Cell culture medium for culturing extracellular vesicles at high concentrations and method for producing conditioned medium containing high levels of extracellular vesicles using the cell culture medium

Abstract

The present invention relates to a cell culture medium for culturing extracellular vesicles containing lactoferrin at a high concentration; a method for producing a conditioned medium containing a high concentration of extracellular vesicles, which comprises culturing cells in the cell culture medium; and the use of lactoferrin for producing a conditioned medium containing a high concentration of extracellular vesicles.

Description

[Technical field]

[0001] The present invention relates to a cell culture medium for culturing extracellular vesicles containing lactoferrin at high concentrations; a method for producing a conditioned medium containing a high concentration of extracellular vesicles, which comprises culturing cells in said cell culture medium; and the use of lactoferrin for use in producing a conditioned medium containing a high concentration of extracellular vesicles. [Background technology]

[0002] Among cell-secreted substances, "extracellular vesicles" have recently been attracting attention. Extracellular vesicles are small spherical objects with sizes of 30 nm to 2 μm (2,000 nm) that act as cell-mimicking delivery vehicles excreted from cells. Extracellular vesicles refer to various vesicles excreted from cells, and the main classifications are " Exosomes The types of extracellular vesicles are given different names by scholars depending on their functions and origins, and are called various names such as ectosomes, microparticles, tolerosomes, prostatosomes, cardiosomes, and vexosomes, but ultimately, according to the principle of formation, they are classified as endosomal intraluminal vesicles. Exosomes It has been reported that they should be appropriately named plasma membrane-derived microvesicles (Non-Patent Document 1). Their main characteristics are that they are enclosed in a lipid bilayer derived from a cell, have membrane proteins, and contain cell regulatory proteins inside.

[0003] One of the reasons why the field of extracellular vesicles has recently become active is that due to the small size of extracellular vesicles, it is difficult to separate them from other secreted proteins, and due to the low content of extracellular vesicles in the culture medium, it is difficult to confirm their effects. Currently, a technology for isolating extracellular vesicles has been secured (Non-Patent Document 2), its effectiveness has been confirmed, and clinical trials are being conducted (Non-Patent Document 3).

[0004] However, it is difficult to confirm the specific effects of extracellular vesicles only in a general conditioned medium obtained by culturing cells in a general cell culture medium for extracellular vesicles, which has a low content of cell secretory substances, and even if extracellular vesicles are separated from the conditioned medium and used, there are limitations due to the lack of absolute amount of extracellular vesicles.

[0005] Currently, research is underway to increase the content of extracellular vesicles in conditioned culture medium by enabling cells to excrete more extracellular vesicles. Basically, a method is used to increase the total concentration of secretions secreted from cells by growing many cells in the same space (Non-Patent Document 4). However, this method does not just increase the ratio of extracellular vesicles, so it is not a method to solve the cause, there is a limit to increasing the amount of secretion, and there are problems in terms of cost.

[0006] Therefore, a method for cells to specifically export extracellular vesicles is needed. t In other words, if only the extracellular vesicles released from the cells are specifically increased, the content of extracellular vesicles in the culture medium increases and the absolute amount also increases. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Nat Rev Drug Discov. 2013 May; 12(5):347-57 [Non-Patent Document 2] Biomed Res Int. 2018 Jan 30; 2018:8545347 [Non-Patent Document 3] J Extracell Vesicles. (2015)31; 4:30087 [Non-Patent Document 4] Sci Rep. 2018 Jan 19; 8(1):1171 Summary of the Invention [Problem to be solved by the invention]

[0008] The core of technology development to improve the production capacity of extracellular vesicles excreted per single cell is that the cell culture medium does not contain substances toxic to the human body and does not require high-cost / inefficient work. In addition, the substances used should not adversely affect the function of extracellular vesicles. Furthermore, if the substances used can improve the function of extracellular vesicles, it will be a more effective method.

[0009] Therefore, the present invention provides a high concentration of Thin The technical objective of the present invention is to develop a cell culture medium for culturing extracellular vesicles and to provide a method for producing a culture medium containing extracellular vesicles at a high concentration. [Means for solving the problem]

[0010] The present inventors have endeavored to solve the above problems, and as a result, have found that a protein called lactoferrin meets all the above conditions and is very effective in increasing the production of extracellular vesicles.Furthermore, the present inventors have found that adding calcium to lactoferrin can further maximize the production ability of extracellular vesicles by lactoferrin, and thus have completed the present invention.

