CN118440893A - Preparation method of high-purity stem cell exosome - Google Patents
Preparation method of high-purity stem cell exosome Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The invention discloses a preparation method of a high-purity stem cell exosome, which relates to the technical field of exosome purification, and comprises the following steps: s1, producing a stem cell culture supernatant liquid for exosomes; s2, filtering the supernatant liquid through a multi-filter to remove cell fragments and large vesicles; s3, adding a chemical reagent for inducing degradation of the hybrid protein to decompose the hybrid protein in the feed liquid so as to obtain a high-purity stock solution; s4, adding nuclease to remove chromatin impurities in the feed liquid; s5, passing through a multiple chromatographic column to obtain high-purity exosome solution; s6, treating the enriched exosome solution through a three-stage tangential flow filtration system; s7, guaranteeing sterility of a final product through filter treatment; the method can realize mass production of high-purity exosomes by combining multiple filtration, multiple tangential flows, chemical induction, serial chromatography and other modes, and the prepared exosomes have high purity and few impurities.
Description
Technical Field
The invention relates to the technical field of exosome purification, in particular to a preparation method of a high-purity stem cell exosome.
Background
Extracellular vesicles are a class of nanoscale vesicles derived from cells that carry a variety of nucleic acids, proteins, and other biomolecules of maternal cell origin. The composition is complex, and can be divided into exosomes (30-150 nm), microvesicles (100-1000 nm) and apoptotic bodies (> 1000 nm) according to the size. Among them, exosomes have been receiving increasing attention in the past decade due to their very important applications in immunosuppression, cancer progression, regenerative medicine, early screening of diseases, etc.
There are many methods for separation of exosomes, such as polymer precipitation, immunoaffinity, size exclusion and currently accepted gold standard ultracentrifugation. Ultracentrifugation is a physical separation method based on different sedimentation rates of particle density, but because lipoproteins, particularly high density lipoproteins, have very overlapping density sizes with exosomes, co-separation of the lipoproteins and exosomes is very easy to affect the purity of a sample when using the ultracentrifugation method, particularly when exosomes are separated in plasma, because the lipoprotein content in plasma is about 6 orders of magnitude higher than that of the exosomes, the exosomes in plasma are very easy to be polluted by lipoproteins, and pathological analysis results are affected. Also when size exclusion chromatography, i.e. a method based on particle size separation, is used, it is difficult to separate lipoproteins, especially low density lipoproteins, from exosomes due to the overlapping of these two species.
Styrene-maleic anhydride (SMA) copolymers have a long history in the life sciences, and were originally used as conjugates of cancer therapeutic drugs. It was later discovered that SMA interacted with phospholipids, and that SMA induced spontaneous formation of a bio-lipid film into disc-like particles of about 10nm in diameter, which solubilized lipoprotein breakdown while maintaining protein homeostasis, and thus could be used to remove lipoproteins from cell supernatants. As SMA was developed, SIGMA ALDRICH (USA) company has produced commercial SMA polymers. The different proportions of styrene and maleic anhydride lead to different properties of the polymer, and the prior data prove that the copolymer has better effect under the physiological pH condition when the proportion of the styrene to the maleic anhydride is 2:1; the Chinese patent application No. 202111509707.1 discloses a purification method of EGF mesenchymal stem cell exosomes, and the preparation method in the prior art does not treat cell supernatant liquid by adding SMA polymer and nuclease, so that the purification method is inconvenient for preparing high-purity exosomes; the method is not combined by multiple filtration, multiple tangential flow, chemical induction, serial chromatography and other modes, is not convenient for realizing the production of large-batch and high-purity exosomes, and the purity of the prepared exosomes needs to be improved; therefore, in view of this current situation, there is an urgent need to develop a method for preparing stem cell exosomes with high purity to meet the demands of practical use.
