Method for promoting cells to secrete exosomes and application

Abstract

The invention belongs to the technical field of biological materials, tissue engineering and regenerative medicine, and discloses a method for promoting cells to secrete exosomes, which comprises the following steps: step 1, placing the cells in a culture medium with the pH value of 6.4-7.1 and the osmotic pressure of 290-320 mmol / L; 2, maintaining the oxygen concentration of the culture environment not to exceed 5% of the volume fraction. By accurately controlling the pH value, the osmotic pressure and the oxygen concentration of a culture environment and combining a small molecule inducer and a gene editing technology, a Rab protein family mediated exosome secretion pathway can be obviously activated. Compared with a conventional method, the exosome secretion amount is increased by 20-30 times, and the clinical requirement for the exosome amount is greatly met. Meanwhile, the obtained exosome is uniform in particle size, the particle size ranges from 50 nanometers to 150 nanometers, the purity reaches up to 95% or above, high quality of the exosome is guaranteed, and a solid foundation is provided for subsequent application.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomaterials, tissue engineering and regenerative medicine, and specifically relates to a method and application for promoting cell secretion of exosomes. Background Art

[0002] In today's medical research, exosomes, as highly promising therapeutic vehicles for disease diagnosis and treatment, are showing remarkable promise. Leveraging their unique biological properties, exosomes can transmit information between cells and regulate cellular function, offering new insights and approaches for treating a wide range of diseases. However, despite this enormous potential, existing exosome production technologies face two critical bottlenecks that severely restrict their widespread clinical application.

[0003] First, low yield is a prominent issue facing current exosome production. Using conventional culture methods, the amount of exosome protein obtained per milliliter of culture medium is extremely limited, less than 1 microgram. This yield falls far short of meeting the needs of large-scale clinical applications. Clinical treatments often require large quantities of exosomes to achieve the desired therapeutic effect, but the current low yield makes clinical supply of exosomes extremely difficult, significantly limiting their further development in disease diagnosis and treatment.

[0004] Secondly, the lack of process standardization is also a key factor hindering the development of the exosome industry. Currently, there are many uncertainties in the existing methods of inducing exosomes. For example, common methods such as hypoxia stimulation or drug treatment can induce the production of exosomes to a certain extent, but the efficiency of these induction methods is not stable. Different experimental conditions, operators, and experimental batches may lead to large differences in the induction effect. In addition, there is a lack of unified standards and specifications in the isolation and purification process of exosomes. Different research teams or production companies often use their own different isolation and purification methods, which makes it difficult to effectively guarantee the quality and purity of exosomes, further affecting the reliability and safety of exosomes in clinical applications.

[0005] While some related patents have attempted to address issues in exosome production, such as authorized patent CN112920991 B, which proposes an exosome inducer scheme, this scheme does not delve into the synergistic enhancement effects of environmental parameters such as pH and osmotic pressure on the inducer. In actual production, environmental parameters often have a significant impact on the effectiveness of the inducer, and the shortcomings of this patent make it difficult to fully realize the inducer's potential in practical applications. Furthermore, while patent CN 117844747 A achieved a 20- to 30-fold increase in exosome secretion, this achievement relied on expensive culture medium components. The high cost of culture medium has led to a significant increase in the production cost of exosomes, placing significant economic pressure on the commercialization of this technology and hindering large-scale production and application. Summary of the Invention

[0006] The purpose of the present invention is to provide a method and application for promoting cell secretion of exosomes to solve the problems raised in the above background technology.

[0007] In order to achieve the above object, the present invention provides the following technical solution: a method for promoting cell secretion of exosomes, the steps of the method for promoting cell secretion of exosomes are: Step 1: placing the cells in a culture medium with a pH of 6.4 to 7.1 and an osmotic pressure of 290 to 320 mmol / L; Step 2: Maintaining the oxygen concentration in the culture environment to no more than 5% by volume; Step 3, adding 50 to 200 micromoles per liter of estrogen and 10 to 30 millimoles per liter of glucose to the culture medium; Step 4: Collect exosomes after 48 to 96 hours of culture.

