A method for culturing and isolating stem cell exosomes

Abstract

This invention relates to stem cell exosomes, specifically providing a method for culturing and isolating stem cell exosomes, aiming to solve the problems of low secretion efficiency, product purity interference from exogenous impurities, and insufficient enrichment of bioactive molecules in the production of stem cell exosomes. To this end, the method for culturing and isolating stem cell exosomes of this invention includes the following steps: S1, culturing stem cells; S2, inducing stem cells to produce exosomes and collecting the exosomes; S3, isolating exosomes from the mixture. This invention achieves efficient and high-purity extraction of exosomes by optimizing the stem cell culture, induction, and isolation processes. It not only significantly increases the exosome yield per unit cell by utilizing specific culture and stimulation methods, but also effectively removes impurities through standardized isolation steps, ensuring that the obtained exosomes have excellent bioactivity and component stability, providing robust process support for the large-scale preparation of high-quality stem cell exosomes.

Description

Technical Field

[0001] This invention relates to stem cell exosomes, and specifically provides a method for culturing and isolating stem cell exosomes. Background Technology

[0002] Mesenchymal stem cell exosomes possess immense potential for application in regenerative medicine and clinical treatment due to their remarkable immunomodulatory, tissue repair, and anti-inflammatory bioactivities. However, in large-scale production using current technologies, effectively mimicking the in vivo physiological microenvironment to stimulate cellular secretory potential while simultaneously increasing exosome yield per unit cell while maintaining stem cell viability remains a major challenge for the industry. Furthermore, since conventional culture processes often require the addition of substances such as fetal bovine serum, thoroughly removing exogenous vesicles and protein impurities from the culture medium to obtain high-purity, highly stable endogenous exosomes is also a critical technological challenge that urgently needs to be addressed.

[0003] Current stem cell culture and exosome isolation technologies often suffer from drawbacks such as low secretion efficiency, insufficient product purity, and unstable biological activity. Single inducing factors or conventional normoxic culture methods are insufficient to fully activate the stress and secretion pathways of stem cells, resulting in exosomes that fail to meet industrial production standards in terms of particle concentration and functional molecule enrichment. Therefore, developing a method that combines synergistic induction by a complex group of factors with precise hypoxic environment simulation and multi-stage purification and separation is of significant technical importance and application value for achieving large-scale, efficient, high-quality, and high-purity preparation of stem cell exosomes. Summary of the Invention

[0004] The present invention aims to solve the above-mentioned technical problems, namely, to solve the problems of low secretion efficiency, product purity interference by exogenous impurities, and insufficient enrichment of bioactive molecules in the existing stem cell exosome production process.

[0005] In a first aspect, the present invention provides a method for culturing and isolating stem cell exosomes, the method comprising the following steps: S1, culturing stem cells; S2 induces stem cells to produce exosomes and collects them; S3, separating exosomes from the mixture.

[0006] In a specific embodiment of the above-mentioned method for culturing and isolating stem cell exosomes, step S1 includes the following steps: S11: Select vigorous P2-P8 generation mesenchymal stem cells and seed them on expansion medium at a density of 1×104cm2 to 5×104cm2. The expansion medium is then cultured in an incubator at 37℃, 5% carbon dioxide concentration, and saturated humidity.

[0007] In a specific embodiment of the above-mentioned method for culturing and isolating stem cell exosomes, the amplification medium is based on Duchenne modified Eagle medium containing 10% fetal bovine serum or a complete medium containing 1% platelet lysate. The amplification medium also includes 1% human serum albumin, 2 µg / ml recombinant insulin, 15 ng / ml epidermal growth factor, 25 ng / ml basic fibroblast growth factor, 0.12% thioglycerol, 1% fructose, 8-12 ng / mL interleukin-1β, interferon, erythropoietin, and platelet-derived growth factor-BB.

[0008] In a specific embodiment of the above-mentioned method for culturing and isolating stem cell exosomes, step S1 further includes the following steps: S12, when the stem cell fusion rate reaches 70% to 90%, the oxygen concentration is adjusted to 1%-5% for 42 to 56 hours of hypoxia induction to simulate the physiological microenvironment in vivo and stimulate exosome secretion.

