Extraction method of sterile extracellular vesicles

Abstract

The invention relates to an extraction method of sterile extracellular vesicles, and belongs to the technical field of extracellular particle extraction. The method provided by the invention comprises the following steps: re-suspending cells, standing at 2-6 DEG C for 2-8 hours, and centrifuging to remove the cells, cell debris and lEV. The method has the advantages that the extracted EVs content is higher, the product integrity is higher, the extraction time is short, the operation difficulty is low, the equipment requirement is low, and the method is more suitable for large-scale industrial application.

Description

Technical Field

[0001] The present application relates to a method for extracting sterile extracellular vesicles, and belongs to the technical field of extracellular particle extraction. Background Art

[0002] Cell therapy based on mesenchymal stromal cells (MSCs) plays a pioneering and leading role in medical revolution in disease treatment and rehabilitation, especially in diseases that cannot be solved by existing medical means. MSCs are an excellent cell source for regenerative therapy, but the potential carcinogenicity, immune stimulation risk and low survival rate of transplanted MSCs have hindered the practical application of MSCs therapy. MSCs mainly exert their effects through the extracellular vesicles (EVs) they secrete. Studies have shown that EVs can replicate the beneficial effects of MSCs. Their advantages of low immunogenicity, low invasiveness and wide clinical applicability make them the first choice for cell-free therapy and have huge market potential and development prospects in regenerative medicine (Distinctive characteristics of extracellular vesicles from feline adipose and placentastromal cells unveil potential for regenerative medicine in cats, J Am Vet Med Assoc, 2024: 1-9).

[0003] EVs are particles with a lipid bilayer membrane structure released from cells. They cannot replicate on their own and can promote intercellular signaling. According to the diameter of the isolated particles, EVs with a diameter less than 200 nm are usually called small EVs (sEVs), and EVs with a diameter greater than 200 nm are called large EVs (lEVs). It is worth noting that "sEV" and "exosomes" are not synonymous. sEVs include exosomes and ectosomes smaller than 200 nm. The Extracellular Vesicle Society (ISEV) recommends the use of the general term "EV" and extensions of this term to replace inconsistent definitions and misleading terms such as exosomes and ectosomes (Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches, J Extracell Vesicles, 2024, 13 (2): e12404.).

[0004] Currently, researchers are increasingly aware that EVs samples separated by traditional methods are heterogeneous mixtures containing EVs and non-vesicular extracellular particles (NVEP) (Reassessment of Exosome Composition, Cell, 2019, 177(2): 428-445.e418). NVEPs are multimolecular assemblies released from cells without a lipid bilayer. NVEPs and EVs have overlapping physical and chemical properties. Previous studies have shown that several proteins marked in small EVs (sEVs) are also abundant in NVEPs. The amount of NVEPs in biological matrices may far exceed that of EVs. A wide range of NVEPs are often co-isolated with EVs. Therefore, when EVs cannot be distinguished from other potential co-isolated products, ISEV recommends the use of the term extracellular particles (EPs), that is, EPs include EVs and NVEPs (Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches, J Extracell Vesicles, 2024, 13(2): e12404.). EPs are cell-derived multimolecular assemblies in the nanometer to micrometer size range and are a general term for all extracellular particles.

[0005] Some of the existing equipment and kits for separating EVs strictly obtain EPs, which are co-isolated products of NVEP / EVs. That is, most of the current EPs separation methods will lead to the co-isolation of NVEP and EVs. Both EPs and EVs contain nucleic acids, proteins, lipids and metabolites, and are believed to be related to several key biological functions (Vesiclepedia 2024: an extracellular vesicles and extracellular particles repository, Nucleic Acids Res, 2024, 52 (D1): D1694-d1698.). In addition, some molecules co-isolated with EVs (such as EPs particles such as exomeres and supermeres) may not be considered as "contaminants" (Comprehensive isolation of extracellular vesicles and nanoparticles, Nat Protoc, 2023, 18(5): 1462-1487), but part of the EVs "corona". Removing corona by size exclusion or ultracentrifugation may result in the loss of its effect on angiogenesis (A functional corona around extracellular vesicles enhance sangiogenesis, skin regeneration and immunomodulation, J Extracell Vesicles, 2022, 11(4): e12207., Opportunities and challenges in studying the extracellular vesicle corona, Nat Cell Biol, 2022, 24(9): 1322-1325). The above studies have changed the requirements for high-purity EVs.

[0006] All types of cells cultured in vitro release EVs and other factors into their culture medium, thereby producing cell-conditioned medium (CCM). Currently, several separation methods for enriching EVs from CCM include differential (ultraspeed) centrifugation, density gradient centrifugation, size exclusion chromatography (SEC), fluid flow-based separation, and separation based on charge and molecular recognition (Minimalinformation for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches, J Extracell Vesicles, 2024, 13 (2): e12404.), all of which collect cell supernatant (or CCM) and then process it.

[0007] The characteristics of various EVs isolation methods in terms of recovery rate (yield) and specificity are as follows: Figure 1 As shown, the blue dotted arrow indicates a combination of methods that can increase specificity. P (precipitation) is precipitation, FC (Filter concention) is filtration concentration, SEC (Size exclusion chromatography) is size exclusion chromatography, dUC (differential ultracentrifugation) is differential (ultraspeed) centrifugation, AP (affinityprecipitation) is affinity precipitation, DG (Density gradients) is density gradient centrifugation; AF4 (Asymmetric flow field-flow fractionation) is asymmetric field flow separation, and IP (immuno-precipitation) is immunoprecipitation.

[0008] P (precipitation method) is to mix the sample with a precipitation solution containing a polymer for extraction. The most common polymer used for precipitation is polyethylene glycol (PEG). However, the mixing of polymer materials will affect downstream analysis, and there is a lack of selective separation mechanism, no specificity, and very low purity. Most of the separated products are also EPs mixtures.

[0009] FC (filtration concentration) has the characteristics of low equipment cost, short time consumption and easy operation, but the purity is not high, the sample is easily consumed and the filter membrane is easily clogged, which can easily shorten the life of the membrane and reduce the separation efficiency. In addition, due to the adhesion of the filter membrane, EVs will be consumed, resulting in reduced yield, and the pressure and shear force during filtration will also cause the EVs structure to deform and damage.

[0010] SEC (size exclusion chromatography) is a method of segmented separation according to molecular particle size. It passes biological samples through SEC fillers with fixed pores to adsorb molecules with smaller particle sizes such as proteins and lipids, while molecules or particles with larger particle sizes cannot enter the pores. Therefore, under the action of the buffer, vesicles with larger particle sizes flow faster and will be eluted into the sample collection tube at a specific stage to obtain experimental samples. The SEC method has moderate specificity and recovery rate, but all EVs types can be recovered together, the processing run time is long, and it is not suitable for processing large numbers of samples. In addition, the purified EVs are severely diluted and require additional enrichment methods. It also requires dedicated equipment, and the equipment cost is relatively high.

[0011] AP (affinity precipitation) is based on the affinity binding between the ligand and the target molecule. It uses centrifugation or filtration technology to recover the precipitate for separation. It has low selectivity and low efficiency. In addition, this method will also affect the activity of the protein.

[0012] DG (density gradient centrifugation) is a method for separating EVs by combining ultracentrifugation with sucrose, iodixanol or iodohexol density gradients. The EVs obtained by DG are of high purity, but the preliminary preparation work is cumbersome, exogenous substances need to be introduced, and the operation is complicated and very time-consuming. The sample processing capacity is also low, and the equipment requirements are high. There is also the problem of high-intensity centrifugal force causing mechanical damage to EVs.