Disclosure of Invention
In view of the above, the present application aims at the defects existing in the prior art, and its main purpose is to provide a preparation method of high purity stem cell exosomes, which provides a better basis for industrial preparation of exosomes by adopting the preparation method of high purity stem cell exosomes provided by the present application, and provides a guarantee for preparing high purity exosomes by adding SMA polymer and nuclease to treat cell supernatant fluid; the method can realize mass production of high-purity exosomes by combining multiple filtration, multiple tangential flows, chemical induction, serial chromatography and other modes, and the prepared exosomes have high purity and few impurities.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A preparation method of a high-purity stem cell exosome comprises the following steps:
S1, producing a stem cell culture supernatant liquid for exosomes;
S2, filtering the supernatant liquid through a multi-filter to remove cell fragments and large vesicles;
S3, treating the feed liquid obtained in the step S2 by adopting a primary tangential flow filtration system, and adding a chemical reagent for inducing degradation of the hybrid protein into the feed liquid before primary tangential flow filtration to decompose the hybrid protein in the feed liquid so as to obtain a high-purity stock solution;
S4, treating the feed liquid by adopting a secondary tangential flow filtration system, and adding nuclease into the feed liquid before the secondary tangential flow filtration to remove chromatin impurities in the feed liquid;
S5, the chromatin-free feed liquid obtained in the step S4 passes through a plurality of chromatographic columns to obtain high-purity exosome solution;
s6, treating the high-purity exosome solution obtained in the step S5 through a three-stage tangential flow filtration system to enrich the exosome solution;
S7, treating the enriched exosome solution obtained in the step S6 through a filter to ensure the sterility of the final product.
As a preferred embodiment: the chemical reagent for inducing the degradation of the hybrid protein in the step S3 is a pre-hydrolyzed styrene-maleic anhydride copolymer, the molar ratio of the styrene to the maleic anhydride is 2:1, the using concentration of the styrene-maleic anhydride copolymer is 5-100nmol, and the mixture is incubated for 8-20 hours on a shaking table at the temperature of 4 ℃; the primary tangential flow filtration system uses a hollow fiber column with a membrane pore size of 750kDa, and the control system pressure is less than 10psi and the ambient temperature is 4 ℃.
As a preferred embodiment: the nuclease in the step S4 is omnipotent nuclease, the use concentration is 2KU-10KU/L, and the use condition is that the incubation is carried out for 30min at room temperature; the secondary tangential flow filtration system uses a hollow fiber column with a membrane pore size of 300kDa, and the control system pressure is less than 4psi and the ambient temperature is 4 ℃.
As a preferred embodiment: the chromatographic column in the step S5 comprises an anion exchange chromatographic column, wherein the filler ligand of the anion exchange chromatographic column is one or a combination of more of quaternary ammonium group, diethylaminopropyl group and diethylaminoethyl group, the eluent is 2M NaCl PBS solution, and the pH value is 7.4-8.0; the chromatographic column volume is 5mL, the chromatographic flow rate is 5mL/min, the buffer system is phosphate buffer solution, the pressure value of the chromatographic system is controlled to be less than 80psi, and the ambient temperature is 4 ℃.
As a preferred embodiment: the chromatographic column in the step S5 also comprises a cation exchange chromatographic column, wherein the filler ligand of the cation exchange chromatographic column is one or a combination of more of carboxymethyl, sulfonic acid methyl and sulfonic acid ethyl, and the eluent is 2M NaCl PBS solution with the pH value of 7.4-8.0.
As a preferred embodiment: the chromatography column in the step S5 also comprises a size exclusion chromatography column, the exosome is separated and purified by adopting a SEC column containing a plurality of holes, capto Core700 or Capto Core400 is used as a filler, and the eluent is PBS buffer solution.
As a preferred embodiment: the three-stage tangential flow filtration system in step S6 uses a hollow fiber column with a membrane pore size of 100kDa, the control system pressure is less than 4psi, and the ambient temperature is 4 ℃.
