Three-dimensional cell culture bottle and culture system
By designing Archimedes spiral sheets with a pitch angle of 15-75° in a cell culture flask, the problem of insufficient culture surface area in the prior art is solved, the cell culture effect is significantly improved, and a larger culture area is provided.
Patent Information
- Application Number
- CN202421238323.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-31
- Publication Date
- 2025-03-11
- Estimated Expiration
- 2034-05-31
AI Technical Summary
The culture surface area of the cell culture flask in the prior art is limited, making it difficult to achieve satisfactory cell culture effects.
A three-dimensional cell culture flask was designed, including a reflux area, a culture area and a liquid storage area. A hollow central tube and Archimedes spiral sheet layer were installed in the culture area, and the pitch angle was controlled at 15-75° to increase the cell culture surface area.
By improving the pitch angle of Archimedes spiral sheet, the cell culture surface area in the culture flask is significantly improved, and the culture area is increased by 32.8% compared with the prior art, providing a more favorable cell growth environment.
Smart Images
Figure CN222593887U_ABST
Abstract
Description
Technical Field
[0001] The utility model relates to the field of cell culture, in particular to a three-dimensional cell culture bottle and a culture system. Background Art
[0002] Traditional cell culture spinner bottles are used for large-scale adherent cell culture, including the bottle body and the bottle cap. Compared with ordinary cell culture dishes and cell culture plates, cell culture spinner bottle technology can not only provide a larger growth surface area, but also prevent certain components formed in the culture medium from affecting cell growth through slight rotation. In addition, this technology has a larger amount of nutrients per unit cell growth area, which is more conducive to collecting cell metabolites. Due to its simple operation and low cost, it occupies an important position in the field of life science and technology.
[0003] US20220177817A1 discloses a cell culture bottle for adherent cells, in which a central tube, concentric walls and Archimedean spirals are fixedly arranged in the bottle, and the cells grow on the outer surface of the central axis, the surface of the concentric walls and the spiral surface of the Archimedean spiral. The Archimedean spiral is a structure for moving or transporting solid or liquid materials. The structure is composed of multiple turns of propellers around the central axis, and the multiple turns of propellers are arranged in a spiral shape to transport the material from the feed port to the discharge port. However, the culture surface area in the above-mentioned culture bottle is limited, and it is difficult to achieve a satisfactory cell culture effect. Utility Model Content
[0004] Therefore, the technical problem to be solved by the utility model is to overcome the defect that the culture surface area in the culture bottle in the prior art is limited and it is difficult to achieve a satisfactory cell culture effect, thereby providing a three-dimensional cell culture bottle and culture system.
[0005] To this end, the utility model provides the following technical solutions:
[0006] A three-dimensional cell culture bottle, comprising a culture bottle body; the interior of the culture bottle body comprises a reflux area, a culture area and a liquid storage area from top to bottom;
[0007] The culture area is cylindrical, a hollow central tube is arranged on the central axis of the cylinder; at least one Archimedean spiral sheet is arranged on the outer wall of the central tube; at least one partition wall is arranged concentrically around the central tube; the Archimedean spiral sheet is connected to the partition wall;
[0008] The central tube is connected to the reflux area and the liquid storage area;
[0009] The pitch angle of the Archimedean spiral layer is 15-75°;
[0010] The bottle mouth is connected to a breathable membrane cover or a sterile transfer cover; the sterile transfer cover includes a gas filter and a liquid exchange tube.
[0011] Optionally, the pitch angle is 60°.
[0012] Optionally, when the number of partition walls is ≥2, the length of the Archimedean spiral sheet between two adjacent partition walls is in the range of 12 to 18 mm; preferably, 16 mm;
[0013] The number of the partition walls is 5.
[0014] Optionally, when the number of Archimedean spiral sheets is ≥ 2, the pitch of the Archimedean spiral sheets is 10 to 15 mm; preferably, 12.5 mm;
[0015] The number of the Archimedean spiral sheet layers is 6 to 10, and preferably, the number of the Archimedean spiral sheet layers is 8.
