CN111349562A - Organ chip culture box capable of adjusting oxygen concentration and using method thereof - Google Patents
Organ chip culture box capable of adjusting oxygen concentration and using method thereof Download PDFInfo
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- 210000000056 organ Anatomy 0.000 title claims abstract description 75
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 61
- 239000001301 oxygen Substances 0.000 title claims abstract description 61
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 12
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 8
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
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- 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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Abstract
The invention belongs to the field of biomedicine and organ chips, and discloses an organ chip culture box capable of adjusting oxygen concentration and a using method thereof. In addition, the cover of the culture box is also provided with a plurality of fluid connecting joints communicated with the inside of the chamber, and the joints are used for injecting culture medium into the organ chip in the culture box and discharging waste liquid. The culture box capable of adjusting the oxygen concentration can simulate the oxygen concentration of different tissues and organs in vivo, so that the organ chip has a more bionic function.
Description
Technical Field
The invention belongs to the field of biomedicine and organ chips, and particularly discloses an organ chip culture box capable of adjusting oxygen concentration and a using method thereof.
Background
With the development of organ chip technology, researchers have developed a variety of organ chips, including: lung chips, liver chips, intestine chips, kidney chips, tumor chips, and the like. These organ chips can provide a biomimetic microenvironment in close proximity to the body for different tissue cells in culture, such as: fluid shear forces, three-dimensional culture, mechanical forces, etc., thereby facilitating the reproduction of cells within the chip that approximate the structure and function of the tissues and organs in vivo. Therefore, researchers think that in the near future, organ chip technology will replace or partially replace existing animal experiments, thereby greatly reducing the cost of biomedicine and new drug development, and effectively shortening the period of new drug development.
Although, existing organ chip technology has been able to provide a biomimetic microenvironment for tissue cells. However, because there are great differences in the oxygen content of different tissues in the human body, for example: the lungs are very oxygen rich, while the intestines are almost anaerobic. The conventional organ chip equipment lacks a device capable of flexibly adjusting the oxygen concentration.
Disclosure of Invention
Aiming at the defects, the invention designs the culture box capable of adjusting the oxygen concentration and the use method thereof on the basis of the organ chip technology. In addition, the invention can be used for investigating the effect on different tissues and organs under different oxygen environments.
The technical scheme of the invention is as follows:
an organ chip culture box capable of adjusting oxygen concentration, which consists of a culture box cover and an upper part and a lower part of a culture box base; the culture box cover can cover the culture box base and form a closed space for cell culture; a ventilation pipeline penetrates through the cover of the culture box; an exhaust pipeline penetrates through the culture box base; a ventilation valve and an exhaust valve are arranged on the ventilation pipeline; the culture box cover is also provided with at least one culture medium outlet box joint and at least one culture medium inlet box joint; an organ chip, at least one culture medium liquid storage tank and a waste liquid tank are arranged in the culture box base; the culture medium liquid storage tank is connected with the culture medium outlet box connector through a pipeline; the organ chip is provided with a chip culture medium inlet joint and a chip culture medium outlet joint respectively, the chip culture medium inlet joint is connected with the culture medium box inlet joint through a pipeline, and the chip culture medium outlet joint is connected with the waste liquid pool through a pipeline.
When the scheme is used, the culture medium liquid storage tank is connected with the culture medium box outlet joint through a pipeline, and the culture medium box outlet joint is connected with the culture medium conveying mechanism through a pipeline, wherein the culture medium conveying mechanism can be various pumps commonly used in a laboratory; the pipeline is connected to a culture medium box inlet joint in a backflow mode, the organ chip inoculated with the cells is placed in a culture box base, a chip culture medium inlet joint is connected with the culture medium box inlet joint through the pipeline, a chip culture medium outlet joint is connected with the waste liquid pool through the pipeline, a culture box cover and the culture box base are sealed, and the sealing of an inner cavity is guaranteed; connecting a gas cylinder required for cell culture, such as a nitrogen gas cylinder, with a filter membrane with a pore size of 0.22 micron, and then connecting the gas cylinder with a vent pipeline; connecting an oxygen detection device, such as a digital display oxygen detector, with a filter membrane with a pore size of 0.22 μm, connecting to an exhaust pipeline or directly placing in a sealed chamber, opening a gas vent valve and an exhaust valve, opening a gas cylinder, allowing new gas to enter the culture box, and discharging the gas in the chamber of the original culture box; ventilating for 3-10 min, or after the oxygen detection device displays that the target concentration is reached, closing the gas cylinder, and adjusting a ventilation valve and an exhaust valve on the culture box to a closed state; and finally, placing the culture box into an incubator, and opening the culture medium conveying mechanism for culture.
