Cell culture medium for producing / preparing mesenchymal stem cell exosome

Abstract

The invention discloses a cell culture medium for producing / preparing mesenchymal stem cell exosomes. The cell culture medium takes DMEM (Dulbecco Modified Eagle Medium) as a basic culture medium and is added with the following components: amino acids, vitamins, inorganic salts, lipids, nucleic acid substances, an EGF (Epidermal Growth Factor), a TGF (Transforming Growth Factor), a growth promoting factor, HCG (Human Chorionic Gonadotropin) and trace elements. The mesenchymal stem cell culture medium is definite in component, free of serum components, controllable in quality and capable of being produced on a large scale, and stem cells can normally grow after being cultured in vitro. Particularly, the cell culture medium for producing / preparing the mesenchymal stem cell exosome can obtain more exosomes with higher purity under the same cell culture condition. The cell culture medium is beneficial to large-scale production of mesenchymal stem cell exosomes with good biological activity, further promotes wide application of the exosomes in medical treatment, anti-aging, beauty treatment and the like, and has good economic benefits.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a serum-free culture medium formulation and preparation method for the efficient production of mesenchymal stem cell exosomes, as well as a cell culture method based on this culture medium. Background Technology

[0002] 1. Current Technological Status

[0003] Mesenchymal stem cells (MSCs) have significant applications in tissue engineering and regenerative medicine due to their self-renewal capacity and multi-lineage differentiation potential. Recent studies have shown that exosomes secreted by MSCs can play unique roles in anti-inflammation, angiogenesis, and neuroprotection by delivering bioactive substances such as proteins, lipids, and RNA, thus becoming a novel therapeutic carrier for alternative cell therapies. However, large-scale exosome production faces the following bottlenecks.

[0004] Culture medium is serum-dependent. Traditional MSC culture generally uses culture medium containing fetal bovine serum (FBS), which carries the risk of contamination by heterologous proteins and bovine vesicles, resulting in limited clinical translation of exosomes, unclear composition, and complex serum composition (containing thousands of proteins, lipids, etc.), making it difficult to obtain high-purity exosomes.

[0005] The product has low purity. The serum contains a large number of vesicle-like particles or aggregates. Even with commercially available exosome-free fetal bovine serum, it is still impossible to completely avoid such impurities, which seriously interferes with the efficiency of downstream purification.

[0006] Scalability barriers. Cell culture yields low-quality exosomes with many impurities and low purification efficiency, making it difficult to meet the demands of large-scale industrial production.

[0007] 2. Deficiencies in existing technology

[0008] While serum-free culture media reduce heterologous proteins, they lack an optimized design for targeted regulation of exosome secretion.

[0009] Adding small chemical molecules can improve cell proliferation efficiency, but has limited effect on increasing EV production.

[0010] Genetic engineering can promote exosome secretion by overexpressing miRNA or modifying ESCRT complex proteins, but safety concerns remain.

[0011] 3. Purpose of the Invention

[0012] To address the shortcomings of existing technologies, this invention aims to provide a serum-free culture medium with clearly defined components that can be mass-produced. By optimizing the combination of nutritional factors and the ratio of growth factors, the secretion efficiency and purity of exosomes can be significantly improved while ensuring the normal physiological function of mesenchymal stem cells, providing key technical support for the clinical-grade preparation of exosomes. Summary of the Invention

[0013] This invention constructs a novel serum-free culture medium system by systematically screening key nutrients that affect exosome biosynthesis. Its core innovations are reflected in the following three aspects:

[0014] 1. Scientific basis for component design

[0015] Based on MSC metabolomics research and exosome biogenesis mechanisms, the culture medium of this invention comprises three major functional modules.

[0016] The basal metabolic support system includes essential amino acids, vitamins, minerals, and trace elements.

[0017] The signaling pathway regulatory network includes multidimensional signaling molecules such as EGF (activating the EGFR pathway), TGF-β (regulating epithelial-mesenchymal transition), and HCG (mimicking the embryonic microenvironment), which synergistically activate exosome synthesis-related pathways (such as MAPK / ERK and PI3K / Akt).

[0018] To ensure the physiological consistency of the cell culture environment, β-mercaptoethanol is added for antioxidant protection, glutathione can protect cells from oxidative stress and help to form a favorable redox environment inside and outside the cells, and HEPES buffer stabilizes the pH and other components.

[0019] 2. Optimization of key process parameters

[0020] DoE experiments were designed for various components to determine the optimal ratio.

[0021] Regulating the carbon-to-nitrogen ratio helps prevent the accumulation of metabolic byproducts.

[0022] Ion strength was optimized, and the Na+ / K+ ratio was maintained to maintain the extracellular fluid environment under physiological conditions.

[0023] Add growth factors and trace elements to stimulate exosome secretion.