[0011] Based on the effect of lactoferrin in increasing the amount of extracellular vesicles produced per single cell, the present inventors have ExosomesIt was confirmed that a conditioned medium containing lactoferrin was spontaneously formed. It was also found that a cell culture medium containing lactoferrin could be produced and a highly concentrated conditioned medium could be produced.

[0012] Thus, the present invention provides a cell culture medium for culturing extracellular vesicles containing lactoferrin at high concentrations.

[0013] The present invention also provides a method for producing a conditioned medium containing a high concentration of extracellular vesicles, comprising culturing cells in the cell culture medium.

[0014] Furthermore, the present invention provides the use of lactoferrin for producing a conditioned culture medium containing a high concentration of extracellular vesicles. Effect of the Invention

[0015] The cell culture medium capable of culturing extracellular vesicles containing lactoferrin of the present invention at high concentrations is safe because it uses lactoferrin that is not toxic to the human body, and it can increase the amount of extracellular vesicles produced. Further aspects of the invention are described below: [Section 1] A cell culture medium for culturing extracellular vesicles at high concentrations, comprising lactoferrin. [Section 2] Item 1. The cell culture medium according to item 1, comprising lactoferrin at a concentration of 0.1 μg / mL to 1 mg / mL. [Section 3] Item 2. The cell culture medium according to item 1, wherein the lactoferrin is selected from the group consisting of Holo-lactoferrin, Apo-lactoferrin and Pis-lactoferrin. [Section 4] Item 1. The cell culture medium according to item 1, further comprising calcium. [Section 5] Item 5. The cell culture medium according to item 4, wherein calcium is added at a concentration of 0.2 μM to 10 mM. [Section 6] Item 1. The cell culture medium according to Item 1, further comprising at least one substance selected from the group consisting of a supplement-free medium, a serum replacement agent, and serum. [Section 7] Item 1. The cell culture medium according to item 1, which is applied to cells of human or animal origin. [Section 8] A method for producing a conditioned culture medium containing extracellular vesicles at a high concentration, comprising culturing cells in the cell culture medium according to any one of items 1 to 7. [Section 9] Item 9. The method of claim 8, wherein the number of extracellular vesicles in the conditioned culture medium is in the range of 1.0 x 108 / mL to 1.0 x 1011 / mL. [Section 10] The manufacturing method according to item 8, wherein the use of the cell culture medium is not set at a specific time point during cell culture, but is used repeatedly. [Brief description of the drawings]

[0016] [Figure 1] These are the results of ELISA confirming the rate of extracellular vesicle production in conditioned culture medium by CD81 and the rate of apoptotic vesicle production by calnexin when lactoferrin was combined at various concentrations in a supplement-free medium. [Diagram 2] These are the results of ELISA confirming the rate of extracellular vesicle production in conditioned culture medium by CD81 and the rate of apoptotic vesicle production by calnexin when transferrin was combined with a supplement-free medium at various concentrations. [Diagram 3] This is the result of confirming the change in intracellular calcium concentration in the conditioned culture medium when a serum replacement agent, lactoferrin, and calcium were combined with a supplement-free medium. [Figure 4] The number of extracellular vesicles produced in conditioned culture medium when serum replacement, lactoferrin, and calcium were combined with a supplement-free medium was measured using a nanoparticle tracking analyzer. [Diagram 5] The size of extracellular vesicles produced in conditioned culture medium when serum replacement, lactoferrin, and calcium were combined with a supplement-free medium was measured using a nanoparticle tracking analyzer. [Figure 6] Calnexin ELISA results showing the relative amount of apoptotic vesicles produced by the same cells in conditioned medium when supplemented with no medium alone, serum replacement alone, or in combination with serum replacement + lactoferrin + calcium. [Figure 7] CD9 ELISA results showing the relative amount of extracellular vesicles produced by the same cells in conditioned medium when supplemented with plain medium alone, serum replacement alone, or serum replacement in combination with lactoferrin and calcium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present inventors have found that lactoferrin is effective in increasing the amount of extracellular vesicles produced by binding to cell receptors.Furthermore, the present inventors have prepared a cell culture medium for producing extracellular vesicles, which contains lactoferrin as an essential component and optionally contains calcium, in order to enhance the calcium delivery ability of lactoferrin, and have confirmed that when the culture medium is used for cell culture, the production ability of extracellular vesicles discharged from cells is rapidly increased.

