Manufacturing device and manufacturing method of chronic hypoxia environment for cell culture

Abstract

The invention relates to the technical field of cell hypoxia culture, in particular to a device for manufacturing a chronic hypoxia environment for cell culture, which comprises a cell culture plate and a plate cover, a circle of connecting piece with an L-shaped section is arranged on the periphery of the bottom of the cell culture plate, and the horizontal end part of the connecting piece is fixed at the bottom of the outer surface of the cell culture plate; an outer cover corresponding to the connecting piece is arranged above the plate cover, the lower end face of the outer cover is downwards connected to the upper end face of the connecting piece, an oxygen consumption layer is formed among the outer cover, the connecting piece, the cell culture plate and the plate cover, and an oxygen consumption agent is arranged in the oxygen consumption layer; the connecting piece and the outer cover are arranged outside the cell culture plate to form the oxygen consumption layer, the outer cover or the plate cover is fixed through the lifting fixing mechanism, the cell culture plate and the plate cover are wrapped with the oxygen permeation membrane, the outer cover and the connecting piece are sealed through the sealing membrane, and the oxygen consumption agent is arranged in the oxygen consumption layer. The oxygen in the oxygen consumption layer and the cell culture plate is gradually consumed, a chronic oxygen-deficient environment is formed, the structure is simple, the cost is low, and the sealing performance is good.

Description

Technical Field

[0001] The invention relates to the technical field of cell hypoxia culture, and in particular to a manufacturing device and a manufacturing method for a chronic hypoxic environment for cell culture. Background Art

[0002] Cell culture refers to a method of simulating the in vivo environment (sterility, suitable temperature, pH and certain nutritional conditions, etc.) in vitro to enable cells to survive, grow, reproduce and maintain their main structures and functions. Cell culture is also called cell cloning technology, and the formal term in biology is cell culture technology. Cell culture technology can transform a cell into a simple single cell or a few differentiated multi-cells through large-scale culture. Cell culture can not only obtain a large number of cells, but also study cell signal transduction, cell anabolism, cell growth and proliferation, etc.

[0003] Some cell cultures need to be placed in an oxygen-deficient environment for the following reasons: (1) reduced energy production in an oxygen-deficient environment will lead to metabolic disorders in cells; (2) a certain degree of oxygen-deficient environment can allow cells to establish a more robust metabolic system and adapt to the oxygen-deficient environment by changing metabolic pathways and increasing the production of antioxidants; (3) certain cell types are more adaptable to the oxygen-deficient environment, which can promote angiogenesis; and (4) hypoxia-inducible factor-1 (HIF-1) will be induced to express in an oxygen-deficient environment, helping cells adapt to new environmental conditions.

[0004] The existing main hypoxic environment devices for cell culture include cell hypoxia incubators and hypoxia chambers. For example, the cell hypoxia incubator is an anaerobic / hypoxic workstation model SD1000. When the cell hypoxia incubator is used, the cell culture plate is placed inside. It has a double working cabin structure and can accommodate 1,200 90mm flat dishes, 140 500mL blue-capped bottles, and a capacity of 25 liters. Therefore, it is very large and will take up a large space. It is also not suitable for small-scale experiments. In addition, the cost of cell hypoxia incubators is very high, and the price is generally between tens of thousands and hundreds of thousands. The carbon dioxide bottle must also be replaced when it is used. The structure of the hypoxia chamber is more like a simplified cell hypoxia incubator. When used, air inlets and outlets are provided at both ends to replace the gas in the chamber to form an hypoxic environment. However, the airtightness of the equipment is not good enough, which is easy to affect the culture of cells. The cost is relatively high, averaging about 20,000. Therefore, the present invention provides a manufacturing device and a manufacturing method for a chronic hypoxic environment for cell culture, which can ensure that the cells are cultured in a hypoxic environment while also ensuring airtightness and low cost. Summary of the invention

[0005] The present invention provides a manufacturing device and a manufacturing method for creating a chronic hypoxia environment for cell culture, aiming to ensure that cells are cultured in a chronic hypoxia environment with good airtightness and to create such an environment at low cost.

[0006] To achieve the above object, the technical solution of the present invention is as follows: A manufacturing device for creating a chronic hypoxia environment for cell culture is provided, including a cuboid-shaped cell culture plate and a plate cover covering the cell culture plate. The innovation lies in that: A ring of L-shaped cross-section connecting members is provided around the bottom periphery of the cell culture plate. The horizontal end of the connecting member is fixed to the bottom surface of the outer surface of the cell culture plate. An outer cover corresponding to the connecting member is provided above the plate cover. The lower end surface of the outer cover is connected downward to the upper end surface of the connecting member. An oxygen-consuming layer is formed between the outer cover, the connecting member, the cell culture plate, and the plate cover. An oxygen-consuming agent is provided in the oxygen-consuming layer;

[0007] It also includes a fixed base, a support rod, a support plate, a lifting and fixing mechanism, and a rotating film-wrapping mechanism. The support rod is in an inverted L shape, and its vertical bottom end is connected to the fixed base. The support plate is provided at the horizontal end of the support rod. The lifting and fixing mechanism is connected to the support plate. The rotating film-wrapping mechanism is provided on the lifting and fixing mechanism. The cell culture plate is located directly below the lifting and fixing mechanism. The rotating film-wrapping mechanism extends to the periphery of the outer cover and can rotate around the outer cover.