The culture box only provides a space, wherein the number of the organ chips, the culture medium liquid storage tank, the number of the pipelines and the connectors on the culture box can be increased or decreased according to the requirement so as to meet the requirement of actual culture, for example, when multiple cell cultures are required, each cell can be correspondingly provided with an independent culture medium liquid storage tank, and pipelines and connectors entering the culture box are added.
Further, the organ chip culture box capable of adjusting the oxygen concentration is characterized in that a sealing cover is arranged above the culture medium liquid storage tank; the sealed cover is provided with a liquid storage tank culture medium outlet connector and an air pressure balancing hole; the culture medium outlet joint of the liquid storage tank is connected with the culture medium outlet box joint through a pipeline; the air pressure balance hole is sealed by a 0.22 micron filter membrane. The filter membrane is used to prevent bacterial contamination.
Furthermore, according to the organ chip culture box capable of adjusting oxygen concentration, a peristaltic pump is arranged outside a cover of the culture box, a pipeline is connected to the culture medium outlet box joint, and the pipeline is connected to the culture medium inlet box joint through the peristaltic pump.
Furthermore, above-mentioned organ chip culture box of adjustable oxygen concentration, still be provided with aseptic water household utensils in the culture box base. Placing a sterile water vessel, placing sterile water therein, and maintaining the humidity in the culture box.
Further, the above-mentioned oxygen concentration adjustable organ chip culturing box, wherein the culture medium outlet box connector or the culture medium inlet box connector is selected from a luer connector or a pagoda-like connector; the number that culture medium goes out box joint or culture medium advances box joint setting is increased and decreased according to the demand of cultivateing.
Further, the above-mentioned organ-chip culturing box capable of regulating oxygen concentration comprises at least a culture medium inlet, a cell culturing area, and a waste liquid outlet.
Furthermore, the cross section of the cover of the organ chip culture box capable of adjusting the oxygen concentration is circular, the inner diameter is 10-30 cm, the height is 10-20 cm, and the wall thickness is 0.5-2 cm; the length of the ventilation pipeline is 5-10 cm, the inner diameter of the pipeline is 2-5 mm, and the outer diameter of the pipeline is 5-15 mm; the cross section of the culture box base is circular, the inner diameter is 10-30 cm, the height is 10-20 cm, and the wall thickness is 0.5-2 cm; the length of the exhaust pipeline is 5-10 cm, the inner diameter of the pipeline is 2-5 mm, and the outer diameter of the pipeline is 5-15 mm.
Furthermore, according to the organ chip culture box capable of adjusting oxygen concentration, the edge of the opening of the culture box cover and the culture box base is provided with a circle of skirt edge, and the upper surface and the lower surface of the skirt edge are hermetically connected by screw holes, screw caps and studs or external clamps matched with sealing gaskets.
Furthermore, the organ chip culture box capable of adjusting the oxygen concentration is made of transparent plastic materials. The culture condition can be observed conveniently from the outside by using the transparent material.
