CN111363680B - Pulse stirring type bioreactor for secretion, separation and collection of exosomes - Google Patents
Pulse stirring type bioreactor for secretion, separation and collection of exosomes Download PDFInfo
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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 separation and collection device is connected with the cell culture device and can separate exosome from culture solution in the cell culture device for collection; the cell culture apparatus comprises a stirring device for applying stirring power to the culture solution in the cell culture apparatus; the cell culture apparatus comprises a pulsing means to which the culture medium may be added in a pulsed manner. The bioreactor can fully mix culture solution and cells to keep sufficient oxygen supply, and the stirring device does not directly contact the cells, so that the shearing force applied to the cells is reduced.
Description
Technical Field
The invention relates to a preparation and separation technology of exosomes, in particular to a pulse stirring type bioreactor for exosome secretion, separation and collection.
Background
Exosomes (exosomes) are extracellular vesicles (extracellular vesicles, EVs) with a lipid bilayer, about 30-150nm in diameter. The exosome can contain various bioactive substances such as protein, lipid, nucleic acids, etc., and most cells of human body such as immune cells (T cells, B cells, kupffer cells and NK cells), endothelial cells, platelets, etc. can secrete the exosome. At present, the research of exosomes in clinical treatment can be roughly summarized as follows: (1) modulating 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 exosomes of the cells serve as a carrier for the mutual communication between the cells, and are also an important mode of paracrine activities of stem cells, and play an important role in tissue regeneration.
CAR-T cell therapy (CAR-T therapy) is the use of genetically engineered T cells to kill cancer cells, a method that has proven to be a promising option when other therapeutic approaches have failed. In a new study, researchers from university of california san diego and minnesota, usa reported that natural killer cells (natural killer cell, NK cells) cultured with human-induced pluripotent stem cells (induced pluripotent stem cell, iPS cells) and modified in a similar manner to CAR-T cells were highly effective against ovarian cancer in a mouse model. The NK cell exosome has the function of NK cell part, is smaller, has better specificity and is expected to play a unique role in cell therapy.
In recent years, research shows 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 a target organ, so that the exosomes can be designed into drug carriers for accurate administration, and specific drugs can be transported to the target organ to play a role. Therefore, the exosomes of NK cells have 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 properties of exosomes, and methods such as differential centrifugation combined with sucrose density gradient centrifugation, ultrafiltration, immunomagnetic bead extraction, exoQuick Precipitation extraction and the like are commonly adopted.
At present, the bioreactor is core equipment for large-scale suspension culture of cells, and can effectively increase the culture density of the cells in unit volume. The animal cell culture bioreactor is divided into a stirred cell culture bioreactor and a non-stirred 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 through 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 large and the cells are extremely easy to damage. At present, the influence on cells can be reduced only by controlling the stirring rotation speed, but the problem still exists. The shearing force generated by the non-stirring cell culture reactor is small, and the culture solution is mainly moved in a vibration, shaking, rolling or swinging mode, so that the suspension state of cells and the gas exchange of the culture solution are maintained. The disadvantage is that it is difficult to ensure the oxygen absorption efficiency. In recent years, methods for solving the problem of oxygen supply in culture solution by air or oxygen injection are also available, but a large amount of foam is easily generated by the method, physical damage is also caused to cells in the process of foam breaking, and the cells are also poisoned by excessive oxygen injection. Precise control of the gas injection also complicates the reactor equipment and increases the cost of cell culture. Thus, how to provide uniform agitation, allowing adequate mixing of the culture broth and cells to maintain adequate oxygen supply, while reducing mechanical shear damage to the cells is an urgent issue to be addressed.