[0008] Furthermore, a groove is provided in the cell culture plate. A plurality of culture grooves are evenly provided in the groove. A plurality of cover rings corresponding to several culture grooves are provided at the inner bottom of the plate cover. When the plate cover covers the cell culture plate, the top of the cell culture groove is placed in the corresponding cover ring;

[0009] An inner connecting ring platform extends upward from the inner part of the upper end surface of the cell culture plate. The part of the upper end surface of the cell culture plate without the connecting ring platform forms a connecting cover platform; The four corners of the cell culture plate and the connecting ring platform are correspondingly provided with chamfers; The four corners of the plate cover are also correspondingly provided with chamfers, and the lower end part of the plate cover correspondingly covers the connecting cover platform.

[0010] Furthermore, the connecting member and the outer cover are provided with chamfer shapes corresponding to the corner parts of the cell culture plate.

[0011] Furthermore, the lifting and fixing mechanism includes a lead screw, a turning handle, a fixed disk, and a pressing disk. The lead screw is vertically arranged and is in transmission connection with the support plate. The turning handle and the pressing disk are respectively provided at the top and bottom ends of the lead screw. The fixed disk is located above the center of the cell culture plate. A rotating groove is provided in the fixed disk, and the turntable is rotatably arranged in the rotating groove.

[0012] Further, the rotating film wrapping mechanism includes a driving lifting plate, a connecting rod, a rotating sleeve and a rotating member; the driving lifting plate is drivingly sleeved on the lead screw, the connecting rod is vertically arranged, and the top end thereof is connected to the driving lifting plate. The rotating sleeve is rotatably sleeved on the connecting rod, and the connecting rod is respectively fixed with limiting rings at the upper and lower ends corresponding to the rotating sleeve; the rotating member is in an inverted L shape, the horizontal end is connected to the rotating sleeve, the vertical part is located outside the outer cover, and a cylindrical film is sleeved at the lower end of the vertical part. The rotating member is provided with fixing rings at the upper and lower end parts corresponding to the cylindrical film. The fixing ring located above is fixedly connected to the rotating member, and the fixing ring located below is threadedly connected to the rotating member; the film on the cylindrical film is an oxygen-permeable film or a sealing film.

[0013] Further, the length of the horizontal part of the rotating member is such that the cylindrical film can rotate along the part where the outer cover and the connecting member are connected.

[0014] Further, it further includes a support base, and the support base is arranged at the bottom of the cell culture plate, and the outer diameter is less than or equal to the outer diameter of the plate cover.

[0015] Further, the cell culture plate, the plate cover, the connecting member, the outer cover, the fixing disk and the pressing disk are all made transparent.

[0016] To achieve the above object, the present invention also provides a manufacturing method of a manufacturing device for a chronic hypoxia environment for cell culture based on the above, and the innovation lies in: specifically including the following steps:

[0017] S1. Cell culture placement: Place the support base directly below the fixing disk, and place the cell culture plate on the support base; place the cells to be cultured in the culture tank, and cover the plate cover on the cell culture plate. The top of the culture tank is placed in the corresponding cover ring, and the plate cover is covered on the connecting cover table of the cell culture plate.

[0018] S2. Plate cover fixation: Rotate the turning handle, so that the lead screw moves downward due to being drivingly connected to the support plate. The turntable rotates in the rotating groove along with the lead screw and moves downward in the rotating groove along with the lead screw, driving the fixing disk downward until it presses the fixed plate cover.

[0019] S3. Oxygen-permeable film wrapping: Place the cylindrical film with the film being an oxygen-permeable film between the two fixing rings, rotate the driving lifting plate, drive the connecting rod and the rotating member to lift and lower until the middle part of the cylindrical film corresponds to the gap between the cell culture plate and the plate cover, press the head end of the oxygen-permeable film at the gap between the cell culture plate and the plate cover, rotate the rotating member, drive the cylindrical film to rotate around the plate cover, and gradually wrap the oxygen-permeable film at the gap between the cell culture plate and the plate cover.

[0020] S4. Outer cover fixation: After wrapping the oxygen-permeable membrane, rotate the turning handle to indirectly move the fixing plate upward. Place the oxygen-consuming agent between the connecting piece and the cell culture plate, cover the outer cover on the connecting piece to form an oxygen-consuming layer; rotate the turning handle, so that the lead screw moves downward due to the transmission connection with the support plate, the turntable rotates in the rotating groove along with the lead screw, and drives the fixing plate downward along with the lead screw in the rotating groove until the outer cover is squeezed and fixed.

[0021] S5. Wrapping the sealing film:

[0022] S51. First wrapping: Remove the lower fixing ring, replace the cylindrical film with the oxygen-consuming film with a cylindrical film with a sealing film, and screw on the fixing ring again; rotate the driving lifting plate to drive the connecting rod and the rotating part to lift and lower until the middle of the cylindrical film corresponds to the gap between the connecting piece and the outer cover connection, and press the head end of the sealing film at the gap between the connecting piece and the outer cover connection, rotate the rotating part to drive the cylindrical film to rotate around the outer cover, and gradually wrap the sealing film at the gap between the connecting piece and the outer cover connection.

