Cell culture method, cell culture container, and cell culture apparatus

Abstract

The present invention places a clean and sterilized one-step container body (1) in a microenvironment, adds a cell culture solution into the one-step container body (1), and gradually adds a replenishing solution into the one-step container body (1) as the culture time becomes longer. The one-step container body (1) has at least two inner surfaces with different areas, and the inner surfaces with different areas form a corresponding number of culture surfaces (2) for culturing cell solutions with different volumes. The cell solution is first placed and cultured on a culture surface (2) with a relatively small area, and when the volume of the cell solution increases, the cell solution is transferred to a culture surface (2) with a relatively large area for culturing, thereby providing a cell culture method. The present invention provides a cell culture container comprising a one-step container body (1). The one-step container body (1) has at least two inner surfaces with different areas, and the inner surfaces with different areas form a corresponding number of culture surfaces (2) for culturing cell solutions with different volumes. This cell culture method and cell culture container can meet the demand for cell solution culture, enhance the cell culture effect, easily realize automated production, and have high production efficiency.

Description

Technical Field

[0001] [Related Application] This application claims priority based on the content of a Chinese patent application with an application date of June 24, 2022, an application number of 202210726988.4, and an invention title of "Cell Culture Method and Cell Culture Vessel", and the full text of this Chinese patent application is incorporated herein by reference in its entirety.

[0002] The present invention relates to the technical fields of food, medicine, and life sciences, and particularly to a cell culture method, a cell culture vessel, and a cell culture device.

Background Art

[0003] Cell culture refers to all in vitro cultures, that is, it means taking out tissues from living animals and placing them under specific in vivo conditions that simulate the physiological environment in the body so that they can survive and grow. General-purpose cell culture bags have a large gas exchange area, are useful for cell culture, and can obtain higher-quality cells. In addition, direct microscopic observation can be performed, and the cell culture process and other operations can be monitored. Since the entire process is aseptic and the operation is simple, it is widely used. As shown in FIG. 10, in the process of cell culture, when a small amount of cell solution is spread in a relatively large-volume cell culture vessel as the initial amount of cell culture, the cell solution will evaporate immediately, the osmotic pressure of the culture medium will become too high, the cells will shrink, fall into cell dysfunction, and ultimately may cause cell apoptosis. On the other hand, when the amount of cell solution is large as the initial amount of cell culture, the cell density may become too low and cell growth may be slow. In addition, in vitro, cell-cell interaction is required for the logarithmic growth of cells. When the cell density is low, the cell proliferation rate will decrease significantly. Furthermore, using a large amount of cell solution as the initial amount of cell culture not only wastes costs but also requires more blood to be collected from patients, which is also undesirable.

[0004] Chinese Patent Application No. 201822257139.0 proposes a novel cell culture bag, in which a seal groove and a seal strip are provided in the center of the culture bag, and half of the culture solution in the culture bag can be quantitatively extracted by using the drainage port on the culture bag.

[0005] Chinese Patent Application No. 201510906678.0 proposes a cell culture bag with a three-dimensional structure. Specifically, it proposes a cell culture bag with a three-dimensional structure integrating a reagent connector, a sensor, and a sampling bag, which can more effectively utilize the sealed culture space compared with a flat culture bag.

[0006] Chinese Patent Application No. 202020480642.7 proposes a variable-volume cell culture bag. The volume of the cell culture bag can be adjusted through the partition clip of the bag body, and it can be applied to a small amount of cell solution as the initial amount of cell culture, and can also collect and monitor PH and DO during the cell culture process.

[0007] Chinese Patent Application No. 202110782370.5 proposes a large-capacity suspension cell culture bottle. By attaching ventilation membranes to the upper and lower parts of the culture bottle, the ventilation volume during the culture process can be increased, and the cell culture effect can be improved.

[0008] The above-mentioned patent technical documents propose some solutions in the cell culture process, but they have the disadvantages of being difficult to meet the culture needs when the volume continues to increase, having a poor culture effect, being difficult to achieve automatic production, and having low production efficiency.

Summary of the Invention

Problems to be Solved by the Invention

[0009] The technical problem to be solved by the present invention is to overcome the drawbacks of the prior art, accommodate an increase in the culture volume of the cell solution, improve the cell culture effect, enable easy cell culture, and realize automated production to enhance production efficiency, and to provide a cell culture method, a cell culture container, and a cell culture device.

