CN111363680A - Pulse stirring type bioreactor for secretion, separation and collection of exosome - Google Patents
Pulse stirring type bioreactor for secretion, separation and collection of exosome Download PDFInfo
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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Abstract
The invention discloses a pulse stirring bioreactor for secretion, separation and collection of exosomes, which comprises a cell culture device and an exosome separation and collection device; the exosome separating and collecting device is connected with the cell culture device and can separate and collect exosomes from the culture solution in the cell culture device; the cell culture device comprises a stirring device, a stirring device and a control device, wherein the stirring device is used for applying stirring power to the culture solution in the cell culture device; the cell culture apparatus includes a pulse device to which the culture solution can be added in a pulsed manner. The bioreactor can ensure that culture solution and cells are fully mixed to keep enough oxygen supply, and the stirring device is not directly contacted with the cells, so that the shearing force borne by the cells is reduced, and meanwhile, the bioreactor can simulate the growth environment of the cells in vivo and stimulate NK cells or other suspension cells to secrete a large amount of cell products.
Description
Technical Field
The invention relates to a preparation and separation technology of exosomes, in particular to a pulse stirring bioreactor for secretion, separation and collection of exosomes.
Background
Exosomes are Extracellular Vesicles (EVs) with lipid bilayers of about 30-150nm in diameter. The exosome can contain various bioactive substances such as protein, lipid, nucleic acid and the like, and most cells of a human body such as immune cells (T cells, B cells, Kupffer cells and NK cells), endothelial cells, platelets and the like can secrete the exosome. At present, the research on clinical treatment of exosome can be roughly summarized as: (1) regulate the release and distribution of exosomes. (2) Exosome-based drug delivery. (3) Exosome-mediated targeted therapy of tumors. (4) Exosomes and immunotherapy. At present, exosomes show great application prospects in immunotherapy of tuberculosis, diagnosis, inhibition or regulation of tumor cell metastasis, prognosis monitoring and treatment of various cancers such as bone tumor, liver cancer, ovarian cancer, prostate cancer and the like. In addition, the cell exosome is used as a carrier for cell-to-cell interaction, is also an important mode of the paracrine activity of stem cells, plays an important role in tissue regeneration, and researches in recent years show that the MSCs exosome has functions similar to MSCs, including repairing and regenerating tissues, inhibiting inflammatory response and regulating body immunity.
CAR-T cell therapy (CAR-T therapy), which utilizes genetically engineered T cells to kill cancer cells, has proven to be a promising option when other therapeutic approaches fail. In a new study, researchers from the university of california, san diego university and the university of minnesota reported that natural killer cells (NK cells), cultured with human induced pluripotent stem cells (iPS cells) and modified in a similar manner to CAR-T cells, were highly effective against ovarian cancer in a mouse model. However, it has been reported that the NK cell exosome has a function of an NK cell portion, is smaller and has better specificity, and is expected to play a unique role in cell therapy.
In recent years, researches show that NK cells can secrete exosomes with NK cell markers and killer proteins to kill tumor cells, and in addition, the exosomes have the characteristic of transferring to targeted target organs, so that the exosomes can be designed into a drug carrier for precise administration to transport specific drugs to the target organs to play a role. Therefore, the exosome of the NK cell has great potential in tumor treatment and application research of drug targeting vectors. At present, the extraction and purification of exosomes are mainly carried out according to the physicochemical characteristics of exosomes, and methods such as a differential centrifugation combined sucrose density gradient centrifugation method, an ultrafiltration method, an immunomagnetic bead extraction method and an ExoQuick Precipitation extraction method are commonly adopted.
At present, a bioreactor is a core device for large-scale suspension culture of cells, and can effectively increase the culture density per unit volume of the cells. Animal cell culture bioreactors are classified into stirred cell culture bioreactors and non-stirred cell culture bioreactors according to whether stirring paddles are present inside the animal cell culture bioreactors. The stirring type cell culture reactor generates vortex by the rotation of the stirring paddle to complete aeration and oxygen supply of the culture solution, but the mechanical shearing force generated by the mode is larger, and cells are easily damaged. At present, the influence on cells can be reduced only by controlling the stirring speed, but the problem still exists. The non-stirring cell culture reactor generates small shearing force, and mainly adopts vibration, shaking, rolling or swinging modes to move the culture solution and keep the suspension state of cells and the gas exchange of the culture solution. The disadvantage is that it is difficult to ensure the absorption efficiency of oxygen. In recent years, air or oxygen injection is adopted to solve the problem of oxygen supply in the culture solution, but the method is easy to generate a large amount of foam, physical damage can be caused to cells in the foam breaking process, and the excessive oxygen injection can also poison the cells. The precise control of gas injection also complicates the reactor equipment, increasing the cost of cell culture. Therefore, it is an urgent problem to provide uniform stirring to mix the culture solution and the cells sufficiently to maintain sufficient oxygen supply and reduce the mechanical shear damage to the cells.
