Method for producing culture supernatant

Abstract

To provide a novel method capable of easily improving the content of bioactive substances in culture supernatant.SOLUTION: A method for producing culture supernatant includes: a thawing process of thawing frozen cells; a first culture process of culturing the cells in a first culture medium; a removal process of removing culture supernatant obtained after the first culture process; a second culture process of further culturing the cells after the removal process in a second culture medium; and a recovery process of recovering culture supernatant obtained after the second culture process, where the first culture medium is a basic culture medium or a serum-free culture medium, and the second culture medium does not contain protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a culture supernatant and a method for increasing the cytokine and / or exosome content in the culture supernatant. [Background technology]

[0002] Mesenchymal stem cell culture supernatant contains bioactive substances such as cytokines and exosomes, which are known to have therapeutic effects such as wound healing and immunosuppressive effects. To enhance the therapeutic effects of culture supernatant, it is important to develop a technology that increases the content of bioactive substances in the culture supernatant.

[0003] The simplest method for increasing the content of biologically active substances in culture supernatants is thought to be stimulating cells with drugs, hormones, etc. However, because drugs, hormones, etc. may remain in the culture supernatant, the collected culture supernatant cannot be directly applied to living organisms. Therefore, an additional step of removing harmful components is required, making this method not necessarily simple.

[0004] Patent Document 1 discloses a method for preventing the contamination of serum components into the main culture that produces the culture supernatant by performing a preculture step in which cells are subcultured in a medium supplemented with serum components such as fetal bovine serum, followed by culturing in a non-human protein-free medium. Patent Document 1 does not disclose a method for further increasing the content of biologically active substances in the culture supernatant, or a method for achieving this without relying on drugs, hormones, etc.

[0005] Therefore, there is a need for a technique that can easily increase the content of biologically active substances in culture supernatants. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6860915 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a new method for simply increasing the content of a biologically active substance in a culture supernatant. [Means for solving the problem]

[0008] In conventional methods for producing culture supernatants containing bioactive substances, it has generally been considered advantageous to use cells in a good state of proliferation that have been subcultured under conditions suitable for growth. Therefore, when producing a culture supernatant by thawing stored frozen cells, it has been common technical knowledge to produce the culture supernatant after at least several passages.

[0009] In order to solve the above-mentioned problems, the present inventors investigated conditions under which the content of biologically active substances can be improved when thawing frozen cells to produce a culture supernatant. As a result, they discovered the surprising effect that, contrary to conventional technical knowledge, a culture supernatant with an improved content of biologically active substances can be obtained from cells with a low passage number after thawing. The method of the present invention is also extremely advantageous in that it does not require stimulation with drugs, hormones, etc., and therefore ensures the safety of the culture supernatant. The present invention is based on the above-mentioned research results and provides the following.

[0010] (1) A method for producing a culture supernatant, comprising: a thawing step of thawing frozen cells; a first culturing step of culturing the cells in a first medium; a removing step of removing the culture supernatant obtained after the first culture step; a second culturing step in which the cells after the removal step are further cultured in a second medium; and a recovery step of recovering the culture supernatant obtained after the second culture step; Including, The first medium is a basal medium or a serum-free medium; and The method, wherein the second medium does not contain protein. (2) The method according to (1), wherein the first medium contains a human protein or a non-human protein. (3) The method according to (1) or (2), wherein the basal medium is selected from the group consisting of EMEM medium, DMEM medium, IMDM medium, GMEM medium, Ham's F10 medium, Ham's F12 medium, RPMI1640 medium, and combinations thereof. (4) The method according to (1) or (2), wherein the serum-free medium is selected from the group consisting of MesenCult, StemPro MSC, BMN211, StemMACS, MSC NutriStem XF, Xuri NSC, PRIME-XV, StemXVivo, Human Mesenchymal-XF Expansion Medium, stemgro, STK2, PLTMax, ProculAD, Mesenchymal Stem Cell Growth Medium XF, Mesenchymal Stem Cell Growth Medium 2, MesenGro, StemFit, CiMS-BM, MSC-Brew GMP Medium, StemXVivo Serum-Free Human MSC Expansion Media, MSC-T4, and any combination thereof. (5) The method according to any one of (1) to (4), wherein the first culture step is carried out for 12 to 120 hours. (6) The method according to any one of (1) to (5), further comprising a growth step of culturing and growing the cells in a serum- or plasma-containing medium after the thawing step and before the first culture step. (7) The method according to (6), which comprises a repeating step of repeating the proliferation step one or more times. (8) The method according to any one of (1) to (7), further comprising a washing step of washing the cells after the removing step and before the second culturing step. (9) The method according to any one of (1) to (8), wherein the second medium is a basal medium. (10) Before the thawing step, a pre-culture step of culturing cells in a pre-culture medium; a replacement step of replacing the pre-culture medium after the pre-culture step with a cryopreservation solution; and a freezing step of freezing the cells after the replacement step; Including, The method according to any one of (1) to (9), wherein the pre-culture medium is a serum- or plasma-containing medium, a serum-free medium, or a basal medium. (11) The method according to (10), wherein the preculture medium contains a human protein or a non-human protein. (12) The method according to (10) or (11), wherein the serum is derived from an animal or is an artificial serum. (13) The method according to (12), wherein the animal-derived serum is selected from the group consisting of fetal bovine serum, newborn bovine serum, bovine serum, and horse serum. (14) The method according to any one of (1) to (13), wherein the cells are mesenchymal stem cells. (15) The method according to (14), wherein the first culture step is carried out for 12 to 48 hours. (16) A composition comprising a culture supernatant produced by the method according to any one of (1) to (15). (17) A method for increasing the content of cytokines and / or exosomes in a culture supernatant, comprising: a thawing step of thawing frozen cells; a first culturing step of culturing the cells in a first medium; a removing step of removing the culture supernatant obtained after the first culture step; a second culturing step in which the cells after the removal step are further cultured in a second medium; and a recovery step of recovering a culture supernatant containing cytokines and / or exosomes obtained after the second culture step; Including, The first medium is a basal medium or a serum-free medium; and The method, wherein the second medium does not contain protein. [Effects of the Invention]