[0023] S52. Second wrapping: After the first wrapping is completed, rotate the driving lifting plate to drive the connecting rod and the rotating part to rise until the middle of the cylindrical film corresponds to the upper end of the sealing film after the first wrapping, and press the head end of the sealing film corresponding to the upper end of the sealing film after the first wrapping on the outer cover and the film after the first wrapping, rotate the rotating part to drive the cylindrical film to rotate around the outer cover, and gradually wrap the sealing film at the upper end of the sealing film after the first wrapping.

[0024] S53. Third wrapping: After the second wrapping is completed, rotate the driving lifting plate to drive the connecting rod and the rotating part to descend until the middle of the cylindrical film corresponds to the lower end of the sealing film after the first wrapping, and press the head end of the sealing film corresponding to the lower end of the sealing film after the first wrapping on the connecting piece and the film after the first wrapping, rotate the rotating part to drive the cylindrical film to rotate around the connecting piece, and gradually wrap the sealing film at the lower end of the sealing film after the first wrapping.

[0025] The third wrapping seals the inside of the whole formed by the connecting piece and the outer cover, thereby creating a chronic hypoxic environment for cell culture upon completion of manufacturing.

[0026] Further, the number of layers of wrapping the oxygen-permeable membrane in step S3 is 4 - 6 layers; the number of layers of wrapping the sealing film each time in step S5 is 3 - 4 layers.

[0027] Compared with the prior art, the beneficial effects produced by the present invention are:

[0028] (1) A manufacturing device for creating a chronic hypoxia environment for cell culture according to the present invention is provided with a connecting member and an outer cover outside the cell culture plate to form an oxygen-consuming layer, and the outer cover or the plate cover is fixed by a lifting and fixing mechanism. The cell culture plate and the plate cover are wrapped with an oxygen-permeable membrane, and the outer cover and the connecting member are sealed with a sealing membrane. An oxygen-consuming agent is provided in the oxygen-consuming layer to gradually consume the oxygen in the oxygen-consuming layer and the cell culture plate, forming a chronic hypoxia environment. The structure is simple, the cost is low, and the sealing performance is good.

[0029] (2) A manufacturing method for creating a chronic hypoxia environment for cell culture according to the present invention, based on the manufacturing device, forms a seal inside the outer cover and the connecting member. The oxygen-consuming agent in the oxygen-consuming layer gradually consumes oxygen, and the oxygen in the plate cover and the cell culture plate can be discharged through the oxygen-permeable membrane into the oxygen-consuming layer to be consumed. Therefore, an anoxic environment, that is, a chronic hypoxia environment, is gradually formed in the plate cover and the cell culture plate, facilitating cell culture under a chronic hypoxia environment. The process of creating a chronic hypoxia environment is convenient and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0031] Figure 1 It is a schematic structural diagram of the cell culture plate in a manufacturing device for creating a chronic hypoxia environment for cell culture according to the present invention.

[0032] Figure 2 It is a schematic structural diagram of the plate cover in a manufacturing device for creating a chronic hypoxia environment for cell culture according to the present invention.

[0033] Figure 3 It is a schematic structural diagram of the plate cover covering the cell culture plate in the present invention.

[0034] Figure 4 It is a schematic structural diagram of the outer cover covering the connecting member in the present invention.

[0035] Figure 5 For the present invention Figure 4 The cross-sectional view after the structure in is wrapped with an oxygen-permeable membrane and a sealing membrane.

[0036] Figure 6 For the present invention Figure 5 The enlarged view of part A in.

[0037] Figure 7 It is a schematic structural diagram of a manufacturing device for creating a chronic hypoxia environment for cell culture according to the present invention when wrapping with an oxygen-permeable membrane.

[0038] Figure 8 The structural schematic diagram when wrapping the sealing film for a device for creating a chronic hypoxia environment for cell culture according to the present invention.

[0039] Figure 9 The cross-sectional view of the connection between the fixed plate and the pressing plate according to the present invention.

[0040] Figure 10 The step flow chart of a method for creating a chronic hypoxia environment for cell culture according to the present invention.

[0041] Wherein, 1, cell culture plate; 2, plate cover; 3, connecting member; 4, outer cover; 5, oxygen-consuming layer; 6, oxygen-consuming agent; 7, lifting and fixing mechanism; 71, lead screw; 72, turning handle; 73, fixed plate; 74, pressing plate; 75, turning groove; 8, rotating film-wrapping mechanism; 81, driving lifting plate; 82, connecting rod; 83, rotating sleeve; 84, rotating member; 85, limiting ring; 86, tubular film; 87, fixing ring; 88, oxygen-permeable membrane; 89, sealing film; 9, groove; 10, fixed base; 11, support rod; 12, support plate; 13, culture tank; 14, cover ring; 15, connecting ring platform; 16, connecting cover platform; 17, support base; 18, rotating ring. Specific embodiments

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] As Figure 1-9 shown, the present invention will be described through the following embodiments.