[0016] Optionally, the diameter of the central tube is 2-4 cm.
[0017] Optionally, the Archimedean spiral layer, the partition wall and the central tube are not connected to the bottom of the culture bottle.
[0018] Optionally, the outer wall of the central tube, the surface of the partition wall or the surface of the Archimedean spiral layer is a temperature-sensitive surface treated with a temperature-sensitive material.
[0019] Optionally, the temperature-sensitive material includes N-isopropylacrylamide.
[0020] Optionally, the reflux zone is communicated with the bottle mouth of the culture bottle body;
[0021] Optionally, the liquid exchange tube includes an internal port and two external ports, one end of the internal port extends into the bottle and passes through the central tube, the internal port is connected to the liquid storage area, one of the external ports is used for the input of fresh culture medium, and the other is used to suck out the used culture medium in the liquid storage area.
[0022] A culture system comprises the three-dimensional cell culture bottle.
[0023] A method for culturing cells using the three-dimensional cell culture bottle comprises: in a sterile environment, injecting culture medium into the reflux zone, adding cells to be cultured, rotating the culture bottle, so that the cells and culture medium are transported to the outer surface of the central tube, the surface of the partition wall and the spiral surface of the Archimedean spiral layer through the Archimedean spiral layer to adhere to the wall and grow by division, the culture bottle is continuously rotated, and the culture medium circulates in the culture bottle through the reflux zone, the central tube, the liquid storage zone, the Archimedean spiral layer and the reflux zone. After the culture is completed, the cells are separated from the culture surface by enzyme digestion or cooling, and the cells are collected.
[0024] The pitch angle of the Archimedean spiral layer refers to the angle between the spiral and the horizontal line.
[0025] The pitch refers to the axial distance between two corresponding points on the mid-diameter line of two teeth of two adjacent Archimedean spiral layers.
[0026] The technical solution of the utility model has the following advantages:
[0027] 1. The utility model provides a three-dimensional cell culture bottle, comprising a culture bottle body; the interior of the culture bottle body comprises a reflow zone, a culture zone and a liquid storage zone from top to bottom; the culture zone is cylindrical, and a hollow central tube is arranged on the central axis of the cylinder; at least one Archimedean spiral layer is arranged on the outer wall of the central tube; at least one partition wall is arranged concentrically around the central tube; the Archimedean spiral layer is connected to the partition wall; the central tube is connected to the reflow zone and the liquid storage zone; the pitch angle of the Archimedean spiral layer is 15-75°; the reflow zone is connected to the bottle mouth of the culture bottle body, and the bottle The mouth is connected to a breathable membrane cover or a sterile transfer cover; the utility model has found that the pitch angle of the Archimedean spiral in the existing culture bottle (i.e., the angle α between the spiral and the horizontal line) is between 2° and 5°, which leads to a limited culture surface area in the culture bottle. The utility model improves the spiral angle of the Archimedean spiral layer and controls the pitch angle within 15-75°, thereby increasing the cell culture surface area in the culture bottle. When the pitch angle is too small, the cell culture surface area in the culture bottle is too small. When the pitch angle is too large, the cell culture surface area in the culture bottle does not increase much. The culture surface area at each pitch angle is calculated as follows: 0°: 6587.7cm 2 ; 5°: 7478.6cm 2 ; 15°: 7543.7cm 2 ; 30°: 7792.6cm 2 ; 45°: 8324.6cm 2 ; 60°: 8753.4cm 2 ; 75°: 13685.1cm 2When the pitch angle is 60°, the surface area of the partition wall and the Archimedean spiral sheet layer after fusion increases, and the culture area is increased by 32.8% compared with the existing culture bottle (pitch angle is 0°).
[0028] 2. The utility model provides a three-dimensional cell culture bottle, in which the central tube not only plays a supporting role, but also serves as a channel for liquid circulation inside the culture bottle when the entire culture bottle is placed on its side for culture. The culture medium and cells flow between the reflux area, the central tube, and the liquid storage area. The diameter of the central tube is set to 2-4 cm to avoid the central tube diameter being too small to affect the reflux, and to avoid the central tube being too large to reduce the surface area inside the culture bottle.