[0018] The cell culture medium of the present invention and the cell culture method using the same are safe in that they use a combination of lactoferrin protein and calcium ions, which are not toxic to the human body, and can efficiently increase the production of extracellular vesicles.

[0019] The present invention relates to a cell culture medium for culturing extracellular vesicles containing lactoferrin at high concentrations.

[0020] The present invention also relates to a method for producing a conditioned medium containing extracellular vesicles at a high concentration, which comprises culturing cells in the cell culture medium.

[0021] Furthermore, the present invention relates to the use of lactoferrin for producing a conditioned culture medium containing a high concentration of extracellular vesicles.

[0022] As used herein, the term "extracellular vesicles" refers to various vesicles that are released from cells. ExosomesThe term "extracellular vesicle" is meant to include "exosomes," "microvesicles," and "microvesicles." The term "extracellular vesicles" is also used to include ectosomes, microparticles, tolerosomes, prostatosomes, cardiosomes, and vexosomes.

[0023] As used herein, the term "cell culture medium" refers to a medium before cells are cultured, and "conditioned medium" refers to a culture medium obtained after cells are cultured.

[0024] In one embodiment, lactoferrin may be added to the cell culture medium at a concentration of 0.1 μg / mL to 1 mg / mL. In one embodiment, the lactoferrin may be selected from the group consisting of Holo-lactoferrin, Apo-lactoferrin and Pis-lactoferrin. Specifically, the lactoferrin may be obtained by synthesis or extraction, and includes both human and non-human animal origin.

[0025] In one embodiment, the number of extracellular vesicles in the conditioned medium obtained by the method for producing a conditioned medium of the present invention is 1.0 × 10 8 / mL ~ 1.0 × 10 11 / mL, specifically, 1.0 × 10 9 / mL ~ 1.0 × 10 11 / mL range.

[0026] According to one embodiment of the present invention, the cell culture medium of the present invention may further contain calcium. The present inventors have confirmed that the production ability of extracellular vesicles by lactoferrin can be further maximized by controlling the concentration of calcium combined with lactoferrin. In one embodiment, the calcium is calcium ion (Ca 2+) and the source of calcium ions includes all salts capable of providing calcium ions.

[0027] In one embodiment, calcium may be added to the cell culture medium at a concentration between 0.2 μM and 10 mM.

[0028] In one embodiment, the cell culture medium of the present invention does not have to be set at a particular point during cell culture and is used repeatedly.

[0029] According to one embodiment of the present invention, the method for producing a cell culture medium or a conditioned medium of the present invention may be combined with a medium used for culturing existing cells, such as a medium containing a serum replacement agent. In one embodiment, the method for producing a cell culture medium and a conditioned medium of the present invention may further include adding one or more substances selected from the group consisting of a basal media, a serum replacement agent, and serum. In one embodiment, the cell culture medium of the present invention may further include one or more substances selected from the group consisting of a basal media, a serum replacement agent, and serum. The "serum replacement agent" refers to a composition that replaces serum to produce a serum-free medium.

[0030] According to one embodiment of the present invention, a combination of lactoferrin, calcium ions and a serum replacement agent can maximize the ability of cells to produce extracellular vesicles.

[0031] Transferrin contained in serum replacement agents is known to increase the production of extracellular vesicles by increasing the concentration of intracellular calcium (J Biol Chem. 2003 May 30; 278(22):20083-90). Lactoferrin is a member of the transferrin family, and its sequence is approximately 60% identical to that of transferrin. Therefore, lactoferrin is similar to transferrin in its structural features and representative functional features such as the ability to bind iron ions. However, lactoferrin has a different isoelectric point from transferrin, and therefore has a different ion-binding ability due to the different degree of positive charge on its surface, and the receptors that the family binds to are also different (Biochem Cell Biol. 2002; 80(1):27-34). In the examples below, the effect of lactoferrin and transferrin showing these differences on the amount of extracellular vesicles produced was confirmed using enzyme-linked immunoprecipitation assay (ELISA), and the production level of extracellular vesicles marker CD81 and apoptotic vesicles was observed using the marker calnexin. As a result, it was observed that the production effect of transferrin on extracellular vesicles was not higher than that of lactoferrin. In the case of lactoferrin, it was observed that it significantly increased intracellular calcium concentration and increased the production amount of extracellular vesicles by exceeding serum replacement agents based on the specific difference in ion binding and ability to bind to cell receptors.

