A cascade stage objective table in-situ cell culture device

Abstract

This invention discloses a cascaded stage in situ cell culture device, relating to the field of incubator technology. It includes a first stage incubator with a top cover and a second stage incubator. A circulation assembly is provided between the second and first stage incubators to enable inter-stage circulation. The flexible combination of the first and second stage incubators can meet the needs of different experimental scenarios—small incubators can be used individually to accommodate large-sized devices, while cascaded micro-culture boxes can fit in confined space, eliminating the need to purchase multiple sets of equipment and reducing costs. The two incubators communicate through airflow circulation, sharing the environmental control system of the first stage incubator to ensure that the internal temperature and gas concentration of the second stage incubator are consistent with those of the first stage incubator, avoiding the challenge of synchronous control of different environments.

Description

Technical Field

[0001] This invention belongs to the field of incubator technology, specifically relating to a cascaded stage in situ cell culture device. Background Technology

[0002] Current cell culture devices struggle to simultaneously meet diverse technical requirements in terms of size and structure. For instance, experiments requiring in-situ perfusion chips or electro-, magnetic, acoustic, and optical excitation devices necessitate a larger in-situ culture chamber (including length, width, and height). However, experiments requiring real-time tracking imaging on inverted microscopes with limited space above the stage (e.g., some confocal microscopes or inverted microscopes with bulky optical components) require an in-situ culture chamber height only slightly higher than a standard culture plate or dish, and less than 30 mm. Traditional stage incubator solutions often consist of a large premixer and a small culture box. The premixer, used to mix gases at specific concentrations and temperatures, does not itself contain a cell culture chamber and its size is significantly larger than the space above the microscope stage. The small culture box, due to its limited size, cannot accommodate the various excitation devices or components required by the user. Therefore, a modular in-situ culture device is needed, allowing the same user to easily combine and modify the device to meet different technical needs or application scenarios. Some foreign staged culture devices can achieve in-situ observation of the single-cell culture process, but it is difficult to achieve the above-mentioned different technical requirements at the same time by simply combining two small culture chambers without gas premixers and at low cost. In addition, if two types of existing products are used, the culture chambers of different sizes will be used independently for different scenarios, which will not only double the cost, but also make it difficult to automatically and synchronously control the internal environment of the two chambers, and there will be too many external auxiliary equipment, making it inconvenient to use. Summary of the Invention

[0003] The purpose of this invention is to provide a cascaded stage in situ cell culture device to solve the above-mentioned problems.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a cascaded stage in situ cell culture device, comprising a first stage incubator, wherein a temperature sensor, a gas concentration sensor, and a heating source are provided inside the first stage incubator to monitor the temperature, gas pressure, and gas concentration inside the first stage incubator for easy subsequent adjustment, and a top cover is provided on the top of the first stage incubator; and further comprising a second stage incubator, wherein a systemic circulation assembly is provided between the second stage incubator and the first stage incubator to achieve circulation communication between the two; The gas circulation assembly includes a small air pump; The small air pump is connected to an air pump suction pipe at its air intake port. One end of the air pump suction pipe is connected to an air port on the top cover to enable the small air pump to communicate with the inside of the first stage incubator. The exhaust port of the small air pump is connected to an exhaust pipe, one end of which is connected to a connecting pipe, and one end of the connecting pipe is connected to the air inlet on the second stage incubator. The second stage incubator is provided with an air outlet, and a connecting pipe is connected to the air outlet. The connecting pipe connects to the air vent on the top cover to achieve communication with the inside of the first stage incubator.

[0005] Preferably, the second connecting pipe includes an air pipe, an air pipe, and an air pump; The air pump's inlet is connected to the second air pipe, and the second air pipe is also connected to the outlet of the second stage incubator. The exhaust port of the air pump is connected to air pipe one, and air pipe one is connected to air port two on the top cover to achieve communication with the inside of the incubator of the first stage.

