Method of producing extracellular vesicles, and extracellular vesicle-containing compositions

Abstract

To provide a method for producing extracellular vesicles that makes it possible to: terminate secretion of extracellular vesicles with appropriate timing allowing a sufficient amount of extracellular vesicles to be secured without requiring complicated means for checking extracellular vesicles in culture; and apply a simple separation method.SOLUTION: In a certain aspect, a method of producing extracellular vesicles includes: a secretion step in which stem cells are made to exist in an extracellular vesicle secretion medium, and extracellular vesicles are secreted from the stem cells; and a determination step in which whether the secretion step can be terminated is determined based on physical indicators of components in the extracellular vesicle secretion medium.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing extracellular vesicles and a composition containing extracellular vesicles. [Background technology]

[0002] Multicellular organisms, including humans, are composed of a wide variety of cells. These cells cooperate with each other through intercellular communication to maintain physiological functions. Known means of intercellular communication include physical means such as cell-cell adhesion, and signal transduction via hormones, cytokines, growth factors, etc.

[0003] In recent years, it has become clear that extracellular vesicles play an important role in this intercellular communication. Extracellular vesicles are particles that are surrounded by a lipid bilayer membrane derived from cells and do not have a nucleus (cannot be replicated), and contain various DNA, RNA, proteins, etc. depending on the type of cell they originate from and the environment. Extracellular vesicles secreted in the body are transported to other cells through blood, etc., and their contents cause various cellular responses. In view of the characteristics of such extracellular vesicles, technical development is currently being actively carried out for application in disease diagnosis and drug delivery systems. Patent Document 1 discloses a technology for introducing foreign substances into target cells using exosomes (a type of extracellular vesicles). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 076347 Summary of the Invention [Problem to be solved by the invention]

[0005] By the way, extracellular vesicles are obtained from cell culture supernatants, but the present inventors have found that culture supernatants obtained by conventional methods contain many extracellular vesicle-like particles having a particle size equivalent to that of extracellular vesicles. Therefore, in order to confirm the presence of extracellular vesicles during culture, a complicated means such as detection of extracellular vesicle markers is required. In addition, it is difficult to remove only these extracellular vesicle-like particles. The present invention has been made in consideration of the above problems, and aims to provide a method for producing extracellular vesicles that can terminate the secretion of extracellular vesicles at an appropriate timing to ensure a sufficient amount of extracellular vesicles without requiring complicated means to confirm the presence of extracellular vesicles during culture, and that has reduced impurities. [Means for solving the problem]

[0006] The present inventors found that these extracellular vesicle-like particles are composed of components derived from the culture medium. Therefore, the present inventors used a culture medium that contains as few such components as possible, and further, determined whether the culture should be terminated by a physical indicator, thereby completing the present invention.

[0007] That is, according to the first aspect of the present invention, A secretion step in which the stem cells are placed in an extracellular vesicle secretion medium and secrete extracellular vesicles from the stem cells; A determination step of determining whether the secretion step is completed based on a physical indicator of a component in the medium for secreting extracellular vesicles, The method for producing extracellular vesicles is provided, wherein the medium for secreting extracellular vesicles is a medium containing TNF-α and TRAIL / APO2L.

[0008] In the first aspect, the medium for extracellular vesicle secretion has a particle size of 1×10 to 1,000 nm before use. 9 The medium may contain less than 10 cells / mL.

[0009] In the first aspect, the medium for extracellular vesicle secretion may be a medium having a total protein amount of 100 μg / mL or less before use.

[0010] In the first aspect, the number of microparticles having a particle diameter of 1 to 1000 nm in the medium prepared as a pre-step of the secretion step is 1×10 9 The method may further include a confirmation step of confirming that the number of particles is less than 1 / mL.

[0011] In the first aspect, the condition for enabling the end of the secretion step in the determination step is The number of fine particles having a particle size of 1 to 1000 nm in 1 mL of culture supernatant may be three times or more as high as that at the start of the secretion step.

[0012] In the first aspect, the condition for enabling the end of the secretion step in the determination step is When the particle size in 1 mL of culture supernatant is plotted on the horizontal axis in 1 nm increments and the particle number on the vertical axis, the particle number is 3 × 10 in the particle size range of 1 to 200 nm. 7 There may be one or more peaks.

[0013] In the first aspect, the stem cells may be mesenchymal stem cells.

[0014] The first aspect may further include a culture step of proliferating the stem cells as a pre-step of the secretion step.

[0015] In the first aspect, the method may further include a recovery step of recovering the extracellular vesicles in the medium for secreting extracellular vesicles, as a subsequent step of the secretion step.

[0016] In the first embodiment, in the extracellular vesicle-containing composition obtained in the recovery step, the expression level of the extracellular vesicle marker relative to the number of microparticles having a particle diameter of 1 to 1000 nm is 5.0 × 10 -9 It may be more than pg / particle.