Disclosure of Invention
In order to solve the above-mentioned problems, the present invention provides a pulse agitation type bioreactor for secretion, separation and collection of exosomes.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a pulse stirring type bioreactor for secretion, separation and collection of exosomes 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 exosome from culture solution in the cell culture device for collection, and cells are left in the cell culture device after separation;
the cell culture apparatus comprises a stirring device configured to apply stirring power to a culture fluid in the cell culture apparatus;
the cell culture apparatus includes a pulsing means for adding a culture fluid to the cell culture apparatus in a pulsed manner to stimulate cells being cultured within the cell culture apparatus.
Further, the cell culture apparatus further comprises a cell culture chamber for performing cell culture and exosome secretion.
Further, an inoculation port, a culture solution filling port and a sample filling 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 rod and magnetic drive device, and magnetic stirring rod can stir under magnetic drive device's drive.
Further, the rotating speed of the magnetic stirring rod can be adjusted through the magnetic field intensity, and the adjusting range is 30-150rpm.
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 preliminary separation device positioned at the bottom of the cell culture cavity, the preliminary separation device comprises a preliminary separation cavity, and the preliminary separation cavity is communicated with the cell culture cavity.
Further, the separation and collection device comprises a multi-layer filter device connected to the preliminary separation chamber through a filter pipe, and an outlet of the multi-layer filter device is connected to 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 CO 2 Content monitoring probe, O 2 Content monitoring probe and 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, and the adjustable range for monitoring the culture condition is as follows50-100% of liquid dissolved oxygen, 0-20% of carbon dioxide and 5-9 of PH; the condition sample adding device is positioned outside the cell culture cavity, the condition sample adding device is communicated with the cell culture cavity, and the condition sample adding device can timely adjust the cell culture environment by adding an adjusting reagent according to the information detected by the probe.
The invention also provides a method for stimulating cells to secrete exosomes in a large quantity, which adopts the bioreactor, monitors and adjusts the culture conditions in the cell culture cavity through a condition monitoring device and a regulating device, simulates the culture conditions in the cell culture cavity through stirring and pulse, enables the cells to be in a cell growth environment dynamically simulating the blood structure in the body, and stimulates the cells to secrete exosomes.
The present invention also provides a method for stimulating secretion of exosomes by cells, the method using the bioreactor described above, comprising the steps of:
(1) Filling the 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 opening of an inoculating port valve, wherein the inoculating density is 2 multiplied by 10 5 NK92mi cells per ml;
(3) Stimulating cells: turning on a magnetic stirring and pulse device, setting the rotating speed of the magnetic stirrer to be 50rmp, adjusting the pulse frequency to be 72 times/min, and simulating the growth environment of cells in a body to form 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 95% of liquid dissolved oxygen, 5% of carbon dioxide concentration and 7.3 PH;
(5) Exosome separation and collection: after the cells were grown in the culture apparatus for 2 days, the filter pump was turned on, and the culture solution was passed through a separation and collection apparatus, and finally the final product was collected.
The beneficial effects of the invention are as follows:
(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 which is communicated with the primary separation cavity and the cell culture cavity, so that the culture solution and the cells can be fully mixed to keep sufficient oxygen supply, the stirring device is not in direct contact with the cells, the shearing force of the cells is reduced, the problems 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 growth environment of NK cells in the body can be fully simulated, NK cells or other suspension cells and adherent cells which can be subjected to suspension culture can be greatly secreted, for example: exosomes, platelets, various cytokines, and the like. The scheme of the invention can be used for NK cells and also used for inoculating other types of cells, such as macrophages, T lymphocytes and B lymphocytes.
(2) The bioreactor and the method can conveniently and massively produce 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 ways, in particular 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 producing 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 diagram of a pulse agitation type bioreactor according to the present invention.
FIG. 2 is a schematic diagram of the structure of a cell culture apparatus.
FIG. 3 is an exploded view of a cell culture apparatus.
Fig. 4 is an exploded view of the multi-layered filter apparatus.
FIG. 5 shows the results of quantitative analysis of the obtained exosome products (where p < 0.001 is very significantly different) by collecting 30ml of reaction solution of stationary culture and culture using a bioreactor and method, respectively, 48 hours after culturing NK92mi cells.
