Medium supplements suitable for promoting cell production of exosomes

Abstract

The present invention provides a culture medium additive composition for promoting cell production of exosomes. The additive composition comprises three subgroup additives. The additive of the present invention is serum-free, low-cost, and has clear ingredients, and is suitable for large-scale production of exosomes.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture medium, and in particular, the present invention relates to a culture medium additive suitable for promoting cells to produce exosomes. Background Art

[0002] Exosomes are a type of nano-scale vesicles secreted by cells, with a diameter between 30 and 150 nm, a double-layer membrane structure and a saucer-shaped morphology. In 1983, exosomes were first discovered in the supernatant of sheep red blood cells cultured in vitro, but they did not attract much attention at the time and were only regarded as "garbage" of cell metabolism. However, with the 2013 Nobel Prize in Physiology or Medicine awarded to scientists who made outstanding contributions in the field of intercellular vesicle transport regulation mechanisms, the research on exosomes has rapidly increased. Exosomes are rich in cholesterol and sphingomyelin, and carry a variety of important biological signal substances such as proteins, RNA, DNA, etc. They are widely distributed in various body fluids such as blood, urine, cerebrospinal fluid, saliva, milk, bile, etc.

[0003] Exosomes show great potential in the fields of medical research and clinical application. First, the RNA or protein components in exosomes can be used as a new type of biomarker for the diagnosis of various diseases. Second, exosomes are wrapped in natural cell membranes and can be used for drug delivery, improving the stability, bioavailability and targeting of drugs and reducing toxic side effects. In addition, exosomes are also involved in processes such as intercellular communication, cell migration, angiogenesis and tumor cell growth, and are an important research direction in the field of regenerative medicine. For example, exosomes have shown significant regenerative ability in cartilage growth, liver damage repair and myocardial damage repair.

[0004] Mesenchymal stem cells (MSCs) and 293 cells (human embryonic kidney cells) exosomes have shown a wide range of application potential in the biomedical field. Mesenchymal stem cell exosomes are widely used in disease treatment and tissue repair due to their self-renewal ability and paracrine function. They can reduce myocardial damage, promote tissue damage repair, promote nerve regeneration, reduce lung damage and regulate the immune system, and show significant therapeutic effects in cancer treatment, anti-aging, skin aging, Alzheimer's disease and other fields. On the other hand, as exosomes secreted by 293 cells, they are widely used as tools for biomedical research and production of recombinant proteins because they are easy to transfect and transform. In addition, 293 cell exosomes can also be used to deliver specific bioactive molecules, such as the mRNA of the anti-inflammatory cytokine IL-10, to regulate immune responses and inhibit inflammatory responses. These exosomes also play an important role in disease diagnosis, disease monitoring and drug efficacy evaluation. In short, mesenchymal stem cells and 293 cell exosomes have broad application prospects in the biomedical field, providing new strategies and tools for disease treatment and tissue repair.

[0005] Given the importance of exosomes in medical research and clinical applications, as well as the current problems of extraction, purification and stability, it is of great significance to develop chemically defined culture medium additives for exosomes. By adding necessary nutrients, growth factors and some components that promote the expression and secretion of exosomes, a more stable environment can be provided for cell culture without adding serum, reducing the adverse factors brought by serum. At the same time, chemically defined culture medium can also simplify the procedures for purifying and identifying various cell products and avoid the harm caused by viral contamination. In addition, the preparation of special chemically defined culture medium for specific cell types can accurately control the proliferation and differentiation process of cells, further improving the yield and quality of exosomes.

[0006] In summary, the development of culture medium additives specifically for exosomes will not only help solve the current technical difficulties in exosome research and application, but also promote the in-depth development of exosomes in medical research and clinical applications, and has significant scientific value and social significance. Summary of the invention

[0007] One object of the present invention is to provide a culture medium additive composition with a clear chemical composition that promotes cell production of exosomes.

[0008] Another object of the present invention is to provide a culture medium for culturing cells to produce exosomes.

