Special culture device for amniotic fluid or chorion cell in-situ culture and application

Abstract

The invention relates to the technical field of cell culture, and discloses a culture device special for amniotic fluid or chorion cell in-situ culture, which comprises a constant-temperature box, a sleeve driven by a driving assembly to rotate is arranged in the constant-temperature box, and a culture bottle is detachably assembled in the sleeve. One end of the culture bottle is positioned in the sleeve, uniformly distributed micropores are formed in the outer wall of the culture bottle, the micropores are opposite to the inner wall of the sleeve to form a waste liquid cavity, a liquid discharge pipe for discharging waste liquid in the waste liquid cavity outwards is mounted at the bottom of the sleeve, a liquid discharge one-way valve is mounted at the outlet end of the liquid discharge pipe, and the inlet end of the liquid discharge one-way valve is communicated with the liquid discharge pipe. The other end of the culture bottle is connected with the liquid storage bag through the liquid inlet one-way valve and the liquid inlet pipe, replacement of culture liquid is achieved under the condition that the culture bottle is not opened, the opportunity that an external pollution source enters the culture bottle is reduced through the design, stability of the culture environment is ensured, the risk that cells are polluted is reduced, and the culture success rate is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, in particular to a special culture device for in situ culture of amniotic fluid or chorionic cells and its application. Background Art

[0002] Currently, when amniotic fluid / chorionic villus cells are cultured in situ, waste products generated by cell metabolism gradually accumulate and affect the quality of the culture medium, requiring frequent replacement of the culture medium. However, if the culture container is opened frequently, the risk of contamination from external sources of contamination increases. For example, contamination by microorganisms such as bacteria and fungi may lead to cell death or culture failure. Therefore, it is necessary to design a culture device that can automatically replace the culture medium. Summary of the Invention

[0003] In order to solve the technical problems existing in the background technology, the present invention proposes a special culture device for in situ culture of amniotic fluid or chorionic villus cells and its application.

[0004] The present invention provides a dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells, comprising a constant temperature chamber, a sleeve driven to rotate by a drive assembly installed in the constant temperature chamber, a culture bottle removably installed in the sleeve, one end of the culture bottle located inside the sleeve, and the outer wall of the sleeve having evenly distributed micropores, the micropores facing the inner wall of the sleeve to form a waste liquid chamber, a drain pipe for discharging waste liquid from the waste liquid chamber installed at the bottom of the sleeve, a drain check valve installed at the outlet end of the drain pipe, and an inlet end of the drain check valve connected to the drain pipe;

[0005] A liquid storage bag is installed above the sleeve inside the constant temperature box. The other end of the culture bottle extends to the top of the sleeve and is equipped with a liquid inlet check valve. The liquid inlet check valve is connected to the liquid outlet at the bottom of the liquid storage bag through a liquid inlet pipe.

[0006] Frequent replacement of culture medium in traditional in situ culture of amniotic fluid / chorionic villus cells can easily cause contamination. This device creates a relatively closed environment through the sleeve and culture bottle, effectively reducing the risk of contamination. The micropores on the outer wall of the culture bottle and the inner wall of the sleeve form a waste liquid cavity. The waste liquid generated by cell metabolism can be discharged through the drainage tube and the drainage check valve, avoiding the accumulation of waste liquid and affecting cell growth. The liquid storage bag is connected to the culture bottle through the liquid inlet tube and the liquid inlet check valve. The culture medium can be replaced without opening the culture bottle, maintaining a stable culture environment and improving the culture success rate.

[0007] As a further optimized solution of the present invention, a bottle cap is threadedly connected to the upper end of the bottle mouth of the culture bottle and is sealed. The liquid inlet one-way valve is fixedly mounted on the bottle cap and its outlet end is connected to the inner cavity of the culture bottle. The inlet end of the liquid inlet one-way valve is located outside the culture bottle and is connected to the liquid inlet tube. The axes of the liquid inlet one-way valve, the liquid inlet tube and the culture bottle coincide.