Detailed Description
The following further details the technical solution of the present invention with reference to the accompanying drawings, it should be noted that the detailed description is only of the present invention and should not be taken as limiting the invention.
In the present invention, the cells to be inoculated are NK cells, but not limited to NK cells, but may be suspension cells such as macrophages, lymphocytes T cells, blood cells, etc., 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 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 enables the cells to be left in the cell culture cavity is a filter membrane, but is not limited to the filter membrane, and can also be 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 may also be generated by other devices having similar functions.
In the invention, the exosome separation and filtration system consists of a membrane filtration system, and the exosome separation work can be finished in other modes, such as: density gradient centrifugation, ultracentrifugation, and the like.
In the present invention, the collected cell product is exosome, but not limited to exosome, and can be various cell secretions: such as platelets, collagen, and the like.
In the present invention, unless explicitly specified and limited otherwise, the term "connected" is to be construed broadly, and for example, may be a fixed connection, may be a removable connection, or may be an integral unit; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communicated with the inside of two elements or the interaction relationship of the two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
Specific examples:
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 exosome from culture solution in the cell culture device for collection, and cells are left in the cell culture device after separation;
the cell culture apparatus comprises a stirring device configured to apply stirring power to a culture fluid in the cell culture apparatus;
the cell culture device comprises a pulse device, wherein the pulse device is used for adding culture solution into the cell culture device in a pulse mode so as to stimulate cells cultured in the cell culture device, and a cell growth environment dynamically simulating an in-vivo blood structure is formed through stirring and pulse action.
In some preferred forms, the cell culture apparatus further comprises a cell culture chamber 1-8, the cell culture chamber 1-8 being for cell culture and exosome secretion.
In some preferred embodiments, the cell culture chambers 1-8 are provided with an inoculation port 1-9, a culture medium filling port 1-10 and a sample filling 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 filling 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 filling the liquid culture medium into the cell culture cavity 1-8; the ports 1-11 may be used to add a conditioning fluid to adjust the pH in the cell culture chambers 1-8.
In some preferred embodiments, the pulsing means is connected to the cell culture chambers 1-8 and the culture medium may be added to the cell culture chambers 1-8 in a pulsed manner to stimulate the cells being cultured within the cell culture chambers 1-8.
In this embodiment, as shown in FIG. 1, the pulsing means comprises a pulsing pump 1-12, the pulsing pump 1-12 being in communication with the cell culture chamber 1-8 through a liquid conduit 1-13. The pulse time and intensity can be adjusted by controlling the stopping, running 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 forms, the stirring device adopts a magnetic stirring device, and the magnetic stirring device 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 be stirred 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 positioned below the cell culture chamber 1-8, and the magnetic stirring rod 1-6 is positioned above the magnetic stirrer 1-2.
In some preferred embodiments, the rotational speed of the magnetic stirring rod 1-6 may be adjusted by the magnetic field strength, which may be adjusted in the range of 30-150rpm.
In some preferred forms, as shown in fig. 2-3, the separation and collection device comprises a preliminary separation device positioned at the bottom of the cell culture chamber 1-8, the preliminary separation device comprises a preliminary separation chamber 1-5, the preliminary separation chamber 1-5 is communicated with the cell culture chamber 1-8, 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, and cells are left in the cell culture chamber 1-8 due to the action of the filter membrane.
In some preferred forms, as shown in FIG. 1, the separation and collection apparatus further comprises a multi-layer filtration apparatus and a collection apparatus, the multi-layer filtration apparatus 2-4 is connected to the preliminary separation chamber 1-5 through a filtration conduit 2-1, and the outlet of the multi-layer filtration apparatus 2-4 is connected to the product collection tank 2-2.
