CN111363680A - A pulse-stirred bioreactor for exosome secretion, isolation and collection - Google Patents

Abstract

The invention discloses a pulse stirring bioreactor for exosome secretion, separation and collection, comprising a cell culture device and an exosome separation and collection device; the exosome separation and collection device is connected to the cell culture device, and can The exosomes are separated from the culture solution in the cell culture device for collection; the cell culture device includes a stirring device, which is used to apply stirring power to the culture solution in the cell culture device; the cell culture device includes a pulse device, which can add a stirring device to the cell culture device. The culture medium was added in pulses. The bioreactor can fully mix the culture medium and the cells to maintain sufficient oxygen supply, and the stirring device does not directly contact the cells, thereby reducing the shear force on the cells. Other suspension cells secrete cellular products in large quantities.

Description

A pulse-stirred bioreactor for exosome secretion, isolation and collection

technical field

The invention relates to the preparation and separation technology of exosomes, in particular to a pulse stirring bioreactor for exosome secretion, separation and collection.

Background technique

Exosomes are extracellular vesicles (EVs) with a lipid bilayer and a diameter of about 30-150 nm. Exosomes can contain a variety of biologically active substances such as proteins, lipids, nucleic acids, and most cells in the human body, such as immune cells (T cells, B cells, Kupffer cells, and NK cells), endothelial cells, and platelets can secrete exosomes. body. At present, the research on exosomes in clinical treatment can be roughly summarized as follows: (1) Regulate the release and distribution of exosomes. (2) Drug delivery based on exosomes. (3) Exosome-mediated tumor targeting therapy. (4) Exosomes and immunotherapy. At present, exosomes have shown great application prospects in the immunotherapy of tuberculosis, the diagnosis of bone tumors, liver cancer, ovarian cancer, prostate cancer and other cancers, inhibition or regulation of tumor cell metastasis, prognosis monitoring and treatment. In addition, as a carrier of cell-to-cell communication, exosomes are also an important way of stem cell paracrine activity, and play an important role in tissue regeneration. Recent studies have shown that MSCs exosomes have similar properties to MSCs. Its functions include repairing and regenerating tissues, inhibiting inflammatory responses and regulating immunity.

CAR-T therapy, the use of genetically engineered T cells to kill cancer cells, has proven to be a promising option when other treatments have failed. In a new study, researchers from the University of California, San Diego and the University of Minnesota reported using human induced pluripotent stem cells (induced pluripotent stem cells, iPS cells) cultured and similar to CAR-T cells. Natural killer cells (NK cells) modified in this way are highly effective against ovarian cancer in a mouse model. However, it has been reported that NK cell exosomes have the function of NK cell part, are smaller and have better specificity, and are expected to play a unique role in cell therapy.

In recent years, studies have shown that NK cells can secrete exosomes with NK cell markers and killer proteins, which can kill tumor cells. In addition, because exosomes have the characteristics of transferring to targeted target organs, they can be designed as A drug carrier for precise drug delivery that transports specific drugs to target organs for action. Therefore, NK cell exosomes have great potential in tumor therapy and the application of drug targeting carriers. At present, the extraction and purification of exosomes are mainly based on the physicochemical properties of exosomes. Differential centrifugation combined with sucrose density gradient centrifugation, ultrafiltration, immunomagnetic bead extraction and ExoQuick Precipitation extraction are commonly used.

