CN117511863A - Method for sequentially separating exosomes and application thereof - Google Patents
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Abstract
The invention relates to a method for sequentially separating exosomes and application thereof, belonging to the technical field of biological macromolecule separation and purification. The method for sequentially separating exosomes comprises the following steps: transferring the cell supernatant containing the exosomes into a hollow fiber column for concentration and preliminary purification until the volume of the cut-off liquid is 10-20 times of the volume of the cell supernatant, wherein the cut-off liquid is the crude purified exosomes; and carrying out composite chromatography on the obtained crude and purified exosomes to obtain purified exosomes. The exosome obtained by separating and purifying the exosome through the hollow fiber column and the protein separating and purifying system has high purity, a large number of exosome and no influence on physiological activity, and reduces the damage to exosome and the loss of activity reduction in the conventional separating method.
Description
Technical Field
The invention relates to the technical field of biological macromolecule separation and purification, in particular to a method for sequentially separating exosomes and application thereof.
Background
Exosomes are a class of extracellular vesicles released by cells and are carriers of various bioactive molecules (such as proteins, nucleic acids and lipids), which are composed of lipid bilayer with particle size between 30-200nm, and which play an important role in intercellular communication, signaling and disease control. The research of exosomes can be traced to the beginning of the 80 s of the 20 th century, and the research related to exosomes was issued by the Nobel physiology and medicine prize in 2013.
More and more studies have shown that exosomes can transmit information and regulate cellular functions through the transport of internal molecules and uptake by target cells. The functions of the method include: 1) Intercellular communication; 2) Cell immunity; 3) Tissue repair and regeneration; 4) Disease control. In order to understand the function and mechanism of exosomes more accurately, to promote the application of exosome research in the basic science and clinical fields, it is necessary to isolate exosome samples with high purity and good integrity. First, high purity exosome samples can exclude interference of other cellular components, thereby accurately researching the components and functions of exosomes. This is important to reveal the mechanisms of exosomes in intercellular communication, disease progression and treatment. Second, maintaining the integrity of exosomes is critical to fully functioning. Exosomes carry abundant bioactive molecules, such as proteins, nucleic acids, lipids, etc., the integrity of which is important to ensure the functionality and bioactivity of the exosomes. Only intact exosomes are able to transmit information between cells, mediate cellular signals and regulate physiological processes. In addition, the exosome sample with high separation purity and good integrity also provides a reliable basis for clinical application. Exosome formulations for therapeutic and diagnostic use require high purification and integrity to ensure safety and efficacy. Therefore, the novel method for efficiently separating the exosomes with high purity and good integrity is of great importance.
Currently, there are various methods for exosome isolation, including: 1) Ultracentrifugation (ultracentrifucation): gradually removing cell fragments and large particles through multiple centrifugation steps, so as to obtain an enriched exosome sample; 2) Density gradient centrifugation (Density Gradient Centrifugation): separating exosomes in layers with different densities by utilizing centrifuge tube chromatography formed by gradients with different densities in the centrifugation process; 3) Size exclusion chromatography (Size Exclusion Chromatography): the exosomes can be separated from other large particulate matters and proteins by size difference using a nano-pore-size column; 4) Immunoaffinity method (immunoaffinity): capturing and enriching with specific proteins on the surface of exosomes, usually isolated using specific antibodies binding to marker proteins on exosomes; 5) Nanofiltration (Nanomembrane Filtration): screening exosomes by using a nano-aperture filter membrane to realize separation and purification, and selecting and screening exosomes with different sizes according to the aperture size; 6) Affinity chromatography (Affinity Chromatography): separating exosomes by utilizing the affinity between the affinity agent and specific components of the exosomes; 7) Magnetic bead separation (Magnetic Bead Separation): exosomes are isolated from complex mixtures by affinity of magnetically specific antibodies.