[0009] In a first aspect of the present invention, an additive composition is provided, the composition comprising:

[0010] (a) additive A, wherein the additive A is composed of the following ingredients and water, including: 1-5 g / L disodium hydrogen phosphate, 0.1-1 g / L potassium dihydrogen phosphate, 5-20 g / L sodium chloride, 0.1-2 g / L potassium chloride, 20-50 g / L alanine glutamine, 20-50 g / L glycine glutamine, 1-10 g / L vitamin C, 0.1-5 g / L glutathione (reduced form), 0.05-1 g / L β-nicotinamide adenine dinucleotide, 0.001-0.04 g / L mercaptoethanol, 50-200 g / L recombinant human albumin, 0.1-1 g / L ethanolamine hydrochloride, 0.1-10 g / L insulin, and 0.0002-0.001 g / L sodium selenite;

[0011] (b) additive B, wherein the additive B is composed of the following ingredients and water, including: 0.005-0.05 g / L basic fibroblast growth factor bFGF, 0.01-0.1 g / L epidermal growth factor EGF, 0.001-0.01 g / L transforming growth factor-β1 TGF-β1, 0.005-0.05 g / L platelet-derived growth factor-BB PDGF-BB, and 0.005-0.05 g / L platelet-derived growth factor-AB PDGF-AB; and

[0012] (c) Additive C, which is composed of the following ingredients and ethanol, including: 1-15 g / L soybean lecithin, 1-50 g / L cholesterol, 0.1-10 g / L linolenic acid, 0.1-10 g / L linoleic acid, and 10-500 g / L Tween-80.

[0013] In another preferred embodiment, the water is ultrapure water, preferably water for injection.

[0014] The water used for preparing the additive provided by the present invention is ultrapure water or water for injection, which is prepared and sterilized and filtered by conventional methods.

[0015] In another preferred embodiment, the ethanol is anhydrous ethanol.

[0016] In another preferred embodiment, the composition comprises the following:

[0017] (a) additive A, wherein the additive A is composed of the following ingredients and water, including: 1-3 g / L disodium hydrogen phosphate, 0.1-0.5 g / L potassium dihydrogen phosphate, 5-15 g / L sodium chloride, 0.1-1 g / L potassium chloride, 30-50 g / L alanine glutamine, 30-50 g / L glycine glutamine, 1-10 g / L vitamin C, 0.1-3 g / L glutathione (reduced form), 0.1-1 g / L β-nicotinamide adenine dinucleotide, 0.001-0.02 g / L mercaptoethanol, 50-150 g / L recombinant human albumin, 0.1-1 g / L ethanolamine hydrochloride, 0.1-5 g / L insulin, and 0.0002-0.001 g / L sodium selenite;

[0018] (b) additive B, wherein the additive B is composed of the following ingredients and water, including: 0.005-0.05 g / L basic fibroblast growth factor bFGF, 0.01-0.1 g / L epidermal growth factor EGF, 0.001-0.01 g / L transforming growth factor-β1 TGF-β1, 0.005-0.05 g / L platelet-derived growth factor-BB PDGF-BB, and 0.005-0.05 g / L platelet-derived growth factor-AB PDGF-AB; and

[0019] (c) Additive C, which is composed of the following ingredients and ethanol, including: 1-15 g / L soybean lecithin, 1-50 g / L cholesterol, 0.1-10 g / L linolenic acid, 0.1-10 g / L linoleic acid, and 10-500 g / L Tween-80.

[0020] In another preferred embodiment, the concentrations of alanyl-glutamine, glycine-glutamine and β-nicotinamide adenine dinucleotide in the additive A are 35-50 g / L, 35-50 g / L and 0.1-1 g / L respectively; and / or the concentration of soybean lecithin in the additive C is 3-15 g / L.

[0021] In the second invention of the present invention, a method for preparing the additive composition according to the first aspect of the present invention is provided.

[0022] The preparation method of the additive A comprises the following steps: dissolving the following ingredients in 1 liter of water, and obtaining the additive A after sterilization and filtration:

[0023] 1-5g disodium hydrogen phosphate, 0.1-1g potassium dihydrogen phosphate, 5-20g sodium chloride, 0.1-2g potassium chloride, 20-50g alanine glutamine, 20-50g glycine glutamine, 1-10g vitamin C, 0.1-5g glutathione (reduced form), 0.05-1g β-nicotinamide adenine dinucleotide, 0.001-0.04g mercaptoethanol, 50-200g recombinant human albumin, 0.1-1g ethanolamine hydrochloride, 0.1-10g insulin and 0.0002-0.001g sodium selenite;

[0024] The preparation method of the additive B comprises the following steps: dissolving the following sterile components in 100 mL of sterile water to obtain the additive B, and storing it at -20°C to -80°C:

[0025] 0.0005-0.005 g basic fibroblast growth factor bFGF, 0.001-0.01 g epidermal growth factor EGF, 0.0001-0.001 g transforming growth factor-β1 TGF-β1, 0.0005-0.005 g platelet-derived growth factor-BB PDGF-BB, and 0.0005-0.005 g platelet-derived growth factor-AB PDGF-AB; and

[0026] The preparation method of the additive C comprises the following steps: dissolving the following components in 100 mL of anhydrous ethanol, obtaining the culture medium additive C after sterile filtration, and storing at -20°C:

[0027] 0.1-1.5g soybean lecithin, 0.1-5g cholesterol, 0.01-1g linolenic acid, 0.01-1g linoleic acid, 1-50g Tween-80.

[0028] In a third aspect of the present invention, a culture medium for culturing cells to produce exosomes is provided, comprising:

[0029] (1) the additive composition according to the first aspect of the present invention; and

[0030] (2) Basic culture medium

[0031] In another preferred embodiment, the basal culture medium is selected from the following group: DMEM, F12, IMEM, MEM, α-MEM, CelerS001, or a combination thereof.

[0032] In another preferred embodiment, the following steps are included: adding the additive composition of claim 1 to the basal culture medium at a ratio of 10 ml of additive A, 1 ml of additive B and 1 ml of additive C per liter of basal culture medium.

[0033] In a fourth aspect of the present invention, there is provided a use of the additive composition described in the first aspect of the present invention for in vitro culturing and promoting cell expression of exosomes.

[0034] In a fifth aspect of the present invention, an in vitro method for promoting cells to express exosomes is provided, characterized in that, during the process of culturing cells, the additive composition as described in the first aspect of the present invention is used.

[0035] In another preferred embodiment, the cells are adherent cells or suspension cells.

[0036] In another preferred embodiment, the cells are of mouse, rabbit, human, camel, gene-edited, or a combination thereof.

[0037] In another preferred embodiment, the cells are selected from the following group: mesenchymal stem cells, such as adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic fluid mesenchymal stem cells, amniotic membrane mesenchymal stem cells, dental pulp mesenchymal stem cells, chorionic mesenchymal stem cells, or a combination thereof, HEK293 cells, 293F cells.

[0038] In another preferred embodiment, the method comprises: adding the additive composition of claim 1 to the basal culture medium during cell culture, wherein 10 ml of additive A, 1 ml of additive B, and 1 ml of additive C are added to each liter of culture medium.

[0039] In the sixth aspect of the present invention, a composition is provided, comprising: 30-50 g / L alanyl-glutamine, 30-50 g / L glycine-glutamine, 0.05-1 g / L β-nicotinamide adenine dinucleotide and 3-15 g / L soybean lecithin.

[0040] In another preferred embodiment, the composition is used to promote cells to express exosomes.

[0041] In another preferred embodiment, the composition is used to promote cells to express exosomes in vitro.

[0042] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The confluence of umbilical cord-derived MSC cells before harvesting exosomes Exo-1 is shown.

[0044] Figure 2 The expansion fold of umbilical cord-derived MSC cells is shown.

[0045] Figure 3 The electron micrograph of particles in the exosomes Exo-1 from umbilical cord-derived MSC cells is shown.

[0046] Figure 4 The particle concentration and size distribution of exosomes Exo-1 from umbilical cord-derived MSC cells are shown.

[0047] Figure 5 The exosome concentrations obtained from umbilical cord-derived MSC cells under different culture conditions are shown.

[0048] Figure 6 Growth curves of HEK293 cells are shown.

[0049] Figure 7 The exosome concentrations obtained from HEK293 cells under different culture conditions are shown. DETAILED DESCRIPTION

[0050] After extensive and in-depth research, the inventors have developed a culture medium additive composition that promotes cell production of exosomes. The additive composition provided by the present invention has clear components, few types, simple preparation method, no need to add serum, and can support various cells to express exosomes efficiently and with high quality. Based on this, the inventors have completed the present invention.

[0051] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terminology used herein is intended only to describe specific embodiments, and is not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0053] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0054] Media additives

[0055] The additive composition provided by the present invention has clear chemical composition and can be added to a conventional basal culture medium to be used for culturing different types of cells to efficiently produce exosomes.