At present, bioreactors are the core equipment for large-scale suspension culture of cells, which can effectively increase the culture density of cells per unit volume. Animal cell culture bioreactors are divided into agitated cell culture bioreactors and non-stirred cell culture bioreactors according to whether there is a stirring paddle inside. The stirred cell culture reactor generates a vortex through the rotation of the stirring paddle to complete the aeration and oxygen supply of the culture medium, but the mechanical shear force generated by this method is relatively large, which can easily damage the cells. At present, the effect on cells can only be mitigated by controlling the stirring speed, but the problem remains. The non-stirred cell culture reactor generates little shear force, and mainly uses vibration, shaking, rolling or swinging to move the culture medium to maintain the suspended state of cells and the gas exchange of the culture medium. The disadvantage is that it is difficult to ensure the absorption efficiency of oxygen. In recent years, there have also been methods of using air or oxygen injection to solve the oxygen supply in the culture medium, but this method is prone to generate a lot of foam, and the bubble bursting process will also cause physical damage to the cells, and excessive oxygen injection will also poison the cells. Precise control of gas injection also complicates reactor equipment and increases the cost of cell culture. Therefore, how to provide uniform agitation to fully mix the culture medium and cells to maintain sufficient oxygen supply while reducing mechanical shear damage to cells is an urgent problem to be solved.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned problems, the present invention provides a pulse-stirred bioreactor for exosome secretion, separation and collection.

In order to achieve the above object, the present invention adopts the following technical solutions:

A pulse-stirred bioreactor for exosome secretion, separation and collection, comprising a cell culture device and an exosome separation and collection device;

The cell culture device is used for culturing cells and stimulating cells to secrete exosomes; the exosome separation and collection device is connected to the cell culture device, and can separate the exosomes from the culture medium in the cell culture device for collection, After separation, the cells remain in the cell culture device;

The cell culture device includes a stirring device configured to apply stirring power to the culture fluid in the cell culture device;

The cell culture device includes a pulse device for adding a culture medium to the cell culture device in a pulsed manner, so as to stimulate the cultured cells in the cell culture device.

Further, the cell culture device further includes a cell culture chamber, and the cell culture chamber is used for cell culture and exosome secretion.

Further, the cell culture cavity is provided with an inoculation port, a culture medium injection port and a sample injection port.

Further, the stirring device is located below the cell culture chamber, and the stirring device adopts a magnetic stirring device, and the magnetic stirring device comprises a magnetic stirring bar and a magnetic driving device, and the magnetic stirring bar can be driven by the magnetic driving device. .

Further, the rotational speed of the magnetic stirring bar can be adjusted by the intensity of the magnetic field, and the adjustment range is 30-150 rpm.

Further, the pulse device includes a pulse pump, and the pulse pump communicates with the cell culture chamber through a liquid pipeline.

Further, the separation and collection device includes a preliminary separation device located at the bottom of the cell culture cavity, the preliminary separation device includes a preliminary separation cavity, and the preliminary separation cavity is communicated with the cell culture cavity.

Further, the separation and collection device includes a multi-layer filter device, the multi-layer filter device is connected to the preliminary separation chamber through a filter pipeline, and the outlet of the multi-layer filter device is connected to a product collection tank.

Further, the bioreactor also includes a condition monitoring device and an adjustment device, the condition monitoring device includes a pH value monitoring probe, a CO ₂ content monitoring probe, an O ₂ content monitoring probe, and a condition sampling device, a pH value monitoring probe, a CO ₂ The content monitoring probe and O ₂ content monitoring probe are placed in the cell culture chamber, and the adjustable range of culture condition monitoring is liquid dissolved oxygen 50-100%, carbon dioxide concentration 0-20%, PH 5-9; Outside the cell culture chamber, the conditional sample addition device communicates with the cell culture chamber, and the conditional sample addition device can adjust the cell culture environment by adding adjustment reagents in time according to the information detected by the probe.

The present invention also provides a method for stimulating cells to secrete a large amount of exosomes. The method adopts the above-mentioned bioreactor, and monitors and adjusts the culture conditions in the cell culture chamber through a condition monitoring device and a regulating device. , and, by simulating the culture conditions in the cell culture chamber by stirring and pulsing, the cells are placed in a cell growth environment that dynamically simulates the blood structure in vivo, and the cells are stimulated to secrete exosomes.