[0044] Embodiment 1

[0045] This embodiment provides a device for creating a chronic hypoxia environment for cell culture, including a cuboid-shaped cell culture plate 1 and a plate cover 2 covering the cell culture plate 1. A connecting member 3 with an L-shaped cross-section is provided around the bottom periphery of the cell culture plate 1. The horizontal end of the connecting member 3 is fixed on the bottom outer surface of the cell culture plate 1. An outer cover 4 corresponding to the connecting member 3 is provided above the plate cover 2. The lower end surface of the outer cover 4 is connected downward to the upper end surface of the connecting member 3. An oxygen-consuming layer 5 is formed between the outer cover 4, the connecting member 3, the cell culture plate 1, and the plate cover 2, and an oxygen-consuming agent 6 is provided in the oxygen-consuming layer 5;

[0046] It also includes a fixed base 10, a support rod 11, a support plate 12, a lifting and fixing mechanism 7, and a rotating film wrapping mechanism 8. The support rod 11 is in an inverted L shape, with its vertical bottom end connected to the fixed base 10. The support plate 12 is arranged at the horizontal end of the support rod 11. The lifting and fixing mechanism 7 is connected to the support plate 12. The rotating film wrapping mechanism 8 is arranged on the lifting and fixing mechanism 7. The cell culture plate 1 is located directly below the lifting and fixing mechanism 7. The rotating film wrapping mechanism 8 extends to the periphery of the outer cover 4 and can rotate around the outer cover 4.

[0047] In this embodiment, a connecting piece 3 and an outer cover 4 are provided outside the cell culture plate 1 to form an oxygen-consuming layer 5. The outer cover 4 or the plate cover 2 is fixed by the lifting and fixing mechanism 7. The cell culture plate 1 and the plate cover 2 are wrapped with an oxygen-permeable membrane 88, and the outer cover 4 and the connecting piece 3 are sealed with a sealing film 89. An oxygen-consuming agent 6 is arranged in the oxygen-consuming layer 5 to gradually consume the oxygen in the oxygen-consuming layer 5 and the cell culture plate 1, forming a chronic hypoxia environment. The structure is simple, the cost is relatively low, and the sealing performance is good.

[0048] In this embodiment, the oxygen-consuming agent 6 is also a deoxidizing agent. The deoxidizing agent can be an iron-based deoxidizing agent or an enzyme-based deoxidizing agent (if water is required during oxygen consumption, an appropriate amount of water can also be added between the connecting piece 3 and the cell culture plate 1). The deoxidizing agent can be placed at the bottom of the oxygen-consuming layer 5 in a breathable bag, and the reaction products are convenient to clean up.

[0049] Embodiment 2

[0050] In the cell culture plate 1 of this embodiment, a groove 9 is provided, and a plurality of culture grooves 13 are evenly arranged in the groove 9. A plurality of cover rings 14 are provided at the inner bottom of the plate cover 2 corresponding to several culture grooves 13. When the plate cover 2 is covered on the cell culture plate 1, the top of the cell culture groove 13 is placed in the corresponding cover ring 14, further fixing the culture groove 13 and the cell culture plate 1, and increasing the stability of the device.

[0051] An inner connection ring platform 15 extends upward from the upper end surface of the cell culture plate 1, and a connection cover platform 16 is formed at the part of the upper end surface of the cell culture plate 1 where the connection ring platform 15 is not provided; the four corners of the cell culture plate 1 and the connection ring platform 15 are all correspondingly provided with chamfers; the four corners of the plate cover 2 are also correspondingly provided with chamfers, and the lower end part of the plate cover 2 is correspondingly covered on the connection cover platform 16.

[0052] The connecting piece 3 and the outer cover 4 are both provided with chamfer shapes corresponding to the corner parts of the cell culture plate 1.

[0053] The rest is the same as Embodiment 1.

[0054] In this embodiment, the chamfers are provided at the corner parts of the cell culture plate 1 and the plate cover 2 for the following purposes: (1) It can reduce contamination: The chamfer design can reduce the formation of condensed water during the culture process. When the culture plate is placed upright, condensed water droplets will form on the surface of the culture medium, and these droplets are likely to flow into the culture medium, resulting in contamination. The chamfer design can reduce the formation of such condensed water, thereby reducing the risk of contamination; (2) The chamfer design helps to maintain the humidity and integrity of the culture medium. When placed upright, the condensed water droplets will flow into the culture medium, causing the culture medium to separate from the culture plate, affecting the growth and reproduction of microorganisms. The chamfer design can reduce the occurrence of this situation, maintain the humidity and integrity of the culture medium, and is beneficial to the growth and reproduction of microorganisms; (3) The chamfer design makes the connection between the plate cover 2 and the cell culture plate 1 more stable. When the cell culture plate 1 is taken, it can maintain its original state and is not prone to the situation where the bottom separates from the lid, thus avoiding the contamination of miscellaneous bacteria in the environment.

[0055] The connecting member 3 and the outer cover 4 are adaptively provided with chamfers also to avoid the formation and inflow of condensed water and to ensure stable connection.