[0056] Specifically, the components of the additive composition of the present invention and their functions are as follows: disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and potassium chloride are used as the buffer system in the additive to ensure that the addition to the basal culture medium will not bring about a huge change in the buffer system of the original culture system and affect the growth of cells. Glutamine is a main component present in the culture medium that provides energy for cells, but this component is extremely unstable in a room temperature aqueous solution and will lose more than 30% in 4-7 days, and a large amount of ammonium ions produced during cell culture have a negative or inhibitory effect on cell growth. Alanine glutamine and glycine glutamine are alternative components of glutamine, which have been proven to completely replace glutamine for cell functions in a variety of cell culture processes and can maintain long-term stability in the culture medium. Vitamin C, reduced glutathione, and β-mercaptoethanol can remove free radicals produced during cell culture and protect cells from oxidative stress damage. This antioxidant effect has a positive effect on maintaining the normal physiological state and survival of cells and can promote cell growth, proliferation and repair. β-Nicotinamide adenine dinucleotide is an important coenzyme that participates in a variety of physiological processes, including energy metabolism, DNA repair, cell signaling and biosynthesis, and is essential for cell growth and function. Serum albumin, insulin, transferrin, ethanolamine and selenium are common components in serum-free culture media. Serum albumin can help maintain the relative stability of the internal environment of the culture system, such as osmotic pressure and pH. As a ligand for a variety of difficult-to-absorb nutrients, it assists in transport and thus meets the needs of cell growth and metabolism; insulin plays a key role in cell metabolism and can promote the absorption and utilization of glucose by cells; transferrin is a protein that transports iron and can transport iron from the extracellular to the intracellular for cell use; ethanolamine can participate in the synthesis of phospholipids; and selenium has an antioxidant effect. The addition of these components can ensure the growth and proliferation of cells under serum-free conditions. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF-AB and PDGF-BB) bind to their receptors respectively, which can activate multiple signaling pathways, regulate cell proliferation, migration and survival, etc., ensure the expression of exosomes or maintain the activity of cells in the stage. Among them, bFGF and PDGF-BB have been found to have a significant effect on promoting the expression of exosomes. Therefore, growth factors promote the expression of exosomes in a direct or indirect way. Cholesterol and soy lecithin are important components of the cell membrane, and they play an important role in promoting the formation of exosome membranes and maintaining the stability of exosome membranes. Linoleic acid and linolenic acid play a role in affecting the formation and secretion of exosomes by affecting and regulating intracellular signal transduction pathways.

[0057] Culture medium

[0058] The culture medium that can be used in the present invention is a conventional basal culture medium in the art, preferably a serum-free culture medium, whose components include amino acids, vitamins, sugars, metal ions and bulk inorganic salt ions necessary for cell growth.

[0059] Culture medium additive composition

[0060] The culture medium additive A is dissolved in water and includes: 1-5 g / L disodium hydrogen phosphate, 0.1-1 g / L potassium dihydrogen phosphate, 5-20 g / L sodium chloride, 0.1-2 g / L potassium chloride, 20-50 g / L alanine glutamine, 20-50 g / L glycine glutamine, 1-10 g / L vitamin C, 0.1-5 g / L glutathione (reduced form), 0.05-1 g / L β-nicotinamide adenine dinucleotide, 0.001-0.04 g / L mercaptoethanol, 50-200 g / L recombinant human albumin, 0.1-1 g / L ethanolamine hydrochloride, 0.1-10 g / L insulin, and 0.0002-0.001 g / L sodium selenite;

[0061] The culture medium additive B is dissolved in water and includes 0.005-0.05 g / L basic fibroblast growth factor (bFGF), 0.01-0.1 g / L epidermal growth factor (EGF), 0.001-0.01 g / L transforming growth factor-β1 (TGF-β1), 0.005-0.05 g / L platelet-derived growth factor-BB (PDGF-BB), and 0.005-0.05 g / L platelet-derived growth factor-AB (PDGF-AB);

[0062] The culture medium additive C is dissolved in anhydrous ethanol and includes 1-15 g / L soybean lecithin, 1-50 g / L cholesterol, 0.1-10 g / L linolenic acid, 0.1-10 g / L linoleic acid, and 10-500 g / L Tween-80.

[0063] During the in vitro culture of mesenchymal stem cells and 293 cells to produce exosomes, the above additives were added in a ratio of 10 ml of culture medium additive A, 1 ml of additive B and 1 ml of additive C per liter of culture medium (basal medium).