[0032] As can be seen in the examples below, the principle that the cell culture medium supplemented with lactoferrin according to the present invention increases the number of extracellular vesicles is not the result of an increase in cell proliferation or apoptosis, but rather the increase in the intracellular calcium concentration increases the ability of cells to directly produce extracellular vesicles (PHYSIOLOGY 20: 22-27, 2005; 10.1152). In addition, the process in which lactoferrin binds to the cell receptor GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and moves into the cell is a phenomenon common to all animal cells, including humans (GAPDH: Biological Properties and Diversity, ISBN 978-94. -007-4716-6). Therefore, the high concentration of lactoferrin in the cell culture medium supplemented with lactoferrin increases the number of extracellular vesicles. Exosomes The production of a conditioned medium containing is a universal method that can be applied to all types of cells, including human.

[0033] In one embodiment, the cells used for the method of producing the cell culture medium and conditioned medium of the present invention may be any kind of cell of human or animal origin.

[0034] A conditioned culture medium containing extracellular vesicles at a high concentration can be easily produced by the method for producing a conditioned culture medium of the present invention. In addition, a cell culture medium containing extracellular vesicles at a high concentration can be easily produced by using the cell culture medium of the present invention.

[0035] As used herein, the term "high concentration of extracellular vesicles" refers to a culture medium obtained by culturing cells, the number of extracellular vesicles in the culture medium being at least 1.0 × 10 8 / mL ~ 1.0 × 10 11 / mL range.

[0036] The conditioned culture medium or cell culture medium obtained by this method contains a high concentration of extracellular vesicles and can therefore be widely and conveniently used as a raw material for cosmetics and therapeutic drugs.

[0037] The following examples are intended to explain the configuration and effects of the present invention in more detail. However, the following examples are intended to only illustrate the present invention, and the scope of the present invention is not limited thereto. EXAMPLES

[0038] Example 1: Increasing extracellular vesicle production using lactoferrin Lactoferrin (LF) is a member of the transferrin (Tf) family, and its sequence is approximately 60% identical to that of transferrin. Thus, lactoferrin is similar to transferrin in its structural features and representative functional features, such as its ability to bind iron ions. On the other hand, lactoferrin has a different isoelectric point from transferrin, and therefore a different degree of positive charge on its surface, leading to different ion-binding abilities, and the receptors to which the family binds are also different (Biochem Cell Biol. 2002; 80(1):27-34). The effect of lactoferrin and transferrin, which show these differences, on the amount of extracellular vesicles generated was confirmed using enzyme-linked immunoprecipitation assay (ELISA), and the generation level of extracellular vesicles marker CD81 and apoptotic vesicles marker calnexin were observed.

[0039] Specifically, human adipose-derived mesenchymal stem cells were cultured for 24 hours in a plate culture dish (48-well plate). After 24 hours, lactoferrin (aspira Scientific, USA) or transferrin (Sigma, USA) was mixed with water to a final concentration of 5, 10, 20, 50, 100, 250, 500, and 1000 μg / mL, and 25 μl of the mixture was inoculated into the basal medium. 25 μL of water was inoculated into the negative control group. After treatment, the cells were cultured for 24 hours in an incubator at 37°C supplying 5% CO2, and then each culture solution was obtained and centrifuged at 1,500 g for 10 minutes to remove dead cells.

[0040] CD81 and calnexin were measured using a CD81 ELISA kit (Mybiosource, USA) and a calnexin ELISA kit (Mybiosource, USA). The specific method is as follows. 100 μL of the obtained culture medium and the standard were added to the coated 96-well plate, and then reacted at 37 °C for 1-2 hours. Then, the standard and the culture medium were removed, and the primary antibody (detection antibody) was added and reacted at 37 °C. After 1 hour, the mixture was washed three times with washing buffer, reacted with the secondary antibody (HRP antibody), and washed again for 0.5-1 hours. Finally, the substrate and stop buffer were added, and the absorbance was measured at 450 nm using an ELISA reader (Molecular Devices, USA), and the ratios calculated based on the negative control group are shown in Figure 1 (lactoferrin) and Figure 2 (transferrin). The result values ​​for CD81 are represented by black bars, and the result values ​​for calnexin are represented by white bars.