[0006] Preferably, the interior of the second stage incubator includes a first wall, a second wall, a microporous structure, a sample culture area, and a sandwich space formed by the first wall, the second wall, and the microporous structure. One end of the air inlet is connected to a connecting pipe and the other end is connected to the interlayer space. The air inlet is perpendicular to the wall surface. The wall surface is provided with an internal opening 1, an internal opening 2 and a microporous structure, and the interlayer space 1 is connected to the sample culture area through the internal opening 1, the internal opening 2 and the microporous structure; One end of the air outlet is connected to the sample culture area and the other end is connected to the connecting tube 2.

[0007] Preferably, the interior of the second stage incubator includes a first wall, a second wall, a microporous structure, a sample culture area, a partition, and a sandwich space formed by the first wall, the second wall, the microporous structure, and the partition. One end of the air inlet is connected to a connecting pipe and the other end is connected to the interlayer space. The air inlet is perpendicular to the wall surface. The second wall surface, located in the interlayer space, has an internal opening one and an internal opening two. The wall surface is located in the second section of the mezzanine space, which has internal openings three and four. The interlayer space 1 is connected to the sample culture area through internal port 1 and internal port 2; The sample culture area is connected to the second interlayer space through internal ports three and four located in the second interlayer space, and is also connected to the air outlet through the second interlayer space.

[0008] Preferably, the interior of the second stage incubator includes a first wall, a second wall, a microporous structure, a sample culture area, a partition, and a sandwich space formed by the first wall, the second wall, the microporous structure, and the partition. One end of the air inlet is connected to a connecting pipe and the other end is connected to the interlayer space. The air inlet is perpendicular to the wall surface. The second wall surface, located in the interlayer space, has an internal opening one and an internal opening two. The second section of the wall surface, located in the interlayer space, has a microporous structure. The interlayer space 1 is connected to the sample culture area through internal port 1 and internal port 2; The sample culture area is connected to the second interlayer space through a microporous structure located in the second interlayer space, and is also connected to the air outlet through the second interlayer space. The sample culture area is connected to the second interlayer space through internal port one and internal port two located in the second interlayer space, and is also connected to the air outlet through the second interlayer space.

[0009] Preferably, the second stage incubator includes a base, a bottom adapter piece with an outer square and an inner circle on the base and a frame fixed on the outside, a top cover on the top of the frame, a circular slide fixing piece embedded in the bottom recessed area formed by the bottom adapter piece and the base, a through hole in the central area of ​​the fixing piece, the diameter of the through hole being able to allow the microscope objective lens to pass through and rise to the circular slide near the top, and the standard culture plate being positioned relative to the top of the circular slide.

[0010] Preferably, the circular glass slide can also be an ultra-thin transparent film. In this case, the transparent film covers the upper part of the fixing plate to replace the circular glass slide, and the film is double-clamped and secured by the contact surface gap between the base and the bottom adapter plate, and the gap between the bottom adapter plate and the fixing plate.

[0011] The technical effects and advantages of this invention are as follows: 1. Modular combination design: By flexibly combining the first stage incubator and the second stage incubator, the needs of different experimental scenarios can be met. The small incubator can be used alone to adapt to the installation of large-size devices, and the micro incubator can be used in cascade to adapt to the narrow stage space. There is no need to purchase multiple sets of equipment, which reduces costs. 2. Stable environmental parameters: The two are interconnected through airflow circulation and share the environmental control system of the first stage incubator, ensuring that the internal temperature and gas concentration of the second stage incubator are consistent with those of the first stage incubator, thus avoiding the problem of synchronous control of different chamber environments; 3. High adaptability: The second stage incubator is as low as 20mm in height, which can be adapted to a variety of inverted microscopes with limited space; the first stage incubator is 45-60mm in height, which can accommodate various excitation devices and perfusion chips, making it widely applicable. 4. Structural optimization: The incubator of the second stage adopts a dual-path gas flow design, combined with a micro-porous structure, to achieve uniform gas distribution.

