Method for separating exosome from heterologous expression cell or primary culture cell, and use of exosome in biological analysis

Abstract

A method for separating an exosome from a heterologous expression cell or a primary culture cell, and a use of an exosome in biological analysis, relating to the technical field of biomedicine. The method for separating the exosome from the heterologous expression cell or the primary culture cell of the present invention comprises: carrying out differential centrifugation on a solution to be extracted that contains an exosome, then taking out a first supernatant, carrying out high-speed centrifugation again, respectively adding iodixanol into an obtained second supernatant and a precipitate, and carrying out spontaneous density gradient centrifugation to obtain a separated purified exosome. According to the present invention, an iodixanol spontaneous density gradient centrifugation method is innovatively used to successfully carry out separation and purification to obtain the exosome having high purity and high yield, and the separated exosome is used as an experimental development universal platform for researching a membrane protein structure and functional characteristics in an original ecological cell membrane environment, and is used for biological analysis, drug screening, and disease diagnosis and treatment.

Description

A method for isolating exosomes from heterologous expression cells or primary cultured cells and its application in biological analysis Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for isolating exosomes from heterologous expression cells or primary cultured cells and its application in biological analysis. Background Art

[0002] Mammalian cells can continuously secrete a variety of extracellular vesicles (EVs) to the extracellular space. Extracellular vesicles are a type of vesicle-like structure enclosed by a double membrane with a diameter ranging from nanometers to micrometers, which contain components derived from secretory cells, such as cell membranes, lipids, proteins, nucleic acids, and various metabolites. Extracellular vesicles can be divided into different types based on their size and composition. The extracellular vesicles secreted from heterologous expression cells or primary cells are mainly of the following types: (1) Exosomes, which are a type of smaller extracellular vesicles released from cells into the extracellular environment mainly through the fusion of multivesicular bodies with the plasma membrane, with a diameter between 30-200 nm. (2) Microvesicles (MVs), which are a type of extracellular vesicles directly produced by the plasma membrane through the process of exocytosis, with a diameter ranging from hundreds to thousands of nanometers.

[0003] To date, exosomes have been widely used as research objects, including: (1) studying the structure and function of membrane proteins in the native cell membrane environment; (2) bioactivity analysis and drug screening; and (3) disease diagnosis and treatment.

[0004] In the prior art, cultured primary cells or heterologous expression cells continuously secrete extracellular vesicles into the culture medium, from which exosomes can be collected and purified. Generally, the formation of exosomes and plasma membrane-derived vesicles, especially the formation of exosomes secreted from heterologous expression cultured cells, is affected by a variety of factors, such as: (1) induction of high-level expression of heterologous membrane proteins. Under these conditions, cells reduce the accumulation of exogenous proteins in the cell by increasing the release of exosomes. (2) Certain compounds can induce the formation of plasma membrane vesicles, such as formaldehyde, relatively low molecular weight aldehydes, thiol blockers (N-ethylmaleimide or dithiothreitol) or cytochalasin B, which can disrupt the interaction between the cytoskeleton and the plasma membrane.

[0005] Through a literature review, we summarized current methods for isolating and purifying exosomes. Currently, mainstream methods for isolating and purifying exosomes are based on size and density, such as ultrafiltration, size exclusion chromatography (SEC), microfluidics, differential ultracentrifugation, and precipitation. While ultrafiltration offers the advantages of speed and convenience, shear forces can damage exosomes when the sample passes through the filter membrane. Furthermore, it can cause exosome aggregation and vesicle clogging, resulting in exosome loss and low separation efficiency. While size exclusion chromatography offers the advantages of high purity, ease of scalability, and automation, it cannot exclude contaminants of similar size to exosomes. While microfluidics offers the advantages of automation and high throughput, it is associated with high instrumentation costs, low sample throughput, and low exosome yields. Furthermore, standardized and scalable sample testing is currently lacking. While differential centrifugation offers the advantages of high purity and large sample throughput, conventional ultracentrifugation can disrupt exosome structure, affecting downstream analysis, resulting in low exosome purity and yield. Furthermore, there is often significant contamination with impurities such as lipoproteins. While precipitation offers the advantages of convenience and the lack of specialized equipment, exosome purity and efficiency are low, and there is significant contamination with impurities. Another approach to isolating exosomes is to separate them based on their component characteristics, such as immunoaffinity capture purification using affinity microbeads coated with capture antibodies targeting specific exosome surface antigens (such as proteins, lipids, and their glycosylation variants). While immunoaffinity capture is highly specific and easy to operate, it suffers from high reagent costs, low sample throughput, and the low activity of exosomes, which is easily affected by pH and salt concentration.

