Cell culture system, bioreactor

Abstract

This application provides a cell culture device and a bioreactor. The culture device includes: a mixer for mixing substances required for cell culture, the mixer including a mixing container and a mixing drive; the mixing container provides a sealed mixing space and is connected to the outside via a first interface and a second interface; the mixing drive is capable of driving the movement of the substances to be mixed; and a bioreactor including a reaction container and multiple culture trays, the reaction container providing a closed space, the multiple culture trays being disposed within the closed space; the closed space is connected to the outside via a third interface and a fourth interface, the third interface being connected to a second interface of the mixer, and the fourth interface being connected to a first interface of the mixer, providing perfusion culture within the bioreactor.

Description

Cell culture systems, bioreactors Technical Field

[0001] The present application relates to a cell culture system and a bioreactor. Background Art

[0002] In the prior art, adherent cell culture in vitro often utilizes two-dimensional static culture in open or semi-open containers, such as culture dishes, culture flasks, multilayer culture flasks, and cell factories. These containers typically undergo a tissue culture (TC) treatment on their bottom surfaces to facilitate adherent cell cultivation. The operator spots the cell suspension into these containers, adds a sufficient amount of culture medium, and uses a "cross" or "figure-of-eight" shaking motion to evenly disperse the cells throughout the container. The cells are then placed in a cell culture incubator for static adherence culture. The cells cling tightly to the bottom of the container, exchanging substances with the outside world through their free surfaces. Operators must regularly replace the culture medium within the container to ensure a stable cell growth environment.

[0003] The inventors have found that the cultivation method of the prior art introduced above has many disadvantages. On the one hand, there are many steps of human intervention in the cultivation process, the operation is cumbersome and causes a greater risk of contamination, the scale of cell culture is limited, and it is not suitable for large-scale cultivation. On the other hand, the cell culture mold is relatively simple and can only allow cells to adhere to the bottom surface of the cell culture device for cultivation, resulting in a large waste of culture space, and such "semi-open containers" are "shared in the same room" in the incubator, which easily causes large-scale cell contamination. In addition, the material exchange efficiency of cell adherence static culture is low, the growth rate is slow, and a large amount of nutrients are wasted in order to maintain a better cell growth environment. At the same time, this static culture method causes the cells to gradually lose their original properties in the body, and they are far from their natural growth state in the body in terms of morphology, structure and function. For example, the cells gradually become flatter, divide abnormally and lose their differentiated phenotype.

[0004] Therefore, the art needs a new cell culture system and bioreactor to solve the problems existing in the prior art. Summary of the Invention

[0005] According to the first aspect of the present application, a cell culture system includes: a mixer for mixing substances required for cell culture, the mixer including a mixing container and a mixing drive, the mixing container providing a sealed mixing space, connected to the outside world through a first interface and a second interface, and the mixing drive capable of driving the substances to be mixed to move and mix; a bioreactor including a reaction container and a plurality of culture plates, the reaction container providing a closed space, and the plurality of culture plates arranged in the closed space; the closed space is connected to the outside world through a third interface and a fourth interface, the third interface being capable of connecting to the second interface of the mixer, and the fourth interface being capable of connecting to the first interface of the mixer, providing perfusion culture in the bioreactor; the direction from the third interface to the fourth interface is defined as a first direction, a plurality of the culture plates are adjacently distributed in the first direction, at least some of the culture plates have through holes, and the through holes extend through the thickness of the culture plates in the first direction; preferably, multiple circles of the through holes are distributed from the center to the periphery of the culture plates, each circle having multiple through holes; more preferably, the bioreactor is a disposable consumable.

[0006] The beneficial effects of the cell culture system described above include, but are not limited to, the structure of the culture system connecting the mixer and the bioreactor, in particular, the third interface can be connected to the second interface of the mixer, and the fourth interface can be connected to the first interface of the mixer, providing perfusion culture in the bioreactor. In addition, the structure of culture plates with through holes is stacked in the bioreactor. The culture plates not only provide more locations for cell growth to attach, but also their arrangement direction and through hole structure further optimize the flow effect of the culture medium, further enhance the effect of perfusion culture of cells in the bioreactor, achieve sufficient material exchange between the culture medium and the cells in the bioreactor, improve the culture effect, make the cell growth rate faster, save culture medium, and also allow the cells to maintain a normal natural growth morphology.