[0017] The method of the first aspect described above provides an extracellular vesicle-containing composition. Effect of the Invention

[0018] According to the present invention, there can be provided a method for producing extracellular vesicles that can terminate the secretion of extracellular vesicles at an appropriate timing to ensure a sufficient amount of extracellular vesicles without requiring a complicated means for checking the extracellular vesicles during culture, and that has reduced impurities. Furthermore, according to the present invention, there can be provided a composition containing extracellular vesicles that has a high expression rate of extracellular vesicle markers. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a schematic flow chart showing the process for producing extracellular vesicles according to this embodiment. [Diagram 2] FIG. 2 is a block diagram showing a schematic configuration of the extracellular vesicle producing system. [Diagram 3] FIG. 3 is a flowchart showing an example of the operation of the extracellular vesicle producing system. [Figure 4] FIG. 4 is a photograph showing the appearance of the medium for extracellular vesicle secretion and the conventional medium before use. [Diagram 5] FIG. 5 shows particle size distributions of the medium for extracellular vesicle secretion and the conventional medium before use. [Figure 6] FIG. 6 is a set of photographs showing the morphology of cells immediately after the addition of the medium for extracellular vesicle secretion and 48 hours after the addition. [Figure 7] FIG. 7 is a graph showing the amount of extracellular vesicle marker per unit volume in each culture supernatant when a basal medium and a medium for extracellular vesicle secretion were added. [Figure 8] FIG. 8 shows the particle size distribution in each culture supernatant at the start of the secretion process and after a certain period of time had elapsed. [Figure 9] FIG. 9 is a graph showing the ratio of the amount of extracellular vesicle marker based on the number of microparticles with a particle diameter of 1 to 1000 nm in each culture supernatant after the start of the secretion process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention will be described in detail below. In this specification, the expression "a to b" in the description of a numerical range means from a to b, unless otherwise specified. In addition, when multiple upper limit values ​​and multiple lower limit values ​​are separately described, it is assumed that all numerical ranges that can be set by freely combining these upper limit values ​​and lower limit values ​​are described.

[0021] The method for producing extracellular vesicles according to this embodiment preferably includes a secretion step of causing stem cells to be present in a culture medium and secreting extracellular vesicles from the stem cells, and a judgment step of judging whether or not the secretion step is complete based on physical indicators of components in the culture medium.

[0022] (stem cells) The cells used in this embodiment are not particularly limited as long as they are stem cells. "Stem cells" are cells that have self-renewal and pluripotency. Stem cells are broadly classified into three types based on their differentiation potential: totipotent stem cells (fertilized eggs, etc.), pluripotent stem cells (ES cells, iPS cells, etc.), and somatic stem cells. As described above, the contents of extracellular vesicles differ depending on the type of cell that secretes them, so the type of stem cells can be selected arbitrarily depending on the extracellular vesicles required. For example, extracellular vesicles secreted by mesenchymal stem cells, which are a type of somatic stem cells, can regulate immune mechanisms and anti-inflammatory effects. Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. Here, for example, adipose tissue-derived mesenchymal stem cells (which may also be referred to as adipose tissue-derived stromal cells) refer to mesenchymal stem cells that exist (or have existed) in adipose tissue. From the viewpoint of effectiveness in treating immune diseases, liver diseases, arthropathy, etc., the mesenchymal stem cells according to this embodiment are preferably adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells, and are preferably adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells. Of these, adipose tissue-derived mesenchymal stem cells are particularly preferred because they can be obtained easily and in large quantities. The mesenchymal stem cells according to this embodiment include mesenchymal stem cells produced by inducing differentiation of pluripotent stem cells such as ES cells and iPS cells.

[0023] (Extracellular vesicle secretion medium) Unless otherwise specified, the medium according to this embodiment refers to a medium for secreting extracellular vesicles used when secreting extracellular vesicles from cells. Hereinafter, the medium according to this embodiment will be specifically referred to as a "medium for secreting extracellular vesicles", and other media, such as media for growing cells, will be referred to as a "growth medium" or the like.

[0024] The basic components constituting the medium for secreting extracellular vesicles according to this embodiment may be the same as the components contained in a medium for proliferation of stem cells (e.g., a medium for proliferation of mesenchymal stem cells). Examples of the basic components of the medium for secreting extracellular vesicles according to this embodiment include amino acids, vitamins, inorganic salts, and other components. Specifically, the amino acids include essential amino acids such as isoleucine, leucine, lysine, methionine, phenylalanine, threonine (threonine), tryptophan, valine, and histidine, and non-essential amino acids such as tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, alanine, and arginine; and the vitamins include ascorbic acid, choline, myoinositol, niacinamide, pantothenate, and the like. Examples of the components in the composition include phosphate, pyridoxine, pyridoxal, thiamine, putrescine, biotin, cyanocobalamin, folic acid and riboflavin; inorganic salts include FeSO4, CuSO4, MnSO4, Na2SiO3, ZnSO4, MgSO4, CaCl2, NaH2PO4, Na2HPO4, NaCl, KCl and NaHCO3; other components include buffers such as HEPES, carbon sources such as glucose and pyruvic acid, and antioxidants such as glutathione (reduced form).

[0025] However, the medium for secreting extracellular vesicles according to the present embodiment is preferably such that the number of microparticles having a particle diameter of 1 to 1000 nm per 1 mL of the medium for secreting extracellular vesicles before use is 1×10 9 Conventional media (such as a medium in which serum is added to a basal medium, or a medium in which hormones and specific nutritional components are added to a basal medium (also called a serum-free medium)) contain many extracellular vesicle-like particles having a particle size equivalent to that of the extracellular vesicles to be secreted. When a medium containing such extracellular vesicle-like particles is used in the secretion step, the presence or absence of secretion of extracellular vesicles and the amount of secretion cannot be confirmed by the physical indicators described below. Therefore, the medium for secreting extracellular vesicles according to this embodiment is a medium in which extracellular vesicle-like particles are eliminated as much as possible, and the number of fine particles having a particle size of 1 to 1000 nm before use is preferably 5×10 8The particle size is less than 10 particles / mL. In addition, the particle size can be set according to the target extracellular vesicles, and in the case of recovering exosomes described later, the particle size is preferably 10 to 500 nm, more preferably 30 to 200 nm, and particularly preferably 50 to 150 nm. When the number of microparticles is within such a range, the degree of interference (extracellular vesicle-like particles being detected together with extracellular vesicles) is within an acceptable range when detecting extracellular vesicles by physical indicators. The method for measuring the number of microparticles present in the medium will be described later.

