Extraction method of tumor stem cell exosome

Abstract

The invention discloses a method for extracting tumor stem cell exosomes. The method comprises the following steps: regulating, enriching and purifying tumor stem cells based on a 3D culture environment and a chemical microenvironment; inducing the tumor stem cells to secrete exosomes, and collecting culture supernatant; performing preliminary separation and concentration on the exosome in the supernatant through tangential flow filtration to obtain the tumor stem cell exosome subjected to preliminary purification; the preliminarily purified tumor stem cell exosomes are purified again through modified double-antibody magnetic bead affinity purification and multi-mode chromatography, high-purity different types of tumor stem cell exosomes are obtained, and efficient and specific capture and purification of epithelial type, mesenchymal type and mixed type tumor stem cell exosomes are achieved; cryopreserving the extracted tumor stem cell exosome through trehalose-DMSO (dimethyl sulfoxide); the yield, the purity and the activity retention rate of the exosome are remarkably improved, the tumor hypoxia microenvironment is effectively simulated, and non-target exosome pollution is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of cell extraction, and in particular to a method for extracting tumor stem cell exosomes. Background Art

[0002] Ordinary stem cells play a key role in tissue homeostasis and repair. Their proliferation is strictly regulated and their differentiation direction is clear, providing an important guarantee for the body to maintain normal physiological functions. However, in special disease models, cancer stem cells (CSCs) are completely different. CSCs have abnormal self-renewal ability and differentiation potential, and are key factors driving tumor growth, metastasis and recurrence. They may carry unique signaling molecules and transmit these stem characteristics to ordinary tumor cells through paracrine effects, thereby promoting tumor heterogeneity and malignant progression.

[0003] Tumor stem cell exosomes are an important bioactive substance secreted by CSCs. Exosomes are tiny vesicles with a diameter of about 30-150 nanometers, containing a variety of bioactive molecules, such as proteins, lipids, RNA, etc. These molecules play an important role in the occurrence, development and metastasis of tumors; especially tumor stem cell exosomes, which may contain special molecules related to CSCs self-renewal and multidirectional differentiation. In terms of tumor diagnosis, the bioactive molecules in tumor stem cell exosomes can be used as biomarkers for early screening, disease monitoring and prognosis evaluation of tumors; these markers are highly specific and sensitive and can more accurately reflect the existence and status of tumors. At the same time, through the analysis of molecular characteristics in exosomes, tumors can also be molecularly classified to provide a basis for individualized treatment.

[0004] However, since CSCs account for an extremely low proportion in tumor tissues, obtaining tumor stem cell exosomes faces huge challenges, which not only increases the difficulty and complexity of research, but also limits the widespread application of exosomes in tumor diagnosis and treatment.

[0005] Therefore, it is necessary to improve a method for extracting tumor stem cell exosomes in the prior art to solve the above problems. Summary of the invention

[0006] The present invention overcomes the shortcomings of the prior art and provides a method for extracting tumor stem cell exosomes, aiming to solve the problems of low yield, insufficient purity, poor activity retention rate and inability to simulate the hypoxic microenvironment of tumors in the prior art.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a method for extracting tumor stem cell exosomes, comprising:

[0008] S1. Enrich and purify tumor stem cells based on 3D culture environment and chemical microenvironment regulation;

[0009] S2, inducing tumor stem cells to secrete exosomes and collecting the culture supernatant;

[0010] S3, preliminarily separating and concentrating the exosomes in the supernatant by tangential flow filtration to obtain preliminarily purified tumor stem cell exosomes;

[0011] S4. The initially purified tumor stem cell exosomes were re-purified by modified double antibody magnetic beads affinity purification and multimodal chromatography to obtain high-purity tumor stem cell exosomes of different types;

[0012] S5. Tumor stem cell exosomes extracted by trehalose-DMSO cryopreservation.

[0013] In a preferred embodiment of the present invention, the 3D culture environment is constructed by the following method:

[0014] Use Matrigel and low-adhesion culture dishes, with a mixing ratio of Matrigel to culture medium of 1:1-2;

[0015] The culture medium formula is DMEM / F12 medium supplemented with 1×B27, 20 ng / mL EGF, 20 ng / mL bFGF, 100 ng / mL Noggin, 2 mM GlutaMAX, 25 mM HEPES, 20-40 mg / mL albumin, and 2-10 mg / mL fibrinogen;

[0016] Chemical microenvironment manipulation included the addition of 100 μM CoCl 2 , 10 μM SB431542, 3 μM CHIR99021 and 10 μM Y-27632.

[0017] In a preferred embodiment of the present invention, the oxygen concentration gradient in the 3D culture environment is dynamically regulated to be 0.5% to 5%, switched once every 24 hours, and 5-10 ng / mL IL-6 and 2-5 ng / mL TGF-α are additionally added.

[0018] In a preferred embodiment of the present invention, in step S2, inducing exosome secretion includes:

[0019] On the fifth day of culture, GW4869 was added at a final concentration of 10 μM for 12 h, and then fresh medium was replaced;

[0020] Tumor stem cells were treated with 1 MHz ultrasound pulses at an intensity of 0.5 W / cm 2 , pulse time 1-2 seconds, interval 3-5 seconds, lasting 30-45 minutes;

[0021] After 36-48 hours of continuous cultivation, the culture medium was collected, centrifuged at 300×g and pre-treated with a 100 nm filter.