[0007] In one or more embodiments of the cell culture system, the interval between adjacent culture plates is 0.5 mm to 10 mm; preferably, the interval between adjacent culture plates is 0.5 mm to 2 mm.

[0008] In one or more embodiments of the cell culture system, the plurality of adjacent culture trays are separated by a support member; or, a mounting shaft is provided in the closed space, and the plurality of adjacent culture trays are fixedly disposed adjacent to each other on the mounting shaft.

[0009] In one or more embodiments of the cell culture system, the reaction container further comprises a pressure interface, and the pressure interface is used to connect to a pressure source; preferably, the pressure source is a filtering mechanism or a pressurizing mechanism.

[0010] In one or more embodiments of the cell culture system, an observation window and an observation device are further included, wherein the observation window provides a visible surface to at least a portion of the enclosed space, and the observation device is optically connected to the visible surface; preferably, the observation device also includes a moving mechanism.

[0011] In one or more embodiments of the cell culture system, the bioreactor further comprises a heat exchange structure disposed on the periphery of the reaction vessel.

[0012] In one or more embodiments of the cell culture system, the reaction container is separately connected to the multiple culture plates, or is integrally connected to the multiple culture plates.

[0013] In one or more embodiments of the cell culture system, a rotation mechanism is further included, capable of driving the bioreactor to rotate around an axis.

[0014] In one or more embodiments of the cell culture system, the mixing container includes a mixing tank body, the mixing drive is arranged inside the mixing tank body, and includes a stirring paddle or a stirring rod; or, the mixing container includes a mixing bag, the mixing drive is arranged outside the mixing bag and connected thereto, and the mixing drive includes an oscillating shaker.

[0015] According to the second aspect of the present application, a bioreactor comprises a reaction vessel and a plurality of culture trays, wherein the reaction vessel provides a closed space, and the plurality of culture trays are arranged in the closed space; the closed space is connected to the outside world through a third interface and a fourth interface, the third interface is arranged below the reaction vessel, and the fourth interface is arranged above the reaction vessel; the direction in which the third interface points to the fourth interface is defined as a first direction, and a plurality of culture trays are adjacently distributed in the first direction, and at least some of the culture trays have through holes, and the through holes penetrate the thickness of the culture trays in the first direction; preferably, the bioreactor is used in the cell culture system as described in the first aspect; more preferably, the bioreactor is a disposable consumable; and still more preferably, the reaction vessel and the culture trays are manufactured integrally.

[0016] The beneficial effects of using the bioreactor introduced above include but are not limited to the structure of stacking culture plates with through holes in the bioreactor. The culture plates not only provide more locations for cell growth to attach, but their arrangement direction and the structure of the through holes also further optimize the flow effect of the culture medium, further enhance the effect of cell perfusion culture in the bioreactor, achieve sufficient material exchange between the culture material and the cells in the bioreactor, improve the culture effect, make the cell growth rate faster, save culture material, and also allow the cells to maintain a normal natural growth morphology.

[0017] According to a biological culture method according to the third aspect of the present application, a cell culture system as described in the first aspect is used, and the culture method includes: after the culture medium is mixed in the mixer, it flows into the reaction container through the second interface and the third interface, flows along the first direction and through the through holes of at least part of the culture plate, flows to the fourth interface, and flows back to the mixer through the first interface.

[0018] In one or more embodiments of the biological culture method, the method further includes cutting off the connection between the bioreactor and the mixer, closing one of the third and fourth interfaces of the bioreactor, and discharging the culture medium from the other interface; then, the other interface is closed, the one interface is opened, and an enzymatic solution is injected; after treatment, the one interface is closed, the other interface is opened, and the cell suspension is discharged.

[0019] The beneficial effects of the above-described biological culture method include, but are not limited to, achieving sufficient material exchange between culture materials and cells, improving culture effects, making cells grow faster, saving culture materials, and allowing cells to maintain their normal natural growth morphology.

