Method for measuring exosome synthesis efficiency

Abstract

The invention relates to the technical field of exosome synthesis, and discloses a method for measuring exosome synthesis efficiency. According to the method, by utilizing the characteristic that four-transmembrane proteins in different types of cells are different in content, various four-transmembrane proteins are selected as calibration substances, and part of the four-transmembrane proteins are used as internal reference, so that in the process of determining the exosome synthesis efficiency, the content of the four-transmembrane proteins in the exosome synthesis efficiency is determined. According to the method, the expression level of the tetratransmembrane protein calibration substance in the cell membrane can be equalized by using the total tetratransmembrane protein, so that the problem of inaccurate determination of the exosome synthesis efficiency caused by difference between cell types is remarkably improved, and accurate determination of the exosome synthesis efficiency of different types of cells can be realized; and the determination method is simple and easy to operate, low in cost, high in speed and suitable for large-scale application in exosome synthesis efficiency determination, and has important significance in large-scale synthesis and commercial application of the exosome.

Description

Technical Field

[0001] The present invention relates to the technical field of biological genetic engineering, mainly to the technical field of exosome synthesis, and particularly to a method for measuring the synthesis efficiency of exosomes. Background Art

[0002] Exosomes are nanoscale membrane vesicles secreted by cells and play a key role in cell - to - cell communication. They are widely present in various body fluids such as blood, saliva, urine, etc., and play important roles in both normal physiological functions and pathological states. The biogenesis mechanism of exosomes is the basis for their function, and the complexity of this mechanism determines the diversity and dynamics of exosomes.

[0003] Research has found that the biogenesis mechanism of exosomes has redundancy, which is the key for them to produce diverse, dynamic and selective exosomes under normal and pathological conditions. Under normal physiological conditions, exosomes participate in various physiological processes such as cell - to - cell signal communication and immune regulation through their diverse compositions and functions. Under pathological conditions, such as during tumorigenesis and development, the composition and secretion pattern of exosomes will change. The existence of their redundancy enables cells to adapt to the pathological environment through multiple pathways, thereby producing exosomes with specific functions. These exosomes may participate in processes such as tumor cell proliferation, migration, invasion, and immune escape. This redundancy provides a guarantee for the adaptability and functionality of exosomes in different environments and is an indispensable characteristic of exosomes in cell - to - cell communication.

[0004] Exosomes have multiple uptake and absorption methods, which more effectively ensure the richness and orderliness of signal communication in the body. Exosomes can be taken up by recipient cells through multiple pathways, including entering cells through endocytosis after binding to cell - surface receptors, directly releasing their contents into the cytoplasm by fusing with the cell membrane, etc. This diverse uptake method enables exosomes to deliver the carried signal molecules to recipient cells through the most appropriate pathway according to different recipient cell types and physiological environments, thereby achieving efficient cell - to - cell communication. For example, in the immune system, exosomes can bind to specific receptors on the surface of immune cells and deliver antigen information to immune cells, thereby activating the immune response; in the nervous system, exosomes can fuse with the nerve cell membrane and deliver signal molecules such as neurotransmitters to nerve cells, participating in the transmission and regulation of nerve signals. This diverse uptake and absorption method is an important guarantee for exosomes to play an important role in cell - to - cell communication.

[0005] The formation of exosomes mainly involves the regulation of the endosomal sorting complex required for transport (ESCRT). The endocytosed plasma membrane (PM) selectively binds to ESCRT, sorting miRNAs and other signaling molecules into the vesicles of early endosomes. This process is a core link in the exosome biogenesis mechanism. Early endosomes play a role in sorting and transporting within cells. Through interaction with ESCRT, different molecules are sorted and packaged into vesicles, providing a basis for exosome formation. miRNAs are a class of important non-coding RNA molecules that play a key role in gene expression regulation. By sorting miRNAs into the vesicles of early endosomes, exosomes can transport these miRNAs into recipient cells, thereby regulating the gene expression of recipient cells. In addition, other signaling molecules such as proteins and lipids are also sorted and packaged through this process, enabling exosomes to carry various types of signaling molecules and thus realizing diverse intercellular communication functions.