Means for Solving the Problem

[0010] To solve the above-mentioned problems, the present invention adopts the following technical solutions. The present invention provides a cell culture method, which comprises placing a clean and sterilized one-step container body in the microenvironment of cell culture, adding a cell culture solution necessary for cell culture into the one-step container body, and gradually adding a replenishing solution necessary for cell culture into the one-step container body as the culture time lengthens. The one-step container body is provided with at least two inner surfaces of different areas, and the inner surfaces of different areas form a corresponding number of culture surfaces for culturing cell solutions of different volumes. The cell solution is placed and cultured on a culture surface with a relatively small area. When the volume of the cell solution increases, the one-step container body is rotated to transfer the cell solution to a culture surface with a relatively large area for culture. The cell culture solution contains liquids necessary for cell culture such as cell fluid and culture solution. The replenishing solution contains a culture medium and other nutrients used for cell growth and proliferation. The cell solution can also be called a cell suspension. The following content is provided as a further improvement measure for the above-mentioned technical solution. During the process of culturing the cell solution on the culture surface, the one-step container body is oscillated to make the cell distribution and temperature of the cell solution in the one-step container body uniform, and to increase the gas exchange rate in the cell solution. During the culturing process of the cell solution, the one-step container body is periodically rotated to the sampling position, left for a certain period of time, and then real-time cell growth information is collected and monitored. After the cell solution reaches a predetermined concentration, the one-step container body is moved to the position for discharging the supernatant. After standing for a certain period of time to allow the cultured cells to settle sufficiently, a part of the supernatant in the one-step container body is discharged to reduce the total volume of the cell solution. Based on the same technical concept, the present invention provides a cell culture container. The cell culture container includes a one-step container body. The one-step container body has at least two inner surfaces with different areas, and the inner surfaces with different areas form a corresponding number of culture surfaces for culturing cell solutions with different volumes. A flow path is provided in the one-step container body, and a ventilation membrane or airway for gas exchange is provided in the one-step container body. The following content is provided as a further improvement measure for the above-mentioned technical solution. The one-step container body is a rigid one-step culture flask. The one-step container body includes two end caps installed opposite to each other, and a rotating seat is provided on the end cap. The culture surface includes polygonal side members arranged parallel to the rotation axis around the rotation axis of the one-step container body. The flow path is provided on the rotation axis of the end cap and extends to the side member. There are at least two flow paths, and one of the flow paths is used for discharging the supernatant. The flow path extends to the culture surface with the smallest area. An observation window for collecting images and / or a sampling port for collecting samples are provided on the one-step container body. A plurality of ventilation holes are provided on the side wall where the culture surface is located, and a ventilation membrane that allows gas to permeate but does not allow the solution to permeate is laid on the culture surface. The one-step container body is a flexible one-step culture bag, and a standard case is installed outside the one-step container body. The standard case fixes the one-step container body so that the one-step container body forms culture surfaces with different areas. A plurality of fixing parts are provided in the fixed case, and the one-step container body is detachably attached in the fixed case by each fixing part. An exchange port is provided on one side of the fixed case, and the fixed case is provided with a sealing lid at the exchange port. A rotating seat is provided on the fixed case. A heating mechanism is provided on the side wall corresponding to the fixed case and each culture surface. Based on the same technical concept, the present invention provides a cell culture device, which includes an incubator and the above-described cell culture container. The one-step container body is detachably attached in the incubator, and a rotation driving mechanism for rotating the one-step container body is provided in the incubator. The following content is provided as a further improvement measure for the above-described technical solution. A driving rotation connector and a driven rotation connector are respectively provided on both sides of the incubator. The one-step container body is detachably attached between the driving rotation connector and the driven rotation connector, and the driving rotation connector is connected to the rotation driving mechanism.

Effects of the Invention

[0011] Compared with the prior art, the advantages of the present invention are as follows. In the cell culture method of the present invention, as the volume of the cell solution increases, by rotating the one-step container body, the cell solution can be transferred onto a culture surface with an area corresponding to its volume for culturing, the depth of the cell solution can be maintained so as not to be too low, and the cell solution can be prevented from evaporating immediately. Also, the depth of the cell solution can be prevented from being too high, preventing the cell density from becoming too low and the growth rate from becoming too slow, meeting the need to culture a cell solution whose volume continues to increase, and enhancing the cell culture effect. Further, this cell culture method is realized by rotating the one-step container body to different culture surfaces, enabling one-step continuous cell culture, capable of using either single-use containers or reusable containers, being convenient for automated production when combined with an automated system, and enhancing production efficiency, thus solving the problems of complicated conventional culture operations, realizing automation, and reducing the usage amount and cost of the one-step container body.