As a preferred embodiment: the sterilization filter equipment used for the enrichment exosome solution in the step S7 after the filter treatment is a capsule filter, and the pore diameter of the filter membrane is 0.22 mu m.
As a preferred embodiment: the stem cell culture supernatant in the step S1 is collected in a 2D culture condition or a 3D bioreactor.
As a preferred embodiment: and (2) filtering the supernatant liquid in the step (S2) by 0.8 mu m and 0.45 mu m to remove cell fragments and large vesicles.
Compared with the prior art, the preparation method has the obvious advantages and beneficial effects, and particularly, the technical scheme shows that the preparation method of the high-purity stem cell exosome provides a good basis for industrial preparation of the exosome, and the preparation method of the high-purity exosome is ensured by adding the SMA polymer and nuclease to treat the cell supernatant fluid; the method can realize the production of large-batch and high-purity exosomes by combining multiple filtration, multiple tangential flows, chemical induction, serial chromatography and other modes, and the prepared exosomes have high purity and few impurities; aims at solving the problems that the existing exosome separation method is difficult to realize scale, the purity of lipoprotein is affected, and the like. The application provides a preparation method of high-purity stem cell exosomes, which is based on an SMA chemical induction method combined with a tangential flow filtration system and a protein purification chromatography system to produce the exosomes with high purity and high yield so as to meet the clinical and industrial requirements.
In order to more clearly illustrate the structural features and efficacy of the present invention, a detailed description thereof will be given below with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a schematic diagram of the final purified exo-particle size distribution in an embodiment of the invention;
FIG. 2 is a schematic diagram of electron microscopy of the final purified exosomes in an embodiment of the invention;
FIG. 3 is a schematic representation of the expression of lipoprotein markers (APOE) of the final purified exosomes in the examples of the invention;
FIG. 4 is a schematic representation of the positive protein marker (CD 63/CD 9) negative marker (GM 130/Calnexin) lipoprotein marker (APOE) and internal reference protein (GAPDH) expression of the final purified exosomes in the examples of the invention;
FIG. 5 is a graph showing the absorption peaks at UV280 for a sample in example 4 of the present invention.
Detailed Description
The invention discloses a preparation method of a high-purity stem cell exosome, which is shown in figures 1 to 5 and comprises the following steps:
S1, producing a stem cell culture supernatant liquid for exosomes;
S2, filtering the supernatant liquid through a multi-filter to remove cell fragments and large vesicles;
S3, treating the feed liquid obtained in the step S2 by adopting a primary tangential flow filtration system, and adding a chemical reagent for inducing degradation of the hybrid protein into the feed liquid before primary tangential flow filtration to decompose the hybrid protein in the feed liquid so as to obtain a high-purity stock solution;
S4, treating the feed liquid by adopting a secondary tangential flow filtration system, and adding nuclease into the feed liquid before the secondary tangential flow filtration to remove chromatin impurities in the feed liquid;
S5, the chromatin-free feed liquid obtained in the step S4 passes through a plurality of chromatographic columns to obtain high-purity exosome solution;
s6, treating the high-purity exosome solution obtained in the step S5 through a three-stage tangential flow filtration system to enrich the exosome solution;
S7, treating the enriched exosome solution obtained in the step S6 through a filter to ensure the sterility of the final product.
The chemical reagent for inducing the degradation of the hybrid protein in the step S3 is a pre-hydrolyzed styrene-maleic anhydride copolymer, the molar ratio of the styrene to the maleic anhydride is 2:1, the using concentration of the styrene-maleic anhydride copolymer is 5-100nmol, and the mixture is incubated for 8-20 hours on a shaking table at the temperature of 4 ℃; the primary tangential flow filtration system uses a hollow fiber column with a membrane pore size of 750kDa, controlling the system pressure to less than 10psi and ambient temperature to 4 ℃.
