CN111235108A - Cell membrane nano vesicle and preparation method thereof - Google Patents
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A—HUMAN NECESSITIES
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- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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Abstract
The invention discloses a cell membrane nano vesicle and a preparation method thereof, wherein the cell membrane nano vesicle is composed of a bilayer lipid membrane and contents wrapped by the bilayer lipid membrane, and the membrane and the contents of the cell membrane nano vesicle are both from mother cells used for preparation. The preparation method of the cell membrane nano vesicle comprises the following steps: and (3) sequentially and repeatedly extruding the single cell suspension through polycarbonate membranes with different apertures of an extruder to obtain a clarified solution, and performing gradient centrifugation, separation and purification to obtain the cell membrane nano vesicles. The preparation method of the cell membrane nano vesicle has the advantages of high yield, stable vesicle property, good product uniformity, simple and convenient operation, controllable quality and high repeatability, is beneficial to large-scale production, and can be applied to treating the Parkinson disease.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a cell membrane nano vesicle and a preparation method thereof.
Background
Parkinson's Disease (PD) is a chronic neurodegenerative Disease that endangers the health of the middle-aged and elderly, with an incidence of about 1% in people over 60 years of age. The main pathological feature of PD is the progressive loss of dopaminergic neurons of the substantia nigra (DN) in the brain, resulting in a deficiency in striatal Dopamine content, which causes dyskinesias. The motor symptoms of PD patients mainly include resting tremor, muscular rigidity, bradykinesia, and dyskinesia. Currently, the main methods for clinical treatment of PD are levodopa preparations, anticholinergics, receptor agonists and DA release promoting drugs, and Deep Brain Stimulation (DBS) surgery. Early dopaminergic drugs were able to control PD symptoms well, but over time, such drugs gradually lost their efficacy due to fluctuations in non-physiological DA concentrations and off-target effects seen with dopaminergic drugs, while producing many side effects, including neuropsychiatric symptoms and dyskinesias. DBS implants tiny electrodes into the brain and connects neurostimulators to improve patient movement disorders by electrically stimulating the associated nuclei. However, DBS surgery is more limited: the treatment cost is high; the application range is limited, and the medicine is more suitable for patients with late PD and no dementia; side effects are large, such as can lead to permanent cognitive impairment. In summary, drugs and DBS treatments only alleviate PD symptoms and do not replace or regenerate lost DN in the brain.
The application of cell transplantation method to carry out replacement therapy on neurodegenerative diseases is a hot problem for research of scientists at home and abroad at present. The cell transplantation therapy is to transplant cells into a focus or a specific part in a specific manner, and cure a disease through tissue regeneration and repair. The study of PD is more suitable for cell replacement therapy due to clear pathological changes and the deletion of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, becomes the earliest model for the treatment of stem cells of neurodegenerative diseases, and is also the earliest central nervous system disease for carrying out clinical tests on the stem cells in human bodies. Stem cells are a class of cells that have the potential for self-renewal and multipotentiality. Different types of Stem Cells from different sources can play a role in nerve regeneration and repair in PD treatment, and currently, clinical tests for treating PD with Stem Cells in research cover Embryonic Stem Cells (ESCs), Neural Stem Cells (NSCs), Mesenchymal Stem Cells (MSCs) and Induced Pluripotent Stem Cells (iPSCs), and thus, the study hopes are brought for the treatment and recovery of PD.
At present, the stem cells can play the functions of tissue repair and regeneration mainly through two ways of replacement action and bystander mechanism. Many studies have found that the survival and transformation number of stem cells after transplantation is small, so that it is presumed that the bystander mechanism plays a major role, namely, the stem cells transplanted into the body regulate the host microenvironment and repair the focus through the paracrine effect, promote cell conduction and activate endogenous stem cells. Further, numerous studies have demonstrated that paracrine action is one of the major effects of stem cell transplantation in treating spinal cord injury, where extracellular vesicles secreted by stem cells play a key role in the paracrine action of stem cells. Extracellular vesicles are vesicular bodies of a double-membrane structure, which are shed from the cell membrane or secreted by the cell and vary in diameter from 40nm to 1000 nm. Extracellular vesicles are composed mainly of microvesicles, exosomes and apoptotic bodies. The vesicles carry proteins and nucleic acids of stem cells and are important mediators of cell-to-cell communication. Unlike stem cells, vesicles fully inherit membrane surface receptors and surface markers of maternal cells; circumventing the ethical challenges faced by stem cells; avoiding the risk of cell neoplasia and disordered differentiation; can be stored for a long time at-80 ℃ and can be conveniently transported in an ice box; concentration detection can be carried out after extraction, time and dosage in the application process can be controlled, and the preparation can be prepared into various dosage forms, so that the defects of long cell culture period and uncontrollable quantity in the cell transplantation process are avoided.