[0041] As shown in Figure 1, lactoferrin was confirmed to increase the production of extracellular vesicles excreted from cells at specific concentrations. CD81, an extracellular vesicle marker, was confirmed to increase by as little as 1.5-fold (50 μg / mL lactoferrin) and as much as 5-fold (5 μg / mL lactoferrin) compared to the untreated group (0 μg / mL) at lactoferrin concentrations of 5 μg / mL, 10 μg / mL, and 50 μg / mL. In comparison, calnexin, a marker of apoptotic vesicles, did not differ significantly with lactoferrin concentration. In conclusion, lactoferrin is a potent inhibitor of two types of extracellular vesicles in the concentration range used. Exosomes and was identified as a protein that specifically increases the production of microvesicles.

[0042] On the other hand, transferrin under the same concentration conditions showed no significant difference in the increase in CD81 compared to lactoferrin (Figure 2), confirming that lactoferrin is a particularly effective substance in increasing the production of extracellular vesicles excreted by cells.

[0043] Example 2: Increasing intracellular calcium levels with lactoferrin / calcium compositions An increase in intracellular calcium concentration is directly related to an increase in the production of extracellular vesicles. An increase in intracellular calcium reverses the asymmetry of cell membrane proteins to symmetry, disrupting the membrane that maintains balance (Physiology (Bethesda) 2005 Feb; 20:22-7). An increase in intracellular calcium concentration is also known to increase the production of intraluminal vesicles in endosomes.

[0044] Serum replacement activates cell metabolism through many nutrients in addition to transferrin, so it is advantageous to include serum replacement in the culture medium for producing extracellular vesicles.On the other hand, it is known that transferrin contained in serum replacement increases the concentration of intracellular calcium, thereby increasing the production of extracellular vesicles (J Biol Chem. 2003 May 30;278(22):20083-90), but the effect of transferrin on the production of extracellular vesicles is not higher than that of lactoferrin, as confirmed by the results of Example 1.

[0045] On the other hand, since lactoferrin may significantly increase intracellular calcium concentration and further increase extracellular vesicle production by exceeding serum replacement agents based on specific differences in its ability to bind ion binding and cellular receptors, the following experiments were designed.

[0046] A calcium colorimetric kit (Biovision, USA) was used to measure the intracellular calcium level according to the lactoferrin concentration. The calcium colorimetric kit measures calcium concentration by measuring the color displayed by the reaction of 0-cresolphthalein with calcium.

[0047] Human adipose-derived mesenchymal stem cells were cultured for 4 days. After removing the culture medium and washing with phosphate buffer solution, calcium and lactoferrin (Aspira Scientific, USA) were mixed at various concentrations (2, 5, 10, 25, 50, 100 μg / mL) in a medium containing serum replacement agent and treated with the cells. Accordingly, experiments were conducted on a group that combined serum replacement agent + calcium only, a group that was treated with 10 μg / mL lactoferrin only, a group that was treated with serum replacement agent only, and a group that was treated with no additives. After 48 hours, the cells were washed with phosphate buffer solution and detached from the culture dish with 1× trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA, welgene, Korea) solution.

[0048] The number of cells in each group was measured using an automatic cell counter (Nucleocounter NC-250, Chemometec, USA), and the same number of cells were dissolved in calcium assay buffer (Biovision, USA). Then, experiments were performed using a calcium colorimetric measurement kit. 50 μL of sample or a fixed amount of calcium, 90 μL of color reagent (0-cresolphthalein), and 60 μL of calcium assay buffer were added per well to a 96-well plate, and the reaction was allowed to proceed for 5 minutes in the dark. Then, the absorbance was measured at 575 nm, and the mesenchymal stem cells cultured in the non-supplemented medium were set as 100% to show the relative increase.

[0049] As shown in FIG. 3, it was confirmed that the lactoferrin / calcium composition in combination with the serum replacement agent significantly increased the intracellular calcium concentration. When only the serum replacement agent was added to the supplement-free medium, it was confirmed that the calcium concentration increased by about 150%, as expected. However, even when more calcium was supplied, the intracellular calcium concentration by the serum replacement agent did not increase further (Tukey's test, p>0.05). On the other hand, when calcium was additionally supplied to 2 μg / mL lactoferrin, the intracellular calcium concentration increased compared to the serum replacement agent, and the highest intracellular calcium concentration was observed at concentrations of 5 μg / mL, 10 μg / mL, and 25 μg / mL. In general, it was confirmed that the lactoferrin 5, 10, and 25 μg / mL and calcium composition can significantly increase the intracellular calcium concentration compared to the supplement-free medium (Tukey's test, p>0.05).