[0020] Summary of the Figures

[0021] In order to make the above-mentioned objects, features and advantages of the present application more clearly understood, the specific embodiments of the present application are described in detail below with reference to the accompanying drawings, wherein:

[0022] FIG1 is a schematic structural diagram of a cell culture system according to an embodiment.

[0023] FIG2 is a schematic structural diagram of a mixer of a cell culture system according to an embodiment.

[0024] FIG3 is a schematic diagram of the internal structure of a bioreactor according to an embodiment.

[0025] FIG4 is a schematic diagram of the internal structure of a reaction container and a culture plate of a bioreactor according to an embodiment.

[0026] FIG5 is a schematic structural diagram of a culture tray of a bioreactor according to an embodiment.

[0027] FIG6 is a schematic structural diagram of a bioreactor and an observation device of a cell culture system according to an embodiment.

[0028] Preferred embodiments of the present invention

[0029] The following discloses a variety of different implementation methods or examples of the subject technical solutions. To simplify the disclosure, specific examples of the various elements and arrangements are described below. Of course, these are merely examples and are not intended to limit the scope of protection of the present invention. For example, a first feature described later in the specification is formed above or on a second feature, which may include an implementation method in which the first and second features are formed in a directly connected manner, or an implementation method in which an additional feature is formed between the first and second features, so that the first and second features may not be directly connected. In addition, the disclosures may repeat the figure marks and / or letters in different examples. This repetition is for brevity and clarity and does not in itself represent the relationship between the various implementation methods and / or structures to be discussed. Further, when a first element is described in a manner connected to or combined with a second element, the description includes an implementation method in which the first and second elements are directly connected or combined with each other, and also includes an implementation method in which one or more other intervening elements are added to indirectly connect or combine the first and second elements.

[0030] It should be understood that, depending on the actual situation, the preceding or following steps are not necessarily performed in exact order. Other steps may also be added to these processes, or one or more steps may be removed from these processes.

[0031] As shown in FIG1 , the cell culture system 100 may include a mixer 1 and a bioreactor 2 , which are separately arranged and connected via a pipeline. In addition, the cell culture system 100 may further include a control unit to control the mixer 1 and the bioreactor 2 to perform relevant steps of cell culture.

[0032] With reference to what is shown in Figure 2, the meaning of mixer 1 here is a device for mixing substances required for cell culture, for example, it can be gas-liquid mixing, or liquid-liquid mixing. Mixer 1 includes a mixing container 11 and a mixing drive 12. Mixing container 11 provides a sealed mixing space and can be communicated with the outside world through a first interface 111 and a second interface 112. The first interface 111 and the second interface 112 can be used for liquid inlet and liquid outlet, respectively. For example, the first interface 111 can be used to add culture medium, cleaning fluid, etc. or liquid flowing out of a bioreactor, and the second interface 112 can be used to flow out the mixed culture medium, cleaning fluid, etc. The mixing drive 12 can drive the substances to be mixed to move and mix. In certain embodiments, the mixing container can be a tank or bag structure, and its material can be metal, glass or plastic. The specific form of the mixing drive 12 can be a stirring paddle, a magnetic stirrer, or a shaking table arranged outside the mixing container, which can be arranged inside the mixing container.

[0033] Preferably, if the culture medium in the mixer 1 is a mixture of liquid and gas, the general mixing container 11 is also provided with a fifth interface and a sixth interface for air intake and air outlet. The gas entering through the fifth interface (air inlet) can be, for example, preferably a mixed gas of CO2, O2, or N2, but is not limited thereto. The sixth interface (air outlet) can be connected to a filter or a waterproof breathable membrane can be provided to balance the internal and external air pressures. When the internal pressure of the mixing container is greater than atmospheric pressure, the gas in the mixing container releases gas to the outside through the air outlet, achieving that the pressure in the mixing container is equal to atmospheric pressure. At the same time, due to the filtering effect of the filter or the waterproof breathable membrane, external particles, pathogens, etc. will not be introduced into the cell culture system.

