Method for extracting exosome through microcarrier culture cells

Abstract

The invention relates to the technical field of exosome extraction, in particular to a method for extracting exosomes by culturing cells through a microcarrier. According to the method, an upstream microcarrier three-dimensional suspension culture process and a downstream differential centrifugal extraction process are creatively combined; the problems of low exosome extraction yield, low purity and incapability of large-scale culture are effectively solved, the problem that a large amount of effective supernatant cannot be collected in a traditional two-dimensional cell culture mode is overcome, the operation is simple, consumables such as kits, gel columns and immunomagnetic beads are not needed, a large amount of exosomes of cell supernatant from different sources can be treated, and the method is suitable for industrial production. The method is suitable for large-scale extraction and concentration and can realize industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of exosome extraction, and particularly relates to a method for extracting exosomes by microcarrier culture cells. BACKGROUND

[0002] As lipid bilayer membrane nanovesicles secreted by cells, exosomes have a diameter of 30-150 nm and contain a large number of bioactive molecules such as proteins, lipids and nucleic acids. They play a key role in cell communication, mediate cell signal transduction, and are involved in important physiological and pathological processes such as immune regulation, angiogenesis and apoptosis. In the treatment of diseases, they can be used as drug carriers to deliver therapeutic substances accurately; in the diagnosis field, specific molecular markers carried by exosomes can help early and accurate diagnosis of diseases, and have great application potential.

[0003] At present, there is a large demand for exosomes in preclinical and clinical development, but the existing extraction methods have many problems. For example, the kit method has limited extraction amount and a time-consuming process; the sucrose density gradient centrifugation method has low recovery rate and cannot meet the purity requirement; the traditional two-dimensional cell culture method cannot collect effective supernatant in large quantities, resulting in low exosome yield and being unable to meet the large-scale extraction and purification demand, which limits the industrial application of exosomes. SUMMARY

[0004] The present application aims to solve the problems of low exosome extraction yield, low purity and the inability of traditional culture methods to collect effective supernatant in large quantities in the prior art, and to realize the large-scale and efficient extraction of exosomes.

[0005] The purpose of the present application is achieved by the following technical solution: a method for extracting exosomes by microcarrier culture cells, comprising the following process steps: Step (1), microcarrier pretreatment: weigh the microcarriers, sterilize them with medical disinfectant alcohol, suck out the alcohol, and add culture medium to swell the microcarriers, Step (2), three-dimensional suspension culture of cells on microcarriers: the cells grow on the three-dimensional surface of the microcarriers and are suspended in the culture medium under stirring: inoculate the cells in the culture bottle with swollen microcarriers, and culture under stirring at 5% CO2 and 37℃, During the initial cell adhesion stage, an intermittent stirring mode is adopted, and the cell adhesion stage usually lasts for 3-6 hours; During the uniform distribution period after the cell adhesion stage (usually lasting for 24-48 hours), the stirring speed is increased, and the stirring is maintained at this speed every day; During the subsequent cell expansion stage (usually lasting for 5-7 days), the stirring is carried out by increasing the stirring speed once a day, Step (3): Collecting exosome cell supernatant: when cell growth reaches the plateau phase (usually 7-10 days), stop stirring, collect the supernatant, Step (4) Differential centrifugation to extract exosomes: four-step differential centrifugation is used to purify and precipitate exosomes, Step (5) Resuspension of exosomes: resuspend the exosome precipitate with PBS buffer for concentration determination and identification.

[0006] The three-dimensional microcarrier three-dimensional suspension culture of cells gives the cells a more physiological state of growth environment, aiming to improve the quality of exosomes, and suspension provides a homogeneous, controllable and easy to scale culture system, aiming to solve the yield and scaling problem of exosomes.