[0013] AF4 (asymmetric field flow fractionation) uses pressure or electric field to screen out EVs from samples through a membrane. This method has a short separation time and can provide EVs of higher purity, but the recovery rate is low and requires special equipment, which is costly.

[0014] IP (immunoprecipitation) is a method that specifically binds to EVs through antibody-coated microspheres. It has the advantages of high purity, good specificity and repeatability, and does not affect the morphological integrity of EVs. However, the reagent cost is high, and a pre-confirmed EVs label (antigen) is required. In addition, the efficiency and recovery rate are low. It is limited to only capturing target EVs that express specific antigens, resulting in a low sample processing volume and therefore a low yield. It is not suitable for obtaining from a large number of samples. In addition, the elution step will destroy the original EVs structure. During the separation process, EVs are easily affected by pH and salt concentration, which is not conducive to downstream experiments and is difficult to be widely popularized.

[0015] The current mainstream method for extracting and isolating EVs is based on the differential (ultraspeed) centrifugation technology proposed by Professor Thery (Isolation and characterization of exosomes from cell culture supernatants and biological fluids, Curr Protoc Cell Biol, 2006, Chapter 3: Unit 3 22.), which is currently a classic and mature method for isolating EVs. This extraction method is simple, and no other markers are introduced during the extraction process. It is suitable for large-dose sample processing, but the operation is cumbersome, time-consuming and labor-intensive, and the recovery rate is unstable. Ultra-high-speed centrifugation is required during the operation, and due to the centrifugal force, the EVs structure may be destroyed, thereby affecting downstream experiments. It is also easy to cause aggregation of lipoproteins, etc., and the equipment requirements are high.

[0016] like Figure 2 As shown in the figure, the steps of its specific scheme are as follows: after inoculating the cells into the culture container, place them in an incubator for culture (if 10% FBS or platelet lysate is not used, EVs depletion supplement needs to be added; if 10% FBS is used, it needs to be replaced with serum-free culture medium 24 to 72 hours in advance when the cell confluence is about 70 to 80%), and then when the cell confluence reaches about 90%, collect the cell supernatant (or CCM), and then perform subsequent 4°C centrifugation treatments in sequence, i.e. 300g×10min, 2000g×10min, 10000g×30min, then discard the precipitate, then the supernatant is 100000g×70min, the precipitate is 100000g×70min, and then the precipitate is collected to obtain EVs.

[0017] Differential (ultraspeed) centrifugation is one of the most widely used and classic methods. However, it has the disadvantages of limited sample volume, time-consuming, and requiring professional and expensive equipment (A review on comparative studies addressing exosome isolation methods from body fluids, Anal Bioanal Chem, 2023, 415(7): 1239-1263). It can also cause structural changes in EVs, destroy EVs and affect their quality (A methodological primer of extracellular vesicles isolation and characterization via different techniques, Biol Methods Protoc, 2024, 9(1): bpae009.). In addition, the EVs isolated and extracted by the above method require a filtration and sterilization step to ensure sterility, but the filtration step will cause the loss of EVs.

[0018] Specifically, 1) the previous EVs isolation methods all used the initial volume of CCM for processing. The maximum volume of the classic differential (ultraspeed) centrifugation method is limited. When the volume of the processed sample exceeds the upper limit, the sample processing time doubles; 2) It is difficult to ensure the sterility of the sample during the differential (ultraspeed) centrifugation process, and filtration sterilization is required. Even if some centrifuge bottles can be sterilized, the entire centrifuge is in a non-sterile state. Similarly, several other separation methods also require filtration sterilization, and filtration will cause sample loss; 3) The high-speed shear force of differential (ultraspeed) centrifugation will destroy the EVs structure and affect product quality; 4) The differential (ultraspeed) centrifugation sample separation and extraction process is time-consuming, generally requiring 3 to 4 hours; 5) Most of the previous EVs isolation methods require expensive equipment and instruments, including ultracentrifuges; 6) In order to avoid the introduction of exogenous EVs, the serum (such as FBS) added to the culture medium for conventional cell culture needs to be replaced with EVs depleted supplements, or the culture medium containing 10% FBS or platelet lysate should be replaced with serum-free culture medium 24 hours in advance.

[0019] Based on the problems existing in the extraction methods of EVs in the prior art, it is necessary to provide a simple method for isolating sterile extracellular vesicles. Summary of the invention

[0020] In order to solve the above problems, a method for extracting sterile extracellular vesicles is provided. In this method, the cells are resuspended in PBS or saline, placed for treatment, and then centrifuged to obtain a PBS solution or saline containing extracellular vesicles. This method can solve the problems of upper limit of sample processing volume, long time consumption and expensive extraction equipment using differential (ultraspeed) centrifugation in the current prior art, and can also solve the problems of sterility and loss of the product. The method provided in this application has obvious industrial application advantages and is more conducive to clinical transformation applications.

[0021] The present application provides a method for extracting sterile extracellular vesicles, which includes placing the resuspended cells at 2 to 6° C. for 2 to 8 hours and then removing cells, dead cells, cell debris and lEVs.

[0022] Optionally, the resuspending cells includes the step of resuspending the cells using PBS or physiological saline.

[0023] In the prior art, EVs extracted from dUC can be resuspended in PBS or saline according to the later experimental conditions. For example, PBS buffer is used to resuspend samples for various experiments in general scientific research, but for clinical experiments, EVs need to be resuspended in saline for clinical research. Therefore, the supernatant finally obtained in the present application scheme is a PBS solution or saline solution containing EVs, which can be directly used in scientific research or clinical practice.

[0024] Optionally, the method for extracting the sterile extracellular vesicles comprises:

[0025] 1) Inoculating cells into a culture container, discarding the cell supernatant, digesting and collecting the cells;

[0026] 2) Resuspend cells;

[0027] 3) Place the resuspended cells at 2-6°C for 2-8 hours;

[0028] 4) Centrifuge at 2-8°C, 300±100g×10±5min, 2000±500g×20±10min, 10000±5000g×40±15min in sequence;

[0029] 5) discarding the precipitate and collecting the supernatant to obtain the sterile extracellular vesicles.

[0030] Optionally, in step 4), centrifugation is performed at 2-6°C, and treatments are performed at 300±50g×10±5min, 2000±300g×20±5min, and 10000±2000g×40±15min in sequence;

[0031] Optionally, in step 4), centrifugation is performed at 3-5°C, and treatments are performed at 300±30g×10±3min, 2000±200g×20±3min, and 10000±1000g×40±10min in sequence.

[0032] Optionally, in step 2), PBS or physiological saline is used to resuspend the cells.

[0033] Optionally, the amount of PBS or saline added in step 2) is 1 / 30 to 1 / 10 of the volume of the culture solution in step 1). If the volume is too small, the cells are crowded and not conducive to EVs secretion. If the volume is too large, the concentration is reduced and the amount of solution processed for subsequent purification is increased. It should be noted that if it is a clinical study, it is mainly to ensure that the maximum concentration ratio of the cells from which EVs are extracted is not greater than 1 / 15 when resuspended, and the minimum concentration ratio does not need to be strictly limited, as long as it does not exceed the volume of the individual infusion volume. This is because for clinical treatment research, the infused EVs are ultimately added to a 50ml or 100ml saline bag for infusion. Therefore, if the individual infusion volume is 50ml, the minimum dilution final volume does not exceed 50ml. Of course, for mass production and mass treatment applications, excessive volume will also be limited by the upper limit of the centrifuge, so the main indicator of concern is that the maximum concentration ratio is not greater than 1 / 15.