The present invention also provides a method for stimulating cells to secrete exosomes, the method utilizes the above-mentioned bioreactor, comprising the following steps:

(1) Filling the culture medium: turn on the switch of the bioreactor, open the valve of the culture medium filling port, add the culture medium, and fill the cell culture chamber;

(2) Inoculation: control the opening of the valve of the inoculation port, and the inoculation density is 2×10 ⁵ /ml of NK92mi cells;

(3) Stimulate cells: turn on the magnetic stirring and pulse device, set the magnetic stirring speed to 50 rmp, and adjust the pulse frequency to 72 times/min, simulate the cell growth environment in vivo, and form turbulent flow to stimulate NK cells to secrete exosomes;

(4) Monitoring and automatic adjustment of culture conditions: The monitoring chip monitors the cell culture environment in real time, and automatically adjusts the culture conditions through the automatic control system controlled by the host. The culture conditions are set to 95% liquid dissolved oxygen, 5% carbon dioxide concentration, and PH 7.3 ;

(5) Separation and collection of exosomes: After the cells were grown in the culture device for 2 days, the filter pump was turned on, and the culture solution was passed through the separation and collection device, and the final product was finally collected.

The beneficial effects of the present invention are:

(1) In the present invention, oxygen and carbon dioxide are introduced into the preliminary separation cavity, and under the action of the magnetic stirring device, the oxygen and carbon dioxide are first dissolved in the culture medium, and then the first filter connecting the preliminary separation cavity and the cell culture cavity is passed through. The membrane enters the cell culture chamber, which can fully mix the culture medium and the cells to maintain sufficient oxygen supply, and the stirring device does not directly contact the cells, which reduces the shear force on the cells, which can effectively solve the problems in the background technology. , The addition of the bidirectional pulse pump can effectively simulate the beating of the pulse, fully simulate the growth environment of NK cells in vivo, and can stimulate NK cells or other suspension cells and adherent cells that can be cultured in suspension to secrete a large amount of cell products, such as: exosomes, platelets , various cytokines, etc. The protocol of the present invention can be used not only for NK cells, but also for inoculation of other types of cells, such as macrophages, T lymphocytes, B lymphocytes.

(2) The bioreactor and method of the present invention can relatively conveniently produce exosomes in large quantities, are ingeniously designed and simple to operate, and can meet the requirements for exosome production in laboratories or factories. More and more studies have confirmed that exosomes play an important role in many aspects, especially in cell therapy and tissue regeneration. In addition, the bioreactor can also be used to culture other suspension cells or suspension culture of individual adherent cells, or to produce other cell products, which has broad application prospects.

(3) The present invention also provides a simple, rapid and mass-producing method for NK cell exosomes or other extracellular products, and has a wide range of applications, ranging from factories to small laboratories, and has wide application prospects.

Description of drawings

FIG. 1 is a schematic structural diagram of a pulse-stirred bioreactor of the present invention.

FIG. 2 is a schematic structural diagram of a cell culture device.

Figure 3 is an exploded view of the cell culture apparatus.

Figure 4 is an exploded view of the multi-layer filter device.

Figure 5 shows the quantitative analysis results of exosome products obtained after culturing NK92mi cells for 48 hours, collecting 30ml of static culture and the reaction solution cultured using the bioreactor and method (where, *** means p<0.001 is very Significant differences).

Detailed ways

The technical solutions of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the specific embodiments are only detailed descriptions of the present invention and should not be regarded as limitations of the present invention.

In the present invention, the inoculated cells are NK cells, but are not limited to NK cells, and may also be suspension cells such as macrophages, lymphoid T cells, blood cells, or adherent cells that can grow in suspension.

In the present invention, the stirring device may be a magnetic stirring device, but is not limited to a magnetic stirring device, and may also be a mechanical stirring device or other stirring methods.

In the present invention, the retention device located between the preliminary separation cavity and the cell culture cavity, and the retention device for keeping the cells in the cell culture cavity, is a filter membrane, but it is not limited to a filter membrane, and can also be other porous materials.