1×107The cells are cultured for 24h to yield about 2.1X 1011Single vesicle, low yield and long time consumption, limit its clinical application. The yield can be properly improved by adding a chemical reagent or a nano material to induce cells, but the formed vesicles are micron-sized, have larger particle size and cannot cross blood brain barriers. In order to better exert the clinical application of the vesicle, the research on more efficiently preparing the nano-scale vesicleThe method has important application value.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a cell membrane nano vesicle and a preparation method and application thereof.
In order to realize the purpose, the invention adopts the following technical scheme:
the invention provides a cell membrane nano vesicle, which consists of a bilayer lipid membrane and contents wrapped by the bilayer membrane; the bilayer lipid membrane is a cell membrane derived from a mother cell; the contents of the vesicles are derived from the mother cell; the blast cells are adherent cells or suspension cells and are in a monodisperse state after being digested.
In the above technical solution, further, the blast cell is a macrophage or a stem cell.
In the above technical scheme, further, the blast cell is mouse mononuclear macrophage RAW264.7, human umbilical cord mesenchymal stem cell or neural stem cell.
In a second aspect, the present invention provides a method of preparing a cell membrane nanovesicle, the method comprising the steps of:
(1) will contain 1.5X 106～5×106Repeatedly extruding the single cell suspension of each cell to pass through polycarbonate membranes with different pore diameters to obtain a clear solution;
(2) and (2) centrifugally separating and purifying the clarified solution obtained in the step (1) to obtain the cell membrane nano vesicles.
In the above technical solution, further, the temperature of the centrifugation in the step (2) is 4 ℃.
In the technical scheme, the polycarbonate membrane with the pore diameter of 10 microns, 5 microns and 1 micron is sequentially extruded for 3-8 times in the step (1) in sequence.
In the above technical solution, further, the centrifugation in the step (2) comprises the following specific steps: centrifuging at 4 deg.C for 15min at 2000g to remove cell debris, centrifuging the supernatant at 4 deg.C for 15min at 20000g, discarding the supernatant, and repeatedly blowing the precipitate with sterile PBS solution to ensure uniform dispersion of vesicles.
The invention has the beneficial effects that: (1) the cell membrane nano vesicle prepared by the method carries bioactive molecules and membrane receptors derived from macrophages and stem cells, can penetrate biological barriers, protects the contents from being degraded and is efficiently taken up by receptor cells.
(2) The cell membrane nano vesicle has no ethical limitation, the vesicle particle size is 100-200nm, the vesicle can be stably stored for 5h at normal temperature, can be stably kept for 48h at 4 ℃, has high preparation yield, stable vesicle property and good product uniformity, is beneficial to large-scale production, is suitable for replacing cell transplantation, and is used for tissue repair and regeneration.
(3) The NVs prepared by the invention has good repairing function in an MPP + induced primary neuron damage PD model, and can be applied to treatment of Parkinson's disease.
Drawings
FIG. 1 is a flow chart of the manufacturing process of the present invention;
FIG. 2 is a transmission electron microscope image of cell membrane nanovesicles P1 prepared from RAW 264.7;
FIG. 3 is a transmission electron microscope image of cell membrane nanovesicles P2 prepared from RAW 264.7;
fig. 4 is a particle size distribution diagram of cell membrane nanovesicles P1 prepared from RAW 264.7;
fig. 5 is a particle size distribution diagram of cell membrane nanovesicles P1 prepared from RAW 264.7;
fig. 6 is an atomic force microscope picture of cell membrane nanovesicles P1 prepared from RAW 264.7;
fig. 7 shows the stability of cell membrane nanovesicles P2 prepared from RAW 264.7;
FIG. 8 is a transmission electron microscope image of cell membrane nanovesicles prepared from umbilical cord mesenchymal stem cells;
FIG. 9 is a diagram of a distribution of the particle size of a cell membrane nanovesicle prepared from umbilical cord mesenchymal stem cells;
FIG. 10 shows the stability of cell membrane nanovesicles prepared from umbilical cord mesenchymal stem cells;
FIG. 11 is a diagram showing a distribution of the particle size of a cell membrane nanovesicle prepared from neural stem cells;
figure 12 is the protective effect of membrane nanovesicles on the MPP + induced neuronal damage PD model.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1
Preparation and characterization of RAW264.7 cell-derived cell membrane nanovesicles
1. Optimal preparation method of RAW264.7 cell membrane nano-vesicles
The mouse mononuclear macrophage leukemia cell RAW264 used in the invention is purchased from the institute of basic medicine of Chinese academy of medical sciences. RAW246.7 cells were cultured to 80% density in 25T flasks, digested, centrifuged at 800g for 5min, the supernatant was discarded, and the pellet was blown off in 1mL PBS to make a single cell suspension.