[0064] As used herein, a method of expressing exosomes comprises the steps of:

[0065] (1) Prepare the culture medium required for cell growth and the basic culture medium with clear chemical composition;

[0066] (2) Cell recovery;

[0067] (3) Cell subculture and seed cell expansion;

[0068] (4) After the cells are inoculated and attached, the culture medium is replaced with the culture medium containing additive A, additive B and additive C with clear chemical composition of the present invention;

[0069] (5) Harvesting cell supernatant;

[0070] (6) Use universal methods to collect, purify, and preserve exosomes.

[0071] As used herein, the "maintenance period" has the same meaning as the "exosome expression period" and the "exosome production period", and refers to the culture period starting from when the culture medium containing the additive of the present invention is replaced after cell inoculation or when the additive is added.

[0072] As used herein, the term "expression medium" is equivalent to the medium in the cell supernatant when the exosomes are harvested.

[0073] Compared with the prior art, the main advantages of the present invention include:

[0074] (1) The chemical composition is clear, which is convenient for downstream separation and purification.

[0075] (2) No need to add serum, no animal-derived ingredients, and reduced (avoided) contamination by exogenous factors.

[0076] (3) There is no need to change the basic culture medium components of the culture system, thus avoiding the drastic changes in the culture environment when the cells enter the exosome expression or maintenance stage, which may lead to the deterioration of cell activity or the performance of expressing exosomes.

[0077] (4) After adding this group of additives, the expression of exosomes in different cells can be effectively improved.

[0078] (5) Low cost, high economic efficiency, and suitable for large-scale production of exosomes.

[0079] (6) The additive combination has a wide range of applications and can be applied to all production processes based on cell culture to harvest exosomes.

[0080] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0081] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0082] Example 1

[0083] The chemical composition of additives A, B and C suitable for efficient secretion and production of exosomes by MSC cells is clearly defined and applied to α-MEM culture medium.

[0084] In this embodiment, the additive A with clear chemical composition contains

[0085]

[0086] The components are fully dissolved in ultrapure water, prepared by conventional methods and sterilized and filtered. This additive is recorded as Add-1.

[0087] Additive B with clear chemical composition, in 100ml of water, contains:

[0088]

[0089] The components are aseptically dissolved in sterile ultrapure water and stored at -20°C to avoid frequent freezing and thawing. After preparation, they can be frozen in multiple portions according to usage habits. This additive is recorded as Add-2.

[0090] Additive C with clear chemical composition, in 100 ml of anhydrous ethanol, contains:

[0091]

[0092] The components were dissolved in anhydrous ethanol, filtered and sterilized, and then frozen at -20° C. The additive was recorded as Add-3.

[0093] The additives were added to the α-MEM basal medium in a manner of adding 10 ml of additive A, 1 ml of additive B and 1 ml of additive C per liter of culture medium to prepare an exosome expression medium.

[0094] A P3 umbilical cord-derived MSC (UMSCs) was revived using Omni Xeno-free MSC medium and subcultured to P4 in the medium. 3 cells / cm 2Cells were seeded in 6-well plates. After the cells adhered for 24-48 hours, the growth medium was removed, the cell layer was washed 2-3 times with PBS, 2 ml of expression medium was added to each well, and the cells were returned to the incubator for incubation. After 48 hours, the supernatant was harvested and immediately transferred to 2-8°C for short-term storage or -20°C for long-term storage. The exosome sample obtained in this experimental group was named Exo-1.

[0095] Use an exosome-specific extraction kit to extract and purify exosomes from 1 ml of Exo-1 sample according to the product instructions, and store them in 100 μL of exosome-specific storage solution. The extracted exosomes should be stored in a -80°C refrigerator. Use a nanoparticle tracking analyzer (ZetaVIEW) to detect the concentration and distribution of nanoparticles.

[0096] After incubation in the expression medium for 48 hours, the cells still maintained a high proliferation rate. When the supernatant was harvested, the cells reached 100% confluence. Figure 1 As shown in the figure, the cell expansion fold was 15.6±0.08 times (with medium replacement), and there was no significant difference in the cell expansion fold (16.8±0.9 times) compared with the culture medium without medium replacement. Figure 2 That is to say, the additive composition of the present invention will not affect the normal growth and proliferation of cells.