[0006] Multivesicular bodies (MVBs) formed by late endosomes contain different aggregated exosome precursors. During exosome biogenesis, MVBs play a key role in transport and sorting. Exosome precursors are either transported to lysosomes for degradation or their cargo is transferred to other organelles, which reflects the fine regulation of the exosome generation process by cells. If exosome precursors are transported to lysosomes for degradation, then these precursors will not form mature exosomes, thus avoiding unnecessary exosome secretion. If the cargo of exosome precursors is transferred to other organelles, then these cargoes may participate in other physiological processes within the cell. The docking of MVBs with PM is a key step in exosome secretion. MVBs limit membrane fusion with PM, enabling exosomes to be released from inside the cell to the outside. This process requires the participation of multiple molecules, including small GTPases (for docking) and SNAREs (for membrane fusion). Small GTPases play a key role in the docking process of MVBs with PM. They bind to specific receptors on the membrane, guiding the docking of MVBs with PM. SNAREs play an important role in the membrane fusion process. They form SNARE complexes to promote the fusion of the limiting membrane of MVBs with PM, thereby realizing exosome secretion. However, many of the regulatory mechanisms are still poorly understood, which also provides a broad space for the study of the exosome biogenesis mechanism.

[0007] Existing studies have also found that calcium ions are essential for exosome secretion in most cases. Calcium ions play an important role in signal transduction within cells. By interacting with various calcium-binding proteins, they regulate multiple physiological processes within the cell. During exosome secretion, calcium ions may affect the fusion process of MVBs with the PM by regulating the activities of small GTPases and SNAREs. In addition, calcium ions may also indirectly affect exosome secretion by regulating other signaling pathways within the cell. Besides calcium ions, small GTPases and SNAREs are also key regulatory molecules during exosome secretion. Small GTPases regulate the docking process of MVBs with the PM through their GTP-binding and hydrolysis activities. SNAREs, on the other hand, promote the membrane fusion process by forming SNARE complexes. The coordinated action of these molecules enables the precise progress of the exosome secretion process. Of course, some of the key regulatory molecules discovered in current research are not all. There are still many regulatory mechanisms during exosome secretion that are unclear to people, which is also one of the directions for future research.

[0008] To study the regulatory mechanism of exosome biogenesis and develop the application of exosomes in the fields of biomedicine, etc., it is inevitable to use culture media for large-scale cultivation to secrete and synthesize exosomes. The secretion and synthesis efficiency of exosomes is surely one of the key factors affecting its large-scale application. In existing technologies, new strategies such as three-dimensional culture and culture with exosome-specific culture media have been proven to accelerate exosome formation and increase exosome yield. During this process, the accurate calculation of exosome synthesis efficiency is one of the key technologies for measuring the feasibility of the strategy.

[0009] Currently, the index for quantifying exosome yield is still the number of exosome particles. However, traditional methods for measuring the number of exosome particles require multiple ultra-high-speed centrifugations and nanoparticle flow cytometer detections. Ultra-high-speed centrifugation is a commonly used method for exosome isolation. By generating centrifugal force through high-speed rotation, it separates exosomes from cell culture fluids or body fluids. However, ultra-high-speed centrifugation requires expensive equipment and complex operation steps, and the separation process is time-consuming. Nanoparticle flow cytometer detection is an instrument for detecting the number and size of exosome particles. It detects exosome particles by detecting the scattered light signal of exosome particles under laser irradiation. Although nanoparticle flow cytometer detection has high sensitivity and accuracy, its equipment cost is high, and the detection process requires professional technical personnel to operate. These factors make the existing methods for detecting the number of exosome particles costly and have low detection efficiency, and cannot quickly obtain the indexes of various exosome synthesis efficiencies.

