CN-122012386-A - Cell culture medium for producing/preparing mesenchymal stem cell exosomes

Abstract

The invention discloses a cell culture medium for producing/preparing mesenchymal stem cell exosomes, which is prepared by adding amino acids, vitamins, inorganic salts, lipids, nucleic acid substances, epidermal Growth Factor (EGF), transforming Growth Factor (TGF), growth promoting factors, human Chorionic Gonadotrophin (HCG) and microelements into a DMEM-based culture medium. In the invention, the mesenchymal stem cell culture medium has definite components, does not contain serum components, has controllable quality, can be produced in large scale, and can normally grow in vitro stem cell culture. In particular, the cell culture medium for producing/preparing the mesenchymal stem cell exosomes can obtain more exosomes with higher purity under the same cell culture condition. The cell culture medium is favorable for large-scale production of mesenchymal stem cell exosomes with good bioactivity, thereby promoting the exosomes to be widely applied in medical treatment, anti-aging, cosmetology and the like, and having good economic benefit.

Inventors

• YANG ZHU

Assignees

• 上海英浛思生物技术有限公司

Dates

Publication Date

20260512

Application Date

20260302

Claims (5)

1. 1. The invention discloses a cell culture medium for producing mesenchymal stem cell exosomes, which takes a DMEM culture medium as a basic culture medium and is added with the following components of amino acids, vitamins, inorganic salts, lipids, nucleic acid substances, epidermal Growth Factor (EGF), transforming Growth Factor (TGF), growth-promoting factor, human Chorionic Gonadotropin (HCG) and microelements.
2. 2. Composition of the components Concentration (mg/L) Glycine (Gly) 1-100 Alanine (Ala) 2-500 Asparagine derivatives 1-2000 Aspartic acid 4-2000 Cystine (cystine) 10-500 Cysteine (S) 10-200 Leucine (leucine) 10-3000 Isoleucine (Ile) 30-500 Glutamic acid 3-200 Glutamine 200-1000 Arginine (Arg) 100-2000 Histidine 10-1000 Lysine 30-1000 Methionine 10-300 Phenylalanine (Phe) 1-2000 Proline (proline) 10-1000 Serine (serine) 15-3000 Threonine (Thr) 20-500 Tryptophan 2-700 Tyrosine 30-600 Valine (valine) 40-600 Vitamin C 1-150 Biotin 0.0002-10 Choline chloride 3-1000 Calcium pantothenate 0.01-30 Folic acid 1.05-90 Vitamin B2 0.002-105 Vitamin B12 0.05-60 Pyridoxine hydrochloride 0.003-50 Thiamine hydrochloride 0.06-80 Inositol (inositol) 1-700 Nicotinamide 0.03-150 Calcium chloride 50-500 Sodium chloride 300-8000 Potassium chloride 100-2000 Lithium chloride 0.00001-1 Pentahydrate copper sulfate 0.001-15 Nickel chloride hexahydrate 0.00015-10 Iron sulfate heptahydrate 0.005-80 Zinc sulfate heptahydrate 0.05-200 Magnesium sulfate 5-1000 Manganese sulfate 0.0015-20 Ferric citrate 10-6000 Sodium butyrate 0.07-10 Ammonium molybdate 0.0005-50 Disodium hydrogen phosphate 50-600 Sodium dihydrogen phosphate 70-300 Uridine (uridine) 0.0008-10 Adenosine 0.0001-10 Hydrocortisone 0.001-20 Human Epidermal Growth Factor (EGF) 0.0007-30 Transforming Growth Factor (TGF) 0.0015-20 Growth promoting factor 0.0001-10 Human Chorionic Gonadotrophin (HCG) 0.001-1 L-glutathione 0.1-100 Beta-mercaptoethanol 0.2-100 Sodium selenite (Se-Na) 0.0001-3 Sodium bicarbonate 1000-3000 Hydroxyethyl piperazine ethyl sulfuric acid (HEPES) 2000-6000
3. 3. Use of the culture medium according to claim 1 for the large-scale culture of stem cell exosomes.
4. 4. A method for culturing mesenchymal stem cell exosomes is characterized by comprising the steps of subculturing umbilical mesenchymal stem cells to the third generation, removing a culture medium, cleaning the cells, and adding the exosome production culture medium according to claim 1 for culturing.
5. 5. A method of culturing mesenchymal stem cell exosomes according to claim 3, wherein the exosome production medium is replaced every 1-3 days after addition of the exosome production medium, and the culture fluid after each replacement is collected to obtain the cell exosomes.