[0006] In order to further optimize and standardize the isolation and purification of exosomes from heterologous expression cells or primary cells, great efforts are still needed. Although there are many methods for exosome isolation, the standards are messy and have obvious shortcomings, and they are prone to producing contradictory results.

[0007] Ideally, the isolation and purification methods used for exosomes derived from heterologous expression cells or primary cells should not adversely affect their biological components. Furthermore, the isolation method should not cause physical damage or aggregation to the exosomes, yielding highly pure and homogenous exosome samples while maximizing yield and reducing experimental sample volume and processing time.

[0008] Summary of the Invention

[0009] To address the shortcomings of the aforementioned prior art, the present invention provides a novel method for isolating and purifying exosomes. This method overcomes the shortcomings of existing exosome isolation methods and uses isolated exosomes as a universal experimental platform for studying the structural and functional characteristics of membrane proteins in their native cell membrane environment, for bioanalysis, drug screening, and disease diagnosis and treatment.

[0010] The present invention utilizes a vertical rotor (Beckman Coulter, VTi 90) to spontaneously form a density gradient in iodixanol after ultracentrifugation. This method allows for the isolation and purification of exosomes by performing differential centrifugation and spontaneous iodixanol density gradient centrifugation on the supernatant of in vitro heterologously expressed cells or primary cell cultures. Because the density gradient spontaneously forms during centrifugation of a homogeneous mixture of iodixanol and EV-containing samples, the method is easy to operate and highly reproducible, making it suitable for heterologously expressing cells or primary cultured cells and potentially scalable to industrial applications.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] On the one hand, the present invention provides a method for isolating exosomes, wherein the liquid to be extracted containing exosomes is centrifuged, the first supernatant is taken and then subjected to high-speed centrifugation, iodixanol is added to the obtained second supernatant and precipitate, and spontaneous density gradient centrifugation is performed to obtain separated and purified exosomes.

[0013] The method specifically comprises the following steps:

[0014] (1) Take the liquid containing exosomes to be extracted, centrifuge it at 300-500g, 2000-2500g, and 10000-12000g in sequence to remove live cells, dead cells and cell debris, respectively, to obtain a first supernatant, centrifuge the first supernatant at 23000-25000g, obtain a second supernatant and precipitate, and place them at 4°C for later use;

[0015] (2) The second supernatant was centrifuged at 100,000-120,000 g for 70-90 min to obtain a precipitate, which was resuspended in 20% iodixanol and then ultracentrifuged at 300,000-350,000 g for 2-4 h. The liquid at the top of the centrifuge tube was collected to obtain exosomes.

[0016] (3) The precipitate obtained in step (1) was resuspended in 20% iodixanol and then ultracentrifuged at 300,000-350,000 g for 4 h. The liquid at the top of the centrifuge tube was taken to obtain exosomes; and the liquid at the bottom of the centrifuge tube was taken to obtain microvesicles.

[0017] The exosomes obtained in step (3) have higher purity and yield than those obtained in step (2).

[0018] In the method, the liquid to be extracted containing exosomes is a culture medium of heterologous expression cells or primary cells.

[0019] The method described is that the centrifugal method is a vertical rotor centrifugal method.

[0020] The method is characterized in that the separated and purified exosomes can be oriented by mechanical force to have two orientations: outside-out and inside-out.