[0022] In a preferred embodiment of the present invention, in step S3, tangential flow filtration uses a 250-300 kDa hollow fiber membrane with a flow rate of 80-100 mL / min and a transmembrane pressure of <15 psi to concentrate to 1 / 20 of the original volume.

[0023] In a preferred embodiment of the present invention, the modified double antibody magnetic beads are prepared by the following method:

[0024] Activating the magnetic beads using one or more activating agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), glutaraldehyde, periodate or chloramine-T;

[0025] Add the washed magnetic beads to the solution containing the activator and mix thoroughly. Keep the magnetic beads suspended at room temperature for 5-30 minutes. After the reaction is completed, remove the supernatant by strong magnetic separation.

[0026] Select the concentration of 10-30 μg / mL EpCAM and CSV to bind to the activated magnetic beads respectively, and add the antibody to the activated magnetic beads; the magnetic beads are divided into small magnetic beads and large magnetic beads; the small magnetic beads are 50-100nm and bind to EpCAM antibodies; the large magnetic beads are 100-200nm and bind to CSV antibodies.

[0027] In a preferred embodiment of the present invention, the step of affinity purification of modified double antibody magnetic beads comprises:

[0028] The modified EpCAM antibody magnetic beads were suspended in 1× PBS buffer;

[0029] The preliminarily purified tumor stem cell exosome sample was fully mixed with the modified double antibody magnetic beads at room temperature for 2-4 hours;

[0030] Place the mixture on a magnetic rack and let it stand for 5-10 minutes to allow the magnetic beads and exosomes to be adsorbed on the magnetic rack. Remove the supernatant and discard the unbound impurities.

[0031] Add 1× PBS buffer and repeat the above steps several times;

[0032] Add glycine buffer at pH 2.5 to elute the exosomes, place on a magnetic rack, let stand for 2-3 minutes, and collect the eluate, which is the tumor stem cell exosomes after affinity purification by modified double antibody magnetic beads.

[0033] In a preferred embodiment of the present invention, the multimodal chromatography uses a CaptoCore700 chromatographic column with a flow rate of 0.3-0.5 mL / min and the eluent is PBS buffer.

[0034] In a preferred embodiment of the present invention, in step S5, the cryopreservation solution consists of 100 mM trehalose and 5% DMSO, the volume ratio of exosomes to the cryopreservation solution is 1:1-3, and the freezing temperature is -80°C.

[0035] In a preferred embodiment of the present invention, the tumor stem cells form microspheres with a diameter of >50 μm in a 3D culture environment and are metabolically screened by 1 nM 2-DG.

[0036] The present invention solves the defects existing in the background technology and has the following beneficial effects:

[0037] (1) The present invention provides a high-fidelity extraction method for tumor stem cell exosomes. Tumor stem cells are enriched and purified by constructing a 3D culture environment and chemical microenvironment regulation. The gradient hypoxic microenvironment, dynamic addition of cytokines and metabolic screening technology are used to significantly improve the stemness characteristics and exosome secretion capacity of tumor stem cells. Exosomes are initially separated and concentrated by tangential flow filtration. Modified double antibody magnetic bead affinity purification and multimodal chromatography technology are combined to achieve efficient and specific capture and purification of epithelial, mesenchymal and mixed tumor stem cell exosomes. Finally, a trehalose-DMSO cryopreservation system is used to protect the capsule structure and functional integrity of the exosomes. The yield, purity and activity retention rate of exosomes can be significantly improved, the tumor hypoxic microenvironment can be effectively simulated, and non-target exosome contamination can be reduced, providing high-quality samples for the research and precision medicine application of tumor stem cell exosomes.

[0038] (2) The present invention simulates peripheral blood circulation by 3D culture. The 3D culture environment uses a specific formula, including DMEM / F12 culture medium, supplemented with 1×B27, 20 ng / mL EGF, 20 ng / mL bFGF, 100 ng / mL Noggin, 2 mM Ingredients such as GlutaMAX, 25mM HEPES, 20-40mg / mL albumin and 2-10mg / mL fibrinogen simulate the biochemical environment of tumor stem cells in the peripheral blood circulation, enabling tumor stem cells to undergo epithelial-mesenchymal transition and form a cell phenotype that is more in line with the actual situation in the body; at the same time, by combining chemical microenvironment regulation, the oxygen concentration is dynamically regulated, and the oxygen concentration gradient is set from 0.5% to 5%, switching every 24 hours, and cytokines are added. Microfluidic chips or rotating culture systems are used to simulate the shear force and dynamic environment of blood flow, accurately simulating the hypoxic microenvironment in tumor tissue and the peripheral blood circulation environment, maintaining the stemness of tumor stem cells, and increasing the expression of key signaling molecules in exosomes, so that they can better reflect the true pathological characteristics of CSCs. Compared with the static culture conditions in the existing technology, the representativeness and research value of the extracted exosomes are further ensured.