[0010] With the continuous in-depth research on exosomes, higher requirements have been put forward for the detection methods of exosome synthesis efficiency. Traditional detection methods can no longer meet the needs of research, so new detection technologies need to be developed to improve the detection efficiency and reduce costs. In recent years, some new detection technologies have been proposed and applied to the detection of exosomes. For example, the detection technology based on fluorescent probes realizes the fluorescent labeling and detection of exosomes by binding fluorescent probes to specific molecules on the surface of exosomes; this method has the advantages of simple operation and low cost, but its detection sensitivity and accuracy are relatively low. Another emerging detection technology is the detection technology based on surface plasmon resonance (SPR), which realizes the detection of exosomes by detecting the interaction between exosomes and the surface of biosensors; although the SPR detection technology has high sensitivity and accuracy, its equipment cost is high and complex sample processing procedures are required. It can be seen that developing a method that can quickly and accurately determine the exosome synthesis efficiency, is simple and easy to operate, and has low cost is of great significance for the large-scale synthesis and application of exosomes. Summary of the Invention

[0011] The object of the present invention is to propose a method for determining the exosome synthesis efficiency in view of the defects of the existing exosome synthesis efficiency detection methods, such as long time, complex operation, and high cost.

[0012] To achieve the above object, the present invention provides a method for determining the exosome synthesis efficiency, including the following steps:

[0013] S1. Add fluorescent antibodies of tetraspanin markers to the cell culture system to be tested, incubate to obtain the first cell tissue; the tetraspanin markers are CD9 and CD63; the fluorescent antibodies of the tetraspanin markers are CD9-PE and CD63-PE;

[0014] S2. Use cell lysis to extract the total protein of the first cell tissue, and determine the total expression level of the tetraspanin markers in the total protein by Western-Blot; detect the positive rate of the fluorescent antibodies on the cell membrane in the first cell tissue by flow cytometry; use cell lysis to extract the cell membrane of the first cell tissue, and determine the expression level of the fluorescent antibodies in the cell membrane by Western-Blot;

[0015] S3. Based on the total expression level of the tetraspanin markers in the first cell tissue and the expression level of the fluorescent antibodies in the cell membrane, calculate the determination result of the exosome synthesis efficiency in the cell culture system to be tested.

[0016] A method for measuring the synthesis efficiency of exosomes according to the present invention utilizes the characteristic that the content of tetraspanins in different types of cells is different, selects a variety of tetraspanins as calibrators, and uses some tetraspanins as internal references. Therefore, during the process of measuring the synthesis efficiency of exosomes, the average value of the total tetraspanins can be used to equalize the expression level of the tetraspanin calibrator in the cell membrane, significantly improving the problem of inaccurate measurement of exosome synthesis efficiency caused by differences between cell types, enabling accurate measurement of the exosome synthesis efficiency of different types of cells, and the measurement method is simple, easy to operate, low-cost, and fast, suitable for large-scale application in the measurement of exosome synthesis efficiency, and is of great significance for the large-scale synthesis and commercial application of exosomes.

[0017] Among them, in step S1, preferably, the cell culture system to be tested is one of stem cells and their differentiated cells.

[0018] Preferably, the concentration of the fluorescent antibody of the tetraspanin calibrator is 10 ± 2 μg / mL.

[0019] Among them, CD9 is a member of the tetraspanin family, can be widely expressed on the cell membrane, is closely related to the formation, release and function of exosomes, and can be separated, purified and quantitatively detected in exosome research, and is one of the classic markers of exosomes.

[0020] Among them, CD63 is a member of the tetraspanin family, is a key factor regulating the production of cell exosomes and the sorting of endosomal cargo, plays an important role in the formation and release of exosomes, is the main determining molecule for endosome formation, and belongs to the specific marker protein of exosomes.

[0021] Among them, CD9-PE is a polyclonal antibody against CD9 labeled with PE fluorescence, is a fluorescently labeled antibody used in flow cytometry (FC), and is widely used to detect CD9 protein on the cell surface.

[0022] Among them, CD63-PE is a polyclonal antibody against CD63 labeled with PE fluorescence, is a fluorescently labeled antibody used in flow cytometry (FC), and is widely used to detect CD63 protein on the cell surface, especially for the detection of CD63 in exosomes.

[0023] Among them, preferably, the incubation temperature is 37 ± 2 °C and the incubation time is 30 ± 5 min.

[0024] Among them, preferably, the tetraspanin calibrator further includes CD81; the fluorescent antibody further includes CD81-APC.