[0021] The outer membrane faces outward (i.e., outside-out) means that the outer side of the membrane of the exosome or vesicle faces outward, which is a natural state.

[0022] The inner membrane faces outward (i.e., inside-out) means that the inside of the exosome or vesicle membrane faces outward, which is the state formed after the membrane is opened by external force.

[0023] In a second aspect, the present invention provides an application of exosomes obtained from heterologous expression cells or primary cell culture fluid using any of the methods described above in biological analysis.

[0024] The application and the biological analysis include native cell membrane structure biological analysis and membrane protein function analysis based on cryo-electron microscopy.

[0025] The application, the biological analysis includes drug screening based on a surface activity test method or a solution detection method;

[0026] Preferably, the surface activity test method is surface plasmon resonance (SPR) or total internal reflection fluorescence (TIRF);

[0027] The solution detection method is a fluorescence, bioluminescence microplate or microfluidics method.

[0028] The application, the membrane protein function analysis includes the binding analysis of the active compound facing outside the membrane (outside-out), the interaction analysis of the inside-out membrane and downstream signaling proteins, and the role analysis of GPCRs and ion channel proteins in drug screening and drug development;

[0029] Preferably, the downstream signal protein is a G protein or an arrestin protein.

[0030] In a third aspect, the present invention provides a composition for medical diagnosis or drug screening, comprising exosomes obtained from the culture fluid of heterologous expression cells or primary cells using any of the methods described above, and a carrier for fixing the exosomes or exosome membrane fragments.

[0031] The present invention efficiently separates exosomes from heterologous expression cells or primary cells and can be applied to: (1) native cell membrane structural biology based on cryo-electron microscopy; (2) membrane protein function screening. The efficiently separated exosomes will be used in two orientations: outside-out and inside-out. It has been reported that the cell-derived exosome membrane contains the cytoplasmic membrane of the original cell source. In inside-out exosomes, the inside membrane flips and becomes interactive with the external environment, allowing the reconstruction of signal complexes and reactions. Normally, these reactions occur on the inside of the cell membrane of the receptor, which is difficult to achieve in outside-out exosomes.

[0032] Efficient isolation of exosomes from heterologous expression cells or primary cells can elucidate the functions of membrane proteins, especially the roles of G protein-coupled receptors (GPCRs) and ion channel proteins in drug screening and drug development. It has been reported that the signaling pathway reactions mediated by ligand-receptor binding can be detected by (1) fluorescence technology or (2) label-free molecular interaction detection technology such as surface plasmon resonance.

[0033] Fluorescence techniques rely on interactions between fluorescently labeled molecules. For example, for GPCRs, one or more interacting components need to be labeled. These components can be genetically engineered to be incorporated into exosomes or added to the reaction environment. The latter is particularly suitable for inside-out exosome experiments. First, fluorescence anisotropy (FA), fluorescence energy transfer (FRET), or fluorescence correlation spectroscopy (FCS) can be used to monitor fluorescence changes on either the inside or outside exosomes to detect interactions between labeled molecules on the exosome membrane. Second, by immobilizing intact exosomes on a support, techniques such as total internal reflection fluorescence can be used to monitor ligand-receptor binding or GPCR-G protein interactions inside-out exosomes. Third, by immobilizing exosomes on micrometer-sized agarose beads and mechanically disrupting the vesicle membrane, a membrane-supported structure is formed that allows for interaction on both sides of the membrane.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention heterologously expresses the membrane proteins NK1R and 5HT3R in human embryonic kidney cells (Expi293F), and successfully separates and purifies exosomes that highly express NK1R and 5HT3R using an innovative iodixanol self-density gradient centrifugation method. Western blot, NTA analysis, cryo-electron microscopy analysis, fluorescence microscopy fluorescence ligand binding analysis and other methods have demonstrated that the method of the present invention can well separate and purify exosomes with high purity and high yield.

[0036] 2. In the present invention, since the iodixanol solution is isotonic, the exosomes will not rupture during the ultracentrifugation process.