[0039] (3) The present invention improves the capture efficiency and specificity of exosomes by using double antibody magnetic beads. In step S4, the double antibody magnetic beads are modified. First, the magnetic beads are activated by using an activator, and then two anti-tumor stem cell exosome antibodies with different antigenic epitopes, EpCAM and CSV, are selected at a concentration of 10-30 μg / mL, and are respectively combined with the activated magnetic beads. Among them, small magnetic beads are combined with EpCAM antibodies to capture epithelial tumor stem cell exosomes, large magnetic beads are combined with CSV antibodies to capture mesenchymal tumor stem cell exosomes, and epithelial-mesenchymal mixed tumor stem cell exosomes are combined with both magnetic beads. This combination of magnetic beads of different particle sizes and specific antibodies reduces the competition between exosomes of different sizes and improves the capture efficiency and specificity; at the same time, the modified antibody magnetic beads are combined with multimodal chromatography technology to achieve the purpose of efficiently removing non-target components and obtain high-quality tumor stem cell exosomes. Compared with the lack of specific capture means in the prior art, which leads to a high contamination rate of non-target exosomes, the present invention further improves the purity of exosomes and the accuracy of capture, which is conducive to the research and application of specific types of tumor stem cell exosomes in precision medicine.

[0040] (4) The present invention adopts a trehalose-DMSO cryopreservation system to enhance the activity retention and long-term storage effect of exosomes. The trehalose-DMSO cryopreservation system is used to dissolve 100mM trehalose and 5% DMSO in PBS buffer, mix with exosomes in a certain volume ratio, and store in an ultra-low temperature refrigerator at -80°C. Trehalose can replace water molecules during the freezing process of exosomes to prevent the formation of ice crystals from damaging the capsule membrane. DMSO can lower the freezing point, increase the osmotic pressure of the solution, reduce the damage of ice crystals to exosomes, effectively protect the capsule membrane structure and functional integrity of the exosomes, and improve the activity retention rate of exosomes. Compared with the common preservation method that may be used in the prior art, this cryopreservation system further ensures that the exosomes can still maintain a high activity and integrity after long-term storage, which is beneficial to the long-term storage of exosomes and various subsequent experimental studies, and ensures the reliability and stability of the research results.

[0041] (5) In the 3D culture environment of step S1, the present invention adds albumin and fibrinogen to the basic culture medium formula. Albumin simulates the nutritional environment in the blood, maintains the stemness characteristics of tumor stem cells, and inhibits their transformation to the mesenchymal type; fibrinogen simulates the coagulation environment in the blood, promotes the adhesion and migration of tumor stem cells, and helps to form a stable microsphere structure. At the same time, the added GlutaMAX provides a stable energy and nitrogen source, and HEPES maintains the pH stability of the culture medium. The synergistic effect of these components, compared with the simple culture medium formula in the prior art, further optimizes the culture conditions of tumor stem cells, is more conducive to the growth of tumor stem cells, the maintenance of stemness and the secretion of exosomes, and improves the quality and yield of exosomes from the source, laying a good foundation for the entire extraction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

[0043] Figure 1 is a flow chart of a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0045] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0046] Application Overview:

[0047] Currently, the extraction of cancer stem cell exosomes mainly relies on general exosome separation technology, including differential ultracentrifugation, polymer precipitation and size exclusion chromatography. Although ultracentrifugation is regarded as the standard method, its recovery efficiency for low-density exosomes is insufficient, and high shear force can easily lead to capsule rupture and leakage of contents; although PEG precipitation can improve the yield, the introduced polymer residues will interfere with the detection of exosome surface markers and affect the reliability of downstream functional experiments. Existing technologies generally use static culture conditions, which cannot simulate the hypoxic microenvironment of tumors, resulting in insufficient expression of key signaling molecules in exosomes, making it difficult to reflect the true pathological characteristics of CSCs. In addition, traditional methods lack specific capture methods for tumor stem cell exosome subpopulations, resulting in a non-target exosome contamination rate of up to 60%, which seriously limits its application in precision medicine.

[0048] In view of the above-mentioned defects, the present invention provides a high-fidelity extraction method for tumor stem cell exosomes, which improves the yield of tumor stem cell exosomes through gradient hypoxia microenvironment regulation and secretion burst induction, and improves the exosome activity retention rate through a trehalose-DMSO cryopreservation system.

[0049] Exemplary methods:

[0050] like Figure 1 As shown, a method for extracting tumor stem cell exosomes comprises the steps of:

[0051] S1. Enrich and purify tumor stem cells based on 3D culture environment and chemical microenvironment regulation;

[0052] S2, inducing tumor stem cells to secrete exosomes and collecting the culture supernatant;

[0053] S3, preliminarily separating and concentrating the exosomes in the supernatant by tangential flow filtration to obtain preliminarily purified tumor stem cell exosomes;

[0054] S4. The initially purified tumor stem cell exosomes were re-purified by modified double antibody magnetic beads affinity purification and multimodal chromatography to obtain high-purity tumor stem cell exosomes of different types;

[0055] S5. Tumor stem cell exosomes extracted by trehalose-DMSO cryopreservation.

[0056] A 3D culture environment refers to the use of physical or biological materials to simulate the three-dimensional growth space of cells in the body, allowing tumor stem cells to form a three-dimensional structure rather than the single-layer adherent growth in traditional 2D culture. Tumor stem cells are usually located in the "stem cell nest" in the body, and the 3D structure can better simulate this hypoxic, high-cell density microenvironment and maintain the self-renewal and differentiation potential of stem cells.