In the present invention, the bidirectional pulse flow is generated by a pulse pump, and the generation of the pulse flow can also be generated by other devices with similar functions.

In the present invention, the exosome separation and filtration system is composed of a membrane filtration system, and the separation of exosomes can also be accomplished in other ways, such as: density gradient centrifugation, ultracentrifugation, and the like.

In the present invention, the collected cell products are exosomes, but are not limited to exosomes, and can also be various types of cell secretions, such as platelets, collagen, and the like.

In the present invention, unless otherwise expressly specified and limited, the term "connection" should be understood in a broad sense, for example, it may be a fixed connection, a detachable connection, or an integral body; it may be a mechanical connection or an electrical connection. Connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be the connection between two elements or the interaction between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

Specific examples:

A pulse stirring bioreactor for exosome secretion, separation and collection of the present invention includes a cell culture device and an exosome separation and collection device;

The cell culture device is used for culturing cells and stimulating cells to secrete exosomes; the exosome separation and collection device is connected to the cell culture device, and can separate the exosomes from the culture medium in the cell culture device for collection, After separation, the cells remain in the cell culture device;

The cell culture device includes a stirring device configured to apply stirring power to the culture fluid in the cell culture device;

The cell culture device includes a pulse device, which is used for adding a culture medium to the cell culture device in a pulsed manner, thereby stimulating the cultured cells in the cell culture device, and forming a cell growth environment that dynamically simulates the blood structure in the body through stirring and pulsing. .

In some preferred manners, the cell culture device further comprises cell culture chambers 1-8, which are used for cell culture and exosome secretion.

In some preferred manners, the cell culture chamber 1-8 is provided with an inoculation port 1-9, a culture medium injection port 1-10 and a sample injection port 1-11.

In this embodiment, as shown in FIG. 1 , the upper surface of the cell culture chamber 1-8 is provided with an inoculation port 1-9, a culture medium filling port 1-10, and a sample addition port 1-11; wherein, the inoculation port 1-9 It can be used to inoculate cells, and the medium filling port 1-10 can be used to add liquid medium to the cell culture chamber 1-8; the sample filling port 1-11 can be used to add conditioning liquid to adjust the cell culture chamber 1 pH in -8.

In some preferred manners, the pulse device is connected to the cell culture chambers 1-8, and the culture medium can be added to the cell culture chambers 1-8 in a pulsed manner to stimulate the cultured cells in the cell culture chambers 1-8.

In this embodiment, as shown in FIG. 1 , the pulse device includes a pulse pump 1-12, and the pulse pump 1-12 communicates with the cell culture chamber 1-8 through a liquid pipeline 1-13. The pulse time and intensity can be adjusted by controlling the stop, operation and intensity of the pulse pumps 1-12.

In some preferred manners, the stirring device is located below the cell culture chambers 1-8.

In some preferred manners, the stirring device adopts a magnetic stirring device, and the magnetic stirring device includes a magnetic stirring bar 1-6 and a magnetic driving device located on the base 1-1, and the magnetic stirring bar 1-6 can be driven by the magnetic force Stirring is carried out under the drive of the device.

In this embodiment, as shown in FIG. 1 , the magnetic drive device is a magnetic stirrer 1-2. The magnetic stirrer 1-2 and the magnetic stirring bar 1-6 are both located below the cell culture chamber 1-8. The magnetic stirring bar 1 -6 is located above the magnetic stirrer 1-2.

In some preferred manners, the rotational speed of the magnetic stirring bars 1-6 can be adjusted by the magnetic field intensity, and the adjustment range of the magnetic field intensity is 30-150 rpm.