[0025] Among them, the CD81 is a member of the tetraspanin protein family, which plays an important role in cell adhesion, activation, proliferation, differentiation, and signal transduction, can affect the production, release, and interaction with target cells of exosomes, and is one of the common surface markers of exosomes; CD81 is a relatively stable transmembrane protein on the cell membrane, with little change in expression on the surface of the same type of cells. In the present invention, it is mainly used to verify whether there are errors in experimental operations and can increase the accuracy of measurement results.

[0026] Among them, the CD81-APC is an APC-fluorescently labeled anti-CD9 polyclonal antibody, which can be used for flow cytometry to detect the CD81 protein on the cell surface.

[0027] Among them, in step S2, the Western-Blot (WB) is a technique widely used in protein analysis; it can quantitatively detect the expression of the measured protein.

[0028] Among them, preferably, the method for extracting the total protein of the first cell tissue by cell lysis includes: The method for extracting the total protein of the first cell tissue by cell lysis includes: washing the first cell tissue with PBS (buffer) at least three times; then, lysing the first cell tissue with RIPA lysis buffer (Beyotime, China) containing 0.5 ± 0.2 mM phenylmethylsulfonyl fluoride; after lysis, obtaining the total protein extract by centrifugation at 12000 ± 1000 rpm for 30 ± 5 minutes.

[0029] Preferably, the method for extracting the cell membrane of the first cell tissue by cell lysis includes: ultrasonically disrupting the first cell tissue in an ice bath; (selecting a power of 30%-50% according to the cell type, with a working time of 10 ± 2 s, an interval of 10 ± 2 s, and cycling at least 5 times), after disruption, centrifuging at 1000 ± 100 g for 10 ± 2 min at 4 ± 1 °C to remove unbroken cells and large fragments; centrifuging at 10000 ± 1000 g for 20 ± 5 min to separate cell organelles; and then centrifuging at 100000 ± 10000 g for 60 ± 10 min to purify and obtain the cell membrane.

[0030] Among them, in step S3, the calculation formula for the exosome synthesis efficiency is:

[0031]

[0032] Among them, preferably, the method for measuring the exosome synthesis efficiency further includes: step S4, verifying and optimizing the measurement results.

[0033] Preferably, the specific methods for verification and optimization include: obtaining the endocytosis efficiency of the cell culture system to be tested and / or the number of exosome precursors MVBs, and verifying and optimizing the determination result of exosome synthesis efficiency based on the endocytosis efficiency of the cell culture system to be tested and / or the number of exosome precursors MVBs.

[0034] Preferably, the method for obtaining the endocytosis efficiency of the cell culture system to be tested includes the following steps:

[0035] 1. Add a fluorescent antibody of a tetraspanin marker to the cell culture system to be tested and incubate under inhibitory conditions to obtain a second cell tissue;

[0036] 2. Detect the positive rates of CD9-PE and CD63-PE on the cell membrane in the second cell tissue by flow cytometry respectively;

[0037] 3. Calculate the endocytosis efficiency of the cell culture system to be tested.

[0038] Among them, preferably, in step 1, the incubation under inhibitory conditions is: incubating at 4±1°C for 30±5 min (low-temperature freezing method), or after adding a targeted dynein inhibitor, incubating at 37±2°C for 6±1 h (inhibitor method).

[0039] Among them, preferably, in step 3, the calculation formula for endocytosis efficiency is:

[0040]

[0041] Preferably, the method for verifying and optimizing the determination result of exosome synthesis efficiency based on the endocytosis efficiency of the cell culture system to be tested includes: comparing the obtained endocytosis efficiency with the predicted endocytosis efficiency. If the difference between the two is significant (the difference rate is greater than 5%), then use cells of the same batch and passage to measure again; the predicted endocytosis efficiency is the ratio of the positive rate of CD63 in the first cell tissue to the positive rate of CD9 in the first cell tissue. The verification and optimization of the determination result of exosome synthesis efficiency based on the endocytosis efficiency of the cell culture system to be tested is mainly for the verification and optimization of the exosome synthesis efficiency of the same type of cells.

