising at least one selected from the group consisting of selenic acid or a salt thereof. The method of claim 1,
The concentration of insulin is 0.01 μg/mL to 100 μg/mL, the concentration of transferrin is 0.01 μg/mL to 100 μg/mL, and the concentration of acetylenic acid or a salt thereof is 0.01 ng/mL to 100 ng/mL, exo A method for promoting the production of vesicles and/or extracellular vesicles or improving productivity. The method of claim 1,
At least one selected from the group consisting of insulin, transferrin, and selenic acid or a salt thereof is insulin, transferrin, or selenic acid or a salt thereof, respectively, or a combination of insulin, transferrin, and selenic acid or a salt thereof, exosome and/or a method for promoting production of extracellular vesicles or enhancing productivity. 4. The method according to any one of claims 1 to 3,
A salt of selenium acid is sodium selenite, exosomes and/or extracellular vesicles production promotion or productivity improvement method. Insulin, transferrin, and at least one selected from the group consisting of selenic acid or a salt thereof, animal cell-derived exosomes and / or a medium for promoting the production of extracellular vesicles or productivity improvement. 6. The method of claim 5,
The concentration of insulin is 0.01 μg/mL to 100 μg/mL, the concentration of transferrin is 0.01 μg/mL to 100 μg/mL, and the concentration of acetylenic acid or a salt thereof is 0.01 ng/mL to 100 ng/mL. A medium for promoting production of cell-derived exosomes and/or extracellular vesicles or improving productivity. 6. The method of claim 5,
Insulin, transferrin, or selenic acid or a salt thereof, respectively, or a combination of insulin, transferrin, and selenic acid or a salt thereof . 8. The method according to any one of claims 5 to 7,
The salt of selenic acid is sodium selenite, a medium for promoting production of animal cell-derived exosomes and/or extracellular vesicles or improving productivity.

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