[0011] According to the present invention, a new method for simply increasing the content of a biologically active substance in a culture supernatant is provided. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a diagram showing each step in one embodiment of the culture supernatant producing method of the present invention. This embodiment includes a thawing step (S0101), a first culturing step (S0106), a removal step (S0107), a second culturing step (S0110), and a recovery step (S0111) as essential steps, and includes a growth step (S0103), a repeating step (S0105), and a washing step (S0109) as optional steps. [Figure 2] 1 is a diagram showing each step in one embodiment of the culture supernatant producing method of the present invention. This embodiment includes a pre-culture step (S0201), a substitution step (S0202), a freezing step (S0203), a thawing step (S0204), a first culture step (S0209), a removal step (S0210), a second culture step (S0213), and a recovery step (S0214) as essential steps, and includes a growth step (S0206), a repeating step (S0208), and a washing step (S0212) as selective steps. [Figure 3] FIG. 1 shows an outline of each method used to produce a culture supernatant in Example 1. Methods A to C involve freezing and thawing. Control 1 is a method that does not involve freezing or thawing. [Figure 4] FIG. 1 shows the results of measuring the content of biologically active substances (HGF, VEGF, progranulin, and exosomes) in cell supernatants produced by methods A to C and a method not involving freezing and thawing (Control 1) in Example 1. [Figure 5] FIG. 1 shows an outline of each method used to produce a culture supernatant in Example 2. Methods D to F involve freezing and thawing. Control 2 is a method that does not involve freezing and thawing. [Figure 6] FIG. 1 shows the results of measuring the content of a biologically active substance (HGF) in cell supernatants produced by methods D to F and a method not involving freezing and thawing (Control 2) in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0013] <Method for producing culture supernatant> In one aspect, the present invention relates to a method for producing a culture supernatant. The method for producing a culture supernatant of the present invention includes, as essential steps, a thawing step, a first culturing step, a removing step, a second culturing step, and a recovering step. It may also include other selection steps, such as a proliferation step, a repeating step, a washing step, and a purification step. One embodiment of the method of this aspect is illustrated in Figure 1. The method of this aspect includes, as additional selection steps, a pre-cultivation step, a replacement step, and a freezing step prior to the thawing step (Figure 2). Each step constituting the method of the present invention will be described in detail below.

[0014] (Pre-culture step) The method of this embodiment includes a pre-culture step of culturing cells in a pre-culture medium as a selection step. As used herein, the term "pre-culture medium" refers to a medium used to culture cells in the pre-culture step. The pre-culture medium is a serum- or plasma-containing medium, a basal medium, or a serum-free medium.

[0015] In the method of this embodiment, the serum or plasma contained in the serum- or plasma-containing medium may be derived from an animal or may be artificial serum. The animal may be any of the animal examples described below, including humans and non-human animals (e.g., mammals such as cows or horses). Examples of human-derived serum or plasma include human serum, human plasma, platelet-rich plasma (PRP), and human AB serum (serum derived from human type AB). Examples of non-human-derived serum or plasma include fetal bovine serum, newborn bovine serum, cow serum, and horse serum.

[0016] As used herein, "artificial serum" refers to a medium additive that can replace serum but does not contain animal-derived or xenogeneic animal-derived components. When added to a medium to culture cells, artificial serum can exhibit growth performance equivalent to or superior to that of animal-derived serum. Specific examples of artificial serum include Artificial Serum (Xeno-free) (Cell Science Institute, #87-081), Artificial Serum (Animal-free) (Cell Science Institute, #87-082), and StemSure Serum Substitute (Fujifilm Wako Pure Chemical Industries, Ltd., #191-18375).

[0017] As used herein, "basal medium" refers to a solution containing components necessary for cell survival, such as inorganic salts, essential amino acids, vitamins, and buffers. Examples of basal media include, but are not limited to, EMEM medium (also referred to as αMEM medium), DMEM medium, IMDM medium, GMEM medium, Ham's F10 medium, Ham's F12 medium, RPMI1640 medium, and combinations thereof.

[0018] As used herein, the term "serum-free medium" refers to a medium that contains components necessary for cell survival and proliferation but does not contain unconditioned or unpurified serum. Components necessary for cell survival and proliferation that may be contained in serum-free medium include hormones and growth factors, as well as serum albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, and the like. Furthermore, serum-free medium may contain purified blood-derived components or animal tissue-derived components. Examples of serum-free media include, but are not limited to, MesenCult, StemPro MSC, BMN211, StemMACS, MSC NutriStem XF, Xuri NSC, PRIME-XV, StemXVivo, Human Mesenchymal-XF Expansion Medium, stemgro, STK2, PLTMax, ProculAD, Mesenchymal Stem Cell Growth Medium XF, Mesenchymal Stem Cell Growth Medium 2, MesenGro, StemFit, MSC-T4, CiMS-BM, MSC-Brew GMP Medium, StemXVivo Serum-Free Human MSC Expansion Media, and any combination thereof. Note that media containing the above-mentioned artificial serum are also considered to be serum-containing media.