Further, the method for using the organ chip culture box capable of adjusting the oxygen concentration comprises the following steps:
1) sterilizing all parts of the culture box by using a wiping method of alcohol, bleaching water and hydrogen peroxide or ultraviolet irradiation sterilization;
2) before use, the ventilation valve and the exhaust valve of the culture box are ensured to be in a closed state;
3) in order to keep the humidity in the culture box, a sterile water vessel containing 10-40 ml of sterile water is placed in the base of the culture box;
4) connecting a culture medium liquid storage tank with the culture medium outlet box connector through a pipeline, connecting the culture medium outlet box connector with a peristaltic pump through a pipeline, and connecting the pipeline with the culture medium inlet box connector in a backflow mode;
5) placing the organ chip inoculated with the cells in a culture box base, wherein a chip culture medium inlet joint is connected with a culture medium inlet joint through a pipeline, and a chip culture medium outlet joint is connected with the waste liquid pool through a pipeline;
6) sealing the cover and the base of the culture box well to ensure the sealing of the inner cavity;
7) connecting a required gas cylinder with a filter membrane with the aperture of 0.22 micron, and then connecting the gas cylinder with a vent pipeline;
8) connecting an oxygen detection device with a filter membrane with the aperture of 0.22 micron and then connecting the oxygen detection device with an exhaust pipeline, wherein optionally, the oxygen concentration detection device can be directly placed in a sealed chamber;
9) opening a ventilation valve and an exhaust valve, opening a gas cylinder, allowing gas to enter from the upper part of the culture box, and discharging the original gas in the culture box cavity from the culture box base;
10) ventilating for 3-10 min, or after the oxygen detection device displays that the target concentration is reached, closing the gas cylinder, and adjusting a ventilation valve and an exhaust valve on the culture box to a closed state;
11) finally, placing the culture box into an incubator, and opening a peristaltic pump for culture;
12) after the culture is finished, the peristaltic pump is closed, and the culture box is taken out of the incubator;
13) opening a ventilation valve and an exhaust valve on the culture box cover;
14) opening the cover of the culture box, and breaking the seal;
15) removing the pipeline connecting the organ chip with the culture medium liquid storage tank and the waste liquid tank, and taking the organ chip out of the culture box base;
16) and cleaning all parts of the culture box, and sterilizing for later use.
According to the technical scheme, the invention has the following beneficial effects: the invention discloses an organ chip culture box capable of adjusting oxygen concentration and a using method thereof, which can simulate the oxygen concentration of different tissues and organs in vivo, such as: hypoxia, anaerobic oxygen and the like, so that tissue cells cultured in the organ chip have a microenvironment which is closer to the in vivo, and the organ chip has a more bionic function. The invention can also be used to examine the effect of different oxygen concentrations on unused tissues and organs.
Drawings
FIG. 1 is a schematic view of an oxygen concentration adjustable organ chip culture cassette according to example 1;
FIG. 2 is a schematic view of an oxygen concentration adjustable organ chip culture cassette according to example 2;
wherein: 10 first culture space, 20 second culture space, 100 culture box cover, 101 vent pipeline, 102 vent valve, 103 culture medium outlet box connector, 104 culture medium inlet box connector, 200 culture box base, 201 vent pipeline, 202 vent valve, 300 organ chip, 301 chip culture medium inlet connector, 302 chip culture medium outlet connector, 400 culture medium liquid storage tank, 410 sealing cover, 411 liquid storage tank culture medium outlet connector, 412 air pressure balancing hole, 500 waste liquid tank, 600 peristaltic pump, 700 sterile water vessel, 800 skirt.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "clockwise", "counterclockwise", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be considered as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, unless otherwise specified, "a plurality" means two or more unless explicitly defined otherwise.
In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature therebetween. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.
Example 1
An oxygen concentration adjustable organ chip incubator as shown in FIG. 1, which is composed of an upper part and a lower part of an incubator lid 100 and an incubator base 200; the culture box cover 100 can cover the culture box base 200 and form a closed space for cell culture; a ventilation pipeline 101 penetrates through the culture box cover 100; an exhaust pipeline 201 is arranged on the culture box base 200 in a penetrating way; the ventilation pipeline 101 is provided with a ventilation valve 102 and an exhaust valve 202; the culture box cover 100 is also provided with a culture medium outlet connector 103 and a culture medium inlet connector 104; an organ chip 300, at least one culture medium liquid storage tank 400 and a waste liquid tank 500 are arranged in the culture box base 200; the culture medium liquid storage tank 400 is connected with the culture medium outlet box connector 103 through a pipeline; the organ chip 300 is provided with a chip culture medium inlet connector 301 and a chip culture medium outlet connector 302 respectively, the chip culture medium inlet connector 301 is connected with the culture medium inlet box connector 104 through a pipeline, and the chip culture medium outlet connector 302 is connected with the waste liquid pool 500 through a pipeline.