[0008] The culture flask is threaded and sealed with a bottle cap to ensure the sealing of the internal environment of the culture flask and prevent external microorganisms from entering and contaminating the cells. The liquid inlet one-way valve is installed on the bottle cap, and it coincides with the axis of the liquid inlet tube and the culture flask. This design ensures that the culture fluid can flow smoothly from the liquid inlet tube into the culture bottle, avoiding poor liquid flow caused by tilted or misaligned pipelines, and ensuring the efficiency and stability of the culture fluid replacement process.

[0009] As a further optimized solution of the present invention, one end of the liquid inlet tube is upward and is threadedly connected to the liquid outlet of the liquid storage bag through a connector, and the other end of the liquid inlet tube is downward and is rotatably and sealedly connected to the inlet end of the liquid inlet one-way valve through a rotary joint;

[0010] The upper end of the rotary joint is fixed to the lower end of the liquid inlet pipe, and the lower end of the rotary joint is rotatably connected to the inlet end of the liquid inlet one-way valve. There is also an inflation joint on the side of the rotary joint, which is connected to the gas generator through a hose. During the culture medium replacement process, the gas generator will input a mixed gas containing 5% CO2 (such as 5% CO2+20% O2+75% N2) into the inlet end of the liquid inlet one-way valve to simulate the physiological environment in the body. The input ratio is adjusted by the solenoid valve to maintain gas stability. The gas must be filtered through a 0.22μm filter membrane before input to prevent microbial contamination, and the gas input must be mixed with the culture medium through a microporous diffuser so that the gas forms tiny bubbles in the culture medium to reduce interference with cell attachment or suspension state;

[0011] The liquid inlet tube is threadedly connected to the liquid storage bag through a connector, which is convenient for installation and disassembly, and easy to replace the liquid storage bag. The rotary joint realizes the rotational sealing connection between the liquid inlet tube and the liquid inlet check valve. When the culture bottle rotates with the sleeve, the connection between the liquid inlet tube and the culture bottle is guaranteed to be sealed to prevent liquid leakage;

[0012] The inflation connector is connected to the gas generator, and a mixed gas that simulates the physiological environment in the body is input into the culture medium. The gas ratio is adjusted by the solenoid valve to maintain a stable gas environment, which is conducive to cell growth. The gas is filtered through a 0.22μm filter membrane to effectively prevent microbial contamination of the culture medium. The microporous diffuser allows the gas to form tiny bubbles that mix with the culture medium, reducing interference with the cell state and providing cells with a growth environment closer to that in the body.

[0013] As a further optimized solution of the present invention, symmetrically distributed limit blocks are installed at the bottom of the inner cavity of the sleeve, and positioning rods corresponding to and adapted to the limit blocks are installed at the bottom of the culture bottle, and the positioning rods and the limit blocks are plugged and assembled; the upper end of the sleeve has an opening adapted to the culture bottle, and a sealing sleeve is installed at the opening, which is sleeved on the outer wall of the culture bottle and is used to seal the joint;

[0014] The limit block at the bottom of the sleeve is plugged into the positioning rod at the bottom of the culture bottle, which can accurately position the culture bottle, ensure the stable position of the culture bottle in the sleeve, avoid shaking or deviation during rotation, and ensure the normal operation of the waste liquid cavity between the micropores of the culture bottle and the inner wall of the sleeve. The sealing sleeve is installed at the opening of the sleeve and is sleeved on the outer wall of the culture bottle, further enhancing the sealing of the culture device, preventing outside air or impurities from entering the waste liquid cavity and affecting the cell culture environment.

