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

Abstract

The invention discloses a method for stimulating cells to secrete exosomes in large quantities. The cells are cultivated in a pulse-stirred bioreactor, which includes the following steps: opening the switch of the bioreactor, opening the valve of the filling port of the culture solution, and filling the culture medium , fill the cell culture chamber; control the opening of the inoculation port valve to inoculate cells; turn on the magnetic stirring and pulse device to simulate the cell growth environment in vivo and stimulate 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 were grown in the culture device for 2 days, the filter pump was turned on to allow the culture medium to pass through the separation and collection device to collect the end product exosomes. The bioreactor can fully mix the culture medium and cells to maintain sufficient oxygen supply, and the stirring device does not directly contact the cells, reducing the shear force on the cells. At the same time, the method of the present invention simulates the growth environment of cells in vivo and stimulates a large number of NK cells. secrete exosomes.

Description

A method for stimulating cells to secrete exosomes in large quantities

technical field

The present invention relates to the preparation and isolation technology of exosomes, in particular to a method for stimulating cells to secrete exosomes in large quantities.

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, and nucleic acids. 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) regulating the release and distribution of exosomes. (2) Exosome-based drug delivery. (3) Exosome-mediated tumor targeting therapy. (4) Exosomes and immunotherapy. At present, exosomes have shown great application prospects in the immunotherapy of tuberculosis, in the diagnosis, inhibition or regulation of tumor cell metastasis, prognosis monitoring and treatment of bone tumors, liver cancer, ovarian cancer, prostate cancer and other cancers. In addition, cellular exosomes, as a carrier of cell-to-cell communication, 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 MSCs functions, including repairing and regenerating tissues, inhibiting inflammatory responses and regulating the body's immunity.

CAR-T cell therapy (CAR-T therapy), which uses genetically modified 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 that human induced pluripotent stem cells (induced pluripotent stem cells, iPS cells) were used to grow cells similar to CAR-T cells. Modified natural killer cells (natural killer cells, NK cells) are highly effective against ovarian cancer in mouse models. However, it has been reported that NK cell exosomes have the function of NK cells, and are smaller and more specific, 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 killing proteins, which can kill tumor cells. In addition, because exosomes have the characteristics of transferring to directional target organs, they can be designed as A drug carrier for precise drug delivery, which transports specific drugs to target organs to play a role. Therefore, the exosomes of NK cells have great potential in the application research of tumor therapy and drug targeting carrier. At present, the extraction and purification of exosomes are mainly carried out based on the physical and chemical 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 stirred cell culture bioreactors and non-stirred cell culture bioreactors according to whether there is a stirring paddle inside. Stirred cell culture reactors generate eddy currents through the rotation of the stirring paddles to complete the aeration and oxygen supply of the culture medium, but the mechanical shear force generated by this method is relatively large, which is very easy to damage the cells. At present, the impact on cells can only be mitigated by controlling the stirring speed, but the problem still exists. The non-stirred cell culture reactor produces low shear force, and mainly uses vibration, shaking, rolling or swinging to move the culture medium to maintain the suspension state of the cells and the gas exchange of the culture medium. Its disadvantage is that it is difficult to ensure the absorption efficiency of oxygen. In recent years, there is also a method of using air or oxygen injection to solve the oxygen supply in the culture medium, but this method is easy to generate a large amount of foam, and the foam will also cause physical damage to the cells during the bubble burst process, and excessive oxygen injection will also poison the cells. The precise control of gas injection also complicates the reactor equipment, increasing the cost of cell culture. Therefore, how to provide uniform stirring 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.

Contents of the invention

In order to solve the above problems, the present invention provides a method for stimulating cells to secrete exosomes in large quantities.

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

A method for stimulating cells to secrete exosomes in large quantities, using a pulse-stirred bioreactor to cultivate cells, comprising the following steps:

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

(2) inoculation: control the valve of the inoculation port to open, and inoculate the cells;

(3) Stimulate cells: turn on the magnetic stirring and pulse device to simulate the cell growth environment of the blood structure in the body, and stimulate the cells to secrete exosomes;

(4) Monitor and adjust the culture conditions: the monitoring chip monitors the cell culture environment in real time, and adjusts the culture conditions through the host;

(5) Separation and collection of exosomes: After the cells grow in the culture device for 2 days, the filter pump is turned on to allow the culture medium to pass through the separation and collection device to collect the end product exosomes.

Further, in step (2), NK92mi cells were inoculated at a density of 2×10 ⁵ /ml.

Further, in step (3), the magnetic stirrer speed is set to 50rmp, and the pulse frequency is adjusted to 72 times/min to simulate the cell growth environment of the blood structure in the body and stimulate the cells to secrete exosomes.

Further, in step (4), the culture conditions are set to liquid dissolved oxygen of 95%, carbon dioxide concentration of 5%, and pH of 7.3.

Further, the bioreactor includes a cell culture device and an exosome separation and collection device;

The cell culture device is used to cultivate cells and stimulate cells to secrete exosomes; the exosome separation and collection device is connected to the cell culture device, and can separate and collect exosomes from the culture medium in the cell culture device, 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 solution in the cell culture device;

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

The cell culture device also includes a cell culture chamber, which is used for cell culture and exosome secretion.

