CN112980791B - Method for producing microvesicles, microvesicles obtained based on the same and use thereof - Google Patents
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Abstract
A production method of microvesicles relates to the technical field of biological medicines, and comprises the following steps: subjecting the cells to ultrasound treatment; placing the cells subjected to ultrasonic treatment into an incubator for culturing for 24-72 hours, and collecting cell supernatants; purifying the cell supernatant to obtain the microvesicles. According to the production method of the microvesicles, cells are stimulated by using an ultrasonic device, so that the generation of the microvesicles can be greatly promoted, the yield of the microvesicles from the cells is improved, and the further clinical popularization and biological application of the microvesicles are facilitated. In addition, the method for producing the microvesicles has no damage to cells by adopting an ultrasonic cell stimulation method, and is suitable for various cells including astrocytes, stem cells and the like. In addition, the microvesicles obtained by the production method of the microvesicles and the application thereof in medicines are also provided.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a production method of microvesicles, microvesicles obtained based on the production method of the microvesicles and application thereof.
Background
Cell therapy has wide application value, including stem cell therapy, immune cell therapy, etc. Exosomes (exosomes), a small lipid microvesicles (microvesicles), are 50-150nm in size and play a vital role in physiology and pathology. As an active biocontainer, exosomes mediate intercellular communication by transferring proteins and genetic information between cells. The exosomes/microvesicles derived from cells can avoid immune rejection reaction, are convenient to store, and have great application value in regenerative medicine.
The current method for collecting exosomes/microvesicles is to culture cells, collect a large amount of cell supernatant, separate and extract, and have high cost. The yield of exosomes severely limits their use in the biomedical field. However, the lower yield of exosomes/microvesicles severely limits their use and further clinical popularization. How to promote the output of exosomes/microvesicles in a noninvasive manner has important scientific research and application values.
The use of shock wave therapy to promote secretion of exosomes has been reported in the art, and the literature is published in Cardiovascular research. In this approach, endothelial cells are stimulated with shock wave therapy and the exosomes produced carry miR-19a-3p, which can improve part of the function of myocardial ischemia.
The disadvantage of the conventional technique is that the mechanical action of the shock wave may damage the cells and the results are shown in the literature to be effective only on endothelial cells.
Disclosure of Invention
In view of the above, it is necessary to provide a method for producing microvesicles which are not damaging to cells and are suitable for use in a variety of cells, microvesicles obtained by the above-mentioned method for producing microvesicles, and uses thereof.
A method of producing microvesicles comprising the steps of:
subjecting the cells to ultrasound treatment;
placing the cells subjected to ultrasonic treatment into an incubator for culturing for 24-72 hours, and collecting cell supernatants;
purifying the cell supernatant to obtain the microvesicles.
In one embodiment, the cell is sonicated further comprising the steps of:
culturing the cells in a carbon dioxide incubator at 37 ℃;
when the concentration of the cells is 70% or more, the cells are placed on a serum-free medium.
In one embodiment, the ultrasonic energy interval is 0.2W/cm 2 -4W/cm 2 The ultrasonic treatment time is 1s-10min.
In one embodiment, an ultrasonic device for ultrasonic treatment comprises a signal generator, a power amplifier and an ultrasonic probe, wherein the power amplifier is connected with the signal generator, the ultrasonic probe is connected with the power amplifier, and the ultrasonic probe is a focusing probe or a non-focusing probe or an array probe, and the frequency is 200KHz-5MHz.
In one embodiment, the step of purifying the cell supernatant to obtain the microvesicles comprises:
centrifuging the cell supernatant for a plurality of times to obtain the microvesicles; or (b)
Performing centrifugal treatment on the cell supernatant for a plurality of times, and then performing ultrafiltration treatment to obtain the microvesicles; or (b)
And (3) after carrying out centrifugal treatment on the cell supernatant for a plurality of times, adding a microvesicle extraction reagent to carry out precipitation reaction, and centrifuging again to obtain the microvesicles.