However, current exosome separation and purification techniques have several drawbacks. Among them, ultracentrifugation and density gradient centrifugation require expensive equipment, are time consuming, and can result in loss of exosomes. Size exclusion chromatography and nanofiltration have limitations on the separation of small-size exosomes. Immunoaffinity methods and affinity chromatography, depending on the selection and specificity of a particular antibody, may have problems of recognition difference and degradation of capturing efficiency. Although convenient, magnetic bead separation is not applicable to large volumes of samples and non-specific adsorption of magnetic beads is a concern. Thus, there remains a need for further improvements and optimizations in exosome separation purification to increase separation efficiency, purity and maintain exosome integrity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for sequentially separating exosomes, which can more accurately obtain exosome samples and reduce the interference of other cell components, and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a method for sequentially isolating exosomes comprising the steps of:
(1) Transferring the cell supernatant containing the exosomes into a hollow fiber column for concentration and primary purification until the volume of the concentrated cell supernatant is 10-20 times of the volume of the cell supernatant before concentration, wherein the obtained concentrated cell supernatant is the crude purified exosomes;
(2) And (3) carrying out composite chromatography on the crude and purified exosomes obtained in the step (1) to obtain purified exosomes.
The method carries out concentration and preliminary purification through the hollow fiber column, and further purifies exosomes by adopting a composite chromatography technology, so that the obtained exosomes have the advantages of more quantity, higher purity and better physiological activity.
As a preferred embodiment of the method of the present invention, in the step (1), the hollow fiber column is used, the molecular weight cut-off is 50-500KD, and the hollow fiber material is polyethersulfone. The hollow fiber column made of polyether sulfone (PES) has good hydrophobicity, can separate exosomes in cell supernatant, and has good exosome concentration and separation effects.
As a preferred embodiment of the method of the present invention, in step (1), the concentration and preliminary purification are performed by circulating the cell supernatant containing exosomes in a hollow fiber column driven by a peristaltic pump; the rotating speed of the peristaltic pump is 20-30 revolutions/min.
As a preferred embodiment of the method of the present invention, in the step (2), the complex chromatography is a complex chromatography of the crude purified exosomes using a protein separation and purification system;
the chromatographic column adopted by the composite chromatography is a highly crosslinked agarose gel column containing core microbeads and multimode octylamine ligands; the core microbeads mainly comprise ligand activated cores and inert shells, and the average molecular weight cut-off of the core microbeads is 600-700KD.
The protein separation and purification system adopted by the invention is matched with the composite chromatographic column and the ultraviolet spectroscopic detection system, and after the sample is loaded, the sample is separated by the composite chromatographic column and detected by ultraviolet spectroscopic detection, and corresponding components are collected according to a response peak at 280nm, so that the protein separation and purification efficiency is improved. The composite chromatographic column adopted by the invention can remove the specific separation and purification exosomes of impurities.
As a preferred embodiment of the method of the invention, the chromatographic column used in the composite chromatography is a HiScreen Capto Core 700 gel column.
As a preferred embodiment of the method of the present invention, the parameter conditions for performing composite chromatography on the crude purified exosomes using a protein separation purification system or peristaltic pump pressurization system are: the pressure before the column is 0.8-1.0MPa, the flow rate is 1.0-1.5mL/min, and the detection wavelength of the ultraviolet absorption detector is 280nm; phosphate buffer is used for column balancing and flushing the pipeline.
As a preferred embodiment of the method according to the invention, the method for sequential isolation of exosomes further comprises the steps of:
(3) Ultrafiltering the purified exosomes obtained in the step (2) by adopting an ultrafiltration tube to obtain refined exosomes; the molecular weight cut-off of the ultrafiltration tube was 100KD.
As a preferred embodiment of the method of the present invention, in the case that the volume of the obtained purified exosome is more than 1L in the step (2), the obtained exosome may be further purified and concentrated according to the method of the step (1), and the hollow fiber column used in this case has a molecular weight cut-off of 50 to 150KD, and the hollow fiber is made of PES.
