Rapid separation and purification method for extracellular vesicles

Abstract

A rapid separation and purification method for extracellular vesicles belongs to the field of extracellular vesicles, and comprises the following steps: collecting a cell culture fluid, obtaining extracellular vesicles through multiple times of centrifugal separation, and optimizing the second centrifugation and the third centrifugation through a first centrifugation parameter optimization equation set and a second centrifugation parameter optimization equation set respectively to obtain the extracellular vesicles. A two-dimensional transformation technology is introduced into the second centrifugal parameter optimization equation set to establish a particle distribution image, the transverse distribution fitness and the longitudinal distribution fitness are calculated, and the centrifugal rotating speed is optimized through a second contribution value calculation equation; the second centrifugal parameter optimization equation set comprises a second mass conservation equation, a second momentum conservation equation, a second sedimentation rate equation, a second separation efficiency equation and the second contribution value calculation equation, and the extracellular vesicle suspension is obtained after membrane filtration, ultrafiltration concentration, separation reagent treatment and ultracentrifugation. The technical problem that a large number of initial samples are consumed in the prior art is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of extracellular vesicles, and in particular relates to a method for rapid separation and purification of extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are small membrane-bound vesicles secreted from the cell membrane. They carry biomolecules such as proteins and nucleic acids from the cell of origin and play an important role in intercellular signaling, immune regulation, and material transport. In recent years, EVs have become a research hotspot in the life sciences due to their unique biological functions and promising applications.

[0003] Currently, the isolation and purification of extracellular vesicles (EVs) remains a key technical bottleneck hindering their further research and application. Existing isolation and purification methods include ultracentrifugation, immunoaffinity chromatography, and density gradient centrifugation. Each has its own advantages and disadvantages: Ultracentrifugation is simple to perform but requires complex instrumentation and equipment, resulting in low separation efficiency; immunoaffinity chromatography offers strong selectivity but low yield and requires specific antibodies; and density gradient centrifugation offers excellent separation results but is cumbersome and time-consuming. Furthermore, these methods often require large initial sample volumes and place high demands on sample content and purity, making them unsuitable for isolating EVs from small sample sizes.

[0004] That is to say, the existing technology has the technical problem of consuming a large number of initial samples. Summary of the Invention

[0005] In view of this, the present invention provides a method for rapid separation and purification of extracellular vesicles, which can improve separation efficiency and product purity while reducing the requirements for initial sample content and purity. The method comprises the following steps:

[0006] The cell culture fluid was collected and extracellular vesicles were obtained by multiple centrifugation separations, wherein the second centrifugation and the third centrifugation were optimized by a first centrifugation parameter optimization equation group and a second centrifugation parameter optimization equation group, respectively. The first centrifugation parameter optimization equation group included a first mass conservation equation, a first momentum conservation equation, a first sedimentation rate equation, and a first separation efficiency equation. The second centrifugation parameter optimization equation group introduced a two-dimensional transformation technology to establish a particle distribution image and calculate the lateral distribution fitness and the longitudinal distribution fitness. The centrifugal speed was optimized by a second contribution value calculation equation. The second centrifugation parameter optimization equation group included a second mass conservation equation, a second momentum conservation equation, a second sedimentation rate equation, a second separation efficiency equation, and the second contribution value calculation equation. The extracellular vesicle suspension was obtained after membrane filtration, ultrafiltration concentration, separation reagent treatment, and ultracentrifugation.

[0007] Among them, a cell culture fluid with a cell density of 1,000,000 to 2,000,000 cells per milliliter and a cell survival rate greater than 90% is selected as a raw material, and the raw material is subjected to a first centrifugation at a centrifugal speed of 1,000 revolutions per minute and a centrifugal time of 10 minutes to obtain a first supernatant.

[0008] Among them, the first centrifugal parameter optimization equation group includes a first mass conservation equation, a first momentum conservation equation, a first sedimentation rate equation, and a first separation efficiency equation. By solving the first centrifugal parameter optimization equation group, the first theoretical sedimentation time and the first optimal centrifugal speed are obtained.

[0009] The input of the first mass conservation equation includes the volume, density, and ambient temperature of the first supernatant, and the output is the first mass distribution function. The input of the first momentum conservation equation includes the first mass distribution function and viscosity, and the output is the instantaneous velocity of the first particle.

[0010] Among them, the input of the first sedimentation rate equation includes the instantaneous velocity of the first particle and the density, and the output is the first theoretical sedimentation time. The input of the first separation efficiency equation includes the first theoretical sedimentation time and the volume of the first supernatant, and the output is the first optimal centrifugal speed.

[0011] Among them, the second centrifugal parameter optimization equation group includes a second mass conservation equation, a second momentum conservation equation, a second sedimentation rate equation, a second separation efficiency equation, and a second contribution value calculation equation. By solving the second centrifugal parameter optimization equation group, the second theoretical sedimentation time and the second optimal centrifugal speed are obtained.

[0012] The input of the second mass conservation equation includes the volume of the second supernatant, the density, and the ambient temperature, and the output is the second mass distribution function. The input of the second momentum conservation equation includes the second mass distribution function and the viscosity, and the output is the second particle instantaneous velocity.

[0013] Among them, the input of the second sedimentation rate equation includes the instantaneous velocity of the second particles and the density, and the output is the second theoretical sedimentation time. The input of the second separation efficiency equation includes the second theoretical sedimentation time, the volume of the second supernatant, the lateral distribution fitness, and the longitudinal distribution fitness. The centrifugal efficiency is calculated based on polynomial fitting, and the output is the initial second optimal centrifugal speed.

[0014] The input of the second contribution value calculation equation includes the lateral distribution fitness, the longitudinal distribution fitness, and the initial second optimal centrifugal speed. The contribution values ​​of different speeds to the separation effect are calculated, and the output is the second optimal centrifugal speed.

[0015] The third supernatant was filtered using a membrane filter with a pore size of 0.22 microns to obtain a filtrate, and the filtrate was concentrated to 1 / 5 to 1 / 10 of the original volume using an ultrafiltration centrifuge tube to obtain a concentrate. A separation reagent was added to the concentrate, wherein the separation reagent included a 30% by mass sucrose solution and an 8% by mass polyethylene glycol solution, and the mixing ratio of the sucrose solution to the polyethylene glycol solution was 2:1. After standing at 4°C for 12 hours, the solution was ultracentrifuged at 100,000 rpm for 60 minutes.

[0016] The effects of the present invention are:

[0017] The rapid extracellular vesicle separation and purification method proposed in this invention optimizes the parameters of multi-step centrifugation separation and combines it with the principle of density gradient separation to obtain high-purity and high-recovery extracellular vesicle products in a relatively short period of time. Specifically, the main technical effects of this method are as follows:

[0018] 1. High separation efficiency. The present method utilizes multiple optimized centrifugation steps combined with density gradient separation to effectively remove cells and large impurities, resulting in a final extracellular vesicle product with a purity exceeding 90%, significantly exceeding existing methods such as single centrifugation or immunoaffinity chromatography. This high-purity of extracellular vesicles provides high-quality samples for subsequent biological function research and clinical applications.

[0019] 2. Low sample volume requirement. The method of the present invention has low requirements for initial cell culture density and viability. Only 1,000,000-2,000,000 cells per milliliter and a viability greater than 90% are required to obtain a satisfactory extracellular vesicle product. This significantly reduces the content and purity requirements of the initial sample, facilitating the isolation and purification of extracellular vesicles from small sample sizes, greatly facilitating life science research.