[0056] Embodiment 3

[0057] The lifting and fixing mechanism 7 of this embodiment includes a lead screw 71, a turning handle 72, a fixing disk 73 and a pressing disk 74. The lead screw 71 is vertically arranged and is in transmission connection with the support plate 12. The turning handle 72 and the pressing disk 74 are respectively arranged at the top and bottom of the lead screw 71. The fixing disk 73 is located above the center of the cell culture plate 1. A rotating groove 75 is provided in the fixing disk 73, and a rotating disk is rotatably arranged in the rotating groove 75.

[0058] The rest is the same as Embodiment 2.

[0059] When the lifting and fixing mechanism 7 of this embodiment is specifically used, by rotating the turning handle 72, the lead screw 71 can move downward relative to the support plate 12, driving the pressing disk 74 to move downward, and further driving the fixing disk 73 to move downward to squeeze and fix the outer cover 4 or the plate cover 2, and the use is relatively flexible.

[0060] The lifting and fixing mechanism 7 is used to fix the outer cover 4 or the plate cover 2 before film wrapping. Because after the cells are placed in the cell culture plate 1 for culture, the cell culture plate 1 cannot be moved or fixed randomly, so as not to affect the cell culture. Therefore, during the film wrapping process, the structure to be film wrapped needs to be fixed to facilitate the realization of an anoxic environment for film wrapping.

[0061] Embodiment 4

[0062] The rotating film wrapping mechanism 8 of this embodiment includes a driving lifting plate 81, a connecting rod 82, a rotating sleeve 83 and a rotating member 84; the driving lifting plate 81 is sleeved on the lead screw 71 in a driving manner, the connecting rod 82 is vertically arranged, and the top end is connected to the driving lifting plate 81. The rotating sleeve 83 is sleeved on the connecting rod 82, and limiting rings 85 are respectively fixed at the upper and lower ends of the connecting rod 82 corresponding to the rotating sleeve 83; the rotating member 84 is in an inverted L shape, the horizontal end is connected to the rotating sleeve 83, the vertical part is located outside the outer cover 4, and a tubular film 86 is sleeved at the lower end of the vertical part. Fixing rings 87 are provided at the upper and lower end parts of the rotating member 84 corresponding to the tubular film 86. The fixing ring 87 located above is fixedly connected to the rotating member 84, and the fixing ring 87 located below is threadedly connected to the rotating member 84; the film on the tubular film 86 is an oxygen-permeable film 88 or a sealing film 89.

[0063] Preferably, the length of the horizontal part of the rotating member 84 is such that the tubular film 86 can rotate along the connection part between the outer cover 4 and the connecting member 3.

[0064] A rotating ring 18 is further provided on the rotating member 84 of this embodiment. The function of the rotating ring 18 is to facilitate holding the rotating ring 18 to rotate the rotating member 84.

[0065] The rest is the same as in Embodiment 3.

[0066] When the rotating film wrapping mechanism 8 of this embodiment is specifically implemented, the outer cover 4 or the plate cover 2 to be film-wrapped is fixed, and then the height of the corresponding rotating film wrapping mechanism 8 is adjusted (by rotating the driving lifting plate 81 to perform the overall lifting of the rotating film wrapping mechanism 8) to adapt to the position to be film-wrapped. The detachable setting of the fixing ring 87 located below facilitates the replacement of the tubular film 86. The length of the rotating member 84 is set to adapt to the outer cover 4 and the plate cover 2, so that during the rotating film wrapping process of the rotating member 84, the film on the tubular film 86 can wrap around the outer cover 4 and the plate cover 2 at the corresponding connection gaps.

[0067] In addition, since the cell culture plate 1, the plate cover 2, the outer cover 4, etc. are all rectangular parallelepipeds, the distance between the vertical part of the rotating member 84 and the side surface of the outer cover 4 or the plate cover 2 can be changed during film wrapping, but it does not affect film wrapping. Only the rotating member 84 needs to rotate horizontally. During specific film wrapping, the film wrapping effect can be ensured through manual cooperation, so that the film fits at the connection between the outer cover 4 and the connecting member 3 or at the connection between the plate cover 2 and the cell culture plate 1.

[0068] Preferably, the oxygen-permeable film 88 can use a cellulose film, a polyimide film or a polyether nitrile film. The sealing film 89 can use a PE film, a PP film, a PA film or a Parafilm sealing film.

[0069] Embodiment 5

[0070] This embodiment further includes a support base 17 , which is disposed at the bottom of the cell culture plate 1 and has an outer diameter that is less than or equal to the outer diameter of the plate cover 2 .

[0071] The rest is the same as Example 4.

[0072] The support base in this embodiment can raise the overall height of the cell culture plate 1, making it easier to wrap the sealing film 89, and preventing the cell culture plate 1 from being directly placed on the platform, which would hinder the tube film 86 from rotating and further affect the film wrapping.

[0073] Example 6

[0074] The cell culture plate 1, the plate cover 2, the connector 3, the outer cover 4, the fixing plate 73 and the pressing plate 74 of this embodiment are all transparent.

[0075] The rest is the same as Example 5.

[0076] In this embodiment, the above components are set to be transparent, so as to facilitate observation of cell culture in the culture tank 13 after the chronic hypoxic environment is created.