The nuclease in the step S4 is omnipotent nuclease, the use concentration is 2KU-10KU/L, and the use condition is that the incubation is carried out for 30min at room temperature; the secondary tangential flow filtration system uses a hollow fiber column with a membrane pore size of 300kDa, controlling the system pressure to be less than 4psi and the ambient temperature to be 4 ℃.
The chromatographic column in the step S5 comprises an anion exchange chromatographic column, wherein the filler ligand of the anion exchange chromatographic column is one or a combination of more of quaternary ammonium group, diethylaminopropyl group and diethylaminoethyl group, the eluent is 2M NaCl PBS solution, and the pH value is 7.4-8.0; the chromatographic column volume is 5mL, the chromatographic flow rate is 5mL/min, the buffer system is phosphate buffer solution, the pressure value of the chromatographic system is controlled to be less than 80psi, and the ambient temperature is 4 ℃.
The chromatographic column in the step S5 also comprises a cation exchange chromatographic column, wherein the filler ligand of the cation exchange chromatographic column is one or a combination of more of carboxymethyl, sulfomethyl and sulfoethyl, and the eluent is 2M NaCl PBS solution with the pH value of 7.4-8.0.
The chromatography column in the step S5 also comprises a size exclusion chromatography column, the exosome is separated and purified by adopting a SEC column containing a plurality of holes, capto Core700 or Capto Core400 is used as a filler, and the eluent is PBS buffer solution.
The three stage tangential flow filtration system in step S6 uses a hollow fiber column with a membrane pore size of 100kDa, a control system pressure of less than 4psi and an ambient temperature of 4 ℃.
The sterilizing and filtering equipment used for the enrichment exosome solution in the step S7 is a capsule filter, and the pore diameter of the filtering membrane is 0.22 mu m.
In step S1, the stem cell culture supernatant is collected under 2D culture conditions or in a 3D bioreactor.
The culture preparation method of the stem cell culture supernatant fluid comprises the steps of using a bioreactor, a cell factory or a culture flask.
The supernatant in step S2 was filtered to remove cell debris and large vesicles by 0.8 μm and 0.45. Mu.m.
Example 1: preparation method of high-purity stem cell exosome
After two 400mL of the supernatant liquid of the mesenchymal stem cell culture (hereinafter referred to as feed liquid A and feed liquid B) were harvested, cell fragments and large vesicles were removed by filtration through a multi-filter (0.8 μm,0.45 μm) at 4℃to obtain a clarified supernatant liquid for use.
Adding 50nmol of polyethylene-maleic anhydride (2:1) copolymer into the feed liquid A obtained in the last step, adding no copolymer into the feed liquid B, incubating for 10 hours at 4 ℃ by a shaking table, concentrating for 80 times by a rappride KrosFlo DEG KMPi tangential flow system, selecting a hollow fiber column for the process, controlling the pore diameter of the membrane to be 750kDa, and controlling the pressure of the system to be less than 4psi. And collecting liquid at the reflux end according to the tangential flow principle for further purification.
The liquid is further washed and filtered, the washing and filtering solution is PBS buffer solution, the washing and filtering multiple is 20 times, impurities in the liquid to be purified are removed, and the supernatant after washing and filtering is collected for the next operation.
Adding 2KU/L omnipotent nuclease into the supernatant after washing and filtering, incubating for 30min at room temperature for a short time, removing enzyme byproducts through a rappride KrosFlo-KMPi tangential flow system, selecting a hollow fiber column for the process, controlling the pore diameter of the membrane to be 100kDa, and controlling the pressure of the system to be less than 4psi. And collecting liquid at the reflux end for further purification.