[0012] 5. Easy to use: The pipeline connection is simple, the combination mode can be quickly switched according to the needs, and there are few external auxiliary materials. It is easy to move and operate, which is conducive to the implementation of automated experiments. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the structure of the present invention; Figure 2 This is a top view of the overall structure of the present invention; Figure 3 This is a side view of the internal structure of the present invention; Figure 4 This is a top view of the internal structure of the present invention; Figure 5 This is a schematic diagram of the airflow in the incubator on the second stage of Embodiment 1 of the present invention; Figure 6 This is a schematic diagram of the airflow in the incubator on the second stage of Embodiment 2 of the present invention; Figure 7 This is a schematic diagram of the airflow in the incubator on the second stage of Embodiment 3 of the present invention; Figure 8 This is a simplified schematic diagram of the internal structure of the second stage incubator of the present invention; Figure 9 This is a three-dimensional cross-sectional view of the second stage incubator of the present invention.

[0014] In the diagram: 1. First stage incubator; 116. Top cover; 131. Air inlet 1; 132. Air inlet 2; 2. Second stage incubator; 201. Air inlet; 202. Air outlet; 204. Interlayer space 1; 205. Interlayer space 2; 301. Wall 1; 302. Wall 2; 303. Microporous structure; 401. Sample culture area; 10. Small air pump; 11. Air pump suction pipe; 12. Exhaust pipe; 13. Connecting pipe 1; 14. Connecting pipe 2; 141. Air pipe 1; 142. Air pipe 2; 17. Circular glass slide; 18. Fixing plate; 19. Bottom adapter plate; 20. Air pump; 21. Base; 22. Frame; 23. Top cover; 24. Standard culture plate; 25. Internal port 1; 26. Internal port 2; 27. Internal port 3; 28. Internal port 4. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] This invention provides, for example Figures 1-9 The cascaded stage in situ cell culture device shown includes a first stage incubator 1 (the first stage incubator 1 is a published patent, patent name: Integrated In situ Observation Stage Cell Culture Apparatus, authorized patent number: ZL2022233948397; patent application number: 2022116300369), the first stage incubator is equipped with a temperature sensor, a gas concentration sensor and a heating source device to monitor the temperature, gas pressure and gas concentration inside the first stage incubator for easy subsequent adjustment, and the top of the first stage incubator is equipped with a top cover 116; it also includes a second stage incubator 2, and a systemic circulation component is provided between the second stage incubator 2 and the first stage incubator 1 to realize the circulation between the two; The gas circulation assembly includes a small air pump 10; The small air pump 10 is connected to an air pump suction pipe 11 at its suction port. One end of the air pump suction pipe 11 is connected to an air port 131 on the top cover 116 to enable the suction end of the small air pump 10 to communicate with the inside of the first stage incubator 1. The exhaust port of the small air pump 10 is connected to an exhaust pipe 12, one end of the exhaust pipe 12 is connected to a connecting pipe 13, and one end of the connecting pipe 13 is connected to the air inlet 201 on the second stage incubator 2. The second stage incubator 2 is provided with an air outlet 202, and a connecting pipe 24 is connected to the air outlet 202. The connecting pipe 202 is connected to the air outlet 232 opened on the top cover 116 to achieve communication with the inside of the first stage incubator.

[0017] Specifically, the second connecting pipe 14 includes a first air pipe 141, a second air pipe 142, and an air pump 20; The air inlet of the air pump 20 is connected to the second air pipe 142, and the air outlet 202 of the second stage incubator 2 is connected through the second air pipe 142. The exhaust port of the air pump 20 is connected to the air pipe 141, and the air pipe 141 is connected to the air port 132 opened on the top cover 116 to achieve communication with the inside of the first stage incubator.