[0019] In one embodiment, the pre-culture medium contains a protein. By including a protein in the pre-culture medium, cell proliferation, adhesion, and differentiation induction (or maintenance of undifferentiated state) can be promoted in this step. The protein may be, but is not limited to, a cell growth factor, an adhesion molecule, and a differentiation-inducing factor (or differentiation-inhibiting factor). Specific examples of proteins include fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), endothelial cell growth factor (ECGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), bone morphogenetic protein (BMP), fibronectin, and vitronectin. Proteins that can be included in the pre-culture medium may be proteins derived from humans (referred to herein as "human proteins") or proteins derived from non-human organisms (referred to herein as "non-human proteins"). Human proteins may be isolated from humans, or may be obtained by expressing a recombinant human-derived protein in a genetically modified organism such as a mammal, insect, yeast, or E. coli. Similarly, non-human proteins may be isolated from the species from which the non-human protein originates, or may be obtained by expressing a recombinant protein from that species in a genetically modified organism such as a mammal, insect, yeast, or E. coli. Examples of non-human proteins include proteins from organisms selected from the group consisting of mammals such as rats, mice, guinea pigs, hamsters, pigs, cows, horses, goats, sheep, rabbits, dogs, cats, and monkeys, amphibians such as Xenopus laevis, birds such as chickens and ostriches, insects, nematodes, yeast, E. coli, and plants, preferably non-human mammals.

[0020] In another embodiment, the pre-culture medium is protein-free. In the methods of the present invention, the biological species from which the cells used may be derived is not limited, and may be, for example, mammals such as rats, mice, guinea pigs, hamsters, pigs, cows, horses, goats, sheep, rabbits, dogs, cats, monkeys, and humans, preferably humans. The specific cell species used is also not specified, and may include, for example, ES cells, iPS cells, somatic stem cells, mesenchymal stem cells (e.g., dental pulp-derived stem cells, adipose-derived stem cells, umbilical cord-derived stem cells, umbilical cord blood-derived stem cells, chorionic villi-derived stem cells, chorionic plate-derived stem cells, decidua-derived stem cells, amniotic membrane-derived stem cells, or bone marrow-derived stem cells), fibroblasts, skin cells, hepatocytes, pancreatic cells, neurons, chondrocytes, endothelial cells, epithelial cells, bone cells, muscle cells, germ cells, and blood cells. The cells may be primary cultured cells or cell lines, and may be genetically modified or unmodified. The shape and structure of these cells are not limited, and may be, for example, a planar bonded sheet or a spherical multilayered aggregate.

[0021] The culture conditions for the pre-culture step are not limited, and for example, the culture temperature may be about 30° C. to about 40° C., the CO 2 concentration may be about 2% to about 10%, and either adhesion culture or suspension culture may be used.

[0022] The duration of the pre-culture step is not particularly limited. For example, the pre-culture step may be carried out for several hours to several days, or may be carried out for several days to several weeks or months after expansion culture or subculture. In this specification, "passage" refers to a process in which a portion of a cell population is removed and transferred to a new medium to maintain cell growth.

[0023] (Replacement process) The method of this embodiment includes a selective step of replacing the pre-culture medium after the pre-culture step with a cryopreservation solution. The replacement step is a step of removing the pre-culture medium and adding the cryopreservation solution to the cells. The pre-culture medium can be removed by a conventional method such as decantation or aspiration.

[0024] As used herein, the term "cryopreservation solution" refers to a liquid containing any cryoprotectant that exhibits cryoprotective properties against cells. Examples of cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, carboxylated polylysine, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, and mannitol, as well as any combination thereof. The concentration of the cryoprotectant contained in the cryopreservation solution can be easily determined by those skilled in the art depending on the type of cryoprotectant. For example, the concentration may be 0.1% to 20% (v / v), 1% to 15% (v / v), or 2% to 10% (v / v).

[0025] Alternatively, the cells may be washed with a washing solution between the removal of the pre-culture medium and the addition of the cryopreservation solution. The washing solution may be a buffer containing additives such as sugar, physiological saline, and / or a cryopreservation solution, if necessary. The number of washes may be, for example, one, two, three, or four times.

[0026] In this step, by replacing the pre-culture medium with a cryopreservation solution, it is possible to prevent a decrease in cell viability and proliferation ability after the freezing and thawing steps described below.

[0027] (freezing process) The method of this embodiment includes a freezing step of freezing the cells after the replacement step as a selection step. The method for freezing the cells in this step is not limited; a cryopreservation solution containing the cells may be placed in a cryopreservation container such as a cryotube and cooled to a temperature below which the cryopreservation solution can freeze. The cooling method is not limited, and examples include freezers such as deep freezers and low-temperature media such as liquid nitrogen. The cooling temperature may be below the freezing temperature of the cryopreservation solution. The specific cooling temperature can be appropriately selected depending on the freezing method, such as slow freezing or vitrification. As used herein, "slow freezing" refers to a method in which a cryoprotectant is infiltrated into the cells and then the temperature is gradually lowered to freeze the cells. The cooling temperature in the slow freezing method may be, for example, 0°C or below, -10°C or below, -20°C or below, -30°C or below, -40°C or below, -50°C or below, -60°C or below, -70°C or below, or -80°C or below. As used herein, "vitrification freezing" refers to a method in which cells are frozen using vitrification. "Vitrification" refers to the phenomenon in which a liquid solidifies rather than crystallizes depending on cooling conditions such as the cooling rate and pressure; it is known that water can also vitrify under certain conditions. Vitrification freezing is often used when efficient preservation using slow freezing is difficult. The cooling temperature used in vitrification freezing is, for example, −100°C or lower, −110°C or lower, −120°C or lower, −130°C or lower, −140°C or lower, −150°C or lower, −160°C or lower, −170°C or lower, −180°C or lower, −190°C or lower, or −200°C or lower. The cooling rate is not limited as long as it does not significantly impair cell viability after freezing and thawing, and can be appropriately selected depending on the freezing method, such as slow freezing or vitrification freezing. In the case of a slow freezing method, the cooling rate may be, for example, -1° C. / min to -0.1° C. / min, -0.8° C. / min to -0.2° C. / min, or -0.6° C. / min to -0.3° C. / min, preferably -0.5° C. / min to -0.4° C. / min. Such cooling rates can be achieved by placing a cryopreservation container such as a cryotube in a further freezing treatment container and subjecting it to cooling in a deep freezer or with liquid nitrogen.In the case of a vitrification freezing method, the freezing rate may be -5°C / min to -250°C / min, -10°C / min to -240°C / min, -15°C / min to -220°C / min, -20°C / min to -200°C / min, -50°C / min to -180°C / min, -100°C / min to -160°C / min, or -120°C / min to -150°C / min, and rapid freezing using liquid nitrogen or the like is preferably used. After the freezing step, the cells can be maintained in a frozen state until the thawing step described below.