[0050] From the above results, it was found that the lactoferrin / calcium composition increases intracellular calcium concentration, and this composition may further increase the amount of extracellular vesicles produced when combined with a serum replacement agent.In addition, by confirming that the lactoferrin / calcium composition increases the amount of extracellular vesicles produced through the general principle of extracellular vesicle production, which is the increase in intracellular calcium concentration, it was found that the range of cells to which lactoferrin can be applied is not only the adipose-derived mesenchymal stem cells used in the above experiment, but also all types of cells.

[0051] One-way ANOVA was performed to identify differences across groups, and differences between individual means were analyzed using Tukey's test. Statistical significance between groups was plotted as *=(p<0.05), NS=(p>0.05). The statistics were analyzed using Prism software version 5.

[0052] Example 3: Increased extracellular vesicle production by lactoferrin / calcium compositions Nanoparticle tracking analysis (NTA) was performed to observe the changes in the number and size of extracellular vesicles in the culture medium depending on the lactoferrin concentration.

[0053] Human adipose-derived mesenchymal stem cells were cultured for 2 days. Lactoferrin concentrations of 5 μg / mL, 10 μg / mL, and 50 μg / mL were combined with calcium, then combined with serum replacement, mixed with supplement-free medium, and replaced. Experiments were also conducted comparing a group containing only a supplement-free medium (alpha-MEM) containing serum replacement with a group containing only a supplement-free medium. After 48 hours, the culture medium was collected and centrifuged at 1500 g for 10 minutes. The number and size of extracellular vesicles in the culture medium were measured using a nanoparticle tracking analyzer (NTA, Nanosight NS300, Malvern, UK), and are shown in Figures 4 and 5.

[0054] Figure 4 is a graph showing the number of extracellular vesicles contained per mL of culture medium. The relative number of extracellular vesicles was calculated based on the unsupplemented medium (100%) and is shown at the top of each graph. As shown in Figure 4, the number of extracellular vesicles contained in the medium was confirmed using a nanoparticle measurement device, and the average number of extracellular vesicles in the unsupplemented medium was 1.1 x 10 8 The serum replacement group had a cell density of approximately 5.6 × 10 8 The number of extracellular vesicles was confirmed to be about 4.5 × 10 8 On the other hand, in the treatment group in which lactoferrin 5 μg / mL + calcium composition was added to the serum replacement agent, the amount of extracellular vesicles produced was approximately 16.1 × 10 8 The cell count was confirmed to be approximately 14 × 10 8 The number of cells / mL increased by 1450% compared to the unsupplemented medium. The lactoferrin 10 μg / mL + calcium composition produced approximately 17 × 10 extracellular vesicles. 8 cells / mL, which was increased by 1590% compared to unsupplemented medium.

[0055] The results show that the lactoferrin+calcium composition can increase production in proportion to the concentration when combined with a serum replacement agent.

[0056] On the other hand, as shown in Figure 5, in the group treated with no supplemented medium, the group treated with serum replacement agent, and the group treated with lactoferrin (5 or 10 μg / mL) + calcium composition, extracellular vesicles were confirmed to exist in the size range of 120 to 140 nm. Images of the extracellular vesicles used for size measurement are shown on the right side of the graph.

[0057] Example 4: Confirmation that the increase in extracellular vesicles caused by lactoferrin / calcium composition is due to an increase in the cells' ability to produce extracellular vesicles Serum replacement affects the production of extracellular vesicles by transferrin, but active metabolism due to nutritional supply increases cell proliferation and increases the production of extracellular vesicles. Experiments were set up to prove that lactoferrin + calcium composition significantly increases the production capacity of extracellular vesicles expressed by cells, not cell proliferation or apoptotic substances.

[0058] To observe the change in the ability to generate extracellular vesicles due to the combination of lactoferrin and calcium, we confirmed it by enzyme-linked immunoprecipitation analysis (ELISA) using CD9, a marker for extracellular vesicles, and calnexin, a marker for apoptotic vesicles.

[0059] Human adipose-derived mesenchymal stem cells were treated with a supplement-free medium (alpha-MEM), a medium containing serum replacement, and a medium containing serum replacement + lactoferrin 10μg / mL + calcium. After 48 hours, the culture medium was centrifuged at 1500g for 10 minutes, and the supernatant was subjected to CD9 ELISA (exoquant overall exosome capture and quantification assay kit, Biovision, USA) and calnexin ELISA (Calnexin ELISA kit, Mybiosource, USA).