[0009] In a specific embodiment of the above-mentioned method for culturing and isolating stem cell exosomes, step S2 further includes the following steps: S21, discard the amplification medium and rinse the stem cells 2-4 times with Hanks balanced salt solution that does not contain calcium and magnesium ions; S22, after adding stem cells to secretory culture medium, culture for 24-72 hours; S23, aspirate the supernatant of the secretion medium, wash the culture dish twice with 1-2 ml of sterile phosphate buffer, and mix the washing solution and supernatant to obtain crude exosome solution.

[0010] In a specific embodiment of the above-mentioned method for culturing and isolating stem cell exosomes, the secretory culture medium is based on a modified version of the minimum essential culture medium α, and the secretory culture medium further includes 0.5% bovine serum albumin, 200 ng / ml epidermal growth factor, 150 ng / ml transforming growth factor, 30 µg / ml coenzyme Q10, 600 µg / ml potassium chloride dissolved in phosphate buffer, 0.6 µg / ml fructose diphosphate sodium and 300 µg / ml histamine dihydrochloride.

[0011] In a specific embodiment of the above-described method for culturing and isolating stem cell exosomes, step S3 further includes the following steps: S31, centrifuge the crude exosome solution at 250-350G for 8-12 minutes; S32, centrifuge the crude exosome solution at 1500-3000G for 18-25 minutes; S33, centrifuge the crude exosome solution at 8000-12000G for 25-35 minutes; S34, the crude exosome fluid was filtered using a 0.22µm filter; S35, using a 10 kDa or 100 kDa ultrafiltration tube, centrifuge at 4°C and 4000g for about 30-45 minutes; S36. Transfer the concentrate to a 30% sucrose heavy water density pad and centrifuge at 4°C and 80,000-120,000g for about 120 minutes.

[0012] By adopting the above technical solution, this invention achieves efficient and high-purity extraction of exosomes by optimizing the culture, induction and separation process of stem cells. It not only significantly increases the exosome yield per unit cell by using specific culture and stimulation methods, but also effectively removes impurities through standardized separation steps, ensuring that the obtained exosomes have excellent biological activity and component stability, providing robust process support for the large-scale preparation of high-quality stem cell exosomes.

[0013] Furthermore, by selecting specific generations of mesenchymal stem cells with vigorous growth and combining them with precisely controlled seeding density and standardized temperature, humidity, and carbon dioxide culture environment, this invention ensures from the source that the seed cells possess excellent proliferative potential and metabolic activity, laying a high-quality cell biology foundation for subsequent large-scale induction of exosome secretion.

[0014] Furthermore, this invention achieves a significant synergistic effect by scientifically combining multiple bioactive factors in the basal culture medium: using IL-1β and interferon for immune pre-stimulation, combined with PDGF-BB to enhance cell proliferation, ensures large-scale expansion of stem cells while significantly reducing cell loss and impurity contamination through anti-apoptotic protection by factors such as EPO and insulin, thereby achieving a leapfrog improvement in exosome particle concentration, purity, and immunomodulatory activity at the product end.

[0015] Furthermore, this invention successfully simulates the physiological stress of the microenvironment, such as bone marrow, by introducing 1%-5% precise hypoxia induction when the fusion degree of stem cells is 70%-90%. While maintaining the balance between mitochondrial function and membrane structure integrity, it maximizes the activation of the hypoxia-inducible factor signaling pathway, thereby significantly increasing the exosome secretion rate per unit number of cells and enriching more bioactive molecules with high tissue repair capabilities.

[0016] Furthermore, this invention eliminates the interference of serum components and exogenous vesicles in the original culture medium on product purity by repeatedly and thoroughly rinsing with D-Hanks solution free of calcium and magnesium ions before switching the secretion medium. Combined with the subsequent combined collection of secretion supernatant and washing solution, this not only ensures that the exosomes have extremely high endogenous purity and accurate composition, but also minimizes exosome loss during the collection process, providing a key process guarantee for obtaining high-quality crude exosome solution with low background impurities.