Disclosure of Invention
In order to solve the existing problems, the present invention provides a pulse agitation type bioreactor for secretion, separation and collection of exosomes.
In order to achieve the purpose, the invention adopts the following technical scheme:
a pulse stirring bioreactor for secretion, separation and collection of exosome comprises a cell culture device and an exosome separation and collection device;
the cell culture device is used for culturing cells and stimulating the cells to secrete exosomes; the exosome separation and collection device is connected with the cell culture device and can separate and collect exosomes from a culture solution in the cell culture device, and cells are left in the cell culture device after separation;
the cell culture apparatus comprises an agitation apparatus configured to apply agitation power to a culture liquid in the cell culture apparatus;
the cell culture device comprises a pulse device which is used for adding culture solution to the cell culture device in a pulse mode so as to stimulate cells cultured in the cell culture device.
Further, the cell culture device also comprises a cell culture cavity, and the cell culture cavity is used for carrying out cell culture and secretion of exosomes.
Furthermore, an inoculation port, a culture solution filling port and a sample adding port are arranged on the cell culture cavity.
Further, agitating unit is located the below in cell culture chamber, agitating unit adopts magnetic stirring device, magnetic stirring device includes magnetic stirring stick and magnetic drive device, and the magnetic stirring stick can stir under magnetic drive device's drive.
Furthermore, the rotating speed of the magnetic stirring rod can be adjusted by the magnetic field intensity, and the adjusting range is 30-150 rpm.
Further, the pulse device comprises a pulse pump which is communicated with the cell culture cavity through a liquid pipeline.
Further, separation collection device is including being located the preliminary separation device of cell culture chamber bottom, and preliminary separation device includes the preliminary separation chamber, and the preliminary separation chamber communicates with the cell culture chamber.
Further, the separation and collection device comprises a multi-layer filtering device, the multi-layer filtering device is connected to the primary separation cavity through a filtering pipeline, and an outlet of the multi-layer filtering device is connected with the product collection tank.
Further, the bioreactor also comprises a condition monitoring device and an adjusting device, wherein the condition monitoring device comprises a pH value monitoring probe and CO2Content monitoring probe, O2Content monitoring probe, condition sample adding device, pH value monitoring probe and CO2Content monitoring probe, O2The content monitoring probe is arranged in a cell culture cavity, and the monitoring adjustable range of the culture condition is that liquid dissolved oxygen is 50-100%, the concentration of carbon dioxide is 0-20%, and the PH is 5-9; the condition application of sample device is located outside the cell culture chamber, and condition application of sample device and cell culture chamber intercommunication, condition application of sample device can be according to the information that the probe detected, in time adjusts the cell culture environment through adding adjusting reagent.
The invention also provides a method for stimulating cells to secrete exosomes in large quantity, which adopts the bioreactor, monitors and adjusts the culture conditions in the cell culture cavity through the condition monitoring device and the adjusting device, and simulates the culture conditions in the cell culture cavity through stirring and pulse, so that the cells are in a cell growth environment dynamically simulating the blood structure in vivo, and the cells are stimulated to secrete exosomes.
The present invention also provides a method for stimulating secretion of exosomes by a cell, the method using the bioreactor described above, comprising the steps of:
(1) filling a culture medium: opening a bioreactor switch, opening a culture solution filling port valve, filling culture medium, and filling a cell culture cavity;
(2) inoculating by controlling the opening of the valve of the inoculating opening and the inoculation densityDegree of 2 × 105NK92mi cells/ml;
(3) stimulating the cells: turning on a magnetic stirring and pulse device, setting the rotating speed of a magnetic stirrer to be 50rmp, adjusting the pulse frequency to be 72 times/min, simulating the in vivo cell growth environment, and forming turbulence to stimulate NK cells to secrete exosomes;
(4) monitoring and automatically adjusting culture conditions: the monitoring chip monitors the cell culture environment in real time, and automatically adjusts the culture conditions through an automatic regulation and control system controlled by a host, wherein the culture conditions are set to be liquid dissolved oxygen of 95%, carbon dioxide concentration of 5% and PH of 7.3;
(5) and (3) separating and collecting exosomes: after the cells are grown in the culture device for 2 days, a filter pump is started, the culture solution passes through the separation and collection device, and finally the final product is collected.