[0077] Example 7

[0078] On the basis of Example 6, this example provides a method for manufacturing a device for manufacturing a chronic hypoxic environment for cell culture based on Example 6, such as Figure 10 As shown, the specific steps include:

[0079] S1. Cell culture placement: Place the support base 17 directly below the fixed plate 73, and place the cell culture plate 1 on the support base 17; Place the cells to be cultured in the culture tank 13, and cover the plate cover 2 on the cell culture plate 1, place the top of the culture tank 13 in the corresponding cover ring 14, and cover the plate cover 2 on the cover platform 16 of the cell culture plate 1;

[0080] S2. Plate cover fixing: Turn the handle 72 so that the screw rod 71 moves downward due to the transmission connection with the support plate 12, and the turntable rotates in the groove 75 along with the screw rod 71, and moves downward along with the screw rod 71 in the groove 75 to drive the fixed plate 73 downward until the plate cover 2 is squeezed and fixed;

[0081] S3. Oxygen-permeable membrane wrapping: Place the cylindrical membrane 86 with the oxygen-permeable membrane 88 between two fixing rings 87. Rotate the driving and lifting plate 81 to drive the connecting rod 82 and the rotating member 84 to move up and down until the middle of the cylindrical membrane 86 corresponds to the gap where the cell culture plate 1 and the plate cover 2 are connected. Press the head end of the oxygen-permeable membrane 88 against the gap where the cell culture plate 1 and the plate cover 2 are connected. Rotate the rotating member 84 to drive the cylindrical membrane 86 to rotate around the plate cover 2, and gradually wrap the oxygen-permeable membrane 88 around the gap where the cell culture plate 1 and the plate cover 2 are connected. During the wrapping process, manual cooperation can be used to ensure that the membrane can completely fit the gap where the cell culture plate 1 and the plate cover 2 are connected, ensuring the wrapping effect;

[0082] S4. Outer cover fixing: After the oxygen-permeable membrane 88 is wrapped (fix the rear end of the membrane on the membrane surface with transparent tape, etc.), rotate the turning handle 72 to indirectly move the fixing disk 73 upward. Place the oxygen-consuming agent 6 between the connecting member 3 and the cell culture plate 1, and cover the outer cover 4 on the connecting member 3 to form an oxygen-consuming layer 5. Rotate the turning handle 72 so that the lead screw 71 moves downward due to the transmission connection with the support plate 12. The turntable rotates in the rotating groove 75 along with the lead screw 71 and moves downward in the rotating groove 75 along with the lead screw 71 to drive the fixing disk 73 downward until it squeezes and fixes the outer cover 4;

[0083] S5. Sealing membrane wrapping:

[0084] S51. First wrapping: Remove the lower fixing ring 87, replace the cylindrical membrane 86 with the oxygen-consuming membrane with the cylindrical membrane 86 with the sealing membrane 89, and screw on the fixing ring 87 again. Rotate the driving and lifting plate 81 to drive the connecting rod 82 and the rotating member 84 to move up and down until the middle of the cylindrical membrane 86 corresponds to the gap where the connecting member 3 and the outer cover 4 are connected. Press the head end of the sealing membrane 89 against the gap where the connecting member 3 and the outer cover 4 are connected. Rotate the rotating member 84 to drive the cylindrical membrane 86 to rotate around the outer cover 4, and gradually wrap the sealing membrane 89 around the gap where the connecting member 3 and the outer cover 4 are connected. During the wrapping process, manual cooperation can be used to ensure that the membrane can completely fit the gap where the connecting member 3 and the outer cover 4 are connected, ensuring the wrapping effect;

[0085] S52. Second wrapping: After the first wrapping is completed, rotate the driving and lifting plate 81 to drive the connecting rod 82 and the rotating member 84 to rise until the middle of the cylindrical membrane 86 corresponds to the upper end of the sealing membrane 89 after the first wrapping. Press the head end of the sealing membrane 89 corresponding to the upper end of the sealing membrane 89 after the first wrapping against the outer cover 4 and the membrane after the first wrapping. Rotate the rotating member 84 to drive the cylindrical membrane 86 to rotate around the outer cover 4, and gradually wrap the sealing membrane 89 around the upper end of the sealing membrane 89 after the first wrapping. During the wrapping process, manual cooperation can be used to ensure that the membrane can completely fit the upper end of the sealing membrane 89 after the first wrapping, ensuring the wrapping effect;

[0086] S53. Third film wrapping: After the second film wrapping is completed, rotate the transmission lifting plate 81 to drive the connecting rod 82 and the rotating member 84 to descend until the middle of the tubular film 86 corresponds to the lower end of the sealing film 89 after the first film wrapping, and press the head end of the sealing film 89 against the lower end of the sealing film 89 after the first film wrapping on the connecting member 3 and the film after the first film wrapping. Rotate the rotating member 84 to drive the tubular film 86 to rotate around the connecting member 3, and gradually wrap the sealing film 89 around the lower end of the sealing film 89 after the first film wrapping. During the film wrapping process, manual cooperation can be used to ensure that the film can be completely attached to the lower end of the sealing film 89 after the first film wrapping, ensuring the film wrapping effect.