[0034] Optionally, in step 1), the culture medium in the culture container is a culture medium containing FBS.

[0035] It should be noted that, for the cell culture process, those skilled in the art can determine it according to the prior art, such as selecting different culture media for different types of cells, which is a routine method for those skilled in the art. For example, the culture medium commonly used in the prior art for mesenchymal stem cells is DMEM / F12 culture medium supplemented with FBS and penicillin-streptomycin.

[0036] Optionally, in step 1), the culture conditions include 37±2°C, 3-7% CO 2 .

[0037] The present application also provides sterile extracellular vesicles prepared by the above-mentioned method for extracting sterile extracellular vesicles.

[0038] The present application also provides a device for separating and extracting sterile extracellular vesicles, wherein the device adopts the above-mentioned method for extracting sterile extracellular vesicles to prepare sterile extracellular vesicles.

[0039] The beneficial effects of this application include but are not limited to:

[0040] 1. According to the method for extracting sterile extracellular vesicles of the present application, EVs are extracted by resuspending cells. Therefore, compared with the extraction of the supernatant in the prior art dUC, the initial volume of the extraction in the present application is significantly smaller, and the supernatant is finally retained, so there is no need for 100,000g centrifugation and concentration, no need for more expensive instruments and equipment, and the operation is convenient, which is more suitable for industrial applications.

[0041] 2. According to the method for extracting sterile extracellular vesicles of the present application, since ultracentrifugation is not required and the amount of liquid to be processed is greatly reduced, sterile centrifuge tubes can be used throughout the entire process of cell processing. However, dUC requires the use of centrifuge tubes / bottles that come with the machine. Even if the centrifuge tubes / bottles can be sterilized, the sterilization process is cumbersome, and the entire centrifuge cannot be guaranteed to be sterile. Currently, several separation methods for enriching EVs, such as ultracentrifugation, density gradient centrifugation, size exclusion chromatography, separation based on fluid flow, and separation based on charge and molecular recognition all require 0.22 μm filtration and sterilization, resulting in sample loss. The technology of the present application does not use ultracentrifugation equipment, and can keep the sample sterile throughout the entire process, so there is no filtration step and no loss.

[0042] 3. According to the method for extracting sterile extracellular vesicles of the present application, for cells cultured with EVs-depleted supplements or serum-free culture, some large-scale production companies on the market will discard this batch of cells, retain only CCM and then use existing technology equipment such as differential ultracentrifugation to conventionally extract EVs. However, using the technology of the present application, the batch of cells to be discarded can be used to extract EVs again, which is equivalent to conducting a batch of cell culture. Two different methods can be used to extract two batches of cells and supernatant, which can make full use of production resources.

[0043] 4. According to the method for extracting sterile extracellular vesicles of the present application, compared with the conventional collection of starvation culture, that is, replacing the serum-free medium with CCM at least 24 hours in advance, the present technology uses PBS or 0.9% NS without serum. After the cells are collected, they are not placed in an incubator, but the cells in the centrifuge tube are placed or gently shaken at 2 to 6°C. Under this condition, EVs secretion can be promoted, and CCM can be collected in 2 to 8 hours for downstream centrifugation processing, which is shorter and more convenient. For example, the centrifugation time of the present technology is about 70 minutes, and each centrifugation does not require a large-scale replacement of sterile centrifuge tubes, with less consumables loss, simple and easy, while the dUC centrifugation time is about 190 minutes, and each centrifugation in the early stage also requires a large-scale replacement of sterile centrifuge tubes, with more consumables loss, time-consuming and labor-intensive.

[0044] 5. According to the method for extracting sterile extracellular vesicles of the present application, the total amount of protein extracted by the present application technology is higher than that of dUC for the final product extracted from the same number of cells, which is 5 to 10 times that of dUC. Specifically, the culture supernatant of the same number of cells, such as 30ml, is concentrated according to 1 / 100 of the culture supernatant by the dUC method to finally obtain EVs with a final volume of 0.3ml, with a maximum protein concentration of about 0.2μg / μl and a total protein amount of 60μg; while the same cells are cultivated, the present technology is concentrated according to 1 / 15 of the cell culture supernatant such as 30ml to obtain EVs with a final volume of 2ml, with a minimum protein concentration of 0.25μg / μl and a total protein amount of up to 500μg. It can be seen that the total amount of protein obtained by this technology for extracting EVs from the same cultured cells is much higher than that of the dUC method.

[0045] 6. According to the method for extracting sterile extracellular vesicles of the present application, the maximum centrifugal force of this technology is significantly lower than that of dUC. In addition, the technology of the present application finally retains the supernatant rather than a solid precipitate, so there is no need to blow hard multiple times to promote the dissolution of the precipitate. The sample processing procedure in the present application scheme is gentler, and the EVs structure will not change due to shear force, and the integrity of EVs is better.

[0046] 7. According to the sterile extracellular vesicle extraction method of the present application, compared with several previous EVs separation methods that all use the initial volume of CCM for processing, this technology is to place the digested cells in a centrifuge tube and add about 1 / 15 of PBS or 0.9% saline. This technology can greatly reduce the volume of the initial CCM, so that the volume of the sample to be processed is reduced to about 1 / 15 of the initial CCM volume, which can significantly improve the extraction efficiency of EVs and the ease of operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0048] Figure 1 It is a method for extracting EVs in the prior art;

[0049] Figure 2 It is an operation flow chart of the EVs extraction method dUC in the prior art;

[0050] Figure 3 This is an operation flow chart of Example 1 of the present application;

[0051] Figure 4 This is a result diagram of the particle size and particle concentration of EVs extracted by the present technology and dUC involved in Test Example 2 of this application;

[0052] Figure 5 This is the ultrastructural morphology of EVs of the present technology and dUC involved in Test Example 3 of this application;

[0053] Figure 6 This is a graph showing the analysis results of EVs phenotypic characteristics extracted by two techniques involved in Test Example 4 of this application;

[0054] Figure 7 The bar graph and pie graph are the result graphs of the subcellular localization of GO involved in Test Example 5 of the present application;

[0055] Figure 8 This is a graph of GO clustering analysis results involved in Test Example 5 of this application;

[0056] Fig. 9 This is a graph showing the results of the GO second-layer functional clustering analysis involved in Test Example 5 of this application;

[0057] Fig.10 This is the result graph of KEGG significance Top20 involved in Test Example 5 of this application;

[0058] Fig.11 This is the result diagram of the WikiPathway significance Top 10 involved in Test Example 5 of this application;

[0059] Fig.12 This is a graph showing the results of the technology and EVs extracted by dUC promoting cell migration involved in Test Example 6 of this application;

[0060] Fig.13 This is a graph showing the protein concentration of EVs extracted at each time point involved in Experimental Example 1 of this application;

[0061] Fig.14 This is a graph showing the results of the sterility culture of EVs involved in Test Example 7 of this application;

[0062] Fig.15 This is a result diagram of EVs promoting reticular blood vessel formation involved in Test Example 8 of the present application;

[0063] Fig.16 This is a graph showing the expression results of factors related to angiogenesis and proliferation promoted by EVs involved in Test Example 9 of this application;

[0064] Fig.17 This is a result diagram of the inhibitory effect of EVs on inflammatory factors involved in Test Example 10 of this application;

[0065] Fig.18 This is a graph showing the expression results of factors related to EVs promoting cell adhesion and cell migration involved in Test Example 11 of this application;

[0066] Fig.19 This is a result diagram of the particle size and particle concentration of other different cell EVs involved in Test Example 13 of this application;

[0067] Fig. 20 This is a diagram showing the ultrastructural results of EVs from other different cells involved in Test Example 13 of this application;

[0068] Fig.21 This is a graph showing the phenotypic characteristics of other different cell EVs involved in Test Example 14 of this application. DETAILED DESCRIPTION

[0069] The present application is described in detail below in conjunction with examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and reagents in the examples of the present application are purchased through commercial channels.