In one embodiment, the centrifugation of the cell supernatant a plurality of times to obtain the microvesicles is performed as follows:
centrifuging the cell supernatant for 3-10 minutes under the condition of the rotating speed of 300-500 g after removing the first precipitate, and obtaining the first liquid after removing the second precipitate;
centrifuging the first liquid for 25-60 minutes at the rotating speed of 1500-4000g, and removing the third precipitate to obtain a second liquid;
centrifuging the second liquid for 45-90 minutes at the rotating speed of 9000-15000 g, and removing the fourth precipitate to obtain a third liquid;
centrifuging the third liquid for 50-120 minutes at the rotating speed of 90000-120000 g, and removing the fifth precipitate to obtain the microvesicles.
In one embodiment, the centrifugation of the cell supernatant is followed by ultrafiltration to obtain the microvesicles by:
centrifuging the cell supernatant for 3-10 minutes under the condition of the rotating speed of 300-500 g after removing the first precipitate, and obtaining the first liquid after removing the second precipitate;
centrifuging the first liquid for 25-60 minutes at the rotating speed of 1500-4000g, and removing the third precipitate to obtain a second liquid;
centrifuging the second liquid for 45-90 minutes at the rotating speed of 9000-12000 g, and removing the fourth precipitate to obtain a third liquid;
centrifuging the third liquid in a 100kDa ultrafiltration tube for 50-120 minutes, and removing the fifth precipitate to obtain the microvesicles.
In one embodiment, the cell supernatant is subjected to a plurality of centrifugation steps, then microvesicle extraction reagent is added to carry out precipitation reaction, and after centrifugation, the microvesicles are obtained by the following steps:
centrifuging the cell supernatant for 3-10 minutes under the condition of the rotating speed of 300-500 g after removing the first precipitate, and obtaining the first liquid after removing the second precipitate;
centrifuging the first liquid for 25-60 minutes at the rotating speed of 1500-4000g, and removing the third precipitate to obtain a second liquid;
centrifuging the second liquid for 60 minutes at the rotating speed of 9000g-12000g, and removing the fourth precipitate to obtain a third liquid;
adding the microvesicle extraction reagent into the third liquid according to the volume ratio of the third liquid to the microvesicle extraction reagent of 1:5, and reacting for 12-24 hours;
centrifuging for 60 minutes at a rotating speed of 3000g-12000g, and reserving fifth sediment;
and adding PBS solution into the fifth precipitate, centrifuging for 60 minutes at the rotating speed of 9000g-12000g, and removing the sixth precipitate to obtain the microvesicles.
A microvesicle obtained by the production method of the microvesicle.
The microvesicles obtained by the production method of the microvesicles are applied to medicines.
According to the production method of the microvesicles, cells are stimulated by using an ultrasonic device, so that the generation of the microvesicles can be greatly promoted, the yield of the microvesicles from the cells is improved, and the further clinical popularization and biological application of the microvesicles are facilitated. In addition, the method for producing the microvesicles has no damage to cells by adopting an ultrasonic cell stimulation method, and is suitable for various cells including astrocytes, stem cells and the like.
Drawings
FIG. 1 is a schematic flow chart of a method for producing microvesicles according to an embodiment;
FIG. 2 is a schematic diagram showing an operation flow of subjecting a cell supernatant to multiple centrifugation to obtain microvesicles according to an embodiment;
FIG. 3 is a schematic diagram showing an operation flow of performing ultrafiltration after subjecting cell supernatants to centrifugation multiple times to obtain microvesicles according to an embodiment;
FIG. 4 is a schematic diagram showing the operation flow of the microvesicles obtained by performing centrifugation on the cell supernatant several times, adding a microvesicle extraction reagent to perform precipitation reaction, and centrifuging again;
FIG. 5 is a graph depicting the number of exosome particles;
FIG. 6 is the total number of microvesicles of example 1 and comparative example 1;
FIG. 7 shows cell proliferation after ultrasonic stimulation.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Furthermore, the terms "first," "second," and the like, are used merely to distinguish between descriptions and should not be construed as indicating or implying relative importance.