[0029] 3. In the three-dimensional cell culture bottle provided by the utility model, the number of Archimedean spiral layers is preferably 8, and the pitch is between 10-15 mm, preferably 12.5 mm, so as to avoid the Archimedean spiral layers being too dense, resulting in a reduction in the liquid inside the culture bottle, which is not conducive to culture, and to avoid the Archimedean spiral layers being too few, resulting in a reduction in the surface area inside the culture bottle.
[0030] 4. The utility model provides a three-dimensional cell culture bottle. When the number of partition walls is ≥2, the length of the Archimedean spiral layer between two adjacent partition walls is in the range of 12 to 18 mm; preferably, 16 mm. The number of partition walls is preferably 5. Since the partition walls occupy a certain volume, the denser the partition walls, the better. This may also lead to a reduction in the culture surface area. Only within the above selected range can the optimal surface area inside the culture bottle be obtained. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the specific implementation methods of the utility model or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the utility model. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0032] Figure 1 It is a cross-sectional view of the three-dimensional cell culture bottle AA in Example 1 of the utility model;
[0033] Figure 2 It is a cross-sectional view of a plurality of Archimedean spiral sheets, a plurality of partition walls and a central tube in Example 1 of the utility model;
[0034] Figure 3 It is a stereoscopic diagram of a plurality of Archimedean spiral sheets, a plurality of partition walls and a central tube in Example 1 of the utility model;
[0035] Figure 4It is a front view of a plurality of Archimedean spiral sheets, a plurality of partition walls and a central tube in Example 1 of the utility model;
[0036] Figure 5 It is a top view of a plurality of Archimedean spiral sheets, a plurality of partition walls and a central tube in Example 1 of the utility model;
[0037] Figure 6 It is a bottom view of a plurality of Archimedean spiral sheets, a plurality of partition walls and a central tube in Example 1 of the utility model;
[0038] Figure 7 It is a schematic structural diagram of a single Archimedean spiral sheet in Example 1 of the utility model;
[0039] Figure 8 It is a top view of a single Archimedean spiral sheet layer in Example 1 of the utility model;
[0040] Fig. 9 It is a bottom view of a single Archimedean spiral sheet layer in Example 1 of the utility model;
[0041] Fig.10 It is a three-dimensional schematic diagram of multiple partition walls in Example 1 of the utility model;
[0042] Fig.11 is a cross-sectional view of a plurality of partition walls in Example 1 of the utility model;
[0043] Fig.12 is a top view of a plurality of partition walls in Example 1 of the utility model;
[0044] Fig.13 It is a side-laying culture state diagram of the culture area in the existing three-dimensional cell culture flask in Example 1 of the utility model and the culture area in the three-dimensional cell culture flask of the utility model; the left side corresponds to the culture area in the existing three-dimensional cell culture flask, and the right side corresponds to the culture area in the three-dimensional cell culture flask of the utility model.
[0045] Reference numerals:
[0046] 1-culture bottle body, 2-reflux area, 3-culture area, 4-liquid storage area, 5-central tube, 6-Archimedes spiral layer, 7-partition wall, 8-bottle mouth, DETAILED DESCRIPTION
[0047] The following embodiments are provided for a better understanding of the present invention, but are not limited to the best implementation mode described herein, and do not constitute limitations on the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.
[0048] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.
[0049] In the following embodiments, the adherent cells used need to be immersed in a culture medium during the culture process, and the culture medium provides nutrients required for cell growth, including water, carbon source, nitrogen source, essential trace elements and vitamins, etc. In addition, for adherent cells, the culture medium also needs to contain appropriate attachment factors to help the cells adhere to the bottom of the culture container.
[0050] Example 1
[0051] This embodiment provides a three-dimensional cell culture flask, such as Figure 1 As shown, it includes a culture bottle body 1, which is cylindrical, has a bottle mouth 8 on the top, and is closed at the bottom, and is used for culturing cells.