[0020] 3. Simple operation. Although the method of the present invention involves multiple steps of centrifugation and density gradient separation, mathematical modeling optimizes key parameters, allowing precise control of the operating conditions of each step. The entire separation process is simple and easy to perform, does not require complex instrumentation, and is highly reproducible, ensuring feasibility for practical application.

[0021] In summary, the rapid separation and purification method of extracellular vesicles proposed in the present invention fully utilizes the advantages of centrifugal separation and density gradient separation. While greatly improving product purity and recovery rate, it also significantly reduces the requirements for initial samples, solving the technical problem of consuming a large amount of initial samples in the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A flow chart of the method provided by the present invention.

[0023] Figure 2 This is a graph showing the relationship between centrifugal speed and separation efficiency in Example 2.

[0024] Figure 3 This is a diagram of the separation process parameter changes in Example 2.

[0025] Figure 4 This is a diagram showing the changes in centrifugal process parameters in Example 2.

[0026] Figure 5 This is the extracellular vesicle size distribution diagram in Example 2. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0028] like Figure 1 As shown, the present invention includes the following steps:

[0029] S01. Collecting cell culture fluid, measuring the cell density and cell viability in the cell culture fluid, and selecting a cell culture fluid with a cell density of 1,000,000 to 2,000,000 cells per milliliter and a cell viability greater than 90% as a raw material;

[0030] S02, centrifuging the raw material for the first time at a centrifugal speed of 1000 rpm for 10 minutes to obtain a first supernatant;

[0031] S03, measuring the volume, density, and viscosity of the first supernatant, recording the ambient temperature, and inputting the volume, density, viscosity, and ambient temperature of the first supernatant into a first centrifugation parameter optimization equation set;

[0032] S04, solving the first centrifugal parameter optimization equation group to obtain a first theoretical sedimentation time and a first optimal centrifugal speed;

[0033] S05, performing a second centrifugation on the first supernatant according to the first theoretical sedimentation time and the first optimal centrifugal speed to obtain a second supernatant;

[0034] S06. Measure the volume, density, and viscosity of the second supernatant, record the ambient temperature, establish a particle distribution image in the second supernatant using a two-dimensional transformation technique, calculate a transverse distribution fitness and a longitudinal distribution fitness of the particle distribution image, and input the volume, density, viscosity, ambient temperature, transverse distribution fitness, and longitudinal distribution fitness of the second supernatant into a second centrifugation parameter optimization equation set;

[0035] S07, solving the second centrifugal parameter optimization equation group to obtain a second theoretical sedimentation time and a second optimal centrifugal speed;

[0036] S08, performing a third centrifugation on the second supernatant according to the second theoretical sedimentation time and the second optimal centrifugal speed to obtain a third supernatant;

[0037] S09, filtering the third supernatant using a membrane filter with a pore size of 0.22 microns to obtain a filtrate;

[0038] S10, concentrating the filtrate using an ultrafiltration centrifuge tube to 1 / 5 to 1 / 10 of the original volume to obtain a concentrated solution;

[0039] S11, adding a separation reagent to the concentrated solution, wherein the separation reagent includes a 30% by mass sucrose solution and an 8% by mass polyethylene glycol solution, wherein the mixing ratio of the sucrose solution to the polyethylene glycol solution is 2 to 1;

[0040] S12, the concentrated solution after adding the separation reagent is allowed to stand at 4°C for 12 hours;

[0041] S13, ultracentrifuging the concentrated solution after standing at a speed of 100,000 rpm for 60 minutes to obtain a precipitate;

[0042] S14, resuspending the precipitate with phosphate buffer to obtain an extracellular vesicle suspension;

[0043] S15. Determine the particle size distribution of particles in the extracellular vesicle suspension, calculate the proportion of particles between 40 and 200 nanometers, and determine that the product is qualified when the proportion is greater than 90%;

[0044] S16. The qualified products are divided into 2 ml cryopreservation tubes, with each tube containing 0.5 ml, and stored at -80°C.

[0045] The first centrifugal parameter optimization equation group includes a first mass conservation equation, a first momentum conservation equation, a first sedimentation rate equation, and a first separation efficiency equation;

[0046] The first mass conservation equation is used to describe the change in mass distribution of extracellular vesicles during the second centrifugation process, the input includes the volume of the first supernatant, the density, and the ambient temperature, and the output is a first mass distribution function;

[0047] The first momentum conservation equation is used to describe the motion state of the particles during the second centrifugation process, the input includes the first mass distribution function and the viscosity, and the output is the instantaneous velocity of the first particle;

[0048] The first sedimentation rate equation is used to calculate the sedimentation velocity of the particles during the second centrifugation process, the input includes the first particle instantaneous velocity and the density, and the output is the first theoretical sedimentation time;

[0049] The first separation efficiency equation is used to evaluate the effect of the second centrifugation, the input includes the first theoretical sedimentation time and the volume of the first supernatant, and the output is the first optimal centrifugal speed;

[0050] The second centrifugal parameter optimization equation group includes a second mass conservation equation, a second momentum conservation equation, a second sedimentation rate equation, a second separation efficiency equation, and a second contribution value calculation equation;

[0051] The second mass conservation equation is used to describe the third centrifugation process.

[0052] The mass distribution change of the extracellular vesicles during the process, the input includes the volume of the second supernatant, the density, and the ambient temperature, and the output is a second mass distribution function;

[0053] The second momentum conservation equation is used to describe the motion state of the particles during the third centrifugation process, the input includes the second mass distribution function and the viscosity, and the output is the second particle instantaneous velocity;

[0054] The second sedimentation rate equation is used to calculate the sedimentation velocity of the particles during the third centrifugation process, the input includes the second particle instantaneous velocity and the density, and the output is the second theoretical sedimentation time;

[0055] The second separation efficiency equation is used to evaluate the effect of the third centrifugation. The input includes the second theoretical sedimentation time, the volume of the second supernatant, the transverse distribution fitness, and the longitudinal distribution fitness. The centrifugal efficiency is calculated based on polynomial fitting, and the output is the initial second optimal centrifugal speed.

[0056] The second contribution value calculation equation is used to optimize the initial second optimal centrifugal speed. The input includes the lateral distribution fitness, the longitudinal distribution fitness, and the initial second optimal centrifugal speed. The contribution values ​​of different speeds to the separation effect are calculated, and the output is the second optimal centrifugal speed.

[0057] The specific implementation of the above steps is described in detail below.

[0058] The specific implementation of step S01 is to determine the quality of the cell culture fluid through cell counting and activity testing. First, a hemocytometer is used to count cells under a microscope. The counts are counted in four large squares, and the average is taken and multiplied by the dilution factor to obtain the cell density. Then, the cell viability is tested using trypan blue staining. 20 μl of cell suspension is mixed with 20 μl of 0.4% trypan blue solution. The number of blue-stained cells and the total number of cells are counted under a microscope to calculate the viability. Finally, qualified cell culture fluids are screened based on the test results. The purpose of this step is to ensure that the raw cell state is good and that the extracellular vesicles obtained in the subsequent separation have good biological activity. If the cell density is too high, excessive cell metabolites will interfere with the separation purity. If the density is too low, the yield will be insufficient. Therefore, the density range is set between 1,000,000 and 2,000,000 cells per milliliter. A cell viability below 90% indicates poor cell state. Dead cells release cell debris that contaminates the product, so a viability of greater than 90% is required.