The invention has the beneficial effects that:
(1) according to the invention, oxygen and carbon dioxide are introduced into the primary separation cavity, under the action of the magnetic stirring device, the oxygen and the carbon dioxide are firstly dissolved in the culture medium, and then enter the cell culture cavity through the first filter membrane communicated with the primary separation cavity and the cell culture cavity, so that culture solution and cells can be fully mixed to keep sufficient oxygen supply, and the stirring device is not in direct contact with the cells, so that the shearing force borne by the cells is reduced, the problem in the background technology can be effectively solved, meanwhile, the pulse beating can be effectively simulated by the addition of the bidirectional pulse pump, the in-vivo NK cell growth environment is fully simulated, NK cells or other suspended cells and adherent cells which can be subjected to suspension culture can be stimulated to secrete a large amount of cell products, for example: exosomes, platelets, various cytokines, etc. The protocol of the invention can be used not only for NK cells but also for seeding other types of cells, such as macrophages, T lymphocytes, B lymphocytes.
(2) The bioreactor and the method can be used for conveniently and massively producing the exosomes, are ingenious in design and simple to operate, and can meet the requirements of laboratories or factories on exosome production. More and more studies have demonstrated that exosomes play an important role in a number of aspects, particularly in cell therapy and tissue regeneration. In addition, the bioreactor can also be used for culturing other suspension cells or suspension culture of individual adherent cells or generating other cell products, and has wide application prospect.
(3) The invention also provides a simple and rapid method for producing a large amount of NK cell exosomes or other extracellular products, and the method has wide application range, can be used in factories and laboratories, and has wide application prospect.
Drawings
FIG. 1 is a schematic structural view of a pulse stirring bioreactor of the present invention.
FIG. 2 is a schematic view of the structure of the cell culture apparatus.
FIG. 3 is an exploded view of the cell culture apparatus.
Fig. 4 is an exploded view of a multi-layer filter device.
FIG. 5 shows the quantitative analysis results of exosome products obtained from 30ml of reaction solutions obtained by static culture and culture using a bioreactor and method, respectively, after culturing NK92mi cells for 48 hours (wherein, represents that p < 0.001 is very significantly different).
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, and it should be noted that the detailed description is only for describing the present invention, and should not be construed as limiting the present invention.
In the present invention, the cells to be seeded are NK cells, but not limited to NK cells, and may be suspension cells such as macrophages, lymphocyte T cells, blood cells, and the like, or adherent cells capable of growing 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 invention, the interception device which is positioned between the primary separation cavity and the cell culture cavity and used for keeping the cells in the cell culture cavity is a filter membrane, but the interception device is not limited to the filter membrane and can be made of other porous materials.
In the present invention, the bi-directional pulse stream is generated by a pulse pump, and the generation of the pulse stream can also be generated by other devices with similar functions.
In the present invention, the exosome separation-filtration system is composed of a membrane filtration system, and the exosome separation work can be completed in other ways, such as: density gradient centrifugation, ultracentrifugation, and the like.
In the invention, the collected cell product is an exosome, but is not limited to an exosome, and can be various cell secretions: such as platelets, collagen, etc.
In the present invention, unless otherwise expressly stated or limited, the term "coupled" is to be construed broadly, e.g., as meaning either a fixed connection or a removable connection, or an integral part; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
The specific embodiment is as follows:
the invention relates to a pulse stirring bioreactor for secretion, separation and collection of exosomes, which comprises a cell culture device and an exosome separation and collection device;
the cell culture device is used for culturing cells and stimulating the cells to secrete exosomes; the exosome separation and collection device is connected with the cell culture device and can separate and collect exosomes from a culture solution in the cell culture device, and cells are left in the cell culture device after separation;
the cell culture apparatus comprises an agitation apparatus configured to apply agitation power to a culture liquid in the cell culture apparatus;
the cell culture device comprises a pulse device, and the pulse device is used for adding culture solution to the cell culture device in a pulse mode, so that cells cultured in the cell culture device are stimulated, and a cell growth environment dynamically simulating in-vivo blood structures is formed through stirring and pulse effects.
In some preferred modes, the cell culture device further comprises cell culture chambers 1-8, and the cell culture chambers 1-8 are used for cell culture and secretion of exosomes.