[0007] Preferably, step (1) further comprises placing the sterilized and alcohol-absorbed microcarriers in a sterile container, adding a coating solution with a concentration of 20-50 μg / mL obtained by dissolving the bioactive substance in PBS buffer, making the liquid surface completely cover and exceed the microcarriers, incubating at 2-8°C for 12-16 hours, discarding the coating solution, washing the microcarriers 2-3 times with pre-cooled 2-8°C basic medium, and then adding preheated 37°C complete medium to swell the microcarriers. Coating the microcarriers with bioactive substances significantly improves the initial adhesion efficiency of cells: shortens the disordered period of cells "finding" anchoring points, making the culture enter the logarithmic growth phase faster. Better maintain cell morphology and function: simulate the natural environment of in vivo cell growth, help cells maintain their physiological state, and possibly secrete more functional exosomes. Improve the final exosome yield: higher cell activity and density are directly related to higher exosome yield.

[0008] More preferably, the bioactive substance is one or any of collagen I, fibronectin, and laminin.

[0009] Preferably, from the stage of swelling the microcarriers until the stage of cell adhesion, it is carried out in complete medium containing a viscosity enhancer selected from the group consisting of trehalose or povidone, after the stage of cell adhesion is completed, the stirring is stopped, and after the microcarrier / cell complex is completely settled by standing for 5-10 minutes, most of the supernatant is aspirated; an equal volume of preheated PBS buffer at 37°C is added to resuspend and wash the microcarriers, and after standing and settling again, the washing liquid is aspirated; finally, preheated complete medium without trehalose or povidone at 37°C is added, and the stirring speed is increased, and the stirring is maintained at this speed every day. The viscosity enhancer is added to create a gentle fluid environment during the cell adhesion stage, reduce shear force, maximize cell-microcarrier contact efficiency, and use a gentle sedimentation displacement method instead of a centrifugation method, which aims to avoid mechanical damage to the cell-microcarrier complex in the cell adhesion stage, ensure cell adhesion rate, and achieve a smooth and damage-free transition from a high-viscosity adhesion environment to a normal-viscosity expansion environment. The special medium for cell adhesion customized for a specific process is changed back to the normal medium after use, which is temporary and functional.

[0010] Preferably, the concentration of the viscosity enhancer in the complete medium is 2%-3% (w / v) of trehalose or 1%-2% (w / v) of povidone.

[0011] Preferably, the microcarriers are porous microcarriers, and the microcarriers are SepLife LX-MC-Dex1. ® The concentration of the microcarriers in the medium is 0.5-2.0 mg / mL.

[0012] Preferably, the cells are human umbilical cord mesenchymal stem cells, and the inoculation amount is 4×10 6 cells / 5L flask.

[0013] Preferably, the base medium is DMEM / F12, and the complete medium is 10% exosome-free fetal bovine serum DMEM / F12.

[0014] Preferably, the intermittent stirring mode in the cell adhesion stage is stirring at a speed of 19-21 rpm for 2-4 minutes, followed by standing for 25-35 minutes and then continuing to stir; after the cell adhesion stage is completed, the stirring speed is increased to 24-26 rpm, and in the subsequent cell expansion stage, the stirring speed is increased by 2-5 rpm per day.

[0015] Preferably, the specific method of the four-step differential centrifugation is as follows: Step A, centrifuge the supernatant at 4°C, 200-500×g, for 10-15 min; Step B, centrifuge the supernatant obtained in Step A at 4℃, 1500-3000xg for 15-25 min, collect the supernatant; Step C, centrifuge the supernatant obtained in Step B at 4℃, 8000-12000xg for 20-40 min, collect the supernatant; Step D, centrifuge the supernatant obtained in Step C at 4℃, 90000-110000xg for 60-90 min, discard the supernatant, and obtain the exosome precipitate.

[0016] Preferably, all PBS buffers in the present application are 1x PBS.

[0017] Coating microcarriers with bioactive substances can significantly improve the initial cell adhesion efficiency: shorten the disordered period of cells "finding" anchoring points, so that the culture enters the logarithmic growth phase faster. Better maintain cell morphology and function: simulate the natural environment of in vivo cell growth, help cells maintain their physiological state, and possibly secrete more functional exosomes. Increase the final exosome yield: higher cell activity and density are directly related to higher exosome yield.

