CN111363679B - Method for stimulating large-scale secretion of exosomes by cells - Google Patents

Method for stimulating large-scale secretion of exosomes by cells Download PDF

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CN111363679B
CN111363679B CN201811600979.0A CN201811600979A CN111363679B CN 111363679 B CN111363679 B CN 111363679B CN 201811600979 A CN201811600979 A CN 201811600979A CN 111363679 B CN111363679 B CN 111363679B
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cell culture
cells
culture
cell
exosomes
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CN111363679A (en
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王书崎
吴建国
吴迪
梁利国
武国华
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Zhejiang University ZJU
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Zhejiang University ZJU
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells

Abstract

The invention discloses a method for stimulating a cell to secrete a large amount of exosomes, which adopts a pulse stirring bioreactor to culture the cell and comprises the following steps: opening a bioreactor switch, opening a culture solution filling port valve, filling culture medium, and filling a cell culture cavity; controlling the opening of an inoculation port valve to inoculate cells; opening a magnetic stirring and pulse device to simulate the in vivo cell growth environment and stimulate the cells to secrete exosomes; the monitoring chip monitors the cell culture environment in real time and adjusts the culture conditions through the host; after the cells are grown in the culture device for 2 days, the filter pump is started, the culture solution passes through the separation and collection device, and the final product exosomes are collected. The bioreactor can ensure that culture solution and cells are fully mixed to keep enough oxygen supply, and the stirring device does not directly contact the cells, so that the shearing force borne by the cells is reduced.

Description

Method for stimulating large-scale secretion of exosomes by cells
Technical Field
The invention relates to a preparation and separation technology of exosomes, in particular to a method for stimulating a cell to secrete a large amount 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) regulating the release and distribution of exosomes. (2) exosome-based drug delivery. (3) exosome-mediated tumor targeted therapy. (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 uses 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 a better specificity, and is expected to exert a unique effect in the aspect of 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 the application research of tumor treatment and 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 of cells per unit volume. The animal cell culture bioreactor is divided into a stirring type cell culture bioreactor and a non-stirring type cell culture bioreactor according to whether a stirring paddle is arranged in the animal cell culture bioreactor. The stirring type cell culture reactor generates vortex by the rotation of the stirring paddle to complete aeration and oxygen supply of 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 above existing problems, the present invention provides a method for stimulating a cell to secrete exosomes in large quantities.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for stimulating a cell to secrete a large amount of exosomes by using a pulse stirring type bioreactor to culture the cell, comprising the following steps:
(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, namely controlling the valve of an inoculating hole to be opened and inoculating cells;
(3) Stimulating the cells: opening the magnetic stirring and pulse device to simulate the cell growth environment of the in vivo blood structure and stimulate the cells to secrete exosomes;
(4) Culture conditions were monitored and adjusted: the monitoring chip monitors the cell culture environment in real time and adjusts the culture conditions through the host;
(5) And (3) separating and collecting exosomes: after the cells are grown in the culture device for 2 days, the filter pump is started, the culture solution passes through the separation and collection device, and the final product exosomes are collected.
Further, in the step (2), the seeding density is 2X 10 5 NK92mi cells per ml.
Further, in the step (3), the rotating speed of the magnetic stirrer is set to be 50rmp, and the pulse frequency is adjusted to be 72 times/min, so that the cell growth environment of the in-vivo blood structure is simulated, and the secretion of exosomes by cells is stimulated.
Further, in the step (4), the culture conditions were set to liquid dissolved oxygen of 95%, carbon dioxide concentration of 5%, and pH of 7.3.
Further, the bioreactor 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.
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, the stirring device is positioned below the cell culture chamber, the stirring device adopts 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; the rotating speed of the magnetic stirring rod can be adjusted through the intensity of the magnetic field.
Further, the pulse device comprises a pulse pump which is communicated with the cell culture cavity through a liquid pipeline.
Further, the separation and collection device comprises a primary separation device positioned at the bottom of the cell culture cavity, the primary separation device comprises a primary separation cavity, and the primary separation cavity is communicated with the cell culture cavity;
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 a 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 CO 2 Content monitoring probe, O 2 Content monitoring probe, condition sample adding device, pH value monitoring probe and CO 2 Content monitoring probe, O 2 The 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.
The beneficial effects of the invention are:
(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 producing the exosomes in large quantities, 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 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 is a graph showing the quantitative analysis results of exosome products obtained by collecting 30ml of reaction solutions obtained by static culture and culture using the bioreactor and method of the present invention, respectively, 48 hours after culturing NK92mi cells (wherein x represents a very significant difference of p < 0.001).
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 embodiments are only for describing the details of 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 the embodiment, as shown in FIG. 1, an inoculation port 1-9, a culture solution filling port 1-10 and a sample adding port 1-11 are arranged on the upper surface of a cell culture cavity 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 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 30-150rpm.
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 CO 2 Content monitoring probe 3-1-1, O 2 Content monitoring probe 3-1-2, condition sample adding device and pH valueValue monitoring probe 3-1-3, CO 2 Content monitoring probe 3-1-1, O 2 The 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 value 2 Content monitoring probe 3-1-1, O 2 The content monitoring probe 3-1-2 monitors the environment in the cell culture cavity 1-8 in time.
In this example, the conditioned sample addition device comprises a conditioning liquid container 1-111 2 1-14 tanks, CO 2 1-15 parts of tank; the adjusting liquid container 1-111 is communicated to the cell culture chamber 1-8, and O 2 Tanks 1-14, CO 2 Tanks 1-15 each passing O 2 1-4 conveying pipe, CO 2 The 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 main machine 3-3, and an O 2 Tank, CO 2 3-4 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 may be any one of the conventional culture media commonly used for culturing NK92mi cells.
(2) Inoculation: controlling the opening of a valve of an inoculation port 1-9 and the inoculation density of 2 multiplied by 10 5 NK92mi cells per 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 controlled 2 、CO 2 And 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 multilayer filtration device 2-4 comprises a plurality of different filter membranes with gradually reduced pore sizes and a plurality of filter cavities, so that the filtrate can be filtered for a plurality of times, and finally, the product is collected.
NK92mi cells were cultured by using the above-described bioreactor and method, and at the same time, cultured by using a conventional bioreactor (i.e., culture using a T25 flask) and static culture, as a comparative example, 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, the other conditions being the same as those in this example) and 30ml of the reaction solution cultured using the bioreactor and method of the present invention, respectively, after culturing NK92mi cells for 48 hours (wherein x represents a very significant difference of p < 0.001). As can be seen from fig. 5, a large number of exosomes can be obtained by using the bioreactor and the method of the present invention for culture, and the bioreactor and the method of the present invention are advantageous for the growth of NK92mi cells and can stimulate NK92mi cells to secrete a large number of exosomes.
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 (8)

