METHOD FOR PROMOTING PRODUCTION OF EXOSOMES AND/OR EXTRACELLULAR VESICLES

Abstract

A method for promoting production of exosomes and/or extracellular vesicles is provided. The method includes culturing animal cells in a medium containing ascorbic acid, an analogue thereof, or a derivative thereof. The method is able to enhance the productivity of exosomes and/or extracellular vesicles secreted or released per cell or cell-derived conditioned medium.

Claims

1. A method for promoting production of exosomes and/or extracellular vesicles, the method comprising culturing animal cells in a medium containing ascorbic acid, an analogue thereof, or a derivative thereof.

2. The method of claim 1, further comprising culturing the animal cells in a medium containing ascorbic acid, an analogue thereof, or a derivative thereof and fetal bovine serum (FBS).

3. The method of claim 2, wherein the fetal bovine serum is one from which FBS-derived exosomes and extracellular vesicles have been removed.

4. The method of claim 1, further comprising culturing the animal cells in a serum-free medium containing ascorbic acid, an analogue thereof, or a derivative thereof

5. The method of claim 1, wherein a concentration of the ascorbic acid, analogue thereof, or derivative thereof is 1 μg/mL to 300 μg/mL.

6. The method of claim 5, wherein the concentration of the ascorbic acid, analogue thereof, or derivative thereof is 10 μg/mL to 100 μg/mL.

7. A medium for promoting production of animal cell-derived exosomes and/or extracellular vesicles, the medium containing ascorbic acid, an analogue thereof, or a derivative thereof

8. The medium of claim 7, wherein a concentration of the ascorbic acid, analogue thereof, or derivative thereof is 1 μg/mL to 300 μg/mL.

9. The medium of claim 8, wherein the concentration of the ascorbic acid, analogue thereof, or derivative thereof is 10 μg/mL to 100 μg/mL.

10. The method of claim 2, wherein a concentration of the ascorbic acid, analogue thereof, or derivative thereof is 1 μg/mL to 300 μg/mL.

11. The method of claim 10, wherein the concentration of the ascorbic acid, analogue thereof, or derivative thereof is 10 μg/mL to 100 μg/mL.

12. The method of claim 3, wherein a concentration of the ascorbic acid, analogue thereof, or derivative thereof is 1 μg/mL to 300 μg/mL.

13. The method of claim 12, wherein the concentration of the ascorbic acid, analogue thereof, or derivative thereof is 10 μg/mL to 100 μg/mL.

14. The method of claim 4, wherein a concentration of the ascorbic acid, analogue thereof, or derivative thereof is 1 μg/mL to 300 μg/mL.

15. The method of claim 14, wherein the concentration of the ascorbic acid, analogue thereof, or derivative thereof is 10 μg/mL to 100 μg/mL.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1A is a graph showing the particle size distribution and the number of particles obtained by performing nanoparticle tracking analysis (NTA) for exosomes obtained after culturing stem cells in a medium not containing ascorbic acid, an analogue thereof, or a derivative thereof (indicated as “0 μg/mL”).

[0032] FIG. 1B is a graph showing the particle size distribution and the number of particles obtained by performing nanoparticle tracking analysis (NTA) for exosomes obtained after pre-treating and culturing stem cells in a medium containing ascorbic acid, an analogue thereof, or a derivative thereof (indicated as “30 μg/mL”).

[0033] FIG. 1C is a graph showing the particle size distribution and the number of particles obtained by performing nanoparticle tracking analysis (NTA) for exosomes obtained after pre-treating and culturing stem cells in a medium containing ascorbic acid, an analogue thereof, or a derivative thereof (indicated as “100 μg/mL”).

[0034] FIG. 2 is a comparative graph showing that when stem cells were pre-treated and cultured in a medium containing ascorbic acid, an analogue thereof, or a derivative thereof, the number of exosome particles per mL (i.e., exosome productivity) remarkably increased compared to that in an untreated control group, i.e., not treated with ascorbic acid, an analogue thereof, or a derivative thereof

[0035] FIG. 3 is a comparative graph showing that when stem cells were pre-treated and cultured in a medium containing ascorbic acid, an analogue thereof, or a derivative thereof, the exosome content (i.e., CD63 content) per mL remarkably increased compared to that in an untreated control group, i.e., not treated with ascorbic acid, an analogue thereof, or a derivative thereof.

EXAMPLES

[0036] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only to illustrate the present invention and are not intended to limit or restrict the scope of the present invention. Those that can be easily inferred by those skilled in the art from the detailed description and examples of the present invention are interpreted as falling within the scope of the present invention. References referred to in the present invention are incorporated herein by reference.

[0037] Throughout the present specification, it is to be understood that, when any part is referred to as “comprising” any component, it does not exclude other components, but may further include other components, unless otherwise specified.

Example 1

Cell Culture

[0038] Human adipose-derived stem cells were suspended in a-MEM culture medium containing 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin, and then seeded into a 6-well plate at a density of 6,000 cells/cm.sup.2 and cultured in an incubator at 37° C. under 5% CO.sub.2. For ascorbic acid-treated groups, 24 hours after seeding, ascorbic acid was added to the culture medium at a concentration of 10 μg/mL, 30 μg/mL and 100 μg/mL, respectively.