[0097] After the exosomes Exo-1 obtained in this group of experiments were collected and purified, an obvious teacup-shaped morphology can be seen under an electron microscope, such as Figure 3 As shown. The diameter of exosomes is 128.8nm. Figure 3 As shown. Converted to the culture supernatant, the exosome concentration is 5.2×10 9 Compared with the control groups, the particle concentration increased by 0.6-2.7 times (e.g. Figure 4 ).

[0098] Example 2

[0099] The culture conditions were the same as in Example 1, and the expression medium consisted of α-MEM, additives A, additives B and additives C, except that:

[0100] The concentrations of alanyl-glutamine, glycine-glutamine, and β-nicotinamide adenine dinucleotide in additive A were 10 g / L, 10 g / L, and 0.01 g / L, respectively; the concentration of soy lecithin in additive C was 0.1 g / L. The exosome sample obtained in this experimental group was named Exo-2.

[0101] Comparative Example 1 (composition without additives)

[0102] The culture conditions were the same as those in Example 1, except that the expression medium was α-MEM basal medium, and no additives were added during the entire cell culture process. The exosome sample in this experimental group was recorded as Con-1-1.

[0103] Comparative Example 2 (basal medium + additive A)

[0104] The culture conditions were the same as those in Example 1, except that the expression medium was α-MEM basal medium + additive A, and the exosome samples in this experimental group were recorded as Con-1-2.

[0105] Comparative Example 3 (basal medium + additive B)

[0106] The culture conditions were the same as in Example 1, except that the expression medium was α-MEM basal medium + additive A + additive B, and the exosome samples in this experimental group were recorded as Con-1-3.

[0107] Comparative Example 4 (basal medium + additive C)

[0108] The culture conditions were the same as in Example 1, except that the expression medium was α-MEM basal medium + additive A + additive B, and the exosome samples in this experimental group were recorded as Con-1-4.

[0109] Comparative Example 5 (basal medium + additives A + B)

[0110] The culture conditions were the same as those in Example 1, except that the expression medium was α-MEM basal medium + additive A + additive B. The exosome samples in this experimental group were recorded as Con-1-5.

[0111] Comparative Example 6 (basal medium + additives A + C)

[0112] The culture conditions were the same as those in Example 1, except that the expression medium was α-MEM basal medium + additive A + additive C, and the exosome samples in this experimental group were recorded as Con-1-6.

[0113] Comparative Example 7 (basal medium + additives B + C)

[0114] The culture conditions were the same as in Example 1, except that the expression medium was α-MEM basal medium + additive B + additive C, and the exosome samples in this experimental group were recorded as Con-1-7.

[0115] Under the culture conditions of Example 1-2 and Comparative Example 1-7, the concentrations of exosomes after collection and purification were: Exo-1 was 5.2×10 9 particles / ml; Exo-2 is 3.4×10 9particles / ml; Con-1-1 is 1.4×10 9 particles / ml; Con-1-2 is 2.9×10 9 particles / ml; Con-1-3 is 3.0×10 9 particles / ml; Con-1-4 is 1.7×10 9 particles / ml; Con-1-5 is 3.3×10 9 particles / ml; Con-1-6 is 3.1×10 9 particles / ml; Con-1-7 is 3.2×10 9 particles / ml. It can be seen that compared with the experimental group without any additives, the additive composition of the present invention can effectively increase the expression of exosomes by 3.7 times. In addition, from the results of the Exo-2 group, it can be seen that the four components of alanyl-glutamine, glycine-glutamine, β-nicotinamide adenine dinucleotide and soy lecithin are essential for the secretion of exosomes. For additives A, B and C in the additive composition, from the present invention Figure 5 It can be seen from the results that the above three groups of additives are indispensable for culturing and obtaining high concentrations of exosomes.

[0116] Example 3

[0117] The additive composition with clear chemical composition of the present invention is applied to HEK293 cells to grow and express exosomes in Celer S001 medium, wherein the components and concentrations of the additive composition are the same as those in Example 1.

[0118] HEK293 suspension cells were revived and cultured using Celer S001 medium. When the cells recovered to stable passage and the viability was above 95%, cells were taken and cultured at 1×10 6 Cells were inoculated into a shake flask at a live cell density of cells / ml, the system was 50 ml, and cultured in 5% CO 2 , 37°C shaker, speed: 110 rpm.