[0028] (Thaw process) The method of this embodiment includes, as an essential step, a thawing step of thawing frozen cells. The method for thawing the cells in this step is not limited, and any thawing method can be used. Specifically, the cells can be thawed by exposing them to a temperature at which the frozen cells can be thawed or higher. Examples of such temperatures include 2°C to 60°C, 4°C to 50°C, 10°C to 45°C, 20°C to 42°C, 30°C to 40°C, 35°C to 38°C, or 36°C to 37°C, and may be, for example, approximately 37°C. The thawing time is not limited as long as it does not reduce the cell viability or proliferation ability, and may be within 4 minutes, 3 minutes, 2 minutes, or 1 minute, and preferably within 30 seconds or 20 seconds.

[0029] The means for subjecting frozen cells to a temperature equal to or higher than the thawing temperature is not limited, and examples include a method in which the cells are placed in a cryotube or the like and warmed in a water bath, incubator, or thermostatic chamber; a method in which the cryotube or the like is warmed by hand; and a method in which the frozen cells are immersed in a liquid such as a culture medium that has been previously heated to the desired temperature.

[0030] The thawed cells can be added to a medium used for culture in the proliferation step or first culture step described below, and the cells may be washed as needed. The washing solution can be a buffer or physiological saline containing additives such as sugar as needed, and / or the medium used for culture in the proliferation step or first culture step described below.

[0031] (Proliferation process) The method of this embodiment includes a selection step of culturing and growing the cells in a serum- or plasma-containing medium.

[0032] The culture conditions for the proliferation step are not limited, and for example, the culture temperature may be about 30° C. to about 40° C., the CO 2 concentration may be about 2% to about 10%, and either adhesion culture or suspension culture may be used.

[0033] Furthermore, the growth time is not particularly limited. For example, the growth step may be carried out for several hours to several days, 1 to 4 days, 2 to 3 days, or 2 days.

[0034] In this step, after growth, the culture supernatant can be removed by a conventional method such as decantation or aspiration.

[0035] (Repeat process) The method of this embodiment includes a repeat step as a selection step, in which the above-mentioned proliferation step is repeated one or more times.

[0036] In the repeating step, the proliferation step is repeated one or more times, for example, two or more times, three or more times, four or more times, or five or more times, and / or five or less times, four or less times, three or less times, or two or less times, and preferably one to three times, one to two times, or one time.

[0037] The total culture time for this step and the proliferation step not included in this step may be 4 hours or more, 8 hours or more, or 24 hours or more, and / or 120 hours or less, 96 hours or less, 72 hours or less, 60 hours or less, 54 hours or less, or 48 hours or less, preferably 8 to 96 hours or 24 to 72 hours.

[0038] (First culture step) The method of this embodiment includes, as an essential step, a first culture step in which cells are cultured in a first culture medium. As used herein, the term "first culture medium" refers to a medium used to culture cells in the first culture step. The first culture medium is a basal medium or a serum-free medium.

[0039] In one embodiment, the first culture medium contains a protein. The protein contained in the first culture medium can promote cell proliferation, adhesion, and cell function induction (including differentiation induction or maintenance of undifferentiated state, suppression of cell senescence, or induction of specific gene expression) in this step. The protein that can be contained in the first culture medium may be either a human protein or a non-human protein, and further examples thereof are similar to those described in the pre-culture step above. Therefore, a detailed description here is omitted.

[0040] In another embodiment, the first medium is protein-free. The culture conditions for the first culture step are not particularly limited. For example, the culture temperature may be about 30°C to about 40°C, the CO2 concentration may be about 2% to about 10%, and either adhesion culture or suspension culture may be used. Furthermore, the duration of the first culture step is not particularly limited. For example, the first culture step may be carried out for several hours to several weeks, several months, or several days. More specifically, the culture time for the first culture step may be 30 minutes or more, 1 hour or more, 1 hour 30 minutes or more, 2 hours or more, 4 hours or more, 8 hours or more, or 24 hours or more, and / or 120 hours or less, 96 hours or less, 72 hours or less, 60 hours or less, 54 hours or less, or 48 hours or less. For example, the duration of the first culture step may be 12 hours to 120 hours, or about 48 hours. When the cells are mesenchymal stem cells, the preferred culture time for the first culture step is 12 hours to 48 hours.

[0041] (Removal process) The method of this embodiment includes, as an essential step, a removal step of removing the culture supernatant obtained after the first culture step.

[0042] The culture supernatant can be removed by a conventional method such as decantation, aspiration, etc. Here, it is preferable to completely remove the culture supernatant from the first culture step so that components of the first culture medium (e.g., human proteins or non-human proteins) are not contaminated into the second culture medium used in the second culture step.

[0043] (Cleaning process) The method of this embodiment includes a washing step as a selective step in which the cells are washed after the removal step and before the second culture step. The washing solution used in the washing step is not limited as long as it does not excessively affect the viability of the cells, and may be, for example, a buffer, physiological saline, the second medium described below, or any of these solutions to which an additive such as sugar has been added. The number of washes may be, for example, one or more times, and may be, for example, one, two, three, or four times.