[0015] As a further optimized solution of the present invention, an annular cover plate is installed on the outer periphery of the upper end of the culture bottle, and the annular cover plate is pressed onto the upper end surface of the sealing sleeve;

[0016] The annular cover is pressed onto the sealing sleeve, which plays a double sealing role and further enhances the sealing performance of the connection between the culture flask and the sleeve. It not only prevents the entry of external pollutants, but also plays a certain protective role for the sealing sleeve, avoiding displacement or damage of the sealing sleeve due to external force or long-term use, ensuring the long-term stability of the sealing performance of the culture device and providing a reliable environmental protection for cell culture.

[0017] As a further optimized solution of the present invention, both sides of the bottom of the inner cavity of the sleeve have inclined surfaces, the two inclined surfaces are symmetrically distributed, and the lower ends of the two inclined surfaces are close to the axis of the sleeve and connected to the opening of the drain pipe;

[0018] The inclined surface design at the bottom of the sleeve cavity facilitates the discharge of waste liquid. When the waste liquid in the culture bottle flows into the waste liquid cavity through the micropores, under the action of gravity, the waste liquid will flow along the inclined surface to the opening of the drain pipe, so that the waste liquid can enter the drain pipe more smoothly, avoiding the accumulation of waste liquid at the bottom of the sleeve, ensuring the efficiency of waste liquid discharge, and maintaining the cleanliness of the culture environment.

[0019] As a further optimized solution of the present invention, the pore size of the micropores is 20-30 μm, which can balance the flow rate and negative pressure stability, and achieve the synergistic effect of efficient liquid replacement during centrifugation and negative pressure-triggered fluid replacement;

[0020] The micropores are carefully designed to have a pore size of 20-30μm. This pore size range ensures that the culture medium can flow out of the culture bottle through the micropores at an appropriate flow rate during centrifugation, and also ensures that a stable negative pressure is formed in the culture bottle. When the culture medium flows out, negative pressure is generated in the bottle, triggering the liquid inlet one-way valve to open, allowing the culture medium in the liquid storage bag to flow in, achieving efficient liquid replacement and fluid replenishment synergy, accurately controlling the culture medium replacement process, and providing a stable growth environment for cells.

[0021] As a further optimized solution of the present invention, a drive box is installed at the bottom of the constant temperature box, and the drive assembly includes a driving shaft, a driven shaft, a driving gear, and a driven gear installed in the drive box. The driving shaft and the driven shaft are both rotatably installed in the drive box, the lower end of the driving shaft is driven by a motor, the upper end of the driving shaft is installed with the driving gear, the upper end of the driven shaft is fixed to the bottom of the sleeve, and the lower end of the driven shaft is installed with the driven gear, and the driven gear and the driving gear are meshed with each other;

[0022] The drive assembly in the drive box provides power for the rotation of the culture bottle. The motor drives the active shaft to rotate, and the active gear on the active shaft drives the driven gear on the driven shaft to rotate, thereby causing the driven shaft to drive the sleeve and culture bottle to rotate. This gear transmission structure is stable and reliable, and can accurately control the rotation speed of the culture bottle, realizing centrifugal operation, so that the culture medium can be replaced by centrifugal force, meeting the demand for culture medium replacement during cell culture.

[0023] Furthermore, there are multiple driven shafts which are annularly symmetrically distributed around the periphery of the driving shaft, and the driving gears are respectively engaged with the multiple driven gears, thereby realizing the rotation and centrifugation of the multiple culture bottles driven by the motor.

[0024] As a further optimization of the present invention, the driven shaft is tubular in structure, one end of the drain pipe is mounted on the bottom of the sleeve, and the other end of the drain pipe passes through the inner cavity of the driven shaft and extends to the bottom of the drive box. The drain pipe and the driven shaft are coaxially aligned. A base is also mounted at the lower end of the drive box, and a removable waste liquid box is disposed within the base. The waste liquid box is a box body with an open top and is located below the liquid outlet of the drain pipe.