The working principle is as follows: connecting a culture medium liquid storage tank 400 with the culture medium outlet box connector 103 through a pipeline, connecting the culture medium outlet box connector 103 with a culture medium conveying mechanism (not shown in the figure) through a pipeline, connecting the pipeline with the culture medium inlet box connector 104 in a backflow mode, placing the organ chip 300 inoculated with cells in the culture box base 200, connecting a chip culture medium inlet connector 301 with the culture medium inlet box connector 104 through a pipeline, connecting a chip culture medium outlet connector 302 with the waste liquid tank 500 through a pipeline, and sealing the culture box cover 100 and the culture box base 200 to ensure the sealing of an internal cavity; connecting a required gas cylinder (not shown in the figure) with a filter membrane with the aperture of 0.22 micron, and then connecting the gas cylinder with the vent pipeline 101; connecting an oxygen detection device with a filter membrane with the aperture of 0.22 micron and then connecting the oxygen detection device with an exhaust pipeline 201, opening a vent valve 102 and an exhaust valve 202, then opening an air bottle to allow air to enter from the upper part of the culture box, and discharging the original air in the cavity of the culture box from the base of the culture box; ventilating for 3-10 minutes, or after the oxygen detection device displays that the target concentration is reached, closing the gas cylinder, and adjusting a ventilation valve 102 and an exhaust valve 202 on the culture box to be in a closed state; finally, the culture cassette is placed in an incubator (not shown in the figure), and a medium conveying mechanism (not shown in the figure) is turned on for culture.
Example 2
An oxygen concentration adjustable organ chip incubator as shown in FIG. 2, which is composed of an upper part and a lower part of an incubator lid 100 and an incubator base 200; the culture box cover 100 can cover the culture box base 200 and form a closed space for cell culture; a ventilation pipeline 101 penetrates through the culture box cover 100; an exhaust pipeline 201 is arranged on the culture box base 200 in a penetrating way; the ventilation pipeline 101 is provided with a ventilation valve 102 and an exhaust valve 202; the culture box cover 100 is also provided with a culture medium outlet connector 103 and a culture medium inlet connector 104; an organ chip 300, at least one culture medium liquid storage tank 400 and a waste liquid tank 500 are arranged in the culture box base 200; the culture medium liquid storage tank 400 is connected with the culture medium outlet box connector 103 through a pipeline; a chip culture medium inlet joint 301 and a chip culture medium outlet joint 302 are respectively arranged on the organ chip 300, the chip culture medium inlet joint 301 is connected with the culture medium box inlet joint 104 through a pipeline, and the chip culture medium outlet joint 302 is connected with the waste liquid pool 500 through a pipeline; in particular, a sealing cover 410 is arranged above the culture medium liquid storage tank 400; the sealed cover is provided with a liquid storage tank culture medium outlet connector 411 and a gas pressure balancing hole 412; the liquid storage tank culture medium outlet connector 411 is connected with the culture medium outlet box connector 103 through a pipeline; the air pressure balance hole 412 is sealed by a 0.22 micron filter membrane; preferably, a peristaltic pump 600 is arranged outside the culture box cover 100, a pipeline is connected to the culture medium outlet box connector 103, and the pipeline is connected to the culture medium inlet box connector 104 through the peristaltic pump 600; further, a sterile water vessel 700 is arranged in the culture box base 200; preferably, the medium outlet box connector 103 or medium inlet box connector 104 is selected from a pagoda-like connector; further, the cross section of the culture box cover 100 is circular, the inner diameter is 10-30 cm, the height is 10-20 cm, and the wall thickness is 0.5-2 cm; the length of the ventilation pipeline 101 is 5-10 cm, the inner diameter of the pipeline is 2-5 mm, and the outer diameter of the pipeline is 5-15 mm; the cross section of the culture box base 200 is circular, the inner diameter is 10-30 cm, the height is 10-20 cm, and the wall thickness is 0.5-2 cm; the length of the exhaust pipeline 201 is 5-10 cm, the inner diameter of the pipeline is 2-5 mm, and the outer diameter of the pipeline is 5-15 mm; particularly, the edge of the opening of the culture box cover 100 and the culture box base 200 is provided with a circle of skirt edge 800, and the upper surface and the lower surface of the skirt edge 800 are hermetically connected by screw holes; preferably, the culture box is made of PMMA.
The working principle is referred to example 1.