[0008] Preferably, it also includes: 50 to 200 micromoles per liter of estrogen, 10 to 30 millimoles per liter of glucose; pH regulator to stabilize the pH of the culture medium at 6.4 to 7.1; Osmotic pressure regulator, maintains osmotic pressure between 290 and 320 mmol / l.

[0009] Preferably, in step three, 0.1 to 1.0 mmol / L β-mercaptoethanol and 1 to 10 μg / mL anti-TGF-β single domain antibody are added simultaneously.

[0010] Preferably, the cells are gene-edited to overexpress Rab11 protein or Rab35 protein.

[0011] Preferably, the cells are selected from mesenchymal stem cells, mammary epithelial cells or tumor cells.

[0012] Preferably, the composition further comprises 1 to 10 micromoles per liter of 5'-adenylic acid and 0.5 to 5 micrograms per milliliter of cofilin-1.

[0013] Preferably, the exosome purification adopts a hollow fiber membrane column combined with anion exchange chromatography technology.

[0014] Preferably, the obtained exosomes have a particle size of 50 to 150 nanometers, and the purity is not less than 95% as determined by nanoflow cytometry.

[0015] Preferably, the exosomes are loaded with the anti-tumor drug doxorubicin or berberine.

[0016] Preferably, exosomes are delivered to the tumor site by surface-modified magnetic nanoparticles.

[0017] The beneficial effects of the present invention are as follows: 1. The methods and culture media provided by this invention for promoting cell secretion of exosomes, through precise control of the pH, osmotic pressure, and oxygen concentration of the culture environment, combined with small molecule inducers and gene editing techniques, can significantly activate the exosome secretion pathway mediated by the Rab protein family. This innovative technology increases exosome secretion by 20 to 30 times compared to conventional methods, greatly meeting the clinical demand for exosome quantity. Furthermore, the obtained exosomes have a uniform particle size ranging from 50 to 150 nanometers and a purity exceeding 95%, ensuring the high quality of the exosomes and providing a solid foundation for subsequent applications.

[0018] 2. The method of the present invention not only achieves breakthroughs in yield and quality, but also demonstrates significant advantages in production and purification. This method supports the use of large-scale production equipment such as hollow fiber bioreactors, enabling the production of exosomes to move from laboratory-scale to industrial production, reducing production costs and improving production efficiency. In terms of exosome purification, the use of hollow fiber membrane columns coupled with anion exchange chromatography technology can more efficiently and accurately separate and purify exosomes, further ensuring the purity and activity of exosomes, and providing strong support for the clinical application of exosomes.

[0019] 3. Exosomes prepared using the methods of this invention have broad application prospects in the field of tumor treatment carriers. These exosomes can be loaded with anti-tumor drugs such as doxorubicin or berberine. By surface-modifying them with magnetic nanoparticles, the exosomes can be delivered to tumor sites, enabling precise treatment, enhancing drug efficacy, and reducing damage to normal tissues. This innovative application provides new insights and methods for tumor treatment, potentially improving treatment outcomes and quality of life for cancer patients, and holds immense clinical and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a diagram showing the steps of the method for promoting cell secretion of exosomes according to the present invention. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] like Figure 1 As shown, the embodiments of the present invention provide a method for promoting cell secretion of exosomes by precisely regulating various parameters of the cell culture environment and combining small molecule inducers with gene editing technology to activate the exosome secretion pathway mediated by the intracellular Rab protein family, thereby achieving a dual improvement in exosome production and quality. The specific implementation steps are as follows: Step 1: Environmental parameter optimization First, the cells are placed in a carefully formulated culture medium. The pH of this medium is strictly controlled within the range of 6.4 to 7.1 to create a suitable acidic microenvironment, which helps enhance cell membrane permeability and creates favorable conditions for subsequent exosome secretion. Furthermore, the osmotic pressure of the culture medium is precisely adjusted to 290 to 320 mmol / L (for mammalian cells) or 250 to 325 mmol / L (for human cells) to ensure cell growth under optimal osmotic pressure conditions.

[0023] Step 2: Oxygen concentration control Next, the oxygen concentration in the culture environment is maintained at no more than 5% by volume. This low-oxygen condition mimics the hypoxic microenvironment of solid tumors, helping to activate the HIF-1α signaling pathway within cells and thereby promoting the secretion of exosomes.