[0037] 3. The present invention generates a spontaneous density gradient of iodixanol via centrifugation in a very short time, and the sample separation distance within the gradient (perpendicular to the axis of the centrifuge tube) is short. Therefore, this method can isolate exosomes in a short time, with high purity and high yield.

[0038] 4. The present invention does not require the sample to be placed at the top or bottom of the centrifuge tube during sample loading. Instead, the sample and iodixanol need only be uniformly mixed at the beginning of the centrifugation process. This ensures high repeatability of the entire operating method and experimental results.

[0039] 5. The spontaneous density gradient centrifugation of the present invention enables exosomes to be separated from MVs with extremely high concentration and purity.

[0040] 6. Since the method of the present invention has high efficiency in isolating exosomes, it is suitable for very small amounts of cell secretion samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 is a schematic flow diagram of a method for isolating exosomes from heterologous expression cells, taking NK1R (neurokinin receptor) expression as an example;

[0042] Figure 2 is a characterization diagram of the results of the heterologous expression of NK1R exosomes in Expi293F cells isolated by the present invention, wherein A and B are Western blot analyses of the NK1R expression of each component of NK1R exosomes and MVs-derived exosomes; C and D are molecular sieve analysis of the separated NK1R exosomes and MVs-derived exosomes; E and F are EGFP fluorescence intensity analysis of the separated NK1R exosomes and MVs-derived exosomes; G and H are SDS-PAGE analysis of the separated NK1R exosomes and MVs-derived exosomes; I and J are negative staining analysis of the separated NK1R exosomes and MVs-derived exosomes;

[0043] FIG3 is a graph showing the biological analysis results of the exosomes derived from Expi293F cells heterologously expressing NK1R and MVs isolated in the present invention;

[0044] FIG4 is a graph showing the results of single particle size analysis of MVs and exosomes isolated from Expi293F cells heterologously expressing 5HT3R of the present invention;

[0045] Figure 5 shows the cryo-electron microscopy results of 5HT3R exosomes isolated from Expi293F cells heterologously expressing the present invention, wherein A is a Western blot analysis of 5HT3R expression in exosomes in each fraction of iodixanol self-density gradient centrifugation; B is a cryo-electron microscopy analysis of the morphology of exosomes isolated from 5HT3R-expressing cells. DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0047] Example 1: Isolation of exosomes from heterologous expressing cells

[0048] 1. Experimental Materials

[0049] (1) Vector construction

[0050] In the present invention, pCDNA3.1+ and pEG BacMam were used as the backbone for constructing mammalian cell expression vectors. pCDNA3.1-5HT3R (ampicin resistance) and pEG BacMam-NK1R-Halotag-EGFP (ampicin resistance) were self-constructed plasmids. Competent cells, including DH5α (KTSM101L), were purchased from Kangti Life Science.

[0051] (2) The main reagents are shown in Table 1 below.

[0052] Table 1 Main reagents

[0053] 2. Experimental methods

[0054] The present invention uses two major classes of membrane protein receptors as experimental subjects, including: (1) the neurokinin receptor (NK1R), a representative G protein-coupled receptor; and (2) the serotonin receptor (5HT3R), a representative ligand-gated ion channel. Using NK1R expression as an example, the process flow of the method for isolating exosomes from heterologous expressing cells is shown in Figure 1.

[0055] (1) Plasmid construction

[0056] The plasmids constructed in the present invention are all constructed using molecular biology methods, using existing plasmid sequences or backbones as templates. The target gene sequence is amplified by DNA polymerase chain reaction (PCR). After purification using a PCR cleanup kit, the target sequence is ligated to the vector backbone using homologous recombination. The target vector is screened for resistance and sequenced for correctness. For example, the amino acid sequence of the 5HT3R target protein is shown in SEQ ID NO. 1, and the amino acid sequence of the NK1R-Halotag-EGFP target protein is shown in SEQ ID NO. 2.

[0057] (2) Mammalian cell culture

[0058] The mammalian cells used in the present invention are human embryonic kidney cells Expi293F.