[0059] Step S02 involves low-speed centrifugation of the raw material to remove cells and large impurities. A temperature-controlled centrifuge is used, and a cantilever rotor is selected to minimize contamination between samples. Before centrifugation, the culture medium is evenly distributed into centrifuge tubes, with each tube filling no more than 80% of its maximum capacity. A protective cap is used during centrifugation to prevent aerosol contamination. After centrifugation, the tubes are gently removed to avoid disturbing the pellet. When aspirating the supernatant with a pipette, care is taken not to touch the pellet. This step is based on the principle of sedimentation, where larger particles preferentially settle under centrifugal force. A speed of 1000 rpm effectively removes intact cells and cell debris, while retaining extracellular vesicles in the supernatant. The centrifugation time is determined by the sample volume; 10 minutes is sufficient for processing samples between 15 and 50 ml.

[0060] The specific implementation of step S03 involves obtaining key parameters of the first supernatant. First, the supernatant volume is measured using a graduated cylinder, ensuring that the liquid level is level with the eye. Density is then determined using the pycnometer method, using a calibrated electronic balance for weighing. Each sample is measured three times and the average is taken. Viscosity is also measured using an Ubbelohde capillary viscometer, maintaining a constant temperature water bath at 25 ± 0.1 degrees Celsius. Furthermore, the ambient temperature is measured using a thermometer. This step aims to provide basic data for subsequent centrifugation parameter optimization, as these parameters influence particle settling behavior and, consequently, separation effectiveness. Volume data is used to calculate sample loading, while density and viscosity data are used to predict particle settling rate. Temperature influences the physical properties of the fluid. The accuracy of the measured data is directly related to the reliability of centrifugation parameter optimization, thus requiring strict adherence to standard operating procedures during the measurement process.

[0061] The specific implementation method of step S04 is to obtain the optimal centrifugal conditions by solving the first centrifugal parameter optimization equation group, which includes the mass conservation equation, the momentum conservation equation, the sedimentation rate equation and the separation efficiency equation. These equations take into account multiple influencing factors such as fluid mechanics and particle dynamics. The solution process adopts a numerical method. First, the mass conservation equation and the momentum conservation equation are solved using the Runge-Kutta method to obtain the motion law of the particles in the flow field; then the theoretical sedimentation time is calculated based on the sedimentation rate equation. The equation takes into account factors such as particle size, density difference, and fluid viscosity; finally, the centrifugal speed is optimized through the separation efficiency equation. The equation introduces correction terms for particle size distribution and density ratio. The equation is solved using an iterative method, and the convergence criterion is set as the relative error of the results of two adjacent iterations is less than 0.1%. The purpose of this step is to obtain the theoretically optimal centrifugal parameters to achieve efficient separation.

[0062] The specific implementation of step S05 involves performing a second centrifugation. Centrifugation conditions are set according to the optimized parameters. Before operation, the centrifuge balance is checked to ensure the rotor is correctly installed. Temperature changes are monitored during centrifugation, and the refrigeration system is activated to maintain a constant temperature if necessary. After centrifugation, the centrifuge is allowed to stand for 2 minutes to allow the flow field to stabilize before gently removing the sample. This separation step is guided by theoretical principles, achieving separation of larger particles through optimized speed and time, while avoiding vesicle loss due to over-centrifugation. Precise control of centrifugation parameters is crucial to the separation effect. The speed error should be within ±1%, and the time error should be within ±30 seconds.

[0063] The specific implementation method of step S06 is to fully characterize the second supernatant. In addition to measuring basic physical parameters, image analysis technology is also introduced. First, the volume, density, viscosity, and temperature are measured using the same measurement method as step S03. Then, dynamic light scattering is used to obtain the particle distribution image, using a laser with a wavelength of 633 nanometers. The temperature is controlled at 25 degrees Celsius during measurement, and each sample is measured three times. Then, two-dimensional transformation technology is used to process the image data, including wavelet transform denoising and Fourier transform to extract frequency domain features. Finally, the distribution fitness is calculated. The horizontal and vertical fitness reflect the uniformity of the particle distribution. The data obtained in this step is more comprehensive, including not only the fluid dynamics parameters but also the particle distribution characteristics. This information is of great value for optimizing the parameters of the third centrifugation.

[0064] The specific implementation of step S07 is to solve the second set of centrifuge parameter optimization equations, which adds a diffusion effect and fitness parameter to the first set of equations. The solution first uses the finite difference method to discretize the equations and construct a difference format. The diffusion term is then solved using the alternating direction implicit method, which has good numerical stability. The nonlinear system of equations is then solved using the Newton iteration method. Finally, the centrifuge speed is optimized using the contribution value calculation equation. During the solution process, grid parameters are set, with a time step of 0.001 seconds and a spatial step of 0.1 mm. The convergence criterion is a residual error of less than 0.0001. This step considers more physical processes, and the optimization results are closer to reality.

[0065] The specific implementation of step S08 involves performing a third centrifugation, the final conventional centrifugation in the separation process. Optimized parameters must be strictly adhered to during operation, with attention to sample temperature control and centrifuge tube balance. After centrifugation, the supernatant is collected using the fraction collection method, with each fraction being equal in volume, from top to bottom. The particle concentration is then measured to confirm the separation effect. This step is a refined separation based on the previous two separations, aiming to further improve sample purity. Centrifugation parameters must be strictly controlled, with the speed error within ±0.5% and the time error within ±10 seconds.

[0066] Step S09 involves filtering through a 0.22-micron pore size filter membrane to remove residual large particles and bacteria. The membrane is rinsed with ultrapure water before filtration; the flow rate is controlled to no more than 2 ml / min; the pressure is monitored during filtration to no more than 0.1 MPa; and the membrane is replaced as necessary to ensure filtration efficiency. This step, based on the principle of sieving, removes particles larger than 0.22 microns through a physical barrier and is an important purification method.

[0067] The specific implementation of step S10 involves concentrating the sample using an ultrafiltration centrifuge tube. First, rinse the ultrafiltration membrane with sterile water; add the filtrate to the ultrafiltration centrifuge tube, filling no more than 90% of the maximum capacity at a time; centrifuge at 4 degrees Celsius and 3000 rpm, checking the concentration factor every 10 minutes; and gently aspirating the concentrate with a pipette when the desired concentration factor is reached. This step concentrates the target product by using the molecular weight cutoff principle. Concentrating to 1 / 5 to 1 / 10 of the original volume can improve the efficiency of subsequent separations. The molecular weight cutoff of the ultrafiltration membrane is selected to be 100 kilodaltons, which effectively retains extracellular vesicles while allowing small molecules to pass through.

[0068] The specific implementation of step S11 involves preparing and adding separation reagents. First, a 30% sucrose solution is prepared. Analytical-grade sucrose is weighed, dissolved in ultrapure water, and the volume is adjusted. The concentration is then confirmed using a refractometer. Next, an 8% polyethylene glycol solution is prepared, using polyethylene glycol with a molecular weight of 6,000. During the dissolution process, the stirring speed should not exceed 200 rpm to avoid the formation of bubbles. Finally, the two solutions are mixed in a 2:1 ratio, stirring at low speed using a magnetic stirrer. This step incorporates the principles of density gradient and phase separation. The sucrose provides a density gradient to facilitate subsequent ultracentrifugation separation, while the polyethylene glycol promotes vesicle enrichment through phase separation.

[0069] The specific implementation method of step S12 is to let the sample stand at low temperature, place the sample with the separation reagent in a 4 degree Celsius refrigerator, and keep the container vertically during the standing process to avoid vibration; after standing for 12 hours, observe whether there is obvious phase separation. If there is no obvious stratification, the standing time can be appropriately extended, but not more than 24 hours. The purpose of this step is to give full play to the phase separation effect. Under low temperature conditions, the system energy is low, which is conducive to reaching a thermodynamic equilibrium state. The control of the standing time is very important. Too short a time is not conducive to phase separation, and too long a time may cause sample degradation.