[0042] Preferably, the method for obtaining the number of exosome precursors MVBs in the cell culture system to be tested includes the following steps:

[0043] a. Digest the first cell tissue and then perform centrifugation to obtain a cell pellet;

[0044] Fix the cell pellet with aqueous glutaraldehyde and osmium tetroxide at 4°C for 2 hours to obtain a cell fixative;

[0045] b. After gradient dehydration of the cell fixative, soak it in acetone, and then successively perform embedding, sectioning, and staining treatments to obtain cell precipitate sections.

[0046] c. Use a scanning electron microscope to capture microscopic images of organelles in the cell precipitate sections, and count the average number of multivesicular bodies (MVBs) of exosome precursors, which is the number of exosome precursors MVBs in the cell culture system to be measured.

[0047] Among them, in step a, preferably, the digestion treatment is as follows: After washing the first cell tissue with PBS at least once, digest it with 0.25 ± 0.1 wt% trypsin-EDTA for 2 ± 1 min.

[0048] Preferably, the rotation speed of the centrifugation treatment is 1000 ± 100 g, and the time is 5 ± 1 min.

[0049] Preferably, the volume fraction of the glutaraldehyde aqueous solution is 2.5 ± 0.5%.

[0050] Preferably, the concentration of osmium tetroxide is 1 ± 0.2% (w / v).

[0051] Among them, in step b, preferably, gradient dehydration is carried out using an ethanol aqueous solution, and the volume percentages of the ethanol aqueous solution are 50%, 70%, 80%, and 90% respectively; soak and dehydrate in each concentration of ethanol aqueous solution for 20 ± 2 minutes.

[0052] Preferably, soak in acetone no less than 2 times, and each time is not less than 15 minutes.

[0053] Preferably, during the staining treatment, the staining agents are uranyl acetate and lead citrate; after staining, wash at least 2 times.

[0054] Preferably, the method for verifying and optimizing the exosome synthesis efficiency measurement result based on the number of exosome precursors MVBs in the cell culture system to be measured includes: Based on the relationship that the exosome synthesis efficiency is proportional to the number of exosome precursors MVBs, verify the synthesis efficiency of different types of cells; in the cell culture system with high exosome synthesis efficiency, the number of exosome precursors MVBs is also correspondingly high; if the verification result does not match, it is necessary to determine whether a large number of cells undergo apoptosis or death during the incubation process (causing enhanced autophagy of cells leading to endocytosis of tetraspanins), resulting in a falsely high exosome synthesis efficiency, and re-measure.

[0055] Compared with the prior art, the beneficial effects of the present invention:

[0056] 1. In the process of measuring the exosome synthesis efficiency, the method for measuring the exosome synthesis efficiency of the present invention can use the total mean of tetraspanins to equalize the expression level of tetraspanin markers in the cell membrane, significantly improving the problem of inaccurate measurement of exosome synthesis efficiency caused by differences between cell types, and enabling accurate measurement of the exosome synthesis efficiency of different types of cells.

[0057] 2. The method for measuring the exosome synthesis efficiency of the present invention can significantly reduce the trial-and-error cost during the measurement process, reduce the waste of culture medium, cell line screening and culture time, reduce the cost of measuring the exosome synthesis efficiency, and the method is simple and easy to operate, suitable for large-scale application in the measurement of exosome synthesis efficiency, which is of great significance for the large-scale synthesis and commercial application of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 It is a schematic diagram of the formation and secretion pathway of exosomes in Example 1 of the present invention;

[0059] Figure 2 It is a result graph of measuring the endocytosis efficiency by the cryogenic freezing method in Example 2 of the present invention;

[0060] Figure 3 It is a result graph of measuring the endocytosis efficiency by the inhibitor method in Example 2 of the present invention;

[0061] Figure 4 It is a result graph of measuring the total expression level of tetraspanin markers in the first cell tissue in Example 1 of the present invention;

[0062] Figure 5 It is a result graph of measuring the expression level of fluorescent antibody in the first cell tissue in Example 1 of the present invention;

[0063] Figure 6 It is an electron micrograph when calculating the number of multivesicular bodies of exosome precursors in Example 2 of the present invention;

[0064] Figure 7 It is a graph showing the relationship between the volume of multivesicular bodies and the number of exosomes contained therein in Example 2 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0065] Next, the technical solutions in some embodiments of the present disclosure will be clearly and completely described in conjunction with the drawings. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present disclosure.