In some preferred manners, as shown in Figures 2-3, the separation and collection device includes a preliminary separation device located at the bottom of the cell culture chamber 1-8, the preliminary separation device includes a preliminary separation chamber 1-5, and the preliminary separation chamber 1- 5 is communicated with the cell culture chamber 1-8. A first filter membrane and a support device 1-7 are arranged between the preliminary separation chamber 1-5 and the cell culture chamber 1-8. Due to the function of the filter membrane, the cells are left in the cell culture chamber. in chambers 1-8.

In some preferred manners, as shown in FIG. 1 , the separation and collection device further includes a multi-layer filter device and a collection device, and the multi-layer filter device 2-4 is connected to the preliminary separation chamber 1-4 through a filter pipe 2-1 5. The outlet of the multi-layer filter device 2-4 is connected to the product collection tank 2-2.

In some preferred manners, as shown in FIG. 4 , the multi-layer filter device 2-4 includes a first filter chamber 2-4-1, a second filter chamber 2-4-3, and a third filter chamber 2-4-5 , the fourth filter chamber 2-4-7, the second filter membrane and support device 2-4-2, the third filter membrane and support device 2-4-4, the fourth filter membrane and support device 2-4-6. The pore diameters of the second filter membrane and the support device 2-4-2, the third filter membrane and the support device 2-4-4, and the fourth filter membrane and the support device 2-4-6 become smaller in turn.

The pressure on the cell culture chambers 1-8 generated by the intermittent operation of the machine-controlled filter pump is the main driving force for driving the liquid through the filter membrane.

In some preferred manners, the bioreactor of the present invention further comprises a condition monitoring device; the condition monitoring device can monitor the state of the cell growth environment in the cell culture chambers 1-8 in real time.

In some preferred manners, the condition monitoring device includes a pH value monitoring probe 3-1-3, a CO ₂ content monitoring probe 3-1-1, an O ₂ content monitoring probe 3-1-2, and a condition sampling device, pH value monitoring probe 3-1-3, CO ₂ content monitoring probe 3-1-1, O ₂ content monitoring probe 3-1-2 are placed in the cell culture chamber 1-8; the conditional sampling device is located in the cell culture chamber 1 In addition to -8, the conditional sample adding device is communicated with the cell culture chambers 1-8, and the conditional sample adding device can add samples to the cell culture chambers 1-8 according to the information detected by the probe.

The condition monitoring device monitors the environment in the cell culture chamber 1-8 in time through the pH value monitoring probe 3-1-3, the CO ₂ content monitoring probe 3-1-1, and the O ₂ content monitoring probe 3-1-2.

In this embodiment, the conditional sample adding device includes a conditioning liquid container 1-111, an O ₂ tank 1-14, and a CO ₂ tank 1-15; the conditioning liquid container 1-111 is connected to the cell culture chamber 1-8, and the O ₂ tank 1-14, _CO2 tank 1-15 are respectively connected with preliminary separation chamber 1-5 through _O2 delivery pipe 1-4, _CO2 delivery pipe 1-3, and preliminary separation chamber 1-5 is communicated with cell culture chamber 1-8 ;

By feeding oxygen and carbon dioxide into the preliminary separation chamber 1-5, under the action of the magnetic stirring device (because the magnetic stirring bar 1-6 is in the preliminary separation chamber 1-5, as shown in Figure 3, the magnetic stirrer 1- 2. The magnetic stirring bar 1-6 is rotated by the magnetic field, so that oxygen and carbon dioxide are first dissolved in the medium, and then enter the cell culture chamber 1 through the first filter membrane connecting the preliminary separation chamber 1-5 and the cell culture chamber 1-8 In -8, the culture medium and cells can be fully mixed to maintain sufficient oxygen supply, and the stirring device does not directly contact the cells, reducing the shear force on the cells.

In some preferred modes, the bioreactor of the present invention further comprises a regulating device, which can regulate and control the culture conditions in the cell culture chamber.