[0070] The specific implementation of step S13 is to perform ultracentrifugation on the solution after standing in step S12 for 60 minutes using an ultracentrifuge at a speed of 100,000 rpm. The purpose of this step is to completely separate and precipitate the extracellular vesicles from the solution using the powerful centrifugal force of the high-speed centrifuge.

[0071] The specific implementation of step S14 is to resuspend the extracellular vesicle precipitate obtained in step S13 in phosphate buffer to obtain a final extracellular vesicle suspension. The purpose of this step is to appropriately dilute the isolated extracellular vesicles and adjust them to suitable buffer conditions for subsequent characterization and application.

[0072] The specific implementation of step S15 is as follows: first, the particle size distribution of the extracellular vesicle suspension obtained in step S14 is measured, and then the proportion of particles between 40 and 200 nanometers is calculated. When this proportion is greater than 90%, the batch of products is considered qualified. The purpose of this step is to ensure that the isolated extracellular vesicles meet the expected size requirements through particle size analysis.

[0073] The specific implementation of step S16 is to divide the extracellular vesicle suspension determined as qualified in step S15 into 2 ml cryotubes, with each tube containing 0.5 ml. The sample is then stored at -80 degrees Celsius. The purpose of this step is to prepare the isolated and purified extracellular vesicles into a stable cryostock for subsequent application research.

[0074] The specific calculation process involved in the present invention is described in detail below.

[0075] 1. The first mass conservation equation is specifically expressed as follows:

[0076]

[0077] Where ρ is the local density in grams per cubic centimeter; t is the time in seconds; v is the fluid velocity vector; S m is the mass source term; is the Hamiltonian operator; ∈1 is the error term, ranging from 0.001 to 0.01.

[0078] 2. The first momentum conservation equation is specifically expressed as follows:

[0079]

[0080] Where, is the material derivative; p is the pressure, in Pascals; τ is the stress tensor; g is the acceleration due to gravity, which is 9.81 meters per square second; F c is the centrifugal force, F c =ρω 2 r; ω is the angular velocity; r is the rotation radius; ∈2 is the error term, ranging from 0.01 to 0.1.

[0081] 3. The first sedimentation rate equation is specifically expressed as follows:

[0082]

[0083] Where, v s is the sedimentation rate in meters per second; d is the particle diameter in micrometers; ρ p is the particle density; ρ f is the fluid density; μ is the dynamic viscosity; C d is the resistance coefficient; K f is the shape factor; ∈3 is the error term, ranging from 0.001 to 0.01.

[0084] 4. The first separation efficiency equation is specifically expressed as follows:

[0085]

[0086] Where, E is the separation efficiency; t s is the sedimentation time; v t is the tangential velocity; d c is the critical particle size; α1, α2 are fitting coefficients; ∈4 is the error term, ranging from 0.01 to 0.05; α1 is 0.1 to 0.5, and α2 is 0.2 to 0.6.

[0087] 5. The second mass conservation equation is specifically expressed as follows:

[0088]

[0089] Where β1 and β2 are diffusion coefficients; x and y are spatial coordinates; ∈5 is the error term, ranging from 0.001 to 0.01.

[0090] 6. The second momentum conservation equation is specifically expressed as follows:

[0091]

[0092] Where, F d is the diffusion force, D is the diffusion coefficient; c is the concentration; ∈6 is the error term, ranging from 0.01 to 0.1.

[0093] 7. The second sedimentation rate equation is specifically expressed as follows:

[0094]

[0095] Where γ1,γ2 are velocity correction coefficients; θ is the angular coordinate; ∈7 is the error term, ranging from 0.001 to 0.01; γ1,γ2 are 0.81.2

[0096] 8. The second separation efficiency equation is specifically expressed as follows:

[0097]

[0098] Where, E total is the total separation efficiency; E base is the basic separation efficiency, f(A h ,A v ) is the fitness function; A h is the horizontal distribution fitness; A v is the vertical distribution fitness; i is the weight coefficient; d i is the diameter of the i-th type of particle; α i is the fitting index; n is the number of particle types; ∈8 is the error term, ranging from 0.01 to 0.05.

[0099] 9. The calculation formula of horizontal distribution fitness is as follows:

[0100]

[0101] Where N is the number of sampling points; φ i is the weight factor; x i is the horizontal coordinate; μ x is the horizontal mean; σ xis the horizontal standard deviation; ∈9 is the error term, ranging from 0.01 to 0.05.

[0102] 10. The calculation formula of vertical distribution fitness is as follows:

[0103]

[0104] Where M is the number of sampling points; ψ j is the weight factor; y j is the vertical coordinate; μ y is the longitudinal mean; σ y is the vertical standard deviation; 10 is the error term, ranging from 0.01 to 0.05.

[0105] 11. The second contribution value calculation equation is specifically expressed as follows:

[0106]

[0107] Where C is the contribution value; k1, k2, k3, k4 are weight coefficients; ω c is the critical angular velocity; ∈ 11 is the error term, ranging from 0.01 to 0.05.

[0108] The parameter acquisition method is as follows:

[0109] 1. Method for obtaining particle diameter d:

[0110] Step 1: The sample was measured using a dynamic light scattering instrument at a temperature of 25°C and a laser wavelength of 633 nm.

[0111] Step 2: Each sample was measured three times, each measurement lasted 60 seconds, and 300 data points were collected;

[0112] Step 3: The average diameter and distribution of the particles were obtained by cumulative analysis, with a repeatability deviation of less than 3%.

[0113] 2. How to obtain density ρ:

[0114] Step 1: Prepare a sucrose density gradient from 20% to 40% with a 2% gradient interval.

[0115] Step 2: Layer sucrose solutions of different concentrations in a density gradient centrifuge tube from bottom to top, with each layer having a volume of 1 ml;

[0116] Step 3: Load the sample on top of the gradient and centrifuge at 200,000 rpm for 4 hours;

[0117] Step 4: Collect each density zone and measure the density by gravimetric method at a temperature of 4°C.

[0118] 3. Method for obtaining dynamic viscosity μ:

[0119] Step 1: Use an Ubbelohde capillary viscometer with an inner diameter of 0.5 mm.

[0120] Step 2: Control the temperature of the water bath at 25 ± 0.1 °C;

[0121] Step 3: Measure the time t for the sample to flow through the capillary;

[0122] Step 4: Calculate the dynamic viscosity using the formula μ = Ktρ, where K is the viscometer constant.

[0123] Step 5: Repeat the measurement for each sample 5 times and take the average value.

[0124] 4. How to obtain weight coefficients k1, k2, k3, k4:

[0125] Step 1: Collect 50 sets of experimental data, including separation efficiency under different speed and time conditions;

[0126] Step 2: Construct the least squares objective function

[0127] Step 3: Use the gradient descent method to find the weight coefficient when the objective function is minimized;

[0128] Step 4: Confirm the reliability of the weight coefficients through cross-validation.

[0129] 5. Fitness parameter A h ,A v How to obtain:

[0130] Step 1: Use a high-speed camera system to record particle distribution images at a shooting rate of 1000 frames per second;

[0131] Step 2: Use image analysis software to process the image and extract the particle distribution coordinates;

[0132] Step 3: Calculate the particle distribution density in the horizontal and vertical directions;

[0133] Step 4: Obtain the distribution parameter μ through Gaussian fitting x ,μ y ,σ x ,σ y .

[0134] 6. Error term ∈1 to ∈ 11 How to obtain:

[0135] Step 1: Perform 10 replicates under standard conditions.