Placing the supernatant into an ultracentrifuge tube for balancing, placing the supernatant into a Beckmann OptimaXPN ultracentrifuge, selecting a SW32Ti rotor, setting the temperature to 4 ℃, setting the rotating speed to 100000g, setting the time to 1 hour for 30 minutes, discarding the supernatant after centrifugation, re-balancing the supernatant after resuspending the sediment at the bottom by using a proper amount of PBS, balancing the parameters of the centrifuge with the parameters of the first time, and filtering the sediment at the bottom after the centrifugation is completed by using a proper amount of PBS and then taking the sediment as an exosome solution after final purification.
Example 2: preparation method of high-purity stem cell exosome
After two 400mL of the supernatant liquid of the mesenchymal stem cell culture (hereinafter referred to as feed liquid A and feed liquid B) were harvested, cell fragments and large vesicles were removed by filtration through a multi-filter (0.8 μm,0.45 μm) at 4℃to obtain a clarified supernatant liquid for use.
Adding 50nmol of polyethylene-maleic anhydride (2:1) copolymer into the feed liquid A obtained in the last step, adding no copolymer into the feed liquid B, incubating for 20 hours at 4 ℃ by a shaking table, concentrating for 100 times by a rappride KrosFlo DEG KMPi tangential flow system, selecting a hollow fiber column for the process, controlling the pore diameter of the membrane to be 750kDa, and controlling the pressure of the system to be less than 4psi. And collecting liquid at the reflux end according to the tangential flow principle for further purification.
The liquid is further washed and filtered, the washing and filtering solution is PBS buffer solution, the washing and filtering multiple is 50 times, impurities in the liquid to be purified are removed, and the supernatant after washing and filtering is collected for the next operation.
Adding 10KU/L omnipotent nuclease into the supernatant after washing and filtering, incubating for 30min at room temperature for a short time, removing enzyme byproducts through a rappride KrosFlo-KMPi tangential flow system, selecting a hollow fiber column for the process, controlling the pore diameter of the membrane to be 100kDa, and controlling the pressure of the system to be less than 4psi. And collecting liquid at the reflux end for further purification.
Adding the supernatant obtained in the last step into an equal volume of PEG solution to ensure that the PEG content in the final solution is 5% -15%, incubating for 20 hours at a4 ℃ in a shaking table, then 1500g of the supernatant is centrifuged for 30 minutes at the 4 ℃, collecting bottom sediment, and re-suspending the sediment by a proper amount of PBS solution, and filtering the sediment through a 0.22 mu m filter membrane to obtain the purified exosome solution.
Example 3: preparation method of high-purity stem cell exosome
After two 400mL of the supernatant liquid of the mesenchymal stem cell culture (hereinafter referred to as feed liquid A and feed liquid B) were harvested, cell fragments and large vesicles were removed by filtration through a multi-filter (0.8 μm,0.45 μm) at 4℃to obtain a clarified supernatant liquid for use.
Adding 50nmol of polyethylene-maleic anhydride (2:1) copolymer into the feed liquid A obtained in the last step, adding no copolymer into the feed liquid B, incubating for 8 hours at 4 ℃ by a shaking table, concentrating for 50 times by a rappride KrosFlo DEG KMPi tangential flow system, selecting a hollow fiber column for the process, controlling the pore diameter of the membrane to be 750kDa, and controlling the pressure of the system to be less than 4psi. And collecting liquid at the reflux end according to the tangential flow principle for further purification.
The liquid is further washed and filtered, the washing and filtering solution is PBS buffer solution, the washing and filtering multiple is 30 times, impurities in the liquid to be purified are removed, and the supernatant after washing and filtering is collected for the next operation.
Adding 2KU/L omnipotent nuclease into the supernatant after washing and filtering, incubating for 30min at room temperature for a short time, removing enzyme byproducts through a rappride KrosFlo-KMPi tangential flow system, selecting a hollow fiber column for the process, controlling the pore diameter of the membrane to be 100kDa, and controlling the pressure of the system to be less than 4psi. And collecting liquid at the reflux end for further purification.