[0070] The present application scheme is described below through specific embodiments.

[0071] Example 1

[0072] like Figure 3 FIG. 1 is an operation flow chart of this embodiment, and the specific operation steps include:

[0073] 1) Cell culture

[0074] In this embodiment, the cells are collected and separated human umbilical cord mesenchymal stromal cells hUC-MSC. The mother and her family were informed and consented before the collection. At the same time, the detection of pathogens such as hepatitis B and AIDS was negative. The experimental plan was approved by the hospital ethics committee. The fetal umbilical cord of 15-20 cm from a healthy mother by cesarean section was collected aseptically, placed in a sealed bottle pre-placed with PBS (containing 2% penicillin + 2% streptomycin) and brought back to the laboratory. In the laboratory clean bench, the umbilical cord was cut into 2-3 cm fragments, cut open longitudinally, and repeatedly rinsed with sterile PBS until the liquid was free of blood stains. The arteries and veins were separated, the outer layer of the umbilical cord amniotic membrane was peeled off, and the Whartongel tissue was separated. After repeated rinsing with PBS, the Whartongel was cut into 1mm×1mm×1mm tissue pieces with tissue scissors and evenly spread in a 10mm culture dish, and a small amount of DMEM / F12 culture medium (containing 10% fetal bovine serum and 1% penicillin + 1% streptomycin) was added. The culture was incubated at 37°C and 5% CO 2 The cells were cultured in an incubator, and after they were fully grown, they were digested and passaged with 0.25% trypsin. The cells of the first passage were recorded as P1.

[0075] 2) Obtaining isolated cells

[0076] Cells from passages P4 to P6 were selected for EV extraction. hUC-MSC cells were cultured in DMEM / F12 medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO. 2Culture in a cell culture incubator until the cell confluence reaches about 90%. Remove the cell culture medium and wash the cells 1 to 2 times with sterile PBS buffer. Add 0.25% trypsin digestion solution to slightly cover the cells and place at room temperature for 30s to 2min. The digestion time for different cells is slightly different and can be determined by observation under a microscope. For example, when the adherent cells become round under a microscope, or when the bottle wall changes from translucent to transparent and dots with the naked eye, that is, small gaps appear, trypsin can be removed and serum-containing cell culture medium can be added to terminate digestion. Then, the cell suspension is centrifuged at 300g×10min to remove the supernatant, and then PBS is added to resuspend the cells. The cells are washed by centrifugation at 300g×10min again, and the supernatant is removed to obtain separated cells.

[0077] 3) Reduce the initial CCM

[0078] The separated cells were placed in a centrifuge tube, and then the cells in the centrifuge tube were resuspended with PBS;

[0079] 4) Promote EVs secretion

[0080] Place the cells in the centrifuge tube that have been resuspended in PBS on a horizontal shaker at 100 rpm and 4°C for 4 h;

[0081] 5) Centrifugation to remove cells, dead cells, cell debris and lEV

[0082] Centrifugation at 4°C, 300 g × 10 min, 2000 g × 20 min, 10000 g × 40 min;

[0083] 6) Collect EVs

[0084] After discarding the precipitate, the supernatant was collected to obtain EVs.

[0085] Example 2

[0086] 1) Cell culture

[0087] In this embodiment, the cells are collected and separated human umbilical cord mesenchymal stromal cells hUC-MSC. Before sampling, the informed consent of the parturient and her family was obtained, and the tests for pathogens such as hepatitis B and AIDS were all negative. The experimental plan was approved by the hospital ethics committee. The umbilical cord of 15 to 20 cm of the fetus from a healthy parturient by cesarean section was collected aseptically, placed in a sealed bottle pre-placed with PBS (containing 2% penicillin + 2% streptomycin) and brought back to the laboratory. In the laboratory clean bench, the umbilical cord was cut into 2 to 3 cm fragments, cut open longitudinally, and repeatedly rinsed with sterile PBS until the liquid was free of blood stains. The arteries and veins were separated, the outer layer of the umbilical cord amniotic membrane was peeled off, and the Whartongel tissue was separated. After repeated rinsing with PBS, the Whartongel was cut into 1mm×1mm×1mm tissue pieces with tissue scissors and evenly spread in a 10mm culture dish, and a small amount of DMEM / F12 culture medium (containing 10% fetal bovine serum and 1% penicillin + 1% streptomycin) was added. The culture was incubated at 37°C and 5% CO 2 The cells were cultured in an incubator, and after they were fully grown, they were digested and passaged with 0.25% trypsin. The cells of the first passage were recorded as P1.

[0088] 2) Obtaining isolated cells

[0089] Cells from passages P4 to P6 were selected for EV extraction. hUC-MSC cells were cultured in DMEM / F12 medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO. 2 Culture in a cell culture incubator until the cell confluence reaches about 90%. Remove the cell culture medium and wash the cells 1 to 2 times with sterile PBS buffer. Add 0.25% trypsin digestion solution to slightly cover the cells and place at room temperature for 30s to 2min. The digestion time for different cells is slightly different and can be determined by observation under a microscope. For example, when the adherent cells become round under a microscope, or when the bottle wall changes from translucent to transparent and dots with small gaps appear, remove the trypsin and add serum-containing cell culture medium to terminate the digestion. Then centrifuge the cell suspension at 300g×10min to remove the supernatant, then continue to add PBS to resuspend the cells, centrifuge and wash the cells again at 300g×10min, remove the supernatant, and obtain separated cells;

[0090] 3) Reduce the initial CCM

[0091] The separated cells were placed in a centrifuge tube, and then the cells in the centrifuge tube were resuspended with 0.9% NS;

[0092] 4) Promote EVs secretion

[0093] The cells in the centrifuge tube that had been resuspended in 0.9% NS were placed on a horizontal shaker at 200 rpm and 8°C for 2 h;

[0094] 5) Centrifugation to remove cells, dead cells, cell debris and lEV

[0095] Centrifugation at 8°C, 400 g for 5 min, 2500 g for 10 min, and 15000 g for 25 min;

[0096] 6) Collect EVs

[0097] After discarding the precipitate, the supernatant was collected to obtain EVs.

[0098] Example 3

[0099] 1) Cell culture

[0100] In this embodiment, the cells are collected and separated human umbilical cord mesenchymal stromal cells hUC-MSC. Before sampling, the informed consent of the parturient and her family was obtained, and the tests for pathogens such as hepatitis B and AIDS were all negative. The experimental plan was approved by the hospital ethics committee. The umbilical cord of 15 to 20 cm of the fetus from a healthy parturient by cesarean section was collected aseptically, placed in a sealed bottle pre-placed with PBS (containing 2% penicillin + 2% streptomycin) and brought back to the laboratory. In the laboratory clean bench, the umbilical cord was cut into 2 to 3 cm fragments, cut open longitudinally, and repeatedly rinsed with sterile PBS until the liquid was free of blood stains. The arteries and veins were separated, the outer layer of the umbilical cord amniotic membrane was peeled off, and the Whartongel tissue was separated. After repeated rinsing with PBS, the Whartongel was cut into 1mm×1mm×1mm tissue pieces with tissue scissors and evenly spread in a 10mm culture dish, and a small amount of DMEM / F12 culture medium (containing 10% fetal bovine serum and 1% penicillin + 1% streptomycin) was added. The culture was incubated at 37°C and 5% CO 2 The cells were cultured in an incubator, and after they were fully grown, they were digested and passaged with 0.25% trypsin. The cells of the first passage were recorded as P1.