In a second aspect, the present invention provides a method for industrially producing mesenchymal stem cell-derived exosomes, comprising the steps of:
s1, mixing mesenchymal stem cells with a microcarrier culture solution, transferring into a rotary bottle, and culturing in a bioreactor until the mesenchymal stem cells completely cover the microcarrier to obtain a microcarrier for loading cells;
s2, dividing the microcarrier loaded with cells obtained in the step S1 into 4 parts averagely, respectively transferring into a rotary bottle, adding a culture medium and the microcarrier not loaded with cells, uniformly mixing, and then placing the rotary bottle into a bioreactor for culturing to obtain mesenchymal stem cell liquid and microcarrier P2 loaded with cells;
s3, continuously rotating the bottle for 2-4 times according to the operation of the step S2 on the microcarrier P2 loaded with cells obtained in the step S2 to obtain mesenchymal stem cell liquid;
s4, removing the mesenchymal stem cells in the mesenchymal stem cell fluid obtained in the steps S2 and S3, and cell fragments and protein aggregates of the mesenchymal stem cells to obtain a mesenchymal stem cell supernatant;
s5, separating and purifying the supernatant of the mesenchymal stem cells obtained in the step S4 according to the method for sequentially separating exosomes, so as to obtain the purified exosomes.
According to the invention, the mesenchymal stem cells are cultured on the microcarrier by adopting a 3D culture technology, and the mesenchymal stem cells can be produced in a large scale by combining a rotating bottle and a bioreactor, so that the number of the obtained mesenchymal stem cells is 17.5 times that of the mesenchymal stem cells obtained by 2D culture. The 3D culture technology is combined with the sequential exosome separation technology, so that a large number of exosomes can be obtained through separation, and the exosomes are complete, high in purity, large in quantity and excellent in physiological activity, so that the industrial production of mesenchymal stem cell exosomes is realized.
As a preferred embodiment of the method of the present invention, in step S1, step S2 and step S3, the microcarrier is subjected to pretreatment before use, and the specific operation is as follows: soaking microcarrier in Du's phosphate buffer for 3 hr, washing with Du's phosphate buffer for 2 times, sterilizing at 121deg.C for 30min, cooling to precipitate, and retaining precipitate; washing the precipitate with culture medium for 2 times in sterile environment, and adding culture medium to constant volume to obtain microcarrier culture solution.
As a preferred embodiment of the method of the present invention, in step S1, the microcarrier content in the microcarrier culture liquid is 0.3-1.0g per liter of culture liquid; the ratio of the mesenchymal stem cells to the microcarriers in the microcarrier culture solution is that the mesenchymal stem cells: microcarrier = (1-3) ×10 6 The following steps: 0.3-1.0g.
As a preferred embodiment of the method of the present invention, in step S2, the ratio of the sum of the cell-loaded microcarrier and the non-cell-loaded microcarrier to the medium is (cell-loaded microcarrier+non-cell-loaded microcarrier): culture medium= (0.3-1.0) g:1L; the ratio of the sum of the cell-loaded microcarrier and the non-cell-loaded microcarrier to the mesenchymal stem cells was (negativeCell-loaded microcarrier + cell-unloaded microcarrier): mesenchymal stem cells = 0.3-1g: (1-3). Times.10 7 And each.
As a preferred embodiment of the method of the present invention, in step S1, the culture conditions are: culturing at 50rpm for 5min and at 0rpm for 24 hr on day 1; culturing at constant speed of 35rpm on day 2-4; the culture temperature is 37 ℃;
in step S2, the culture conditions are: culturing at constant speed at 37 deg.C at 35rpm for 4-8 days.
Compared with the prior art, the invention has the beneficial effects that:
1. the exosome obtained by separating and purifying the exosome through the hollow fiber column and the protein separating and purifying system has high purity, a large number of exosome and no influence on physiological activity, and reduces the damage to exosome and the loss of activity reduction in the conventional separating method.
2. The invention combines a 3D culture technology and a sequential exosome separation technology, provides a method capable of producing exosomes in a large scale, the 3D culture technology can prepare a large number of mesenchymal stem cells, the number of the obtained mesenchymal stem cells is 17.5 times that of 2D culture, and the sequential exosome separation technology can greatly improve the number and purity of exosomes and realize industrial production of exosomes derived from the mesenchymal stem cells.
Drawings
FIG. 1 is a hollow fiber column used in example 1;
FIG. 2 is a schematic diagram of a protein isolation and purification system used in example 1;
FIG. 3 is a roller bottle and bioreactor used in example 2;
FIG. 4 shows the DiO staining results of mesenchymal stem cells of different sources in effect example 1;
FIG. 5 is an electron microscope view of the exosome of example 1 of effect example 2;
FIG. 6 is a graph showing the particle size concentration of exosomes in example 2 of effect example 2;
FIG. 7 shows the results of the measurement of the content of the exosome immune modulator in effect example 3 in example 2 and example 3;
FIG. 8 shows the results of measuring the content of the exosome antibacterial peptide LL37 in effect example 3 in example 2 and example 3.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
In the following examples and comparative examples, the mesenchymal stem cells used were, unless otherwise specified, mesenchymal stem cells of foreskin, isolated from waste foreskin tissue generated by a prepuce excision operation in children.