[0026] The medium for secreting extracellular vesicles according to the present embodiment preferably does not contain lipids or contains less than 100 nM lipids (preferably less than 50 nM, more preferably less than 10 nM). This is because lipids can cause the formation of fine particles in the medium. In addition, lipids are required as components of cell membranes when cells grow, but the medium for secreting extracellular vesicles according to the present embodiment is not required in the first place because it is not intended for cell growth. Here, lipids that can be contained in the cell growth medium are typically at least one selected from unsaturated fatty acids, saturated fatty acids, and sterols. In addition, main lipids that constitute the cell membrane of mesenchymal stem cells include oleic acid, arachidonic acid, linoleic acid, stearic acid, and palmitic acid. Therefore, in the medium for extracellular vesicle secretion according to this embodiment, the total concentration of oleic acid, arachidonic acid, linoleic acid, stearic acid and palmitic acid is preferably less than 100 nM, more preferably less than 50 nM, particularly preferably less than 10 nM, and most preferably less than 5 nM.

[0027] In addition, the medium for secreting extracellular vesicles according to the present embodiment preferably does not contain a surfactant or contains a surfactant at less than the critical micelle concentration. In conventional media, surfactants are added to solubilize insoluble components such as the above-mentioned lipids and some nutritional components in the medium or to promote cell growth. In the detection of extracellular vesicles using physical indicators, micelles formed by this solubilization can be detected together with extracellular vesicles as extracellular vesicle-like particles. In addition, the present inventors have confirmed the formation of extracellular vesicle-like particles even when the surfactant in the medium is less than the critical micelle concentration. This is presumably due to the formation of micelle-like bodies (like micelles formed to wrap around lipids in the medium). In order to suppress the influence of the formation of the micelle-like body on the detection of extracellular vesicles, the concentration of the surfactant is preferably 10 times the critical micelle concentration (1 / 10 of the critical micelle concentration) or less, more preferably 50 times or less, even more preferably 100 times or less, particularly preferably 500 times or less, and particularly preferably 1000 times or less. Examples of the surfactant include Tween 80, SDS, Tergitol 7, Irgasan, and monesin. The critical micelle concentration of the surfactant can be measured, for example, by the following method: A surface tensiometer Sigma (manufactured by KSV Instruments) is used to perform analysis using an analysis program in the Sigma system. The surfactant is dropped into an aqueous medium in 0.01% increments, and the interfacial tension after stirring and standing is measured. From the obtained surface tension curve, the surfactant concentration at which the interfacial tension does not decrease even when the surfactant is dropped is calculated as the critical micelle concentration. Using this method, the critical micelle concentration of Tween 80 in aqueous media was measured using a Sigma surface tensiometer and found to be 15 mg / L.

[0028] Furthermore, the medium for secreting extracellular vesicles according to the present embodiment preferably contains a cytokine belonging to the TNF (tumor necrosis factor) family. Specifically, the medium for secreting extracellular vesicles according to the present embodiment preferably contains TNF-α and TRAIL / APO2L as cytokines. These cytokines contribute to the induction of cell death, and are not added to culture media for growing or maintaining cells. Usually, when secreting extracellular vesicles, a medium suitable for growing or maintaining cells, in which the cells show a high survival rate, is used. However, as described above, the medium for secreting extracellular vesicles according to the present embodiment does not assume cell growth. By containing these cytokines in the medium for secreting extracellular vesicles, the amount of extracellular vesicles secreted from the cells is increased, although the growth and maintenance of cells are suppressed compared to the case where these cytokines are not contained. The contents of TNF-α and TRAIL / APO2L before use are preferably 0.0001 to 2.0 mg / L, and particularly preferably 0.001 to 1.0 mg / L, respectively. When the contents of TNF-α and TRAIL / APO2L are within such ranges, damage to cells is minimized and extracellular vesicles are efficiently secreted from the cells. For the reasons described above, the medium for secreting extracellular vesicles according to this embodiment does not need to contain growth factors (e.g., bFGF, EGF, PDGF and / or TGF-β1) that may be contained in conventional media. When the components of the medium are unknown, the presence or absence of the above proteins can be determined by a conventionally known protein detection method (e.g., ELISA method, Western blotting method) or the like.

[0029] The medium for secreting extracellular vesicles according to the present embodiment preferably has a total protein amount of 100 μg / mL or less before use. Conventional media contain a variety of proteins in large amounts for cell growth or maintenance. However, these proteins may remain in the product as impurities when purifying extracellular vesicles. Therefore, the medium for secreting extracellular vesicles according to the present embodiment is a medium in which proteins unnecessary for secretion of extracellular vesicles are removed as much as possible, and the total protein amount per 1 mL of the medium for secreting extracellular vesicles before use is preferably 50 μg / mL or less, more preferably 30 μg / mL or less, and particularly preferably 20 μg / mL or less. When the total protein amount is within such a range, the impurities during purification are within an acceptable range. The total protein amount in the medium can be measured by a conventionally known measurement method. Examples of methods for measuring the total protein amount include the Bradford method, the WST method, the Biuret method, the Lowry method, and the BCA method. Note that these measurement methods may be interfered with by surfactants and the like in the medium, so the medium needs to be appropriately diluted before measurement.

[0030] The medium for secreting extracellular vesicles according to the present embodiment is preferably free of animal-derived components (e.g., bovine serum such as fetal bovine serum, human serum). Serum contains a large amount of protein, and in addition, performance varies between lots, and there is a risk of infection with mad cow disease, etc. By making the medium for secreting extracellular vesicles free of such components that may have adverse effects on the living body, the extracellular vesicles produced by the production method according to the present embodiment can also be used in clinical trials, etc.