[0057] In step S1, Matrigel and low-adhesion culture dishes are used to construct a 3D environment. The surface characteristics of the low-adhesion culture dishes can reduce the adhesion between cells and the culture dishes, which is conducive to the formation of microsphere structures by tumor stem cells. Matrigel is a commonly used basement membrane component that can provide extracellular matrix support and promote cell growth and tissue formation.

[0058] The basic formula of the culture medium for constructing the 3D environment is: use DMEM / F12 culture medium, add 1×B27, 20ng / mL EGF, 20ng / mL bFGF, and 100ng / mL Noggin;

[0059] Studies have shown that tumor stem cells undergo epithelial-mesenchymal transition when entering the peripheral blood circulation, resulting in different phenotypes, including epithelial, mesenchymal, and mixed epithelial-mesenchymal tumor stem cells;

[0060] In order to make the 3D environment more suitable for the growth of tumor stem cells and simulate the changes of tumor stem cells in peripheral blood circulation through the 3D environment, the above basic formula was modified to add 1×B27, 20ng / mL EGF, 20ng / mL bFGF, 100ng / mL Noggin, 2mM GlutaMAX, 25mM HEPES, 20-40mg / mL albumin and 2-10mg / mL fibrinogen to DMEM / F12 medium;

[0061] GlutaMAX is a glutamine substitute that can more stably provide the energy and nitrogen source required for cell growth, promote the proliferation and metabolism of tumor stem cells, and facilitate their better growth and maintenance of stemness in a 3D culture environment. HEPES is a buffer that can maintain the pH stability of the culture medium. In addition, albumin and fibrinogen are added to simulate the biochemical environment in peripheral blood. Albumin is the most abundant protein in the blood and can provide the nutrients required for cell growth. Fibrinogen is a key component of blood coagulation and is closely related to cell adhesion and migration. Fibrinogen can simulate the coagulation environment in the blood in 3D culture, promote cell adhesion and migration, help tumor stem cells form microspheres in a 3D environment, and simulate changes in peripheral blood circulation, so that tumor stem cells can undergo epithelial-mesenchymal transition in a 3D environment.

[0062] Chemical microenvironment regulation of 3D environment by 100 μM CoCl 2 A hypoxic environment was simulated, and 10μM SB431542, 3μM CHIR99021 and 10μM Y-27632 were combined to maintain cell stemness. The above chemical microenvironment regulation added Y-27632 to the basic substance. Y-27632 is a Rho kinase inhibitor that can inhibit the contraction of the cytoskeleton, reduce the adhesion of cells, and promote the formation of microsphere structures of tumor stem cells in a 3D culture environment. It also helps to maintain the stemness of cells, improve the purity of tumor stem cells and the content of key signaling molecules in exosomes.

[0063] Use Matrigel as the matrix material, mix Matrigel with culture medium at a mixing ratio of 1:1-2; evenly spread the mixed matrix on the bottom of the low-adhesion culture dish to form a thin matrix layer, and shake it authoritatively to avoid bubbles;

[0064] In the above 3D environment, the oxygen concentration is dynamically regulated, and the oxygen concentration gradient is set from 0.5% to 5%, which is switched every 24 hours to simulate the hypoxic microenvironment in tumor tissue; and IL-6 and TNF-α at a concentration of 5-10 ng / mL and 2-5 ng / mL are dynamically added during the culture period. These cytokines can further regulate the microenvironment of tumor stem cells and further simulate the peripheral blood circulation environment, which is conducive to maintaining stemness characteristics, thereby indirectly promoting the proliferation of tumor stem cells, improving the quality of exosomes, and reducing the proportion of non-functional exosomes caused by cell apoptosis; in addition, during the culture process, a microfluidic chip or a rotating culture system is used to rotate the low-adhesion culture dish to simulate the shear force and dynamic environment of blood flow.

[0065] 1 nM 2-DG was added to the culture medium to inhibit the proliferation of non-CSCs for metabolic screening. Through metabolic screening, the enrichment of tumor stem cells was further improved, which is conducive to obtaining purer tumor stem cell exosomes.

[0066] The single cell suspension of tumor cells was inoculated into the constructed 3D culture environment to form tumor microspheres with a diameter of >50 μm. The low-adhesion culture dish was placed at 37°C and 5% CO. 2 Culture in an incubator for more than one week.

[0067] Through the regulation of 3D culture environment and chemical microenvironment, tumor stem cells are enriched and purified, so that they form a stable microsphere structure in the 3D culture environment and have higher stemness and exosome secretion ability, which lays a good foundation for the subsequent extraction and research of exosomes. In step S2, tumor stem cells are induced to secrete exosomes, and the culture supernatant is collected to further extract and purify tumor stem cell exosomes.