[0018] Specifically, the interior of the second stage incubator 2 includes a first wall 301, a second wall 302, a microporous structure 303, a sample culture area 401, and a sandwich space 204 formed by the first wall 301, the second wall 302, the microporous structure 303, and the inner wall of the outer shell of the second stage incubator 2. One end of the air inlet 201 is connected to the connecting pipe 13 and the other end is connected to the interlayer space 204. The air inlet 201 is perpendicular to the wall surface 301. The wall 301 is provided with an internal port 25, an internal port 26 and a microporous structure 303, and the interlayer space 204 is connected to the sample culture area 401 through the internal port 25, the internal port 26 and the microporous structure 303. One end of the air outlet 202 is connected to the sample culture area 401 and the other end is connected to the connecting tube 14. The second stage incubator 2 includes a base 21, on which a bottom adapter 19 with an outer square and inner circle is provided and a frame 22 is fixed on the outside. A circular glass slide fixing piece 18 is embedded in the bottom recessed area formed by the bottom adapter 19 and the base 21. A through hole is provided in the central area of ​​the fixing piece 18. The diameter of the through hole allows the microscope objective lens to pass through and rise to the circular glass slide 17 near the top. The standard culture plate 24 is positioned on top of the circular glass slide 17. The air inlet of the air outlet 202 is connected only to the sample culture area 401, and the air outlet of the air inlet 201 is connected only to the interlayer space 204. The circular glass slide 17 can also be an ultra-thin transparent film. In this case, the transparent film covers the upper part of the fixing piece 18 to replace the circular glass slide, and the film is double-clamped and fastened by the contact seam between the base 21 and the bottom adapter piece 19, and the seam between the bottom adapter piece 19 and the fixing piece 18.

[0019] Example 1: As Figure 1 , 2 3, 4 and Figure 5As shown, when the air pressure at the inlet 201 is greater than the air pressure at the outlet 202, the airflow circulation path inside the second stage incubator 2 is formed by the interlayer space 204 and the sample culture area 401, which are connected by the opposing internal ports 25 and 26 and the microporous structure 303. Furthermore, the resistance of the microporous structure 303 to airflow is greater than the resistance of the internal ports 25 and 26 to airflow. The inlet 201 and outlet 202 of the second stage incubator are respectively connected to the interlayer space 204. It is connected to the sample culture area 401; since the air outlet 202 is close to the microporous structure 303, in order to prevent a large amount of gas from entering the sample culture area 401 through the microporous structure 303 and flowing directly away from the air outlet 202 without covering the sample culture area 401, the diameter of the micropores of the microporous structure 303 is set to be much smaller than the pore diameter of the inner port 1 25 and the inner port 26, so that the resistance of the microporous structure 303 to the airflow is greater than the resistance of the inner port 1 25 and the inner port 26 to the airflow. The interior of the second stage incubator 2 includes a first wall 301, a second wall 302, a microporous structure 303, a sample culture area 401, a partition 212, and a first interlayer space 204 and a second interlayer space 205 formed by the first wall 301, the second wall 302, the microporous structure 303, the partition 212, and the inner wall of the outer shell of the second stage incubator 2; One end of the air inlet 201 is connected to the connecting pipe 13 and the other end is connected to the interlayer space 204. The air inlet 201 is perpendicular to the wall surface 301. The wall surface 301 is provided with an internal opening 25 and an internal opening 26 in the interlayer space 204. The wall surface 301 is provided with internal openings 27 and 28 in the interlayer space 205. The interlayer space 204 is connected to the sample culture area 401 through internal port 25 and internal port 26. The sample culture area 401 is connected to the interlayer space 205 through the internal port 3 27 and internal port 4 28 located in the interlayer space 205, and is also connected to the air outlet 202 through the interlayer space 205. The air outlet of the air inlet 201 is connected only to the interlayer space 204, and is connected to the sample culture area 401 through the internal port 25 and the internal port 26. The air inlet of the air outlet 202 is only connected to the interlayer space 205, and is connected to the sample culture area 401 through the internal port 3 27 and internal port 4 28 located in the interlayer space 205. Example 2: Figure 1 , 2 3, 4 and Figure 6As shown, when the air pressure at the inlet 201 is equal to the air pressure at the outlet 202: the inlet 201 and outlet 202 are located at the lower right corner. The inlet 201 is only connected to the interlayer space 1 204. The gas flows to the inner port 1 25 and the inner port 26, and then enters the sample culture area 401. The gas flows out of the sample culture area 401 through the inner port 3 27 and the inner port 4 28 to the sample culture area 2 205, and finally flows out through the outlet 202. The outlet 202 is only connected to the interlayer space 2 205. The microporous structure 303 can be made of porous ceramics, sintered glass or porous plastics, because the resistance of the gas flow through the inner port 1 25 and the inner port 26 is basically equal to the resistance of the gas flow through the inner port 3 27 and the inner port 4 28. The second stage incubator 2 includes a first wall 301, a second wall 302, a microporous structure 303, a sample culture area 401, a partition 212, and a first interlayer space 204 and a second interlayer space 205 formed by the first wall 301, the second wall 302, the microporous structure 303 and the partition 212. One end of the air inlet 201 is connected to the connecting pipe 13 and the other end is connected to the interlayer space 204. The air inlet 201 is perpendicular to the wall surface 301. The wall surface 302 is provided with an internal opening 25 and an internal opening 26 in the interlayer space 204. The wall surface 301 is provided with a microporous structure 303 in the interlayer space 205; The interlayer space 204 is connected to the sample culture area 401 through internal port 25 and internal port 26; The sample culture area 401 is connected to the interlayer space 205 through the microporous structure 303 located in the interlayer space 205 and is also connected to the air outlet 202 through the interlayer space 205. The air outlet of the air inlet 201 is only connected to the interlayer space 204, and is connected to the sample culture area 401 through the internal port 25 and the internal port 26. The air inlet of the air outlet 202 is only connected to the interlayer space 205, and is connected to the sample culture area 401 through the microporous structure 303 located in the interlayer space 205. Example 3: Figure 1 , 2 3, 4 and Figure 7As shown, when the air pressure at the inlet 201 is lower than that at the outlet 202, the inlet 201 and outlet 202 are located at the lower right corner. The inlet 201 is only connected to the interlayer space 204, and the gas flows to the inner port 25 and the inner port 26, and then enters the sample culture area 401. The outlet 202 is only connected to the interlayer space 205. The gas in the sample culture area 401 flows through the microporous structure 303 into the interlayer space 205, and finally flows out through the outlet 202, eventually flowing to the second stage incubator 2. The microporous structure 303 can be made of porous ceramics, sintered glass, or porous plastics.

