Microgravity culture dish and use method thereof

Abstract

The invention discloses a microgravity culture dish and a using method, and relates to the technical field of biological culture devices.The microgravity culture dish comprises a culture dish body and a culture dish cover, the culture dish body comprises a culture bottom shell and a sealing cover, a breathable separation ring is arranged in the middle of the culture bottom shell, and a culture area is arranged on the inner side of the breathable separation ring; a humidifying area is arranged on the outer side of the breathable separation ring, and a combined filter membrane is arranged on the inner side of the breathable separation ring; a culture medium channel is arranged on the side face of the culture bottom shell, a sealing plug is arranged on the culture medium channel, the culture bottom shell is provided with a gas exchange channel, and a water absorption body is arranged in the humidification area. Meanwhile, the invention discloses a using method based on the microgravity culture dish, a liquid dynamic exchange function and a gas exchange function are achieved at the same time in a suspension culture mode, cell development morphology observation and judgment can be effectively improved, and evaporation of a culture medium is inhibited in a humidification area. A partitioning mechanism is arranged, and culture of different regions is carried out according to the size of an actual sample.

Description

Technical Field

[0001] The present invention relates to the technical field of biological culture devices, and particularly to a microgravity culture dish and a usage method thereof. Background Art

[0002] A microgravity culture dish is a container for culturing biological samples, and is widely used in fields such as bioengineering, pharmaceutical research and development, tissue engineering, and biotechnology.

[0003] In the field of biological sample culture, static culture methods and semi-closed culture methods are usually adopted for culturing biological samples. In the static culture method, when the biological sample cells develop into cell aggregates, due to insufficient nutrient exchange between the cells and the culture solution and the culture environment not meeting the requirements of the original organism conditions, the development of the biological sample cells will be retarded or the cells will die. The static culture method is a semi-closed culture (the sample is placed in a non-hermetic culture dish, and the sample will absorb oxygen and discharge carbon dioxide gas during the development process, and needs to exchange oxygen and carbon dioxide with the environment in real time to maintain the constant concentration of oxygen and carbon dioxide, otherwise it will cause the death or abnormal development of the sample). It will be affected by harmful substances in the culture environment and affect the development of biological samples, especially dust particles and microbial contamination. When using the static culture method, the cells have the property of wall absorption during development (the cells adhere to the wall of the culture dish), which affects the natural morphological changes of the cells and affects the observation and determination of developmental morphology. After retrieval, the patent with the publication number CN220132229U discloses a microgravity cell culture dish, which includes a culture dish main body and a sealing mechanism. The setting of the ventilation hole provides a closed environment suitable for microgravity cell culture for the entire culture dish, and at the same time facilitates the supply of oxygen, nutrients, etc. to the cells during the microgravity cell culture process. The setting of the oscillator provides a closed environment suitable for microgravity cell culture for the entire culture dish, and at the same time can prevent the nutrients remaining at the bottom of the culture dish from affecting the cell growth during the microgravity cell culture process, and stir the liquid regularly to ensure the full distribution of nutrients. The structure of the existing microgravity culture dish is simple and has few functions. Summary of the Invention

[0004] The purpose of the present invention is to provide a microgravity culture dish and a usage method thereof to solve the above technical problems.

[0005] To achieve the above-mentioned purpose, the present invention provides a microgravity culture dish, including a culture dish body and a culture dish cover, the culture dish body including a culture bottom shell and a sealing cover, the sealing cover is installed above the culture bottom shell, a breathable separation ring is arranged in the middle of the culture bottom shell, the inner side of the breathable separation ring is a culture area, the outer side of the breathable separation ring is arranged with a humidification area, and the inner side of the breathable separation ring is arranged with a combined filter membrane; a culture medium channel communicating with the breathable separation ring is arranged on the side of the culture bottom shell, a sealing plug is arranged on the culture medium channel, a gas exchange channel is arranged on the culture bottom shell arranged opposite to the humidification area, a water absorbent body is arranged in the humidification area, and a water inlet is opened on the sealing cover.