In the following examples and comparative examples, the culture medium used includes complete medium and serum-free medium; the complete culture medium is DMEM-F12 containing 10v/v% of fetal bovine serum, and the serum-free culture medium is DMEM-F12.
In the following examples and comparative examples, the cell culture conditions used were 37℃and 5% CO 2 。
The hollow fiber column was purchased from Repligen under the trade designation K05-E500-05N.
Microcarriers were purchased from cytova under the accession number 17044802.
Legendplex TM Multi-factor magnetic beads were purchased from BioLegend, legendplex TM Human IL-4Capture Bead A4,13X (740933), legendplex TM Human IL-1 beta Capture Bead A7,13X (accession number 740936), legendplex TM Human TNF-alpha Capture Bead A8,13X (accession number 740937), legendplex TM Human IL-6Capture Bead B3,13X (740940), legendplex TM Human IL-10Capture Bead B4,13X (740941), legendplex TM Human IL-12p70 Capture Bead B6,13X (740943), legendplex TM Human Free Active TGF-beta 1Capture Bead B9,13X (product number 740944), legendplex TM Human IFN-. Gamma.Capture Bead B5,13X (cat. No. 740942).
Exosome lysates were purchased from the next holy of Shanghai under the accession number 41211ES20.
Antibacterial peptide LL37 detection kit was purchased from Reddot Biotech under the designation RDR-CAMP-Hu.
Example 1
The present embodiment provides a method for sequentially separating exosomes, comprising the steps of:
a1, culturing mesenchymal stem cells by adopting a complete culture medium, collecting supernatant, centrifuging the obtained supernatant at 4 ℃ for 10min at 3000 Xg to remove large cell fragments, further removing large protein aggregates and small cell fragments by a 0.45 mu m filter membrane, and then passing through a 0.22 mu m filter membrane to obtain filtrate which is cell supernatant containing exosomes;
a2, transferring the cell supernatant containing the exosomes obtained in the step A1 into a hollow fiber column, starting a peristaltic pump to circulate the cell supernatant into the hollow fiber column (PES, 500 KD) at a rotating speed of 25 revolutions/min, and after circulation for 2 hours, wherein the volume of the cell supernatant is 10-20 times that of the cell supernatant before concentration, and the obtained concentrated cell supernatant is a crude purified exosomes; the hollow fiber column is shown in figure 1;
a3, loading the crude purified exosome obtained in the step A2 to a protein separation and purification system, separating and purifying by adopting a HiScreen Capto Core gel column, wherein the pre-column pressure is 0.8MPa, the flow rate is 1.5mL/min, performing column balancing and flushing a pipeline by adopting 1 x phosphate buffer, and collecting a component with an absorption peak at 280nm, wherein the component is the purified exosome; the protein separation and purification system is shown in figure 2;
a4, transferring the purified exosomes obtained in the step A3 into a 50mL ultrafiltration tube (100 KD), and centrifuging at 4 ℃ and 3500 Xg for 20min to obtain the precise exosomes.