[0024] Step 3: Add inducer combination In addition to basic nutrients, the culture medium also contains 50 to 200 micromoles per liter of estrogen and 10 to 30 millimoles per liter of glucose. These small molecule inducers work synergistically to enhance the activity of the intracellular Rab protein family. Furthermore, to further optimize the induction effect, 0.1 to 1.0 millimoles per liter of β-mercaptoethanol and 1 to 10 micrograms per milliliter of an anti-TGF-β single-domain antibody are also added. These ingredients synergistically enhance the activity of the Rab35 protein, thereby promoting exosome secretion.

[0025] Step 4: Cultivation and collection After culturing the cells under the optimized culture conditions for 48 to 96 hours, exosomes can be collected. The exosomes collected at this time not only have a significantly higher yield but also a significantly improved quality.

[0026] It is made by adding specific inducers and regulators to the basal culture medium and exosome-free serum. The culture medium contains the following key components: 50 to 200 micromoles per liter of estrogen: As one of the important inducers of exosome secretion, estrogen can activate related signaling pathways within cells and promote the secretion of exosomes.

[0027] 10 to 30 mmol / L of glucose: provides energy for cell growth and metabolism, and also facilitates the secretion of exosomes.

[0028] pH regulator: used to stabilize the pH value of the culture medium within the range of 6.4 to 7.1 to form a suitable acidic microenvironment.

[0029] Osmotic pressure regulator: Used to maintain the osmotic pressure of the culture medium at 290 to 320 mmol / L (for mammalian cells) or 250 to 325 mmol / L (for human cells) to ensure that cells grow under optimal osmotic pressure conditions.

[0030] In addition, according to actual needs, other components can be further added to the culture medium, such as 1 to 10 micromoles per liter of 5'-adenosine monophosphate and 0.5 to 5 micrograms per milliliter of cofilin-1, to further optimize the induction effect.

[0031] In step three, not only estrogen and glucose are added as inducers, but also 0.1 to 1.0 mmol / L of β-mercaptoethanol and 1 to 10 μg / mL of anti-TGF-β single-domain antibody are added simultaneously. These ingredients work synergistically to further enhance the activity of the intracellular Rab35 protein, thereby promoting exosome secretion. This combination of inducers significantly improves exosome production and quality.

[0032] To further enhance the efficiency of exosome secretion, cells can also be genetically edited. Specifically, CRISPR / Cas9 technology can be used to overexpress Rab11 or Rab35 proteins. These Rab proteins play a key role in exosome secretion, and overexpression of these proteins can activate the exosome secretion pathway, significantly increasing exosome production.

[0033] These include but are not limited to mesenchymal stem cells, mammary epithelial cells, or tumor cells. These cells are capable of secreting exosomes under specific culture conditions, and the production and quality of exosomes can be further improved by optimizing the culture environment and adding inducers.

[0034] The exosome secretion induction medium of claim 2 may contain, in addition to the basal medium, exosome-free serum, estrogen, glucose, pH regulator, and osmotic pressure regulator, other ingredients to optimize the induction effect. For example, 1 to 10 micromoles per liter of 5'-adenosine monophosphate and 0.5 to 5 micrograms per milliliter of cofilin-1 may be added. These ingredients can act synergistically to further improve the secretion efficiency and quality of exosomes.

[0035] The collected exosomes require purification to remove impurities and unsecreted cellular components. To this end, a hollow fiber membrane column coupled with anion exchange chromatography can be used for purification. This purification technique is efficient, rapid, and reproducible, enabling the production of highly purified exosome samples. The exosomes prepared by the method of claim 1 exhibit the following notable properties: uniform particle size ranging from 50 to 150 nanometers; and high purity, with a purity of at least 95% as determined by nanoflow cytometry. These properties hold great promise for the application of exosomes in biomedical research and clinical applications.