[0101] 2) Obtaining isolated cells

[0102] Cells from passages P4 to P6 were selected for EV extraction. hUC-MSC cells were cultured in DMEM / F12 medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO. 2Culture in a cell culture incubator until the cell confluence reaches about 90%. Remove the cell culture medium and wash the cells 1 to 2 times with sterile PBS buffer. Add 0.25% trypsin digestion solution to slightly cover the cells and place at room temperature for 30s to 2min. The digestion time for different cells is slightly different and can be determined by observation under a microscope. For example, when the adherent cells become round under a microscope, or when the bottle wall changes from translucent to transparent and dots with small gaps appear, remove the trypsin and add serum-containing cell culture medium to terminate digestion. Then centrifuge the cell suspension at 300g×10min to remove the supernatant, then continue to add PBS to resuspend the cells, centrifuge again at 300g×10min, remove the supernatant, and obtain separated cells;

[0103] 3) Reduce the initial CCM

[0104] The separated cells were placed in a centrifuge tube, and then the cells in the centrifuge tube were resuspended with PBS;

[0105] 4) Promote EVs secretion

[0106] The cells in the centrifuge tube that had been resuspended in PBS were placed at 4°C for 8 h;

[0107] 5) Centrifugation to remove cells, dead cells, cell debris and lEV

[0108] Centrifugation at 2°C, 200 g for 15 min, 1500 g for 30 min, and 5000 g for 55 min;

[0109] 6) Collect EVs

[0110] After discarding the precipitate, the supernatant was collected to obtain EVs.

[0111] Example 4

[0112] 1) Cell culture

[0113] Human pulmonary artery endothelial cells (HPAEC, purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd.) were selected for EV extraction. HPAEC cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO. 2Culture in a cell culture incubator until the cell confluence reaches about 90%. Remove the cell culture medium and wash the cells 1 to 2 times with sterile PBS buffer. Add 0.25% trypsin digestion solution to slightly cover the cells and place at room temperature for 30s to 2min. The digestion time for different cells is slightly different and can be determined by observation under a microscope. For example, when the adherent cells become round under a microscope, or when the bottle wall changes from translucent to transparent and dots with small gaps appear, remove the trypsin and add serum-containing cell culture medium to terminate digestion. Then centrifuge the cell suspension at 300g×10min to remove the supernatant, then continue to add PBS to resuspend the cells, centrifuge again at 300g×10min, remove the supernatant, and obtain separated cells;

[0114] 2) Reduce the initial CCM

[0115] The separated cells were placed in a centrifuge tube, and then the cells in the centrifuge tube were resuspended with PBS;

[0116] 3) Promote EVs secretion

[0117] Place the cells in the centrifuge tube that have been resuspended in PBS on a horizontal shaker at 100 rpm and 4°C for 4 h;

[0118] 4) Centrifugation to remove cells, dead cells, cell debris and lEV

[0119] Centrifugation at 4°C, 300 g × 10 min, 2000 g × 20 min, 10000 g × 40 min;

[0120] 5) Collect EVs

[0121] After discarding the precipitate, the supernatant was collected to obtain EVs.

[0122] Example 5

[0123] 1) Cell culture

[0124] Human breast cancer cells (SK-Br3, purchased from Peking Union Medical College Cell Resource Center) were selected for EVs extraction. SK-Br3 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO. 2Culture in a cell culture incubator until the cell confluence reaches about 90%. Remove the cell culture medium and wash the cells 1 to 2 times with sterile PBS buffer. Add 0.25% trypsin digestion solution to slightly cover the cells and place at room temperature for 30s to 2min. The digestion time for different cells is slightly different and can be determined by observation under a microscope. For example, when the adherent cells become round under a microscope, or when the bottle wall changes from translucent to transparent and dots with small gaps appear, remove the trypsin and add serum-containing cell culture medium to terminate digestion. Then centrifuge the cell suspension at 300g×10min to remove the supernatant, then continue to add PBS to resuspend the cells, centrifuge again at 300g×10min, remove the supernatant, and obtain separated cells;

[0125] 2) Reduce the initial CCM

[0126] The separated cells were placed in a centrifuge tube, and then the cells in the centrifuge tube were resuspended with PBS;

[0127] 3) Promote EVs secretion

[0128] Place the cells in the centrifuge tube that have been resuspended in PBS on a horizontal shaker at 100 rpm and 4°C for 4 h;

[0129] 4) Centrifugation to remove cells, dead cells, cell debris and lEV

[0130] Centrifugation at 4°C, 300 g × 10 min, 2000 g × 20 min, 10000 g × 40 min;

[0131] 5) Collect EVs

[0132] After discarding the precipitate, the supernatant was collected to obtain EVs.

[0133] Example 6

[0134] 1) Cell culture

[0135] Human embryonic kidney cells (293T, purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were selected for EVs extraction. 293T cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO. 2Culture in a cell culture incubator until the cell confluence reaches about 90%. Remove the cell culture medium and wash the cells 1 to 2 times with sterile PBS buffer. Add 0.25% trypsin digestion solution to slightly cover the cells and place at room temperature for 30s to 2min. The digestion time for different cells is slightly different and can be determined by observation under a microscope. For example, when the adherent cells become round under a microscope, or when the bottle wall changes from translucent to transparent and dots with small gaps appear, remove the trypsin and add serum-containing cell culture medium to terminate digestion. Then centrifuge the cell suspension at 300g×10min to remove the supernatant, then continue to add PBS to resuspend the cells, centrifuge again at 300g×10min, remove the supernatant, and obtain separated cells;

[0136] 2) Reduce the initial CCM

[0137] The separated cells were placed in a centrifuge tube, and then the cells in the centrifuge tube were resuspended with PBS;

[0138] 3) Promote EVs secretion

[0139] Place the cells in the centrifuge tube that have been resuspended in PBS on a horizontal shaker at 100 rpm and 4°C for 4 h;

[0140] 4) Centrifugation to remove cells, dead cells, cell debris and lEV

[0141] Centrifugation at 4°C, 300 g × 10 min, 2000 g × 20 min, 10000 g × 40 min;

[0142] 5) Collect EVs

[0143] After discarding the precipitate, the supernatant was collected to obtain EVs.

[0144] Comparative Example 1

[0145] like Figure 2 As shown in the figure, it is a traditional differential (ultra) centrifugation technology (dUC), using the human umbilical cord mesenchymal stromal cells hUC-MSC separated and extracted above, and then inoculating the mesenchymal stromal cells into the culture container, and then placing it in an incubator for culture (if 10% FBS is not used, EVs depletion supplement needs to be added; if 10% FBS or platelet lysate is used, it needs to be replaced with serum-free culture medium 24 to 72 hours in advance when the cell confluence is about 70 to 80%), and then when the cell confluence reaches about 90%, the cell supernatant or CCM is collected, and then subsequent 4°C centrifugation treatments are performed in sequence, that is, 300g×10min, 2000g×10min, 10000g×30min, and then the precipitate is discarded, and then the supernatant is 100000g×70min, and after discarding the supernatant, the precipitate is 100000g×70min, and finally the precipitate is collected to obtain EVs.