[0068] In step S2, on the fifth day of culture, a final concentration of 10 μM is added to the culture medium. After 12 hours of treatment, the drug is withdrawn and replaced with fresh culture medium, which is consistent with the original matrix, thereby inducing cells to secrete a large amount of exosomes; GW4869 is a cell-permeable, non-competitive neutral sphingomyelinase inhibitor, which inhibits the synthesis and release of exosomes by blocking ceramide-mediated multivesicular body budding and mature exosome release from MVBs;

[0069] After replacing the fresh culture medium, the tumor stem cells were treated with 1 MHz ultrasonic pulses for 30-45 minutes at an intensity of 0.5 W / cm 2 , the pulse time is 1-2s, and the pulse interval is 3-5s; ultrasonic stimulation can promote the metabolic activity of cells and the release of exosomes, and improve the secretion efficiency of exosomes;

[0070] After inducing exosome secretion, tumor stem cells were cultured for 36-48 h to allow the cells to have sufficient time to secrete exosomes. The culture medium in the culture dish was transferred to a sterile container and pre-treated by centrifugation at 300 × g for 10-15 minutes to remove cell debris and other large particle impurities to obtain the culture supernatant.

[0071] In step S3, before tangential flow filtration, the culture supernatant is pretreated by removing large particle impurities such as apoptotic bodies through a 100 nm nylon filter membrane in series to ensure that the liquid entering the tangential flow filtration system is purer, reduce clogging and contamination of the filter membrane, and improve filtration efficiency and exosome recovery rate.

[0072] In step S3, the culture supernatant is preliminarily purified using a tangential flow filtration system combined with a hollow fiber membrane with a pore size of 250-300 kDa. Tangential flow filtration is a highly efficient separation and concentration technology, particularly suitable for the treatment of nano-sized particles such as exosomes. For example, preferably, the TFF system of Sartorius can effectively reduce membrane blockage and contamination, improve filtration efficiency and recovery rate compared with traditional ultrafiltration methods. By using a hollow fiber membrane with a pore size of 250-300 kDa, it can ensure that the exosomes are effectively retained while allowing small molecules and impurities to pass through, thereby achieving preliminary separation and concentration of exosomes.

[0073] Pour the pretreated culture supernatant into the liquid storage tank of the tangential flow filtration system, start the circulation pump, and allow the culture supernatant to flow tangentially through the hollow fiber membrane surface. Set the flow rate to 80-100 mL / min and the transmembrane pressure to <15 psi. Ensure that the liquid forms a stable tangential flow on the membrane surface to reduce membrane blockage and contamination, and improve filtration efficiency and recovery rate. Continue tangential flow filtration until the volume of the culture supernatant is concentrated to 1 / 20 of the original volume to obtain preliminarily purified tumor stem cell exosomes. During the concentration process, the transmembrane pressure and flow rate need to be continuously monitored to ensure the stability of the filtration process. When the membrane pressure is too high, the hollow fiber membrane needs to be cleaned or replaced to ensure the filtration effect.

[0074] In step S4, in order to improve the adsorption effect of the double antibody magnetic beads on tumor stem cell exosomes, a modification treatment was performed. First, the magnetic beads were activated using one or more activators such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), glutaraldehyde, periodate or chloramine-T. Specifically, the EDC concentration is set to 5-50mM, and the preferred range is 10-20mM. The activation process includes adding the washed magnetic beads to a solution containing an activator and mixing them thoroughly, reacting at room temperature for 5-30 minutes while keeping the magnetic beads suspended. After the reaction, the supernatant is removed by strong magnetic separation.

[0075] Next, two anti-tumor stem cell exosome antibodies with different antigenic epitopes, EpCAM and CSV, were selected at a concentration of 10-30 μg / mL and respectively bound to the activated magnetic beads, and the antibodies were added to the activated magnetic beads;

[0076] In a certain embodiment, the magnetic beads are divided into small magnetic beads and large magnetic beads; the small magnetic beads are 50-100nm, combined with EpCAM antibodies, epithelial tumor stem cell exosomes specifically bind to EpCAM antibodies; the large magnetic beads are 100-200nm, combined with CSV antibodies, mesenchymal tumor stem cell exosomes specifically bind to CSV antibodies; epithelial-mesenchymal mixed tumor stem cell exosomes are combined with both magnetic beads; magnetic beads of different particle sizes can reduce competition between exosomes of different sizes and improve capture efficiency. Specific antibody binding can ensure the specific binding of magnetic beads to target exosomes, distinguish exosome types, increase capture rate, and improve capture purity.

[0077] After adding the antibody to the activated magnetic beads and incubating them at room temperature for 4-6 hours, the antibody forms a stable covalent bond with the active groups on the surface of the magnetic beads. This activation of the magnetic bead surface increases the number of its active groups, thereby improving its binding ability with the antibody. The antibody coupled in this way can specifically capture tumor stem cell exosomes and enhance the specificity and affinity of adsorption.

[0078] The modified antibody magnetic beads have significantly improved adsorption effects on tumor stem cell exosomes, can more effectively capture and enrich exosomes, improve the purity and recovery rate of exosomes, and provide more reliable samples for subsequent exosome research and applications.

[0079] In step S4, the step of affinity purification of modified double antibody magnetic beads includes:

[0080] Suspend the modified double antibody magnetic beads in 1× PBS buffer and mix gently to ensure that the magnetic beads are evenly dispersed;

[0081] The preliminarily purified tumor stem cell exosome sample was fully mixed with the modified double antibody magnetic beads at room temperature for 2-4 hours to ensure the specific binding of the antibody to the antigen on the surface of the exosome;

[0082] Place the mixture on a magnetic rack and let it stand for 5-10 minutes to allow the magnetic beads and exosomes to be adsorbed on the magnetic rack. Remove the supernatant and discard the unbound impurities.

