CN116355742A - Promote culture apparatus that exosome secretes - Google Patents
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- CN116355742A CN116355742A CN202310396820.6A CN202310396820A CN116355742A CN 116355742 A CN116355742 A CN 116355742A CN 202310396820 A CN202310396820 A CN 202310396820A CN 116355742 A CN116355742 A CN 116355742A
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- 210000001808 exosome Anatomy 0.000 title claims abstract description 50
- 238000004113 cell culture Methods 0.000 claims abstract description 77
- 210000004027 cell Anatomy 0.000 claims abstract description 51
- 230000000638 stimulation Effects 0.000 claims abstract description 34
- 230000028327 secretion Effects 0.000 claims abstract description 28
- 230000005540 biological transmission Effects 0.000 claims abstract description 24
- 230000001737 promoting effect Effects 0.000 claims abstract description 24
- 238000001914 filtration Methods 0.000 claims abstract description 22
- 239000012531 culture fluid Substances 0.000 claims abstract description 11
- 230000001105 regulatory effect Effects 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 230000008859 change Effects 0.000 abstract description 6
- 210000003722 extracellular fluid Anatomy 0.000 abstract description 3
- 238000001125 extrusion Methods 0.000 description 6
- 238000007789 sealing Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
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- 230000008569 process Effects 0.000 description 3
- 238000004891 communication Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
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- 238000012546 transfer Methods 0.000 description 2
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- 229910002601 GaN Inorganic materials 0.000 description 1
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- 230000002900 effect on cell Effects 0.000 description 1
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- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
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- 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
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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- 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
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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Abstract
The invention relates to the technical field of bioengineering, and discloses culture equipment for promoting secretion of extracellular fluid, which comprises a cell culture device, a gas transmission device, an electric stimulation device, a filtering device and a control device, wherein a culture fluid is contained in the cell culture device, the gas transmission device is used for introducing gas into the cell culture device or extracting gas from the cell culture device so as to change the in-bottle pressure of the cell culture device, the electric stimulation device applies current to the culture fluid through a cable so as to stimulate cells in the culture fluid to produce the extracellular fluid, the filtering device is connected with the cell culture device so as to filter the culture fluid, and the control device is respectively electrically connected with the gas transmission device and the electric stimulation device so as to control starting and stopping of the gas transmission device and the electric stimulation device. Compared with the prior art, the culture equipment promotes the cells to accelerate secretion of exosomes by increasing the gas pressure and the electrical stimulation, the production mode is novel and reliable, the yield of the exosomes is improved, and the culture equipment is simple in structure and easy to popularize.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to culture equipment for promoting secretion of an extracellular secretion.
Background
The extracellular secretion is a bioactive substance which is naturally secreted by cells and consists of lipid, protein, nucleic acid and the like, is a vesicle with the diameter of 30-150nm, is an important way for the stem cells to exert biological effects, has the advantages of low immunogenicity, high stability, nanoscale penetration of blood brain barrier, high activity, difficult tumorigenicity and the like compared with the stem cells, and transmits the expression information of the donor cells to the receptor cells through multiple mechanisms such as combination with the target cells, endocytosis, membrane fusion and the like, so that the target cells are reprogrammed to exert biological regulation functions, and has the mechanism characteristics of regulating inflammatory reaction, promoting cell proliferation migration, promoting angiogenesis, regulating matrix reconstruction and the like.
However, the quantity of exosomes produced by cells under the condition of no external inducement stimulus is low, and the requirements of research and development and mass production of the exosome drugs and biological agents at present cannot be met.
The existing technology for improving the yield of the exosomes of cells adopts mechanical energy to stimulate the cells so as to achieve the purpose of promoting the secretion of the exosomes. For example, chinese patent CN110093274B discloses a technique for increasing the yield of exosomes by extruding cells using an extrusion device. The technology utilizes a steering engine to drive a gallium nitride porous extrusion plate to extrude the cell surface, thereby improving the content of extracellular vesicles in a culture device. For another example, chinese patent CN110777142a discloses a method for promoting secretion of exosomes by low-intensity pulsed ultrasound stimulation, under pulsed ultrasound stimulation, exosomes secreted by cells can reach 150% -200% of exosome yield under non-stimulated conditions.