[0146] Test Example 1

[0147] The difference in protein concentration of EVs extracted by this protocol and traditional differential (ultra) centrifugation (dUC) technology was analyzed, and the results are shown in the table below.

[0148] Among them, sample A is EVs extracted by the technology of this scheme; sample B is the cell supernatant collected after replacing the serum-free medium 24h to 72h in advance, and then EVs extracted by dUC; and sample C is based on the extraction process of sample B, collecting the unused cells after dUC extraction of cell supernatant, and then using the cells and the technical method of this application to extract EVs. Sample C is extracted by this technology, which is used to verify the feasibility of integrating the technology of this application into the dUC extraction process in the current existing technology.

[0149] Table 1 Comparison of protein concentration extracted by this protocol and dUC

[0150] Grouping Concentration ratio Protein concentration (μg / μl) Sample A 1:15 0.3348±0.033 Sample B 1:100 0.1335±0.017 Sample C 1:15 0.2350±0.026

[0151] According to the results in the above table, compared with the traditional dUC technology, even if the protein concentrations of samples A and C are extracted at a lower concentration ratio, the protein concentrations of sample A and sample C are higher than the protein concentration of sample B extracted by dUC.

[0152] The results of sample C also show that for those who have previously purchased extraction equipment using the dUC method, the technology of the present application can be added to achieve a batch of cell culture and two extractions using dUC and the present technology at the same time, that is, the same batch of cells can use dUC to extract sample B from the cell supernatant, and the technology of the present application can also be used to extract sample C from the cells. Although the protein concentration of the extracted sample C is slightly lower than that of sample A, it is still higher than the concentration of sample B extracted using traditional technology.

[0153] Test Example 2

[0154] Nano FCM was used to analyze the particle size and concentration of EVs extracted by this protocol and dUC. The results are as follows Figure 4 As shown. Figure 4 A and B are freeze-dried samples with the same number of cells. The testing company is Shenzhen Huixin Biomedical Technology Co., Ltd. Figure 4 C and D in the middle are non-lyophilized samples with the same cell number. The testing company is Shandong Jiehelix Biotechnology Co., Ltd.

[0155] The results showed that under the condition that the number of cells for extracting EVs was basically the same (the number of cells in A and B was the same, the number of cells in C and D was the same, and the number of cells in A / B was twice the number of cells in C / D), the particle size range of EVs detected by two different testing companies was within 30-150nm, and the concentration of EVs particles extracted by this technology was higher than that of EVs particles extracted by dUC; and the average particle size of EVs extracted by this technology was smaller than that of EVs extracted by dUC technology.

[0156] It should be noted that the particle size of the EVs samples extracted by the two technologies increased after freeze-drying, and the concentration of freeze-dried EVs particles was seriously reduced. Therefore, it is not recommended to use freeze-dried samples for subsequent experiments or storage of EVs products. In addition, compared with the D sample (1:100 concentration) extracted by dUC, the particle concentration of the C sample (1:15 concentration) extracted by this technology is also significantly higher than that of the D sample.

[0157] By comparing and analyzing the measurements of EVs particle size and concentration under different cell amounts, different concentration ratios, and different storage conditions by different testing companies, the results showed that the particle concentration of EVs extracted by this technology was higher than that of EVs extracted by dUC technology.

[0158] Test Example 3

[0159] The morphology of EVs extracted by this protocol and traditional dUC was analyzed. Specifically, the morphology of EVs extracted by this technology (concentration ratio 1:15) and dUC extraction (concentration ratio 1:300) was analyzed using transmission electron microscopy. The results are as follows Figure 5 shown.

[0160] According to the results of the pictures, there is no obvious difference in morphology between the two. Both show a saucer-like structure and the particle size is less than 200nm.

[0161] Test Example 4

[0162] The phenotypes of EVs extracted by this technology and traditional dUC were analyzed. Specifically, the MACSplex kit was used to analyze the expression of CD9, CD63, and CD81 in EVs by flow cytometry; Western-blot was used to analyze the protein expression of TSG101, HSP70, etc. The results are shown in Figure 6 As shown, FCM in A analyzed the protein expression of CD9, CD63, and CD81 in EVs, and Western-blot in B analyzed the protein expression of CD63, CD81, TSG101, HSP70, and calnexin.

[0163] According to the results in the picture, the phenotypic analysis results of EVs extracted by this method (concentration ratio 1:15) and dUC (concentration ratio 1:300) showed that the EVs extracted by the two technologies were positive for CD9, CD63, CD81, TSG101 and HSP70, and negative for calnexin, which met the ISEV2023 standards.

[0164] Test Example 5

[0165] The clustering characteristics of EVs extracted by this technology and dUC were analyzed by sequencing. Specifically, proteomics sequencing was used to analyze the GO, KEGG and WikiPathway of EVs extracted by this technology and dUC. The number of cells cultured for EVs extracted by dUC was twice that of this technology to ensure the minimum protein content required for sequencing.

[0166] The GO database describes genes and gene products from three aspects: biological process (BP), molecular function (MF) and cellular component (CC). These three aspects are the first level (level 1) of GO annotation. Each branch is further divided into more specific aspects, namely level 2, level 3, etc. The results of this study include the first level (level 1) and second level (level 2) of GO annotation.

[0167] like Figure 7 As shown, A and B are the results of this technology, C and D are the results of dUC. According to the GO subcellular localization annotation statistics, the subcellular localization of EVs extracted by this technology and dUC is completely consistent, and the protein clustering in EVs is most highly distributed in the cytoplasm and plasma membrane. The number of proteins in the subcellular clustering of this technology is actually significantly higher than that of EVs extracted by dUC.

[0168] like Figure 8As shown, A, B and C are the results of this technology, D, E and F are the results of dUC, the horizontal axis in the figure represents the negative logarithmic transformation of p-value, the color of the circle represents the enrichment factor (rich factor), and the size of the circle represents the number (count). The GO cluster analysis Top10 or Top20 enrichment results show that the clustering of EVs extracted by this technology and dUC in terms of cellular components (CC, Cellular Component), molecular functions (MF, Molecular Function) and biological processes (BP, Biological Process) is basically consistent. Among them, EVs are completely consistent in the Top10 clustering of cellular components, and the main components are extracellular vesicles, vesicles, exosomes, organelles, etc. In terms of molecular functions, they are mainly involved in the binding of proteins, RNA, cadherins and cell adhesion molecules.

[0169] like Fig. 9 As shown in the figure, the horizontal axis is the aspect of level 2, the vertical axis on the left is the count, which is the number of proteins annotated to this aspect, and the vertical axis on the right is the percentage, which is the ratio of the number of proteins annotated to this term to the total number of proteins with GO annotations. The analysis of the results of GO second-level functional clustering annotation showed that the clustering of EVs extracted by this technology and dUC in terms of biological process (BP), cellular component (CC) and molecular function (MF) was completely consistent. The number of proteins clustered in each component of EVs actually separated and extracted by this technology was higher than that of EVs.