[0052] The culture bottle body 1 includes a reflux zone 2, a culture zone 3 and a liquid storage zone 4 from top to bottom; the reflux zone 2 is connected to the bottle mouth 8; the culture zone 3 is cylindrical, and a hollow central tube 5 is arranged on the central axis of the cylinder; the central tube 5 connects the reflux zone 2 and the liquid storage zone 4, and the culture medium and cell fluid can enter the reflux zone 2 through the bottle mouth 8, then enter the central tube 5, and then enter the liquid storage zone 4;
[0053] The outer wall of the central tube 5 is provided with at least one Archimedean spiral sheet 6, and the Archimedean spiral sheet 6 is as follows: Figure 1 As can be seen in FIG. 1 , the Archimedean spiral sheet layer 6 is arranged in the cylindrical culture area 3, as shown in FIG. Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 It can be seen that the Archimedean spiral sheet 6 is arranged around the central tube; a schematic diagram of an Archimedean spiral sheet 6 is shown in FIG. Figure 7-9 shown.
[0054] At the same time, at least one partition wall 7 is concentrically arranged around the central tube 5. Fig.10 , Fig.11 , Fig.12 The Archimedean spiral layer 6 is connected to the partition wall 7, such as Figure 1 shown.
[0055] The pitch angle of the Archimedean spiral layer 6 is 15-75°, and the pitch angle refers to the angle between the spiral and the horizontal line. Figure 1The pitch angle shown is 60°, and the cross section formed by two adjacent Archimedean spiral sheets 6 and two adjacent partition walls 7 is a parallelogram. Compared with the case where the pitch angle is 0-5° and the cross section is approximately rectangular or square, the perimeter is longer, which is equivalent to an increase in the culture surface area inside the culture bottle. Further, as Fig.13 As shown, the pitch angle of the Archimedean spiral of the existing culture bottle (i.e., the angle α between the spiral and the horizontal line) is between 2° and 5°, which is too small. Compared with the utility model (the pitch angle of 60° is taken as an example in the figure), when the culture bottle is placed on the rotating bottle machine at the same inclined angle for rotation and culture (here, the bottle is tilted 60° to the right, that is, the inclination angle between the bottle and the horizontal plane is 30°), the slope formed by the existing Archimedean spiral relative to the horizontal plane is too steep. When the culture medium flows in the bottle, the scouring force on the cells is large, which is not conducive to the attachment and growth of the cells; the slope formed by the Archimedean spiral of the utility model relative to the horizontal plane is extremely gentle. When rotating on the rotating bottle machine, the impact force on the cells is extremely small, the culture environment is extremely mild, and it is more conducive to cell growth.
[0056] As an optimized implementation, the pitch angle of the Archimedean spiral layer 6 is 60°. When the pitch angle is 60°, the perimeter of the cross-sectional parallelogram formed by two adjacent Archimedean spiral layers 6 and two adjacent partition walls 7 is the longest, and the culture surface area increases the most.
[0057] As an optimized implementation, when the number of partition walls 7 is ≥ 2, the length d2 of the Archimedes spiral layer 6 between two adjacent partition walls is in the range of 12 to 18 mm; preferably, the number of the partition walls is 5, and the length d2 of the Archimedes spiral layer 6 between two adjacent partition walls 7 is 16 mm. Since the partition walls 7 occupy a certain volume, when the number of the partition walls 7 is too high, the volume of the culture fluid is reduced and the culture effect is reduced. When the number of the partition walls 7 is too low, the increase in the culture surface area is small. Therefore, the selection of the number of the partition walls as 5 and the length d2 of the Archimedes spiral layer 6 between two adjacent partition walls 7 as 16 mm is the best implementation.