[0025] The driven shaft is designed as a tubular structure to facilitate the passage of the drain pipe, so that the drain pipe and the driven shaft are coaxially aligned, ensuring smooth discharge of waste liquid and preventing the drain pipe from twisting or clogging when the driven shaft rotates. The waste liquid box in the base is used to collect waste liquid discharged from the drain pipe. The outward-removable design facilitates the cleaning of waste liquid, keeps the device clean and hygienic, and avoids the accumulation of waste liquid affecting the experimental environment.

[0026] An application of a dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells, comprising the following specific steps:

[0027] S1: Place amniotic fluid or chorionic villus cells into a culture bottle, then add an appropriate amount of culture medium and cover the bottle cap to form a sealed environment. After the culture bottle is assembled into the sleeve and secured, the door can be closed for constant temperature culture.

[0028] This step is the preparation stage for cell culture. After placing cells and culture medium into the culture bottle and sealing it, it can prevent external microbial contamination. The culture bottle is firmly installed in the sleeve to ensure its stability during the culture process. The constant temperature box door is closed for constant temperature culture to provide a suitable temperature environment for cell growth and meet the basic conditions for cell growth.

[0029] S2: After a certain period of culture, when the culture medium in the culture bottle needs to be replaced, the drive assembly drives the sleeve to drive the culture bottle to rotate centrifugally at high speed, so that the culture medium in the culture bottle moves toward the peripheral micropores under the influence of centrifugal force, and the culture medium in the culture bottle gradually flows out of the micropores. Since the culture bottle is a closed cavity as a whole, negative pressure is formed inside and triggers the liquid inlet one-way valve to open, so that the culture medium in the liquid storage bag flows into the interior of the culture bottle under the action of the pressure difference, thereby achieving dynamic balanced supply of culture medium;

[0030] As cell culture progresses, the culture medium will deteriorate due to cell metabolism and need to be replaced. The drive component drives the culture bottle to rotate centrifugally at high speed, using centrifugal force to make the culture medium flow out from the micropores. The negative pressure formed in the culture bottle automatically triggers the liquid inlet one-way valve to open, allowing fresh culture medium in the liquid storage bag to flow in, realizing automatic replacement of the culture medium, maintaining the quality of the culture medium and the stability of the cell growth environment, reducing the risk of contamination caused by manual operation, and ensuring that the cells are always in a good nutritional environment.

[0031] S3: When the culture fluid in the liquid storage bag is transferred to the inlet end of the liquid inlet check valve through the liquid inlet tube, the gas generator injects the culture gas into the liquid inlet check valve through the inflation connector, so that the culture gas and the culture fluid are mixed and injected into the culture bottle simultaneously;

[0032] While replacing the culture medium, a mixed gas that simulates the physiological environment in the body is injected into the culture bottle. The gas and culture medium mix and then enter the culture bottle together, providing cells with a gas environment closer to that in the body, promoting cell metabolism and growth. In this way, the gas environment in the body is simulated, the quality of cell culture is improved, and it is more conducive to cell proliferation and experimental research.

[0033] S4: During the entire culture period, repeat steps S2 and S3 several times to complete the in situ culture of amniotic fluid or chorionic cells. After the culture is completed, remove the culture bottle;

[0034] Cell culture is an ongoing process that requires regular replacement of culture medium and replenishment of gas. Repeating steps S2 and S3 can continuously provide cells with fresh culture medium and a suitable gas environment to meet the needs of cell growth. After the culture is completed, the culture flask is removed to facilitate subsequent observation, analysis and research of the cells.

[0035] The dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells and its application proposed in the present invention have the following beneficial effects:

[0036] (1) In the culture device of the present invention, the sleeve and culture bottle in the constant temperature box form a relatively closed culture environment. One end of the culture bottle is located in the sleeve, and a waste liquid cavity is formed by the micropores and the inner wall of the sleeve. The waste liquid is discharged through the drain pipe and the drain check valve, thereby preventing the accumulation of waste liquid in the culture bottle. At the same time, the other end of the culture bottle is connected to the liquid storage bag through the liquid inlet check valve and the liquid inlet pipe, so that the culture liquid can be replaced without opening the culture bottle. This design reduces the chance of external contamination sources entering the culture bottle, ensures the stability of the culture environment, reduces the risk of cell contamination, and improves the success rate of culture.