[0044] (Second culture step) The method of this embodiment includes, as an essential step, a second culture step in which the cells after the removal step are further cultured in a second culture medium. As used herein, the term "second culture medium" refers to the medium used to culture the cells in the second culture step. The second culture medium is a medium that does not contain protein (hereinafter referred to as a "protein-free medium").

[0045] As used herein, the term "protein-free medium" refers to a medium that is free of or substantially free of protein. Examples of protein-free medium include protein-free serum-free medium and protein-free basal medium.

[0046] The culture conditions for the second culture step are not limited, and for example, the culture temperature may be about 30° C. to about 40° C., the CO 2 concentration may be about 2% to about 10%, and either adhesion culture or suspension culture may be used.

[0047] The duration of the second culture step can be appropriately selected depending on the target product to be obtained after the recovery step described below. The second culture step may be, for example, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, or 42 hours or more, or 120 hours or less, 96 hours or less, 72 hours or less, 60 hours or less, or 54 hours or less. For example, the duration of the second culture step may be 12 to 120 hours, 24 to 72 hours, 42 to 54 hours, or approximately 48 hours.

[0048] As used herein, the term "product of interest" refers to a product contained in the culture supernatant obtained after the second culture step by the method of the present invention, and examples thereof include therapeutic proteins, small molecules such as hormones, exosomes, viruses, or combinations thereof. Examples of therapeutic proteins include, but are not limited to, enzymes, hormones such as peptide hormones, blood coagulation factors, cytokines, growth factors, antibodies or antibody fragments, as well as progranulin and semaphorin. Examples of growth factors include hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDGF), placenta growth factor (PLGF), and bone morphogenetic protein (BMP).

[0049] (Recovery process) The method of this embodiment includes, as an essential step, a recovery step of recovering the culture supernatant obtained after the second culture step. The recovery method is not particularly limited. The recovered culture supernatant may be subjected to centrifugation and / or membrane filtration to remove cells and cell-derived debris.

[0050] (purification process) The method of this embodiment includes a selection step of further purifying the target product from the culture supernatant obtained after the recovery step. For example, if the target product is a protein, it can be purified by standard methods such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse-phase column chromatography, HPLC, ammonium sulfate fractionation, ultrafiltration, and immunoadsorption. If the target product is an exosome, it can be purified by ultracentrifugation, immunoprecipitation, ultrafiltration, polymer precipitation, field-flow fractionation, and centrifugal field-flow fractionation.

[0051] (effect) According to the method of this embodiment, a culture supernatant containing a high concentration of a biologically active substance can be produced. The method of this embodiment can produce a culture supernatant containing a high concentration of a biologically active substance without stimulating the cells with drugs, hormones, or the like, and therefore has the advantage of easily ensuring safety when the culture supernatant is used for therapeutic purposes, etc. Therefore, when applied to humans, there is a low risk of side effects such as allergic reactions, and the culture supernatant can be used in at least one of the following applications: medical use, cosmetics, and health food use in humans.

[0052] <Culture supernatant> In one aspect, the present invention relates to a culture supernatant produced by the method described herein. The culture supernatant of the present invention may contain various substances depending on the type of cells cultured and the culture conditions. For example, the culture supernatant may contain natural proteins and / or exosomes secreted by non-genetically modified cells, or recombinant proteins and / or exosomes secreted by genetically modified cells. Examples of proteins contained in the culture supernatant include therapeutic proteins, such as enzymes, hormones, blood coagulation factors, cytokines, growth factors, antibodies, etc., or combinations thereof.

[0053] The culture supernatant of the present invention may be used as is, or may be formulated as necessary by lyophilization or the like. Formulation can be carried out according to standard methods using pharmaceutically acceptable carriers and additives (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). For example, compositions containing the culture supernatant of the present invention, and compositions (e.g., pharmaceutical compositions) containing the culture supernatant of the present invention and pharmaceutically acceptable carriers and additives are also provided.

[0054] Examples of pharmaceutically acceptable carriers and additives include, but are not limited to, water, pharmaceutically acceptable organic solvents, thickeners such as polyethylene glycol, diglycerin, glycerin, propylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, water-soluble dextran, sodium carboxymethyl starch, methylcellulose, ethylcellulose, and xanthan gum; lipids such as petrolatum and paraffin; sugar alcohols and sugars such as mannitol, sorbitol, and lactose; surfactants such as Tween 80 and Tween 20; and the like. The additives may be used alone or in appropriate combination.

[0055] The culture supernatant of this embodiment provides a method for treating a disease, a method for healing an injury in a subject, and a method for suppressing immunity in a subject, each of which comprises the step of administering the culture supernatant of this embodiment to a subject. Examples of diseases include hepatitis, liver cirrhosis, nerve regeneration, spinal cord injury, osteoarthritis, chronic pain, cerebral infarction, Alzheimer's disease, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, hypoxic-ischemic encephalopathy, peripheral neuropathy, diabetes, diabetic neuropathy, diabetic nephropathy, ischemic heart disease, heart failure, interstitial pneumonia, bone defect, osteonecrosis of the jaw, periodontal disease, allergic dermatitis, alopecia, peripheral arterial disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, collagen disease, chronic renal failure, stress urinary incontinence, depression, chronic headache, insomnia, eye strain, bedsore, severe burns, stress urinary incontinence, osteoporosis, intractable fractures, menopausal disorders, edema, and Guillain-Barré syndrome.

[0056] Furthermore, the culture supernatant of this embodiment provides a culture supernatant for use in disease treatment, a culture supernatant for use in wound healing, and a culture supernatant for use in immunosuppression.

[0057] Also provided is the use of the culture supernatant of this embodiment in the manufacture of a medicament for treating a disease, healing a wound, or suppressing immunity.