The following describes specific embodiments of the present invention in detail with reference to the drawings.
As shown in fig. 1, the method for producing microvesicles according to an embodiment includes the following steps:
s10, performing ultrasonic treatment on the cells.
In one embodiment, the cell is sonicated further comprising the steps of:
s1, culturing the cells in a carbon dioxide incubator.
S2, placing the cells on a serum-free culture medium when the concentration of the cells is more than or equal to 70%.
In one embodiment, in S10, the cells are sonicated on serum-free medium.
In one embodiment, the cells may be cultured in a petri dish or flask.
It will be appreciated that the cells may be suspension cells or adherent cells. The cell type may be nerve cell, glial cell, stem cell, adipocyte, tumor cell, immune cell, etc.
Further, in S1, the temperature at which the cells were cultured in a carbon dioxide incubator was 37 ℃.
In one embodiment, an ultrasonic device for ultrasonic treatment comprises a signal generator, a power amplifier and an ultrasonic probe, wherein the power amplifier is connected with the signal generator, and the ultrasonic probe is connected with the power amplifier. The ultrasound probe may be a focused probe or a non-focused probe or an array probe.
Further, the frequency of the ultrasonic probe may be 200KHz-5MHz.
In one embodiment, the ultrasonic energy interval may be 0.2W/cm 2 -4W/cm 2 The ultrasonic treatment time can be 10s-10min.
In one embodiment, the ultrasound waves may be continuous waves or pulsed waves.
S20, placing the cells subjected to ultrasonic treatment in an incubator for culturing for 24-72 hours, and collecting cell supernatants.
In one embodiment, the incubator is a carbon dioxide incubator.
Preferably, the incubation time may be 24 hours, 48 hours or 72 hours.
S30, purifying the cell supernatant to obtain the microvesicles.
In one embodiment, microvesicles refer to exosomes.
It will be appreciated that in S30, the step of purifying the cell supernatant to obtain microvesicles may be:
A. centrifuging the cell supernatant for a plurality of times to obtain microvesicles; or (b)
B. Centrifuging the cell supernatant for a plurality of times, and then performing ultrafiltration treatment to obtain microvesicles; or (b)
C. And (3) after carrying out centrifugal treatment on the cell supernatant for a plurality of times, adding a microvesicle extraction reagent to carry out precipitation reaction, and centrifuging again to obtain microvesicles.
Further, please refer to fig. 2, a, the operations of centrifuging the cell supernatant multiple times to obtain microvesicles are as follows:
SA10, centrifuging the cell supernatant at a rotating speed of 300g-500g for 3-10 minutes after removing the first precipitate, and obtaining the first liquid after removing the second precipitate.
SA20, centrifuging the first liquid for 25-60 minutes at the rotating speed of 1500-4000g, and removing the third precipitate to obtain the second liquid.
SA30, centrifuging the second liquid for 45-90 minutes at the rotating speed of 9000-15000 g, and removing the fourth precipitate to obtain a third liquid.
SA40, centrifuging the third liquid for 50-120 minutes at the rotating speed of 90000-120000 g, and removing the fifth precipitate to obtain the microvesicles.
Further, referring to fig. 3, b, the operations of performing centrifugation on the cell supernatant several times, and then performing ultrafiltration to obtain microvesicles are as follows:
SB10, after removing the first precipitate from the cell supernatant, centrifuging for 3-10 minutes at a rotation speed of 300-500 g, and removing the second precipitate to obtain the first liquid.
SB20, centrifuging the first liquid for 25-60 minutes at the rotating speed of 1500-4000g, and removing the third precipitate to obtain the second liquid.
SB30, centrifuging the second liquid for 45-90 minutes under the condition of the rotating speed of 9000-12000 g, and removing the fourth precipitate to obtain a third liquid.