Example 2
The embodiment provides a method for industrially producing mesenchymal stem cell-derived exosomes, comprising the following steps:
b1, performing adherent culture on mesenchymal stem cells by adopting a complete culture medium, removing the culture medium after the mesenchymal stem cells grow to more than 90% of confluence, and cleaning by adopting PBS (phosphate buffer solution) to prepare a cell suspension;
b2, pretreatment of the microcarrier: transferring the microcarrier into a Du's Phosphate Buffer Solution (DPBS) according to the proportion of 3g microcarrier 1L, soaking for 3h in the DPBS, washing for 2 times by using the DPBS, sterilizing for 30min at 121 ℃, cooling and precipitating, and retaining the precipitate; washing the precipitate in a biosafety cabinet for 2 times by using a serum-free culture medium, and adding the serum-free culture medium to fix the volume to 1L to obtain a microcarrier culture solution;
b3, mixing the mesenchymal stem cell suspension obtained in the step B1 with the microcarrier culture solution obtained in the step B2 according to the ratio of 1.0g/L microcarrier culture solution: (1-3). Times.10 6 Mixing the mesenchymal stem cells in proportion, transferring into a rotary bottle, culturing for 4-8 days in a bioreactor, and observing to obtain a microcarrier which completely covers the mesenchymal stem cells to obtain a microcarrier carrying cells; the rotating speed of the bottle is 30 revolutions per minute; the roller bottle and the bioreactor are shown in figure 3;
b4, uniformly dividing the microcarrier of the loaded cells obtained in the step B3 into 4 parts, respectively transferring into a rotating bottle, adding the microcarrier culture solution (microcarrier without the loaded cells) obtained in the step B2 into the rotating bottle, wherein the total content of the microcarrier in the rotating bottle is 1.0g, adding a serum-free culture medium to a volume of 1L, uniformly mixing, and placing the rotating bottle in a bioreactor for culturing for 4-8 days to obtain mesenchymal stem cell solution and microcarrier P2 with the loaded cells; the rotating speed of the bottle is 30 revolutions per minute;
b5, carrying out continuous bottle rotating for 2-4 times on the microcarrier P2 loaded with cells obtained in the step B4 according to the operation of the step B4 to obtain mesenchymal stem cell liquid;
b6, collecting cell supernatant in the mesenchymal stem cell fluid obtained in the step B4 and the step B5, centrifuging the obtained supernatant at 4 ℃ for 10min at 3000 Xg to remove large cell fragments, further removing large protein aggregates and small cell fragments through a 0.45 mu m filter membrane, and then passing through a 0.22 mu m filter membrane to obtain filtrate which is cell supernatant containing exosomes;
transferring the cell supernatant containing the exosomes obtained in the step B6 into a hollow fiber column, starting a peristaltic pump to circulate the cell supernatant into the hollow fiber column (PES, 500 KD) at a rotation speed of 25 revolutions per minute, and after 2 hours of circulation, wherein the volume of the intercepting liquid is 10-20 times of the volume of the cell supernatant, and the intercepting liquid is the crude purified exosomes;
b8, loading the crude purified exosome obtained in the step B7 to a protein separation and purification system, separating and purifying by adopting a HiScreen Capto Core gel column, wherein the pre-column pressure is 0.8MPa, the flow rate is 1.5mL/min, performing column balancing and flushing pipelines by adopting phosphate buffer solution, and collecting components with absorption peaks at 280nm, wherein the components are purified exosome;
b9, transferring the purified exosomes obtained in the step B8 into a 50mL ultrafiltration tube (100 KD), and centrifuging at 4 ℃ and 3500 Xg for 20min to obtain the precise exosomes.
Example 3
The present example provides a method for industrially producing mesenchymal stem cell-derived exosomes, which is similar to example 2 in steps except that the mesenchymal stem cells used are umbilical cord mesenchymal stem cells, and the remaining steps and parameters thereof are unchanged.
Example 4
The present example provides a method for industrially producing mesenchymal stem cell-derived exosomes, which is similar to example 2 in steps except that the mesenchymal stem cells used are adipose-derived mesenchymal stem cells, and the remaining steps and parameters thereof are unchanged.
Comparative example 1
The comparative example provides a method for separating and purifying exosomes by using an overspeed separation method, comprising the following steps:
c1, culturing mesenchymal stem cells by adopting a complete culture medium, collecting supernatant, and centrifuging the obtained supernatant at 4 ℃ for 10min at 300 Xg to remove large cell fragments;
c2, centrifuging the supernatant at 4 ℃ for 10min at 2000 Xg to remove cell debris;
c3, centrifuging the supernatant at 4 ℃ and 10000 Xg for 10min to remove impurities such as microbubbles;
and C4, centrifuging the supernatant at the temperature of 4 ℃ and the temperature of 100000-200000 Xg for 70min to settle exosomes, wherein the sediment is the exosomes.