[0018] From the swelling of the microcarriers to the cell adhesion stage, using complete medium containing viscosity enhancer can reduce the interference of fluid shear force on cell adhesion: high viscosity medium reduces the sedimentation speed and collision energy of cells and microcarriers when stirring, creating a more gentle initial contact environment. Increase the effective collision frequency: the increase in medium viscosity makes cells and microcarriers not quickly settle after stirring is paused, increasing their chances of contact. Synergistic effect with intermittent stirring mode: this physical thickening method perfectly adapts and enhances the effect of the intermittent stirring mode, achieving gentle mixing during the stirring period and promoting stable adhesion during the resting period. The synergistic effect ensures efficient and stable cell adhesion during the cell adhesion stage. High initial adhesion rate directly translates to higher initial cell density and better cell activity, which enables the culture system to enter the logarithmic growth phase and plateau phase faster, thereby providing a fundamental guarantee for obtaining high yield of exosomes.

[0019] Advantages of the present application: The present application provides a method for extracting exosomes by culturing cells on microcarriers, which effectively solves the problems of low extraction yield, low purity, and inability to large-scale culture of exosomes by creatively combining the upstream microcarrier three-dimensional suspension culture process with the downstream differential centrifugation extraction process. In particular, it overcomes the problem that traditional two-dimensional cell culture methods cannot collect effective supernatant in large quantities. The method is simple to operate and does not require reagent kits, gel columns, and immunomagnetic beads and other consumables, can process a large amount of exosomes from supernatant of different source cells, is suitable for large-scale extraction and concentration, and can realize industrialized production. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 An electron microscope image of the exosomes extracted by the extraction method of the present application; Figure 2 A result image of the Western blot experiment of the present application. DETAILED DESCRIPTION

[0023] The present application is further described in conjunction with the following examples.

[0024] Example 1

[0025] A method for extracting exosomes by culturing cells on microcarriers, comprising the following process steps: Step (1), microcarrier pretreatment: weigh the microcarriers, sterilize them with medical sterilized alcohol, aspirate the alcohol, and add culture medium to swell the microcarriers, Step (2), three-dimensional suspension culture of cells on microcarriers: inoculate the cells into a 5L culture flask containing swollen microcarriers, the 5L culture flask contains 2.5L of culture medium, and the culture is stirred at 5% CO2, 37℃, During the initial cell adhesion stage of the culture, an intermittent stirring mode is used; During the uniform distribution period after the end of the cell adhesion stage, the stirring speed is increased, and the stirring is maintained at this speed every day; During the subsequent cell expansion stage, the stirring speed is increased every day, Step (3), collection of exosome cell supernatant: when the cell growth reaches the plateau phase, stop stirring, and collect the supernatant, Step (4), differential centrifugation to extract exosomes: four-step differential centrifugation is used to purify and precipitate exosomes, Step (5), resuspension of exosomes: resuspend the exosome precipitate with PBS buffer, and perform concentration determination and identification.

[0026] The microcarriers are porous microcarriers, and the microcarriers are Seplife ® LX-MC-Dex1, the concentration of the microcarriers in the culture medium is 1.0 mg / mL.

[0027] The cells are human umbilical cord mesenchymal stem cells, and the inoculation amount is 4×10 6 cells / 5L culture flask.

[0028] The basic culture medium is DMEM / F12, and the complete culture medium is 10% exosome-free fetal bovine serum DMEM / F12.

[0029] The intermittent stirring mode of the cell adhesion stage is stirring at a speed of 20 rpm for 3 minutes, then standing for 30 minutes and continuing to stir; after the cell adhesion stage ends, the speed is increased to 25 rpm, and in the subsequent cell expansion stage, the stirring speed is increased by 3 rpm per day.

[0030] The specific method of the four-step differential centrifugation is: Step A, centrifuge the supernatant at 4℃, 400xg for 12min; Step B, centrifuge the supernatant obtained in step A at 4℃, 2000xg for 20min, and collect the supernatant; Step C, centrifuge the supernatant obtained in step B at 4℃, 10000xg for 30min, and collect the supernatant; Step D, centrifuge the supernatant obtained in step C at 4℃, 100000xg for 75min, discard the supernatant, and obtain the exosome precipitate.

[0031] All PBS buffers in the present application are 1x PBS.