Description

Cell culture medium for producing/preparing mesenchymal stem cell exosomes Technical Field The invention relates to the technical field of cell culture, in particular to a serum-free culture medium formula special for high-efficiency production of mesenchymal stem cell exosomes, a preparation method thereof and a cell culture method based on the culture medium. Background 1. State of the art The mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCS) have important application value in the fields of tissue engineering, regenerative medicine and the like due to self-renewal capacity and multidirectional differentiation potential. Recent researches show that Exosomes (Exosomes) secreted by MSCs can play a unique role in anti-inflammatory, pro-angiogenic, neuroprotection and the like by delivering bioactive substances such as proteins, lipids, RNAs and the like, and become a novel therapeutic carrier for replacing cell therapies. However, large-scale exosome production faces the following bottlenecks. The culture medium is serum dependent. The traditional MSCs are cultured by adopting a culture medium containing Fetal Bovine Serum (FBS), so that the pollution risk of heterologous proteins and bovine-derived vesicles exists, the clinical transformation of exosomes is limited, the components are undefined, the serum components are complex (containing thousands of proteins, lipids and the like), and the high-purity exosomes are difficult to obtain. The purity of the product is low. The serum contains a large amount of vesicle-like particles or aggregates, and even if commercial exosome-removed bovine serum is used, such impurities cannot be completely avoided, and the downstream purification efficiency is seriously disturbed. Scale disorders. The yield of the exosome obtained by cell culture is low, the impurity is more, the purification efficiency is low, and the industrial large-scale requirement is difficult to meet. 2. Defects of the prior art Serum-free media reduce heterologous proteins, but lack optimal designs for directed regulation of exosome secretion. The addition of small chemical molecules can increase cell proliferation efficiency, but has limited yield enhancement for EVs. Genetic engineering, by over-expressing mirnas or modifying ESCRT complex proteins to promote exosome secretion, is a safety issue. 3. Object of the invention Aiming at the defects of the prior art, the invention aims to provide a serum-free culture medium with definite components and large-scale production, and by optimizing the nutrition factor combination and the growth factor proportion, the secretion efficiency and purity of the exosomes are obviously improved on the premise of ensuring the normal physiological function of the mesenchymal stem cells, thereby providing a key technical support for clinical-grade preparation of the exosomes. Disclosure of Invention The invention constructs a novel serum-free culture medium system by systematically screening key nutrient elements influencing exosome biosynthesis, and the core innovation points are shown in the following three aspects: 1. scientific basis for component design Based on MSCs metabonomics research and exosome biogenesis mechanism, the culture medium of the invention comprises three functional modules. The basic metabolism ensuring system includes essential amino acids, vitamins, inorganic salts and trace elements. A signal pathway regulation network, multidimensional signal molecules such as EGF (activating EGFR pathway), TGF-beta (regulating epithelial-mesenchymal transition), HCG (simulating embryo microenvironment) and the like, and cooperatively activate exosome synthesis related pathways (such as MAPK/ERK and PI 3K/Akt); To ensure the physiological consistency of the cell culture environment, the beta-mercaptoethanol is added for antioxidation protection, the glutathione can protect cells from being influenced by oxidative stress, and is favorable for forming favorable redox environment inside and outside the cells, and the HEPES buffer solution stabilizes components such as pH and the like. 2. Key process parameter optimization And (3) carrying out DoE experiment design on various components, and establishing an optimal proportioning scheme. And the carbon nitrogen ratio is regulated, so that the accumulation of metabolic byproducts is avoided. The ionic strength is optimized, and the Na+/K+ ratio maintains the extracellular fluid environment under the physiological condition. Growth promoting factors and microelements are added to stimulate secretion of exosomes. 3. Technical Effect verification The culture medium of the invention realizes the following breakthrough indexes. Yield enhancement, 8.2-fold (8.2X10. 10 9 parts/mL) higher exosome yield than medium containing exosome-free serum (1.0X10. 10 9 parts/mL); Nanoparticle analysis shows that the vesicle particle size is intensively distributed in the range of 50-120 nm, and a Transmission Electron Microscop