[0066] In the following experimental methods of the embodiments, unless otherwise specified, they are all conventional methods; the reagents, biological materials, and detection kits used in the experiments, unless otherwise specified, can all be obtained from commercial channels.

[0067] Brief Introduction of Some Biomaterials and Reagents in the Embodiments of the Present Invention:

[0068] Cells: Human umbilical cord mesenchymal stem cells (hUCMSCs) are derived from the applicant's clinical placental samples;

[0069] Materials: The hUCMSCs culture medium is DMEM / F12 medium containing 10% fetal bovine serum;

[0070] Direct flow antibodies CD9-PE, CD63-PE and CD81-APC;

[0071] Targeted dynein inhibitor dyngo 4a;

[0072] Monoclonal rabbit anti-HRS, TSG101, Rab27a, Rab27b and Lamp1.

[0073] For the schematic diagram of the formation and secretion pathway of exosomes, see Figure 1 .

[0074] Example 1:

[0075] A method for measuring the synthesis efficiency of exosomes, comprising the following steps:

[0076] S1. Add fluorescent antibodies of tetraspanin markers to the cell culture system to be tested, and incubate (the incubation temperature is 37 °C and the incubation time is 30 min) to obtain the first cell tissue; the tetraspanin markers are CD9, CD81 and CD63; the fluorescent antibodies of the tetraspanin markers are CD9-PE, CD81-APC and CD63-PE;

[0077] S2. Use cell lysis to extract the total protein of the first cell tissue, and measure the total expression level of the tetraspanin markers in the first cell tissue by Western-Blot ( Figure 4 ); Detect the positive rate of the fluorescent antibodies on the cell membrane in the first cell tissue by flow cytometry; Use cell lysis to extract the cell membrane of the first cell tissue, and measure the expression level of the fluorescent antibodies in the cell membrane by Western-Blot ( Figure 5 );

[0078] Among them, the method for using cell lysis to extract the total protein of the first cell tissue includes: washing the first cell tissue with PBS (buffer) at least three times; then, lysing the first cell tissue with RIPA lysis buffer (Beyotime, China) containing 0.5 ± 0.2 mM phenylmethanesulfonyl fluoride; after the lysis is completed, obtain the total protein extract by centrifuging at 12000 ± 1000 rpm for 30 ± 5 minutes.

[0079] Among them, the method for extracting the cell membrane of the first cell tissue by cell lysis includes: ultrasonically disrupting the first cell tissue under ice bath; (power 40%, working time 10 s, interval 10 s, cycle 5 times). After disruption, centrifuge at 1000 g for 10 min at 4°C to remove unbroken cells and large fragments; then centrifuge at 10000 g for 20 min to separate cell organelles; and then centrifuge at 100000 g for 60 min for purification to obtain the cell membrane.

[0080] S3. Calculate the exosome synthesis efficiency in the cell culture system to be measured based on the total expression level of the tetraspanin marker in the first cell tissue and the expression level of the fluorescent antibody in the cell membrane.

[0081] Among them, the calculation formula for the exosome synthesis efficiency is:

[0082]

[0083] Example 2

[0084] A method for measuring the exosome synthesis efficiency includes the following steps:

[0085] S1, S2, and S3 are the same as in Example 1, and the difference is only that it further includes:

[0086] S4. Obtain the endocytosis efficiency and / or the number of exosome precursors MVBs in the cell culture system to be measured, and verify and optimize the measurement result of the exosome synthesis efficiency based on the endocytosis efficiency and / or the number of exosome precursors MVBs in the cell culture system to be measured.

[0087] Among them, the method for obtaining the endocytosis efficiency of the cell culture system to be measured includes the following steps:

[0088] 1. Add the fluorescent antibody of the tetraspanin marker to the cell culture system to be measured, and incubate under inhibitory conditions (incubate at 4°C for 30 min (low-temperature freezing method); after adding the targeted dynein inhibitor, incubate at 37°C for 6 h (inhibitor method)) to obtain the second cell tissue.