[0059] Expi293F cell culture, passage, transfection, and cryopreservation: Expi293F cells from Sino Biological were used; the culture medium was SMM 293-TII complete medium (Sino biological, M293TII); the transfection agent was Polyethylenimine Linear (PEI) MW40000 (Yeasen, 40816ES03), and the storage solution concentration was 1 mg / mL. When the cell concentration reached 2-3×10 6 When the concentration of PEI was 1:1, transient transfection was performed according to the PEI manufacturer's instructions at a ratio of 3:1.

[0060] The frozen suspension cells were thawed at 37℃ and the cells were divided into 0.3-0.5×10 6 / mL concentration was diluted with serum-free medium in a 100mL cell shake flask (the volume of the medium should not exceed 20mL), culture temperature was 37℃, CO2 concentration was 8%, humidity was 60%, and the shaker speed was 130rpm. The doubling time of suspended cells was about 24 hours. When the cell concentration reached 3×10 6 / mL, the cells can be passaged or frozen again.

[0061] During passage, cells were cultured in fresh serum-free medium at a certain concentration (0.5 × 10 6 / mL) was diluted in a cell shake flask of the corresponding volume. When frozen, the cells were collected by centrifugation at 300g for 5 minutes, and the supernatant was discarded and the volume was adjusted to 1×10 7 Add the corresponding volume of cell freezing solution (10% DMSO plus cell suspension culture medium) to a concentration of / mL, mix thoroughly by pipetting, and then dispense into 2mL cryovials. Place the cryovials in a cryostat box containing isopropanol and store at -80℃ for more than 4 hours before storing in a liquid nitrogen tank.

[0062] (3) Heterologous expression of 5HT3R and NK1R proteins

[0063] NK1R and 5HT3R receptors were heterologously expressed in human embryonic kidney cells (Expi293F). For cell transfection, when the cell concentration increased to 2-3×10 6 When the concentration of 5HT3R or NK1R is less than 1 mL, the cells are in a rapid growth phase and are suitable for transfection. Transfection method: Transfect suspension cells at a transfection volume of 1 μg DNA (5HT3R or NK1R) per mL, using a transfection agent to DNA ratio of 3:1. Experiments can generally be performed 72 hours after transfection.

[0064] (4) Iodixanol self-density gradient centrifugation to extract exosomes

[0065] Cells transfected with 5HT3R or NK1R were centrifuged at 300g, 2000g, and 10000g to remove live cells, dead cells, and cell debris. The supernatant after centrifugation at 10000g was centrifuged again at 23000g, and the resulting supernatant and precipitate were placed at 4°C for later use. The supernatant after centrifugation at 23000g was centrifuged again at 100000g for 70 min, and the resulting precipitate was treated with 20% iodixanol (OptiPrep TM The pellet was resuspended in 20% iodixanol (OptiPrep) and ultracentrifuged at 350,000 g for 4 hours. A 500 μL sample of each fraction was taken from the top of the centrifuge tube for subsequent western blot analysis. Similarly, the pellet after centrifugation at 23,000 g was resuspended in 20% iodixanol (OptiPrep) and ultracentrifuged at 350,000 g for 4 hours. A 500 μL sample of each fraction was taken from the top of the centrifuge tube and aliquoted for subsequent experiments.

[0066] As shown in Figure 1, using the method of the present invention, extracellular vesicles (EVs) in the culture medium can be successfully separated into plasma membrane-enclosed microvesicles (MVs) and smaller exosomes. The isolated exosomes have the following characteristics.