[0012] In the cell culture container of the present invention, during the cell culture process, when the volume of the cell solution increases, by rotating the one-step container body, the cell solution can be transferred onto a culture surface with an area corresponding to its volume for culturing, the depth of the cell solution is maintained so as not to be too low, preventing the cell solution from evaporating rapidly, and also its depth can be controlled so as not to be too high, thus preventing the cell density from becoming too low or the growth rate from becoming too slow, meeting the need for culturing when the volume increases, and enhancing the cell culture effect. Furthermore, this cell culture container can be realized by rotating the one-step container body to different culture surfaces, is convenient for automated production when combined with an automated system, and has high production efficiency.

[0013] When the volume of the cell solution in the cell culture container of the present invention increases, it rotates at a relatively small rotation angle, positions a culture surface with an area corresponding to its volume to be horizontally arranged at the bottom for culturing the cell solution, and is highly efficient and low energy-consuming.

[0014] In the cell culture container of the present invention, a plurality of ventilation holes and ventilation membranes are provided on the side wall where the culture surface is located, which together form a ventilation system for the cell culture container, and the ventilation volume during the culture process can be increased.

[0015] Since the standard case of the cell culture container of the present invention has a rigid structure, this cell culture container is a soft-rigid composite, and is easy to manufacture.

[0016] In the cell culture container of the present invention, the one-step container body is detachably attached to the inside of the standard case by each fixing part, which is convenient for replacing the one-step container body. The standard case is provided with a sealing lid at the replacement port. When removing the one-step container body, after opening the sealing lid, the one-step container body can be taken out by releasing the fixation of the fixing part to the one-step container body. Therefore, the cell culture container of the present invention has a simple structure and convenient operation.

[0017] The cell culture device of the present invention has all the technical features of the cell culture container and all the advantages of the cell culture container.

Brief Description of the Drawings

[0018]

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Explanation of Reference Numerals

[0019] 1, One-step container body; 11, Sapwood; 12, End cap; 121, Flow path; 13, Flow path; 14, Air passage; 2, Culture surface; 21, Vent hole; 22, Observation window; 23, Sampling port; 24, Virus injection port; 3, Rotating base; 4, Ventilation membrane; 5, Standard case; 6, Fixed part; 7, Exchange port; 8, Sealing lid; 9, Heating mechanism; 91, Incubator; 92, Rotation drive mechanism; 93, Main driving rotation connector; 94, Driven rotation connector.

Embodiments for Carrying Out the Invention

[0020] Hereinafter, the present invention will be described in detail in conjunction with the drawings and specific embodiments. In the following description, terms such as "upper", "lower", "front", "rear", "side", "bottom", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings. For convenience, they are used to make it easier to explain the present invention and simplify it. They do not imply that the devices or components mentioned must be located in a specific orientation or indicate the structure and operation in a specific direction, and are not intended to limit the present invention.

[0021] Also, in the following description, "a plurality" means two or more unless explicitly and particularly limited. In this specification, unless specifically described and limited otherwise, terms such as "combination", "connection", "attachment", "fixation" and other terms should be understood in a broad sense and can be interpreted, for example, as a fixed connection, a removable connection, or an integrated structure. Furthermore, the connection can be either a mechanical connection or an electrical connection. Additionally, the connection can be a direct connection or can be made through another structure between two structures. It can also be interpreted as a connection within two structures or an interaction between two structures. A person skilled in the art can understand the specific meaning of the above terms in this specification according to specific situations.

[0022] Embodiment 1 In the cell culture method of this embodiment, first, a clean and sterile (cleaned and sterilized) one-step container body 1 is loaded into a microenvironment for cell culture. After adding a cell culture medium necessary for cell culture to the one-step container body 1, as the culture time elapses (as the culture time becomes longer), a supplementary solution necessary for cell culture is gradually added to the one-step container body 1. The one-step container body 1 has at least two inner surfaces with different areas, and the inner surfaces with different areas form a corresponding number of culture surfaces 2 for culturing cell solutions with different volumes. The cell solution is first cultured on the culture surface 2 with a small area. When the volume of the cell solution increases, the one-step container body 1 is rotated, and the cell solution is transferred to the culture surface 2 with a large area for culturing.