[0060] The specific method of CD9 ELISA is as follows. Each standard and 100 μL of the obtained culture medium were added to the coated 96-well plate and reacted at 37°C for 20 hours. After that, the standard and culture medium were removed and washed three times, and then the primary antibody (detection antibody) was added and reacted at 37°C. After 2 hours, the plate was washed three times with washing buffer, and the secondary antibody (HRP antibody) was added and reacted, and after 1 hour, it was washed again. Finally, the substrate and stop buffer were added, and the absorbance was measured at 450 nm using an ELISA reader (Molecular Devices, USA).

[0061] The measured values ​​of calnexin and CD9 were divided by the proliferation rate (MTT assay) measured for the cells when the culture medium was obtained. The MTT assay method is as follows. After removing the medium, the cells were washed three times with phosphate buffer solution. The MTT reagent was diluted with the medium so that the treatment concentration was 0.5 mg / mL, and then reacted at 37°C for 2 hours. After washing with phosphate buffer solution, the cells were stirred in 500 μL of isopropyl alcohol solution for 30 minutes, and the absorbance was measured at 570 nm.

[0062] The results of the apoptosis index were shown in Figure 6 using 100% supplement-free medium. As shown in Figure 6, apoptosis factors were not specifically overexpressed in the generated extracellular vesicles. Overall, it was confirmed that the expression levels of calnexin were similar in the supplement-free medium, the medium containing serum replacement, and the medium containing serum replacement + lactoferrin + calcium composition (one-way ANOVA, F(2,3)=1.797, p>0.05).

[0063] However, CD9, a marker of extracellular vesicles, was confirmed to be significantly increased in the lactoferrin + calcium composition. As shown in Figure 7, the medium containing serum replacement agent using 100% of the supplement-free medium did not significantly increase the expression of CD9 per cell compared to the supplement-free medium, confirming that the effect of the serum replacement agent is not an increase in the amount of extracellular vesicles produced per cell, but an increase in cell number, which increases the production of extracellular vesicles (Tukey's test, p>0.05).

[0064] On the other hand, in the case of the medium using the lactoferrin + calcium composition containing serum replacement, CD9 expression per cell increased by 1463% compared to the medium without supplementation, a result similar to the increase rate of extracellular vesicles shown in Example 3 (1590%). These results clearly show that the lactoferrin + calcium composition increased the production ability of extracellular vesicles produced by single cells (Tukey's test, p>0.05). In addition, the lactoferrin + calcium composition was able to reduce the two classifications of extracellular vesicles in the concentration range used. Exosomes and a composition that specifically increases the amount of microvesicles produced.

[0065] Differences across groups were identified by one-way ANOVA, and differences between individual means were analyzed using Tukey's test. Statistical significance between groups was plotted as *=(p<0.05), NS=(p>0.05). The statistics were analyzed using Prism software version 5.

Claims

1. A cell culture medium for culturing extracellular vesicles at a high concentration, comprising lactoferrin and calcium at a concentration of 0.1 μg / mL to 1 mg / mL, wherein the number of extracellular vesicles in the cell culture medium obtained by culturing cells is 1.0 x 10 / mL to 1.0 x 10 １１ / mL, the extracellular vesicles are exosomes or microvesicles, and the cells are mesenchymal stem cells.

2. 2. The cell culture medium of claim 1, wherein the lactoferrin is selected from the group consisting of Holo-lactoferrin, Apo-lactoferrin and Pis-lactoferrin.

3. 2. The cell culture medium of claim 1, wherein calcium is added at a concentration of 0.2 μM to 10 mM.

4. 2. The cell culture medium of claim 1, further comprising at least one substance selected from the group consisting of a supplement-free medium, a serum replacement agent, and serum.

5. A method for producing a conditioned culture medium containing extracellular vesicles at a high concentration, comprising culturing cells in the cell culture medium according to any one of claims 1 to 4, wherein the number of extracellular vesicles in the conditioned culture medium is 1.0 x 10 9 / mL to 1.0 x 10 １１ / mL, the extracellular vesicles are exosomes or microvesicles, and the cells are mesenchymal stem cells.

6. The method of claim 5, wherein the use of the cell culture medium is not set at a specific time point during cell culture, but is used repeatedly.

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