[0017] Furthermore, this invention provides an optimized secretory microenvironment for stem cells by scientifically compounding a variety of nutrients and protective factors in a modified α-MEM basal medium: using BSA to maintain osmotic pressure and protect the structural integrity of exosomes, combining active ingredients such as EGF, TGF, and coenzyme Q10 to alleviate metabolic stress in cells under serum-free conditions, and combining histamine dihydrochloride and potassium chloride to regulate ion balance and enhance secretory dynamics. Thus, while ensuring cell viability, it significantly improves the exosome yield per unit number of cells and the stability of the products during the collection process.

[0018] Furthermore, this invention achieves highly efficient purification and enrichment of exosomes through a combination of gradient centrifugation, microfiltration, ultrafiltration concentration, and sucrose heavy water density pad ultracentrifugation: physical impurities such as intact cells, cell debris, dead cells, and large vesicles are precisely removed sequentially by graded centrifugation and filtration, followed by high-rate concentration of the product through ultrafiltration, and finally, non-exosome components such as proteins and nucleic acids are completely separated by density pad ultracentrifugation technology, ensuring that the obtained exosomes have extremely high purity, structural integrity, and biological activity. Detailed Implementation

[0019] Preferred embodiments of the present invention are described below. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0020] It should be noted that in the description of this invention, terms such as "upper," "lower," "left," "right," "inner," and "outer," which indicate directional or positional relationships, are merely for ease of description and do not indicate or imply that the device or element must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0021] Furthermore, it should be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "setting," and "connection" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a direct connection, an indirect connection through an intermediate medium, or a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0022] To address the problems of low secretion efficiency, product purity interference from exogenous impurities, and insufficient enrichment of bioactive molecules during stem cell exosome production, this embodiment discloses a method for culturing and isolating stem cell exosomes, comprising the following steps: S1, culture stem cells.

[0023] S2 induces stem cells to produce exosomes and collect them.

[0024] S3, separating exosomes from the mixture.

[0025] Step S1 includes the following steps: S11, selected from highly vigorous P2-P8 generation mesenchymal stem cells, at a concentration of 1×10⁻⁶. 4 cm 2 Up to 5×10 4 cm 2 The amplification medium was inoculated at a density on the amplification medium and then incubated in an incubator at 37°C, 5% carbon dioxide concentration, and saturated humidity.

[0026] The amplification medium used was Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, or a complete medium containing 1% platelet lysate. The amplification medium also included 1% human serum albumin, 2 µg / ml recombinant insulin, 15 ng / ml epidermal growth factor (EGF), 25 ng / ml basic fibroblast growth factor (bFGF, also known as FGF-2), 0.12% thioglycerol, 1% fructose, 8-12 ng / mL interleukin-1β (IL-1β), interferon, erythropoietin (EPO), and platelet-derived growth factor-BB (PDGF-BB).

[0027] S12, when the stem cell fusion rate reaches 70% to 90%, the oxygen concentration is adjusted to 1%-5% for 42 to 56 hours of hypoxia induction to simulate the physiological microenvironment in vivo and stimulate exosome secretion.

[0028] Step S2 includes the following steps: S21, discard the original amplification medium and use Hanks' Balanced Salt Solution without calcium and magnesium ions. 2+ and Mg 2+ Rinse stem cells 2-4 times with D-Hanks solution (abbreviated as D-Hanks solution) to thoroughly remove interference from exogenous vesicles in the serum.

[0029] S22: Stem cells were added to the secretory medium and cultured for 24-72 hours. The secretory medium was based on Minimum Essential Medium Eagle - Alpha Modification (α-MEM), with the addition of 0.5% bovine serum albumin (BSA), 200 ng / ml epidermal growth factor (EGF), 150 ng / ml transforming growth factor (TGF), 30 µg / ml coenzyme Q10 (CoQ10), 600 µg / ml potassium chloride dissolved in phosphate buffer, 0.6 µg / ml fructose diphosphate sodium, and 300 µg / ml histamine dihydrochloride to increase the exosome yield per unit number of cells, which is beneficial for large-scale production.