[0083] Add 1× PBS buffer and repeat the above steps several times;

[0084] Add pH 2.5 glycine buffer to elute the exosomes, place on a magnetic rack, let stand for 2-3 minutes, and collect the eluate, which is the tumor stem cell exosomes after affinity purification by modified double antibody magnetic beads;

[0085] In step S4, for further purification, a CaptoCore700 multimode chromatography column is used, and PBS buffer is used for balancing at a flow rate of 0.3-0.5 mL / min until the baseline is stable; the tumor stem cell exosomes after affinity purification by modified double antibody magnetic beads are loaded into the chromatography column at the same flow rate, and then eluted with PBS buffer to obtain the final high-purity tumor stem cell exosomes; it can not only specifically capture the target exosomes, but also remove residual impurities such as lipoproteins and free proteins, greatly improving the purity and quality of the exosomes; through the combined application of modified double antibody magnetic bead affinity purification and multimode chromatography technology, the purpose of efficiently removing non-target components is achieved, and high-quality tumor stem cell exosomes are obtained.

[0086] In step S5, 100mM trehalose and 5% DMSO are dissolved in PBS buffer to prepare trehalose-DMSO cryopreservation solution, the volume ratio of exosomes to cryopreservation solution is 1:1-3, and the freezing temperature is -80°C. Trehalose is a natural sugar substance with good moisturizing and protective effects. It can replace water molecules during the freezing process of exosomes to prevent the formation of ice crystals from damaging the capsule membrane; DMSO is a commonly used cryoprotectant that can lower the freezing point, increase the osmotic pressure of the solution, and reduce the damage of ice crystals to exosomes. The use of trehalose-DMSO cryopreservation system can effectively protect the capsule structure and functional integrity of exosomes and improve the activity retention rate of exosomes.

[0087] Embodiment 1:

[0088] This embodiment provides a method for extracting tumor stem cell exosomes, comprising the following steps:

[0089] S1. Enrich and purify tumor stem cells based on 3D culture environment and chemical microenvironment regulation;

[0090] Matrigel and low-adhesion culture dishes were used to construct a 3D environment. The mixing ratio of Matrigel to culture medium was 1:1. Culture medium formula: DMEM / F12 culture medium, supplemented with 1×B27, 20ng / mL EGF, 20ng / mL bFGF, 100ng / mL Noggin, 2mM GlutaMAX, 25mM HEPES, 30mg / mL albumin, and 5mg / mL fibrinogen. Chemical microenvironment regulation: Add 100μM CoCl 2 , 10μM SB431542, 3μM CHIR99021, 10μM Y-27632; dynamic oxygen gradient control: oxygen concentration gradually increased from 0.5% to 5%, switched every 24 hours; additional cytokines: 7ng / mL IL-6 and 4ng / mL TGF-α; metabolic screening: 1nM 2-DG was added to the culture medium and cultured for 7 days; a microfluidic chip was used to simulate the peripheral blood circulation environment, and single cell suspensions were inoculated into a 3D culture system to form tumor microspheres with a diameter of >50μm, which were cultured at 37°C and 5% CO 2 Cultured under the same conditions for 7 days;

[0091] S2, induce exosome secretion and collect culture supernatant;

[0092] On day 5, GW4869 was added at a final concentration of 10 μM, and the medium was replaced with fresh medium after 12 h of treatment;

[0093] Ultrasonic treatment parameters: 1 MHz frequency, 0.5 W / cm 2, pulse time 1 second, interval 3 seconds, last 30 minutes, continue to culture for 48 hours, then collect the culture medium; centrifuge at 300×g for 15 minutes to remove cell debris, filter through 100nm nylon filter membrane to remove large impurities such as apoptotic bodies;

[0094] S3, preliminary purification;

[0095] The culture supernatant was concentrated to 1 / 20 of the original volume by filtration using a 270 kDa hollow fiber membrane through a Sartorius TFF system with a flow rate set at 90 mL / min and a transmembrane pressure controlled at 10 psi;

[0096] S4, modified double antibody magnetic beads affinity purification and multimodal chromatography;

[0097] Activate magnetic beads with 15mM EDC for 40 minutes, and couple antibodies according to particle size; 20μg / mL EpCAM and CSV, 100nm small magnetic beads coupled with EpCAM antibody, 200nm large magnetic beads coupled with CSV antibody, incubate at room temperature for 5 hours; mix the preliminarily purified exosomes with modified magnetic beads, incubate at room temperature for 3 hours, after magnetic separation, elute the exosomes with pH2.5 glycine buffer, use CaptoCore700 column, flow rate 0.4mL / min, equilibrate with PBS, load and elute, and collect the target peak;

[0098] S5, exosome cryopreservation;

[0099] 100 mM trehalose and 5% DMSO were dissolved in PBS buffer, and the exosomes were mixed with the freezing solution at a volume ratio of 1:2; the mixture was directly stored in a -80°C ultra-low temperature refrigerator.

[0100] Embodiment 2:

[0101] This embodiment provides a method for extracting tumor stem cell exosomes. The same points as those in the first embodiment are not described in detail. The difference between this embodiment and the first embodiment is that;

[0102] The culture medium was supplemented with 20 mg / mL albumin and 2 mg / mL fibrinogen.