[0023] In the cell culture method of this embodiment, when the volume of the cell solution increases, the one-step container body 1 is rotated to transfer the cell solution onto the culture surface 2 that can face it for culture (when the volume of the same cell solution is the same, as the area of the culture surface 2 increases, the height of the cell solution decreases). As a result, the depth of the cell solution can be maintained so that it is not too low, and the cell solution can be prevented from evaporating immediately. Also, the depth of the cell solution being too high can be avoided, preventing the cell density from becoming too low and the growth rate from becoming slow, and preventing the cells in the cell solution from being unable to come into contact with air. Thus, the conditions required for culturing the cell solution with a continuously increasing volume can be satisfied, and the cell culture effect can be improved. Also, this cell culture method is realized by rotating the one-step container body 1 to use different culture surfaces 2. During the culture process, even if the volume of the cell solution increases, there is no need to replace the container, and one-step (using multiple locations individually in the same container for cell culture from start to finish) continuous cell culture can be completed. Note that in this cell culture method, a single-use container can be used, but a reusable container can also be used. Combining it with an automated system is convenient for automated production and can increase production efficiency.

[0024] In this embodiment, during the process of culturing the cell solution on the culture surface 2, by rocking the one-step container body 1, the cell distribution of the cell solution in the one-step container body 1 and the temperature of the cell solution can be made uniform, and the cell culture effect can be improved.

[0025] In this embodiment, during the process of culturing the cell solution on the culture surface 2, the one-step container body 1 is periodically rotated to a position where the culture surface 2 with the minimum area is in the lower horizontal position, allowed to stand for a certain period of time, and then real-time cell growth information can be collected and monitored. Alternatively, the cell solution can also be extracted as a sample through the sampling port 23. After the cell solution reaches a predetermined concentration, the one-step container body 1 is rotated to a position where the culture surface 2 with the minimum area is in the lower horizontal position. In this case, the one-step container body 1 is in a state where the supernatant can be discharged. After standing for a certain period of time to sufficiently precipitate the cultured cells, a part of the supernatant in the one-step container body 1 can be discharged to reduce the total volume of the cell solution. As shown in FIG. 2, a virus injection port 24 is provided near the sampling port 23. The virus injection port 24 adds lentivirus at the initial stage of cell culture to perform T cell transfection, enabling effector T cells to stably express CAR.

[0026] Embodiment Two: FIGS. 1 to 7 show an embodiment of the cell culture container of the present invention. This cell culture container includes a one-step container body 1. The one-step container body 1 has at least two inner surfaces with different areas, and the inner surfaces with different areas form a corresponding number of culture surfaces 2 for culturing cell solutions with different volume capacities.

[0027] Cell culture process: When the volume of the cell solution increases, the one-step container body 1 is rotated to transfer the cell solution onto the culture surface 2 with an area corresponding thereto for culturing, thereby maintaining the depth of the cell solution from being too low and avoiding the cell solution from evaporating immediately. Also, it can be avoided that the depth of the cell solution is too high, preventing the cell density from becoming too low and the growth rate from being too slow. This meets the conditions required for culturing the cell solution with a continuously increasing volume and improves the cell culture effect. In addition, in this cell culture container, it is realized by rotating the one-step container body 1 to adapt to different culture surfaces 2, which is advantageous for automatic production when combined with an automated system and can improve production efficiency.

[0028] In this embodiment, a rotating base 3 is provided on the one-step container body 1, and each culture surface 2 is sequentially arranged surrounding the rotation axis of the one-step container body 1, and the culture surfaces 2 are sequentially connected and the areas are sequentially enlarged. In this way, when the volume of the cell solution increases, by only rotating at a small angle, the culture surface 2 with a larger area can be positioned at the bottom correspondingly, and the culture surface 2 can be horizontally arranged so that the cell solution is placed on the culture surface 2, which is highly efficient and consumes less energy.

[0029] In this embodiment, a plurality of ventilation holes 21 are provided on the side wall where the culture surface 2 is located, and a ventilation membrane 4 through which air permeates but the solution does not permeate is laid on the culture surface 2. The plurality of ventilation holes 21 and the ventilation membrane 4 on the side wall where the culture surface 2 is located constitute a ventilation system of the cell culture container, and the ventilation volume during the culture process can be increased.