[0032] Example 2 A method for extracting exosomes by culturing cells with microcarriers, comprising the following process steps: Step (1), microcarrier pretreatment: weigh the microcarriers, sterilize them with medical sterilized alcohol, suck out the alcohol, and add culture medium to swell the microcarriers, Step (2), three-dimensional suspension culture of cells on microcarriers: inoculate the cells in a 5L culture flask containing 2.5L of culture medium, and culture under the condition of 5% CO2 and 37℃, During the initial cell adhesion stage, an intermittent stirring mode is used; During the uniform distribution period after the cell adhesion stage ends, the stirring speed is increased, and the stirring speed is maintained every day; In the subsequent cell expansion stage, the stirring speed is increased every day, Step (3), collect the exosome cell supernatant: when the cell growth reaches the plateau phase, stop stirring, and collect the supernatant, Step (4), differential centrifugation to extract exosomes: four-step differential centrifugation is used to purify and precipitate exosomes, Step (5), resuspension of exosomes: resuspend the exosome precipitate with PBS buffer, and perform concentration determination and identification.

[0033] The microcarriers are porous microcarriers, and the microcarriers are Seplife ® LX-MC-Dex1, the concentration of the microcarriers in the culture medium is 1.0mg / mL.

[0034] The cell is a human umbilical cord mesenchymal stem cell, and the inoculation amount is 4 x 10 6 cells / 5L culture flask.

[0035] The basic culture medium is DMEM / F12, and the complete culture medium is 10% exosome-free fetal bovine serum DMEM / F12.

[0036] The intermittent stirring mode of the cell adhesion stage is stirring at a speed of 20 rpm for 3 minutes, then standing for 30 minutes and continuing to stir; after the cell adhesion stage ends, the stirring speed is increased to 25 rpm, and in the subsequent cell expansion stage, the stirring speed is increased by 3 rpm per day.

[0037] The specific method of the four-step differential centrifugation is as follows: Step A, centrifuge the supernatant at 4°C, 400xg for 12min; Step B, centrifuge the supernatant obtained in step A at 4°C, 2000xg for 20min, and collect the supernatant; Step C, centrifuge the supernatant obtained in step B at 4°C, 10000xg for 30min, and collect the supernatant; Step D, centrifuge the supernatant obtained in step C at 4°C, 100000xg for 75min, discard the supernatant, and obtain the exosome precipitate.

[0038] All PBS buffers in the present application are 1x PBS.

[0039] Further, step (1) further comprises placing the sterilized microcarriers that have absorbed alcohol into a sterile container, adding a coating solution obtained by dissolving a biological active substance in a PBS buffer to a concentration of 30μg / mL, so that the liquid surface is completely covered and exceeds the microcarriers, incubating at 6°C for 14 hours, aspirating and discarding the coating solution, washing the microcarriers 3 times with pre-cooled 6°C basic culture medium, and then adding a complete culture medium preheated to 37°C to swell the microcarriers. The biological active substance is collagen I.

[0040] Example 3 A method for extracting exosomes by culturing cells on microcarriers, comprising the following process steps: Step (1), microcarrier pretreatment: weigh the microcarriers, sterilize them with medical disinfectant alcohol, aspirate the alcohol, and add culture medium to swell the microcarriers, Step (2), three-dimensional suspension culture of cells on microcarriers: inoculate the cells into a 5L culture flask containing swollen microcarriers, and the 5L culture flask contains 2.5L of culture medium, and culture under stirring at 5% CO2, 37°C, In the initial cell adhesion stage of the culture, an intermittent stirring mode is adopted; The uniform distribution period after the cell adhesion stage ends, the rotation speed is increased, and the stirring is maintained at this rotation speed every day; The subsequent cell expansion stage is stirred in the manner of increasing the stirring speed by 5 rpm every day, Step (3): collecting exosome cell supernatant: when the cell growth reaches the plateau phase, stop stirring, and collect the supernatant, Step (4) differential centrifugation to extract exosomes: four-step differential centrifugation is used to purify and precipitate exosomes, Step (5) resuspension of exosomes: PBS buffer is used to resuspend the exosome precipitate for concentration determination and identification.