The method I comprises the following steps: the cell suspension was extruded continuously 11 times through a 100 μm polycarbonate membrane filter using a Mini Extruder Mini-Extruder supplied by Avanti Polar Lipids, USA. Separation and purification: centrifuging at 4 deg.C and 20000g for 15min, collecting supernatant at 4 deg.C and 100000g, ultraseparating for 1 hr, discarding supernatant, and repeatedly blowing precipitate with sterile PBS solution to ensure uniform vesicle dispersion. The final product is designated as P1. Store at-80 ℃.
Method II: the cell suspension was continuously extruded 5 times through 10, 5 and 1 μm polycarbonate membrane filters using a mini-extruder (FIG. 1). Separation and purification: centrifuging at 4 deg.C for 15min to remove cell debris, centrifuging the supernatant at 4 deg.C for 20000g for 15min, discarding the supernatant, and repeatedly blowing the precipitate with sterile PBS solution to ensure uniform vesicle dispersion. The final product is designated as P2. Store at-80 ℃.
2. Characterization of cell membrane nanovesicles
Transmission Electron Microscope (TEM) characterization: performing ultrasonic treatment on 10 mu L of the cell membrane nano vesicle suspension for 20min, fixing the cell membrane nano vesicle suspension with 4% paraformaldehyde of the same volume at room temperature for 30min, settling the cell membrane nano vesicle suspension on a copper net, dyeing the cell membrane nano vesicle suspension with phosphotungstic acid for 5min, removing redundant liquid, drying, and collecting and observing the size and the appearance of the cell membrane nano vesicle through a TEM. The TEM observes that the cell membrane nano vesicles are all of lipid bilayer structures and are round or cup-mouth-shaped. The agglomeration phenomenon of P1 is obvious (figure 2), and the average grain diameter is 165.3 nm; the P2 particles were uniform in size and dispersed relatively uniformly, and had an average particle size of 54.8nm (FIG. 3).
Dynamic Light Scattering (DLS) characterization: and detecting the hydrated particle size and the distribution of the cell membrane nano vesicles by adopting a DLS (digital Living SoftSpecification) technology. And (3) carrying out water-proof ultrasonic treatment on 1mL of cell membrane nano vesicle suspension for 20min, and transferring the cell membrane nano vesicle suspension into a measuring cell for measurement. After the measurement, the average hydraulic diameter and the polydispersity index (PDI) of the nanovesicles are obtained and analyzed for particle size distribution. The results show that: the average particle size of P1 was 385.6nm, and the PDI was 0.554 (FIG. 4); the average particle size of P2 was 123.2nm, and the PDI was 0.316 (FIG. 5). The PDI represents the uniformity of particle size and is an important index for particle size characterization. When the PDI value is 0.08-0.7, the system has moderate dispersity, and is the optimal application range of the algorithm. The above data indicate that the dispersibility of P2 is superior to P1. Since DLS measures the hydrated particle size of vesicles, it is larger than TEM.
Atomic Force Microscope (AFM) characterization: the morphology of the nanovesicles was studied with AFM. And (3) carrying out ultrasonic treatment on the nano vesicle suspension for 20min, placing a drop of sample on a silicon chip, drying under argon flow, and finally obtaining a measurement image. The obtained images were subjected to Analysis processing using the NanoScope Analysis software. The results show that P1 is a small protrusion on the nanometer scale under AFM. The average height of NVs was 14nm and the average particle size was 90nm (FIG. 6).
Cell membrane nanovesicle stability: selecting 1ml of the nano vesicle PBS suspension prepared under the optimal condition (method II), placing at normal temperature, measuring by DLS technology at 0.5h, 1h, 2h, 3h and 5h respectively, and analyzing the obtained data. P2 ultrasonic water isolation for 20min, placing in a measuring cell, and performing DLS measurement at 0, 0.5, 1, 2, 3, 5h respectively. The results show that the NVs mean particle sizes were 123.2, 137.9, 136.4, 133.6, 130.5, and 125.1nm, respectively, and were relatively stable over time (fig. 7), indicating that NVs were stable in the PBS solution when left at room temperature.