[0006] Preferably, the combined filtration membrane comprises a gas exchange membrane and a microbial filtration membrane; Gas exchange membranes are used to isolate liquid from penetrating while exchanging water vapor and gas; The microbial filtration membrane is used for allowing liquid to pass through while blocking the passage of microbial samples. The microbial filtration membrane is arranged opposite to the culture medium channel.

[0007] Preferably, the air permeable separation ring is provided with a number of evenly distributed air permeable openings, and the outer circumference of the air permeable separation ring is provided with supporting convex ridges, so that an air permeable gap can be left between the air permeable opening and the water absorbing body, and the water absorbing body is placed to completely fit the air permeable separation ring, thereby blocking the air permeable opening. If the water absorbing body adheres to the air permeable separation ring after absorbing water, the air permeability of the air permeable opening will be reduced due to the adhesive property of water.

[0008] Preferably, a supplementary channel is provided on the culture dish cover, and a sealing plug is provided on the supplementary channel.

[0009] Preferably, the outer side of the sealing plug is wrapped with silicone material and includes an opening end and an outlet end. The opening end is circular, the outlet end is strip-shaped and an elastic support member is arranged inside. When liquid passes through, the water pressure will open the outlet end for liquid replenishment. When there is no liquid, the outlet end is closed under the action of the elastic support member.

[0010] Preferably, a partition mechanism is provided in the culture area, and the partition mechanism includes a culture plate, which is made of nanoporous alumina ceramic material (a nanoporous structure with a pore size of 10-200nm can be prepared by sol-gel method or anodic oxidation (such as AAO template)). If it is made of 0.2-0.5um (200-500nm) pore size material, it has the function of liquid exchange and sample isolation. The culture plate is provided with a first culture tank distributed in a circumference and provided with digital markings, and a second culture tank is provided in the middle of the culture plate.

[0011] Based on the above method for using a microgravity culture dish, the specific steps are as follows: Step S1: replenishing water in the humidification area; The humidification area is replenished with water through the water addition hole. After a set stationary time, the water absorbent completely adsorbs the replenished liquid water, so that there is no liquid water in the humidification area; Step S2: Add the initial culture medium; Use a syringe to inject the culture medium into the culture area through the replenishment channel. The gas in the culture area is discharged successively through the gas exchange membrane, the air vent, the humidification area, and the gas exchange channel, and the bubble removal operation is carried out; Step S3: Balance the culture medium environment; Place the microgravity culture dish in an incubator with set environmental parameters and balance for a set time, so that the culture medium in the culture area has the same temperature and gas concentration as the culture environment; at the same time, prepare a spare balanced culture medium; Step S4: Place the culture sample; Step S5: After culturing for a set time, replenish the culture medium; Lay the microgravity culture dish flat, and use a syringe to inject the spare balanced culture medium from the sealing plug on the side of the microgravity culture dish at a set flow rate.

[0012] Preferably, in step S2, while the syringe injects the culture medium into the culture area, the microgravity culture dish is shaken. The specific bubble removal operation is as follows: Install the microgravity culture dish on a self-rotating device and maintain a set rotation speed for a set time to vibrate the bubbles in the culture area out.

[0013] Preferably, in step S4, unified culture or zoned culture is carried out according to actual needs; Unified culture: Open the culture dish lid, use a syringe to suck out the culture medium and put the sample into the culture area, and then cover the culture dish lid; Zoned culture: Use a syringe to suck out the culture medium, place the culture medium plate in the culture area, put the sample with a diameter not greater than 60um into the first culture tank, and put the sample with a diameter greater than 60um into the second culture tank. When the microgravity culture dish is at a constant rotation speed, the sample in the first culture tank moves in a reciprocating straight line; the sample in the second culture tank moves in a synchronous rotation along the center of the microgravity culture dish.