[0031] (Method of Producing Extracellular Vesicles) The method for producing extracellular vesicles according to the present embodiment will be described with reference to FIG. 1. In the drawings referred to below, the dimensional ratios are exaggerated for the convenience of explanation and may differ from the actual ratios. In addition, the drawings cited in the following embodiments show simplified or schematic configurations or omit some components for the convenience of explanation. FIG. 1 is a schematic process diagram for producing extracellular vesicles according to the present embodiment.

[0032] First, as shown in FIG. 1(a), a stem cell 10, a growth medium 20, and a culture vessel 30 are prepared, and a culture step for growing the cells is performed. However, this step may be omitted. The growth medium 20 in this step may be a conventionally known medium suitable for growing stem cells. Examples of the growth medium 20 that can be used include a medium in which serum is added to a basal medium, and a serum-free medium. The serum also includes a cell culture additive {a medium made from bovine serum containing growth factors and cytokines: for example, NeoSERA (registered trademark) manufactured by JAPAN BIOMEDICAL Co., Ltd.}. In addition, during the culture step, the medium may be cultured in a basal medium in which serum is added, and then the medium may be replaced with a serum-free medium and cultured again. A general cell culture flask or dish may be used as the culture vessel 30. For the culture, an incubator or a thermostatic bath adjusted to a desired temperature and a desired carbon dioxide concentration may be used. One embodiment of the culture step includes, for example, a step of preparing a proliferation medium 20 by adding 2% NeoSERA (registered trademark) to a DMEM / Ham's F-12 medium, a step of warming the proliferation medium 20 to room temperature to 37°C before use, a step of suspending stem cells 10 in the proliferation medium 20 to a desired density, a step of seeding the stem cells in a culture vessel 30 such as a cell culture flask, and a step of culturing the culture vessel 30 under conditions of 37°C and 5% CO2. In another embodiment, the step further includes a step of replacing the proliferation medium 20 with a serum-free medium (for example, KBM ADSC-4 manufactured by Kohjin Bio Co., Ltd.). When replacing the proliferation medium 20, the existing medium may be removed and a new medium may be added directly to the culture vessel 30, or the stem cells 10 may be suspended in the new medium and seeded in a new culture vessel 30. When a medium containing serum is used as the proliferation medium 20, it is preferable to include such an acclimation operation to the serum-free medium. In this step, it is preferable to grow the stem cells 10 to 80 to 90% confluence. Note that "confluence" means that the ratio of the area occupied by the cells to the entire culture surface of the culture vessel is about 100%, that is, the state in which the cells have grown over the entire culture surface with no gaps.

[0033] 1(b), a medium 40 for secreting extracellular vesicles is prepared, and a secretion step is performed. Here, as a pre-step of the secretion step, a confirmation step may be performed to confirm that the prepared medium is the medium 40 for secreting extracellular vesicles. As a means for the confirmation step, for example, (1) the number of microparticles having a particle diameter of 1 to 1000 nm per 1 mL of medium is 1×10 9 (1) the medium contains less than 100 μg / mL of TNF-α and TRAIL / APO2L; and (2) the medium contains 100 μg / mL or less of total protein per mL of medium. In the secretion step, for example, the stem cells 10 grown in the culture step are cultured in the medium 40 for secreting extracellular vesicles to secrete extracellular vesicles from the stem cells 10. When replacing the medium from the medium 20 for proliferation to the medium 40 for secreting extracellular vesicles, for example, the same method as that for replacing the medium 20 for proliferation described above can be used.

[0034] Next, at least a part of the medium 40 for secreting extracellular vesicles during the secretion step in FIG. 1(b) is collected, and a judgment step is performed. Specifically, in the judgment step, it is judged whether or not the secretion step can be terminated based on the physical indicators of the components in the collected medium 40 for secreting extracellular vesicles. In this specification, the term "physical indicator" refers to, for example, particle size, particle size distribution, etc., and excludes biological indicators such as detection of extracellular vesicle markers. Specifically, for example, the termination condition is (1) the number of fine particles with a particle size of 1 to 1000 nm in 1 mL of culture supernatant is three times or more as high as that at the start of the secretion step, and (2) when the particle size in 1 mL of culture supernatant is plotted on the horizontal axis in 1 nm increments and the number of particles on the vertical axis, the number of particles is 3×10 in the region of particle size 1 to 200 nm. 7If at least one of the above peaks, preferably all of the above peaks are present, it is determined that the secretion process can be completed. In addition, the "start time of the secretion process" in (1) refers to before the secretion process or immediately after the start of the secretion process, and either the medium 40 for secreting extracellular vesicles before use or the medium 40 for secreting extracellular vesicles collected immediately after the start of the secretion process may be used as the reference. In addition, the particle size in (1) can be set according to the target extracellular vesicles, and in the case of recovering exosomes described later, it is preferably 10 to 500 nm, more preferably 30 to 200 nm, and particularly preferably 50 to 150 nm. Furthermore, in (1), the number of fine particles with a particle size of 1 to 1000 nm in 1 mL of culture supernatant can be 5 times or more, 10 times or more, 15 times or more, 20 times or more, or 30 times or more based on the start time of the secretion process. When the extracellular vesicle secretion medium 40 according to this embodiment is used, for example, 24 to 72 hours after the start of the secretion step (start of culture), the number of microparticles with a particle diameter of 1 to 1000 nm in 1 mL of culture supernatant becomes three times or more compared to the number at the start of culture. According to this embodiment, the secretion step can be terminated at an appropriate timing that ensures a sufficient amount of extracellular vesicles.