[0030] S23, aspirate the supernatant from the secretion medium, wash the culture dish twice with 1-2 ml of sterile phosphate-buffered saline (PBS), and mix the washing solution and supernatant to obtain crude exosome solution.

[0031] Step S3 includes the following steps: S31, centrifuge the crude exosome solution at 250-350G for 8-12 minutes to remove intact cells.

[0032] S32, centrifuge the crude exosome solution at 1500-3000G for 18-25 minutes to remove cell debris and dead cells.

[0033] S33, centrifuge the crude exosome solution at 8000-12000G for 25-35 minutes to remove large vesicles.

[0034] S34, the crude exosome fluid was filtered through a 0.22µm filter to remove any remaining large vesicles and bacteria.

[0035] S35, using a 10 kDa or 100 kDa ultrafiltration tube, centrifuge at 4°C and 4000g for approximately 30-45 minutes.

[0036] S36. Transfer the concentrate to a 30% sucrose heavy water density pad and centrifuge at 4°C and 80,000-120,000g for about 120 minutes.

[0037] The technical effects of this method are illustrated below through several embodiments.

[0038] Example 1 Select vigorous P2-P8 generation mesenchymal stem cells, at a ratio of 1×10⁸ 4 cm 2 Up to 5×10 4 cm 2 The amplification medium was inoculated at a specific density onto the amplification medium, which was then incubated at 37°C with 5% carbon dioxide and saturated humidity. The amplification medium was based on Duchenne modified Eagle medium containing 10% fetal bovine serum, supplemented with 1% human serum albumin, 2 µg / ml recombinant insulin, 15 ng / ml epidermal growth factor, 25 ng / ml basic fibroblast growth factor, 0.12% thioglycerol, and 1% fructose.

[0039] After the amplification medium was cultured in an incubator at 37°C, 5% carbon dioxide concentration, and saturated humidity until the stem cell confluence reached 70%, the particle concentration of exosomes per unit volume, the total production of exosome proteins, the number of exosomes produced by a single cell, the LDH content, the anti-inflammatory miRNA content, the content of immunomodulatory factors in exosomes, the proportion of M2 macrophages, and the T cell inhibition rate were measured.

[0040] Example 2 The difference between this embodiment and Example 1 is the addition of 8-12 ng / mL of interleukin-1β to the amplification culture medium. Comparison of the experimental results of Example 1 and Example 2 demonstrates that interleukin-1β, as a core pretreatment factor, activates the stress and secretion pathways of stem cells by mimicking the inflammatory microenvironment. Its effects are mainly reflected in: firstly, significantly increasing NTA particle concentration and secretion efficiency per unit cell through NF-κB signal transduction; secondly, enhancing cell membrane stability, effectively reducing the incorporation of cell debris and endoplasmic reticulum proteins by decreasing LDH release and apoptosis rate, thus ensuring high product purity; and most importantly, IL-1β induces the enrichment of exosomes in immunomodulatory molecules such as miR-146a, resulting in superior biological activity in promoting M2 macrophage polarization and inhibiting inflammatory responses.

[0041] Example 3 The difference between this embodiment and Example 1 is the addition of interferon to the amplification culture medium. Specifically, the interferon in this embodiment is recombinant human interferon-γ (IFN-γ). Comparison of the experimental results from Examples 1 and 3 demonstrates that interferon, as a key immunomodulatory pretreatment factor, significantly enhances the immunogenicity regulation of stem cell exosomes and the antiviral / antitumor activity of cells. By activating the intracellular JAK-STAT signaling pathway, interferon can induce stem cells to secrete exosomes enriched with specific microRNAs (miRNAs) and cytokines, thereby more effectively inhibiting inflammatory responses and regulating T cell activity.