Example 3
The effect of hypoxia on neurons was examined by performing a neuron-like chip culture in a hypoxic environment using the oxygen concentration-adjustable organ chip culture cassette described in example 1. The mouse hippocampal neuron cells are inoculated in an axon culture micro-fluidic organ chip capable of perfusing, and are placed in an incubator for static culture first, so that the neuron cells are adhered in the organ chip to grow.
The culture box is used according to the following steps:
1) sterilizing all parts of the culture box by using a wiping method of alcohol, bleaching water and hydrogen peroxide or ultraviolet irradiation sterilization;
2) before use, the ventilation valve 102 and the exhaust valve 202 of the culture box are ensured to be in a closed state;
3) the culture medium liquid storage tank 400 is connected with the culture medium outlet box connector 103 through a pipeline, the culture medium outlet box connector 103 is connected with a culture medium conveying mechanism (not shown in the figure) through a pipeline, and the pipeline is connected with the culture medium inlet box connector 104 in a backflow mode;
4) placing the organ chip 300 inoculated with the neuron cells in a culture box base 200, wherein a chip culture medium inlet joint 301 is connected with a culture medium inlet joint 104 through a pipeline, and a chip culture medium outlet joint 302 is connected with the waste liquid pool 500 through a pipeline;
5) the culture box cover 100 and the culture box base 200 are sealed, so that the sealing of an internal cavity is ensured;
6) connecting a required gas cylinder with a filter membrane with the aperture of 0.22 micron, and then connecting the gas cylinder with a vent pipeline 101;
7) connecting an oxygen detection device with a filter membrane with the aperture of 0.22 micron and then connecting the oxygen detection device with an exhaust pipeline 201;
8) opening the vent valve 102 and the exhaust valve 202, then opening the gas cylinder to allow gas to enter from the upper part of the culture box, and discharging the original gas in the culture box cavity from the culture box base 200;
9) ventilating for 3-10 minutes, or after the oxygen detection device displays that the target concentration is reached, closing the gas cylinder, and adjusting a ventilation valve 102 and an exhaust valve 202 on the culture box to be in a closed state;
10) finally, the culture box is placed in an incubator, and a culture medium conveying mechanism (not shown in the figure) is opened for culture;
11) after the culture is finished, the culture box is taken out from the incubator after a culture medium conveying mechanism (not shown in the figure) is closed;
12) opening the vent valve 102 and the vent valve 202 on the lid 100 of the incubator;
13) opening the lid 100 of the incubator to break the seal;
14) removing the tube connecting the organ chip 300 inoculated with the neuronal cells with the culture medium reservoir 400 and the waste liquid reservoir 500, and then taking the organ chip 300 out of the culture cassette base 200;
15) and cleaning all parts of the culture box, and sterilizing for later use.
Example 4
The culture of the intestinal chip in an anaerobic environment was performed using the oxygen concentration adjustable organ chip culture cassette described in example 2. Caco-2 cells and vascular endothelial cells were seeded in an organ chip 300 of a perfusable culture of the upper and lower layers to construct an intestinal chip.
1) Sterilizing all parts of the culture box by using a wiping method of alcohol, bleaching water and hydrogen peroxide or ultraviolet irradiation sterilization;
2) before use, the ventilation valve 102 and the exhaust valve 202 of the culture box are ensured to be in a closed state;
3) in order to maintain the humidity inside the incubator, a sterile water vessel 700 containing 10 ml of sterile water is placed in the incubator base 200;
4) placing two culture medium liquid storage tanks 400 containing 10 ml of culture medium in a culture box base 200;
5) connecting each culture medium liquid storage tank 400 with the culture medium outlet box connector 103 through a pipeline, connecting the culture medium outlet box connector 103 with the peristaltic pump 600 through a pipeline, and connecting the pipeline with the culture medium inlet box connector 104 in a backflow mode;
6) the cultured intestinal chips 300 are assembled and placed in a culture box base 200, the upper layers and the lower layers of the chips are respectively and independently connected with a culture medium inlet connector 301 through pipelines, the culture medium inlet connectors 301 are respectively connected with a culture medium inlet connector 104 through pipelines (two culture medium inlet connectors 104 are arranged at the positions, and can respectively and independently fill culture mediums for the upper layers and the lower layers of the chips), the upper layers and the lower layers of the chips are respectively and independently connected with chip culture medium outlet connectors 302 through pipelines, and the chip culture medium outlet connectors 302 are connected with a waste liquid pool 500 through pipelines (the waste liquid pools can be shared and can also be respectively and independently arranged);