[0036] The exosomes prepared in claim 1 offer significant advantages in preparing tumor therapeutic vectors. These exosomes can be loaded with anti-tumor drugs such as doxorubicin or berberine, forming targeted tumor therapeutic vectors. By loading drugs into exosomes, sustained release and targeted delivery of the drugs can be achieved, thereby improving therapeutic efficacy and reducing side effects.

[0037] In the application described in claim 9, to further enhance the targeting and delivery efficiency of exosomes, surface-modified magnetic nanoparticles can be used to deliver exosomes to tumor sites. By modifying the exosomes with magnetic nanoparticles, an external magnetic field can be used to guide the exosomes precisely to tumor tissue, thereby achieving precise drug delivery and treatment. This approach offers new insights and approaches for tumor treatment.

[0038] Example 1: Efficient secretion of exosomes from mesenchymal stem cells The pH value of the DMEM / F12 medium was adjusted to 6.8, the osmotic pressure was adjusted to 300 mmol / L, and a mixed gas containing 3% oxygen and 5% carbon dioxide by volume was introduced; Add 100 μmol / L estradiol, 20 mmol / L glucose, and 0.5 mmol / L β-mercaptoethanol; After transfection with Rab35 overexpression plasmid, the cell supernatant was collected after 72 h of culture; Exosomes were purified using a hollow fiber membrane column, and nanoflow cytometry analysis confirmed that the yield reached 3.5 times 1011 particles per milliliter.

[0039] Example 2: Engineering preparation of tumor cell exosomes Using CRISPR / Cas9 technology to knock out the p62 gene in cells, reducing the intracellular retention of miR-122; L-α-phosphatidylserine was added to the acidic medium at pH 6.5 to promote exosome release; Combined with anion exchange chromatography purification, functionalized exosomes with a drug loading rate of 0.25 mg doxorubicin per ml were obtained.

[0040] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0041] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for promoting cell secretion of exosomes, characterized by: The steps of the method for promoting cell secretion of exosomes are: Step 1: placing the cells in a culture medium with a pH of 6.4 to 7.1 and an osmotic pressure of 290 to 320 mmol / L; Step 2: Maintaining the oxygen concentration in the culture environment to no more than 5% by volume; Step 3, adding 50 to 200 micromoles per liter of estrogen and 10 to 30 millimoles per liter of glucose to the culture medium; Step 4: Collect exosomes after 48 to 96 hours of culture.

2. An exosome secretion induction medium comprising a basal medium and exosome-free serum, characterized in that Also includes: 50 to 200 micromoles per liter of estrogen, 10 to 30 millimoles per liter of glucose; pH regulator to stabilize the pH of the culture medium at 6.4 to 7.1; Osmotic pressure regulator, maintains osmotic pressure between 290 and 320 mmol / l.

3. The method for promoting cell secretion of exosomes according to claim 1, wherein: In the step 3, 0.1 to 1.0 mmol / L β-mercaptoethanol and 1 to 10 μg / mL anti-TGF-β single domain antibody are added simultaneously.

4. The method for promoting cell secretion of exosomes according to claim 1, wherein: The cells are gene-edited to overexpress Rab11 protein or Rab35 protein.

5. The method for promoting cell secretion of exosomes according to claim 1, wherein: The cells are selected from mesenchymal stem cells, mammary epithelial cells or tumor cells.

6. The exosome secretion induction medium according to claim 2, characterized in that: Also included are 1 to 10 micromoles per liter of 5'-adenylic acid and 0.5 to 5 micrograms per milliliter of cofilin-1.

7. The method for promoting cell secretion of exosomes according to claim 1, characterized in that: The exosome purification adopts the hollow fiber membrane column combined with anion exchange chromatography technology.

8. The method for promoting cell secretion of exosomes according to claim 1, characterized in that: The obtained exosomes have a particle size of 50 to 150 nanometers, and the purity is no less than 95% after nanoflow cytometry detection.

9. Use of the exosomes prepared according to claim 1 in preparing a tumor treatment carrier, characterized in that: The exosomes are loaded with the anti-tumor drug doxorubicin or berberine.

10. The use of exosomes according to claim 9 in preparing a tumor treatment carrier, characterized in that: Targeted delivery of exosomes to tumor sites via surface-modified magnetic nanoparticles.

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