[0035] The medium 40 for secreting extracellular vesicles used for measuring the physical indicators may be collected and used as is, or may be one from which cell debris and the like have been removed. For measuring the number and size of particles in the medium 40 for secreting extracellular vesicles, for example, a measurement method using light (e.g., nanotracking method, dynamic light scattering method), electrons (e.g., scanning electron microscope), electricity {Tunable resistive pulse sensing method; measuring the electrical resistance when nanoparticles pass through nano-sized holes}, etc. can be used, and specifically, NANOSIGHT (registered trademark) LM10-HS and Zetasizer (registered trademark) Nano ZS, etc., manufactured by Malvern Panalytical, can be used for measurement. Therefore, according to this embodiment, in order to confirm the presence of extracellular vesicles, there is no need for a complicated means such as detection of extracellular vesicle markers by ELISA method, Western blotting method, etc.

[0036] Furthermore, the method for producing extracellular vesicles according to this embodiment may further include a recovery step of recovering extracellular vesicles in the medium for secreting extracellular vesicles as a post-step of the secretion step. The recovery step includes a step of recovering the culture supernatant (extracellular vesicle-containing composition) from the culture vessel 30 after the secretion step. Usually, extracellular vesicle-like particles, proteins, etc. contained in the medium cannot be removed. However, according to this embodiment, a composition containing extracellular vesicles with relatively few impurities can be obtained.

[0037] (Extracellular vesicle-containing composition) The composition containing extracellular vesicles according to the present embodiment contains extracellular vesicles. Here, "extracellular vesicles" refer to vesicles produced in stem cells and secreted from stem cells. Examples of extracellular vesicles include membrane particles, membrane vesicles, microvesicles, nanovesicles, microvesicles (average particle size 30 to 1000 nm), exosome-like vesicles, exosomes (average particle size 30 to 200 nm), ectosome-like vesicles, ectosomes, and exovesicles. Among these, exosomes encapsulate nucleic acid substances such as miRNA, proteins, etc., and the extracellular vesicles according to the present embodiment are preferably exosomes from the viewpoint of clinical research, etc. Therefore, the size of the extracellular vesicles according to the present embodiment is a particle diameter of 1 to 1000 nm, preferably 10 to 500 nm, more preferably 30 to 200 nm, and particularly preferably 50 to 150 nm. In addition, extracellular vesicles derived from stem cells can be distinguished based on their intracellular origin, density, shape, sedimentation rate, lipid composition, marker proteins, and secretion pattern of the extracellular vesicles in sucrose. Furthermore, the extracellular vesicles contain any of phosphatidylserine, phosphatidylcholine, cholesterol, sphingomyelin, and ceramide as their constituent lipids.

[0038] The composition containing extracellular vesicles according to the present embodiment preferably has an expression level of the extracellular vesicle marker relative to the number of microparticles having a particle diameter of 1 to 1000 nm of 5.0 × 10 -9 pg / particle or more, and more preferably 1.0×10 -8pg / particle or more. Conventional culture media contain many extracellular vesicle-like particles that do not have extracellular vesicle markers, so the amount of extracellular vesicle markers relative to the number of microparticles in the obtained extracellular vesicle-containing composition is relatively low. On the other hand, the extracellular vesicle-containing composition according to the present embodiment contains almost no extracellular vesicle-like particles derived from the culture medium, so the amount of extracellular vesicle markers relative to the number of microparticles in the obtained extracellular vesicle-containing composition is relatively high. In other words, the extracellular vesicle-containing composition according to the present embodiment contains high-purity extracellular vesicles. The presence or absence of extracellular vesicle markers can be measured by conventionally known ELISA methods, Western blotting methods, and the like. In addition, the "amount of extracellular vesicle marker" in this specification is a measurement value by sandwich ELISA using CD9 / CD63 fusion protein (standard protein) as a standard, specifically, a measurement value using CD9 / CD63 Exosome ELISA Kit (EXH0102EL; manufactured by Cosmo Bio Co., Ltd.).

[0039] (Extracellular vesicle manufacturing system) 2 is a block diagram showing a schematic configuration of the extracellular vesicle production system 100 according to the present embodiment. As shown in FIG. 2, the extracellular vesicle production system 100 includes an extracellular vesicle production apparatus 200 and an extracellular vesicle production management apparatus 300.

[0040] The extracellular vesicle-producing apparatus 200 is a culture device capable of maintaining a predetermined temperature inside, such as an incubator or a thermostatic bath. A culture vessel 30 is stored inside the extracellular vesicle-producing apparatus 200. The culture vessel 30 contains a medium 40 for secreting extracellular vesicles and stem cells 10.

[0041] The extracellular vesicle production management apparatus 300 is a general personal computer (hereinafter also referred to as PC) device, a notebook PC, a smartphone, a tablet terminal, etc. The extracellular vesicle production management apparatus 300 includes a medium information input unit 310, a secretion process termination possibility determination unit 320, and a display unit 340. In one embodiment, the extracellular vesicle production management apparatus 300 further includes an extracellular vesicle secretion medium confirmation unit 330.

[0042] The culture medium information input unit 310 may be, for example, an operation input means such as a keyboard, a mouse, a touch panel, etc. In addition, in an embodiment, the culture medium information input unit 310 may be a memory connected to an interface circuit for communicating with the culture medium information acquisition unit 210 described later.

[0043] The secretion process termination determination unit 320 and the extracellular vesicle secretion medium confirmation unit 330 are configured with a CPU (Central Processing Unit), a semiconductor memory such as a ROM (Read Only Memory) or a RAM (Random Access Memory), a non-volatile memory including a HDD (Hard Disk Drive) or an SSD (Solid State Drive), etc. The memory stores an operating system program, a driver program, an application program, and data used when determining whether the secretion process can be terminated or when confirming the medium for extracellular vesicle secretion. The program stored in the memory may be provided by a non-transitory recording medium (not shown) on which the program is readably recorded. Examples of the recording medium include a portable memory such as a CD-ROM, a USB (Universal Serial Bus) memory, a SD (Secure Digital) card, a micro SD card, and a Compact Flash (registered trademark).