SB40, centrifuging the third liquid in a 100kDa ultrafiltration tube for 50-120 minutes, and removing the fifth precipitate to obtain the microvesicles.
Further, please refer to fig. 4, c, after performing centrifugation on the cell supernatant for a plurality of times, adding a microvesicle extraction reagent to perform precipitation reaction, and performing centrifugation again to obtain microvesicles:
SC10, after removing the first precipitate from the cell supernatant, centrifuging for 3-10 minutes at a rotation speed of 300-500 g, and after removing the second precipitate, obtaining the first liquid.
And SC20, centrifuging the first liquid for 25-60 minutes at the rotating speed of 1500-4000g, and removing the third precipitate to obtain the second liquid.
And SC30, centrifuging the second liquid for 60 minutes at the rotating speed of 9000g-12000g, and removing the fourth precipitate to obtain a third liquid.
SC40, adding the microvesicle extraction reagent into the third liquid according to the volume ratio of the third liquid to the microvesicle extraction reagent of 1:5, and reacting for 12-24 hours.
In one embodiment, the microvesicle extraction reagent is Exoquick-TC.
SC50, centrifuging for 60 minutes at a rotational speed of 3000g-12000g, and retaining a fifth precipitate.
SC60, PBS solution is added into the fifth precipitate, and the mixture is centrifuged for 60 minutes under the condition that the rotating speed is 9000g-12000g, and the sixth precipitate is removed, so that microvesicles are obtained.
Ultrasonic waves are mechanical waves that are capable of propagating in both solids and liquids (elastic media) and have a variety of biological effects (including mainly thermal effects, mechanical effects, cavitation effects, etc.). According to the production method of the microvesicles, cells are stimulated by using an ultrasonic device, so that the generation of the microvesicles can be greatly promoted, the yield of the microvesicles from the cells is improved, and the further clinical popularization and biological application of the microvesicles are facilitated. In the above production method of microvesicles, the cell is not damaged by the ultrasonic stimulation method, the cell morphology is not changed under a microscope after ultrasonic stimulation, the cell proliferation condition is detected by cck-8 after continuous culture, and the proliferation activity is unchanged from a control untreated group, as shown in fig. 7, wherein ctrl represents a control group which is not subjected to ultrasonic treatment, and us represents an experimental group after ultrasonic treatment. Is suitable for various cells including astrocytes, stem cells, etc.
The microvesicles prepared by the production method of the microvesicles are quantitatively analyzed by the following method.
The particle number of the microvesicles under different sizes can be precisely obtained by directly imaging and observing specific microvesicles in the diameter range of 50nm-1000nm in suspension in real time according to the Nanoparticle Tracking Analysis (NTA) technology by using a NanoSight instrument of Markov. The secretion of cell microvesicles is increased after ultrasonic stimulation, and the NTA measurement quantity is increased, as shown in a graph five, wherein HA-EXO is microvesicles derived from astrocytes without ultrasonic stimulation, and US-HA-EXO is microvesicles derived from astrocytes after ultrasonic stimulation.
The microvesicles obtained by the production method of the microvesicles can be applied to medicines, and can also be applied to scientific researches and clinic.
The following is a detailed description of embodiments.
Example 1
Glial cells were cultured in a carbon dioxide incubator at 37℃until cell density reached 80%, and after replacing the medium with serum-free medium, the flask or dish was placed on an ultrasonic rack. The degassed water is used as a coupling to transfer acoustic energy.
The ultrasonic device used for ultrasonic treatment comprises a signal generator, a power amplifier and an ultrasonic probe, wherein the power amplifier is connected with the signal generator, the ultrasonic probe is connected with the power amplifier, and the ultrasonic probe is a focusing probe. The frequency of the ultrasonic probe is 200KHz. Ultrasonic energy of 2.5W/cm 2 . The ultrasonic treatment time is 5min. The ultrasonic wave is a continuous wave.
The sonicated cells were placed in a carbon dioxide incubator for 24 hours and the cell supernatant was collected.