1. A method for stimulating a cell to secrete a large amount of exosomes is characterized in that a pulse stirring type bioreactor is used for culturing the cell, and the method comprises the following steps:
(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, namely controlling a valve of an inoculating port to be opened and inoculating cells;
(3) Stimulating the cells: opening a magnetic stirring and pulse device to simulate the in vivo cell growth environment and stimulate the cells to secrete exosomes;
(4) Culture conditions were monitored and adjusted: the monitoring chip monitors the cell culture environment in real time and adjusts the culture conditions through the host;
(5) And (3) separating and collecting exosomes: after the cells grow in the culture device for 2 days, starting a filter pump, enabling the culture solution to pass through a separation and collection device, and collecting final product exosomes;
the bioreactor 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, a pulse device and a control device, wherein the pulse device 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;
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.
2. The method according to claim 1, wherein in step (3), the magnetic stirrer is set to rotate at 50rmp and the pulse frequency is adjusted to 72 times/min to simulate the cell growth environment of the blood structure in vivo and stimulate the cells to secrete exosomes.
3. The method according to claim 1, wherein in step (4), the culture conditions are set to 95% dissolved oxygen, 5% carbon dioxide and 7.3 pH.
4. The method according to claim 1, wherein the cell culture chamber is provided with an inoculation port, a culture solution feeding port and a sample adding port.
5. The method according to claim 1, wherein the stirring device is positioned 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; the rotating speed of the magnetic stirring rod can be adjusted through the intensity of the magnetic field.
6. A method according to claim 1, wherein the pulsing device comprises a pulse pump in fluid communication with the cell culture chamber via a fluid conduit.
7. The method according to claim 1, wherein the separation and collection device 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; 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 a product collection tank.
8. A method according to claim 4, further comprising condition monitoring means including a pH monitoring probe, a CO regulator and a CO regulator 2 Content monitoring probe, O 2 Content monitoring probe, condition sample adding device, pH value monitoring probe and CO 2 Content monitoring probe, O 2 The 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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