Adding sucrose solutions with different concentrations into a centrifuge tube, sequentially adding 0.25mol/L,0.5mol/L,0.8mol/L,1.16mol/L,1.3mol/L and 2mol/L from top to bottom, slowly adding the supernatant into the uppermost layer of the sucrose solution, placing the uppermost layer in a Beckmann OptimaXPN ultracentrifuge, selecting a SW32Ti rotor, setting the temperature to 4 ℃, setting the rotating speed to 100000g, setting the time to 1 hour for 30 minutes, slightly taking out an ultracentrifuge tube after centrifugation, sucking the solution from the 4 th layer to the 9 th layer, collecting the solution into an ultrafilter tube with the cutoff aperture of 10KDa, concentrating the solution to 500 mu L through centrifugation at the temperature of 4000g and the temperature of 4 ℃, collecting the solution in the upper tube, and filtering the solution through a filter membrane with the temperature of 0.22 mu m to be regarded as a finally purified exosome solution.
Example 4: preparation method of high-purity stem cell exosome
After two 400mL of the supernatant liquid of the mesenchymal stem cell culture (hereinafter referred to as feed liquid A and feed liquid B) were harvested, cell fragments and large vesicles were removed by filtration through a multi-filter (0.8 μm,0.45 μm) at 4℃to obtain a clarified supernatant liquid for use.
Adding 50nmol of polyethylene-maleic anhydride (2:1) copolymer into the feed liquid A obtained in the last step, adding no copolymer into the feed liquid B, incubating for 8 hours at 4 ℃ by a shaking table, concentrating for 60 times by a rappride KrosFlo DEG KMPi tangential flow system, selecting a hollow fiber column for the process, controlling the pore diameter of the membrane to be 750kDa, and controlling the pressure of the system to be less than 4psi. And collecting liquid at the reflux end according to the tangential flow principle for further purification.
The liquid is further washed and filtered, the washing and filtering solution is PBS buffer solution, the washing and filtering multiple is 30 times, impurities in the liquid to be purified are removed, and the supernatant after washing and filtering is collected for the next operation.
Adding 8KU/L omnipotent nuclease into the supernatant after washing and filtering, incubating for 30min at room temperature for a short time, removing enzyme byproducts through a rappride KrosFlo-KMPi tangential flow system, selecting a hollow fiber column for the process, controlling the pore diameter of the membrane to be 100kDa, and controlling the pressure of the system to be less than 4psi. And collecting liquid at the reflux end for further purification.
Buffer preparation ：Buffer A(50mM HEPES,20mM NaCl,pH=7.0),Buffer B(50mM HEPES,2M NaCl,pH=7.0),Buffer C(1M NaOH,2M NaCl),20% ethanol, calibration BufferA/B pH to 7.
The buffer solution, ultrapure water and the supernatant were filtered through a 0.45 μm filter membrane for use.
Starting the protein purifier, connecting the computer with the NGC purification system, cleaning the pipeline with ultrapure water, evacuating air in the pipeline, and connecting the chromatographic column.
The pipeline is connected with the chromatographic column in a liquid-liquid connection mode, so that no bubbles enter the column in the process, and the sequence of the Capto core700 chromatographic column and the anion exchange column is adopted.
Balance: the ultraviolet detector was zeroed using Buffer a balance line until the system baseline was stationary.
Loading: the system selects a manual operation mode, a sampling needle sucks 1CV supernatant, the supernatant is injected into a loading valve, and the sample is waited for hanging by low flow speed operation.
Eluting: running at a lower flow rate, eluting with a linear gradient (0-100%) by using Buffer B, collecting elution peaks, changing the collected liquid into PBS Buffer solution by using a Reeplerian KrosFlo DEG KR2i tangential flow system, concentrating to a proper volume, collecting liquid at a reflux end, and filtering by using a 0.22 mu m filter membrane to obtain the final purified exosome solution.