[0089] 2. Detect the positive rates of CD9-PE and CD63-PE in the cell membrane of the second cell tissue by flow cytometry respectively.

[0090] 3. Calculate the endocytosis efficiency of the cell culture system to be measured (for the low-temperature freezing method is Figure 2 , and for the inhibitor method is Figure 3 ).

[0091]

[0092] A method for verifying and optimizing the determination result of exosome synthesis efficiency based on the endocytosis efficiency of a cell culture system to be tested includes: comparing the obtained endocytosis efficiency with the predicted endocytosis efficiency. If the difference between the two is significant (the difference rate is greater than 5%), then use cells of the same batch and passage to perform the determination again;

[0093] The predicted endocytosis efficiency is the ratio of the positive rate of CD63 in the first cell tissue to the positive rate of CD9 in the first cell tissue.

[0094] Among them, the method for obtaining the number of exosome precursor MVBs in the cell culture system to be tested includes the following steps:

[0095] a. After digesting the first cell tissue (the first cell tissue is washed once with PBS and then digested with 0.25 wt% trypsin-EDTA for 2 min), perform centrifugation (rotation speed: 1000 g, time: 5 min) to obtain a cell pellet; fix the cell pellet with an aqueous glutaraldehyde solution (volume fraction: 2.5%) and osmium tetroxide (volume fraction: 1%) at 4°C for 2 hours to obtain a cell fixative.

[0096] b. After gradient dehydration of the cell fixative (using aqueous ethanol solutions for gradient dehydration, the volume percentages of the aqueous ethanol solutions are 50%, 70%, 80%, and 90% respectively; soak and dehydrate in each concentration of aqueous ethanol solution for 20 minutes), soak with acetone (3 times, 15 minutes each time), then perform embedding, sectioning, and staining (uranyl acetate and lead citrate) in sequence, and wash 3 times to obtain a cell pellet section;

[0097] c. Use a scanning electron microscope to capture microscopic images of organelles in the cell pellet section, and count the average number of multivesicular bodies of exosome precursor MVBs, which is the number of exosome precursor MVBs in the cell culture system to be tested;

[0098] A method for verifying and optimizing the determination result of exosome synthesis efficiency based on the number of exosome precursor MVBs in the cell culture system to be tested includes: based on the relationship that the exosome synthesis efficiency is proportional to the number of exosome precursor MVBs (the volume of multivesicular bodies is related to the number of exosomes contained, see Figure 7 ), verify the synthesis efficiency of different types of cells; for a cell culture system with a high exosome synthesis efficiency, the number of exosome precursor MVBs is correspondingly high; if the verification result does not match, it is necessary to determine whether a large number of cells undergo apoptosis or death during the incubation process (resulting in enhanced autophagy of cells leading to endocytosis of tetraspanins), causing the exosome synthesis efficiency to be falsely high, and perform a re-determination.

[0099] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any one or more embodiments or examples in a suitable manner.

[0100] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claimed rights.

Claims

1. A method for determining the efficiency of exosome synthesis, characterized in that: The following steps are involved: S1. Adding fluorescent antibodies for tetraspanin markers to the cell culture system to be tested, incubating, and obtaining a first cell tissue; the tetraspanin markers are CD9 and CD63; the fluorescent antibodies for tetraspanin markers are CD9-PE and CD63-PE; S2, extracting the total protein of the first cell tissue by cell lysis, and determining the total expression level of the tetraspanin marker in the total protein by Western-Blot; detecting the positive rate of the fluorescent antibody of the cell membrane in the first cell tissue by flow cytometry; extracting the cell membrane of the first cell tissue by cell lysis, and determining the total expression level of the fluorescent antibody in the cell membrane by Western-Blot; S3. Based on the total expression level of the tetraspanin marker in the first cell tissue and the expression level of the fluorescent antibody in the cell membrane, the determination result of the exosome synthesis efficiency in the cell culture system to be tested is calculated; The calculation formula of the exosome synthesis efficiency is:

2. The method for determining the exosome synthesis efficiency according to claim 1, characterized in that: In step S1, the cell culture system to be tested is one of stem cells and differentiated cells thereof.