Comparative example 2
The comparative example provides a method for separating and purifying exosomes by ultrafiltration, which has the similar operation steps to those of comparative example 1, except that after the end of step C4, the obtained exosomes are resuspended in PBS, transferred into an ultrafiltration tube (100 KD), and centrifuged at 4 ℃ for 20min at 3500×g, to obtain the pure exosomes.
Comparative example 3
The comparative example provides a method for separating and purifying exosomes, which is similar to example 1 in operation steps, except that the step A2 is not performed, and the cell supernatant containing exosomes obtained in step A1 is directly subjected to composite chromatography according to the operation of step A3.
Comparative example 4
The comparative example provides a method for separating and purifying an exosome, which is similar to example 1 in operation steps, except that the crude purified exosome obtained in step A2 is directly subjected to ultrafiltration according to the operation of step A4 without going through step A3.
Effect example 1
DiO cell membrane staining was performed on the mesenchymal stem cells obtained in examples 2 to 4, and the specific procedure was as follows:
the cells were collected, washed 2-3 times with PBS for 5min each, incubated with DiO working solution (5. Mu.M) at room temperature for 30min, centrifuged to remove the supernatant, washed 2-3 times with PBS, resuspended in PBS, and observed under a fluorescence microscope, as shown in FIG. 4.
As shown in FIG. 4, the method for industrially producing the exosome of the mesenchymal stem cells can be applied to the mesenchymal stem cells of different sources, and the mesenchymal stem cells obtained by culture are complete and have good states.
In addition, the method for industrially producing the exosome of the mesenchymal stem cells can culture the mesenchymal stem cells in a large scale, and the number of the obtained mesenchymal stem cells can reach 17.5 times of that of 2D culture by adopting a 3D culture technology to culture the mesenchymal stem cells. One 2000mL 3D flask (roller bottle) can collect 1500mL of cell culture supernatant at a time and 4 times, thus 6000mL of cell supernatant can be obtained; the bottle is turned 3-4 times, and finally can be turned into 64-256 3D culture bottles, a 3D culture system can be used for obtaining a bottle-to-4 bottle 3D culture bottle, 6000mL of cell culture supernatant can be collected once, and according to the bottle turning 3 times, the bottle is finally turned into 64 3D culture bottles, 510000mL of cell supernatant can be collected in the whole process for extracting and purifying exosomes, and the method is suitable for large-scale industrial production of exosomes.
Effect example 2
The exosomes obtained in examples 1-2 and comparative examples 1-4 were characterized as follows:
1. transmission electron microscope observation
The transmission electron microscope negative staining method is a method commonly used for identifying exosomes, and can visually observe the membrane capsule structure and size of the exosomes: mixing phosphotungstic acid serving as a staining agent of an electron microscope negative staining method with 50 mu L of exosome sample, and incubating for 2-5min so that the staining agent is combined with the exosome; then stopping the dyeing reaction and fixing the sample by adding the fixing agent glucuronic acid; finally, the fixed exosome sample is taken out to prepare a slice. The sheet was placed in a transmission electron microscope, and the morphology and structure of the exosome were observed using the transmission electron microscope. The exosome electron microscopy image obtained in example 1 is shown in FIG. 5.
As shown in FIG. 5, the method for sequentially separating exosomes can successfully extract and obtain complete exosomes.
3. Nanoparticle tracking analysis
Nanoparticle tracking analysis (Nanoparticle Tracking Analysis, NTA) is a high resolution technique that is widely used to detect the size, concentration and distribution of exosomes and other nanoparticles. Before NTA detection of exosomes is performed, the sample is first filtered using a 0.22 μm filter membrane; next, the exosome samples were diluted into an appropriate amount of PBS to ensure that the particles were within the NTA analysis range; then, opening NTA equipment and starting related software to ensure normal operation of parameters of the equipment such as a laser, a camera, a lens and the like, and performing optical calibration of the equipment by using standard microbeads (with the diameter of 100 nm) to ensure the accuracy of a tracking algorithm; after that, when the sample is loaded, the diluted exosome sample is injected into the sample chamber by a syringe, and the sample is ensured to be full of the whole observation area, and in the loading process, air bubbles and particle agglomeration are required to be avoided so as to keep tracking precision; after the sample loading is completed, proper acquisition time and frame rate are set, data acquisition is started, and the equipment tracks and records the movement track of the particles. The analysis of the exosome particle size concentration in example 2 is shown in FIG. 6, and the analysis data of exosome NTA in example 2 and comparative examples 1 to 4 are shown in Table 1.