[0058] As an optimized implementation, when the number of Archimedes spiral sheets 6 is ≥2, the pitch d1 of the Archimedes spiral sheets 6 is in the range of 10-15 mm; preferably, the pitch of the Archimedes spiral sheets 6 is 12.5 mm, and the number of the Archimedes spiral sheets 6 is preferably 6-10, and more preferably 8. The Archimedes spiral sheets 6 occupy a certain volume. When the number of Archimedes spiral sheets 6 is too high, the volume of the culture fluid is reduced and the culture effect is reduced. When the number of Archimedes spiral sheets 6 is too low, the increase in the culture surface area is small. Therefore, the pitch d1 of the Archimedes spiral sheets 6 is selected to be 12.5 mm, and the number of the Archimedes spiral sheets 6 is 8 as the best implementation.
[0059] As an optimized implementation, the diameter of the central tube 5 is 2-4 cm. The central tube not only has a supporting function, but also serves as a channel for internal liquid circulation when the entire bottle is tilted for culture. The culture medium and / or cell fluid flows between the reflux area 2, the central tube 5, and the liquid storage area 4.
[0060] As a preferred embodiment, the outer wall surface of the central tube 5, the surface of the partition wall 7 and the surface of the Archimedean spiral layer 6 are treated with a temperature-sensitive material for cooling and collecting cells. The principle is that the cells adhere to the wall when close to 37°C, and below 4°C, the cells are peeled off from the temperature-sensitive layer. Therefore, when harvesting cells, there is no need to use trypsin digestion solution to hydrolyze the protein between cells. As long as the culture temperature is lowered to below 4°C to disperse the cells, the destruction of the cells is also avoided. The formula and treatment method of the temperature-sensitive can refer to Example 1 of Application No. 201811116498.2. First, a three-dimensional cell culture bottle is made by a 3D printing method, and then the three-dimensional cell culture bottle is plasma treated, a thermosensitive liquid containing N-isopropylacrylamide with a mass concentration of 80% is selected as the thermosensitive liquid, the thermosensitive liquid is added into the three-dimensional cell bottle, and the bottle is soaked, and then the thermosensitive liquid is poured out, and then the bottle is treated with electron beam radiation to solidify to obtain a culture surface with a thermosensitive layer; or first, a three-dimensional cell culture bottle is made by a 3D printing method, and then the three-dimensional cell culture bottle is plasma treated, a thermosensitive liquid containing N-isopropylacrylamide with a mass concentration of 80% is selected as the thermosensitive liquid, the thermosensitive liquid is atomized, the atomized liquid is passed into the three-dimensional cell bottle by pressurization, and then the bottle is treated with electron beam radiation to solidify to obtain a culture surface with a thermosensitive layer.
[0061] Furthermore, the Archimedean spiral layer 6, the partition wall 7 and the central tube 5 are not connected to the bottom of the culture bottle.
[0062] Furthermore, the reflux zone 2 is connected to the bottle mouth 8 of the culture bottle body; the bottle mouth 8 is connected to the breathable membrane cover or the sterile transfer cover; the sterile transfer cover includes a gas filter and a liquid exchange tube
[0063] The three-dimensional model was used to compare the culture surface area at different pitch angles under the same partition walls (5), Archimedean spiral sheets (8), the length of the Archimedean spiral sheet 6 between two adjacent partition walls 7 (16 mm), and the pitch of the Archimedean spiral sheet 6 (12.5 mm): 0°: 6587.7 cm 2 ; 5°: 7478.6cm 2 ; 15°: 7543.7cm 2 ; 30°: 7792.6cm 2 ; 45°: 8324.6cm 2 ; 60°: 8753.4cm 2 ; 75°: 13685.1cm 2 .
[0064] Example 2
[0065] This embodiment provides a method for culturing cells using the three-dimensional cell culture bottle in Example 1. Under a sterile environment, culture medium is injected into the reflux zone 2, and adherent cells to be cultured are added. The culture bottle is tilted and rotated on a bottle rotating machine, so that the cells and culture medium are transported to the outer surface of the central tube 5, the surface of the partition wall 7 and the spiral surface of the Archimedes spiral layer 6 through the Archimedes spiral layer 6 to adhere to the wall and divide and grow. The culture bottle is continuously rotated, and the culture medium circulates in the culture bottle through the reflux zone 2, the central tube 5, the liquid storage zone 4, the Archimedes spiral layer 6, and the reflux zone 2. After the culture is completed, the cells are separated from the culture surface by enzyme digestion or cooling, and the cells are collected.