[0119] After 48 hours of culture, A (Add-1), Additive B (Add-2), and Additive C (Add-3) were added to the system at a ratio of 10 ml of Additive A, 1 ml of Additive B, and 1 ml of Additive C per liter of culture medium, i.e., 0.5 ml of A, 0.05 ml of B, and 0.05 ml of C. Thereafter, samples were taken every 24 hours to detect the density and activity of the cells. 1 ml of the culture fluid was centrifuged and collected to collect the culture supernatant 72 hours (i.e., 120 hours after cell inoculation) and 96 hours (i.e., 144 hours after cell inoculation) after adding the additives, and the culture supernatant was immediately transferred to 2-8°C for short-term storage or -20°C for long-term storage. This experimental group was recorded as Exo-3.

[0120] Using an exosome-specific extraction kit, extract and purify exosomes from 1 ml of culture supernatant according to the product instructions, and store in 100 μl of exosome preservation solution. The extracted exosomes should be stored in a -80°C refrigerator. The concentration and distribution of nanoparticles were detected by a nanoparticle tracking analyzer (ZetaVIEW).

[0121] like Figure 6 As shown, the growth curve of HEK293 cells was plotted against the viable cell density. It can be seen that the additives had no effect on cell growth (viable cell density, VCD) and survival rate (cell viability, Via).

[0122] like Figure 7 As shown in the figure, the concentration of exosomes in the culture supernatant of the Exo-3 experimental group was higher than that of Exo-4 and Con-3-8 at the sampling time point. Taking 144h as an example, the concentration of exosomes in Exo-3 reached 8.86×10 particles per milliliter. 10 , which were increased by 1.76 times and 0.73 times compared with Exo-4 and Con-3-8, respectively.

[0123] Example 4

[0124] The expression medium is composed of Celer S001 medium, additives A, additive B and additive C. The difference is that the concentrations of alanyl-glutamine, glycine-glutamine and β-nicotinamide adenine dinucleotide in additive A are 10 g / L, 10 g / L and 0.01 g / L respectively, and the other components remain unchanged. The additive is named Add-4; the concentration of soybean lecithin in additive C is 0.1 g / L, and the other components remain unchanged. The additive is named Add-5.

[0125] The culture conditions were the same as those in Example 3, except that after 48 h of cell culture, A (Add-4), additive B (Add-2), and additive C (Add-5) were directly added to the system, i.e., 0.5 ml A (Add-4), 0.05 ml B (Add-2), and 0.05 ml C (Add-5). The exosome sample obtained in this experimental group was recorded as Exo-4.

[0126] Comparative Example 8 (composition without additives)

[0127] The culture conditions were the same as those in Example 2, except that the expression medium was Celer S001 medium, and no additives were added during the entire cell culture process. The exosome sample in this experimental group was recorded as Con-3-8.

[0128] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An additive composition, characterized in that The composition comprises the following: (a) additive A, wherein the additive A is composed of the following ingredients and water, including: 1-5 g / L disodium hydrogen phosphate, 0.1-1 g / L potassium dihydrogen phosphate, 5-20 g / L sodium chloride, 0.1-2 g / L potassium chloride, 20-50 g / L alanine glutamine, 20-50 g / L glycine glutamine, 1-10 g / L vitamin C, 0.1-5 g / L glutathione (reduced form), 0.05-1 g / L β-nicotinamide adenine dinucleotide, 0.001-0.04 g / L mercaptoethanol, 50-200 g / L recombinant human albumin, 0.1-1 g / L ethanolamine hydrochloride, 0.1-10 g / L insulin, and 0.0002-0.001 g / L sodium selenite; (b) additive B, wherein the additive B is composed of the following ingredients and water, including: 0.005-0.05 g / L basic fibroblast growth factor bFGF, 0.01-0.1 g / L epidermal growth factor EGF, 0.001-0.01 g / L transforming growth factor-β1 TGF-β1, 0.005-0.05 g / L platelet-derived growth factor-BB PDGF-BB, and 0.005-0.05 g / L platelet-derived growth factor-AB PDGF-AB; and (c) Additive C, which is composed of the following ingredients and ethanol, including: 1-15 g / L soybean lecithin, 1-50 g / L cholesterol, 0.1-10 g / L linolenic acid, 0.1-10 g / L linoleic acid, and 10-500 g / L Tween-80.

2. The additive composition according to claim 1, characterized in that The water is ultrapure water, preferably water for injection.