However, mechanical energy stimulates cells to secrete exosomes and simultaneously causes a certain damage to cells, which affects the yield of exosomes. Therefore, development of a novel device for promoting cell secretion of exosomes is needed to achieve the effects of increasing exosome yield and promoting the industrialization of exosome technology.
Disclosure of Invention
The invention aims to provide a culture device for promoting secretion of an exosome, which can improve the production efficiency of the exosome and increase the yield of the exosome.
Based on this, the present invention provides a culture apparatus for promoting secretion of an extracellular body, comprising:
the cell culture device is internally provided with a culture solution;
a gas delivery device for varying the pressure of a gas within the cell culture device;
an electrical stimulation device for applying an electrical current to the culture fluid to stimulate cells within the culture fluid to produce exosomes;
a filter device for filtering the culture liquid;
and the control device controls the operation of the gas transmission device and the electric stimulation device.
In some embodiments of the present application, the gas delivery device comprises a gas conduit in communication with the interior of the cell culture device.
In some embodiments of the present application, the electrical stimulation device comprises a cable having one end inserted into the cell culture device and submerged within the culture fluid.
In some embodiments of the present application, the filtration device and the cell culture device are in communication via a drain tube, and a flow limiting valve is provided on the drain tube.
In some embodiments of the present application, the air duct includes an air inlet pipe and an air outlet pipe, and air pressure regulating valves are arranged on the air inlet pipe and the air outlet pipe.
In some embodiments of the present application, the electrical stimulation device applies a voltage of 8-12V.
In some embodiments of the present application, the gas pressure within the cell culture apparatus is between 0.03MPa and 0.05MPa.
In some embodiments of the present application, an air pressure sensing device for monitoring air pressure in the bottle is disposed in the cell culture device, and the air pressure sensing device is electrically connected with the control device.
In some embodiments of the present application, a current sensing device for detecting the magnitude of the receiving current of the culture solution is disposed in the cell culture device, and the current sensing device is electrically connected with the control device.
In some embodiments of the present application, a storage device is included in connection with the cell culture device, the storage device configured to store the filtered culture fluid comprising exosomes.
The embodiment of the invention provides a culture device for promoting secretion of an extracellular body, which has the beneficial effects that compared with the prior art:
the embodiment of the invention provides a culture device for promoting secretion of cell exosomes, which comprises a cell culture device, a gas transmission device, an electric stimulation device, a filtering device and a control device, wherein a culture solution is contained in the cell culture device, the gas transmission device is arranged outside the cell culture device and communicated with the cell culture device through a gas duct, the gas transmission device can be used for introducing gas into the cell culture device or extracting gas from the cell culture device through the gas duct so as to change the bottle pressure of the cell culture device, the gas duct obviously comprises at least one gas inlet pipe and at least one gas outlet pipe, the gas inlet pipe and the gas outlet pipe are both connected with the cell culture device and the gas transmission device, the electric stimulation device is arranged beside the cell culture device and connected with a cable, the other end of the cable is immersed in the cell culture device, the electric stimulation device can apply current to the culture solution through the cable so as to stimulate cells in the culture solution to produce exosomes, the filtering device is arranged in the cell culture device so as to filter the culture solution so as to extract the exosomes in the culture solution, and the control device is respectively electrically connected with the gas transmission device and the electric stimulation device so as to control normal start and stop of the cell culture device and the cell exosomes. Based on above-mentioned structure, this application promotes the cell secretion exosome through the external stimulus of two kinds of differences, can effectively promote the quantity that acquires the exosome in the same time, specifically, gas transmission device can be to the interior gas pressure that lets in gas or extraction cell culture device in the cell culture device in this change cell culture device, the cell in the culture solution adheres to on the platelike plane, the cell receives gaseous extrusion equally when the device internal pressure increases, continue to let in the microcurrent stimulation cell through the electrostimulation device to the culture solution this moment can cooperate the gas pressure and act on the cell simultaneously and promote the cell secretion exosome, also this application is through increasing the extrusion cell of aerated air pressure as the main, microcurrent stimulation cell is as the assistance, both act on the cell and then promote it to produce more exosome in the same time simultaneously. In addition, the culture equipment is also provided with a filtering device, the filtering device is positioned in the cell culture device, the culture solution can be simultaneously filtered in the process of gas extrusion and electric stimulation so as to separate exosomes from other impurities such as organelles and the like, the independent filtering process can be reduced on the premise of not affecting the air tightness, and the construction process is optimized. Thus, the production mode of the culture equipment is novel and reliable, the yield of exosomes is improved, and the culture equipment is simple in structure and easy to popularize.