After removing the first precipitate from the cell supernatant, the supernatant was centrifuged at 400g for 5 minutes to remove the second precipitate, thereby obtaining a first liquid.
The first liquid was centrifuged at 2000g for 30 minutes to remove the third precipitate and obtain a second liquid.
The second liquid was centrifuged at 10000g for 60 minutes to remove the fourth precipitate, and a third liquid was obtained.
Centrifuging the third liquid at 100000g for 60 min, and removing the fifth precipitate to obtain microvesicles.
The number of microvesicles obtained as described above was examined according to the Nanoparticle Tracking Analysis (NTA) technique described above, and reference is made to fig. six, which shows that the average total number of microvesicles secreted by the sonicated group was about 6.067 ×10 10 /mL。
Example 2
After the cell density reaches 60%, the culture medium is replaced by serum-free medium, and the flask or dish is placed on an ultrasonic frame. The degassed water is used as a coupling to transfer acoustic energy.
The ultrasonic device used for ultrasonic treatment comprises a signal generator, a power amplifier and an ultrasonic probe, wherein the power amplifier is connected with the signal generator, the ultrasonic probe is connected with the power amplifier, and the ultrasonic probe is a focusing probe. The frequency of the ultrasonic probe was 1MHz. Ultrasonic energy of 4.0W/cm 2 . The sonication time was 10s. The ultrasonic wave is a continuous wave.
The sonicated cells were placed in a carbon dioxide incubator for 72 hours and the cell supernatant was collected.
After removing the first precipitate from the cell supernatant, the supernatant was centrifuged at 400g for 5 minutes to remove the second precipitate, thereby obtaining a first liquid.
The first liquid was centrifuged at 2000g for 30 minutes to remove the third precipitate and obtain a second liquid.
The second liquid was centrifuged at 10000g for 60 minutes to remove the fourth precipitate, and a third liquid was obtained.
The third liquid was centrifuged in a 100kDa ultrafiltration tube for 60 min and the fifth precipitate was removed to give microvesicles.
The number of microvesicles obtained above was examined according to the Nanoparticle Tracking Analysis (NTA) technique described above, and the average total number of microvesicles secreted by the sonicated group was about 5.8X10 × 10 /mL。
Example 3
After the cell concentration reaches 70%, the culture medium is replaced by serum-free culture medium, and the culture flask or the culture dish is placed on an ultrasonic frame. The degassed water is used as a coupling to transfer acoustic energy.
The ultrasonic device used for ultrasonic treatment comprises a signal generator, a power amplifier and an ultrasonic probe, wherein the power amplifier is connected with the signal generator, the ultrasonic probe is connected with the power amplifier, and the ultrasonic probe is a focusing probe. The frequency of the ultrasonic probe was 5MHz. Ultrasonic energy of 0.2W/cm 2 . The ultrasonic treatment time is 10min. The ultrasonic wave is a pulse wave.
The sonicated cells were placed in a carbon dioxide incubator for 48 hours and the cell supernatant was collected.
After removing the first precipitate from the cell supernatant, the supernatant was centrifuged at 400g for 5 minutes to remove the second precipitate, thereby obtaining a first liquid.
The first liquid was centrifuged at 2000g for 30 minutes to remove the third precipitate and obtain a second liquid.
The second liquid was centrifuged at 10000g for 60 minutes to remove the fourth precipitate, and a third liquid was obtained.
The Exoquick-TC was added to the third liquid at a ratio of 1:5 by volume of the third liquid to the Exoquick-TC, and reacted for 12 hours.
Centrifugation was carried out at 12000g for 60 minutes, leaving a fifth precipitate.
Adding PBS solution into the fifth precipitate, centrifuging at 12000g for 60 min, and removing the sixth precipitate to obtain microvesicles.
Pairs according to Nanoparticle Tracking Analysis (NTA) techniques described aboveThe number of microvesicles obtained above was measured, and the average total number of microvesicles secreted by the sonicated group was about 6.2X10 10 /mL。
Example 4
Glial cells were cultured in a carbon dioxide incubator at 37℃until cell density reached 80%, and after replacing the medium with serum-free medium, the flask or dish was placed on an ultrasonic rack. The degassed water is used as a coupling to transfer acoustic energy.