In this embodiment, the adjustment device includes a liquid release switch (numerical control) 3-2, a host 3-3, an _O2 tank, a _CO2 tank gas valve (numerical control) 3-4, and a pulse pump regulator (numerical control) 3-5.

The host 3-3 controls the automatic regulation system to automatically adjust the culture conditions in the cell culture chambers 1-8.

The present invention also provides a method for stimulating cells to secrete a large amount of exosomes, using the above-mentioned bioreactor, comprising the following steps:

(1) Filling medium: turn on the switch of the bioreactor, control the valve of the medium filling port 1-10 to open, add the medium, and fill the cell culture chambers 1-8; the medium can be selected from those commonly used for culturing NK92mi cells The existing culture medium can be;

(2) Inoculation: control the opening of the valves of inoculation ports 1-9, and inoculate NK92mi cells with a density of 2×10 ⁵ /ml;

(3) Stimulate cells: turn on the magnetic stirring and pulse device, set the magnetic stirring speed to 50 rmp, and adjust the pulse frequency to 72 times/min, simulate the cell growth environment in vivo, and form turbulent flow to stimulate NK cells to secrete exosomes;

(4) Monitoring and automatic adjustment of culture conditions: The monitoring chip 3-1 monitors the cell culture environment in real time, and automatically adjusts the culture conditions through the automatic control system controlled by the host 3-3. The culture conditions are set to 95% of liquid dissolved oxygen and the concentration of carbon dioxide is 5%, PH is 7.3, in this embodiment, the control and adjustment of the culture conditions can be achieved by controlling O ₂ , CO ₂ and adjusting the amount and speed of adding the adjusting liquid in the liquid container 1-111;

(5) Separation and collection of exosomes: After the cells grow in the cell culture chambers 1-8 for 2 days, turn on the filter pump 2-3, so that the culture solution passes through the separation and collection device, and finally collects the final product exosomes; in this example , the culture solution first enters the preliminary separation chamber 1-5 through the first filter membrane and the support device 1-7, then the culture solution enters the filter pipe 2-1, passes through the multi-layer filter device 2-4, and performs layer-by-layer filtration, and finally, The final product exosomes enter the product collection tank 2-2;

(6) Clean and disinfect the bioreactor for the next use.

The device of the invention is characterized in that: through mechanical stirring and pulse action, a dynamic cell growth environment simulating the blood structure in the body is formed, and the condition monitoring device and the adjusting device are used to monitor and adjust the state of the cell growth environment in the culture chamber in real time, and finally pass the membrane The filtration system separates and collects the final product. The mechanical stirring and pulse action means that, by placing the magnetic stirring rod 1-6 in the preliminary separation chamber 1-5, the magnetic stirring rod 1-6 is rotated by the magnetic field, and the liquid in the preliminary separation chamber 1-5 follows. Rotate and affect the environment of cell culture chambers 1-8 to generate continuous fluctuations, and at the same time, cooperate with pulse pumps 1-12 to intermittently pump nutrient solution to generate pulsed fluctuations in the culture chambers. The rotating speed of the magnetic stirring bar 1-6 can be adjusted by the magnetic field intensity, the adjustable range is 30-150rpm, and the pulse time and intensity can be adjusted by controlling the stop, operation and intensity of the pulse pump 1-12.

The separation and collection device includes a preliminary separation device, a multi-layer filter device and a collection device; the preliminary separation device includes a preliminary separation cavity 1-5, the preliminary separation cavity 1-5 is communicated with the cell culture cavity 1-8, and the preliminary separation cavity 1-5 A first filter membrane and a supporting device 1-7 are arranged between the cell culture chamber 1-8. Due to the function of the filter membrane, the cells are kept in the cell culture chamber 1-8; the membrane filtration of the multi-layer filter device 2-4 The separation system includes several different filter membranes with gradually smaller pore size and multiple filter chambers, which can filter the filtrate for many times and finally collect the product.