[0020] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cascaded stage in situ cell culture device, comprising a first stage incubator, wherein a temperature sensor, a gas concentration sensor, and a heating source are disposed inside the first stage incubator to monitor the internal temperature, gas pressure, and gas concentration, and a top cover is disposed on the top of the first stage incubator; characterized in that: It also includes a second stage incubator, and a gas circulation component is provided between the second stage incubator and the first stage incubator to achieve circulation between the two. The gas circulation assembly includes a small air pump; The small air pump is connected to an air pump suction pipe at its air intake port. One end of the air pump suction pipe is connected to an air port on the top cover to enable the small air pump to communicate with the inside of the first stage incubator. The exhaust port of the small air pump is connected to an exhaust pipe, one end of which is connected to a connecting pipe, and one end of the connecting pipe is connected to the air inlet on the second stage incubator. The second stage incubator is provided with an air outlet, and a connecting pipe is connected to the air outlet. The connecting pipe connects to the air vent on the top cover to achieve communication with the inside of the first stage incubator.

2. The cascaded stage in situ cell culture device according to claim 1, characterized in that: The second connecting pipe includes an air pipe, an air pipe, and an air pump; The air pump's inlet is connected to the second air pipe, and the second air pipe is also connected to the outlet of the second stage incubator. The exhaust port of the air pump is connected to air pipe one, and air pipe one is connected to air port two on the top cover to achieve communication with the inside of the incubator of the first stage.