[0014] The micron (um)-sized bubbles generated by rotational oscillation will dissolve into the culture medium. They are invisible to the naked eye but can be seen under a microscope, which will affect the sample observation. For example, they may adsorb onto the sample or block the sample field of view during microscope observation. Although the combined filter membrane is hydrophobic, after rotational oscillation and long-term immersion in the culture medium, a layer of culture medium will be adsorbed on the combined filter membrane. At this time, there are basically no bubbles between the adsorbed culture medium and the combined filter membrane. When adding fresh culture medium, the adsorbed culture medium and the newly added culture medium will fuse with each other without hydrophobic properties and there are no bubbles either. Using a hydrophobic combined filter membrane can also avoid liquid blocking the air holes of the exchange membrane and damaging the air permeability.

[0015] Therefore, by adopting the above-mentioned microgravity culture dish and its usage method, the present invention has the following beneficial effects: (1) There is a humidification area which is provided with a water absorbent. Under the action of natural evaporation, a saturated humidity environment is provided during gas exchange, greatly inhibiting the evaporation of the culture medium and preventing the water molecules in the culture medium from volatilizing and changing the osmotic pressure (liquid concentration of the culture medium) between the culture medium and the sample, thus avoiding insufficient nutrient exchange of the sample due to the change of osmotic pressure.

[0016] (2) A zoning mechanism is provided for culturing in different areas according to the size of the actual sample.

[0017] (3) The microgravity culture dish adopts a suspension culture method, which can effectively solve the problem of cell wall-attached growth, enabling the cells to be in a natural development state, and can effectively improve the observation and determination of cell development morphology. The microgravity culture dish can realize the functions of liquid dynamic exchange and gas exchange on the premise of providing a closed culture environment for biological sample culture, and can effectively isolate the influence of harmful substances in the culture environment.

[0018] The technical solution of the present invention will be further described in detail below with reference to the drawings and embodiments. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the overall structure of a microgravity culture dish of the present invention; Figure 2 It is a cross-sectional view of a microgravity culture dish of the present invention; Figure 3 It is a schematic diagram of the structure of the culture bottom shell of the present invention; Figure 4 It is an exploded view of the culture dish cover and the sealing plug of the present invention; Figure 5 It is a cross-sectional view of the culture bottom shell of the present invention; Figure 6 It is a schematic diagram of the structure of the sealing plug of the present invention; Figure 7 It is a schematic diagram of the structure of the culture medium plate of the present invention.

[0020] Reference numerals 1. Petri dish body; 11. Culture bottom shell; 111. Ventilation partition ring; 112. Culture area; 113. Gas exchange channel; 114. Ventilation opening; 115. Humidification area; 116. Culture medium channel; 117. Petri dish buckle; 118. Water absorbent body; 119. Support rib; 12. Sealing cover; 121. Water filling port; 13. Sealing plug; 131. Open end; 132. Outlet end; 14. Composite filter membrane; 141. Gas exchange membrane; 142. Microorganism filter membrane; 2. Petri dish cover; 21. Supplementary channel; 3. Culture medium plate; 31. First culture tank; 32. Second culture tank. Detailed implementation manners

[0021] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship in which the product of the present invention is usually placed during use. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention. In the description of the present invention, it should also be noted that unless otherwise clearly specified and limited, the terms "set", "installed", "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, or an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0022] The following will describe the implementation manners of the present invention in detail with reference to the drawings.

[0023] As Figure 1 - Figure 2 shown, a microgravity Petri dish includes a Petri dish body 1 and a Petri dish cover 2. A supplementary channel 21 is provided on the Petri dish cover 2, and the sealing plug 13 is inserted into the supplementary channel 21 in an interference fit manner to ensure that the culture area 112 is in a closed state. A Petri dish buckle 117 is provided at the bottom of the microgravity Petri dish for installation and auxiliary positioning.