[0058] <Method for increasing cytokine and / or exosome content> In one aspect, the present invention relates to a method for increasing the content of cytokines and / or exosomes in a culture supernatant. The method of this aspect includes, as essential steps, a thawing step, a first culturing step, a removal step, a second culturing step, and a recovery step. The method may also include other selection steps, such as a proliferation step, a repeating step, a washing step, and a purification step. Additional selection steps may include a pre-culturing step, a replacement step, and a freezing step prior to the thawing step. Of the steps constituting the method of the present invention, the steps other than the recovery step are similar to those in the method for producing a culture supernatant described above. Therefore, a description of these steps will be omitted, and only the recovery step in the method of this aspect will be described below.

[0059] (Recovery process) In the method of this embodiment, the recovery step is a step of recovering the culture supernatant containing cytokines and / or exosomes obtained after the second culture step.

[0060] The culture supernatant containing cytokines and / or exosomes recovered in this step may be subjected to centrifugation and / or membrane filtration to remove cells and cell-derived debris. Alternatively, cytokines and / or exosomes in the culture supernatant may be purified by standard methods. For example, cytokines can be purified by chromatography such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse-phase column chromatography, and HPLC, ammonium sulfate fractionation, ultrafiltration, and immunoadsorption. Alternatively, exosomes can be purified by ultracentrifugation, immunoprecipitation, ultrafiltration, polymer precipitation, field-flow fractionation, centrifugal field-flow fractionation, and the like.

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0062] <Example 1: Production of culture supernatant using thawed cells> (the purpose) We will investigate the conditions under which the content of biologically active substances can be improved when producing culture supernatant using thawed cells.

[0063] (method) In this example, culture supernatants were produced by methods including freezing and thawing (Methods A to C) and a method not including freezing and thawing (Control 1). The steps involved in each method are described below, and an overview is shown in Figure 3.

[0064] Method A: Method A includes the following steps: (1) pre-culture, (2) freezing of cells, (3) thawing of cells, (4) culture in serum-free medium and / or serum-containing medium (4-1) Method A, (5) culture in basal medium, and (6) collection of culture supernatant (Figure 3, "Method A").

[0065] Method B: Method B includes the following steps: (1) pre-culture, (2) freezing of the cells, (3) thawing of the cells, (4) culture in serum-free medium and / or serum-containing medium (4-2) Method B, (5) culture in basal medium, and (6) collection of the culture supernatant (Figure 3, "Method B").

[0066] Method C: Method C includes the following steps: (1) pre-culture, (2) freezing of the cells, (3) thawing of the cells, (4) culture in serum-free medium and / or serum-containing medium (4-3) Method C, (5) culture in basal medium, and (6) collection of the culture supernatant (Figure 3, "Method C").

[0067] Control 1: The control method did not involve (2) cell freezing or (3) cell thawing. This control method (Control 1) included (1) pre-culture followed by culture in serum-free medium, followed by (5) culture in basal medium, and (6) collection of the culture supernatant (Figure 3, "Control 1").

[0068] Each step will be described below. (1) Preculture Subcutaneous adipose-derived mesenchymal stem cells (MSCs) were cultured in serum-containing medium, PRIME-XV MSC Expansion XSFM (Irvine Scientific, #91149-1L) supplemented with FBS.

[0069] After the mesenchymal stem cells reached a cell density of approximately 80% in the culture vessel, they were washed with metal ion-free phosphate buffered saline (PBS(-)). Then, the cells were detached from the culture vessel using a cell detachment solution, Actase (Innovative Cell Technologies, #AT104-500). Culture medium was added, and the cells were transferred to a centrifuge tube.

[0070] The cells were separated from the solution by low-speed centrifugation (1,200 rpm, 5 minutes), and the supernatant was discarded. The cells were washed by resuspending them in PBS(-). A portion of the cell suspension was taken and the cell number was counted. The cells were separated again by low-speed centrifugation.

[0071] (2) Freezing of cells Cell density is 1–3 × 10 6 Bambanker (GC Lymphotec, #CS-02-001) was added to give a concentration of cells / mL, and 1 mL of the solution was dispensed into a 1.5 mL cryotube.

[0072] The cryotubes containing the aliquots were placed in a BICELL (registered trademark) (Nippon Freezer) freezing container pre-cooled to 4°C, and the container was placed in a -80°C freezer for at least 3 hours to freeze the cells at a cooling rate of -0.5°C / min to -0.4°C / min. The frozen cells were stored at -80°C or in a storage tank using liquid nitrogen until thawed.

[0073] (3) Thawing of cells The cells cryopreserved in (1) above were quickly thawed at 37° C. 1 mL of the thawed cells was transferred to a centrifuge tube, and 10 volumes of Dulbecco Minimum Essential Media (DMEM) were slowly added.

[0074] The cells were separated from the solution by low-speed centrifugation. PBS(-) was added to suspend the cells. A portion of the cell suspension was taken and the number of cells was counted. The cells were separated by low speed centrifugation.

[0075] (4) Culture in serum-free medium and / or serum-containing medium The cells isolated in (3) above were cultured. The culture methods differ for Method A, Method B, and Method C, and are specifically described in each of (4-1) to (4-3) below.

[0076] (4-1) Method A The cells isolated in (3) above were resuspended in serum-free medium. PRIME-XV MSC Expansion XSFM without FBS was used as the serum-free medium. Approximately 2.7 × 10 cells were then resuspended in the same serum-free medium in a culture vessel. 4 cells / cm 2 The cells were seeded so that

[0077] After 48 hours from the start of culture, when the cell density in the culture vessel reached about 95%, the medium was removed.