3. The additive composition according to claim 1, characterized in that The composition comprises the following: (a) additive A, wherein the additive A is composed of the following ingredients and water, including: 1-3 g / L disodium hydrogen phosphate, 0.1-0.5 g / L potassium dihydrogen phosphate, 5-15 g / L sodium chloride, 0.1-1 g / L potassium chloride, 30-50 g / L alanine glutamine, 30-50 g / L glycine glutamine, 1-10 g / L vitamin C, 0.1-3 g / L glutathione (reduced form), 0.1-1 g / L β-nicotinamide adenine dinucleotide, 0.001-0.02 g / L mercaptoethanol, 50-150 g / L recombinant human albumin, 0.1-1 g / L ethanolamine hydrochloride, 0.1-5 g / L insulin, and 0.0002-0.001 g / L sodium selenite; (b) additive B, wherein the additive B is composed of the following ingredients and water, including: 0.005-0.05 g / L basic fibroblast growth factor bFGF, 0.01-0.1 g / L epidermal growth factor EGF, 0.001-0.01 g / L transforming growth factor-β1 TGF-β1, 0.005-0.05 g / L platelet-derived growth factor-BB PDGF-BB, and 0.005-0.05 g / L platelet-derived growth factor-AB PDGF-AB; and (c) Additive C, which is composed of the following ingredients and ethanol, including: 1-15 g / L soybean lecithin, 1-50 g / L cholesterol, 0.1-10 g / L linolenic acid, 0.1-10 g / L linoleic acid, and 10-500 g / L Tween-80.

4. The additive composition according to claim 1, characterized in that In the additive A, the concentrations of alanyl-glutamine, glycine-glutamine and β-nicotinamide adenine dinucleotide are 35-50 g / L, 35-50 g / L and 0.1-1 g / L respectively; and / or the concentration of soybean lecithin in the additive C is 3-15 g / L.

5. A method for preparing the additive composition according to claim 1, characterized in that: The preparation method of the additive A comprises the following steps: dissolving the following ingredients in 1 liter of water, and obtaining the additive A after sterilization and filtration: 1-5g disodium hydrogen phosphate, 0.1-1g potassium dihydrogen phosphate, 5-20g sodium chloride, 0.1-2g potassium chloride, 20-50g alanine glutamine, 20-50g glycine glutamine, 1-10g vitamin C, 0.1-5g glutathione (reduced form), 0.05-1g β-nicotinamide adenine dinucleotide, 0.001-0.04g mercaptoethanol, 50-200g recombinant human albumin, 0.1-1g ethanolamine hydrochloride, 0.1-10g insulin and 0.0002-0.001g sodium selenite; The preparation method of the additive B comprises the following steps: dissolving the following sterile components in 100 mL of sterile water to obtain the additive B, and storing it at -20°C to -80°C: 0.0005-0.005 g basic fibroblast growth factor bFGF, 0.001-0.01 g epidermal growth factor EGF, 0.0001-0.001 g transforming growth factor-β1 TGF-β1, 0.0005-0.005 g platelet-derived growth factor-BB PDGF-BB, and 0.0005-0.005 g platelet-derived growth factor-AB PDGF-AB; and The preparation method of the additive C comprises the following steps: dissolving the following components in 100 mL of anhydrous ethanol, obtaining the culture medium additive C after sterile filtration, and storing at -20°C: 0.1-1.5g soybean lecithin, 0.1-5g cholesterol, 0.01-1g linolenic acid, 0.01-1g linoleic acid, 1-50g Tween-80.

6. A culture medium for culturing cells to produce exosomes, characterized in that: include: (1) The additive composition according to claim 1; and (2) Basic culture medium 7. The culture medium according to claim 6, characterized in that The basic culture medium is selected from the group consisting of DMEM, F12, IMEM, MEM, α-MEM, Celer S001, or a combination thereof.

8. The method for preparing a culture medium according to claim 6, wherein: The method comprises the following steps: adding the additive composition of claim 1 to the basic culture medium at a ratio of 10 ml of additive A, 1 ml of additive B and 1 ml of additive C per liter of basic culture medium.

9. A method for promoting cell expression of exosomes in vitro, characterized in that: In the process of culturing cells, the additive composition according to claim 1 is used.

10. The method according to claim 9, characterized in that The cells are selected from the following group: mesenchymal stem cells, such as adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic fluid mesenchymal stem cells, amniotic membrane mesenchymal stem cells, dental pulp mesenchymal stem cells, chorionic mesenchymal stem cells, or a combination thereof, HEK293 cells, 293F cells.

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