[0042] Example 4 The difference between this embodiment and Example 1 lies in the addition of erythropoietin to the amplification culture medium. Comparison of the experimental results from Examples 1 and 4 demonstrates that erythropoietin primarily functions as a potent cell-protective and anti-apoptotic factor: by binding to the EPO receptor on the surface of stem cells, it activates the PI3K / Akt pro-survival signaling pathway. Under hypoxia induction in the S1 phase and high-intensity secretory pressure in the S2 phase, it significantly enhances the tolerance of stem cells and reduces spontaneous apoptosis, thereby effectively reducing large vesicles and protein impurities generated by cell lysis. Simultaneously, EPO can induce exosomes to accumulate bioactive molecules with angiogenesis-promoting and tissue antioxidant capacity, resulting in the final prepared exosomes exhibiting stronger therapeutic efficacy in clinical applications such as repairing ischemic injuries, accelerating wound healing, and neuroprotection.

[0043] Example 5 The difference between this embodiment and Example 1 is the addition of platelet-derived growth factor-BB to the amplification culture medium. Comparison of the experimental results from Examples 1 and 5 demonstrates that platelet-derived growth factor-BB primarily functions as a potent mitogen and chemokine: by activating the PDGFR-β receptor on the surface of stem cells and its downstream MAPK / ERK signaling pathway, it significantly enhances the proliferation and colony-forming ability of mesenchymal stem cells in the S1 phase, ensuring the basic cell base required for large-scale production. Simultaneously, PDGF-BB can induce exosomes to accumulate high levels of pro-angiogenic and fibroblast migration-related active proteins and miRNAs, resulting in the final exosomes exhibiting excellent clinical effects in skin regeneration, bone repair, and accelerated chronic wound healing.

[0044] Example 6 The difference between this embodiment and Example 1 is the addition of 8-12 ng / mL of interleukin-1β, interferon, erythropoietin, and platelet-derived growth factor-BB to the amplification medium. Comparison of the experimental results from Examples 1 to 5 demonstrates that this embodiment achieves a significant synergistic effect across the entire cell chain: single components often only improve specific steps such as proliferation or anti-apoptosis, while this complex factor group, through the interaction and superposition of multiple signaling pathways, not only overcomes the limitations of insufficient induction intensity of single factors, but also, while ensuring large-scale cell proliferation through the PDGF-BB effect, significantly reduces cell damage during the secretion phase through immune pre-stimulation achieved by IL-1β and interferon effects and anti-stress protection provided by the EPO effect. This results in a significant improvement in particle concentration, purity, and biological activity at the product end, constructing a highly efficient and high-quality exosome production system that is unmatched by conventional culture methods.

[0045] Example 7 In an environment with an oxygen concentration of 21%, stem cells that have reached 80% fusion after step S11 are cultured for 50 hours.

[0046] Example 8 The difference between this embodiment and Embodiment 7 is that the stem cells with 80% fusion after step S11 were cultured for 50 hours in an oxygen concentration of 0.5%. This embodiment aims to explore the effect of more severe physiological stress on stem cell secretion behavior by reducing the oxygen concentration to 0.5%. The experimental results show that although extremely low oxygen theoretically produces stronger stress stimulation, compared with Embodiments 9-11, a concentration of 0.5% may lead to impaired cell viability or premature apoptosis due to excessive inhibition of cellular aerobic metabolism. Therefore, it is slightly inferior to the optimal induction range of 1%-5% in maintaining product purity and stability of unit cell secretion efficiency.

[0047] Example 9 The difference between this embodiment and Embodiment 7 is that, in an environment with an oxygen concentration of 1%, stem cells with an 80% fusion rate after step S11 were recultured for 50 hours. This embodiment, by adjusting the oxygen concentration to 1%, effectively simulates the physiological microenvironment of deep ischemia and hypoxia in vivo, thereby activating a strong hypoxia-inducible factor signaling pathway within the stem cells. While ensuring basic cell viability, it significantly increases the exosome secretion rate per unit number of cells and the enrichment of angiogenesis-related factors in the products, laying a crucial environmental foundation for the subsequent preparation of therapeutic exosomes with high tissue repair capabilities.