[0067] Example 2: Characterization of exosomes

[0068] 1. Western blotting to detect 5HT3R or NK1R protein in exosomes

[0069] Western blotting was used to detect the expression of 5HT3R or NK1R in the exosomes obtained in Example 1 and the effects of density gradient centrifugation. Transfected and untransfected cells were collected and added to lysis buffer to fully lyse the cells. After centrifugation, the supernatant was added with a certain amount of loading buffer to prepare a sample of the target protein expressed by the cells. 20 μL of each isolated exosome was taken and added with a certain amount of loading buffer to prepare a sample of the target protein contained in the exosomes. The prepared cell and exosome samples were heated at 100°C for 10 minutes and set aside. Depending on the experimental requirements, the primary antibody against strep, his, or TSG 101 was diluted to a specific ratio (1:1000) with 5% skim milk and incubated on the blocked PVDF membrane. Incubation conditions were either overnight in a 4°C refrigerator or 1 hour at room temperature. The membrane was then washed with 1x PBST for 10 minutes three times. After washing, the membrane was placed in the corresponding secondary antibody diluted 1:5000 and incubated on a shaker for 1 hour. Finally, wash three times with TBST. Mix color development solution A and solution B in a ratio of 1:1 and add them to the box. After the membrane is fully exposed to the color development solution, expose it on the Invitrogen iBright and take a photo of it.

[0070] 2. Molecular sieve purification and fluorescence intensity determination of NK1R exosomes

[0071] After Western blotting, exosomes with high target protein content were collected and diluted with PBS using a Milipore Amicon Ultra-15100KD to remove iodixanol. Finally, 1 mL of exosomes was centrifuged at 2000 g to remove large precipitates, and the supernatant was injected into a fast protein liquid chromatography (AKTA pure) column with PBS as the mobile phase. The column used was Superose 6 3.2 / 300, with a flow rate of 0.1 mL / min, and 0.1 mL was collected per tube. After molecular sieve separation, 50-100 μL of the sample was added to a 96-well opaque white plate. The fluorescence intensity of each sample was measured using a microplate reader (Molecular Devices) with 488 nm excitation.

[0072] Using Example 1, exosomes were isolated from Expi293F cells heterologously expressing NK1R. Western blot gel electrophoresis showed that the isolated exosomes all contained the heterologously expressed membrane protein NK1R (Figures 2A, B). This indicates that the isolated exosomes are suitable for structural function studies and drug screening of heterologously expressed proteins in their native membrane environment. Separation molecular weight (SEC) analysis showed that the obtained exosomes exhibited a single peak (Figures 2C, D), indicating high homogeneity of the isolated exosomes. It was also found that MV-derived exosomes had a higher OD280. Because the NK1R fusion protein C-terminus carries the EGFP fluorescent protein, SEC fractions were tested for EGFP fluorescence intensity. The results showed that the isolated exosomes had a higher EGFP fluorescence intensity at the peak position, and the EGFP fluorescence intensity of the MV-derived exosomes was higher (Figures 2E, F). Further polyacrylamide gel electrophoresis (PAGE) analysis of the SEC fractions showed that protein was observed at the peak position in the isolated exosomes, and the protein concentration in the MV-derived exosomes was higher (Figures 2G, H). The above experimental results show that the exosomes separated by the present invention are uniform in size and have a higher yield.

[0073] Example 3: Activity detection of NK1R exosomes on microbeads

[0074] In 2011, Professor Horst Vogel published research in JACS using wheat germ agglutinin-coated agarose beads (WGA-beads) to detect GPCR-G protein interactions. Fluorescence microscopy was used to observe the binding of fluorescently labeled agonists to GPCRs. Subsequently, a fluorescent miniG protein (a protein-engineered small G protein) was added to monitor the interaction between the GPCR and miniG protein. This experimental method was used to examine the biological activity of purified and isolated exosomes.

[0075] The isolated exosomes of the present invention can be immobilized on a solid support in the form of intact exosomes or exosome membrane fragments (by applying gentle mechanical force, such as gentle shaking or shear force from a flowing fluid). This support is achieved by coating a type of protein that interacts with the exosomes onto micrometer-scale agarose or sepharose. For intact, immobilized exosomes, the transport function of membrane proteins can only be studied through interaction with the outer membrane. For exosome membrane fragments, both the outer and inner membranes can be used to study the function of membrane proteins. For example, GPCRs can be used to study the binding of active compounds to the extracellular side of the cell, as well as the interaction of the inner side with downstream signaling proteins.