[0034] Inside the mixing container 11 of the mixer 1, gases required for cell culture (such as air, oxygen and carbon dioxide) and / or reaction liquids (such as culture medium, cleaning solution or enzymatic solution) are mixed, for example, the culture medium is mixed with CO2, O2, N2 gases, or the culture medium is mixed with glucose, acid-base buffer, growth factors, etc.

[0035] Referring to Figure 2, in some embodiments, the specific structure of the mixer 1 can be that the mixing container 11 includes a mixing tank body 113 and a mixing tank cover 114, and the mixing tank cover 114 is provided with a first interface 111, the first interface 111 is connected to the liquid inlet pipe 118 and extends into the interior of the mixing tank body 113 to achieve liquid inlet. A second interface 112 is provided below the mixing tank body 113, and a sealing gasket 115 is also provided at the connection position between the mixing tank body 113 and the mixing tank cover 114, and can be fixedly connected by bolts. A stirring paddle 116 and its corresponding motor 117 are integratedly installed in the mixing tank cover 114. The number of stirring paddles 116 is not limited to the one group shown in the figure, and can also be multiple groups. For example, two groups of stirring paddles are provided along the direction of their installation axis to promote gas-liquid mixing in the mixing container. The upper blades can also prevent the generation of bubbles. The stirring paddle 116 is driven to rotate by the motor 117, and its rotation speed can be adjusted as needed. The rotation speed is preferably below 200rpm. It is understood that in some embodiments, the mixing drive 12 can also be a stirrer or a stirring paddle corresponding to a magnetic stirrer, which generates a rotating magnetic field through a motor to drive the stirring paddle or the stirring paddle inside the mixing tank to rotate, thereby achieving stirring, and the rotating speed can be adjusted as needed, and the rotating speed is preferably below 200rpm. In addition, in some embodiments, the mixing container can also be a mixing bag, such as a disposable mixing bag, the material of which is EVA (ethylene-vinyl acetate copolymer), and the mixing bag can also have the above-mentioned first interface and second interface for liquid inlet and outlet, as well as the fifth interface and sixth interface for air inlet and outlet, and the specific function is similar to that described above, and no further details are given here. If a mixing bag is used as the mixing container 11, then the corresponding mixing drive 12 can be a shaking table, which refers to a biological shaking table here, and the specific meaning is similar to that of the biological field, and no further introduction is given here.

[0036] In addition, a temperature sensor, and / or a dissolved oxygen sensor, and / or a pH sensor may be provided inside the mixer 1 to detect various parameters of the culture medium in the mixer 1, and corresponding adjustments may be made based thereon.

[0037] 3 to 6 , the bioreactor 2 is used to provide nutrients and an environment required for the growth of adherent cells, so as to perform various biological reactions of cell culture.

[0038] The bioreactor 2 may include a reaction vessel 21 and a plurality of culture trays 22. The reaction vessel 21 provides an enclosed space, and the plurality of culture trays 22 are disposed within the enclosed space. The reaction vessel 21 may include a reaction tank body 213 and a reaction tank cover 214. The material of the reaction vessel 21 may be stainless steel or plastic, such as polystyrene, polycarbonate, polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, polyvinyl pyrrolidone, polybutadiene, polyethylene oxide, polypyrrole, and polypropylene oxide, and may be disposable.

[0039] The enclosed space can be connected to the outside world through the third interface 211 and the fourth interface 212. For example, the third interface 211 is arranged at the lower part of the reaction tank body 213 and can be connected to the second interface 112 (i.e., the liquid outlet) of the mixer 1. The fourth interface 212 is arranged on the reaction tank cover 214 and can be connected to the first interface 111 (i.e., the liquid inlet) of the mixer 1, providing perfusion culture of the cells in the bioreactor 2. The meaning of perfusion culture here is similar to that in the art, that is, after the cells and culture medium are added to the reactor, part of the culture medium is continuously taken out during the cell growth and product formation process, and new culture medium is continuously perfused. It can be understood that the role of the third interface 211 is not limited to being connected to the second interface 112 to realize the input of the culture medium flowing out of the mixer, but can also be used to harvest the product of cell culture, which function will be described in detail later. The function of the fourth interface 212 located at the top is also not limited to outputting the culture medium to the first interface 111 of the mixer 1. For example, it can also be an interface for inoculating cells, adding cleaning solution, enzymatic solution, etc.