[0024] The Petri dish body 1 includes a culture bottom shell 11 and a sealing cover 12. The sealing cover 12 is installed above the culture bottom shell 11 in an interference fit manner, as Figure 3As shown in the figure, an air-permeable partition ring 111 is provided in the middle of the culture bottom shell 11. A number of uniformly distributed air vents 114 are provided on the air-permeable partition ring 111. A circumferentially distributed support rib 119 is provided on the circumferential side of the air-permeable partition ring 111. The inner side of the air-permeable partition ring 111 is the culture area 112, and a humidification area 115 is provided on the outer side of the air-permeable partition ring 111. A combined filter membrane 14 is provided on the inner side of the air-permeable partition ring 111. The combined filter membrane 14 includes a gas exchange membrane 141 and a microorganism filter membrane 142. The gas exchange membrane 141 is used to isolate liquid penetration while exchanging water vapor and gas, and can also isolate dust and microorganisms. In this embodiment, a breathable and water-impermeable filter membrane with a pore size ≤ 0.2um is used. The microorganism filter membrane 142 is used to allow liquid to pass through while blocking the passage of microorganism samples. The microorganism filter membrane 142 is disposed opposite to the culture medium channel 116. The microorganism filter membrane 142 uses a filter membrane with a pore size of 0.8um - 5um, which can allow liquid to pass through while blocking the passage of microorganism samples. The culture medium passes through the culture medium channel 116 and the microorganism filter membrane 142 and enters the culture area 112, and then flows out of the culture dish through the microorganism filter membrane 142 from the culture medium channel 116.

[0025] A culture medium channel 116 communicating with the air-permeable partition ring 111 is provided on the side of the culture bottom shell 11, as Figure 4 - Figure 5 shown. A sealing plug 13 is provided on the culture medium channel 116. A gas exchange channel 113 is provided on the culture bottom shell 11 opposite to the humidification area 115. An absorbent body 118 (such as a water-absorbing sponge or a high-molecular water-absorbing resin, etc.) is provided in the humidification area 115. A water filling port 121 is provided on the sealing cover 12. Through the natural evaporation effect, water is evaporated into water vapor. The water vapor and the culture environment gas exchange gas with the environment in the culture area 112 through the gas exchange membrane 141, which can provide a saturated humidity culture environment to inhibit the evaporation of the culture solution.

[0026] As Figure 6 shown, the outer side of the sealing plug 13 is wrapped with a silica gel material and includes an open end 131 and an outlet end 132. The open end 131 is circular, and the outlet end 132 is strip-shaped and an elastic support member is provided inside. When liquid passes through, the water pressure flushes open the outlet end 132 for liquid replenishment. When there is no liquid, the outlet end 132 is closed under the action of the elastic support member. It can ensure that gas or liquid can only enter from direction A and exit from direction B.

[0027] The microgravity culture dish has two culture medium channels 116. The sealing plug 13 is assembled in the culture medium channel 116 by interference fit. The input end of the liquid exchange pipe (with an electric telescopic function or manual insertion) can be inserted into the sealing plug 13 on either side by interference fit. The output end of the liquid exchange pipe can be inserted into the sealing plug 13 on the other side by interference fit and passes through the open end 131 to perform liquid exchange on the culture area 112.

[0028] A partition mechanism is provided in the culture area 112, and the partition mechanism includes a culture plate 3, such as Figure 7 As shown, the culture substrate 3 is provided with first culture grooves 31 distributed circumferentially and provided with digital marks, and the corresponding samples can be placed according to the digital marks to facilitate sample coding and record to prevent sample confusion. A second culture groove 32 is provided in the middle of the culture substrate 3.

[0029] Based on the above method for using a microgravity culture dish, the specific steps are as follows: Step S1: replenishing water in the humidifying area 115 .

[0030] The humidification area is replenished with water through the water adding hole. A syringe with a volume of ≤20 ml is inserted into the water adding port 121 to inject 10-15 ml of water. The water is left to stand for 10-12 hours until the water absorbing body 118 is full of water. The water absorbing body 118 completely absorbs the replenished liquid water, so that there is no liquid water in the humidification area 115. This prevents the liquid water from scratching the gas exchange membrane 141 and leaking through the gas exchange channel 113 due to the movement of the microgravity culture dish.

[0031] Step S2: Adding initial culture medium.