TABLE 1 NTA analytical data for separation and purification of exosomes by different methods
As shown in fig. 6 and table 1, the recovery rate, the exosome purity and the number of the extraction obtained by the method of example 2 are more, which indicates that the method of the present invention can culture a large number of mesenchymal stem cells to generate more exosomes, and simultaneously obtain a refined exosomes by a method of sequentially separating exosomes, which is suitable for industrial production of exosomes.
Effect example 2
In order to verify the physiological activity of the extracted exosomes, the immune regulation capacity and the antibacterial peptide content of the exosomes are measured, and the specific scheme is as follows:
1. identification of exosome immune regulation ability
The exosomes extracted in example 2 and example 3 were tested for inflammatory factors IL-4, IL-1. Beta. TNF-. Alpha., IL-6, IL-10, IL-12p70, TGF-. Beta.1 and IFN-. Gamma.by LegendplexTM multifactor bead-flow cytometry as subjects, as follows:
1.1 the exosomes obtained in example 2 and example 3 were mixed with lysate in a volume ratio of 1:1, uniformly mixing, placing on ice for 10min, centrifuging at 4 ℃ for 5min at 3500 Xg, and collecting a lysate supernatant as a sample to be detected of exosomes;
1.2, performing ultrasonic treatment on the magnetic beads for 1min, and performing vortex vibration for 30s; dissolving the standard substance with 250 μl of Assay Buffer, mixing, standing for 10min, adding into EP tube, and marking as C7; in addition, taking EP pipes (C6/C5/C4/C3/C2/C1/C0), adding 75 mu L of Assay Buffer into each pipe, taking 25 mu L of the EP pipes from C7 to C6 for gradient dilution, and so on until the EP pipes are diluted to C1, wherein C0 is Assay Buffer (0 pg/ml);
1.3 adding 25. Mu.L of Assay Buffer to each sample tube; adding 25 mu L of each standard substance and the exosome to-be-detected sample prepared by the method 1.1 to corresponding standard substance tubes and sample tubes; then add 25 μl capture beads to each tube;
1.4 vortex shaking and incubation for 2 hours, 1 XWash Buffer (200. Mu.L) was added to each well and washed once, centrifuged (1000 Xg, 5min normal temperature) and the supernatant removed;
1.5 adding 25 mu L of SA-PE into each tube, shaking in a dark place, and incubating for 30min at room temperature;
1.6 washing with 1 XWash Buffer (200. Mu.L) once per well, centrifuging (1000 Xg, 5min at normal temperature), removing supernatant, and washing again;
1.7 Add 200-300. Mu.L of 1 XWash Buffer, vortex beads, transfer beads to flow tube, prepare for on-press detection, at least 3 replicates per sample. The experimental results are shown in FIG. 7.
2. Antibacterial peptide content determination
The exosomes extracted in example 2 and example 3 were used as subjects, and the content of the exosome antibacterial peptide was detected by using an ELISA kit, and the specific procedures were as follows:
2.1 the exosomes obtained in example 2 and example 3 were mixed with lysate in a volume ratio of 1:1, uniformly mixing, placing on ice for 10min, centrifuging at 4 ℃ for 5min at 3500 Xg, and collecting a lysate supernatant as a sample to be detected of exosomes;
2.2 measuring the amount of the antibacterial peptide LL37 of the sample species to be tested according to the LL37-ELISA kit instructions, at least 3 replicates of each sample were performed, and the measurement results are shown in FIG. 8.
As shown in fig. 7 and 8, the exosomes of example 2 and example 3 are capable of secreting anti-inflammatory factors, pro-inflammatory factors and antimicrobial peptides LL37, which indicates that the method for sequentially separating exosomes of the present invention can extract exosomes with high purity while maintaining the physiological activity of exosomes.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A method for sequentially isolating exosomes, comprising the steps of:
(1) Transferring the cell supernatant containing the exosomes into a hollow fiber column for concentration and primary purification until the volume of the concentrated cell supernatant is 10-20 times of the volume of the cell supernatant before concentration, wherein the obtained concentrated cell supernatant is the crude purified exosomes;
(2) And (3) carrying out composite chromatography on the crude and purified exosomes obtained in the step (1) to obtain purified exosomes.