[0041] Preferably, step (1) further comprises placing the sterilized and alcohol-absorbed microcarriers in a sterile container, adding a coating solution with a concentration of 40 μg / mL obtained by dissolving the bioactive substance in PBS buffer, so that the liquid surface is completely covered and exceeds the microcarriers, incubating at 8°C for 15 hours, discarding the coating solution, washing the microcarriers 3 times with pre-8°C basic medium, and then adding preheated to 37°C complete medium to swell the microcarriers.

[0042] The bioactive substance is fibronectin.

[0043] The microcarriers are porous microcarriers, and the microcarriers are SepLife LX-MC-Dex1. ® The concentration of the microcarriers in the culture medium is 2.0 mg / mL.

[0044] The cells are human umbilical cord mesenchymal stem cells, and the inoculation amount is 4×10 6 cells / 5L flask.

[0045] The basic medium is DMEM / F12, and the complete medium is 10% exosome-free fetal bovine serum DMEM / F12.

[0046] The intermittent stirring mode of the cell adhesion stage is stirring at a rotation speed of 21 rpm for 4 minutes, followed by standing for 30 minutes and then continuing to stir; after the cell adhesion stage ends, the rotation speed is increased to 26 rpm, and in the subsequent cell expansion stage, the stirring speed is increased by 5 rpm every day.

[0047] The specific method of the four-step differential centrifugation is as follows: Step A, centrifuge the supernatant at 4°C, 400×g, for 15 min; Step B, centrifuge the supernatant obtained in step A at 4°C, 2500×g, for 25 min, and collect the supernatant; Step C, centrifuge the supernatant obtained in step B at 4°C, 11000×g, for 40 min, and collect the supernatant; Step D, centrifuge the supernatant obtained in step C at 110000xg for 70 min at 4℃, discard the supernatant, and obtain the exosome precipitate.

[0048] All PBS buffers in the present application are 1x PBS.

[0049] Example 4 A method for extracting exosomes by culturing cells on microcarriers, comprising the following process steps: Step (1), microcarrier pretreatment: weigh the microcarriers, sterilize them with medical sterilized alcohol, aspirate the alcohol, and add culture medium to swell the microcarriers, Step (2), three-dimensional suspension culture of cells on microcarriers: inoculate the cells in a 5L culture flask containing 2.5L of culture medium, and culture under stirring at 5% CO2 and 37℃, During the initial cell adhesion stage, use an intermittent stirring mode; During the uniform distribution period after the cell adhesion stage, increase the stirring speed, and maintain the stirring speed every day; During the subsequent cell expansion stage, increase the stirring speed every day, Step (3), collection of exosome cell supernatant: when the cell growth reaches the plateau phase, stop stirring, and collect the supernatant, Step (4), differential centrifugation to extract exosomes: use four-step differential centrifugation to purify and precipitate exosomes, Step (5), resuspension of exosomes: resuspend the exosome precipitate with PBS buffer, and perform concentration determination and identification.

[0050] The microcarriers are porous microcarriers, and the microcarriers are Seplife ® LX-MC-Dex1, the concentration of the microcarriers in the culture medium is 1.0 mg / mL.

[0051] The cells are human umbilical cord mesenchymal stem cells, and the inoculation amount is 4x10 6 cells / 5L culture flask.

[0052] The basic culture medium is DMEM / F12, and the complete culture medium is 10% exosome-free fetal bovine serum DMEM / F12.

[0053] The intermittent stirring mode during the cell adhesion stage is to stir at a speed of 20 rpm for 3 minutes, then stand for 30 minutes and continue to stir; after the cell adhesion stage, increase the stirring speed to 25 rpm, and during the subsequent cell expansion stage, increase the stirring speed by 3 rpm every day.

[0054] The specific method of the four-step differential centrifugation is as follows: Step A, centrifuge the supernatant at 4°C, 400xg for 12 min; Step B, centrifuge the supernatant obtained in step A at 4°C, 2000xg for 20 min, and collect the supernatant; Step C, centrifuge the supernatant obtained in step B at 4°C, 10000xg for 30 min, and collect the supernatant; Step D, centrifuge the supernatant obtained in step C at 4°C, 100000xg for 75 min, discard the supernatant, and obtain the exosome precipitate.