[0044] The medium information input unit 310 may be input by a user with information about the medium 40 for secreting extracellular vesicles in the culture vessel 30. Examples of information about the medium 40 for secreting extracellular vesicles include the number of microparticles in the medium, components and concentrations, and total protein amount. In addition, information about the stem cells 10 (e.g., type of stem cells 10, seeding number, etc.) may be input. The extracellular vesicle producing apparatus 200 may further be provided with a medium information acquisition unit 210 for detecting the information about the medium 40 for secreting extracellular vesicles.

[0045] The culture medium information acquisition unit 210 includes a culture medium sampling unit 212 and a culture medium information detection unit 214. A part of the culture medium 40 for secreting extracellular vesicles in the culture vessel 30 is sampled by the culture medium sampling unit 212, and information such as the number of particles, components, concentration, and total protein amount in the culture medium is acquired by the culture medium information detection unit 214. Alternatively, the information on the culture medium 40 for secreting extracellular vesicles may be acquired by placing a sensor or the like directly in the culture medium 40 for secreting extracellular vesicles. Information acquired by these methods may be input to the culture medium information input unit 310 that is connected to be able to communicate wirelessly or by wire.

[0046] The secretion process termination determination unit 320 determines whether the secretion process can be terminated based on the information input to the medium information input unit 310. The determination of whether the secretion process can be terminated is made based on physical indicators, specifically, for example, when (1) the number of fine particles with particle diameters of 1 to 1000 nm in 1 mL of culture supernatant is more than doubled based on the time of the start of the secretion process, and (2) when the particle diameters in 1 mL of culture supernatant are plotted on the horizontal axis in 1 nm increments and the number of particles on the vertical axis, the number of particles is 3×10 in the region of particle diameters of 1 to 200 nm. 7 If at least one, and preferably all, of the above conditions are met, it is determined that the secretion process can be terminated.

[0047] In one embodiment, the medium confirmation unit 330 for secreting extracellular vesicles confirms whether the medium is a medium for secreting extracellular vesicles based on the information input to the medium information input unit 310. Specifically, the determination of whether the medium is a medium for secreting extracellular vesicles is made, for example, when (1) the number of microparticles having a particle diameter of 1 to 1000 nm per 1 mL of medium is 1×10 9 It is confirmed that the medium satisfies at least one, preferably two or more, and more preferably all of the following criteria: (1) the medium contains less than 100 μg / mL of TNF-α and TRAIL / APO2L; and (2) the medium contains a total protein amount of 100 μg / mL or less per mL of medium.

[0048] The display unit 340 displays necessary information on a display (not shown). The information displayed by the display unit 340 includes the determination result determined by the secretion process termination possibility determination unit 330, the confirmation result, and the like.

[0049] 3 is a flowchart showing an example of the operation of the extracellular vesicle production system 100. The example shown below is an embodiment including the confirmation steps (S10 and S20), but in another embodiment, the confirmation steps (S10 and S20) may be omitted. First, medium information before the start of the secretion process obtained by the medium information acquisition unit 210 is input to the medium information input unit 80 (S10). The medium information before the start of the secretion process may include the number of microparticles with a particle diameter of 1 to 1000 nm per 1 mL of medium, the presence or absence of TNF-α and TRAIL / APO2L, the total protein amount per 1 mL of medium, etc. The medium information before the start of the secretion process may be input to the medium information input unit 310 by the user. Next, based on the medium information before the start of the secretion step that was input, it is determined whether the medium is a medium for secreting extracellular vesicles (specifically, for example, (1) the number of microparticles having a particle diameter of 1 to 1000 nm per 1 mL of medium is 1×10 9 It is confirmed that at least one, preferably two or more, and more preferably all of the following conditions are met (S20): (1) the number of cells in the medium is less than 100, (2) the medium contains TNF-α and TRAIL / APO2L, and (3) the total amount of protein per 1 mL of medium is 100 μg / mL or less. If the medium is a medium for secreting extracellular vesicles (yes in S20), the secretion process is started (S30). If the medium is not a medium for secreting extracellular vesicles (no in S20), the production of extracellular vesicles is terminated. Next, the medium information during the secretion process obtained by the medium information acquisition unit 210 is input to the medium information input unit 80 (S40). Examples of the medium information during the secretion process include the number of microparticles with a particle size of 1 to 1000 nm in 1 mL of culture supernatant, the number of particles in 1 nm increments with a particle size range of 1 to 200 nm in 1 mL of culture supernatant, etc. The medium information during the secretion process may be input to the medium information input unit 310 by the user. Next, based on the inputted medium information during the secretion process, whether or not the secretion process termination conditions are met (specifically, for example, termination conditions (1) the number of particles with a particle diameter of 1 to 1000 nm in 1 mL of culture supernatant is more than doubled based on the time of the start of the secretion process, and (2) when the particle diameter in 1 mL of culture supernatant is plotted on the horizontal axis in 1 nm increments and the particle number on the vertical axis, the particle number in the region of particle diameter 1 to 200 nm is 3×10 7 It is determined whether at least one, preferably all, of the conditions for terminating the secretion process are met (S50). If the conditions for terminating the secretion process are met (yes in S50), the production of extracellular vesicles is terminated. If the conditions for terminating the secretion process are not met (no in S50), the process returns to inputting medium information during the secretion process in S40.