[0076] To demonstrate the feasibility of the experiment, we immobilized exosomes containing NK1R on WGA-coated agarose beads and treated them with Cy3-labeled substance P (SP, synthesized by Jier Biochemical (Shanghai) Co., Ltd.) to characterize the binding of the ligand to NK1R.

[0077] Specifically, three groups of WGA-beads were incubated with the exosomes isolated from the present invention, the experimental expression group, the blank control group, and the experimental treatment group (the experimental expression group to be treated with fluorescently labeled ligands), and incubated at 4°C for 30 minutes to allow the WGA-beads to fully bind to the exosomes. After incubation, the mixture was centrifuged at 100g for 3 minutes, and the exosomes or other components not bound to the WGA-beads in the supernatant were removed. 1 mL of PBS was added for resuspending, and the exosome-coated WGA-beads were washed by pipetting. This was repeated twice to obtain WGA-beads coated with exosomes. The exosome-coated WGA-beads were transferred to a glass slide, and the EGFP- and Cy3-labeled ligands were excited with excitation wavelengths of 488 nm and 532 nm, respectively, and images were collected using a fluorescence microscope.

[0078] As shown in Figure 3, compared to existing methods that immobilize living cell membranes onto microbeads, immobilizing exosomes onto microbeads is faster, more uniform, and has higher reproducibility and yield. Because the entire experiment does not require living cells, the prepared exosomes can be stored frozen for several months and used at any time, which is more conducive to their application in medical diagnosis and drug screening.

[0079] Example 4: Negative staining analysis of exosomes

[0080] The prepared vesicle samples were analyzed for size and morphology using a negative staining technique. 10 μL of exosome sample was placed on a hydrophilic-treated copper grid covered with a carbon film and incubated at room temperature for approximately 1 minute. Excess sample was then removed with filter paper. Next, 10 μL of 1% uranyl acetate was placed on the grid and stained for 1 minute, after which the excess stain was removed with filter paper. After air-drying, the sample was observed and inspected for quality using a room-temperature transmission electron microscope (Hitachi).

[0081] As shown in Figure 2I, J, the exosomes derived from the second supernatant and the exosomes derived from MVs separated by the present invention have a clear background and are uniform in size.

[0082] Example 5: Single Particle Size Analysis (NTA) of Exosomes

[0083] Single particle size analysis (NTA) was performed on exosomes isolated from Expi293F cells heterologously expressing the 5HT3R using the present invention. A 10 μL exosome sample was diluted to 500 μL with PBS, pipetted to mix, and then added to the particle size analysis sample pool. Particle diameter analysis was selected according to the Malvern particle size analyzer operating manual.

[0084] The results are shown in Figure 4 . The size distribution of exosomes is between 100-250 nm, with a maximum value of around 192 nm, while the size distribution of microvesicles MVs is between 300-1300 nm, with a maximum value of around 885 nm.

[0085] Example 6: Frozen Sample Preparation and Data Acquisition for Cryo-EM

[0086] The purified vesicles were analyzed for morphological analysis using cryo-electron microscopy. Quantifoil R1.2 / 1.3 cryo-EM copper grids were hydrophilized using a glow discharge apparatus and then prepared using a FEI Vitrobot Mark IV instrument. The following parameters were used: temperature 4°C, humidity 100%, sample volume 3 μL, blot force 3 seconds, blot force 0, and wait time 0 seconds. After removing excess sample using filter paper, the grids were quickly plunged into liquid ethane for freezing. The frozen grids were then transferred to a FEI Titan Krios transmission electron microscope for observation and examination of freezing quality, including ice thickness, ice contamination, and particle size. Freezing parameters such as sample concentration, volume, and humidity were adjusted to optimize freezing conditions. After sample selection, data were collected on a FEI Titan Krios cryo-EM equipped with a GIF energy filter and operated at 300 kV. A Gatan K3 Summit direct electron detector was used for imaging.