Example 2
1. Preparation method of NVs (human umbilical cord mesenchymal stem cell) -derived
Culture of Umbilical cord Mesenchymal Stem Cells (UMSCs): fully rinsing umbilical cord tissues by PBS buffer solution, shearing the tissues into sections of 2-3 cm by sterile scissors, washing blood clots by the PBS buffer solution, and repeating for 2-3 times until the washing solution is clear. The umbilical cord is cut open and the two arteries and one vein are removed in sequence. The tissue is cut to a surface area of about 10mm225 mm-2 tissue blocks, spreading on the bottom of a culture dish with the colloid surface facing downwards, standing for 15-20 min, slowly adding appropriate amount of culture medium along the side wall, standing at 37 deg.C and 5% CO2Culturing in an incubator. When the cell fusion reaches 80-90%, the culture medium is discarded, and after being cleaned by PBS buffer solution, pancreatin is added to digest the cells until most of the cells become round and begin to fall off. The digestion was stopped by adding the appropriate amount of medium. The cells were blown up and transferred to a centrifuge tube and centrifuged at 1000r/min for 5 min. The supernatant was discarded and PBS was added and blown up to form a single cell suspension.
1.5X 10 by using a small-sized extruder6The single cell suspension of umbilical cord mesenchymal stem cells is continuously extruded for 5 times respectively through a 10, 5 and 1 mu m polycarbonate membrane filter (figure 1). Separation and purification: centrifuging at 4 deg.C for 15min to remove cell debris, centrifuging the supernatant at 4 deg.C for 20000g for 15min, discarding the supernatant, and repeatedly blowing the precipitate with sterile PBS solution to ensure uniform vesicle dispersion. The final product is designated as P3. Store at-80 ℃.
2. Characterization of cell membrane nanovesicles
TEM representation: and (3) carrying out ultrasonic treatment on 10 mu L P3 for 20min, settling on a copper net, dyeing for 5min by using phosphotungstic acid, removing redundant liquid, drying, and collecting and observing the size and the shape of the cell membrane nano vesicle by using a TEM (transmission electron microscope). TEM observed that the cell membrane nanovesicles are all lipid bilayer structures, are round and have an average particle size of 80.3nm (FIG. 8).
Nanoparticle Tracking Analysis (NTA): take 1mLP3, the syringe injector pushes NTA while detecting the particle size and number of P3. The result showed that the average particle size of P3 was 176.4nm (FIG. 9). 1.5X 1061.16X 10 cells can be prepared107730 cell membrane nano vesicles can be prepared by a single cell, and the result shows that the cell membrane nano vesicles prepared by the method are high in yield and suitable for industrial production.
Cell membrane nanovesicle stability: and carrying out ultrasonic treatment on 1mL of P3 in a water-proof way for 5min, measuring the hydration particle size of the mixture by using a DLS (digital Living system) technology at 0 hour, 1 hour, 2 hours, 3 hours and 48 hours respectively, and carrying out result analysis on the obtained data. Wherein the nano vesicles are placed at room temperature at 0, 1, 2 and 3 hours. The storage time is changed to 4 ℃ between 3h and 48 h. The results show that the particle size of NVs is relatively stable over time, with good stability in PBS solution (figure 10). The average NVs particle size was 166.7, 169.3, 173.1, 178.8 and 174.8nm, respectively. Therefore, the cell membrane nano vesicle prepared by the method has good stability.
Example 3
1. Preparation method of cell membrane nano vesicle derived from neural stem cell
Neural Stem cell (Neural Stem Cells, NSCs) culture: the tissue is 7-14 weeks of gestation of aborted fetus (medical waste), the cerebral cortex is separated, placed in NSCs complete culture medium, and divided into small tissue pieces. Blowing and sucking 20-30 times, standing for 10min, sucking supernatant, centrifuging at 820rpm for 5min, and discarding supernatant. Adding special digestive juice into the precipitate, placing the culture dish into an incubator for digestion for 1-2min, centrifuging at 820rpm for 5min, and removing the supernatant. Adding NSCs complete culture solution into the precipitate, adding into a centrifuge tube, gently sucking for 10-15 times to form single cell suspension, and inoculating. Placing into carbon dioxide incubator at 35 deg.C and CO2The concentration was 5%. And (3) centrifuging at 820rpm for 5min after the NSCs grow into neurospheres with proper sizes, collecting precipitates, adding the NSC complete culture solution, flicking the cell precipitates, and sucking the cells for 30 times by using a 1ml pipette tip to obtain a single-cell suspension.