[0055] All PBS buffers in the present application are 1x PBS.

[0056] Further, from the microcarrier swelling stage to the cell adhesion stage, the complete medium containing a viscosity enhancer selected from trehalose or povidone is used, after the cell adhesion stage ends, stop stirring, stand for 8 minutes until the microcarrier / cell complex completely settles, and then aspirate most of the supernatant; add an equal volume of preheated PBS buffer at 37°C to resuspend and wash the microcarriers, and after standing and settling again, aspirate the washing liquid; finally, add preheated complete medium without trehalose or povidone at 37°C, and increase the rotation speed, and keep the rotation speed every day for stirring.

[0057] The concentration of the viscosity enhancer in the complete medium is: trehalose is 2.5% (w / v).

[0058] Example 5 A method for extracting exosomes by culturing cells on microcarriers, which comprises the following process steps: Step (1), microcarrier pretreatment: weigh the microcarriers, sterilize them with medical sterilized alcohol, aspirate the alcohol, and add culture medium to swell the microcarriers, Step (2), three-dimensional suspension culture of cells on microcarriers: inoculate the cells into a 5L culture flask containing 2.5L of culture medium in which the microcarriers are swollen, and culture under stirring at 5% CO2 and 37°C, During the initial cell adhesion stage, use an intermittent stirring mode; During the uniform distribution period after the cell adhesion stage ends, increase the rotation speed, and keep the rotation speed every day for stirring; During the subsequent cell expansion stage, increase the stirring speed every day, Step (3), collect the exosome cell supernatant: when the cell growth reaches the plateau phase, stop stirring, and collect the supernatant, Step (4), differential centrifugation to extract exosomes: use four-step differential centrifugation to purify and precipitate exosomes, Step (5) Resuspension of exosomes: Resuspend the exosome pellet with PBS buffer for concentration determination and identification.

[0059] The microcarrier is a porous microcarrier, and the microcarrier is SepLife LX-MC-Dex1. ® The concentration of the microcarrier in the culture medium is 1.0 mg / mL.

[0060] The cell is a human umbilical cord mesenchymal stem cell, and the inoculation amount is 4*10 6 cells / 5L flask.

[0061] The basic medium is DMEM / F12, and the complete medium is 10% exosome-free fetal bovine serum DMEM / F12.

[0062] The intermittent stirring mode in the cell adhesion stage is stirring at a speed of 20 rpm for 3 minutes, then standing for 30 minutes and continuing to stir; after the cell adhesion stage is over, the speed is increased to 25 rpm, and in the subsequent cell expansion stage, the stirring speed is increased by 3 rpm per day.

[0063] The specific method of the four-step differential centrifugation is as follows: Step A, centrifuge the supernatant at 4°C, 400* g, for 12 min; Step B, centrifuge the supernatant obtained in step A at 4°C, 2000* g, for 20 min, and collect the supernatant; Step C, centrifuge the supernatant obtained in step B at 4°C, 10000* g, for 30 min, and collect the supernatant; Step D, centrifuge the supernatant obtained in step C at 4°C, 100000* g, for 75 min, discard the supernatant, and obtain the exosome pellet.

[0064] All PBS buffers in the application are 1* PBS.

[0065] Further, step (1) further comprises placing the sterilized microcarriers that have absorbed alcohol into a sterile container, adding a coating solution obtained by dissolving a biological active substance in a PBS buffer to a concentration of 30 μg / mL, so that the liquid surface is completely covered and exceeds the microcarriers, incubating at 6°C for 14 hours, discarding the coating solution, washing the microcarriers 3 times with pre-cooled 6°C basic medium, and then adding pre-heated 37°C complete medium to swell the microcarriers. The biological active substance is collagen I.