[0103] Embodiment three:

[0104] This embodiment provides a method for extracting tumor stem cell exosomes. The same points as those in the first embodiment are not described in detail. The difference between this embodiment and the first embodiment is that;

[0105] The culture medium was supplemented with 30 mg / mL albumin and 10 mg / mL fibrinogen.

[0106] Embodiment 4:

[0107] This embodiment provides a method for extracting tumor stem cell exosomes. The same points as those in the first embodiment are not described in detail. The difference between this embodiment and the first embodiment is that;

[0108] Cytokines were added: 5 ng / mL IL-6 and 3 ng / mL TGF-α.

[0109] Embodiment five:

[0110] This embodiment provides a method for extracting tumor stem cell exosomes. The same points as those in the first embodiment are not described in detail. The difference between this embodiment and the first embodiment is that;

[0111] Cytokines were added: 10 ng / mL IL-6 and 5 ng / mL TGF-α.

[0112] Embodiment 6

[0113] This embodiment provides a method for extracting tumor stem cell exosomes. The same points as those in the first embodiment are not described in detail. The difference between this embodiment and the first embodiment is that;

[0114] 10 μg / mL EpCAM and CSV.

[0115] Embodiment 7

[0116] This embodiment provides a method for extracting tumor stem cell exosomes. The same points as those in the first embodiment are not described in detail. The difference between this embodiment and the first embodiment is that;

[0117] 30 μg / mL EpCAM and CSV.

[0118] Comparative Example 1:

[0119] This comparative example provides a method for extracting tumor stem cell exosomes. The same points as those in Example 1 are not repeated here. The difference between this example and Example 1 is that;

[0120] No albumin and fibrinogen were added.

[0121] Comparative Example 2:

[0122] This comparative example provides a method for extracting tumor stem cell exosomes. The same points as those in Example 1 are not repeated here. The difference between this example and Example 1 is that;

[0123] No cytokines were added.

[0124] Comparative Example 3:

[0125] This comparative example provides a method for extracting exosomes from tumor stem cells. The same points as those in Example 1 are not described in detail. The difference between this example and Example 1 is that;

[0126] No distinction was made between magnetic bead-antibody coupling.

[0127] Experimental Example 1:

[0128] This experimental example selected Examples 1 to 5 and Comparative Examples 1 to 2, and conducted physical and chemical analysis on the extracted tumor stem cell exosomes to verify the epithelial-mesenchymal transition and quality of the tumor stem cell exosomes.

[0129] Table 1 Transformation and quality of tumor stem cell exosomes

[0130]

[0131]

[0132] The proportion of epithelial exosomes in Example 1 is 45%, the proportion of mesenchymal exosomes is 35%, and the proportion of mixed exosomes is 20%. Compared with other examples, the proportion of epithelial exosomes in Example 1 is higher and the proportion of mesenchymal exosomes is lower, indicating that its 3D culture environment and chemical microenvironment regulation effect is the best.

[0133] In Comparative Examples 1 and 2, the proportion of epithelial exosomes was significantly reduced, and the proportion of interstitial exosomes was significantly increased, indicating that the absence of albumin, fibrinogen and cytokines would inhibit the formation of epithelial exosomes.

[0134] The purity of the exosomes in Example 1 was 95%, the recovery rate was 90%, and the integrity was 98%, all of which were at a relatively high level, indicating that its extraction method can efficiently obtain high-quality exosomes.

[0135] Albumin and fibrinogen are important components in the 3D culture environment, simulating the biochemical conditions of tumor stem cells in the in vivo microenvironment; in the 3D culture environment, albumin can simulate the nutritional environment in the blood, maintain the stemness characteristics of tumor stem cells, and inhibit their transformation to the mesenchymal type; fibrinogen can simulate the coagulation environment in the blood, promote the adhesion and migration of tumor stem cells, and help form a stable microsphere structure. The addition of albumin and fibrinogen can maintain the stemness characteristics of tumor stem cells and promote their secretion of high-quality exosomes; IL-6 is a pleiotropic cytokine that can promote the self-renewal and stemness maintenance of tumor stem cells by activating the STAT3 signaling pathway; TGF-α is a ligand of the epidermal growth factor receptor that can promote the proliferation and survival of tumor stem cells.

[0136] The synergistic effects of albumin, fibrinogen and cytokines enable cancer stem cells to better maintain their stemness characteristics in a 3D culture environment, reduce the formation of mesenchymal exosomes, and thus improve the purity and quality of exosomes.

[0137] Experimental Example 1:

[0138] This experimental example selected Examples 1, 6 and 7, and Comparative Example 3, and conducted physical and chemical analysis on the extracted tumor stem cell exosomes to verify the capture of different tumor stem cell exosomes.

[0139] Table 2 Capture of exosomes from different cancer stem cells

[0140]

[0141] In Example 1, Example 1 uses 20 μg / mL EpCAM and CSV antibodies, and the antibody concentration is moderate, which can ensure the full binding of the antibody to the magnetic beads, while avoiding nonspecific binding caused by excessive antibodies; the magnetic beads are divided into small magnetic beads, coupled with EpCAM antibodies and large magnetic beads, coupled with CSV antibodies, which specifically capture epithelial and interstitial exosomes respectively; EpCAM antibodies: specifically bind to epithelial exosomes, and increase the capture rate of epithelial exosomes; CSV antibodies: specifically bind to interstitial exosomes, and increase the capture rate of interstitial exosomes; In Comparative Example 3, the antibody coupling is not distinguished, resulting in competitive binding between different exosomes, reducing the capture efficiency and specificity.