3. The method for determining the exosome synthesis efficiency according to claim 1, characterized in that: In step S1, the incubation temperature is 37±2°C and the incubation time is 30±5min.

4. The method for determining the exosome synthesis efficiency according to claim 1, characterized in that: In step S1, the tetraspanin marker also includes CD81; the fluorescent antibody also includes CD81-APC.

5. The method for determining the exosome synthesis efficiency according to claim 1, characterized in that: In step S2, the method of extracting total protein of the first cell tissue by cell lysis includes: washing the first cell tissue with PBS at least three times; then, lysing the first cell tissue with RIPA lysis buffer containing 0.5±0.2 mM phenylmethylsulfonyl fluoride; after lysis, obtaining a total protein extract by centrifugation at 12000±1000 rpm for 30±5 minutes; The method of extracting the cell membrane of the first cell tissue by cell lysis includes: ultrasonically disrupting the first cell tissue in an ice bath; after the disruption, centrifuging at 1000±100g for 10±2min at 4±1°C to remove unbroken cells and large fragments; centrifuging at 10000±1000g for 20±5min to separate organelles; and further centrifuging at 100000±10000g for 60±10min to purify the cell membrane.

6. The method for determining the exosome synthesis efficiency according to any one of claims 1 to 5, characterized in that: The method for determining the exosome synthesis efficiency also includes: step S4, verifying and optimizing the determination result; the specific method of the verification and optimization includes: obtaining the endocytosis efficiency of the cell culture system to be tested and / or the number of exosome precursor MVBs, and verifying and optimizing the determination result of the exosome synthesis efficiency based on the endocytosis efficiency of the cell culture system to be tested and / or the number of exosome precursor MVBs.

7. The method for determining the exosome synthesis efficiency according to claim 6, characterized in that: The method for obtaining the endocytosis efficiency of the cell culture system to be tested comprises the following steps:

1. Add fluorescent antibodies of tetraspanin markers to the cell culture system to be tested, incubate under inhibitory conditions, and obtain a second cell tissue; 2. Detect the positive rates of CD9-PE and CD63-PE on the cell membrane of the second cell tissue by flow cytometry; 3. Calculate the endocytosis efficiency of the cell culture system to be tested; The calculation formula for endocytosis efficiency is:

8. The method for determining the exosome synthesis efficiency according to claim 7, characterized in that: The method for verifying and optimizing the exosome synthesis efficiency measurement results based on the endocytosis efficiency of the cell culture system to be tested includes: comparing the obtained endocytosis efficiency with the predicted endocytosis efficiency, and if the difference between the two is significant, using cells of the same batch and generation for re-measurement; the predicted endocytosis efficiency is the ratio of the positive rate of CD63 in the first cell tissue to the positive rate of CD9 in the first cell tissue.

9. The method for determining the exosome synthesis efficiency according to claim 6, characterized in that: The method for obtaining the number of exosome precursor MVBs in the cell culture system to be tested comprises the following steps: a. digesting the first cell tissue and then centrifuging it to obtain a cell precipitate; fixing the cell precipitate with a glutaraldehyde aqueous solution and osmium acid at 4° C. for 2 hours to obtain a cell fixative; b. After gradient dehydration, the cell fixative is soaked in acetone, and then embedded, sliced ​​and stained in sequence to obtain cell precipitation slices. c. Use a scanning electron microscope to capture the microscopic image of the organelles in the cell sediment slices, and count the average number of exosome precursor MVBs multivesicular bodies, which is the number of exosome precursor MVBs in the cell culture system to be tested.

10. The method for determining the exosome synthesis efficiency according to claim 9, characterized in that: The method for verifying and optimizing the results of the exosome synthesis efficiency determination based on the number of exosome precursor MVBs in the cell culture system to be tested includes: verifying the synthesis efficiency of different types of cells based on the proportional relationship between the exosome synthesis efficiency and the number of exosome precursor MVBs; the cell culture system with high exosome synthesis efficiency also has a correspondingly high number of exosome precursor MVBs; if the verification results are inconsistent, it is necessary to determine whether a large number of cells have undergone apoptosis or death during the incubation process, which makes the exosome synthesis efficiency artificially high, and re-measure.

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