[0048] Example 10 The difference between this embodiment and Embodiment 7 is that, in an environment with an oxygen concentration of 2%, stem cells with an 80% fusion rate after step S11 were cultured for another 50 hours. This embodiment, by setting the oxygen concentration to 2% for moderate hypoxia induction, achieved an excellent balance between simulating the physiological microenvironment and maintaining cellular metabolic homeostasis. This not only significantly enhanced the kinetic efficiency of stem cell exosome secretion through moderate oxidative stress, resulting in a substantial increase in particle concentration compared to Embodiment 7, but also, compared to the lower oxygen concentration group, the 2% concentration better maintained the integrity of mitochondrial function and membrane structure. Thus, while ensuring high exosome production, it effectively improved the stability and functional consistency of bioactive molecules in the product.

[0049] Example 11 The difference between this embodiment and Embodiment 7 is that the stem cells with 80% fusion after step S11 are cultured for 50 hours in an oxygen concentration of 5%. This embodiment, by adjusting the oxygen concentration to 5%, more accurately simulates the real physical stress of mesenchymal stem cells in the bone marrow and other microenvironments in vivo. This concentration setting significantly induces exosome secretion while minimizing the cellular metabolic burden that hypoxia may cause. This results in exosomes that, while maintaining extremely high biocompatibility, are enriched with more stable growth factors and anti-inflammatory factors, making them particularly suitable for clinical applications where the cell environment is highly sensitive and long-term biological activity of the product is required.

[0050] Example 12 The difference between this embodiment and Embodiment 7 is that, in an environment with an oxygen concentration of 10%, stem cells with an 80% fusion rate after step S11 were recultured for 24 hours. While setting the oxygen concentration to 10% in this embodiment activated the cellular stress response to some extent compared to a normoxic environment, its concentration was still significantly higher than the range of physiological hypoxia, resulting in insufficient stimulation of hypoxia-inducible factors and their downstream secretory pathways. Experimental results showed that the particle concentration and bioactive molecule enrichment of exosomes at this concentration were significantly lower than in the 5% oxygen concentration group, failing to fully leverage the core advantages of hypoxia induction in increasing exosome production and immunomodulatory efficacy, and making it difficult to achieve the highly efficient secretion state required for industrial production.

[0051] Example 13 After step S1, the original culture medium was aspirated, and stem cells were added to the secretory medium and cultured for 24-72 hours. The secretory medium was based on a modified version of the minimum essential medium α, with the following added: 200 ng / ml epidermal growth factor, 150 ng / ml transforming growth factor, 30 µg / ml coenzyme Q10, 600 µg / ml potassium chloride dissolved in phosphate buffer, 0.6 µg / ml fructose diphosphate sodium, and 300 µg / ml histamine dihydrochloride. Then, the supernatant of the secretory medium was aspirated, and the culture dishes were washed twice with 1-2 ml of sterile phosphate buffer. The washings and supernatant were mixed to obtain crude exosome solution.

[0052] Example 14 The difference between this embodiment and Embodiment 13 is that after discarding the original culture medium, the stem cells are washed 2-4 times with a calcium- and magnesium-free Hanks balanced salt solution. This embodiment, by adding a D-Hanks solution washing step after discarding the amplification culture medium, effectively utilizes a calcium- and magnesium-free balanced salt solution to remove residual serum proteins and exogenous small vesicles from the cell surface and intercellular spaces, eliminating contamination of product purity by additives such as fetal bovine serum at the source. Compared with Embodiment 13, this washing process significantly reduces the background impurity level in the crude exosome solution, ensuring that the exosomes obtained after subsequent separation have extremely high endogenous purity and precise composition. It is an indispensable quality control step in achieving high-standard, research-grade exosome extraction.

[0053] Example 15 The difference between this embodiment and Embodiment 13 is the addition of 0.5% bovine serum albumin to the secretion medium. This embodiment provides necessary physical protection and osmotic pressure support to stem cells by adding 0.5% bovine serum albumin to the secretion medium, effectively alleviating metabolic stress in cells under serum-free secretion pressure. This significantly improves the stability and dispersibility of exosomes during collection while maintaining cell viability, reducing losses due to adsorption or aggregation, and ultimately achieving a higher density of exosomes per unit volume, ensuring process robustness for large-scale production.