Further, the cell culture chamber is provided with an inoculation port, a culture solution filling port and a sampling 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 includes a magnetic stirring rod and a magnetic drive device, and the magnetic stirring rod can be stirred under the drive of the magnetic drive device ; The rotating speed of the magnetic stirring bar can be adjusted through the magnetic field strength.

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 chamber, the preliminary separation device includes a preliminary separation chamber, and the preliminary separation chamber communicates with the cell culture chamber;

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 pipe, 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 a regulating device, and the condition monitoring device includes a pH value monitoring probe, a _CO content monitoring probe, _{an O} content monitoring probe and a condition sampling device, a pH value monitoring probe, The CO ₂ content monitoring probe and the O ₂ content monitoring probe are placed in the cell culture chamber; the conditional sampling device is located outside the cell culture chamber, and the conditional sampling device is connected with the cell culture chamber, and the conditional sampling device can Load the cell culture chamber.

The beneficial effects of the present invention are:

(1) In the present invention, oxygen and carbon dioxide are introduced into the preliminary separation chamber, and under the action of a magnetic stirring device, the oxygen and carbon dioxide are first dissolved in the culture medium, and then passed through the first filter connecting the preliminary separation chamber and the cell culture chamber. When the membrane enters the cell culture chamber, the culture medium and the cells can be fully mixed 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, and at the same time , the addition of a bidirectional pulse pump can effectively simulate the beating of the pulse, fully simulate the growth environment of NK cells in vivo, and 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 inoculating other types of cells, such as macrophages, T lymphocytes, and B lymphocytes.

(2) The bioreactor and method of the present invention can be used to produce exosomes in large quantities more conveniently, with ingenious design and simple operation, which can meet the requirements of laboratories or factories for exosome production. 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 produce other cell products, and has broad application prospects.

(3) The present invention provides a simple and rapid method for producing NK cell exosomes or other extracellular products in large quantities, and the method has a wide range of applications, ranging from large factories to small laboratories, and has a wide application prospect .

Description of drawings

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

Fig. 2 is a schematic diagram of the structure of the cell culture device.

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

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

Figure 5 is the quantitative analysis results of exosome products obtained by collecting 30ml of static culture and using the bioreactor and method of the present invention after culturing NK92mi cells for 48 hours, respectively (wherein, *** represents p<0.001 There are very significant differences).

Detailed ways

The technical solution of the present invention will be described in further detail below in conjunction with the accompanying drawings. It should be noted that the specific implementation is only a detailed description of the present invention and should not be regarded as a limitation of the present invention.

In the present invention, the inoculated cells are NK cells, but are not limited to NK cells, and can also be macrophages, lymphoid T cells, blood cells and other suspension cells or adherent cells capable of suspension growth.

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 retaining device located between the preliminary separation chamber and the cell culture chamber, and keeps the cells in the cell culture chamber is a filter membrane, but not limited to the 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 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 completed in other ways, such as: density gradient centrifugation, ultracentrifugation, etc.

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

In the present invention, unless otherwise specified and limited, the term "connection" should be understood in a broad sense, for example, it can be a fixed connection, or a detachable connection, or integrated; it can be a mechanical connection, or an electrical connection. Connection; it can be a direct connection or an indirect connection through an intermediary, and it can be an internal connection between two elements or an interaction relationship between two elements. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present invention according to specific situations.

Specific examples:

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

The cell culture device is used to cultivate cells and stimulate cells to secrete exosomes; the exosome separation and collection device is connected to the cell culture device, and can separate and collect exosomes from the culture medium in the cell culture device, 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 solution in the cell culture device;

The cell culture device includes a pulse device, which is used to add culture fluid 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 pulse action .

In some preferred manners, the cell culture device further includes cell culture chambers 1-8, and the cell culture chambers 1-8 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 solution filling port 1-10 and a sampling port 1-11.

In this embodiment, as shown in Figure 1, the upper surface of the cell culture cavity 1-8 is provided with an inoculation port 1-9, a culture solution filling port 1-10, and a sampling port 1-11; wherein, the inoculation port 1-9 It can be used to inoculate cells, and the culture 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 regulating liquid to adjust the cell culture chamber 1 pH in -8.

In some preferred manners, the pulse device is connected to the cell culture chamber 1-8, and culture fluid can be pulsed into the cell culture chamber 1-8 to stimulate the cultured cells in the cell culture chamber 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 a 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 pump 1-12.

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

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

In this embodiment, as shown in Figure 1, the magnetic driving device is a magnetic stirrer 1-2, the magnetic stirrer 1-2 and the magnetic stirring rod 1-6 are all 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 manners, the rotational speed of the magnetic stirring rods 1-6 can be adjusted through the magnetic field strength, and the adjustment range of the magnetic field strength is 30-150rpm.

In some preferred modes, as shown in Figure 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 1-8 5 communicates with the cell culture chamber 1-8, and a first filter membrane and a supporting device 1-7 are arranged between the preliminary separation chamber 1-5 and the cell culture chamber 1-8. Due to the effect of the filter membrane, the cells are left in the cell culture chamber chambers 1-8.