[0030] The one-step container body 1 can realize the rocking during the culture process, and can improve the cell distribution, temperature uniformity and optimal ventilation effect of the culture solution at different rotation angles.

[0031] In this embodiment, the one-step container body 1 is a rigid one-step culture bottle. The one-step container body 1 includes a polygonal side member 11 and two end caps 12 respectively arranged at both ends of the side member 11. The culture surface 2 is formed on the inner surface of the side member 11, the rotating base 3 is provided at the center of the end cap 12, and a flow path 121 is communicated between the outside of the rotation center of the end cap 12 and the inside of the narrowest culture surface 2. The flow path 121 extends from the center of the end cap 12 to the side member 11, and one end of the rotating base 3 and the flow path 121 are both provided at the center of the end cap 12. The flow path 121 is arranged on the rotation axis so that the position of the end of the flow path does not change during rotation, whereby a replenishing solution can be added during the culture. An observation window 22 and a sampling port 23 for collecting samples are provided on the side wall where the narrowest culture surface 2 is provided.

[0032] The number of channels 121 is two, one of which is used to discharge the supernatant liquid. Since the supernatant liquid is located above the cell solution, the channel 121 extends above the culture surface 2 and has a certain distance from the culture surface 2. The other channel 121 is used for operations such as inflow and outflow of liquid. In order to discharge all the solution, the channel 121 extends to the culture surface 2. For cell cultures that require sampling, if the sample is collected from the channel 121, there is a possibility that the cell solution will be wasted because the channel 121 is too long. As shown in FIG. 3, the two channels 121 are arranged at 180° along the rotation axis, one of which is connected to the small-area culture surface 2, the connection port of the channel 121 is located on the small-area culture surface 2, and the connection port of the other channel 121 is located on the opposite small-area culture surface 2. The connection port has a certain height from the small area culture surface 2 and is used to drain the upper supernatant liquid after the culture is completed.

[0033] Due to the structure of the flow path 121 and the rotating seat 3, the one-step container body 1 can realize automatic addition of replenishment liquid, automatic discharge of supernatant liquid, and automatic collection during the rotation process, and can function as an alternative to manual operation.

[0034] In the process of culturing the cell solution on the culture surface 2, the one-step container body 1 is periodically rotated to a position where the culture surface 2 with the minimum area is in a lower horizontal position, and after leaving it for a certain period of time, real-time cell proliferation information is collected through the observation window 22. The collection position is a position where information can be collected from the observation window 22 using an information collecting device such as a camera. The observation window 22 in this embodiment is disposed on the culture surface 2 with the minimum area, and after the cell solution reaches a predetermined concentration, the one-step container body 1 is rotated to a position where the culture surface 2 with the minimum area is in a lower horizontal position, in which case the one-step container body 1 is in a state where the supernatant liquid can be discharged, and after leaving it for a certain period of time to allow the cultured cells to fully settle, a part of the supernatant liquid in the one-step container body 1 is discharged through the flow channel 121 to reduce the total volume of the cell solution.

[0035] As shown in Fig. 7, in this embodiment, the culture surface 2 is configured such that the culture area increases in the order of small surface, medium surface, and large surface. Fig. 7(a) shows the case of culturing the cell solution using the small surface, Fig. 7(b) shows the case of culturing the cell solution using the medium surface, and Fig. 7(c) shows the case of culturing the cell solution using the large surface. As the number of culture days increases, the volume of the cell solution also gradually increases, and the angle of the one-step container body 1 is adjusted to change the culture surface 2. That is, after culturing using the small surface, it is changed to culture using the medium surface, and finally, culture is performed using the large surface. During cell culture, the one-step container body 1 is oscillated.

[0036] Table 1, Table 2, and Table 3 show the number of cultures, survival rates, and densities for different culture days on different culture surfaces. The first group, the second group, the third group, and the fourth group each represent four identical one-step container bodies 1. From Table 1, Table 2, Table 3, Fig. 12, Fig. 13, and Fig. 14, during culture, as the number of culture days increases, the culture volume increases. By changing the culture surface 2, it is possible to prevent the decrease in the cell oxygen dissolution rate of intermediate cells due to the increase in volume and the accumulation of the cell solution, meet the requirement of increasing the need to culture a larger amount of the cell solution as the volume increases, and improve the cell culture effect.