7) the culture box cover 100 and the culture box base 200 are sealed, so that the sealing of an internal cavity is ensured;
8) connecting a required gas cylinder with a filter membrane with the aperture of 0.22 micron, and then connecting the gas cylinder with a vent pipeline 101;
9) connecting an oxygen detection device with a filter membrane with the aperture of 0.22 micron and then connecting the oxygen detection device with an exhaust pipeline 201;
10) opening the vent valve 102 and the exhaust valve 202, then opening the gas cylinder to allow gas to enter from the upper part of the culture box, and discharging the original gas in the culture box cavity from the culture box base 200;
11) ventilating for 3-10 minutes, or after the oxygen detection device displays that the target concentration is reached, closing the gas cylinder, and adjusting a ventilation valve 102 and an exhaust valve 202 on the culture box to be in a closed state;
12) finally, placing the culture box into an incubator, and opening a peristaltic pump 600 for culture;
13) after the culture is finished, the peristaltic pump 600 is closed, and then the culture box is taken out from the incubator;
14) opening the vent valve 102 and the vent valve 202 on the lid 100 of the incubator;
15) opening the lid 100 of the incubator to break the seal;
16) after the pipeline connecting the intestinal organ chip 300 with the culture medium liquid storage tank 400 and the waste liquid tank 500 is released, the organ chip 300 is taken out from the culture box base 200 for subsequent related experiments;
17) and cleaning all parts of the culture box, and sterilizing for later use.
The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.
Claims (10)
1. An organ chip culture box capable of adjusting oxygen concentration is characterized in that the culture box consists of an upper part and a lower part of a culture box cover (100) and a culture box base (200); the culture box cover (100) can cover the culture box base (200) and form a closed space for cell culture; a ventilation pipeline (101) penetrates through the culture box cover (100); an exhaust pipeline (201) penetrates through the culture box base (200); a ventilation valve (102) and an exhaust valve (202) are arranged on the ventilation pipeline (101); the culture box cover (100) is also provided with at least one culture medium outlet box connector (103) and at least one culture medium inlet box connector (104); an organ chip (300), at least one culture medium liquid storage tank (400) and a waste liquid tank (500) are arranged in the culture box base (200); the culture medium liquid storage tank (400) is connected with the culture medium outlet box connector (103) through a pipeline; the organ chip (300) is provided with a chip culture medium inlet joint (301) and a chip culture medium outlet joint (302) respectively, the chip culture medium inlet joint (301) is connected with the culture medium inlet box joint (104) through a pipeline, and the chip culture medium outlet joint (302) is connected with the waste liquid pool (500) through a pipeline.
2. The oxygen concentration adjustable organ chip culture box according to claim 1, wherein a sealing cover (410) is provided above the culture medium reservoir (400); the sealed cover is provided with a liquid storage tank culture medium outlet connector (411) and an air pressure balancing hole (412); the liquid storage tank culture medium outlet joint (411) is connected with the culture medium outlet box joint (103) through a pipeline; the air pressure balance holes (412) are closed with a 0.22 micron filter.
3. The oxygen concentration adjustable organ chip culture box according to claim 2, characterized in that a peristaltic pump (600) is arranged outside the cover (100) of the culture box, a pipeline is connected to the culture medium outlet box joint (103), and the pipeline is connected to the culture medium inlet box joint (104) through the peristaltic pump (600).
4. The oxygen concentration adjustable organ chip incubator according to claim 3, wherein a sterile water vessel (700) is further provided in the incubator base (200).
5. The oxygen concentration adjustable organ chip culture box according to claim 3, wherein the medium outlet connector (103) or the medium inlet connector (104) is selected from a luer connector or a pagoda-like connector; the number of the culture medium outlet connectors (103) or the culture medium inlet connectors (104) is increased or decreased according to the culture requirement.
6. The oxygen concentration adjustable organ chip culture cassette of claim 1, wherein the organ chip (300) comprises at least a culture medium inlet, a cell culture section, and a waste liquid outlet.