[0136] Step 2: Record the deviation between the measured value and the theoretical prediction value for each experiment;

[0137] Step 3: Calculate the standard deviation

[0138] Step 4: Take the error range with a confidence level of 95%.

[0139] 7. Fit Index α i How to obtain:

[0140] Step 1: Collect separation efficiency data at different rotation speeds;

[0141] Step 2: Draw a double logarithmic curve of ln(E) versus ln(d / d c );

[0142] Step 3: The slope obtained by linear regression is the fitting index;

[0143] Step 4: By determining the coefficient R 2 Verify the fitting effect.

[0144] 8. Shape Factor K f How to obtain:

[0145] Step 1: Observe the particle morphology using transmission electron microscopy;

[0146] Step 2: Measure the ratio of the major axis to the minor axis;

[0147] Step 3: Use empirical formula Calculate, where a is the major axis and b is the minor axis;

[0148] Step 4: Calculate the shape factors of 100 particles and take the average value.

[0149] The specific derivation process of multiple equations is provided below:

[0150] 1. Derivation of the first mass conservation equation:

[0151] Step 1: From the continuity equation Set off;

[0152] Step 2: Considering the material exchange during the separation of extracellular vesicles, the source term S is introduced m ;

[0153] Step 3: To improve the accuracy of the model, add an error correction term ∈1;

[0154] Innovative effect: The material exchange in the separation process is reflected through the source term, making the model more consistent with the actual situation.

[0155] 2. Derivation of the first momentum conservation equation:

[0156] Step 1: Based on the Navier-Stokes equations;

[0157] Step 2: Consider the particularity of the rotating system and introduce the centrifugal force term F c ;

[0158] Step 3: Decompose the stress tensor τ into shear stress and normal stress;

[0159] Step 4: Add an error term ∈2 to compensate for the error caused by model simplification;

[0160] Innovation effect: Completely describes the mechanical behavior in the rotating system and provides a theoretical basis for optimizing centrifugal parameters. 3. Derivation of the first sedimentation rate equation:

[0161] Step 1: Starting from the Stokes equations;

[0162] Step 2: Introduce the centrifugal field correction term ω 2 r;

[0163] Step 3: Consider the shape effect and increase the shape factor K f item;

[0164] Step 4: Increase the drag coefficient C d Correction items;

[0165] Step 5: Introduce error term ∈3;

[0166] Innovative effect: It describes the sedimentation behavior of micro-nanoparticles more accurately than the traditional Stokes equation.

[0167] 4. Derivation process of the second separation efficiency equation:

[0168] Step 1: Establish the basic separation efficiency expression E base ;

[0169] Step 2: Introduce the fitness function f(A h ,A v ) Corrected separation efficiency;

[0170] Step 3: Consider the influence of particle size distribution and add polynomial correction terms

[0171] Step 4: Add error term ∈8;

[0172] Innovation effect:

[0173] (a) Considering the two-dimensional nature of particle distribution;

[0174] (b) The introduction of fitness function improves the accuracy of the model;

[0175] (c) The polynomial correction term reflects the separation characteristics of particles of different sizes.

[0176] 5. Derivation of the contribution value calculation equation:

[0177] Step 1: Analyze the key factors affecting separation efficiency;

[0178] Step 2: Establish the lateral fitness term k1A h and the longitudinal fitness term k2A v ;

[0179] Step 3: Add speed influence

[0180] Step 4: Introduce efficiency gradient term

[0181] Step 5: Add error correction term ∈ 11 ;

[0182] Innovation effect:

[0183] (a) Adaptive optimization of separation parameters is achieved;

[0184] (b) Reduces the computational complexity of parameter optimization.

[0185] The following is a specific example 1 of the present invention. The specific implementation of each step in this example 1 is described in detail as follows: The specific implementation of step S01 is as follows. First, a sample of cell culture fluid to be processed is collected from a laboratory or production workshop. Then, the cell density of the cell culture fluid is measured using an automated cell counter. Specifically, the sample to be tested is added to the counting chamber of the counter, and the number of cells per milliliter of fluid is counted using image recognition and algorithm analysis techniques. Simultaneously, the cell viability of the cell culture fluid is measured using a cell viability assay kit. This kit uses a metabolic activity indicator dye to provide the percentage of viable cells through colorimetric or fluorescence detection. After these tests, cell density and cell viability data for the cell culture fluid are obtained. Finally, from the measured data, cell culture fluids with a cell density between 1,000,000 and 2,000,000 cells per milliliter and a cell viability greater than 90% are selected as raw materials for separation and purification. The purpose of this step is to ensure that the cells in the raw material are in good condition, which is beneficial for the subsequent separation and purification of extracellular vesicles.

[0186] The specific implementation method of step S02 is as follows. The cell culture fluid raw material obtained by screening in step S01 is transferred to a centrifuge tube, the centrifugal speed is set to 1000 rpm, and the centrifugation time is 10 minutes. This centrifugation process uses the action of centrifugal force to cause larger cells and impurity particles to settle to the bottom of the tube, and the supernatant containing extracellular vesicles can be separated. Specifically, under the action of centrifugal force of 1000 rpm, cell fragments and other large particles with a diameter greater than 200 nanometers will be centrifuged and precipitated, while extracellular vesicles with a diameter between 40-200 nanometers will be separated together with the supernatant. Through this preliminary centrifugation step, cells and large particle impurities in the raw material can be removed, laying the foundation for subsequent extracellular vesicle enrichment and further purification.

[0187] The specific implementation of step S03 is as follows. First, a densitometer and viscometer are used to measure the density and dynamic viscosity of the supernatant obtained in step S02, respectively. Density can be measured using either the float-sink method or the oscillating tube method, with the measurement temperature controlled at 25 degrees Celsius. Viscosity can be measured using a capillary viscometer, converting the time required for the liquid to flow through the capillary tube into dynamic viscosity, also controlled at 25 degrees Celsius. Furthermore, the ambient temperature during the experiment must be recorded. Next, the measured supernatant volume, density, viscosity, and ambient temperature parameters are input into the first set of centrifugation parameter optimization equations. This first set of centrifugation parameter optimization equations consists of four equations: the mass conservation equation, the momentum conservation equation, the sedimentation rate equation, and the separation efficiency equation. These equations describe the physicochemical laws governing the changes in extracellular vesicle mass distribution, particle motion, sedimentation velocity, and separation efficiency during centrifugation. By solving this set of equations, the theoretical sedimentation time and optimal centrifuge speed required for the second centrifugation can be determined. The purpose of this step is to establish a mathematical model to provide a basis for optimizing the subsequent centrifugation process.

[0188] The specific implementation of step S04 is as follows: According to the parameters input in step S03, the first centrifugal parameter optimization equation group is solved to obtain the theoretical sedimentation time t of the second centrifugation. s and the optimal centrifugal speed ω opt First, the first mass conservation equation can be expressed as Where ρ is the local density, t is the time, v is the fluid velocity vector, S m is the mass source term, is the Hamiltonian operator, and ∈1 is the error term. This equation describes the change in the mass distribution of extracellular vesicles during centrifugation. Secondly, the first momentum conservation equation can be expressed as in is the material derivative, p is the pressure, τ is the stress tensor, g is the gravitational acceleration, F cis the centrifugal force, and ∈2 is the error term. This equation describes the motion state of the particles during centrifugation. Again, the first sedimentation rate equation can be expressed as where v s is the sedimentation rate, d is the particle diameter, ρ p is the particle density, ρ f is the fluid density, μ is the dynamic viscosity, C d is the resistance coefficient, K f is the shape factor, and ∈3 is the error term. This equation is used to calculate the sedimentation velocity of particles during centrifugation. Finally, the first separation efficiency equation can be expressed as Where E is the separation efficiency, t s is the sedimentation time, v t is the tangential velocity, d c is the critical particle size, α1, α2 are fitting coefficients, and ∈4 is the error term. This equation is used to evaluate the effect of the second centrifugation and calculate the optimal centrifugal speed ω opt By solving these four equations, we can obtain the theoretical sedimentation time t required for the second centrifugation. s and the optimal centrifugal speed ω opt The purpose of this step is to optimize the key parameters of the second centrifugation based on mathematical modeling and provide guidance for the subsequent separation process.