[0078] (4-2) Method B The cells isolated in (3) above were resuspended in serum-containing medium. PRIME-XV MSC Expansion XSFM supplemented with FBS was used as the serum-containing medium. Approximately 0.8–1.3 × 10 cells were placed in a culture vessel containing the same serum-containing medium. 4 cells / cm 2 The cells were seeded so that

[0079] Approximately 48 hours after the start of culture, when the cell density in the culture vessel reached approximately 80%, the cells were washed with PBS(-), then detached from the culture vessel with acetase, medium was added, and the cells were transferred to a centrifuge tube.

[0080] The cells were separated from the solution by low-speed centrifugation and the supernatant was discarded. The cells were washed by resuspending them in PBS(-). A portion of the cell suspension was taken and the cell number was counted. The cells were separated again by low-speed centrifugation.

[0081] The isolated cells were then resuspended in serum-free medium (PRIME-XV MSC Expansion XSFM without FBS). 4 cells / cm 2 The cells were seeded in a culture vessel containing the same serum-free medium so that the

[0082] After 48 hours from the start of culture, when the cell density in the culture vessel reached about 95%, the medium was removed.

[0083] (4-3) Method C The cells isolated in (3) above were resuspended in serum-containing medium. PRIME-XV MSC Expansion XSFM supplemented with FBS was used as the serum-containing medium. Approximately 0.8–1.3 × 10 cells were placed in a culture vessel containing the same serum-containing medium. 4 cells / cm 2 The cells were seeded so that

[0084] Approximately 48 hours after the start of culture, when the cell density in the culture vessel reached approximately 80%, the cells were washed with PBS(-), then detached from the culture vessel with acetase, medium was added, and the cells were transferred to a centrifuge tube.

[0085] The cells were separated from the solution by low-speed centrifugation and the supernatant was discarded. The cells were washed by resuspending them in PBS(-). An aliquot of the cell suspension was taken and the cell number was counted. The cells were re-separated by low-speed centrifugation.

[0086] The re-isolated cells were then resuspended in serum-containing medium, and the same procedure as above was repeated from about 48 hours of culture in serum-containing medium to re-isolation.

[0087] The cells were then resuspended in serum-free medium (PRIME-XV MSC Expansion XSFM without FBS). 4 cells / cm 2 The cells were seeded in a culture vessel containing the same serum-free medium so that the

[0088] After 48 hours from the start of culture, when the cell density in the culture vessel reached about 95%, the medium was removed.

[0089] (5) Culture in basal medium The culture medium was removed from the flasks, and the cells were washed four times with gradually increasing volumes of PBS(-) (10 mL, 20 mL, 30 mL, and 40 mL). Basal medium was added to each flask, and the cells were cultured for 48 hours. DMEM medium without FBS was used as the basal medium.

[0090] (6) Collection of culture supernatant and evaluation of bioactive substance content After 48 hours of culture in basal medium, the culture medium was harvested and centrifuged at high speed to remove debris. Culture supernatants were then obtained. HGF, VEGF, and progranulin contents were quantified using sandwich ELISA. HGF was measured using the Quantikine ELISA Human HGF Immunoassay (R&D Systems, #DHG00B), VEGF was measured using the Quantikine ELISA Human VEGF Immunoassay (R&D Systems, #DVE00), and progranulin was measured using the Progranulin (human) ELISA kit (AdipoGen Life Science, #AG-45A-0018YEK-KI01). Exosome content was measured using the CD63-Capture Human Exosome ELISA Kit (Fujifilm Wako Pure Chemical Industries, #290-83601). Measurements were performed according to the kit instructions. Briefly, the required amount of culture supernatant was added to each well of a 96-well plate, as instructed by the kit. After the specified incubation time, the wells containing the culture supernatant were washed four times with the provided washing solution. The detection antibody provided with each kit was added, and after further incubation, the wells were washed. After washing, TMB substrate was added to each well and the color reaction was carried out for 10 or 30 minutes. The color reaction was stopped with the reaction stop solution, and the absorbance at 450 nm was measured.

[0091] (result) The contents of bioactive substances in the culture supernatants produced by each of Methods A to C and Control 1 were measured, and the results are shown in FIG.

[0092] The results shown in Figure 4 indicate that the culture supernatants produced by Methods A to C, which include freezing and thawing, contained higher amounts of bioactive substances than did Control 1, which did not. Furthermore, a comparison of Methods A to C showed that the fewer the number of passages after thawing, the higher the content of bioactive substances in the culture supernatant. In particular, the content of bioactive substances in the culture supernatant produced by Method A was significantly higher than that of Methods B and C and Control 1. Specifically, the content of HGF, VEGF, progranulin, and exosomes was highest in the cell supernatant produced by Method A.

[0093] The results of this example demonstrated that the culture supernatant of frozen-thawed cells contains high concentrations of biologically active substances. This surprising result contradicts the common general knowledge in the art that it is preferable to produce a culture supernatant using cells that have been passaged for at least several generations after thawing.

[0094] Example 2: Culture conditions after thawing (the purpose) We will further investigate the conditions for culturing the thawed cells. Specifically, we will investigate the effects of the type of medium in which the thawed cells are cultured and whether or not they are passaged on the content of biologically active substances in the culture supernatant.

[0095] (method) Culture supernatants were produced using methods including freezing and thawing (Methods D to F) and a method not including freezing and thawing (Control 2). The steps involved in each method are explained below and outlined in Figure 5.

[0096] Method D: Method D includes the steps of (1) preculture, (2) cell freezing, (3) cell thawing, (4) (4-2) Method B of culturing in serum-free medium and / or serum-containing medium, (5) culturing in basal medium, and (6) recovery of culture supernatant, as described in Example 1. However, in (4-2) Method B, the culturing time in serum-containing medium and serum-free medium is 2 days each ( Figure 5 , "Method D").