[0142] The above is based on the ideal embodiment of the present invention. Through the above description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the content in the specification, and the technical scope must be determined according to the scope of the claims.

Claims

1. A method for extracting tumor stem cell exosomes, characterized in that: Includes steps: S1. Enrich and purify tumor stem cells based on 3D culture environment and chemical microenvironment regulation; S2, inducing tumor stem cells to secrete exosomes and collecting the culture supernatant; S3, preliminarily separating and concentrating the exosomes in the supernatant by tangential flow filtration to obtain preliminarily purified tumor stem cell exosomes; S4. The initially purified tumor stem cell exosomes were re-purified by modified double antibody magnetic beads affinity purification and multimodal chromatography to obtain high-purity tumor stem cell exosomes of different types; S5. Tumor stem cell exosomes extracted by trehalose-DMSO cryopreservation.

2. The method for extracting tumor stem cell exosomes according to claim 1, characterized in that: The 3D culture environment is constructed in the following way: Use Matrigel and low-adhesion culture dishes, with a mixing ratio of Matrigel to culture medium of 1:1-2; The culture medium formula is DMEM / F12 medium supplemented with 1×B27, 20 ng / mL EGF, 20 ng / mL bFGF, 100 ng / mL Noggin, 2 mM GlutaMAX, 25 mM HEPES, 20-40 mg / mL albumin, and 2-10 mg / mL fibrinogen; Chemical microenvironment manipulation included the addition of 100 μM CoCl2, 10 μM SB431542, 3 μM CHIR99021, and 10 μM Y-27632.

3. The method for extracting tumor stem cell exosomes according to claim 1, characterized in that: The oxygen concentration gradient in the 3D culture environment is dynamically regulated from 0.5% to 5%, switched every 24 hours, and 5-10 ng / mL IL-6 and 2-5 ng / mL TGF-α are additionally added.

4. The method for extracting tumor stem cell exosomes according to claim 1, characterized in that: In step S2, inducing exosome secretion includes: On the fifth day of culture, GW4869 was added at a final concentration of 10 μM for 12 h, and then fresh medium was replaced; Tumor stem cells were treated with 1 MHz ultrasound pulses at an intensity of 0.5 W / cm 2 , pulse time 1-2 seconds, interval 3-5 seconds, lasting 30-45 minutes; After 36-48 hours of continuous cultivation, the culture medium was collected, centrifuged at 300×g and pre-treated with a 100 nm filter.

5. The method for extracting tumor stem cell exosomes according to claim 1, characterized in that: In step S3, tangential flow filtration uses a 250-300 kDa hollow fiber membrane with a flow rate of 80-100 mL / min and a transmembrane pressure of <15 psi, and is concentrated to 1 / 20 of the original volume.

6. The method for extracting tumor stem cell exosomes according to claim 1, characterized in that: The modified double antibody magnetic beads are prepared by the following method: Activating the magnetic beads using one or more activating agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), glutaraldehyde, periodate or chloramine-T; Add the washed magnetic beads to the solution containing the activator and mix thoroughly. Keep the magnetic beads suspended at room temperature for 5-30 minutes. After the reaction is completed, remove the supernatant by strong magnetic separation. Select the concentration of 10-30 μg / mL EpCAM and CSV to bind to the activated magnetic beads respectively, and add the antibody to the activated magnetic beads; the magnetic beads are divided into small magnetic beads and large magnetic beads; the small magnetic beads are 50-100nm and bind to EpCAM antibodies; the large magnetic beads are 100-200nm and bind to CSV antibodies.

7. The method for extracting tumor stem cell exosomes according to claim 1, characterized in that: The steps of affinity purification of modified double antibody magnetic beads include: The modified EpCAM antibody magnetic beads were suspended in 1× PBS buffer; The preliminarily purified tumor stem cell exosome sample was fully mixed with the modified double antibody magnetic beads at room temperature for 2-4 hours; Place the mixture on a magnetic rack and let it stand for 5-10 minutes to allow the magnetic beads and exosomes to be adsorbed on the magnetic rack. Remove the supernatant and discard the unbound impurities. Add 1× PBS buffer and repeat the above steps several times; Add glycine buffer at pH 2.5 to elute the exosomes, place on a magnetic rack, let stand for 2-3 minutes, and collect the eluate, which is the tumor stem cell exosomes after affinity purification by modified double antibody magnetic beads.

8. The method for extracting tumor stem cell exosomes according to claim 1, characterized in that: The multi-mode chromatography used a CaptoCore700 column with a flow rate of 0.3-0.5 mL / min and the eluent was PBS buffer.

9. The method for extracting tumor stem cell exosomes according to claim 1, characterized in that: In step S5, the freezing solution consists of 100 mM trehalose and 5% DMSO, the volume ratio of exosomes to freezing solution is 1:1-3, and the freezing temperature is -80°C.

10. The method for extracting tumor stem cell exosomes according to claim 1, characterized in that: The tumor stem cells formed microspheres with a diameter of >50 μm in a 3D culture environment and were metabolically screened by 1 nM 2-DG.

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