[0037] Table 1: Number of cultures for different culture days on different culture surfaces 2 JPEG2025520777000002.jpg41154

[0038] Table 2: Cell survival rates for different culture days on different culture surfaces 2 JPEG2025520777000003.jpg41149

[0039] Table 3: Cell densities for different culture days on different culture surfaces 2 JPEG2025520777000004.jpg42147

[0040] Embodiment Three: Figures 8 and 9 show another embodiment of the cell culture container of the present invention. This cell culture container comprises a one-step container body 1. The one-step container body 1 has at least two inner surfaces with different areas. The inner surfaces with different areas form a corresponding number of culture surfaces 2 for culturing cell solutions with different volumes. The outside of the one-step container body 1 is surrounded by a standard case 5. The one-step container body 1 is a flexible one-step culture bag. The one-step culture bag is shaped by the standard case 5 to form at least two inner surfaces with different areas. In the present invention, the number of inner surfaces includes, but is not limited to, two, three, four, or five. Each culture surface 2 is sequentially connected and its area increases sequentially. The standard case 5 is provided with a rotating seat 3, and each culture surface 2 is sequentially arranged around the rotation center of the one-step container body 1. The standard case 5 has a rigid structure, and this cell culture container is an assembly of a soft material and a hard material, which is easy to manufacture. In another embodiment, the standard case 5 is made of a non-metallic material such as metal or plastic rubber. The standard case 5 is used in combination with the one-step container body 1. In some cases, the culture volume may be from several tens of milliliters to several thousand milliliters, or from several liters to several thousand liters.

[0041] The one-step container body 1 is provided with a flow path 13 and a gas exchange airway 14. The flow path 13 consists of two parts. One is used for sample collection, and the other is used for the entry and exit of the solution. Similarly, the airway 14 also consists of two parts. One is used for air supply, and the other is used for exhaust.

[0042] In this embodiment, a plurality of fixing parts 6 are provided on the standard case 5. The one-step container body 1 is detachably fixed in the standard case 5 by each fixing part 6. An exchange port 7 is provided on one side of the standard case 5, and a sealing lid 8 is provided at the exchange port 7 of the standard case 5. The one-step container body 1 is detachably fixed to the standard case 5 by each fixing part 6, and with this structure, it is easy to remove and replace the one-step container body 1. The standard case 5 is provided with a sealing lid 8 at the exchange port 7. When removing the one-step container body 1, the sealing lid 8 is opened and the fixing of the fixing part 6 to the one-step container body 1 is released, so that the one-step container body 1 can be removed. The structure of the present invention is simple and the operation is also convenient.

[0043] In this embodiment, a heating mechanism 9 is provided on the side wall of the standard case 5 corresponding to the culture surface 2. Thereby, it is convenient to heat the cell solution.

[0044] Embodiment Four: FIG. 11 shows an embodiment of the cell culture device of the present invention. The cell culture device of this embodiment includes an incubator 91 and the cell culture container of Embodiment One. The one-step container body 1 is detachably mounted in the incubator 91. A rotation drive mechanism 92 for rotationally driving the one-step container body 1 is provided in the incubator 91.

[0045] Driven by the rotation drive mechanism 92, the one-step container body 1 rotates, and the cell solution can be cultured on the culture surfaces 2 with different corresponding areas. This cell culture device has all the technical features of the cell culture container and all the advantages of the cell culture container.

[0046] In this embodiment, a main drive connector 93 and a driven rotation connector 94 are respectively provided on both sides of the incubator 91. The one-step container body 1 is detachably attached between the main drive connector 93 and the driven rotation connector 94, and the main drive connector 93 is connected to the rotation drive mechanism 92. The one-step container body 1 is detachably attached between the main drive connector 93 and the driven rotation connector 94. For example, both sides of the one-step container body 1 are fixed between the main drive connector 93 and the driven rotation connector 94 in a manner that allows for easy disassembly and assembly. The rotation drive mechanism 92 is used to rotationally drive the main drive connector 93 to rotate the one-step container body 1. An entrance / exit for the one-step container body 1 to enter and exit is provided on the side of the incubator 91.

[0047] As described above, the preferred embodiments of the present invention have been explained, but the present invention is not limited thereto. Those skilled in the art can make many changes and modifications to the technical solution of the present invention, or modify the technical solution of the present invention into equivalent changes, without departing from the scope of the technical solution of the present invention by using the technical content disclosed above. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical solution of the present invention without departing from the technical solution of the present invention are included within the scope of protection of the technical solution of the present invention.