[0170] like Fig.10 As shown in the figure, the KEGG (Kyoto Encyclopedia of Genes and Genomes) database is used to understand the advanced functions and uses of cells, organisms, and ecosystems from information at the molecular level. The bubble chart shows the top 20 pathways with the highest significance. The horizontal axis represents the negative logarithmic transformation of the p-value, the circle color represents the enrichment factor, and the circle size represents the number. The results of the top 20 pathways of the KEGG clustering show that the pathways of the KEGG clustering of EVs extracted by this technology and dUC are mostly the same.

[0171] like Fig.11As shown, the results of the top 10 pathways of WikiPathway clustering show that the pathways of the EVs clustering extracted by this technology and dUC are mostly the same, and the number of proteins clustered by this technology is higher than that of the dUC group. Some different clustering is due to the fact that the EVs protein clustering extracted by dUC is not among the top 10, and the number of clustered proteins is lower than that of this technology group. The VEGFA-VEGFR2 signaling pathway, which is closely related to angiogenesis, is at the top of the clusters in both this technology and the dUC group.

[0172] In summary, the morphology and particle size of EVs extracted by this scheme are consistent with those of EVs extracted by differential (ultraspeed) centrifugation. In addition, the particle and protein concentration extracted by this technology is higher than that of dUC.

[0173] Test Example 6

[0174] The effects of this technique and EVs extracted from dUC on the lateral and longitudinal migration of cells were studied by scratch and transwell migration experiments. Fig.12 shown.

[0175] According to the results in the picture, compared with the Control group, EVs extracted by this technology and dUC can promote cell migration, and the ability of EVs extracted by this technology to promote cell migration is higher than that of the dUC group.

[0176] Test Example 7

[0177] The final product of this application does not need to be sterilized and filtered at 0.22 μm for sterility testing. Specifically, the EVs sample extracted by this technology is inoculated in a Columbia blood plate culture dish and incubated at 37°C and 5% CO 2 Incubate in an incubator for 72 hours and determine whether the sample is contaminated based on bacterial growth. Fig.14 shown.

[0178] according to Fig.14 The results show that no colonies grew in the blood plate culture dish after 72 hours of culture, proving that the EVs extracted by this technology are sterile. This technology does not require 0.22μm sterile filtration, and there will be no product loss due to sterile filtration.

[0179] Test Case 8

[0180] Using Matrigel TM Matrigel was used to study the effects of EVs extracted by this technique and dUC on HUVEC cell angiogenesis. The results are shown in Fig.15 shown.

[0181] according to Fig.15The results showed that compared with the control group, the addition of EVs extracted by this technology and dUC could promote HPAEC cells to form vascular cavity-like reticular structures, as shown by the total area, number and number of intersections of the tubes being significantly higher than those in the control group.

[0182] Test Example 9

[0183] qPCR and western-blot were used to study the effects of EVs extracted by this technique and dUC on angiogenesis and proliferation-related factors. The results are as follows Fig.16 As shown, *p<0.05, **p<0.01 vs.Control.

[0184] according to Fig.16 The results showed that compared with the Control group, EVs extracted by this technology and dUC could promote the mRNA expression of CD105 and TGFB, which was consistent with the results of WikiPathway cluster analysis in sequencing. In addition, the expression of CD105 in EVs extracted by this technology was significantly higher than that in the dUC extraction group; there was no significant difference in the expression of TGFB in EVs extracted by the two different methods, proving that EVs can activate signaling pathways closely related to angiogenesis.

[0185] Test Example 10

[0186] Many previous literature reports have shown that EVs extracted from dUC have anti-inflammatory effects. The EVs extracted by this technology have also been tested to have anti-inflammatory effects. Fig.17 As shown, *p<0.05, **p<0.01 vs. LPS group.

[0187] according to Fig.17 The results showed that compared with the control group, LPS (2 μg / ml) could significantly induce the expression of inflammatory factors TNF-α, IL-6, and IL-1β in HPAEC cells. When treated with EVs (20 μg / ml) extracted by this technology, the expression of inflammatory factors in HPAEC cells induced by LPS could be significantly inhibited.

[0188] Test Example 11

[0189] EVs carry a large amount of information and macromolecules from their source, easily fuse with the cell membranes of neighboring cells, and can cross biological barriers to mediate intercellular information transmission of bioactive molecules. They play an important role in cell-cell communication, and cell adhesion and cell migration play an important role in this process.

[0190] After testing, it was found that EVs extracted by this technology can promote the expression of factors related to cell adhesion (ITGA5, ITGB1, E-cadherin) and cell migration (MMP2), and contribute to signal transduction and interaction between cells. Fig.18 shown.

[0191] Experimental Example 1

[0192] The cell activity at different time points and the protein concentration of extracellular vesicles EVs extracted using this technology were analyzed to determine the optimal time for collecting and extracting EVs. Specifically, the cells were collected by digestion, resuspended in PBS, and then placed at 4°C. The cell activity was tested at 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, and 8h. The results are shown in the following table and Fig.13 shown.

[0193] Table 2 Effects of different time points on cell activity and protein concentration

[0194] Grouping Protein concentration (μg / μl) Dead cells (%) 0.5h 0.2245±0.0155 0％ 1h 0.2394±0.0199* 0％ 2h 0.2661±0.017** 0％ 3h 0.2997±0.0154** 0％ 4h 0.3132±0.0143** 0％ 5h 0.283±0.0514** 0％ 6h 0.2342±0.0228 2.83％±0.0073 8h 0.2833±0.0856* 3.15％±0.0021

[0195] Note: *p<0.05, **p<0.01 vs.0.5h

[0196] According to the results in the table and pictures above, there were no dead cells in each group from 0.5h to 5h, and dead cells began to appear from 6h. The number of dead cells could be kept low from 6h to 8h. The protein concentration increased at 8h compared with 6h, which was mainly caused by the release of EVs by the rupture of dead cells. Compared with 8h and 4h, 4h not only ensured 0% dead cells, but also more EVs secreted by simple cells, and the processing time was halved, with higher efficiency.

[0197] In addition, by detecting the protein concentration of EVs extracted at each time point, it was found that the protein content was the highest when placed at 4°C for 4 hours. Therefore, in the actual operation process, the cells can be placed at 4°C for 4 hours for treatment. Based on the ideas of this application scheme, technicians in this field can explore and adjust for different cell types to ensure a lower cell mortality rate and a higher protein content. At present, the application time of domestic cell therapy products is generally no more than 12 hours from the preparation of fresh cell preparations to the reinfusion treatment at 4°C. In view of this, the operating conditions of this technical method can ensure cell activity, and there is basically no or very little cell death.

[0198] Experimental Example 2

[0199] The experimenters also optimized the optimal volume of the initial cell-conditioned medium (CCM) by using this technology. Specifically, the initial CCM volume was reduced to 1 / 30, 1 / 15 and 1 / 10. The effects of different resuspension volumes on protein concentration are shown in the following table.

[0200] Table 3 Effect of different resuspension volumes on protein concentration

[0201] Grouping Protein concentration (μg / μl) Dead cells (%) 1 / 30 volume 0.4657±0.0053 0％ 1 / 15 volume 0.2627±0.0026 0％ 1 / 10 volume 0.1379±0.0105 0％

[0202] According to the results in the table above, the protein concentration of EVs increases with the increase of the concentration ratio. However, although the protein concentration of the highest concentration ratio (1 / 30 volume group) is the highest, it is not doubled compared with the 1 / 15 volume group, and the protein concentration of the 1 / 15 concentration ratio group is not doubled compared with the 1 / 30 volume group. In addition, compared with the 1 / 30 volume group and the 1 / 15 volume group, the protein concentration of the 1 / 10 volume group is greatly reduced. Therefore, in the actual operation process, the initial CCM can be reduced by 1 / 15, and the protein extraction effect is better than that of the 1 / 10 ratio.