The ultrasonic device used for ultrasonic treatment comprises a signal generator, a power amplifier and an ultrasonic probe, wherein the power amplifier is connected with the signal generator, the ultrasonic probe is connected with the power amplifier, and the ultrasonic probe is a focusing probe. The frequency of the ultrasonic probe is 200KHz. Ultrasonic energy of 2.5W/cm 2 . The ultrasonic treatment time is 5min. The ultrasonic wave is a continuous wave.
The sonicated cells were placed in a carbon dioxide incubator for 24 hours and the cell supernatant was collected.
After removing the first precipitate from the cell supernatant, the supernatant was centrifuged at 500g for 3 minutes to remove the second precipitate, thereby obtaining a first liquid.
The first liquid was centrifuged at 4000g for 25 minutes to remove the third precipitate and obtain a second liquid.
The second liquid was centrifuged at 9000g for 60 minutes to remove the fourth precipitate, and a third liquid was obtained.
The third liquid was centrifuged at 90000g for 50 minutes to remove the fifth precipitate, and the microvesicles were obtained.
The number of microvesicles obtained as described above was examined according to the Nanoparticle Tracking Analysis (NTA) technique described above, and reference is made to fig. six, in which the average total number of microvesicles secreted by the sonicated group was about 6.1×10 10 /mL。
Example 5
After the cell density reaches 60%, the culture medium is replaced by serum-free medium, and the flask or dish is placed on an ultrasonic frame. The degassed water is used as a coupling to transfer acoustic energy.
The ultrasonic device used for ultrasonic treatment comprises a signal generator, a power amplifier and an ultrasonic probe, wherein the power amplifier is connected with the signal generator, the ultrasonic probe is connected with the power amplifier, and the ultrasonic probe is a focusing probe. The frequency of the ultrasonic probe was 1MHz. Ultrasonic energy of 4.0W/cm 2 . The sonication time was 10s. The ultrasonic wave is a continuous wave.
The sonicated cells were placed in a carbon dioxide incubator for 72 hours and the cell supernatant was collected.
After removing the first precipitate from the cell supernatant, the supernatant was centrifuged at 300g for 100 minutes to remove the second precipitate, thereby obtaining a first liquid.
The first liquid was centrifuged at 1500g for 60 minutes to remove the third precipitate and obtain a second liquid.
The second liquid was centrifuged at 12000g for 45 minutes to remove the fourth precipitate, and a third liquid was obtained.
The third liquid was centrifuged in a 100kDa ultrafiltration tube for 120 min and the fifth precipitate was removed to give microvesicles.
The number of microvesicles obtained above was examined according to the Nanoparticle Tracking Analysis (NTA) technique described above, and the average total number of microvesicles secreted by the sonicated group was about 5.7X10 10 /mL。
Example 6
After the cell concentration reaches 70%, the culture medium is replaced by serum-free culture medium, and the culture flask or the culture dish is placed on an ultrasonic frame. The degassed water is used as a coupling to transfer acoustic energy.
The ultrasonic device used for ultrasonic treatment comprises a signal generator, a power amplifier and an ultrasonic probe, wherein the power amplifier is connected with the signal generator, the ultrasonic probe is connected with the power amplifier, and the ultrasonic probe is a focusing probe. The frequency of the ultrasonic probe was 5MHz. Ultrasonic energy of 0.2W/cm 2 . The ultrasonic treatment time is 10min. The ultrasonic wave is a pulse wave.
The sonicated cells were placed in a carbon dioxide incubator for 48 hours and the cell supernatant was collected.
After removing the first precipitate from the cell supernatant, the supernatant was centrifuged at 500g for 10 minutes to remove the second precipitate, thereby obtaining a first liquid.