[0040] The direction from the third interface 211 to the fourth interface 212 is defined as a first direction, for example, as indicated by the arrows from bottom to top in Figures 3 and 4 . Multiple culture trays 22 are adjacently arranged in this first direction. The culture trays 22 can be disc-shaped, but are not limited thereto. As shown in Figure 5 , at least some of the culture trays 22 have through-holes 221 extending through the thickness of the culture tray 22 in this first direction. Preferably, all of the culture trays 22 have through-holes 221. The through-holes 221 allow cells or liquid in the reaction vessel to pass through, facilitating uniform distribution of the cells or culture medium across the culture trays 22. Preferably, as shown in Figure 5 , the through-holes 221 can be arranged in multiple circles from the center to the periphery of the culture tray 22, with each circle having multiple through-holes. For example, if the culture tray 22 is circular, multiple circles of through-holes 221 can be arranged from the center to the circumference. Each circle of through-holes is substantially the same size, with the outer circles having more through-holes due to their greater length.

[0041] The cell culture dish 22 can be made of a material with good biocompatibility, such as polystyrene, polycarbonate, polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, polyvinyl pyrrolidone, polybutadiene, polyethylene oxide, polypyrrole, polypropylene oxide, or glass. The dish can be disposable. Preferably, one or both sides of the cell culture dish 22 can be treated with a TC surface treatment to further facilitate the attachment and growth of adherent cells.

[0042] Through the above structure, especially the structure in which the culture system 100 is connected through the mixer 1 and the bioreactor 2, especially the third interface 211 can be connected to the second interface 112 of the mixer 1, and the fourth interface 212 can be connected to the first interface 111 of the mixer 1, perfusion culture is provided in the bioreactor 2, and a structure in which culture plates 22 with through holes 221 are stacked in the bioreactor 2. The culture plates 22 not only provide more locations for cell growth to attach, but also their arrangement direction (for example, up and down direction) and the structure of the through holes 221 make the culture medium dispersed better in the reaction vessel, further optimize the flow effect of the culture medium, further enhance the effect of cell perfusion culture in the bioreactor, realize sufficient material exchange between culture materials and cells in the bioreactor, improve the culture effect, make the cell growth rate faster, save culture materials, and also enable the cells to maintain a normal natural growth morphology.

[0043] The distribution structure of multiple culture plates 22 can be that the cell culture plates 22 are separated by supporting members, such as legs or gaskets. In some embodiments, referring to Figures 3 and 4, a central axis 215 can be provided inside the reaction tank body 213, and the central hole of the disc-shaped culture plate 22 passes through the central axis 215 and is fixed on the central axis to limit and separate the cell culture plates. For example, a slot structure can be provided on the central axis for fixing. Generally speaking, the interval between adjacent culture plates 22 is 0.5mm to 10mm, preferably 0.5mm to 2mm, to achieve high-density attachment and proliferation of adherent cells.

[0044] It can be understood that the reaction vessel 21 and the culture plate 22 can be split structures, which are processed separately and then assembled into the bioreactor 2. At this time, the reaction vessel 21 can generally be reused after being fully washed and disinfected, while the culture plate is a disposable consumable. However, this is not limited to the above. For example, the two can be an integrally formed structure. During manufacturing, the reaction vessel 21 and the culture plate 22 are directly manufactured as one body. In this case, there is no need to assemble the two. At this time, the entire bioreactor 2 is a disposable consumable.

[0045] As shown in FIG3 , in some embodiments, the bioreactor 2 further includes a heat exchange structure 23 disposed on the periphery of the reaction vessel 21. The heat exchange structure 23 generally functions to provide heating. For example, it may be in the form of a water bath thermostat and may include a temperature sensor and a heating device to achieve constant temperature heating of the reaction vessel 21. However, this is not intended to be limiting. For example, the heat exchange structure 23 may also cool the reaction vessel 21. For example, when the ambient temperature is too high, the reaction vessel 21 may need to be cooled to maintain a suitable temperature for the bioreactor.