In some preferred modes, the cell culture chamber 1-8 is provided with an inoculation port 1-9, a culture solution filling port 1-10 and a sample adding port 1-11.
In this embodiment, as shown in FIG. 1, an inoculation port 1-9, a culture solution filling port 1-10, and a sample addition port 1-11 are provided on the upper surface of a cell culture chamber 1-8; wherein, the inoculation port 1-9 can be used for inoculating cells, and the culture solution filling port 1-10 can be used for adding liquid culture medium into the cell culture cavity 1-8; the sample addition ports 1-11 can be used to add a conditioning fluid to adjust the pH in the cell culture chambers 1-8.
In some preferred modes, the pulse device is connected with the cell culture cavities 1-8, and culture solution can be added into the cell culture cavities 1-8 in a pulse mode to stimulate the cells cultured in the cell culture cavities 1-8.
In this embodiment, as shown in FIG. 1, the pulse device comprises a pulse pump 1-12, and the pulse pump 1-12 is communicated with the cell culture chamber 1-8 through a liquid pipe 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 forms, the agitation means is located below the cell culture chambers 1-8.
In some preferred modes, the stirring device is a magnetic stirring device which comprises a magnetic stirring rod 1-6 and a magnetic driving device positioned on the base 1-1, and the magnetic stirring rod 1-6 can stir under the driving of the magnetic driving device.
In this embodiment, as shown in FIG. 1, the magnetic driving device is a magnetic stirrer 1-2, the magnetic stirrer 1-2 and the magnetic stirring rod 1-6 are both located below the cell culture chamber 1-8, and the magnetic stirring rod 1-6 is located above the magnetic stirrer 1-2.
In some preferred modes, the rotating speed of the magnetic stirring rods 1-6 can be adjusted through the magnetic field intensity, and the adjusting range of the magnetic field intensity is 30-150 rpm.
In some preferred modes, as shown in fig. 2-3, the separation and collection device comprises a primary separation device positioned at the bottom of the cell culture cavity 1-8, the primary separation device comprises a primary separation cavity 1-5, the primary separation cavity 1-5 is communicated with the cell culture cavity 1-8, a first filter membrane and a support device 1-7 are arranged between the primary separation cavity 1-5 and the cell culture cavity 1-8, and cells are retained in the cell culture cavity 1-8 due to the action of the filter membrane.
In some preferred modes, as shown in figure 1, the separation and collection device further comprises a multi-layer filtering device and a collection device, wherein the multi-layer filtering device 2-4 is connected to the primary separation cavity 1-5 through a filtering pipeline 2-1, and the outlet of the multi-layer filtering device 2-4 is connected to the product collection tank 2-2.
In some preferred modes, as shown in FIG. 4, the multi-layer filtering device 2-4 comprises a first filtering chamber 2-4-1, a second filtering chamber 2-4-3, a third filtering chamber 2-4-5, a fourth filtering chamber 2-4-7, a second filtering membrane and supporting device 2-4-2, a third filtering membrane and supporting device 2-4-4 and a fourth filtering membrane and supporting device 2-4-6. The aperture of the second filter membrane and the support device 2-4-2, the aperture of the third filter membrane and the support device 2-4-4, and the aperture of the fourth filter membrane and the aperture of the support device 2-4-6 are reduced in sequence.
The pressure generated by the intermittent operation of the mechanical control filter pump to the cell culture chambers 1-8 is the main power for driving the liquid to pass through the filter membrane.
In some preferred forms, the bioreactor of the present invention further comprises condition monitoring means; the condition monitoring device can monitor the cell growth environment state in the cell culture cavities 1-8 in real time.
In some preferred modes, the condition monitoring device comprises a pH value monitoring probe 3-1-3 and CO2Content monitoring probe 3-1-1, O2A content monitoring probe 3-1-2, a condition sample adding device, a pH value monitoring probe 3-1-3, and CO2Content monitoring probe 3-1-1, O2The content monitoring probe 3-1-2 is arranged in the cell culture cavity 1-8; the condition sample adding device is positioned outside the cell culture chambers 1-8 and is communicated with the cell culture chambers 1-8, and the condition 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 probe 3-1-3 and CO through the pH value2Content monitoring probe 3-1-1, O2Content monitoring probe 3-1-2 cell cultureThe environment in the breeding chambers 1-8 is monitored in time.