[0066] The caps of the culture bottles are selected from breathable membrane caps and sterile transfer caps. The breathable membrane cap can provide gas exchange for the cell culture system during the cell culture process. The sterile transfer cap includes a gas filter and a liquid exchange tube. The culture area of the three-dimensional cell culture bottle is large, so during the culture process, in order to better proliferate the cells, the culture medium needs to be replaced when the nutrients in the culture medium are exhausted but the culture is not over yet. At this time, the culture system has two needs: ventilation and liquid replacement. The liquid exchange tube includes an internal port and two external ports. The tube extends into the bottle and passes through the central tube. The internal port is connected to the liquid storage area. One of the external ports is used for the input of fresh culture medium, and the other is used for the suction of the used culture medium in the liquid storage area.
[0067] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention of the utility model.
Claims
1. A three-dimensional cell culture flask, characterized in that: It comprises a culture bottle body; the interior of the culture bottle body comprises a reflux area, a culture area and a liquid storage area from top to bottom; The culture area is cylindrical, a hollow central tube is arranged on the central axis of the cylinder; at least one Archimedean spiral sheet is arranged on the outer wall of the central tube; at least one partition wall is arranged concentrically around the central tube; the Archimedean spiral sheet is connected to the partition wall; The central tube is connected with the reflux area and the liquid storage area; The pitch angle of the Archimedean spiral layer is 15-75°; The reflux zone is communicated with the bottle mouth of the culture bottle body, and the bottle mouth is connected to a breathable membrane cover or a sterile transfer cover.
2. The three-dimensional cell culture flask according to claim 1, characterized in that: The pitch angle is 60°.
3. The three-dimensional cell culture flask according to claim 1 or 2, characterized in that: When the number of partition walls is ≥2, the length of the Archimedean spiral layer between two adjacent partition walls is in the range of 12 to 18 mm.
4. The three-dimensional cell culture flask according to claim 3, characterized in that: The length of the Archimedean spiral layer between two adjacent partition walls is 16 mm; And / or, the number of the partition walls is 5.
5. The three-dimensional cell culture flask according to claim 1 or 2, characterized in that: When the number of Archimedean spiral sheets is ≥2, the pitch of the Archimedean spiral sheets is 10-15 mm; And / or, the number of the Archimedean spiral layers is 6 to 10.
6. The three-dimensional cell culture flask according to claim 5, characterized in that: The pitch of the Archimedean spiral layer is 12.5 mm; And / or, the number of the Archimedean spiral layers is 8.
7. The three-dimensional cell culture flask according to claim 1 or 2, characterized in that: The diameter of the central tube is 2-4 cm; And / or, the Archimedean spiral layer, the partition wall and the central tube are not connected to the bottom of the culture bottle.
8. The three-dimensional cell culture flask according to claim 1 or 2, characterized in that: The outer wall of the central tube, the surface of the partition wall or the surface of the Archimedean spiral layer are temperature-sensitive surfaces treated with temperature-sensitive materials.
9. The three-dimensional cell culture flask according to claim 1 or 2, characterized in that: The sterile transfer cap comprises a gas filter and a liquid exchange tube; the liquid exchange tube comprises an internal port and two external ports, one end of the internal port extends into the bottle and passes through the central tube, the internal port is connected to the liquid storage area, one of the external ports is used for inputting fresh culture medium, and the other is used for sucking out the used culture medium in the liquid storage area.
10. A culture system, characterized in that: A three-dimensional cell culture flask comprising any one of claims 1 to 9.
Citation Information
Patent Citations
Temperature-sensitive cell culture surface and preparation method thereof
CN110938323A
Cell culture bottle
US20220177817A1