[0054] Example 16 The difference between this embodiment and Embodiment 13 is that after discarding the original culture medium, the stem cells are rinsed 2-4 times with a calcium- and magnesium-free Hanks balanced salt solution; and 0.5% bovine serum albumin is added to the secretory culture medium. This embodiment achieves synergistic effects in the process by combining thorough rinsing with D-Hanks solution with the addition of 0.5% bovine serum albumin to the secretory culture medium: the rinsing step eliminates contamination from exogenous vesicles, ensuring high product purity, while the addition of BSA provides an optimized secretory microenvironment for the cells, effectively protecting the integrity of the exosome structure and preventing its loss due to adsorption. Experimental results demonstrate that this combined strategy significantly improves exosome purity while further ensuring the yield efficiency and bioactivity per unit cell, making it the optimal process for the industrial preparation of high-quality exosomes.

[0055] The technical solution of the present invention has been described in conjunction with preferred embodiments. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions resulting from these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A method for culturing and isolating stem cell exosomes, characterized in that, Includes the following steps: S1, culturing stem cells; S2 induces stem cells to produce exosomes and collects them; S3, separating exosomes from the mixture.

2. The method for culturing and isolating stem cell exosomes according to claim 1, characterized in that, Step S1 includes the following steps: S11, selected from highly vigorous P2-P8 generation mesenchymal stem cells, at a concentration of 1×10⁻⁶. 4 cm 2 Up to 5×10 4 cm 2 The amplification medium was inoculated at a density on the amplification medium and then incubated in an incubator at 37°C, 5% carbon dioxide concentration, and saturated humidity.

3. The method for culturing and isolating stem cell exosomes according to claim 2, characterized in that, The amplification medium is based on Duchenne modified Eagle medium containing 10% fetal bovine serum or complete medium containing 1% platelet lysate. The amplification medium also includes 1% human serum albumin, 2 µg / ml recombinant insulin, 15 ng / ml epidermal growth factor, 25 ng / ml basic fibroblast growth factor, 0.12% thioglycerol, 1% fructose, 8-12 ng / mL interleukin-1β, interferon, erythropoietin, and platelet-derived growth factor-BB.

4. The method for culturing and isolating stem cell exosomes according to claim 2, characterized in that, Step S1 also includes the following steps: S12, when the stem cell fusion rate reaches 70% to 90%, the oxygen concentration is adjusted to 1%-5% for 42 to 56 hours of hypoxia induction to simulate the physiological microenvironment in vivo and stimulate exosome secretion.

5. The method for culturing and isolating stem cell exosomes according to claim 2, characterized in that, Step S2 also includes the following steps: S21, discard the amplification medium and rinse the stem cells 2-4 times with Hanks balanced salt solution that does not contain calcium and magnesium ions; S22, after adding stem cells to secretory culture medium, culture for 24-72 hours; S23, aspirate the supernatant of the secretion medium, wash the culture dish twice with 1-2 ml of sterile phosphate buffer, and mix the washing solution and supernatant to obtain crude exosome solution.

6. The method for culturing and isolating stem cell exosomes according to claim 5, characterized in that, The secretory medium is based on a modified version of the minimum essential medium α, and the secretory medium also includes 0.5% bovine serum albumin, 200 ng / ml epidermal growth factor, 150 ng / ml transforming growth factor, 30 µg / ml coenzyme Q10, 600 µg / ml potassium chloride dissolved in phosphate buffer, 0.6 µg / ml fructose diphosphate sodium and 300 µg / ml histamine dihydrochloride.

7. The method for culturing and isolating stem cell exosomes according to claim 1, characterized in that, Step S3 also includes the following steps: S31, centrifuge the crude exosome solution at 250-350G for 8-12 minutes; S32, centrifuge the crude exosome solution at 1500-3000G for 18-25 minutes; S33, centrifuge the crude exosome solution at 8000-12000G for 25-35 minutes; S34, the crude exosome fluid was filtered using a 0.22µm filter; S35, using a 10 kDa or 100 kDa ultrafiltration tube, centrifuge at 4°C and 4000g for about 30-45 minutes; S36. Transfer the concentrate to a 30% sucrose heavy water density pad and centrifuge at 4°C and 80,000-120,000g for about 120 minutes.