In this example, the conditioned sample adding device comprises a regulating liquid container 1-111, O21-14 tanks, CO21-15 parts of tank; the regulating liquid container 1-111 is communicated to the cell culture cavity 1-8, and O21-14 tanks, CO2Tanks 1-15 each passing O21-4 conveying pipe, CO2The conveying pipe 1-3 is communicated with the primary separation cavity 1-5, and the primary separation cavity 1-5 is communicated with the cell culture cavity 1-8;
by introducing oxygen and carbon dioxide into the primary separation chamber 1-5, under the action of a magnetic stirring device (because the magnetic stirring rod 1-6 is positioned in the primary separation chamber 1-5, as shown in fig. 3, the magnetic stirring rod 1-6 is rotated by the magnetic stirrer 1-2 through a magnetic field), the oxygen and the carbon dioxide are firstly dissolved in the culture medium and then enter the cell culture chamber 1-8 through a first filter membrane which is communicated with the primary separation chamber 1-5 and the cell culture chamber 1-8, so that the culture solution and the cells can be fully mixed to keep enough oxygen supply, and the stirring device is not directly contacted with the cells, thereby reducing the shearing force applied to the cells.
In some preferred modes, the bioreactor of the present invention further comprises a regulating device, and the regulating device can regulate and control the culture conditions in the cell culture chamber.
In this embodiment, the adjusting device comprises a liquid release switch (numerical control) 3-2, a host 3-3, and an O2Tank, CO23-4 parts of a tank gas valve (numerical control) and 3-5 parts of a pulse pump regulator (numerical control).
The host machine 3-3 controls the automatic regulating system to automatically regulate the culture conditions in the cell culture cavities 1-8.
The invention also provides a method for stimulating a cell to secrete a large amount of exosomes, which adopts the bioreactor and comprises the following steps:
(1) filling a culture medium: opening a bioreactor switch, controlling a valve of a culture solution filling port 1-10 to be opened, filling a culture medium, and filling a cell culture cavity 1-8; the culture medium can be selected from the existing culture medium commonly used for culturing NK92mi cells;
(2) inoculating with inoculating density of 2 × 10 by controlling the opening of valves of inoculating ports 1-95NK92mi cells/ml;
(3) stimulating the cells: turning on a magnetic stirring and pulse device, setting the rotating speed of a magnetic stirrer to be 50rmp, adjusting the pulse frequency to be 72 times/min, simulating the in vivo cell growth environment, and forming turbulence to stimulate NK cells to secrete exosomes;
(4) monitoring and automatically adjusting culture conditions: the monitoring chip 3-1 monitors the cell culture environment in real time, and automatically adjusts the culture conditions through an automatic regulation and control system controlled by the host 3-3, wherein the culture conditions are set to be 95% of liquid dissolved oxygen, 5% of carbon dioxide concentration and 7.3 of PH, and in the embodiment, the O can be controlled2、CO2And adjusting the sample adding amount and the sample adding speed of the adjusting liquid in the liquid containers 1-111 to realize the control and adjustment of the culture conditions;
(5) and (3) separating and collecting exosomes: after the cells grow in the cell culture cavities 1-8 for 2 days, starting a filter pump 2-3, enabling the culture solution to pass through a separation and collection device, and finally collecting the final product exosomes; in the embodiment, a culture solution firstly enters a primary separation cavity 1-5 through a first filter membrane and a supporting device 1-7, then enters a filter pipeline 2-1, is filtered layer by layer through a multilayer filter device 2-4, and finally, a final product exosome enters a product collection tank 2-2;
(6) cleaning and disinfecting the bioreactor for the next use.
The equipment of the invention is characterized in that: through mechanical stirring and pulse action, a dynamic cell growth environment simulating a blood structure in vivo is formed, the cell growth environment state in a culture cavity is monitored and adjusted in real time through a condition monitoring device and an adjusting device, and finally, the cell growth environment state is separated through a membrane filtration system and a final product is collected. The mechanical stirring and pulse action means that a magnetic stirring rod 1-6 is placed in a primary separation cavity 1-5, the magnetic stirring rod 1-6 is rotated through a magnetic field, liquid in the primary separation cavity 1-5 rotates along with the magnetic stirring rod and influences the environment of a cell culture cavity 1-8, continuous fluctuation is generated, and meanwhile, a pulse type fluctuation is generated in the culture cavity by intermittently pumping nutrient solution in cooperation with a pulse pump 1-12. The rotating speed of the magnetic stirring rods 1-6 can be adjusted through the magnetic field intensity, the adjustable range is 30-150rpm, and the pulse time and the pulse intensity can be adjusted through controlling the stop, the operation and the intensity of the pulse pumps 1-12.