[0050] According to the extracellular vesicle production system 100 of this embodiment, the secretion process of extracellular vesicles can be managed according to medium information. That is, according to the extracellular vesicle production system 100, extracellular vesicles can be obtained by a simple operation.

[0051] The present invention is not limited to the above-described embodiments, and various modifications such as design changes may be made based on the knowledge of those skilled in the art. Such modified embodiments are also included in the scope of the present invention. EXAMPLES

[0052] 1. Measurement of particle and total protein content in culture medium before use Each medium component was measured before use. Specifically, the particle size distribution and total protein amount were measured for the medium for secreting extracellular vesicles according to the present embodiment (see Table 1 for the composition per 1000 mL of medium) and the conventional medium {serum-free medium: KBM (registered trademark) ADSC-4 (manufactured by Kohjin Bio Co., Ltd.)}. Note that, as shown in Table 1, the contents of TNF-α and TRAIL / APO2L in the medium for secreting extracellular vesicles before use were 0.01 mg / L, respectively.

[0053] [Table 1]

[0054] (Measurement of particle size distribution) For each medium, the number and particle size of particles in the medium were measured using NANOSIGHT (registered trademark) LM10-HS (manufactured by Malvern Panalytical). Each medium was diluted 20 to 200 times. Figure 4 shows photographs of each medium observed during measurement. From Figure 4, it was found that the conventional medium contained a large amount of particles before use, while the medium for extracellular vesicle secretion contained almost no particles. In addition, the particle size distribution in the particle size range of 1 to 1000 nm per 1 mL of medium at this time (particle size in 1 nm increments on the horizontal axis and particle number on the vertical axis) is shown in Figure 5, and the number of particles in each particle size range is shown in Table 2.

[0055] [Table 2]

[0056] As shown in Figure 5, the conventional medium contained a large amount of microparticles with a particle diameter of 1 to 1000 nm before use, whereas the medium for extracellular vesicle secretion contained almost no such microparticles. Specifically, as shown in Table 2, the medium for extracellular vesicle secretion contained 1 x 10 microparticles with a particle diameter of 1 to 1000 nm before use. 9 The number was less than 1 / mL.

[0057] (Measurement of total protein amount) The total protein amount was measured for each medium using a total protein measurement kit Qubit (registered trademark) protein assay kit (manufactured by Thermo Fisher Scientific). Specifically, 0.5 to 5% of the total protein measurement reagent was added and mixed with a vortex mixer. At this time, the total protein measurement reagent was also added to the standard protein concentration solution and its serial dilution solution, and mixed in the same manner. The fluorescence intensity of each sample was measured using a Qubit 4 Fluorometer (manufactured by Thermo Fisher Scientific). The total protein concentration of each sample was calculated from the fluorescence intensity of the standard protein concentration solution and its serial dilution solution. The results are shown in Table 3.

[0058] [Table 3]

[0059] As can be seen from Table 3, before use, the conventional medium contained a large amount of protein, whereas the total protein amount in the medium for extracellular vesicle secretion was below the detection limit (12.5 μg / mL).

[0060] 2. Secretion of extracellular vesicles using the extracellular vesicle secretion medium A proliferation medium, which was prepared by adding 2% of a cell culture additive {NeoSERA (registered trademark) (JAPAN BIOMEDICAL)} to a basal medium {D-MEM / Ham's F-12 (Fujifilm Wako Pure Chemical Industries, Ltd.)}, was heated to room temperature to 37°C. Next, adipose-derived mesenchymal stem cells (samples 1 and 2) collected from humans were added at 3 × 10 3 cells / cm 2 The cells were suspended at a density of 100 μg / ml and seeded in a cell culture flask. Each cell culture flask was placed in an incubator adjusted to 37° C. and 5% CO2, and the culture process was started.

[0061] Next, each adipose-derived mesenchymal stem cell was detached with trypsin, suspended in serum-free medium {KBM (registered trademark) ADSC-4 (manufactured by Kohjin Bio Co., Ltd.)}, and seeded in a new cell culture flask. At this time, the cell density was 3 × 10 4 cells / cm 2 Each cell culture flask was placed in an incubator adjusted to 37°C and 5% CO2, and cultured again until the cells were grown to 80 to 90% confluent.

[0062] After removing the medium from each cell culture flask, washing with PBS buffer (addition and removal of PBS buffer) was performed twice. After washing, the medium for extracellular vesicle secretion (see Table 1 for composition) was added to sample 1, and the basal medium was added to sample 2. Each cell culture flask was left standing in an incubator adjusted to 37°C and 5% CO2. After 48 hours, a part of each culture supernatant (extracellular vesicle-containing composition) was collected (hereinafter, the extracellular vesicle-containing composition samples sampled from samples 1 and 2 are referred to as Example 1 and Comparative Example 1, respectively). Here, FIG. 6 shows the morphology of the cells immediately after the addition of the medium for extracellular vesicle secretion to sample 1 and after 48 hours. In addition, the cell viability of sample 1 after 48 hours was measured using the trypan blue method and was 96%. Therefore, it was found that TNF-α and TRAIL / APO2L contained in the medium for extracellular vesicle secretion hardly damage the cells.

[0063] After removing cell debris from each sample, extracellular vesicle markers were detected by ELISA. Specifically, extracellular vesicles expressing both CD9 and CD63 were detected using the CD9 / CD63 Exosome ELISA Kit (Cosmo Bio Co., Ltd.). The amount of extracellular vesicle markers per unit volume at this time is shown in Figure 7.

[0064] FIG. 7 shows that when the medium for extracellular vesicle secretion was added, the amount of extracellular vesicles increased compared to the basal medium not containing TNF-α and TRAIL / APO2L.