[0097] Method E: Method E includes the steps of (1) preculture, (2) cell freezing, (3) cell thawing, (4) culture in serum-free medium and / or serum-containing medium (4-1) Method A, (5) culture in basal medium, and (6) collection of culture supernatant, as described in Example 1. However, in (4-1) Method A, the culture time in serum-free medium was set to 4 days ( Figure 5 , "Method E").

[0098] Method F: In Method F, culture was performed in a serum-containing medium instead of the serum-free medium used in Method E (Figure 5, "Method F"). The serum-containing medium used was PRIME-XV MSC Expansion XSFM supplemented with FBS, and the culture time was 4 days.

[0099] Control 2: The control method did not involve (2) freezing and thawing the cells, as described in Example 1. This control method (Control 2) included the steps of (1) preculture followed by culture in serum-free medium, (5) culture in basal medium, and (6) recovery of the culture supernatant. However, the culture period for (1) preculture was 2 days, and the culture period in serum-free medium was 2 days (Figure 3, "Control 2").

[0100] (result) The contents of bioactive substances in the culture supernatants produced by Methods D to F and Control 2 were measured, and the results are shown in FIG.

[0101] The results shown in Figure 6 demonstrate that the HGF content in the cell supernatants produced by methods D and E is significantly higher than that of method F. This indicates that culturing in a serum-free medium after thawing significantly increases the content of bioactive substances.

[0102] Furthermore, comparing the results of Method D and Method E in the results shown in Figure 6, it was also shown that when culturing is performed for the same period after thawing, culturing in a serum-containing medium before culturing in a serum-free medium can increase the content of biologically active substances.

Claims

1. A method for producing a culture supernatant, comprising: a thawing step of thawing frozen cells; a first culturing step of culturing the cells after the thawing step in a first medium for 96 hours or less; a removing step of removing the culture supernatant obtained after the first culture step; a second culturing step in which the cells after the removal step are further cultured in a second medium; and a recovery step of recovering the culture supernatant obtained after the second culture step; Including, the first medium is a basal medium or a serum-free medium; the second medium is protein-free; and The method, wherein the method does not include culturing other than the first culturing step and the second culturing step after the thawing step.

2. The method described in claim 1, wherein the first culture step is carried out for 72 hours or less.

3. The method described in claim 1, wherein the first culture step is carried out for 12 to 48 hours.

4. A method for producing a culture supernatant, comprising: a thawing step of thawing frozen cells; a proliferation step in which the cells after the thawing step are cultured and proliferated in a serum- or plasma-containing medium for 1 to 4 days; a first culturing step of culturing the cells after the proliferation step in a first medium for 72 hours or less; a removing step of removing the culture supernatant obtained after the first culture step; a second culturing step in which the cells after the removal step are further cultured in a second medium; and a recovery step of recovering the culture supernatant obtained after the second culture step; Including, the first medium is a basal medium or a serum-free medium; the second medium is protein-free; and The method, wherein the thawing step does not include any culturing other than the proliferation step, the first culturing step, and the second culturing step.

5. A method for producing a culture supernatant, comprising: a thawing step of thawing frozen cells; a proliferation step of culturing and proliferating the cells after the thawing step in a serum- or plasma-containing medium; a repeating step of repeating the proliferation step one or more times; a first culturing step of culturing the cells after the repeating step in a first medium for 72 hours or less; a removing step of removing the culture supernatant obtained after the first culture step; a second culturing step in which the cells after the removal step are further cultured in a second medium; and a recovery step of recovering the culture supernatant obtained after the second culture step; Including, The total culture time for the proliferation step and the repeating step is 96 hours or less; the first medium is a basal medium or a serum-free medium; the second medium is protein-free; and The method, wherein the thawing step does not include any culturing other than the proliferation step, the repeating step, the first culturing step, and the second culturing step.

6. The method according to any one of claims 1 to 5, wherein the first culture medium comprises a human protein or a non-human protein.

7. The method according to any one of claims 1 to 6, wherein the basal medium is selected from the group consisting of EMEM medium, DMEM medium, IMDM medium, GMEM medium, Ham's F10 medium, Ham's F12 medium, RPMI1640 medium, and combinations thereof.

8. The method according to any one of claims 1 to 7, further comprising a washing step of washing the cells after the removing step and before the second culturing step.

9. The method according to any one of claims 1 to 8, wherein the second culture medium is a basal medium.

10. Before the thawing step, a pre-culture step of culturing cells in a pre-culture medium; a replacement step of replacing the pre-culture medium after the pre-culture step with a cryopreservation solution; and a freezing step of freezing the cells after the replacement step; Including, The method according to any one of claims 1 to 9, wherein the pre-culture medium is a serum- or plasma-containing medium, a serum-free medium, or a basal medium.

11. The method of claim 10 , wherein the pre-culture medium comprises a human protein or a non-human protein.

12. 12. The method of claim 10 or 11, wherein the serum is of animal origin or is an artificial serum.

13. 13. The method of claim 12, wherein the serum from an animal is selected from the group consisting of fetal bovine serum, newborn bovine serum, bovine serum, and horse serum.

14. The method according to any one of claims 1 to 13, wherein the cells are mesenchymal stem cells.

15. The method according to claim 14, wherein the first culturing step is carried out for 12 to 48 hours.

16. A method for increasing the content of cytokines and / or exosomes in a culture supernatant, comprising: a thawing step of thawing frozen cells; a first culturing step of culturing the cells after the thawing step in a first medium for 96 hours or less; a removing step of removing the culture supernatant obtained after the first culture step; a second culturing step in which the cells after the removal step are further cultured in a second medium; and a recovery step of recovering a culture supernatant containing cytokines and / or exosomes obtained after the second culture step; Including, the first medium is a basal medium or a serum-free medium; the second medium is protein-free; and The method, wherein the method does not include culturing other than the first culturing step and the second culturing step after the thawing step.

17. The method described in claim 16, wherein the first culture step is carried out for 12 to 48 hours.

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