The separation and collection device comprises a preliminary separation device, a multi-layer filtering device and a collection device; the primary separation device comprises a primary separation cavity 1-5, the primary separation cavity 1-5 is communicated with the cell culture cavity 1-8, a first filter membrane and a support device 1-7 are arranged between the primary separation cavity 1-5 and the cell culture cavity 1-8, and cells are retained in the cell culture cavity 1-8 under the action of the filter membrane; the membrane filtration separation system of the multi-layer filtration device 2-4 comprises a plurality of different filtration membranes with gradually reduced pore sizes and a plurality of filtration cavities, and can filter the filtrate for a plurality of times and finally collect the product.
NK92mi cells were cultured in the bioreactor and the method described above, and at the same time, the cells were cultured in a conventional bioreactor (i.e., in a T25 flask) by static culture, and the other culture conditions of the comparative example were the same as those of the present example.
FIG. 5 shows the quantitative analysis results of the exosome products obtained from 30ml of the reaction solution cultured in the conventional cell culture mode (i.e., static culture, using T25 flasks, and under the same conditions as in the present example) and 30ml of the reaction solution cultured by using the bioreactor and method of the present invention, respectively, after culturing NK92mi cells for 48 hours (wherein, represents that p < 0.001 is very significantly different). As can be seen from fig. 5, a large number of exosomes can be obtained by culturing using the bioreactor and the bioreactor method of the present invention, which are advantageous for the growth of NK92mi cells and for the secretion of exosomes from NK92mi cells.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A pulse stirring bioreactor for secretion, separation and collection of exosome is characterized by comprising a cell culture device and an exosome separation and collection device;
the cell culture device is used for culturing cells and stimulating the cells to secrete exosomes; the exosome separation and collection device is connected with the cell culture device and can separate and collect exosomes from a culture solution in the cell culture device, and cells are left in the cell culture device after separation;
the cell culture apparatus comprises an agitation apparatus configured to apply agitation power to a culture liquid in the cell culture apparatus;
the cell culture device comprises a pulse device which is used for adding culture solution to the cell culture device in a pulse mode so as to stimulate cells cultured in the cell culture device.
2. The pulse stirring bioreactor for secretion, separation and collection of exosomes according to claim 1, wherein the cell culture apparatus further comprises a cell culture chamber for cell culture and secretion of exosomes.
3. The pulse stirring bioreactor for secretion, separation and collection of exosome according to claim 2, wherein the cell culture cavity is provided with an inoculation port, a culture solution filling port and a sample adding port.
4. The pulse stirring bioreactor for secretion, separation and collection of exosomes according to claim 2, wherein the stirring device is located below the cell culture chamber, the stirring device is a magnetic stirring device, the magnetic stirring device comprises a magnetic stirring rod and a magnetic driving device, and the magnetic stirring rod can stir under the driving of the magnetic driving device.
5. A pulse stirred bioreactor for secretion, separation and collection of exosomes according to claim 4, wherein the rotation speed of the magnetic stirring rod can be adjusted by the intensity of the magnetic field.
6. A pulse stirred bioreactor for secretion, separation and collection of exosomes according to claim 2, wherein said pulse means comprises a pulse pump in communication with the cell culture chamber through a liquid conduit.
7. A pulse stirred bioreactor for secretion, separation and collection of exosomes according to claim 2, wherein the separation and collection means comprises a preliminary separation device at the bottom of the cell culture chamber, the preliminary separation device comprising a preliminary separation chamber, the preliminary separation chamber being in communication with the cell culture chamber.
8. The pulse stirred bioreactor for secretion, separation and collection of exosomes according to claim 7, wherein the separation and collection device comprises a multi-layer filtering device, the multi-layer filtering device is connected to the primary separation chamber through a filtering pipeline, and the outlet of the multi-layer filtering device is connected to a product collection tank.
9. The pulse stirring bioreactor for secretion, separation and collection of exosomes according to claim 2, further comprising condition monitoring device and adjusting device, wherein the condition monitoring device comprises pH value monitoring probe, CO2Content monitoring probe, O2Content monitoring probe, condition sample adding device, pH value monitoring probe and CO2Content monitoring probe, O2The content monitoring probe is arranged in the cell culture cavity; the condition application of sample device is located outside the cell culture chamber, and condition application of sample device and cell culture chamber intercommunication, condition application of sample device can carry out the application of sample to the cell culture chamber according to the information that the probe detected.
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