Drawings
FIG. 1 is a schematic diagram showing the structure of a culture apparatus for promoting secretion of an extracellular body according to some embodiments of the present invention.
In the figure, 1, a cell culture device; 101. a drainage tube; 102. a flow limiting valve; 2. a gas transmission device; 3. an electrical stimulation device; 4. a filtering device; 5. a control device; 6. an air pressure sensing device; 7. a current sensing device; 8. a storage device; 9. an air duct; 901. an air inlet pipe; 902. an air outlet pipe; 10. an air pressure regulating valve; 11. a cable.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
It should be understood that the terms "front", "rear", etc. are used in the present invention to describe various information, but the information should not be limited to these terms, which are only used to distinguish the same type of information from each other. For example, "front" information may also be referred to as "rear" information, and "rear" information may also be referred to as "front" information, without departing from the scope of the invention.
As shown in fig. 1, an embodiment of the present invention provides a culture apparatus for promoting secretion of extracellular fluid, which includes a cell culture device 1, a gas transmission device 2, an electrical stimulation device 3, a filtering device 4, and a control device 5, wherein a culture solution is contained in the cell culture device 1, the gas transmission device 2 is disposed outside the cell culture device 1 and is communicated with the cell culture device 1 through a gas duct 9, and the gas transmission device 2 can supply gas to the cell culture device 1 through the gas duct 9 or exhaust gas in the cell culture device 1 to change the pressure in a bottle of the cell culture device 1. The air duct 9 comprises at least one air inlet pipe 901 and at least one air outlet pipe 902, and the air inlet pipe 901 and the air outlet pipe 902 are connected with the cell culture device 1 and the air transmission device 2. The electro-stimulation device 3 is arranged beside the cell culture device 1 and connected with one end of a cable 11, and the other end of the cable 11 is inserted into the cell culture device 1 and immersed in the culture solution. The electro-stimulation device 3 may apply an electric current to the culture broth via the cable 11 to stimulate cells within the culture broth to produce exosomes. The cell culture apparatus 1 is further connected to a filter device 4 for filtering the culture medium. The control device 5 is electrically connected with the gas transmission device 2 and the electro-stimulation device 3 to control the start and stop of the two devices.
In a preferred embodiment, the cells in the culture broth are adherent cells, such as mesenchymal stem cells, dermal fibroblasts, and the like. In another preferred embodiment, the cells in the culture are suspension cells, such as T cells, and the like.
Based on above-mentioned structure, the exosome is promoted through the external stimulus of two kinds of differences to this application, can effectively promote the quantity of acquireing the exosome in unit time. Specifically, the gas transmission device 2 can introduce gas into the sealed cell culture device 1 or extract gas from the cell culture device 1 to change the gas pressure in the cell culture device 1, cells in the culture solution are adhered to microcarriers or cultured in a suspension state, and when the pressure in the device is increased, the cells are extruded by the gas, and at the moment, the cells are stimulated by continuously introducing micro-current into the culture solution through the electric stimulation device 3, so that the cells can be matched with the gas pressure to promote the cells to secrete exosomes. Preferably, the gas pressure within the cell culture apparatus 1 is between 0.03MPa and 0.05MPa. The cell is extruded by adding the air pressure to the cell, the microcurrent stimulates the cell to be auxiliary, and the microcurrent stimulates the cell to act on the cell simultaneously so as to promote the cell to generate more exosomes in the same time. The culture apparatus is further provided with a filter device 4 which is capable of filtering the culture liquid after the end of the culture cycle of the cell culture apparatus 1 to separate exosomes from other impurities such as organelles and the like. Thus, the production mode of the culture equipment is novel and reliable, the yield of exosomes is improved, and the culture equipment is simple in structure and easy to popularize.