7. The oxygen concentration adjustable organ chip culture box according to claim 1, wherein the cross section of the culture box cover (100) is circular, the inner diameter is 10-30 cm, the height is 10-20 cm, and the wall thickness is 0.5-2 cm; the length of the ventilation pipeline (101) is 5-10 cm, the inner diameter of the pipeline is 2-5 mm, and the outer diameter of the pipeline is 5-15 mm; the cross section of the culture box base (200) is circular, the inner diameter is 10-30 cm, the height is 10-20 cm, and the wall thickness is 0.5-2 cm; the length of the exhaust pipeline (201) is 5-10 cm, the inner diameter of the pipeline is 2-5 mm, and the outer diameter of the pipeline is 5-15 mm.
8. The oxygen concentration adjustable organ chip culture box of claim 1, wherein the opening of the culture box cover (100) and the culture box base (200) is provided with a skirt (800) at the edge, and the sealing connection is performed by screw hole sealing connection, nut stud connection or external clamp matching with sealing gasket at the upper and lower surfaces of the skirt (800).
9. The oxygen concentration adjustable organ chip culture box according to claim 1, wherein the culture box is made of transparent plastic material.
10. The method of using the oxygen concentration adjustable organ chip culture box according to claim 4, comprising the steps of:
1) sterilizing all parts of the culture box by using a wiping method of alcohol, bleaching water and hydrogen peroxide or ultraviolet irradiation sterilization;
2) before use, ensuring that a ventilation valve (102) and an exhaust valve (202) of the culture box are in a closed state;
3) in order to keep the humidity in the culture box, a sterile water vessel (700) containing 10-40 ml of sterile water is placed in a base (200) of the culture box;
4) connecting a culture medium liquid storage tank (400) with the culture medium outlet box connector (103) through a pipeline, connecting the culture medium outlet box connector (103) with the peristaltic pump (600) through a pipeline, and connecting the pipeline with the culture medium inlet box connector (104) in a backflow mode;
5) placing the organ chip (300) inoculated with the cells in a culture box base (200), wherein a chip culture medium inlet joint (301) is connected with a culture medium inlet joint (104) through a pipeline, and a chip culture medium outlet joint (302) is connected with the waste liquid pool (500) through a pipeline;
6) sealing the culture box cover (100) and the culture box base (200) well to ensure the sealing of the internal cavity;
7) connecting a required gas cylinder with a filter membrane with the aperture of 0.22 micron, and then connecting the gas cylinder with a vent pipeline (101);
8) connecting an oxygen detection device with a filter membrane with the aperture of 0.22 micron and then connecting the oxygen detection device with an exhaust pipeline (201), wherein optionally, the oxygen concentration detection device can also be directly placed in a sealed chamber;
9) opening a ventilation valve (102) and an exhaust valve (202), opening a gas cylinder, allowing new gas to enter the culture box, and exhausting gas in the cavity of the original culture box;
10) ventilating for 3-10 minutes, or after the oxygen detection device displays that the target concentration is reached, closing the gas cylinder, and adjusting a ventilation valve (102) and an exhaust valve (202) on the culture box to be in a closed state;
11) finally, the culture box is placed in an incubator, and a peristaltic pump (500) is started to culture;
12) after the culture is finished, the peristaltic pump (600) is closed, and then the culture box is taken out from the incubator;
13) opening a vent valve (102) and an exhaust valve (202) on a lid (100) of the incubator;
14) opening the lid (100) of the culture box, and breaking the seal;
15) removing the pipeline connecting the organ chip (300) with the culture medium liquid storage tank (400) and the waste liquid storage tank (500), and taking the organ chip (300) out of the culture box base (200);
16) and cleaning all parts of the culture box, and sterilizing for later use.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010325673.XA CN111349562A (en) | 2020-04-22 | 2020-04-22 | Organ chip culture box capable of adjusting oxygen concentration and using method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010325673.XA CN111349562A (en) | 2020-04-22 | 2020-04-22 | Organ chip culture box capable of adjusting oxygen concentration and using method thereof |
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| CN111349562A true CN111349562A (en) | 2020-06-30 |
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Cited By (1)
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|---|---|---|---|---|
| CN113930339A (en) * | 2021-10-21 | 2022-01-14 | 中国科学院大连化学物理研究所 | General type organ chip perfusion device |
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Application publication date: 20200630 |