[0037] (2) The drive assembly drives the sleeve and culture bottle to rotate centrifugally at high speed. The culture fluid in the culture bottle flows out from the micropores under the action of centrifugal force. The negative pressure formed in the bottle triggers the opening of the liquid inlet one-way valve, and the culture fluid in the liquid storage bag flows into the culture bottle, realizing dynamic balanced supply. The whole process does not require frequent manual operation, which not only reduces labor costs, but also can replace the culture fluid in time, ensuring that the cells are always in a good growth environment, improving the culture efficiency, and providing convenience for the long-term culture of amniotic fluid or chorionic cells.

[0038] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a front cross-sectional structural diagram of the present invention;

[0040] Figure 2 Schematic diagram of the front cross-sectional structure of the sleeve and culture bottle of the present invention;

[0041] Figure 3 Schematic diagram of the top view of the inner cavity of the sleeve of the present invention;

[0042] Figure 4 It is a schematic diagram of the top view of the inner cavity of the drive box of the present invention.

[0043] Description of the accompanying drawings: 1. Constant temperature box; 2. Sleeve; 3. Culture bottle; 4. Micropore; 5. Drain pipe; 6. Drain check valve; 7. Liquid storage bag; 8. Liquid inlet check valve; 9. Liquid inlet pipe; 10. Bottle cap; 11. Rotary joint; 12. Limit block; 13. Positioning rod; 14. Inclined surface; 15. Sealing sleeve; 16. Annular cover; 17. Connector; 18. Driving shaft; 19. Driven shaft; 20. Driving gear; 21. Driven gear; 22. Drive box; 23. Base; 24. Waste liquid box; 25. Inflatable joint. DETAILED DESCRIPTION

[0044] The following describes embodiments of the present invention in detail. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar symbols throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present invention, and are not to be construed as limiting the present invention.

[0045] In the present invention, unless otherwise expressly specified or limited, when a first feature is "above" or "below" a second feature, it may mean that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediary. Furthermore, when a first feature is "above," "above," or "above" a second feature, it may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. When a first feature is "below," "below," or "below" a second feature, it may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.

[0046] In the field of cell culture technology, in situ culture of amniotic fluid or chorionic villus cells faces the problem of easy contamination when the culture medium is replaced. The culture device and application of the present invention effectively solve this problem through innovative design. The specific implementation method is as follows:

[0047] like Figure 1 and Figure 4 As shown, the core part of the culture device is located inside the constant temperature box 1. A driving assembly is installed in the constant temperature box 1, and its function is to drive the sleeve 2 to rotate. The driving assembly includes a driving shaft 18, a driven shaft 19, a driving gear 20 and a driven gear 21 installed in the driving box 22. The driving shaft 18 and the driven shaft 19 are both rotatably installed in the driving box 22. The lower end of the driving shaft 18 is driven by the motor, and the driving gear 20 is installed at the upper end. The upper end of the driven shaft 19 is fixed to the bottom of the sleeve 2, and the driven gear 21 is installed at the lower end. The driven gear 21 and the driving gear 20 are meshed with each other. This gear transmission structure ensures that the sleeve 2 can rotate stably, and the rotation speed can be accurately controlled by the motor.

[0048] like Figure 1 and Figure 2 As shown, the culture bottle 3 is detachably mounted inside the sleeve 2. A positioning rod 13 is installed at the bottom of the culture bottle 3, which is plugged into and assembled with the limit blocks 12 symmetrically distributed at the bottom of the inner cavity of the sleeve 2. This design can accurately position the culture bottle 3 and prevent it from shaking or deflecting during the rotation of the sleeve 2.