1.0X 10 by using a small-sized extruder6The single cell suspension of the neural stem cells sequentially passes through 10, 5 and 1 mu m polycarbonThe acid ester membrane filters were extruded continuously 5 times each (fig. 1). Separation and purification: centrifuging at 4 deg.C for 15min to remove cell debris, centrifuging the supernatant at 4 deg.C for 20000g for 15min, discarding the supernatant, and repeatedly blowing the precipitate with sterile PBS solution to ensure uniform vesicle dispersion. The final product is designated as P4. Store at-80 ℃.
2. Characterization of cell membrane nanovesicles
NTA detection: take 1mLP4, the syringe injector pushes NTA while detecting the particle size and number of P4. The result showed that the average particle size of P4 was 142.9nm (FIG. 11).
Example 4
Cell membrane nanovesicles for treatment of MPP+PD model for inducing neuronal injury
1、MPP+PD model for inducing neuronal injury
The ventral midbrain tissue of a newborn SD rat within 24h is cut into pieces and is digested by adding trypsin. The digestion was stopped with DMEM/F12, fetal bovine serum, horse serum, glutamine and streptomycin inoculum, filtered three times through a filter screen, counted and inoculated into polylysine-coated well plates. After 4h, the neurons were attached to the wall, the inoculum was aspirated and replaced with maintenance fluid consisting of Neurobasal-A, B27, glutamine and penicillin, after which half of the maintenance fluid was replaced every 2 days. Day 7, 100. mu.M MPP+Adding into neuron, and after 48 hr, the neuron survival rate is 48.15 + -1.16%, and MPP with half lethal dose+The prepared neuron damage is a PD model.
2. Cell membrane nanovesicles
Inoculating the neuron cells of the midbrain on a 96-well plate and a 24-well plate with a creeping plate, culturing for 5 days, dividing the cells into blank groups without any drug intervention, and giving 100 mu M MPP+Control group for intervention, P2 cell membrane nanovesicles of example 1 and 100. mu.M MPP were sequentially administered+The experimental group of (1). Wherein after the experiment group is administered with the cell membrane nano vesicle for 2h, the control group and the experiment group are simultaneously administered with 100 μ M MPP+Intervening, placing the mixture in an incubator for continuous culture, measuring the OD value at 450nm by using a CCK8 kit through a microplate reader after 48 hours, and calculating the survival rate of the neurons. The results show that cell membrane nanovesicles are responsible for MPP+Induced nervesThe neuron has protective effect, the neuron survival rate of the control group is 65.37 +/-6.37%, and the neuron survival rate of the experimental group is 81.45 +/-3.67%. And MPP+Neuronal survival was significantly increased in the NVs group compared to the group (figure 12).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, it is still within the scope of the present invention if they fall within the scope of the claims of the present invention and their equivalents.
Claims (7)
1. A cell membrane nanovesicle, which is characterized in that the cell membrane nanovesicle is composed of a bilayer lipid membrane and a content wrapped by the bilayer lipid membrane; the bilayer lipid membrane is a cell membrane derived from a mother cell; the contents of the vesicles are derived from the mother cell; the blast cell is an adherent cell or a suspension cell.
2. The cell membrane nanovesicle of claim 1, wherein the parent cell is a macrophage or a stem cell.
3. The cell membrane nanovesicle of claim 2, wherein the blast is mouse mononuclear macrophage RAW264.7, human umbilical cord mesenchymal stem cell or neural stem cell.
4. A method of making a cell membrane nanovesicle, comprising the steps of:
(1) will contain 1.5X 106～5×106Repeatedly extruding the single cell suspension of each cell to pass through polycarbonate membranes with different pore diameters to obtain a clear solution;
(2) and (2) centrifugally separating and purifying the clarified solution obtained in the step (1) to obtain the cell membrane nano vesicles.
5. The method for preparing cell membrane nanovesicles according to claim 4, wherein the temperature of centrifugation in step (2) is 4 ℃.
6. The method for preparing cell membrane nanovesicles according to claim 4, wherein the step (1) comprises repeatedly squeezing the polycarbonate membranes with 10 μm, 5 μm and 1 μm pore size for 3-8 times.
7. The method for preparing cell membrane nanovesicles according to claim 4, wherein the centrifugation step in step (2) comprises the following steps: centrifuging at 4 deg.C for 15min at 2000g to remove cell debris, centrifuging the supernatant at 4 deg.C for 15min at 20000g, discarding the supernatant, and repeatedly blowing the precipitate with sterile PBS solution to ensure uniform dispersion of vesicles.
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