[0203] It should be noted that, based on the inventive concept of the present application, those skilled in the art can use similar methods to explore the optimal volume of the appropriate reduced initial cell conditioned medium (CCM) for different types of cells or specific cells, or select other suitable resuspension volumes according to the ideas of the present application, and both can achieve better EVs extraction effects. Those skilled in the art can also select a suitable resuspension volume according to the requirements for protein concentration.

[0204] Experimental Example 3

[0205] In this application, based on the idea that cells may still secrete EVs under non-culture conditions, the inventors try to verify whether it is possible to place cells under non-culture conditions, such as in two solvents commonly used in experimental research or clinical practice, namely PBS or saline, so that cells can secrete EVs under these conditions, so that PBS or saline solution containing EVs can be directly obtained after the cells are separated, and the purification method is simpler. However, whether this idea is feasible, how to extract, and how much to extract are still to be studied and verified.

[0206] In particular, based on the general understanding of the current state of the art, it is unclear whether cells can secrete EVs or even secrete in amounts that are valuable for purification when placed in non-culture conditions, and there is currently no research. In this application, it is demonstrated that cells can still secrete EVs in PBS or saline solution, and the extraction is less difficult and the extracted EVs concentration is higher.

[0207] In order to increase the concentration of EVs as much as possible, the experimenters found through experimental research that adding a certain amount of mechanical stress can promote the increase of EVs protein concentration. The protein concentrations of gentle shaking and static state at 4°C were specifically compared. The results are shown in the table below.

[0208] Table 4 Effect of mechanical stress on protein concentration

[0209] Grouping Protein concentration (μg / μl) Gently shake (horizontal shaker 100rpm) 0.3348±0.033* Stand 0.2641±0.036

[0210] Note: *p<0.05vs. static

[0211] According to the results in the table above, good protein concentration results can be obtained regardless of gentle shaking or static state. By comparing the EVs protein content extracted from cells under mechanical stress or static state, it can be seen that the protein concentration of EVs can be further increased by adding a certain amount of mechanical stress.

[0212] It should be noted that those skilled in the art can make adjustments based on the concept of the present application. For example, standing still itself can achieve relatively good results. Those skilled in the art can also further adjust and determine the gentle shaking method based on needs and specific circumstances, combined with the method of increasing protein concentration by increasing mechanical stress.

[0213] Test Example 12

[0214] As described in Examples 4 to 6, the experimenters also used this technology to extract EVs from human pulmonary artery endothelial cells (HPAEC), human breast cancer cells (SK-Br3) and human embryonic kidney cells (293T). In view of the different growth rates and EVs secretion efficiencies of various cells, 0.4 ml (the concentration ratio is approximately 1 / 12 to 1 / 15) of PBS was added to each 1×10^6 cell and resuspended, and EVs were extracted by gently shaking at 4°C for 4 hours. The EVs protein concentration results are shown in the following table.

[0215] Table 5 Protein concentration of cell-derived EVs (μg / μl)

[0216] Grouping mean±SD HPAEC 0.2130±0.0053 sk-Br3 0.2239±0.0068 293T 0.6521±0.0340

[0217] According to the results in the above table, based on the common characteristics of vesicle secretion in each cell, the extraction scheme provided in this application is universal and suitable for extracting EVs from different cells. Specifically, with respect to the results in the above table, it can be seen that EVs from various cell sources can be effectively obtained by using this technology, and the protein concentration from 293T is higher. This is because 293T cells are a high-yield protein expression system in scientific research, and their high protein concentration has a certain relationship with their rapid proliferation characteristics.

[0218] Test Example 13

[0219] NanoFCM was also used to analyze the particle size and particle concentration (partical / ml) of EVs extracted from human pulmonary artery endothelial cells (HPAEC), human breast cancer cells (SK-Br3), and human embryonic kidney cells (293T). The results are as follows: Fig.19 In addition, the ultrastructure of human pulmonary artery endothelial cells (HPAEC), human breast cancer cells (SK-Br3) and human embryonic kidney cells (293T) extracted by this technology was analyzed by electron microscopy. The results are shown in Fig. 20 shown.

[0220] according to Fig. 20 Electron microscopy results show that EVs all have a saucer-like structure and a particle size of less than 200 nm. Fig.19 The results show that since the proliferation rate and type of EVs secreted by each cell may be different, the particle concentration is also inconsistent due to different secretion situations, but the particle size is less than 200nm. It can be seen that the method of this application can efficiently extract cell EVs.

[0221] Test Case 14

[0222] For the EVs extracted from Examples 4 to 6, the protein expressions of CD63, CD81, TSG101, and HSP70 were analyzed by Western-blot. Fig.21 As shown, in the figure, 1 is human pulmonary artery endothelial cells (HPAEC), 2 is human breast cancer cells (SK-Br3), and 3 is human embryonic kidney cells (293T).

[0223] According to the results in the pictures, the EVs derived from HPAEC, SK-Br3 and 293T cells extracted by this technology all showed positive expression of CD63, CD81, TSG101 and HSP70, and had the same characteristics as the EVs of mesenchymal stem cells.

[0224] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for extracting sterile extracellular vesicles, characterized in that: The method comprises the steps of placing the resuspended cells at 2 to 6° C. for 2 to 8 hours and then removing cells, dead cells, cell debris and lEVs.

2. The method for extracting sterile extracellular vesicles according to claim 1, characterized in that: The resuspending of cells comprises the step of resuspending the cells with PBS or physiological saline.

3. The method for extracting sterile extracellular vesicles according to claim 1, characterized in that: The method comprises: 1) Inoculating cells into a culture container, discarding the cell supernatant, digesting and collecting the cells; 2) Resuspend cells; 3) Place the resuspended cells at 2-6°C for 2-8 hours; 4) Centrifuge at 2-8°C, 300±100g×10±5min, 2000±500g×20±10min, 10000±5000g×40±15min in sequence; 5) discarding the precipitate and collecting the supernatant to obtain the sterile extracellular vesicles.

4. The method for extracting sterile extracellular vesicles according to claim 3, characterized in that: In the step 4), centrifugation is performed at 2-6°C, and treatments are performed successively at 300±50g×10±5min, 2000±300g×20±5min, and 10000±2000g×40±15min.

5. The method for extracting sterile extracellular vesicles according to claim 4, characterized in that: In the step 4), centrifugation is performed at 3-5°C, and treatments are performed at 300±30g×10±3min, 2000±200g×20±3min, and 10000±1000g×40±10min in sequence.

6. The method for extracting sterile extracellular vesicles according to claim 3, characterized in that: In the step 2), PBS or physiological saline is used to resuspend the cells.

7. The method for extracting sterile extracellular vesicles according to claim 6, characterized in that: The amount of PBS or physiological saline added in step 2) is 1 / 30 to 1 / 10 of the volume of the culture solution in step 1).

8. The method for extracting sterile extracellular vesicles according to claim 1, characterized in that: In the step 1), the culture conditions include 37±2° C. and 3-7% CO 2 . 9 . Sterile extracellular vesicles prepared by the method for extracting sterile extracellular vesicles according to any one of claims 1 to 8 .

10. A device for separating and extracting sterile extracellular vesicles, characterized in that: The device adopts the method for extracting sterile extracellular vesicles as described in any one of claims 1 to 8 to prepare sterile extracellular vesicles.

Images