The final exosome samples obtained in examples 1 to 4 showed no significant difference in size distribution and morphology of exosomes, and the different preparation methods did not affect the basic exosome properties, but as can be seen from fig. 4, the APOE of lipoprotein markers in exosome samples isolated by four methods from SMA pretreatment supernatant was significantly reduced, indicating that SMA could be used for lipoprotein removal during exosome purification. As can be seen from FIG. 5, the APOE expression level in the sample of the exosome purified by the SMA pretreatment combined with the CC mode is the least, and the exosome negative protein GM130 and Calnexin expression level are also lower, which can be deduced that the purity of the separation method is higher. The expression level of the sample exosome marker protein separated by the PEG precipitation method is lower than that of exosomes separated and purified by UC, DGUC and CC methods, which indicates that the PEG method is not suitable for separating high-purity exosomes.
Meanwhile, in order to avoid the phenomenon of the co-separation of lipoprotein and exosome, the exosome prepared by the chromatography is superior to the conventional super-separation method. Although co-separation of lipoprotein and exosome can be avoided as much as possible by density gradient ultracentrifugation, column chromatography is preferred in view of ease of handling and scale-up. Further, the extent of lipoprotein co-separation is reduced by the electron microscopy result, which is sufficient to indicate that the mode of adding SMA and combining multi-column chromatography can prepare the exosomes with higher purity, and is a mode of producing the exosomes in a large scale.
The invention provides a preparation method of a high-purity stem cell exosome, which comprises the following steps: harvesting culture supernatant from large-scale stem cell culture as a raw material for separating exosomes; removing cell debris and large vesicles from the supernatant by a multiple filter; adding nuclease to remove chromatin impurities; selectively decomposing lipoprotein based on chemical induction method to obtain stock solution with higher purity; filtering the stock solution through multiple tangential flows to improve the yield of exosomes; passing the filtered stock solution through a multiple chromatographic column to obtain high-purity exosomes; concentrating and enriching the exosome after chromatography through a tangential flow filtration system; finally, sterilization by a filter ensures sterility of the final product.
The invention can realize the purification of exosomes with different volumes, improves the purity of exosomes by decomposing lipoprotein by a chemical induction method, and anion exchange chromatography, cation exchange chromatography and molecular sieve chromatography, is a production method capable of realizing high-purity stem cell exosomes, and provides support for the transformation of clinical application of exosomes.
The design of the invention is focused on that compared with the prior art, the invention does not need to use an ultracentrifuge, and reduces the cost. The chromatography is a separation method suitable for automatic mass production, provides a good basis for industrial preparation of exosomes, and provides a guarantee for preparing high-purity exosomes by adding the SMA polymer and nuclease to treat cell supernatant fluid. The method can realize the production of large-batch and high-purity exosomes by combining multiple filtration, multiple tangential flows, chemical induction, serial chromatography and other modes, and the prepared exosomes have high purity and few impurities; aims at solving the problems that the existing exosome separation method is difficult to realize scale, the purity of lipoprotein is affected, and the like. The invention provides a preparation method of high-purity stem cell exosomes, which is based on an SMA chemical induction method combined with a tangential flow filtration system and a protein purification chromatography system to produce the exosomes with high purity and high yield so as to meet the clinical and industrial requirements.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention, so any minor modifications, equivalent changes and modifications made to the above embodiments according to the technical principles of the present invention still fall within the scope of the technical solutions of the present invention.