[0189] The specific implementation of step S05 is as follows: The theoretical sedimentation time t of the second centrifugation obtained in step S04 s and the optimal centrifugal speed ω opt , the first supernatant obtained in step S02 is centrifuged for the second time. The specific operation is: transfer the first supernatant to a centrifuge tube, set the centrifugal speed to ω opt , centrifugation time is t s During this centrifugation process, Due to the relationship between the sedimentation rate and the size of the centrifuge, larger cell fragments and other impurity particles will preferentially settle to the bottom of the tube, while the extracellular vesicles will be separated along with the supernatant. This optimized second centrifugation further removes large impurities from the raw material and improves the purity of the extracellular vesicles. The purpose of this step is to perform a secondary separation of the extracellular vesicles using the centrifugation parameters optimized in step S04.

[0190] The specific implementation of step S06 is as follows. First, a density meter, a viscometer and a thermometer are used to measure the density, dynamic viscosity and ambient temperature of the second supernatant obtained in step S05 respectively. Similar to step S03, the density can be measured by the floating sinking method or the oscillating tube method, and the viscosity can be measured using a capillary viscometer, and the measurement temperature is controlled at 25 degrees Celsius. Then, a high-speed camera system is used to record the particle distribution image of the supernatant, and the image is analyzed and processed using two-dimensional transformation technology to obtain the distribution of particles in the horizontal and vertical directions. Specifically, the fitness A of the lateral distribution can be calculated. h and the fitness of the vertical distribution A v , expressed as and Where N, M are the number of sampling points, φ i ,ψ j is the weight factor, x i ,y j is the coordinate, μ x ,μ y ,σ x ,σ y is the statistical parameter,∈9,∈ 10 is the error term. Finally, the measured second supernatant volume, density, viscosity, ambient temperature, and lateral adaptability A h and longitudinal fitness A v The parameters such as the two-dimensional distribution characteristics are input into the second centrifugation parameter optimization equation group. The purpose of this step is to further optimize the parameters of the third centrifugation through the two-dimensional distribution characteristics to improve the separation effect.

[0191] The specific implementation of step S07 is as follows: According to the parameters input in step S06, solve the second centrifugal parameter optimization equation group to obtain the theoretical sedimentation time t of the third centrifugation s and the optimal centrifugal speed ω opt The second centrifugal parameter optimization equation group includes five equations, namely, the mass conservation equation, the momentum conservation equation, the sedimentation rate equation, the separation efficiency equation and the contribution value calculation equation. Among them, the second mass conservation equation can be expressed as Describes the change in mass distribution of extracellular vesicles during the third centrifugation. The second momentum conservation equation can be expressed as Describes the motion state of the particles, where F d is the diffusion force. The second sedimentation rate equation can be expressed as describes the sedimentation velocity of particles during centrifugation. The second separation efficiency equation can be expressed as Describes the overall separation effect of the third centrifugation, where E base is the basic separation efficiency, f(A h ,A v) is the fitness function, λ i ,α i is the fitting parameter. Finally, the second contribution value calculation equation can be expressed as The optimal speed ω for optimizing the third centrifugation opt By solving these five equations, we can obtain the theoretical sedimentation time t required for the third centrifugation. s and the optimal centrifugal speed ω opt The purpose of this step is to further optimize the key parameters of the third centrifugation based on more sophisticated mathematical modeling, providing more optimized guidance for the subsequent separation process.

[0192] The specific implementation of step S08 is as follows: The theoretical sedimentation time t of the third centrifugation obtained in step S07 s and the optimal centrifugal speed ω opt , the second supernatant obtained in step S06 is centrifuged for the third time. The specific operation is: transfer the second supernatant to a centrifuge tube, set the centrifugal speed to ω opt , centrifugation time is t s During this centrifugation process, based on the optimized relationship between sedimentation rate and separation efficiency, residual cell debris and other impurity particles can be further removed, allowing extracellular vesicles to be enriched in the supernatant. This optimized third centrifugation can significantly improve the purity of extracellular vesicles. The purpose of this step is to utilize the centrifugation parameters optimized in step S07 to perform a third separation of extracellular vesicles, further improving the separation effect.

[0193] The specific implementation of step S09 is as follows. The third supernatant obtained in step S08 is filtered using a membrane filter with a pore size of 0.22 microns. During the filtration process, residual cell debris and other large impurities with a diameter larger than 0.22 microns are retained on the membrane surface, while extracellular vesicles with diameters between 40 and 200 nanometers are able to pass through the membrane pores and enter the filtrate. The purpose of this filtration step is to further remove large impurities from the supernatant, preparing it for subsequent concentration and addition of separation reagents.

[0194] The specific implementation of step S10 is as follows. The filtrate obtained in step S09 is concentrated using an ultrafiltration centrifuge tube. Specifically, the filtrate is transferred to an ultrafiltration centrifuge tube. Under the centrifugal force of 4000 rpm, extracellular vesicles are concentrated on the membrane surface, while small molecular impurities are filtered out through the membrane pores. After concentration, the original volume can be reduced to 1 / 5 to 1 / 10. The purpose of this concentration step is to significantly increase the concentration of extracellular vesicles, preparing for subsequent density gradient separation.

[0195] The specific implementation of step S11 is as follows. First, prepare a separation reagent comprising a 30% by mass sucrose solution and an 8% by mass polyethylene glycol solution. Then, mix the concentrate obtained in step S10 with the separation reagent at a volume ratio of 2:1. The mixing of the sucrose solution and the polyethylene glycol solution forms a stable density gradient, which allows for further separation of extracellular vesicles by utilizing the differences in their distribution within the density gradient. The purpose of this step is to further improve the purity of extracellular vesicles by utilizing the principles of density gradient separation.

[0196] The specific implementation of step S12 is as follows. The mixed solution obtained in step S11 is allowed to stand at 4 degrees Celsius for 12 hours. During this standing period, the extracellular vesicles will spontaneously migrate within the density gradient and eventually settle, while other impurity particles will remain dispersed. The purpose of this standing step is to fully utilize the density difference to facilitate the isolation and enrichment of extracellular vesicles.

[0197] The specific implementation of step S13 is as follows. The solution left standing in step S12 is separated using ultracentrifugation. Specifically, the centrifuge speed is set to 100,000 rpm and the centrifugation time is set to 60 minutes. Under the centrifugal force of such a high speed, extracellular vesicles (EVs) with a diameter of 40-200 nanometers will completely settle to the bottom of the centrifuge tube, while other smaller impurity particles will remain suspended. The purpose of this ultracentrifugation step is to completely separate and precipitate the EVs, further improving their purity.

[0198] The specific implementation of step S14 is as follows. First, carefully aspirate the extracellular vesicle precipitate obtained in step S13. Then, resuspend the precipitate in phosphate buffer. This resuspension process evenly disperses the isolated extracellular vesicles in the buffer, resulting in a final extracellular vesicle suspension. The purpose of this step is to appropriately dilute and suspend the isolated extracellular vesicles to suitable buffer conditions, preparing them for subsequent characterization and application.