[0049] The upper opening of the sleeve 2 is adapted to the culture bottle 3, and a sealing sleeve 15 is installed at the opening. The sealing sleeve 15 is sleeved on the outer wall of the culture bottle 3 to seal the connection, further enhancing the sealing of the culture environment;

[0050] An annular cover plate 16 is also installed on the outer periphery of the upper end of the culture bottle 3, which is pressed on the upper end surface of the sealing sleeve 15 to play a double sealing role, protecting the sealing sleeve 15 and further preventing external contaminants from entering;

[0051] One end of the culture bottle 3 is located inside the sleeve 2. The outer wall of this end is provided with evenly distributed micropores 4. A waste liquid chamber is formed between the micropores 4 and the inner wall of the sleeve 2. Both sides of the bottom of the inner chamber of the sleeve 2 have symmetrically distributed inclined surfaces 14. The lower ends of the two inclined surfaces 14 are close to the axis of the sleeve 2 and connected to the opening of the drainage pipe 5. When the waste liquid in the culture bottle 3 flows into the waste liquid chamber through the micropores 4, it will flow along the inclined surface 14 to the drainage pipe 5 under the action of gravity. A drainage check valve 6 is installed at the outlet end of the drainage pipe 5, which can effectively prevent the waste liquid from flowing back and ensure that the waste liquid is discharged and collected smoothly.

[0052] like Figure 1 As shown, one end of the drain pipe 5 is installed at the bottom of the sleeve 2, and the other end passes through the inner cavity of the driven shaft 19 and extends to the bottom of the drive box 22. A base 23 is installed at the lower end of the drive box 22. A waste liquid box 24 that can be removed outward is provided inside the base 23. The waste liquid box 24 is a box body with an open upper end, which is located below the liquid outlet of the drain pipe 5 and is used to collect waste liquid, facilitate cleaning, and keep the device clean and hygienic.

[0053] like Figure 1 As shown, a liquid storage bag 7 located above the sleeve 2 is also installed in the constant temperature box 1, and the other end of the culture bottle 3 extends to the top of the sleeve 2. The bottle mouth is threadedly connected to a bottle cap 10 and sealed. The liquid inlet one-way valve 8 is fixedly installed on the bottle cap 10, and its outlet end is connected to the inner cavity of the culture bottle 3. The inlet end is located outside the culture bottle 3 and is connected to the liquid inlet pipe 9. One end of the liquid inlet pipe 9 faces upward and is threadedly connected to the liquid outlet at the bottom of the liquid storage bag 7 through a connector 17, which facilitates the installation and replacement of the liquid storage bag 7. The other end of the liquid inlet pipe 9 faces downward and is rotatably and sealedly connected to the inlet end of the liquid inlet one-way valve 8 through a rotary joint 11.

[0054] An inflation joint 25 is also provided on the side of the rotary joint 11, and the inflation joint 25 is connected to the gas generator through a hose. During the culture process, the gas generator injects a mixed gas that simulates the physiological environment in the body, such as 5% CO2+20% O2+75% N2, into the liquid inlet one-way valve 8 through the inflation joint 25. The gas must be filtered through a 0.22μm filter membrane before input to prevent microbial contamination, and is mixed with the culture solution through a microporous diffuser to form tiny bubbles before entering the culture bottle 3, providing a more suitable growth environment for the cells.

[0055] The working principle and operation process of the culture device are as follows:

[0056] 1. Preparation stage: Before cell culture, first put the amniotic fluid or chorionic villus cells into the culture bottle 3, add appropriate amount of culture medium and cover the bottle with the cap 10 to form a sealed environment. This step effectively prevents external microorganisms from contaminating the cells. Then, the culture bottle 3 is passed through the following steps: Figure 3 The positioning rod 13 is plugged into and assembled with the stopper 12 at the bottom of the sleeve 2 and securely installed in the sleeve 2. The door of the incubator 1 is then closed to begin constant temperature cultivation, providing suitable temperature conditions for cell growth.