In some preferred forms, as shown in FIG. 4, the multi-layer filter apparatus 2-4 includes a first filter chamber 2-4-1, a second filter chamber 2-4-3, a third filter chamber 2-4-5, a fourth filter chamber 2-4-7, a second filter membrane and support apparatus 2-4-2, a third filter membrane and support apparatus 2-4-4, and a fourth filter membrane and support apparatus 2-4-6. The pore diameters of the second filter membrane and the supporting device 2-4-2, the third filter membrane and the supporting device 2-4-4, and the fourth filter membrane and the supporting device 2-4-6 become smaller in sequence.
The pressure on the cell culture chambers 1-8 generated by intermittent operation of the mechanically controlled filter pump is the primary motive force for driving the liquid through the filter membrane.
In some preferred forms, the bioreactor of the invention further comprises condition monitoring means; the condition monitoring device can monitor the growth environment state of cells in the cell culture chambers 1-8 in real time.
In some preferred forms, the condition monitoring device comprises a pH monitoring probe 3-1-3, CO 2 Content monitoring probe 3-1-1, O 2 Content monitoring probe 3-1-2 and condition sample loading device, pH value 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 cavity 1-8, the condition sample adding device is communicated with the cell culture cavity 1-8, and the condition sample adding device can sample the cell culture cavity 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 chamber 1-8 in time.
In this embodiment, the conditional sample addition means comprises a regulating liquid container 1-111, O 2 Tanks 1-14, CO 2 1-15 parts of a tank; the regulating liquid container 1-111 is communicated with the cell culture cavity 1-8, O 2 Tanks 1-14, CO 2 Tanks 1-15 are each filled with O 2 Conveying pipe 1-4, 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 the 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 field through the magnetic stirring device 1-2), the oxygen and the carbon dioxide are dissolved in the culture medium first, and then enter the cell culture chamber 1-8 through the 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 sufficient oxygen supply, and the stirring device is not in direct contact with the cells, thereby reducing the shearing force applied to the cells.
In some preferred forms, the bioreactor of the present invention further comprises a regulating device that can regulate and control the culture conditions within the cell culture chamber.
In this embodiment, the regulating device comprises a liquid release switch (numerical control) 3-2, a host computer 3-3, O 2 Tank, CO 2 3-4 parts of tank gas valve (numerical control) and 3-5 parts of pulse pump regulator (numerical control).
The host 3-3 controls the automatic regulating system to automatically regulate the culture conditions in the cell culture chambers 1-8.
The invention also provides a method for stimulating cells to secrete exosomes in large quantities, the bioreactor is adopted, and the method comprises the following steps:
(1) Filling the 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 conventional culture medium commonly used for culturing NK92mi cells;
(2) Inoculating: controlling the opening of the 1-9 valves of the inoculation port, and the inoculation density is 2 multiplied by 10 5 NK92mi cells per ml;
(3) Stimulating cells: turning on a magnetic stirring and pulse device, setting the rotating speed of the magnetic stirrer to be 50rmp, adjusting the pulse frequency to be 72 times/min, and simulating the growth environment of cells in a body to form 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 by an automatic regulation and control system controlled by the host computer 3-3, wherein the culture conditions are set to 95% of liquid dissolved oxygen, 5% of carbon dioxide concentration and 7.3 of PH, and in the embodiment, O can be controlled 2 、CO 2 And adjusting the sample addition amount and the sample addition speed of the adjustment liquid in the liquid containers 1 to 111 to realize the control and adjustment of the culture conditions;
(5) Exosome separation and collection: after the cells grow in the cell culture cavity 1-8 for 2 days, starting the filter pump 2-3 to enable the culture solution to pass through the separation and collection device, and finally collecting the final product exosomes; in the embodiment, the culture solution firstly enters the primary separation cavity 1-5 through the first filter membrane and the supporting device 1-7, then enters the filter pipeline 2-1, is filtered layer by layer through the multi-layer filter device 2-4, and finally, the final product exosome enters the product collection tank 2-2;
(6) The disinfection bioreactor is cleaned for the next use.