[0087] The third film wrapping seals the interior of the whole formed by the connecting member 3 and the outer cover 4, thereby creating a chronic hypoxic environment for cell culture after manufacturing is completed.

[0088] The structure after the third film wrapping in this embodiment is as Figure 5 and 6 shown, further ensuring the sealing performance inside the outer cover 4 and the connecting member 3.

[0089] Based on the manufacturing device in this embodiment, a seal is formed inside the outer cover 4 and the connecting member 3. The oxygen-consuming agent 6 in the oxygen-consuming layer 5 gradually consumes oxygen, and the oxygen in the plate cover 2 and the cell culture plate 1 can be discharged through the oxygen-permeable membrane 88 into the oxygen-consuming layer 5 and consumed. Therefore, an anoxic environment, that is, a chronic hypoxic environment, is gradually formed inside the plate cover 2 and the cell culture plate 1, facilitating cell culture in the chronic hypoxic environment. The manufacturing process of the chronic hypoxic environment is convenient and fast.

[0090] The specific process of realizing the chronic hypoxic environment in this embodiment is as follows: After the outer cover 4 and the connecting member 3 are sealed, the oxygen-consuming agent 6 will first gradually consume the oxygen in the oxygen-consuming layer 5, making the oxygen content in the oxygen-consuming layer 5 lower than the oxygen content in the cell culture plate 1. The oxygen in the cell culture plate 1 will gradually move outward through the oxygen-permeable membrane 88 into the oxygen-consuming layer 5 and be gradually consumed by the oxygen-consuming agent 6 in the oxygen-consuming layer 5. Therefore, an anoxic environment is gradually formed, creating a chronic hypoxic environment for cell culture.

[0091] Example 8

[0092] Based on Example 7 in this embodiment, the number of layers of the oxygen-permeable membrane 88 wrapped in step S3 is 4 - 6 layers; the number of layers of each sealing film 89 wrapped in step S5 is 3 - 4 layers.

[0093] The above-described embodiments are only described as the preferred embodiments of the present invention, and do not limit the concept and scope of the present invention. Without departing from the design concept of the present invention, various modifications and improvements made by ordinary engineering and technical personnel in the field to the technical solutions of the present invention should fall within the protection scope of the present invention. The technical content claimed by the present invention has been fully recorded in the technical requirements.

Claims

1. A device for manufacturing a chronic hypoxic environment for cell culture, comprising a rectangular cell culture plate and a plate cover covering the cell culture plate, characterized in that: The bottom periphery of the cell culture plate is provided with a circle of connecting pieces with an L-shaped cross section, the horizontal end of the connecting piece is fixed to the bottom of the outer surface of the cell culture plate, an outer cover corresponding to the connecting piece is provided above the plate cover, the lower end face of the outer cover is downwardly connected to the upper end face of the connecting piece, an oxygen-consuming layer is formed between the outer cover, the connecting piece, the cell culture plate and the plate cover, and an oxygen-consuming agent is provided in the oxygen-consuming layer; It also includes a fixed base, a support rod, a support plate, a lifting and fixing mechanism and a rotating film wrapping mechanism, wherein the support rod is an inverted L-shape, and the vertical bottom end is connected to the fixed base, the support plate is arranged at the horizontal end of the support rod, the lifting and fixing mechanism is connected to the support plate, the rotating film wrapping mechanism is arranged on the lifting and fixing mechanism, the cell culture plate is located directly below the lifting and fixing mechanism, and the rotating film wrapping mechanism extends to the periphery of the outer cover and can rotate around the outer cover.

2. The device for manufacturing a chronic hypoxic environment for cell culture according to claim 1, characterized in that: The cell culture plate is provided with a groove, and a plurality of culture grooves are evenly arranged in the groove. A plurality of cover rings are arranged at the bottom of the plate cover corresponding to the plurality of culture grooves. When the plate cover is covered on the cell culture plate, the top of the cell culture groove is placed in the corresponding cover ring. A connecting ring platform is extended upward from the interior of the upper end surface of the cell culture plate, and a portion of the upper end surface of the cell culture plate where the connecting ring platform is not provided forms a connecting cover platform; the four corners of the cell culture plate and the connecting ring platform are correspondingly chamfered; the four corners of the plate cover are also correspondingly chamfered, and the lower end of the plate cover is correspondingly covered on the connecting cover platform.

3. The device for manufacturing a chronic hypoxic environment for cell culture according to claim 2, characterized in that: The corners of the connecting piece and the outer cover corresponding to the cell culture plate are both chamfered.

4. The device for manufacturing a chronic hypoxic environment for cell culture according to claim 3, characterized in that: The lifting and fixing mechanism includes a screw rod, a rotating handle, a fixed plate and a pressure plate. The screw rod is vertically arranged and is transmission-connected to the support plate. The rotating handle and the pressure plate are respectively arranged at the top and bottom ends of the screw rod. The fixed plate is located above the center of the cell culture plate. A rotating groove is arranged in the fixed plate, and the rotating plate rotates in the rotating groove.