[0057] 2. Culture medium replacement and gas replenishment stage: As cell culture proceeds, the culture medium will need to be replaced due to waste generated by cell metabolism. At this time, the drive assembly is started, the motor drives the driving shaft 18 to rotate, and the driving gear 20 rotates accordingly, which in turn drives the driven gear 21 and the driven shaft 19 to rotate, so that the sleeve 2 drives the culture bottle 3 to rotate centrifugally at high speed. Under the action of centrifugal force, the culture medium in the culture bottle 3 moves toward the peripheral micropores 4 and gradually flows out of the micropores 4 into the waste liquid chamber. Since the culture bottle 3 is a closed cavity, a negative pressure will be formed inside. When the negative pressure reaches a certain level, the liquid inlet one-way valve 8 is triggered to open. At this time, the culture medium in the liquid storage bag 7 flows into the culture bottle 3 through the liquid inlet pipe 9 and the liquid inlet one-way valve 8 under the action of the pressure difference, thereby realizing the dynamic balance replenishment of the culture medium;

[0058] While the culture fluid in the liquid storage bag 7 is being transferred to the culture bottle 3, the gas generator injects the culture gas into the liquid inlet check valve 8 via the inflation connector 25. Before entering the liquid inlet check valve 8, the gas is first filtered through a 0.22 μm filter membrane, then mixed with the culture fluid through a microporous diffuser to form tiny bubbles, which then enter the culture bottle 3 together. In this way, during the process of replacing the culture fluid, the cells are also provided with gas that simulates the physiological environment in the body, promoting cell metabolism and growth.

[0059] 3. Continuous culture and termination stage: During the entire culture period, the above-mentioned culture medium replacement and gas replenishment steps are repeated several times according to the needs of cell growth. By regularly replacing the culture medium and replenishing the gas, a stable growth environment is provided for the cells to meet the needs of cell growth. When the culture is completed, the operation of the drive component is stopped, the constant temperature box 1 is opened, and the culture bottle 3 is taken out for subsequent observation, analysis and research of the cells.

[0060] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells, comprising a constant temperature box (1), characterized in that: A sleeve (2) driven to rotate by a driving assembly is installed in the constant temperature box (1), and a culture bottle (3) is detachably installed in the sleeve (2). One end of the culture bottle (3) is located inside the sleeve (2) and the outer wall is provided with evenly distributed micropores (4). The micropores (4) are opposite to the inner wall of the sleeve (2) to form a waste liquid cavity. A drain pipe (5) for discharging waste liquid in the waste liquid cavity is installed at the bottom of the sleeve (2). A drain check valve (6) is installed at the outlet end of the drain pipe (5), and the inlet end of the drain check valve (6) is connected to the drain pipe (5). A liquid storage bag (7) located above the sleeve (2) is installed inside the constant temperature box (1); the other end of the culture bottle (3) extends to the top of the sleeve (2) and is installed with a liquid inlet check valve (8); and the liquid inlet check valve (8) is connected to the liquid outlet at the bottom of the liquid storage bag (7) through a liquid inlet pipe (9).

2. A dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells according to claim 1, characterized in that: The upper end of the culture bottle (3) is threadedly connected to a bottle cap (10) and sealed. The liquid inlet one-way valve (8) is fixedly mounted on the bottle cap (10) and its outlet end is connected to the inner cavity of the culture bottle (3). The inlet end of the liquid inlet one-way valve (8) is located outside the culture bottle (3) and is connected to the liquid inlet pipe (9). The axes of the liquid inlet one-way valve (8), the liquid inlet pipe (9) and the culture bottle (3) coincide with each other.

3. A dedicated culture device for in situ culture of amniotic fluid or chorionic cells according to claim 2, characterized in that: One end of the liquid inlet pipe (9) faces upward and is threadedly connected to the liquid outlet of the liquid storage bag (7) through a connector (17), and the other end of the liquid inlet pipe (9) faces downward and is rotationally sealedly connected to the inlet end of the liquid inlet one-way valve (8) through a rotary joint (11).