The device of the invention is characterized in that: through mechanical stirring and pulse action, a dynamic cell growth environment simulating the blood structure in the body is formed, the condition of the cell growth environment in the culture cavity is monitored and regulated in real time through a condition monitoring device and a regulating device, and finally, the cell growth environment is separated through a membrane filtration system, and the final product is collected. The mechanical stirring and pulse action means that the magnetic stirring rod 1-6 is placed in the 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 affects the environment of the cell culture cavity 1-8, so that continuous fluctuation is generated, and meanwhile, the pulse pump 1-12 is matched for intermittently pumping nutrient solution, so that pulse fluctuation is generated in the culture cavity. The rotating speed of the magnetic stirring rod 1-6 can be adjusted by the intensity of the magnetic field, the adjustable range is 30-150rpm, and the pulse time and the intensity can be adjusted by controlling the stop, the operation and the intensity of the pulse pump 1-12.
The separation and collection device comprises a primary 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 a cell culture cavity 1-8, a first filter membrane and a supporting device 1-7 are arranged between the primary separation cavity 1-5 and the cell culture cavity 1-8, and cells are left in the cell culture cavity 1-8 due to the action of the filter membrane; the membrane filtration separation system of the multi-layer filtration device 2-4 comprises a plurality of different filter membranes with gradually smaller pore diameters and a plurality of filter cavities, so that the filtrate can be filtered for a plurality of times, and finally the product is collected.
The NK92mi cells were cultured using the above-described bioreactor and method, and at the same time, using a conventional bioreactor (i.e., using T25 flask culture), the culture was performed by a stationary culture method, and other culture conditions of the comparative example were the same as those of the present example as a comparative example.
FIG. 5 shows the results of quantitative analysis of the obtained exosome products (where p < 0.001 is very significantly different) by collecting 30ml of the conventional cell culture (i.e., stationary culture, culture in T25 flask, and other conditions similar to the present example) and 30ml of the reaction solution cultured using the bioreactor and method of the present invention, respectively, after 48 hours of culturing NK92mi cells. As can be seen from FIG. 5, more 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 secretion of exosomes by NK92mi cells.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
1. The pulse stirring type bioreactor for secretion, separation and collection of exosomes 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 exosome from culture solution in the cell culture device for collection, and cells are left in the cell culture device after separation;
the cell culture apparatus comprises a stirring device configured to apply stirring power to a culture fluid 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, wherein the cell culture cavity is used for cell culture and exosome secretion;
the cell culture cavity is provided with an inoculation port, a culture solution filling port and a sample filling port;
the stirring device is positioned below the cell culture cavity, adopts a magnetic stirring device, and comprises a magnetic stirring rod and a magnetic driving device, wherein 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;
the pulse device comprises a pulse pump, and the pulse pump is communicated with the cell culture cavity through a liquid pipeline;
the separation and collection device comprises a primary separation device positioned at the bottom of the cell culture cavity, wherein 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, wherein 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.
2. The pulse agitation type bioreactor for secretion, separation and collection of exosomes according to claim 1, further comprising a condition monitoring device and an adjusting device, wherein the condition monitoring device comprises a pH monitoring probe, a CO 2 Content monitoring probe, O 2 Content monitoring probe and 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 sample adding device is located outside the cell culture cavity, the condition sample adding device is communicated with the cell culture cavity, and the condition sample adding device can sample the cell culture cavity according to information detected by the probe.
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| CN114107057A (en) * | 2021-12-01 | 2022-03-01 | 重庆医科大学 | Reactor for separating extracellular vesicles |
| CN115521914B (en) * | 2022-10-12 | 2024-04-19 | 西北工业大学 | In-vitro amplification system and method for human primary natural killer cells |
| CN116716288B (en) * | 2023-04-28 | 2024-04-26 | 四川大学 | Method for improving exosome yield by acoustic wave vibration |
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