In particular, the cell culture apparatus 1 should be in an airtight state together with the gas transfer apparatus 2, the electro-stimulation apparatus 3, and the filtration apparatus 4, so that the gas pressure in the cell culture apparatus 1 can be changed by the gas transfer apparatus 2. As such, the cell culture apparatus 1 of the present application needs to ensure its own air tightness to the greatest extent, and the sealing parts are used to seal each inlet, outlet and part connection place of the cell culture apparatus 1, including but not limited to a safety valve sleeve sealing sleeve and a rubber sealing sleeve, specifically, the through hole part for setting the pipeline structure on each apparatus is sealed by using the safety valve sleeve sealing sleeve, and the outer wall of the inlet/outlet is connected with other parts of the apparatus by using the rubber sealing sleeve in a sealing manner. Of course, a differential pressure bypass valve may also be used in order to control the air pressure balance within the device 5.
Further, the cell culture apparatus 1 and the filter apparatus 4 of the present application are communicated through a drain pipe 101, and a flow limiting valve 102 is provided on the drain pipe 101. Based on the above structure, the operator can start the gas transmission device 2 and the electro-stimulation device 3 by using the control device 5 to perform electro-stimulation and pneumatic extrusion treatment on the culture solution in the cell culture device 1. After the culture period in the cell culture apparatus 1 is completed, an operator can open the flow limiting valve 102 and adjust the flow rate of the cell culture apparatus 1 flowing to the filtering apparatus 4 by controlling the flow limiting valve 102, and the culture solution enters the filtering apparatus 4 for filtering, so that the exosomes are separated from other impurities such as organelles.
Optionally, in some embodiments of the present application, an air pressure regulating valve 10 is provided on the air duct 9, and an operator can control the flow of air into and out of the cell culture apparatus 1 through the air pressure regulating valve 10. As shown in fig. 1, in a preferred embodiment, the air duct 9 in the present application includes an air inlet pipe 901 and an air outlet pipe 902, and air pressure regulating valves 10 are disposed on the air inlet pipe 901 and the air outlet pipe 902.
Further, a plurality of plate-like flat surfaces are provided in the cell culture apparatus 1 of the present application. In a preferred embodiment, the cable 11 of the electrostimulation device 3 is directly connected to the plate-like plane, enabling an effective electrostimulation of the cells. In order to realize normal production of the extracellular body, the current of the electric stimulation device is required to be controlled, so that the stimulation effect on cells is ensured, and damage to the cells is avoided. In a preferred embodiment of the present application, the voltage applied by the electro-stimulation device 3 to the plate-like plane is 8-12V. In order to realize the above precise control, the control device 5 in the present application is preferably a single chip microcomputer.
Further, in some embodiments of the present application, the cell culture apparatus 1 is provided with an air pressure sensing device 6 for monitoring the air pressure in the bottle, and the air pressure sensing device 6 is electrically connected to the control device 5. When the device is operated, the air pressure in the cell culture device 1 can be monitored through the air pressure sensing device 6, and when the air pressure meets the requirement, the control device 5 instructs the electric stimulation device 3 to introduce micro-current into the cell culture device 1 to stimulate cells, so that secretion of exosomes is promoted.
Similar to the air pressure sensing device 6, in some embodiments of the present application, a current sensing device 7 for detecting the micro-current level to which the cells are subjected is further provided in the cell culture device 1, and the current sensing device 7 is electrically connected to the control device 5. When the device operates, the current sensing device 7 can be used for measuring the microcurrent received by cells, so that the damage to the cells caused by excessive microcurrent is avoided, and the problem that the effective stimulation effect cannot be achieved due to excessive microcurrent is also avoided.