Claims

1. A cell culture method, comprising: placing a clean and sterilized one-step container body (1) in a microenvironment for cell culture, adding a cell culture solution necessary for cell culture into the one-step container body (1), and gradually adding a replenishing solution necessary for cell culture into the one-step container body (1) as the culture time increases; the one-step container body (1) is provided with at least two inner surfaces of different areas, and the inner surfaces of different areas form a corresponding number of culture surfaces (2) for culturing cell solutions of different volumes. The cell solution is placed and cultured on a culture surface (2) with a relatively small area. When the volume of the cell solution increases, the one-step container body (1) is rotated to transfer the cell solution to a culture surface (2) with a relatively large area for culture. Each of the culture surfaces (2) is installed so as to surround the axis of rotation of the one-step container body (1). A cell culture method characterized by this.

2. During the process of culturing the cell solution on the culture surface (2), the one-step container body (1) is oscillated to make the cell distribution and temperature of the cell solution in the one-step container body (1) uniform. The cell culture method according to claim 1, characterized by this.

3. During the culture process of the cell solution, the one-step container body (1) is periodically rotated to a sampling position, left for a certain period of time, and then real-time cell proliferation information is collected and monitored. After the cell solution reaches a predetermined concentration, the one-step container body (1) is rotated to a position for discharging the supernatant. After standing for a certain time to allow the cultured cells to settle sufficiently, a part of the supernatant in the one-step container body (1) is discharged to reduce the total volume of the cell solution. The cell culture method according to claim 1, characterized by this.

4. A cell culture container comprising a one-step container body (1), the one-step container body (1) is provided with at least two inner surfaces of different areas, and the inner surfaces of different areas form a corresponding number of culture surfaces (2) for culturing cell solutions of different volumes. A flow path (121, 13) is provided in the one-step container body (1), and an air-permeable membrane (4) or an airway (14) for gas exchange is provided in the one-step container body (1). Each of the culture surfaces (2) is installed so as to surround the axis of rotation of the one-step container body (1). A cell culture container characterized by this.

5. The one-step container body (1) is a rigid one-step culture flask. The one-step container body (1) includes two end caps (12) installed opposite to each other. A rotating base (3) is provided on the end cap (12). The culture surface (2) includes a polygonal side member (11) arranged parallel to the rotation axis around the rotation axis of the one-step container body (1). The cell culture container according to claim 4, characterized in that.

6. The flow path (121) is provided on the rotation axis of the end cap (12) and extends to the side member (11). There are at least two flow paths (121). One of the flow paths (121) is used to discharge the supernatant. The cell culture container according to claim 5, characterized in that.

7. The flow path (121) extends to the culture surface (2) with the smallest area. The one-step container body (1) is provided with an observation window (22) for collecting images and / or a sampling port (23) for collecting samples. The cell culture container according to claim 6, characterized in that.

8. The one-step container body (1) is a flexible one-step culture bag. An outer mold case (5) is installed outside the one-step container body (1). The mold case (5) fixes the one-step container body (1) so that the one-step container body (1) forms culture surfaces (2) with different areas. The cell culture container according to claim 4, characterized in that.

9. A plurality of fixing parts (6) are provided in the mold case (5). The one-step container body (1) is detachably attached to the inside of the mold case (5) by each fixing part (6). An exchange port (7) is provided on one side of the mold case (5). The mold case (5) is provided with a sealing lid (8) at the exchange port (7). The mold case (5) is provided with a rotating base (3). The cell culture container according to claim 8, characterized in that.

10. The mold case (5) is provided with a heating mechanism (9). The cell culture container according to claim 8 or 9, characterized in that.

11. Comprising: An incubator (91) and the cell culture container according to any one of claims 4 to 9. The one-step container body (1) is detachably attached in an incubator (91), and the incubator (91) is provided with a rotation drive mechanism (92) for rotationally driving the one-step container body (1). A cell culture device characterized by this.

12. On both sides of the incubator (91), a driving rotary connector (93) and a driven rotary connector (94) are respectively provided. The one-step container body (1) is detachably attached between the driving rotary connector (93) and the driven rotary connector (94), and the driving rotary connector (93) is connected to the rotation drive mechanism (92). The cell culture device according to claim 11, characterized by this.

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