4. The dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells according to claim 1, characterized in that: The bottom of the inner cavity of the sleeve (2) is provided with symmetrically distributed limiting blocks (12), and the bottom of the culture bottle (3) is provided with a positioning rod (13) corresponding to and adapted to the limiting blocks (12), and the positioning rod (13) is plugged and assembled with the limiting blocks (12); The upper end of the sleeve (2) has an opening adapted to the culture bottle (3), and a sealing sleeve (15) sleeved on the outer wall of the culture bottle (3) is installed at the opening for sealing the joint.

5. The dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells according to claim 4, characterized in that: An annular cover plate (16) is installed on the outer periphery of the upper end of the culture bottle (3), and the annular cover plate (16) is pressed onto the upper end surface of the sealing sleeve (15).

6. The dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells according to claim 1, characterized in that: Both sides of the bottom of the inner cavity of the sleeve (2) are provided with inclined surfaces (14), the two inclined surfaces (14) are symmetrically distributed, and the lower ends of the two inclined surfaces (14) are close to the axis of the sleeve (2) and connected to the opening of the discharge pipe (5).

7. The dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells according to claim 1, characterized in that: The pore size of the micropores (4) is 20-30 μm.

8. The dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells according to claim 1, characterized in that: A driving box (22) is installed at the bottom of the constant temperature box (1). The driving assembly includes a driving shaft (18), a driven shaft (19), a driving gear (20), and a driven gear (21) installed in the driving box (22). The driving shaft (18) and the driven shaft (19) are both rotatably installed in the driving box (22). The lower end of the driving shaft (18) is driven by a motor. The driving gear (20) is installed at the upper end of the driving shaft (18). The upper end of the driven shaft (19) is fixed to the bottom of the sleeve (2). The driven gear (21) is installed at the lower end of the driven shaft (19), and the driven gear (21) and the driving gear (20) are meshed with each other.

9. A dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells according to claim 8, characterized in that: The driven shaft (19) is a tubular structure. One end of the drain pipe (5) is installed at the bottom of the sleeve (2). The other end of the drain pipe (5) passes through the inner cavity of the driven shaft (19) and extends to the bottom of the drive box (22). The drain pipe (5) and the driven shaft (19) are coaxially aligned.

10. Application of a dedicated culture device for in situ culture of amniotic fluid or chorionic villus cells, characterized in that: The specific steps are as follows: S1: Place amniotic fluid or chorionic villus cells into a culture bottle (3), then add an appropriate amount of culture fluid and cover the bottle with a cap (10) to form a sealed environment. After the culture bottle (3) is assembled into the sleeve (2) and secured, the door of the box can be closed for constant temperature culture. S2 After a certain period of culture, when the culture fluid in the culture bottle (3) needs to be replaced, the drive assembly drives the sleeve (2) to drive the culture bottle (3) to rotate centrifugally at high speed, so that the culture fluid in the culture bottle (3) is affected by the centrifugal force and moves toward the peripheral micropores (4), and the culture fluid in the culture bottle (3) gradually flows out of the micropores (4). Since the culture bottle (3) is a closed cavity as a whole, a negative pressure is formed inside and the liquid inlet one-way valve (8) is triggered to open, so that the culture fluid in the liquid storage bag (7) flows into the interior of the culture bottle (3) under the action of the pressure difference, thereby achieving a dynamic balanced supply of the culture fluid; S3 When the culture solution in the liquid storage bag (7) is transferred to the inlet end of the liquid inlet check valve (8) through the liquid inlet pipe (9), the gas generator injects the culture gas into the liquid inlet check valve (8) through the inflation connector (25), so that the culture gas is mixed with the culture solution and is simultaneously injected into the culture bottle (3); S4 During the entire culture period, steps S2 and S3 are repeated several times to complete the in situ culture of the amniotic fluid or chorionic cells. After the culture is completed, the culture bottle (3) is removed.

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