[0032] Use a syringe with a volume of ≤10ml to inject 10.0-10.5ml of culture medium into the culture area 112 through the supplementary channel 21. While the syringe injects the culture medium into the culture area 112, shake the microgravity culture dish. The gas in the culture area 112 is discharged through the gas exchange membrane 141, the air vent 114, the humidification area 115 and the gas exchange channel 113 in sequence, and perform a bubble removal operation: install the microgravity culture dish on the autorotation device, and maintain the autorotation balance at a speed of 8-16RPM / min for 3.5-6 hours to vibrate out the bubbles in the culture area 112. Prevent bubbles from interfering with and impacting the sample during the culture process, and prevent bubbles from wearing the combined filter membrane 14 during the culture process. The combined filter membrane 14 is damaged, causing the culture medium in the culture area 112 to penetrate into the humidification area 115 through the air vent 114.

[0033] Step S3: Equilibrating the culture medium environment.

[0034] Place the microgravity culture dish in an incubator with set environmental parameters and balance for the set time. For example, the operating temperature parameter of the mouse embryo incubator is set to 37.0°C, and the operating carbon dioxide concentration and oxygen concentration parameters are set to 5.0%. Place the prepared microgravity culture dish in the incubator to balance the liquid for 10 to 16 hours. Make the culture medium in the culture area 112 maintain the same temperature and gas concentration as the culture environment; and prepare a standby balanced culture medium at the same time.

[0035] Step S4: placing the culture sample.

[0036] Cultivate uniformly or in zones according to actual needs; Uniform cultivation: Open the culture dish lid 2, use a 10 ml syringe to suck out the culture medium and place the sample in the culture area 112, then cover the culture dish lid 2; Zonal cultivation: Use a syringe to suck out the culture medium, place the culture medium plate 3 in the culture area 112, place the sample with a diameter not greater than 60 μm in the first culture tank 31, and place the sample with a diameter greater than 60 μm in the second culture tank 32. Under a constant rotation speed (low-speed motion state of 5 - 30 RPM / min and constant rotation speed state) of the microgravity culture dish, the sample in the first culture tank 31 makes a reciprocating linear motion. The sample in the second culture tank 32 makes a synchronous rotational motion around the center of the microgravity culture dish (makes a regular elliptical trajectory motion with the center of the culture dish as the reference, and the rotation speed is the same as that of the culture dish).

[0037] Place the microgravity culture dish into a culture instrument (a device that can maintain the culture environment of operating temperature, operating oxygen concentration, and operating carbon dioxide concentration, and can maintain a self-rotation of 0 - 30 RPM / min with the center point of the culture dish as the center) for cultivation.

[0038] Step S5: After cultivating for a set time, supplement the culture medium.

[0039] During automatic liquid change, lay the microgravity culture dish flat. The input end with an electric telescopic function can be inserted into any one of the sealing plugs 13 through interference fit. The output end of the liquid exchange pipeline can be inserted into the other sealing plug 13 through interference fit and pass through the opening end 131 to perform liquid exchange on the culture area 112. Use a 10 ml volumetric syringe to inject the standby balanced culture medium from the sealing plug 13 on the side of the microgravity culture dish at a flow rate of 60 ml / min. The culture medium is injected into the culture area 112 through the sealing plug 13 at a flow rate of ≤60 ml / min, which can solve the risk that the sample adheres to the combined filter membrane 14 and cannot naturally detach from the combined filter membrane 14.

[0040] During manual liquid change, place the microgravity culture dish vertically, insert a syringe into any one of the sealing plugs 13, first suck out most of the culture solution, and then inject some new culture solution. During the injection process, pay attention to pressing the sealing plug 13 tightly with the syringe to perform the operation of injecting the culture medium, preventing the culture medium from leaking from the contact port between the syringe and the sealing plug 13. After placing the sample in the culture area 112, avoid injecting the culture medium from the supplementary channel 21, as the liquid impact force generated by the drop of the culture medium falling from the supplementary channel 21 to the bottom of the dish may disperse the sample and cause the sample to be dispersed and adhered to the combined filter membrane 14.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A microgravity culture dish, comprising a culture dish body and a culture dish cover, characterized in that: The culture dish body comprises a culture bottom shell and a sealing cover, which is installed above the culture bottom shell. A breathable separation ring is arranged in the middle of the culture bottom shell, the inner side of the breathable separation ring is a culture area, the outer side of the breathable separation ring is arranged with a humidification area, and the inner side of the breathable separation ring is arranged with a combined filter membrane; a culture medium channel communicating with the breathable separation ring is arranged on the side of the culture bottom shell, a sealing plug is arranged on the culture medium channel, a gas exchange channel is arranged on the culture bottom shell arranged opposite to the humidification area, a water absorbent body is arranged in the humidification area, and a water inlet is opened on the sealing cover.