[0039] In all of the ascorbic acid-treated groups and the untreated group, when the adipose-derived stem cells reached a confluency of 80% or more, the culture medium was replaced with α-MEM medium containing 100 units/mL penicillin and 100 μg/mL streptomycin (hereinafter referred to as “serum-free medium” or “SFM”), or α-MEM medium containing 2% FBS from which FBS-derived exosomes and extracellular vesicles have been removed, 100 units/mL penicillin and 100 μg/mL streptomycin (hereinafter referred to as “extracellular vesicles-depleted medium” or “EDM”). In the ascorbic acid-treated groups, ascorbic acid was added to each of the serum-free medium and the extracellular vesicles-depleted medium at a concentration of 10 μg/mL, 30 μg/mL and 100 μg/mL, respectively. Thereafter, the cells were cultured for 24 hours, followed by recovery of the conditioned medium of the cells. After completion of recovery of the conditioned medium, the cells were counted using a cell counter.

Example 2

Isolation and Purification of Exosomes

[0040] To isolate exosome from the conditioned medium recovered in Example 1, ultracentrifugation was used. The recovered conditioned medium was subjected to sequential centrifugation at 4° C. to isolate exosomes as follows.

[0041] First, to remove cells from the recovered conditioned medium, the conditioned medium was centrifuged at 300×g for 10 minutes, and then the supernatant was collected. In addition, to remove cell debris from the supernatant, the supernatant was centrifuged at 2,000×g for 20 minutes, and then the supernatant was collected. Then, to remove microvesicles from the supernatant, the supernatant was centrifuged at 16,500×g for 10 minutes, and then the supernatant was collected. The finally obtained supernatant was centrifuged at 120,000×g for 120 minutes, and then the supernatant was discarded and the pellets were collected. Then, the pellets were washed with phosphate-buffered saline, and centrifuged again at 120,000×g for 120 minutes, and the supernatant was discarded. Next, the pellets were suspended in phosphate-buffered saline, thereby isolating exosomes.

[0042] Meanwhile, as methods of isolating exosomes from a conditioned medium of animal cells including stem cells, various methods known in the art may be used in addition to the isolation method as described above. For example, for isolation of exosomes, known isolation methods may be used, such as ultrafiltration, density gradient centrifugation, tangential flow filtration (TFF), size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, polymer based precipitation and the like. However, the method for isolating exosomes is not limited to the above-described methods, and it is of course possible to use various isolation methods that are being used in the art or may be used in the future.

Example 3

Characterization of Isolated Exosomes and Evaluation of Exosome Productivity

[0043] The particle size and concentration of the isolated exosomes were measured by nanoparticle tracking analysis (NTA) instrument (purchased from Malvern). FIG. 1 shows the results of NTA of the exosomes isolated by the isolation method according to one embodiment of the present invention. As shown in FIG. 1, it could be seen that the exosomes obtained after pre-treating and culturing the stem cells in the medium containing ascorbic acid according to one embodiment of the present invention (hereinafter referred to “experimental group 1”) had a more uniform particle size distribution than the exosomes obtained after culturing the stem cells in the medium not containing ascorbic acid (hereinafter referred to “experimental group 2”).

[0044] As shown in FIG. 2, from a comparative analysis result of NTA, it was confirmed that the productivity (i.e., the number of exosome particles per mL) of the exosomes derived from the stem cells pre-treated and cultured in the medium containing ascorbic acid (experimental group 1) increased by approximately four times or more as compared with the control group not treated with ascorbic acid (experimental group 2).

[0045] In addition, for accurate comparative analysis of exosome productivity, the exosomes of each of experimental groups 1 and 2 and Exosome-Human CD63 Flow Detection Reagent (Invitrogen™) were mixed together overnight. Then, each of the mixtures was allowed to react with PE mouse anti-Human CD63 (BD Parmigen™), and the PE mean fluorescence intensity (MFI) thereof was measured using flow cytometry.

[0046] Meanwhile, since CD63 is a representative positive marker of exosomes, the CD63 content and the exosome content are linearly proportional to each other, and an increase in the CD63 content means a proportional increase in the exosome content. According to this principle, human CD63 protein of known concentration was serially diluted to prepare human CD63 protein solutions having different human CD63 protein concentrations, and each human CD63 protein solution and Exosome-Human CD63 Flow Detection Reagent (Invitrogen™) were mixed together overnight. Then, each of the mixtures was allowed to react with PE mouse anti-human CD63 (BD Parmigen™), and the PE mean fluorescence intensity (MFI) thereof was measured using flow cytometry. Then, linear regression analysis was performed on the human CD63 protein concentration values and the corresponding MFI measurement values, and a standard quantitative analysis graph satisfying a linearity of 0.99 or higher was generated. Then, using the generated standard quantitative analysis graph, the CD63 content in each of experimental groups 1 and 2 was determined.

[0047] As a result, it was confirmed that the content of the exosomes (i.e., the content of CD63) derived from the stem cells pre-treated and cultured in the medium containing ascorbic acid remarkably increased compared to that in the untreated control group (see FIG. 3). From this result, it is understood that the present invention is able to enhance the productivity of exosomes secreted or released per cell (or cell-derived conditioned medium) by culturing cells in a medium containing ascorbic acid, an analogue thereof, or a derivative thereof.

[0048] Thus, the present invention is able to enhance the productivity of exosomes and/or extracellular vesicles secreted or released per cell (or cell-derived conditioned medium). Therefore, the present invention has advantages that it can economically and efficiently produce exosomes and/or extracellular vesicles, which can be utilized commercially and/or clinically, in high yield, and in particular, it can produce a large amount of exosomes and/or extracellular vesicles as compared with conventional methods.

[0049] Although the present invention has been described with reference to the embodiments, the scope of the present invention is not limited to these embodiments. Any person skilled in the art will appreciate that various modifications and changes are possible without departing from the spirit and scope of the present invention and these modifications and changes also fall within the scope of the present invention.