5. The device for manufacturing a chronic hypoxic environment for cell culture according to claim 4, characterized in that: The rotating film wrapping mechanism includes a transmission lifting plate, a connecting rod, a rotating sleeve and a rotating part; the transmission lifting plate is sleeved on the screw rod, the connecting rod is vertically arranged, and the top end is connected to the transmission lifting plate, the rotating sleeve is rotationally sleeved on the connecting rod, and the upper and lower ends of the connecting rod corresponding to the rotating sleeve are respectively fixed with limiting rings; the rotating part is in an inverted L shape, the horizontal end is connected to the rotating sleeve, the vertical part is located at the periphery of the outer cover, and the vertical lower end is sleeved with a tubular film, the upper and lower ends of the rotating part corresponding to the tubular film are provided with fixing rings, the fixing ring located at the top is fixedly connected to the rotating part, and the fixing ring located at the bottom is threadedly connected to the rotating part; the film on the tubular film is an oxygen permeable film or a sealing film.

6. The device for manufacturing a chronic hypoxic environment for cell culture according to claim 5, characterized in that: The length of the horizontal part of the rotating part is such that the tubular film can rotate along the part where the outer cover and the connecting part are connected.

7. The device for manufacturing a chronic hypoxic environment for cell culture according to claim 6, characterized in that: It also includes a supporting base, which is arranged at the bottom of the cell culture plate and has an outer diameter that is less than or equal to the outer diameter of the plate cover.

8. The device for manufacturing a chronic hypoxic environment for cell culture according to claim 7, characterized in that: The cell culture plate, plate cover, connector, outer cover, fixed plate and pressure plate are all transparent.

9. A method for manufacturing a device for manufacturing a chronic hypoxic environment for cell culture according to claim 8, characterized in that: The specific steps include: S1. Cell culture placement: Place the support base directly below the fixed plate, and place the cell culture plate on the support base; Place the cells to be cultured in the culture tank, and cover the plate cover on the cell culture plate, place the top of the culture tank in the corresponding cover ring, and cover the plate cover on the cover platform of the cell culture plate; S2. Plate cover fixation: Turn the handle so that the screw rod moves downward due to the transmission connection with the support plate, and the turntable rotates in the turn slot along with the screw rod, and moves downward in the turn slot along with the screw rod to drive the fixed plate downward until the plate cover is squeezed and fixed; S3. wrapping with oxygen permeable film: placing a tube film with an oxygen permeable film between two fixed rings, rotating the transmission lifting plate, driving the connecting rod and the rotating part to rise and fall until the middle of the tube film corresponds to the gap between the cell culture plate and the plate cover, and pressing the head end of the oxygen permeable film against the gap between the cell culture plate and the plate cover, rotating the rotating part, driving the tube film to rotate around the plate cover, and gradually wrapping the oxygen permeable film in the gap between the cell culture plate and the plate cover; S4. Fixing the outer cover: After wrapping the oxygen permeable film, turn the handle to indirectly move the fixed plate upward, put the oxygen consuming agent between the connecting piece and the cell culture plate, and cover the outer cover on the connecting piece to form an oxygen consuming layer; turn the handle so that the screw rod moves downward due to the transmission connection with the support plate, and the rotating disk rotates in the rotating groove with the screw rod, and moves downward in the rotating groove with the screw rod to drive the fixed plate downward until the outer cover is squeezed and fixed; S5. Sealing film wrapping: S51. Wrapping the film once: remove the fixing ring at the bottom, replace the film with the oxygen-consuming film with the film with the sealing film, and screw the fixing ring on again; rotate the transmission lifting plate to drive the connecting rod and the rotating member to move up and down until the middle of the film corresponds to the gap between the connecting member and the outer cover, and press the head end of the sealing film to the gap between the connecting member and the outer cover, rotate the rotating member, drive the film to rotate around the outer cover, and gradually wrap the sealing film in the gap between the connecting member and the outer cover; S52. Secondary film wrapping: After the primary film wrapping is completed, the transmission lifting plate is rotated to drive the connecting rod and the rotating member to rise until the middle of the tube film corresponds to the upper end of the sealing film after the primary film wrapping, and the head end of the sealing film corresponding to the upper end of the sealing film after the primary film wrapping is pressed on the outer cover and the film of the primary film wrapping, and the rotating member is rotated to drive the tube film to rotate around the outer cover, and gradually wrap the sealing film at the upper end of the sealing film after the primary film wrapping; S53. Three-time film wrapping: After the second film wrapping is completed, the transmission lifting plate is rotated to drive the connecting rod and the rotating member to descend until the middle of the tube film corresponds to the lower end of the sealing film after the first film wrapping, and the head end of the sealing film corresponds to the lower end of the sealing film after the first film wrapping and is pressed on the connecting member and the film of the first film wrapping, and the rotating member is rotated to drive the tube film to rotate around the connecting member, and gradually wrap the sealing film at the lower end of the sealing film after the first film wrapping; The triple wrapping makes the interior of the whole formed by the connector and the outer cover sealed, thereby completing the manufacture of a chronic hypoxic environment for cell culture.

10. The method for producing a chronic hypoxic environment for cell culture according to claim 9, characterized in that: The number of layers of the oxygen permeable film wrapping in step S3 is 4-6 layers; the number of layers of the sealing film wrapping each time in step S5 is 3-4 layers.

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