The first liquid was centrifuged at 1500g for 60 minutes to remove the third precipitate and obtain a second liquid.
The second liquid was centrifuged at 9000g for 60 minutes to remove the fourth precipitate, and a third liquid was obtained.
The Exoquick-TC was added to the third liquid at a ratio of 1:5 by volume of the third liquid to the Exoquick-TC, and reacted for 24 hours.
Centrifugation was carried out at 3000g for 60 minutes, leaving a fifth precipitate.
Adding PBS solution into the fifth precipitate, centrifuging at 9000g for 60 min, and removing the sixth precipitate to obtain microvesicles.
The number of microvesicles obtained above was examined according to the Nanoparticle Tracking Analysis (NTA) technique described above, and the average total number of microvesicles secreted by the sonicated group was about 5.5X10 10 /mL。
Comparative example 1
Culturing glial cells in a carbon dioxide incubator at 37 ℃, and replacing the culture medium with a serum-free culture medium for continuous culture for 5min after the cell density reaches 60% -80%.
The cells were then placed in a carbon dioxide incubator for continued culture for 24 hours, and the cell supernatants were collected.
After removing the first precipitate from the cell supernatant, the supernatant was centrifuged at 400g for 5 minutes to remove the second precipitate, thereby obtaining a first liquid.
The first liquid was centrifuged at 2000g for 30 minutes to remove the third precipitate and obtain a second liquid.
The second liquid was centrifuged at 10000g for 60 minutes to remove the fourth precipitate, and a third liquid was obtained.
Centrifuging the third liquid at 100000g for 60 min, and removing the fifth precipitate to obtain microvesicles.
The number of microvesicles obtained as described above was examined according to the Nanoparticle Tracking Analysis (NTA) technique described above, and reference is made to fig. six, in which the average total number of microvesicle particles of comparative example 1 was about 1.467×10 10 /mL。
It can be seen that examples 1 to 6, since cells were stimulated with ultrasound, cells promoted secretion of microvesicles, and the number of particles of microvesicles was much larger than that of comparative example 1. The total number of particles of the microvesicles produced in example 1 was 4.13 times the total number of particles of the microvesicles produced in comparative example 1.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (2)
1. A method for producing microvesicles, comprising the steps of:
subjecting the cells to ultrasound treatment;
placing the cells subjected to ultrasonic treatment in an incubator for culturing for 48 hours, and collecting cell supernatants;
purifying the cell supernatant to obtain the microvesicles;
ultrasonic energy of 0.2W/cm 2 The ultrasonic treatment time is 10min;
the ultrasonic device used for ultrasonic treatment comprises a signal generator, a power amplifier and an ultrasonic probe, wherein the power amplifier is connected with the signal generator, the ultrasonic probe is connected with the power amplifier, the ultrasonic probe is a focusing probe, the frequency of the ultrasonic probe is 5MHz, and ultrasonic waves are pulse waves;
the cells are glial cells;
the step of purifying the cell supernatant to obtain the microvesicles comprises the following steps:
centrifuging the cell supernatant for 5 minutes at a rotation speed of 400g after removing the first precipitate, and obtaining a first liquid after removing the second precipitate;
centrifuging the first liquid for 30 minutes at a rotating speed of 2000g, and removing the third precipitate to obtain a second liquid;
centrifuging the second liquid for 60 minutes at the rotation speed of 10000g, and removing the fourth precipitate to obtain a third liquid;
adding Exoquick-TC into the third liquid according to the volume ratio of the third liquid to the Exoquick-TC of 1:5, and reacting for 12 hours;
centrifuging for 60 minutes at a speed of 12000g, and retaining a fifth precipitate;
adding PBS solution into the fifth precipitate, centrifuging at 12000g for 60 min, and removing the sixth precipitate to obtain microvesicles.
2. The method of claim 1, wherein the step of sonicating the cells further comprises the step of:
culturing the cells in a carbon dioxide incubator at 37 ℃;
when the concentration of the cells is 70% or more, the cells are placed on a serum-free medium.
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