2. The method according to claim 1, wherein in the step (1), the hollow fiber column has a molecular weight cut-off of 50-500KD and the hollow fiber is polyethersulfone.
3. The method of claim 1, wherein in step (1), the concentrating and preliminary purifying is performed by circulating cell supernatant containing exosomes in a hollow fiber column using peristaltic pump; the rotating speed of the peristaltic pump is 20-30 revolutions/min.
4. The method of claim 1, wherein in step (2), the complex chromatography is performed on the crude purified exosomes using a protein separation purification system;
the chromatographic column adopted by the composite chromatography is a highly crosslinked agarose gel column containing core microbeads and multimode octylamine ligands; the core microbeads mainly comprise ligand activated cores and inert shells, and the average molecular weight cut-off of the core microbeads is 600-700KD.
5. The method of claim 4, wherein the parameters of the complex chromatography of the crude purified exosomes using the protein separation and purification system are: the pressure before the column is 0.8-1.0MPa, the flow rate is 1.0-1.5mL/min, and the detection wavelength of the ultraviolet absorption detector is 280nm; phosphate buffer is used for column balancing and flushing the pipeline.
6. The method of claim 1, wherein the method of sequentially isolating exosomes further comprises the steps of:
(3) Ultrafiltering the purified exosomes obtained in the step (2) by adopting an ultrafiltration tube to obtain refined exosomes; the molecular weight cut-off of the ultrafiltration tube was 100KD.
7. A method for industrially producing mesenchymal stem cell-derived exosomes, comprising the following steps:
s1, mixing mesenchymal stem cells with a microcarrier culture solution, transferring into a rotary bottle, and culturing in a bioreactor until the microcarrier completely covers the mesenchymal stem cells to obtain a microcarrier for loading cells;
s2, dividing the microcarrier loaded with cells obtained in the step S1 into 4 parts averagely, respectively transferring into a rotary bottle, adding a culture medium and the microcarrier not loaded with cells, uniformly mixing, and then placing the rotary bottle into a bioreactor for culturing to obtain mesenchymal stem cell liquid and microcarrier P2 loaded with cells;
s3, continuously rotating the bottle for 2-4 times according to the operation of the step S2 on the microcarrier P2 loaded with cells obtained in the step S2 to obtain mesenchymal stem cell liquid;
s4, removing the mesenchymal stem cells in the mesenchymal stem cell fluid obtained in the steps S2 and S3, and cell fragments and protein aggregates of the mesenchymal stem cells to obtain a mesenchymal stem cell supernatant;
s5, separating and purifying the supernatant of the mesenchymal stem cells obtained in the step S4 according to the method for sequentially separating exosomes according to any one of claims 1-6, so as to obtain the purified exosomes.
8. The method according to claim 7, wherein in step S1, the microcarrier content in the microcarrier culture liquid is 0.3-1.0g microcarrier per liter of culture liquid; the ratio of the mesenchymal stem cells to the microcarriers in the microcarrier culture solution is that the mesenchymal stem cells: microcarrier = (1-3) ×10 6 The following steps: 0.3-1.0g.
9. The method according to claim 7, wherein in step S2, the ratio of the sum of the cell-loaded microcarrier and the non-cell-loaded microcarrier to the medium is (cell-loaded microcarrier+non-loadedMicrocarriers of cells): culture medium= (0.3-1.0) g:1L; the ratio of the sum of cell-loaded microcarriers and non-cell-loaded microcarriers to mesenchymal stem cells is (cell-loaded microcarriers + non-cell-loaded microcarriers): mesenchymal stem cells = 0.3-1g: (1-3). Times.10 7 And each.
10. The method of claim 7, wherein in step S1, the culture conditions are: culturing at 50rpm for 5min and at 0rpm for 24 hr on day 1; culturing at constant speed of 35rpm on day 2-4; the culture temperature is 37 ℃;
in step S2, the culture conditions are: culturing at constant speed at 37 deg.C at 35rpm for 4-8 days.
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