3. The cascaded stage in situ cell culture device according to claim 2, characterized in that: The interior of the second stage incubator includes a first wall, a second wall, a microporous structure, a sample culture area, and a sandwich space formed by the first wall, the second wall, and the microporous structure. One end of the air inlet is connected to a connecting pipe and the other end is connected to the interlayer space. The air inlet is perpendicular to the wall surface. The wall surface is provided with an internal opening 1, an internal opening 2 and a microporous structure, and the interlayer space 1 is connected to the sample culture area through the internal opening 1, the internal opening 2 and the microporous structure; One end of the air outlet is connected to the sample culture area and the other end is connected to the connecting tube 2.

4. The cascaded stage in situ cell culture device according to claim 3, characterized in that: The air inlet of the air outlet is connected only to the sample culture area, and the air outlet of the first air inlet is connected only to the interlayer space.

5. The cascaded stage in situ cell culture device according to claim 2, characterized in that: The interior of the second stage incubator includes wall one, wall two, a microporous structure, a sample culture area, a partition, and a sandwich space one and a sandwich space two enclosed by wall one, wall two, the microporous structure, and the partition; One end of the air inlet is connected to a connecting pipe and the other end is connected to the interlayer space. The air inlet is perpendicular to the wall surface. The second wall surface, located in the interlayer space, has an internal opening one and an internal opening two. The wall surface is located in the second section of the mezzanine space, which has internal openings three and four. The interlayer space 1 is connected to the sample culture area through internal port 1 and internal port 2; The sample culture area is connected to the second interlayer space through internal ports three and four located in the second interlayer space, and is also connected to the air outlet through the second interlayer space.

6. The cascaded stage in situ cell culture device according to claim 5, characterized in that: The outlet of the air inlet is connected only to the interlayer space and is connected to the sample culture area through the inner inlet and the inner inlet. The air inlet of the outlet is connected only to the second interlayer space, and is connected to the sample culture area through the internal port three and internal port four located in the second interlayer space.

7. The cascaded stage in situ cell culture device according to claim 2, characterized in that: The interior of the second stage incubator includes wall one, wall two, a microporous structure, a sample culture area, a partition, and a sandwich space one and a sandwich space two enclosed by wall one, wall two, the microporous structure, and the partition; One end of the air inlet is connected to a connecting pipe and the other end is connected to the interlayer space. The air inlet is perpendicular to the wall surface. The second wall surface, located in the interlayer space, has an internal opening one and an internal opening two. The second section of the wall surface, located in the interlayer space, has a microporous structure. The interlayer space 1 is connected to the sample culture area through internal port 1 and internal port 2; The sample culture area is connected to the second interlayer space through a microporous structure located in the second interlayer space, and is also connected to the air outlet through the second interlayer space.

8. The cascaded stage in situ cell culture device according to claim 7, characterized in that: The outlet of the first air inlet is connected only to the first interlayer space, and is connected to the sample culture area through the first internal inlet and the second internal inlet. The air inlet of the outlet is connected only to the second interlayer space, and is connected to the sample culture area through the microporous structure located in the second interlayer space.

9. The cascaded stage in situ cell culture device according to claim 1, characterized in that: The second stage incubator includes a base, a bottom adapter plate with an outer square and an inner circle on the base, and a frame fixed on the outside. The top of the frame is provided with a top cover. A circular glass slide fixing plate is embedded in the bottom recessed area formed by the bottom adapter plate and the base. A through hole is provided in the central area of ​​the fixing plate. The diameter of the through hole is sufficient to allow the microscope objective lens to pass through and rise to the circular glass slide near the top. The standard culture plate is positioned on top of the circular glass slide.

10. The cascaded stage in situ cell culture device according to claim 9, characterized in that: The circular glass slide can also be an ultra-thin transparent film. In this case, the transparent film covers the upper part of the fixing plate to replace the circular glass slide, and the film is double-clamped and secured by the contact surface gap between the base and the bottom adapter plate, as well as the gap between the bottom adapter plate and the fixing plate.

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