[0065] 3. Size distribution of microparticles during secretion As in 2., adipose-derived mesenchymal stem cells (samples 3 to 7) were cultured using proliferation medium, then suspended in serum-free medium and cultured again in an incubator adjusted to 37°C and 5% CO2, until they were grown to 80-90% confluence.

[0066] After removing the medium from each cell culture flask, washing with PBS buffer (addition and removal of PBS buffer) was performed twice. After washing, extracellular vesicle secretion medium (see Table 1 for composition) was added to samples 3 to 5, and conventional medium {KBM (registered trademark) ADSC-4 (manufactured by Kohjin Bio Co., Ltd.)} was added to samples 6 and 7. Each cell culture flask was placed in an incubator adjusted to 37°C and 5% CO2 to start the secretion process.

[0067] For each specimen, a portion of each culture supernatant (extracellular vesicle composition) was sampled at the start of the secretion process and at specified time intervals (hereinafter, the culture supernatant samples sampled from specimens 3 to 7 are referred to as Examples 2 to 4, Comparative Example 2 and Comparative Example 3, respectively).

[0068] After removing the cell debris from each sample, the number of particles and particle diameter were measured in the same manner as in 1. Figure 8 shows the particle size distribution in the particle diameter range of 1 to 1000 nm per 1 mL of sample (particle diameter is plotted on the horizontal axis in 1 nm increments, and particle number is plotted on the vertical axis). Figure 8 shows that since the conventional medium already contains a large amount of particles at the start of the secretion process, particles derived from the medium and extracellular vesicles cannot be distinguished. On the other hand, since the medium for secreting extracellular vesicles contains almost no particles derived from the medium, it was found that the secretion of extracellular vesicles can be detected by physical indicators. Specifically, in 1 mL of culture supernatant, a particle number of 3×10 7It was found that the secretion of a sufficient amount of extracellular vesicles can be detected using the presence of one or more peaks as an indicator. In this example, based on the above indicators, it was found that a sufficient amount of extracellular vesicles was secreted after 48 hours in Example 3, and after 24 hours in Examples 4 and 5.

[0069] Table 4 shows the number of particles in each particle size range at the start of the secretion process and the ratio of the number of particles after each specified time has elapsed based on this number.

[0070] [Table 4]

[0071] From Table 4, it was found that in the medium for secreting extracellular vesicles, the secretion of extracellular vesicles can be detected by using the number of fine particles with a particle diameter of 1 to 1000 nm in 1 mL of culture supernatant as an indicator of three times or more the number at the start of the secretion process. In other words, it was found that it is possible to determine whether the secretion process has ended.

[0072] 4. Measuring the amount of extracellular vesicle markers relative to particle number As in 2. above, samples 8 and 9 were subjected to the culture step and the secretion step. However, the secretion step was carried out by adding an extracellular vesicle secretion medium (see Table 1 for composition) to sample 6 and a conventional medium {KBM (registered trademark) ADSC-4 (manufactured by Kohjin Bio Co., Ltd.)} to sample 7. A portion of each culture supernatant (extracellular vesicle-containing composition) was collected at predetermined time intervals from the start of the secretion step (hereinafter, the extracellular vesicle-containing composition samples sampled from samples 8 and 9 are referred to as Example 5 and Comparative Example 4, respectively).

[0073] After removing cell debris from each sample, the number of particles and particle size were measured in the same manner as in 1. Furthermore, extracellular vesicle markers were detected for each sample by ELISA. Specifically, extracellular vesicles expressing both CD9 and CD63 were detected using a CD9 / CD63 Exosome ELISA Kit (EXH0102EL; manufactured by Cosmo Bio Co., Ltd.). The ratio of the amount of extracellular vesicle markers based on the number of particles with a particle diameter of 1 to 1000 nm is shown in FIG. 9.

[0074] 9, the composition containing extracellular vesicles according to the present embodiment had fewer extracellular vesicle-like particles and was highly pure, compared to the extracellular vesicle composition obtained using a conventional medium. Specifically, the composition containing extracellular vesicles according to the present embodiment had an expression level of extracellular vesicle markers of 5.0×10 relative to the number of microparticles with a particle diameter of 1 to 1000 nm. -9 It was found to be more than pg / particle. [Explanation of symbols]

[0075] 10 Stem cells, 20 Growth medium, 30 Culture vessel, 40 Medium for secreting extracellular vesicles, 100 Extracellular vesicles production system, 200 Extracellular vesicles production device, 210 Medium information acquisition unit, 212 Medium sampling unit, 214 Medium information detection unit, 300 Extracellular vesicles production management device, 310 Medium information input unit, 320 Secretion process completion possibility determination unit, 330 Medium for secreting extracellular vesicles confirmation unit, 340 Display unit

Claims

1. A process of culturing stem cells in an extracellular vesicle secretion medium containing TNF-α and TRAIL / APO2L and having a total protein content of 100 μg / mL or less, and secreting extracellular vesicles; and a recovery step of recovering the extracellular vesicles in the medium for secreting extracellular vesicles.

2. The method for producing extracellular vesicles according to claim 1, wherein in the extracellular vesicle-containing composition obtained in the recovery step, the expression levels of CD9 and CD63 relative to the number of microparticles with a particle diameter of 1 to 1000 nm are 5.0 x 10-9 pg / particle or more.

3. A method for producing extracellular vesicles as described in claim 1 or 2, wherein the stem cells are mesenchymal stem cells.

4. A composition containing extracellular vesicles obtained by the method described in claim 1 or 2.

5. A method for promoting the secretion of extracellular vesicles from stem cells, comprising culturing stem cells in a medium containing TNF-α and TRAIL / APO2L.

6. The method described in claim 5, wherein the stem cells are mesenchymal stem cells.

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