In addition, in some embodiments of the present application, the culture apparatus comprises a storage device 8 connected to the filtering device 4, the storage device 8 being operable to collect and store the filtered culture fluid; the filtered culture solution can be used for preparing other exosome products.
In summary, the present invention provides a culture apparatus for promoting secretion of exosomes, which comprises a cell culture device, a gas transmission device, an electrical stimulation device, a filtering device and a control device, wherein the cell culture device is filled with a culture solution, the gas transmission device is used for introducing gas into the cell culture device or extracting gas from the cell culture device to change the in-bottle pressure of the cell culture device, the electrical stimulation device applies current to the culture solution through a cable to stimulate cells in the culture solution to produce exosomes, the filtering device is connected with the cell culture device to filter the culture solution, and the control device is respectively electrically connected with the gas transmission device and the electrical stimulation device to control starting and stopping of the gas transmission device and the electrical stimulation device. Compared with the prior art, the culture equipment promotes the cells to accelerate secretion of exosomes by increasing the gas pressure and the electrical stimulation, the production mode is novel and reliable, the yield of the exosomes is improved, and the culture equipment is simple in structure and easy to popularize.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and substitutions can be made by those skilled in the art without departing from the technical principles of the present invention, and these modifications and substitutions should also be considered as being within the scope of the present invention.
Claims (10)
1. A culture apparatus for promoting secretion of an exosome, comprising:
the cell culture device is internally provided with a culture solution;
a gas delivery device for varying the pressure of a gas within the cell culture device;
an electrical stimulation device for applying an electrical current to the culture fluid to stimulate cells within the culture fluid to produce exosomes;
a filter device for filtering the culture liquid;
and the control device controls the operation of the gas transmission device and the electric stimulation device.
2. The culture apparatus for promoting secretion of extracellular bodies according to claim 1, wherein the gas delivery means comprises a gas duct that communicates with the interior of the cell culture means.
3. The culture apparatus for promoting secretion of extracellular bodies according to claim 1, wherein the electrical stimulation means comprises a cable having one end inserted into the cell culture means and immersed in the culture solution.
4. The culture apparatus for promoting secretion of extracellular bodies according to claim 1, wherein the filtering means and the cell culture means are communicated through a drainage tube provided with a flow limiting valve.
5. The culture apparatus for promoting secretion of extracellular bodies according to claim 2, wherein the air duct comprises an air inlet duct and an air outlet duct, and air pressure regulating valves are provided on the air inlet duct and the air outlet duct.
6. The culture apparatus for promoting secretion of extracellular bodies according to claim 1, wherein the voltage applied by the electrical stimulation means is 8-12V.
7. The culture apparatus for promoting secretion of extracellular bodies according to claim 1, wherein the gas pressure in the cell culture device is 0.03MPa to 0.05MPa.
8. The culture apparatus for promoting secretion of extracellular bodies according to claim 1, wherein a pressure sensing device for monitoring the pressure in the bottle is provided in the cell culture device, and the pressure sensing device is electrically connected to the control device.
9. The culture apparatus according to claim 1 or 8, wherein a current sensing device for detecting the magnitude of a receiving current of the culture solution is provided in the cell culture device, and the current sensing device is electrically connected to the control device.
10. The culture apparatus for promoting secretion of an exosome according to claim 1, comprising a storage device connected to the cell culture device for storing the filtered culture solution containing the exosome.
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| CN202310396820.6A CN116355742A (en) | 2023-04-13 | 2023-04-13 | Promote culture apparatus that exosome secretes |
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| CN202310396820.6A CN116355742A (en) | 2023-04-13 | 2023-04-13 | Promote culture apparatus that exosome secretes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| TWI840269B (en) * | 2023-07-06 | 2024-04-21 | 浩河未來實業有限公司 | Exosome manufacturing system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| TWI840269B (en) * | 2023-07-06 | 2024-04-21 | 浩河未來實業有限公司 | Exosome manufacturing system |
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