In some preferred modes, as shown in Figure 1, the separation and collection device also 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 1-1 through a filter pipeline 2-1. 5. 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 Figure 4, the multi-layer filter device 2-4 includes a first filter cavity 2-4-1, a second filter cavity 2-4-3, and a third filter cavity 2-4-5 , The fourth filter cavity 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 filter membrane apertures of the second filter membrane and support device 2-4-2, the third filter membrane and support device 2-4-4, and the fourth filter membrane and support device 2-4-6 are successively reduced.

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 modes, the bioreactor of the present invention also includes a condition monitoring device; the condition monitoring device can monitor the state of the cell growth environment in the cell culture chamber 1-8 in real time.

In some preferred modes, the condition monitoring device includes a pH value monitoring probe 3-1-3, a _CO content monitoring probe 3-1-1, an _O content monitoring probe 3-1-2 and a conditional sampling device, The pH value monitoring probe 3-1-3, the _CO2 content monitoring probe 3-1-1, and the _O2 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 -8, the conditional sampling device is in communication with the cell culture chamber 1-8, and the conditional sampling device can add samples to the cell culture chamber 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 sampling device includes a regulating liquid container 1-111, an O ₂ tank 1-14, and a CO ₂ tank 1-15; the regulating liquid container 1-111 is connected to the cell culture chamber 1-8, and the O ₂ tank 1-14, _CO2 tanks 1-15 are respectively connected to the preliminary separation chamber 1-5 through the _O2 delivery pipe 1-4 and _CO2 delivery pipe 1-3, and the preliminary separation chamber 1-5 is connected to the cell culture chamber 1-8 ;

By passing into oxygen and carbon dioxide in the preliminary separation chamber 1-5, under the effect 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 a magnetic field), so that oxygen and carbon dioxide are first dissolved in the culture 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, which reduces the shear force on the cells.

In some preferred modes, the bioreactor of the present invention further includes an adjustment device, which can adjust and control the culture conditions in the cell culture chamber.

In this embodiment, the regulating device includes a liquid release switch (numerical control) 3-2, a host machine 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 machine 3-3 controls the automatic regulation system and can automatically adjust the culture conditions in the cell culture chambers 1-8.

The present invention also provides a method for stimulating cells to secrete exosomes in large quantities, using the bioreactor described above, comprising the following steps:

(1) Filling medium: turn on the switch of the bioreactor, control the opening of valves 1-10 of the medium filling port, fill the medium, and fill the cell culture chamber 1-8; the medium can be commonly used when cultivating NK92mi cells existing culture medium.

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

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

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

(5) Separation and collection of exosomes: After the cells grow in the cell culture chamber 1-8 for 2 days, the filter pump 2-3 is turned on, so that the culture medium passes through the separation and collection device, and the end product exosomes are finally collected; in this embodiment , 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 pipeline 2-1, and is filtered layer by layer through the multi-layer filter device 2-4, finally, The end product exosomes enter the product collection tank 2-2;

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

The device of the present 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 adjustment device are used to monitor and adjust the state of the cell growth environment in the culture chamber in real time, and finally through the membrane The filtration system separates and the final product is collected. 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 a magnetic field, and the liquid in the preliminary separation chamber 1-5 is then The rotation affects the environment of the cell culture chamber 1-8 to produce continuous fluctuations. At the same time, the nutrient solution is pumped intermittently with the pulse pump 1-12 to generate pulsed fluctuations in the culture chamber. The speed of magnetic stirring rod 1-6 can be adjusted by the strength of the magnetic field, and the adjustable range is 30-150rpm. The pulse time and intensity can be adjusted by controlling the stop, operation and intensity of 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 chamber 1-5, the preliminary separation chamber 1-5 communicates with the cell culture chamber 1-8, and the preliminary separation chamber 1-5 A first filter membrane and a supporting device 1-7 are arranged between the cell culture chamber 1-8, and the cells are left in the cell culture chamber 1-8 due to the effect of the filter membrane; the membrane filtration of the multi-layer filter device 2-4 The separation system includes a number of different filter membranes with gradually smaller pore sizes and multiple filter chambers. The filtrate can be filtered multiple times and the product is finally collected.

Adopt above-mentioned bioreactor and method, cultivate NK92mi cell, simultaneously, adopt traditional bioreactor (i.e. adopt T25 culture bottle to cultivate), the method for static culture is cultivated, as comparative example, other culture conditions of comparative example and This embodiment is the same.

Fig. 5 is after cultivating NK92mi cells for 48 hours, respectively collecting 30ml of traditional cell culture methods (i.e. static culture, culturing with T25 culture flasks, other conditions are the same as in this example) and 30ml of the reactions cultivated using the bioreactor and method of the present invention Liquid, the quantitative analysis results of the obtained exosome products (wherein, *** 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 method of the present invention for cultivation. The bioreactor and method of the present invention are beneficial to the growth of NK92mi cells and can stimulate NK92mi cells to secrete a large amount of exosomes.

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 modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within 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.

Images