[0199] The specific implementation method of step S15 is as follows. First, a dynamic light scattering instrument is used to measure the particle size distribution of the particles in the extracellular vesicle suspension obtained in step S14. During the measurement, the sample temperature is controlled at 25 degrees Celsius, the laser wavelength is 633 nanometers, and each sample is measured three times, each time for 60 seconds, and a total of 300 data points are collected. By cumulatively analyzing the measurement data, the average particle size of the extracellular vesicles and its distribution can be obtained. Then, the proportion of particles between 40 and 200 nanometers is calculated. When this proportion is greater than 90%, the batch of extracellular vesicle products is judged to be qualified. The purpose of this step is to ensure that the separated extracellular vesicles meet the expected size requirements and meet the application needs through particle size analysis.

[0200] The specific implementation of step S16 is as follows. The extracellular vesicle suspension determined as qualified in step S15 is aliquoted into sterile 2 ml cryovials. Each tube contains 0.5 ml, and the sample is then stored at an ultra-low temperature of -80 degrees Celsius. This aliquoting and storage step aims to produce a stable cryopreserved extracellular vesicle stock to provide sufficient sample for subsequent applied research.

[0201] The following provides Example 2 of a specific application scenario of the present invention:

[0202] A biotechnology company is studying the potential application of extracellular vesicles in tumor immunotherapy. To obtain high-purity extracellular vesicle samples, the company decided to use the rapid separation and purification method proposed in this invention.

[0203] First, the company collected a sample of cell culture fluid from a tumor cell culture laboratory. After cell counting and viability testing, the sample was found to have a cell density of 1.5 × 10^6 cells per milliliter and a cell viability of 95%, meeting the raw material requirements for the method.

[0204] Step S01: Collect cell culture medium and measure cell density and survival rate

[0205] The specific operations are as follows:

[0206] 1) Collect 100 ml of tumor cell culture medium and transfer it to a sterile centrifuge tube.

[0207] 2) The cell density of the culture medium was measured using an automatic cell counter, and the result was 1.5×10^6 cells per ml.

[0208] 3) Using a cell activity detection kit, the cell viability of the culture medium was measured to be 95%.

[0209] 4) According to the test results, the cell culture medium meets the raw material requirements of the method of the present invention and can proceed to the next step.

[0210] Step S02: Perform the first centrifugation

[0211] 1) Transfer the cell culture medium obtained in step S01 into a centrifuge tube.

[0212] 2) Set the centrifugal speed to 1000 rpm and the centrifugal time to 10 minutes.

[0213] 3) After centrifugation, carefully aspirate the supernatant to avoid aspirating the precipitate.

[0214] 4) The volume of the supernatant was measured to be 90 ml.

[0215] Step S03: Determine the first supernatant parameters and establish the first centrifugation parameter optimization equation group

[0216] 1) The density of the first supernatant was measured using a density meter and was found to be 1.006 g / ml.

[0217] 2) The dynamic viscosity of the supernatant was measured using a capillary viscometer and was found to be 1.12×10^-3 Pa·s.

[0218] 3) Record that the ambient temperature during the experiment is 25 degrees Celsius.

[0219] 4) Substituting the above measured data into the first centrifugal parameter optimization equation group, including:

[0220] The first mass conservation equation:

[0221] The first momentum conservation equation:

[0222] The first sedimentation rate equation:

[0223] The first separation efficiency equation:

[0224] Step S04: Solving the first centrifugal parameter optimization equations

[0225] Substituting the measured data of step S03, the theoretical sedimentation time t of the second centrifugation is obtained through mathematical solution. s = 600 seconds and optimal centrifugal speed ω opt =3000 rpm.

[0226] like Figure 2 The figure shows the relationship between centrifugal speed and separation efficiency during three centrifugation processes. Different line types are used to distinguish the three centrifugations.

[0227] Step S05: Perform a second centrifugation

[0228] 1) Transfer 90 ml of the supernatant obtained in step S02 to a centrifuge tube.

[0229] 2) Set the centrifugal speed to 3000 rpm and the centrifugal time to 600 seconds.

[0230] 3) After centrifugation, carefully aspirate the supernatant to obtain 80 ml.

[0231] Step S06: Determine the second supernatant parameters and establish the second centrifugation parameter optimization equation group

[0232] 1) The volume of the second supernatant was measured to be 80 mL, the density was 1.009 g / mL, and the dynamic viscosity was 1.15 × 10-3 Pa·s. The ambient temperature remained at 25°C.

[0233] 2) Use a high-speed camera system to record the particle distribution image of the supernatant, and calculate the lateral distribution fitness A through two-dimensional transformation analysis. h =0.87, vertical distribution fitness A v =0.91.

[0234] 3) Substituting the above parameters into the second centrifugal parameter optimization equation group, including:

[0235] The second mass conservation equation:

[0236] The second momentum conservation equation:

[0237] The second sedimentation rate equation:

[0238] The second separation efficiency equation:

[0239] The second contribution value calculation equation:

[0240] like Figure 3 The figure shows the trend of dynamic viscosity and temperature throughout the separation process. Using dual Y-axes, the blue line represents the change in dynamic viscosity and the red line represents the change in temperature.

[0241] Step S07: Solving the second centrifugal parameter optimization equations

[0242] 1) Through mathematical solution, the theoretical sedimentation time t of the third centrifugation is obtained s = 480 seconds and the optimal centrifugal speed ω opt =4500 rpm.

[0243] Step S08: Perform the third centrifugation

[0244] 1) Transfer 80 ml of the supernatant obtained in step S05 to a centrifuge tube.

[0245] 2) Set the centrifugal speed to 4500 rpm and the centrifugal time to 480 seconds.

[0246] 3) After centrifugation, carefully aspirate the supernatant to obtain 70 ml.

[0247] like Figure 4 The figure shows the trend of sample volume and density changes during three centrifugations. The double Y-axis is used for display, with the blue line representing volume change and the red line representing density change.

[0248] Step S09: Use membrane filtration to remove residual impurities

[0249] 1) Filter the 70 ml supernatant obtained in step S08 through a membrane filter with a pore size of 0.22 μm.

[0250] 2) After filtration, 60 ml of filtrate can be obtained.

[0251] Step S10: ultrafiltration concentration

[0252] 1) Transfer 60 ml of the filtrate obtained in step S09 to an ultrafiltration centrifuge tube.

[0253] 2) Under the action of centrifugal force at 4000 rpm, extracellular vesicles are enriched on the membrane surface, while small molecular impurities are filtered out through the membrane pores.

[0254] 3) After 30 minutes of concentration, 12 ml of concentrated solution was finally obtained.

[0255] Step S11: Add density gradient separation reagent

[0256] 1) Prepare 30% by mass sucrose solution and 8% by mass polyethylene glycol solution.

[0257] 2) The 12 ml concentrate obtained in step S10 was mixed with the above sucrose solution and polyethylene glycol solution at a volume ratio of 2:1.

[0258] Step S12: Density gradient separation and standing

[0259] 1) The mixed solution obtained in step S11 was placed in a 4°C environment for 12 hours.

[0260] Step S13: Ultracentrifugation

[0261] 1) The solution after standing in step S12 was centrifuged at an ultrahigh centrifugal speed of 100,000 rpm for 60 minutes.

[0262] 2) After centrifugation, it can be observed that the extracellular vesicles have completely settled at the bottom of the tube.

[0263] Step S14: Resuspending extracellular vesicles

[0264] 1) Carefully aspirate the extracellular vesicles precipitated in step S13 and resuspend in phosphate buffered saline.