The above-mentioned bioreactor and method were used to cultivate NK92mi cells. At the same time, traditional bioreactors (that is, T25 culture flasks were used for culture) were used for static culture. As a comparative example, other culture conditions of the comparative example were the same as This embodiment is the same.

Fig. 5 is after cultivating NK92mi cells for 48 hours, respectively collecting 30ml of traditional cell culture method (i.e. static culture, cultured with T25 culture flask, other conditions are the same as in this example) and 30ml of the reaction cultured using the bioreactor and method of the present invention Quantitative analysis results of the obtained exosome products (where *** represents a very significant difference at p<0.001). It can be seen from FIG. 5 that more exosomes can be obtained by using the bioreactor and the biological reaction method of the present invention for culturing, and the bioreactor and the biological reaction method of the present invention are beneficial to the growth of NK92mi cells and to the secretion of exosomes by NK92mi cells. body.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (9)

1. a pulse-stirred bioreactor for exosome secretion, separation and collection, is characterized in that, comprises cell culture device, exosome separation and collection device; The cell culture device is used for culturing cells and stimulating cells to secrete exosomes; the exosome separation and collection device is connected to the cell culture device, and can separate the exosomes from the culture medium in the cell culture device for collection, After separation, the cells remain in the cell culture device; The cell culture device includes a stirring device configured to apply stirring power to the culture fluid in the cell culture device; The cell culture device includes a pulse device for adding a culture medium to the cell culture device in a pulsed manner, so as to stimulate the cultured cells in the cell culture device. 2. A pulse-stirred bioreactor for exosome secretion, separation and collection according to claim 1, wherein the cell culture device further comprises a cell culture cavity, and the cell culture cavity is used for conducting Cell culture and exosome secretion. 3. a kind of pulse-stirred bioreactor for exosome secretion, separation and collection according to claim 2, is characterized in that, described cell culture cavity is provided with inoculation port, culture liquid filling port and Injection port. 4. a kind of pulse stirring bioreactor for exosome secretion, separation and collection according to claim 2, is characterized in that, described stirring device is located below the cell culture cavity, and described stirring device adopts magnetic force A stirring device, the magnetic stirring device includes a magnetic stirring bar and a magnetic driving device, and the magnetic stirring bar can be stirred under the driving of the magnetic driving device. 5. The pulse-stirred bioreactor for exosome secretion, separation and collection according to claim 4, wherein the rotating speed of the magnetic stirring bar can be adjusted by the magnetic field intensity. 6. a kind of pulse stirring bioreactor for exosome secretion, separation and collection according to claim 2, is characterized in that, described pulse device comprises pulse pump, and pulse pump passes through liquid pipeline and cell culture cavity Connected. 7. A pulse-stirred bioreactor for exosome secretion, separation and collection according to claim 2, wherein the separation and collection device comprises a preliminary separation device positioned at the bottom of the cell culture cavity, and a preliminary The separation device includes a preliminary separation chamber, and the preliminary separation chamber is communicated with the cell culture chamber. 8. A pulse-stirred bioreactor for exosome secretion, separation and collection according to claim 7, wherein the separation and collection device comprises a multi-layer filter device, and the multi-layer filter device It is connected to the preliminary separation chamber through a filter pipe, and the outlet of the multi-layer filter device is connected to a product collection tank. 9. a kind of pulse stirring bioreactor for exosome secretion, separation and collection according to claim 2, is characterized in that, also comprises condition monitoring device and regulating device, and described condition monitoring device comprises pH value The monitoring probe, CO ₂ content monitoring probe, O ₂ content monitoring probe and conditional sampling device, pH value monitoring probe, CO ₂ content monitoring probe, and O ₂ content monitoring probe are placed in the cell culture chamber; the conditional sampling device is located in the cell culture chamber Outside the cavity, the conditional sample adding device is communicated with the cell culture chamber, and the conditional sample adding device can add samples to the cell culture chamber according to the information detected by the probe.

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