[0024] 3. Verification of technical effectiveness

[0025] The culture medium of this invention achieves the following breakthrough indicators: Increased yield, compared to culture medium containing exosome-free serum (1.0 × 10⁻⁶). 9 Compared to (particles / mL), exosome production increased by 8.2 times (8.2 × 10⁻⁶). 9(particles / mL)

[0026] Nanoparticle analysis showed that the vesicle size was concentrated in the range of 50-120 nm, and transmission electron microscopy (TEM) revealed the classic exosome saucer or cup-shaped structure. Detailed Implementation

[0027] Example 1: Culture medium formulation

[0028] 1. Weigh each component accurately according to Table 1:

[0029]

[0030]

[0031]

[0032]

[0033] 2. Dissolving step:

[0034] The amino acid and vitamin components were dissolved in 800 mL of ultrapure water and magnetically stirred for 2 hours.

[0035] Add the inorganic salt solution dropwise, stirring continuously until completely dissolved;

[0036] Adjust the pH to 7.2 ± 0.1 using 0.1 M NaOH;

[0037] Bring the volume to 1 L, then filter and sterilize using a 0.22 μm polyethersulfone filter cartridge;

[0038] Example 2: Culture medium formulation

[0039]

[0040]

[0041]

[0042]

[0043] Dissolving steps:

[0044] The amino acid and vitamin components were dissolved in 800 mL of ultrapure water and magnetically stirred for 2 hours.

[0045] Add the inorganic salt solution dropwise, stirring continuously until completely dissolved;

[0046] Adjust the pH to 7.2 ± 0.1 using 0.1 M NaOH;

[0047] Bring the volume to 1 L, then filter and sterilize using a 0.22 μm polyethersulfone filter cartridge;

[0048] Example 3: Culture medium formulation

[0049]

[0050]

[0051]

[0052]

[0053] Dissolving steps:

[0054] The amino acid and vitamin components were dissolved in 800 mL of ultrapure water and magnetically stirred for 2 hours.

[0055] Add the inorganic salt solution dropwise, stirring continuously until completely dissolved;

[0056] Adjust the pH to 7.2 ± 0.1 using 0.1 M NaOH;

[0057] Bring the volume to 1 L, then filter and sterilize using a 0.22 μm polyethersulfone filter cartridge;

[0058] Comparative example: DMEM basal medium supplemented with 10% exosome-free serum.

[0059] Exosome production process

[0060] 1. Cell resuscitation: Umbilical cord mesenchymal stem cells were removed from the liquid nitrogen tank, rapidly thawed in a 37°C water bath, and transferred to a T25 culture flask containing 5 mL of pre-warmed culture medium;

[0061] 2. Amplification culture: Incubate at 37℃ in a 5% CO2 incubator, changing the medium every 3 days;

[0062] 3. Induction phase: When the cell confluence reaches 70%, discard the original solution and add freshly prepared culture medium of this invention;

[0063] 4. Harvesting cycle: Collect the culture supernatant every 48 hours, and harvest three times consecutively;

[0064] 5. Separation and purification: Differential centrifugation was used (300×g 10 mins → 2000×g 20 mins → 10000×g 30 mins → 100000×g 70 mins), and the precipitate was collected and resuspended in PBS buffer.

[0065] Particle size analysis was performed to determine the particle size distribution of exosomes. The results showed that the vesicle particle size was concentrated in the range of 50-120 nm.

[0066] Transmission electron microscopy (TEM) can reveal the classic exosome saucer or cup-shaped structure.

[0067] Exosome yield per unit volume in culture medium in Examples 1-3

[0068]

[0069] Adding growth factors and a balanced range of nutrients to the culture medium significantly increases the secretion of exosomes, thus enabling large-scale production of exosomes from mesenchymal stem cells.

[0070] Experimental conclusion: The serum-free culture medium of this invention is specifically designed for the efficient production of exosomes from mesenchymal stem cells. Through formulation optimization, it promotes the secretion of exosomes in the MSC culture system, and the expression level is increased by 5-8 times. Attached Figure Description

[0071] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention, and are illustrative of the invention.

[0072] The examples and descriptions are used to explain the present invention and do not constitute an undue limitation of the present invention.

[0073] Figure 1 This is a transmission electron microscope image of exosomes.

[0074] Figure 2 This is a diagram showing the analysis of exosome nanoparticles.

[0075] Figure 3 The exosome yield per unit volume was compared for culture media containing exosome-free serum, as shown in Examples 1, 2, and 3.

Claims

1. This invention discloses a cell culture medium for the production of mesenchymal stem cell exosomes, which is based on DMEM medium and supplemented with the following components: amino acids, vitamins, inorganic salts, lipids, nucleic acids, epidermal growth factor (EGF), transforming growth factor (TGF), growth-promoting factors, human chorionic gonadotropin (HCG), and trace elements.

2.

3. The application of the culture medium according to claim 1 in the large-scale culture of stem cell exosomes.

4. A method for culturing mesenchymal stem cell exosomes, characterized in that, It includes the following steps: after passage and culturing umbilical cord mesenchymal stem cells to the third generation, the culture medium is removed, the cells are washed, and then the exosome production culture medium described in claim 1 is added for further culture.

5. The method for culturing mesenchymal stem cell exosomes according to claim 3, characterized in that, After adding the exosome production medium, the exosome production medium is changed every 1-3 days, and the culture medium after each change is collected to obtain cell exosomes.

Images