[0066] Further, from the swelling of the microcarriers until the cell adhesion stage, it is carried out in complete culture medium containing a viscosity enhancer selected from trehalose or povidone, after the end of the cell adhesion stage, the stirring is stopped, after 8 minutes of rest for the complete sedimentation of the microcarrier / cell complex, most of the supernatant is aspirated; an equal volume of PBS buffer preheated at 37°C is added to resuspend and wash the microcarriers, after again resting and sedimenting, the washing liquid is aspirated; finally, preheated at 37°C, complete culture medium without trehalose or povidone is added, the rotation speed is increased again and the stirring is maintained daily at this rotation speed. The concentration of the viscosity enhancer in the complete culture medium is: trehalose 2.5% (w / v).

[0067] Experimental data 1、 Figure 1 The electron microscope image of the exosomes extracted by the extraction method of the present application. The exosomes extracted by the method of the present application are typical tea tray or cup-shaped spherical structures, with complete morphology, clear boundary and few background impurities, which proves that the method of the present application can effectively obtain exosomes with complete structure and high purity.

[0068] 2, Western Blot is used to detect the specific protein markers of the extracted exosome samples, TSG is a common marker of exosomes, Figure 2 From right to left are the results of Western Blot experiments of exosome protein extracted by the extraction method of Example 1, Example 2 and Example 5 of the present application, which shows that the exosomes are successfully extracted. Compared with Example 1 without coating treatment, Example 2 coated with collagen I shows a stronger positive marker signal, and Example 5 coated with collagen I and trehalose viscosity enhancement shows the strongest positive marker signal. This shows that the optimization scheme (surface coating combined with viscosity enhancement) of the present application not only improves the yield of exosomes, but also significantly improves the quality of exosomes.

[0069] 3, BCA kit is used to determine the protein concentration, exosome solutions extracted by the methods of Examples 1-5 of the present application are taken, added to a 96-well plate, and the protein concentration is determined by BCA kit. The results are shown in Table 1.

[0070] Table 1 As can be seen from Table 1, compared with Example 1 (uncoated), the cell adhesion rate of Example 2 (collagen I coating) increased from 65.2% to 85.7%, and the exosome yield increased by about 48%. Reason analysis: Collagen I, as a major component of the extracellular matrix, provides a biologically active surface that can be specifically recognized by cells, greatly promoting the initial adhesion, spreading and survival of cells, thereby laying a solid foundation for subsequent high-density culture and high-level exosome secretion. The yield of Example 3 (2.0 mg / mL microcarriers) reached 2264.6 μg / mL under fibronectin coating, which was significantly higher than that of Example 2 under the same coating conditions and low concentration of microcarriers. Reason analysis: Increasing the concentration of microcarriers directly increases the total growth surface area per unit volume, enabling the culture system to support the growth of a larger number of cells and achieve linear scaling of production capacity. Data comparison: The cell adhesion rate and yield of Example 4 (only adding trehalose) were significantly higher than those of Example 1 without coating. Reason analysis: During the cell adhesion stage, trehalose increases the viscosity of the culture medium, effectively reducing fluid shear stress and creating a more gentle and stable physical microenvironment for the initial contact between cells and microcarriers, thereby independently improving adhesion efficiency. Data comparison: Example 5 (collagen I coating + trehalose) achieved the best overall effect, with the highest cell adhesion rate (91.5%) and the most significant marker expression intensity, and even exceeded the exosome yield of Example 3 with higher microcarrier concentration (2489.7 μg / mL).

[0071] Reason analysis: This reflects the greatest creativity of the present application. Surface coating (biochemical signal) and viscosity enhancement (physical environment optimization) simultaneously act on the cell adhesion process from two different dimensions, producing a "1+1>2" synergistic effect. Coating ensures the quality of adhesion (firm, directional), while viscosity enhancement guarantees the quantity of adhesion (efficient, stable), and the combination of the two maximizes cell utilization and production efficiency.