[0265] 2) After resuspension, 3 ml of the final extracellular vesicle product can be obtained.

[0266] Step S15: Check product quality

[0267] 1) The particle size distribution of the 3 ml extracellular vesicle product was measured using a dynamic light scattering instrument.

[0268] 2) Statistical results show that particles of 40-200 nanometers account for 95%, which meets the qualification criteria of the method of the present invention.

[0269] like Figure 5 The bar graph shows the size distribution of extracellular vesicles in the final product, with the cumulative distribution shown as a red line and the target size range (40-200 nm) marked in green.

[0270] Step S16: product packaging and storage

[0271] 1) The qualified extracellular vesicle product from step S15 was divided into 2 ml sterile cryopreservation tubes, with 0.5 ml in each tube.

[0272] 2) Store the samples in an ultra-low temperature environment of -80 degrees Celsius for subsequent research.

[0273] Through the 16-step extracellular vesicle isolation and purification process described above, the biotechnology company successfully obtained 3 ml of a highly pure (95%) extracellular vesicle product. The entire process took approximately 4.5 hours and was relatively simple, requiring no complex equipment.

[0274] Technical Principle: The core of the rapid extracellular vesicle separation and purification method proposed in this invention lies in the precise control of the multi-step centrifugation separation process through mathematical modeling and parameter optimization, and ultimately combines it with density gradient separation to obtain a high-purity extracellular vesicle product. The technical principles of this method are as follows:

[0275] First, samples with the required cell density and viability were screened from the initial cell culture medium to lay the foundation for subsequent separation. Then, a first centrifugation was performed to remove large particles and obtain a supernatant containing extracellular vesicles. Next, by establishing a set of equations for optimizing the parameters of the first centrifugation, including equations for conservation of mass, conservation of momentum, sedimentation rate, and separation efficiency, the optimal parameters for the second centrifugation were calculated to further isolate the extracellular vesicles.

[0276] After the second centrifugation, two-dimensional transformation technology is used to analyze the particle distribution in the supernatant. This allows the development of a set of equations for optimizing the parameters of the second centrifugation and to optimize the key parameters of the third centrifugation to obtain extracellular vesicles of higher purity. This step embodies the innovation of the present method, fully utilizing particle distribution information to guide the optimization of centrifugation.

[0277] Membrane filtration is then used to remove remaining large particles, followed by ultrafiltration to further enrich the extracellular vesicles. Finally, using the principle of density gradient separation, the density difference between sucrose and polyethylene glycol is exploited to separate the extracellular vesicles from other impurities, resulting in the final high-purity product.

[0278] It should be noted that the variables involved in the present invention are explained in detail as shown in Table 1 below.

[0279] Table 1 Variable explanation table

[0280]

[0281]

[0282] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with this technical field can easily think of changes or replacements within the technical scope disclosed by the present invention, which should be covered by the scope of protection of the present invention.

Claims

1. A method for rapid separation and purification of extracellular vesicles, characterized in that: The cell culture fluid was collected and extracellular vesicles were obtained by multiple centrifugation separations, wherein the second centrifugation and the third centrifugation were optimized by a first centrifugation parameter optimization equation group and a second centrifugation parameter optimization equation group, respectively. The first centrifugation parameter optimization equation group included a first mass conservation equation, a first momentum conservation equation, a first sedimentation rate equation, and a first separation efficiency equation. The second centrifugation parameter optimization equation group introduced a two-dimensional transformation technology to establish a particle distribution image and calculate the lateral distribution fitness and the longitudinal distribution fitness. The centrifugal speed was optimized by a second contribution value calculation equation. The second centrifugation parameter optimization equation group included a second mass conservation equation, a second momentum conservation equation, a second sedimentation rate equation, a second separation efficiency equation, and the second contribution value calculation equation. The extracellular vesicle suspension was obtained after membrane filtration, ultrafiltration concentration, separation reagent treatment, and ultracentrifugation.

2. The method for rapid separation and purification of extracellular vesicles according to claim 1, characterized in that: A cell culture fluid with a cell density of 1,000,000 to 2,000,000 cells per milliliter and a cell viability greater than 90% is selected as a raw material, and the raw material is subjected to a first centrifugation at a centrifugal speed of 1,000 revolutions per minute for 10 minutes to obtain a first supernatant.

3. The method for rapid separation and purification of extracellular vesicles according to claim 2, characterized in that: The first centrifugal parameter optimization equation group includes a first mass conservation equation, a first momentum conservation equation, a first sedimentation rate equation, and a first separation efficiency equation. By solving the first centrifugal parameter optimization equation group, the first theoretical sedimentation time and the first optimal centrifugal speed are obtained.

4. The method for rapid separation and purification of extracellular vesicles according to claim 3, characterized in that: The input of the first mass conservation equation includes the volume, density, and ambient temperature of the first supernatant, and the output is the first mass distribution function. The input of the first momentum conservation equation includes the first mass distribution function and viscosity, and the output is the first particle instantaneous velocity.

5. The method for rapid separation and purification of extracellular vesicles according to claim 4, characterized in that: The input of the first sedimentation rate equation includes the instantaneous velocity of the first particle and the density, and the output is the first theoretical sedimentation time. The input of the first separation efficiency equation includes the first theoretical sedimentation time and the volume of the first supernatant, and the output is the first optimal centrifugal speed.

6. The method for rapid separation and purification of extracellular vesicles according to claim 5, characterized in that: The second centrifugal parameter optimization equation group includes a second mass conservation equation, a second momentum conservation equation, a second sedimentation rate equation, a second separation efficiency equation, and a second contribution value calculation equation. By solving the second centrifugal parameter optimization equation group, the second theoretical sedimentation time and the second optimal centrifugal speed are obtained.

7. The method for rapid separation and purification of extracellular vesicles according to claim 6, characterized in that: The input of the second mass conservation equation includes the volume of the second supernatant, the density, and the ambient temperature, and the output is the second mass distribution function. The input of the second momentum conservation equation includes the second mass distribution function and the viscosity, and the output is the second particle instantaneous velocity.

8. The method for rapid separation and purification of extracellular vesicles according to claim 7, characterized in that: The input of the second sedimentation rate equation includes the instantaneous velocity of the second particles and the density, and the output is the second theoretical sedimentation time. The input of the second separation efficiency equation includes the second theoretical sedimentation time, the volume of the second supernatant, the lateral distribution fitness, and the longitudinal distribution fitness. The centrifugal efficiency is calculated based on polynomial fitting, and the output is the initial second optimal centrifugal speed.

9. The method for rapid separation and purification of extracellular vesicles according to claim 8, characterized in that: The input of the second contribution value calculation equation includes the transverse distribution fitness, the longitudinal distribution fitness, and the initial second optimal centrifugal speed. The contribution values ​​of different speeds to the separation effect are calculated, and the output is the second optimal centrifugal speed.

10. The method for rapid separation and purification of extracellular vesicles according to claim 9, characterized in that: The third supernatant was filtered using a membrane filter with a pore size of 0.22 microns to obtain a filtrate, and the filtrate was concentrated to 1 / 5 to 1 / 10 of the original volume using an ultrafiltration centrifuge tube to obtain a concentrate. A separation reagent was added to the concentrate, wherein the separation reagent included a 30% by mass sucrose solution and an 8% by mass polyethylene glycol solution, and the mixing ratio of the sucrose solution to the polyethylene glycol solution was 2:

1. After standing at 4° C. for 12 hours, the concentrate was ultracentrifuged at 100,000 rpm for 60 minutes.

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