2. A microgravity culture dish according to claim 1, characterized in that: The combined filtration membrane includes a gas exchange membrane and a microbial filtration membrane; Gas exchange membranes are used to isolate liquid from penetrating while exchanging water vapor and gas; The microbial filtration membrane is used for allowing liquid to pass through while blocking the passage of microbial samples. The microbial filtration membrane is arranged opposite to the culture medium channel.

3. The microgravity culture dish according to claim 1, characterized in that: The air-permeable separation ring is provided with a plurality of air-permeable openings which are evenly distributed, and the outer side of the air-permeable separation ring is provided with supporting convex ridges which are circumferentially distributed.

4. The microgravity culture dish according to claim 1, characterized in that: The culture dish cover is provided with a supplementary channel, and a sealing plug is provided on the supplementary channel.

5. The microgravity culture dish according to claim 1, characterized in that: The outside of the sealing plug is wrapped with silicone material and includes an opening end and an outlet end. The opening end is circular, the outlet end is strip-shaped and an elastic support member is arranged inside. When liquid passes through, the water pressure will open the outlet end for liquid replenishment. When there is no liquid, the outlet end is closed under the action of the elastic support member.

6. The microgravity culture dish according to claim 1, characterized in that: A detachable partition mechanism is arranged in the culture area, and the partition mechanism comprises a culture plate made of nanoporous alumina ceramic material. The culture plate is provided with first culture grooves distributed in a circumference and provided with digital marks, and a second culture groove is arranged in the middle of the culture plate.

7. A method for using a microgravity culture dish according to any one of claims 1 to 6, characterized in that: The specific steps are as follows: Step S1: replenishing water in the humidification area; The humidification area is replenished with water through the water adding hole. After a set period of static time, the water absorbing body completely absorbs the replenished liquid water, so that there is no liquid water in the humidification area. Step S2: adding initial culture medium; The culture medium is injected into the culture area through the supplementary channel using a syringe, and the gas in the culture area is discharged through the gas exchange membrane, the air permeable port, the humidification area and the gas exchange channel in sequence, and a bubble removal operation is performed; Step S3: balancing the culture medium environment; The microgravity culture dish is placed in an incubator with set environmental parameters, and the set time is balanced so that the culture medium in the culture area maintains the same temperature and gas concentration as the culture environment; meanwhile, a standby balanced culture medium is prepared; Step S4: placing the culture sample; Step S5: After the set culture time, add supplementary culture medium; The microgravity culture dish was placed flat, and the standby equilibrated culture medium was injected through the sealing plug on the side of the microgravity culture dish using a syringe at a set flow rate.

8. The method for using a microgravity culture dish according to claim 7, characterized in that: In step S2, while the syringe injects the culture medium into the culture area, the microgravity culture dish is shaken to remove bubbles. The specific operation is as follows: The microgravity culture dish is mounted on a rotating device and rotated at a set speed for a set time to vibrate out the bubbles in the culture area.

9. The method for using a microgravity culture dish according to claim 7, characterized in that: In step S4, uniform cultivation or partitioned cultivation is performed according to actual needs; Unified culture: open the lid of the culture dish, use a syringe to aspirate the culture medium and place the sample in the culture area, then cover the lid of the culture dish; Partition culture: Use a syringe to suck out the culture medium, place the culture plate in the culture area, place samples with a diameter of no more than 60um in the first culture tank, and samples with a diameter of more than 60um in the second culture tank. The microgravity culture dish is kept at a constant speed, and the samples in the first culture tank perform reciprocating linear motion; The samples in the second culture tank perform synchronous rotational motion along the center of the microgravity culture dish.

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