[0072] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A method for extracting exosomes from cells cultured via microcarriers, characterized in that: It includes the following process steps: Step (1), Microcarrier pretreatment: Weigh the microcarriers, add medical disinfectant alcohol for sterilization, remove the alcohol, add culture medium to make the microcarriers swell. Step (2), three-dimensional suspension culture of cells on microcarriers: Cells were seeded into culture flasks containing swollen microcarriers and cultured with stirring at 37°C and 5% CO2. During the initial cell adhesion stage of culture, an intermittent stirring mode was used; During the uniform distribution phase after the cell adhesion stage, increase the stirring speed and maintain this speed for stirring every day. In the subsequent cell expansion phase, the stirring speed was increased once a day. Step (3): Collect exosome cell supernatant: When cell growth reaches the plateau phase, stop stirring and collect the supernatant. Step (4) Differential centrifugation to extract exosomes: Four-step differential centrifugation was used to purify and precipitate exosomes. Step (5) Exosome resuspension: Resuspend the exosome precipitate using PBS buffer for concentration determination and identification.

2. The method for extracting exosomes by culturing cells using microcarriers according to claim 1, characterized in that: Step (1) also includes placing the sterilized microcarriers, after the alcohol has been absorbed, into a sterile container, adding a coating solution with a concentration of 20-50 μg / mL obtained by dissolving the bioactive substance in PBS buffer, so that the liquid completely covers and exceeds the microcarriers, incubating at 2-8°C for 12-16 hours, aspirating the coating solution, washing the microcarriers 2-3 times with pre-cooled basal medium at 2-8°C, and then adding complete medium preheated to 37°C to swell the microcarriers.

3. The method for extracting exosomes by culturing cells using microcarriers according to claim 2, characterized in that: The bioactive substance is one or more of collagen I, fibronectin, and laminin.

4. A method for extracting exosomes from cells cultured via microcarriers according to claim 1 or 2, characterized in that: From the swelling of the microcarriers to the cell adhesion stage, the process is carried out in a complete culture medium containing a viscosity enhancer selected from trehalose or povidone. After the cell adhesion stage is completed, stirring is stopped, and the mixture is allowed to stand for 5-10 minutes to allow the microcarrier / cell complex to settle completely. Most of the supernatant is then discarded. An equal volume of preheated 37°C PBS buffer is added to resuspend and wash the microcarriers. After settling again, the washing solution is discarded. Finally, preheated 37°C complete culture medium without trehalose or povidone is added, and the stirring speed is increased. This stirring speed is maintained daily.

5. The method for extracting exosomes by culturing cells using microcarriers according to claim 4, characterized in that: The concentration of the viscosity enhancer in the complete culture medium is: trehalose 2%-3% (w / v), or povidone 1%-2% (w / v).

6. The method for extracting exosomes by culturing cells using microcarriers according to claim 1, characterized in that: The microcarrier is Seplife. ® The concentration of LX-MC-Dex1 microcarriers in the culture medium is 0.5-2.0 mg / mL.

7. The method for extracting exosomes by culturing cells using microcarriers according to claim 1, characterized in that: The cells were human umbilical cord mesenchymal stem cells, with an inoculum size of 4 × 10⁻⁶. 6 1 cell / 5L culture flask 8. The method for extracting exosomes by culturing cells using microcarriers according to claim 2, characterized in that: The basal culture medium was DMEM / F12, and the complete culture medium was 10% exosome-free fetal bovine serum in DMEM / F12.

9. The method for extracting exosomes by culturing cells using microcarriers according to claim 1, characterized in that: The intermittent stirring mode during the cell adhesion stage involves stirring at 19-21 rpm for 2-4 minutes, followed by standing for 25-35 minutes before stirring again. After the cell adhesion stage, the stirring speed is increased to 24-26 rpm. During the subsequent cell expansion stage, the stirring speed is increased by 2-5 rpm per day.

10. The method for extracting exosomes by culturing cells using microcarriers according to claim 1, characterized in that: The specific method for the four-step differential centrifugation is as follows: Step A: Centrifuge the supernatant at 4℃, 200-500×g for 10-15 min; Step B: Centrifuge the supernatant obtained in Step A at 4℃, 1500-3000×g for 15-25 min, and collect the supernatant. Step C: Centrifuge the supernatant obtained in step B at 4℃, 8000-12000×g for 20-40 min, and collect the supernatant. Step D: Centrifuge the supernatant obtained in step C at 4°C, 90,000-110,000 × g for 60-90 min, discard the supernatant, and obtain the exosome precipitate.

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