Claims (10)
1. A preparation method of a high-purity stem cell exosome is characterized by comprising the following steps: the method comprises the following steps:
S1, producing a stem cell culture supernatant liquid for exosomes;
S2, filtering the supernatant liquid through a multi-filter to remove cell fragments and large vesicles;
S3, treating the feed liquid obtained in the step S2 by adopting a primary tangential flow filtration system, and adding a chemical reagent for inducing degradation of the hybrid protein into the feed liquid before primary tangential flow filtration to decompose the hybrid protein in the feed liquid so as to obtain a high-purity stock solution;
S4, treating the feed liquid by adopting a secondary tangential flow filtration system, and adding nuclease into the feed liquid before the secondary tangential flow filtration to remove chromatin impurities in the feed liquid;
S5, the chromatin-free feed liquid obtained in the step S4 passes through a plurality of chromatographic columns to obtain high-purity exosome solution;
s6, treating the high-purity exosome solution obtained in the step S5 through a three-stage tangential flow filtration system to enrich the exosome solution;
S7, treating the enriched exosome solution obtained in the step S6 through a filter to ensure the sterility of the final product.
2. The method for preparing high purity stem cell exosomes according to claim 1, wherein: the chemical reagent for inducing the degradation of the hybrid protein in the step S3 is a pre-hydrolyzed styrene-maleic anhydride copolymer, the molar ratio of the styrene to the maleic anhydride is 2:1, the using concentration of the styrene-maleic anhydride copolymer is 5-100nmol, and the mixture is incubated for 8-20 hours on a shaking table at the temperature of 4 ℃; the primary tangential flow filtration system uses a hollow fiber column with a membrane pore size of 750kDa, and the control system pressure is less than 10psi and the ambient temperature is 4 ℃.
3. The method for preparing high purity stem cell exosomes according to claim 1, wherein: the nuclease in the step S4 is omnipotent nuclease, the use concentration is 2KU-10KU/L, and the use condition is that the incubation is carried out for 30min at room temperature; the secondary tangential flow filtration system uses a hollow fiber column with a membrane pore size of 300kDa, and the control system pressure is less than 4psi and the ambient temperature is 4 ℃.
4. The method for preparing high purity stem cell exosomes according to claim 1, wherein: the chromatographic column in the step S5 comprises an anion exchange chromatographic column, wherein the filler ligand of the anion exchange chromatographic column is one or a combination of more of quaternary ammonium group, diethylaminopropyl group and diethylaminoethyl group, the eluent is 2M NaCl PBS solution, and the pH value is 7.4-8.0; the chromatographic column volume is 5mL, the chromatographic flow rate is 5mL/min, the buffer system is phosphate buffer solution, the pressure value of the chromatographic system is controlled to be less than 80psi, and the ambient temperature is 4 ℃.
5. The method for preparing high purity stem cell exosomes according to claim 1, wherein: the chromatographic column in the step S5 also comprises a cation exchange chromatographic column, wherein the filler ligand of the cation exchange chromatographic column is one or a combination of more of carboxymethyl, sulfonic acid methyl and sulfonic acid ethyl, and the eluent is 2M NaCl PBS solution with the pH value of 7.4-8.0.
6. The method for preparing high purity stem cell exosomes according to claim 1, wherein: the chromatography column in the step S5 also comprises a size exclusion chromatography column, the exosome is separated and purified by adopting a SEC column containing a plurality of holes, capto Core700 or Capto Core400 is used as a filler, and the eluent is PBS buffer solution.
7. The method for preparing high purity stem cell exosomes according to claim 1, wherein: the three-stage tangential flow filtration system in step S6 uses a hollow fiber column with a membrane pore size of 100kDa, the control system pressure is less than 4psi, and the ambient temperature is 4 ℃.
8. The method for preparing high purity stem cell exosomes according to claim 1, wherein: the sterilization filter equipment used for the enrichment exosome solution in the step S7 after the filter treatment is a capsule filter, and the pore diameter of the filter membrane is 0.22 mu m.
9. The method for preparing high purity stem cell exosomes according to claim 1, wherein: the stem cell culture supernatant in the step S1 is collected in a 2D culture condition or a 3D bioreactor.
10. The method for preparing high purity stem cell exosomes according to claim 1, wherein: and (2) filtering the supernatant liquid in the step (S2) by 0.8 mu m and 0.45 mu m to remove cell fragments and large vesicles.
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