[0087] As shown in Figure 5, cryo-electron microscopy (cryo-EM) analysis revealed that the isolated exosomes had clear boundaries and were free of background contamination. The average size of the exosomes was approximately 200 nm, consistent with the results of single-particle size analysis. The exosomes exhibited a distinct bilayer membrane structure, with large protrusions on the surface likely representing the extracellular domain of the heterologously expressed 5HT3R. This demonstrates that the isolated exosomes are suitable for high-resolution structural studies of membrane proteins using single-particle cryo-EM imaging and cryo-electron tomography.

[0088] The MVs isolated by the present invention can also be used for cryo-electron microscopy structural studies. If necessary, the size of MVs can be reduced by ultrasound to meet the optimal conditions for cryo-electron microscopy. In addition to reducing the size of MVs, ultrasonic treatment of MVs has an additional effect, namely that the smaller exosomes produced will contain two orientations: an outside-out orientation and an inside-out orientation. In the inside-out orientation, the inner membrane can interact with downstream signaling pathway proteins. This is very beneficial for GPCR research because it provides a possibility to interact heterotrimeric G proteins or monomeric miniG proteins with the inner side of the cell membrane of GPCRs, so as to perform structural studies on activated GPCR / G protein complexes in the original cell membrane environment. In addition, inside-out exosomes are suitable for functional screening of potential agonists of GPCR / G protein complexes.

Claims

1. A method for isolating exosomes, characterized in that: After centrifuging the liquid to be extracted containing exosomes, the first supernatant was taken and subjected to high-speed centrifugation again. Iodixanol was added to the obtained second supernatant and precipitate respectively for spontaneous density gradient centrifugation to obtain the separated and purified exosomes.

2. The method according to claim 1, characterized in that The specific steps include: (1) taking the solution containing exosomes to be extracted, centrifuging it at 300-500g, 2000-2500g, 10000-12000g, or only centrifuging it at 10000-12000g, to obtain a first supernatant, centrifuging the first supernatant at 23000-25000g, obtaining a second supernatant and a precipitate, and placing them at 4°C for later use; (2) taking the second supernatant, centrifuging at 100,000-120,000 g for 70-90 min to obtain a precipitate, adding 20% ​​iodixanol to resuspend it, and then ultracentrifuging at 300,000-350,000 g for 2-4 h, taking the liquid at the top of the centrifuge tube to obtain exosomes; (3) The precipitate obtained in step (1) was resuspended in 20% iodixanol, and then ultracentrifuged at 300,000-350,000 g for 2-4 h, and the liquid at the top of the centrifuge tube was collected to obtain exosomes.

3. The method according to claim 1, characterized in that The liquid to be extracted containing exosomes is the culture liquid of heterologous expression cells or primary cells.

4. The method according to claim 1, characterized in that The centrifugal method is a vertical rotor centrifugal method.

5. The method according to claim 1, characterized in that The separated and purified exosomes can be oriented in two directions, one with the outer membrane facing outwards and the other with the inner membrane facing outwards, by mechanical force.

6. Use of exosomes obtained from heterologous expression cells or primary cell culture fluid using the method according to any one of claims 1 to 5 in biological analysis.

7. The use according to claim 6, characterized in that The biological analysis includes native cell membrane structure biological analysis or membrane protein function analysis based on cryo-electron microscopy.

8. The use according to claim 6, characterized in that The bioanalysis includes drug screening based on surface activity test methods or solution detection methods; Preferably, the surface activity test method is surface plasmon resonance or total internal reflection fluorescence; The solution detection method is a fluorescence, bioluminescence microplate or microfluidics method.

9. The use according to claim 7, characterized in that The membrane protein function analysis includes the analysis of the binding of the outer membrane to the active compound, the interaction analysis of the inner membrane to the downstream signaling protein, and the role analysis of GPCRs and ion channel proteins in drug screening and drug development; Preferably, the downstream signal protein is a G protein or an arrestin protein.

10. A composition for medical diagnosis or drug screening, characterized in that: The method comprises exosomes obtained from the culture fluid of heterologous expression cells or primary cells using the method according to any one of claims 1 to 5, and a carrier for fixing the exosomes or exosome membrane fragments.

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