[0046] Referring to FIG4 , in some embodiments, the reaction vessel 21 further includes a pressure port 217 for connecting to a pressure source, such as a filter or a waterproof breathable membrane, and for connecting to the outside atmospheric pressure. Its function can be similar to that of the gas outlet of the mixer 1. When the internal pressure of the reaction vessel 21 is greater than atmospheric pressure, the gas in the tank is released to the outside through the filter or the waterproof breathable membrane, thereby achieving an equilibrium between the pressure inside the reaction vessel 21 and atmospheric pressure. Simultaneously, due to the filtering effect of the filter or the waterproof breathable membrane, external particles, pathogens, and the like are prevented from entering the reaction vessel 21. Furthermore, the pressure port 217 can also be connected to other pressure sources, such as a pressurizing mechanism, to provide periodic pressure fluctuations to the liquid culture medium inside the reaction vessel 21. The pressure fluctuations can be, for example, 110 kPa to 115 kPa, for simulating cell culture under human blood pressure conditions. The pressurizing mechanism can be a common piston cylinder, peristaltic pump, etc., but is not limited thereto.

[0047] With reference to what is shown in Figure 6, in some embodiments, the reaction vessel 21 may further include an observation window 218, such as an observation window of glass or other transparent material (such as PMMA) provided on the reaction tank cover 214, wherein the observation window 218 provides a visible surface to at least part of the enclosed space. In addition, the culture system 100 may further include an observation device 4, which is optically connected to the visible surface provided by the above-mentioned observation window 218. For example, the observation device may be a microscope observation system for online real-time observation of cell growth status, such as cell implantation, cell division growth, cell planar distribution density, etc. The microscopic observation system is arranged above the reaction vessel 21, and the reaction vessel 21 has an observation window, which may be one or more observation windows. The microscopic observation system may include a coaxial light source microscope 41, which can automatically focus and shoot the culture status of cells on the culture dish 22 through the observation window. Preferably, there are multiple observation windows. Preferably, the observation device 4 may further include a moving mechanism to drive the coaxial light source microscope 41 to move and observe the cell culture status in different observation windows. It is understood that the observation device 4 may be in other forms as long as it can obtain the cell production status inside the reaction container 21 through the observation window 218. The observation results obtained may be static images or dynamic images, without limitation.

[0048] Continuing with FIG. 5 , in some embodiments, the culture system 100 may further include a rotation mechanism 5 capable of driving the bioreactor 2 to rotate about an axis. For example, this could be a one-dimensional rotary motor that provides a certain rotation angle range, such as ±30°. This mechanism can be used to ensure uniform cell seeding during the cell seeding phase through inversion and shaking. During cell harvesting after the culture phase, the bioreactor can be inverted and disturbed to facilitate separation of adherent cells from the culture dish 22.

[0049] It can be understood that the present application also provides a bioreactor, whose structure is similar to the bioreactor 2 of the cell culture system 100 described in the above embodiment, and will not be repeated here.

[0050] According to the cell culture system 100 introduced above, the specific cell culture process can be.

[0051] Pre-cleaning: Inject the culture medium into the mixing container 11 through the pipeline, start the first peristaltic pump to rotate forward, and pump the culture medium into the reaction container 21. After the reaction container 21 is full, the culture medium flows back to the mixing container 11 from the fourth interface 212 at the top. Repeat for 1 hour, collect the culture medium and test it to ensure that the collected culture medium is not contaminated by bacteria or viruses.

[0052] Cell seeding: The cell suspension is injected into the input bag. The third port 211 of the reaction vessel 21 is closed. A second peristaltic pump is then used to inject the cell suspension into the reaction vessel 21 through the fourth port 212 via the input bag. The reaction vessel 21 can then be shaken for approximately 10 minutes using the rotation mechanism 5 to ensure uniform cell seeding. For example, for human cells, the reaction vessel can be left in a water bath at a constant temperature of 37°C. Cell production can be monitored using the observation device 4 to ensure complete cell attachment.

[0053] Cell culture: The culture medium is injected into the mixing container 11 from the first interface 111 through the pipeline, connecting the third interface 211 of the reaction container 21 with the second interface 112 of the mixing container 11, and starting the first peristaltic pump to rotate forward. The culture medium is mixed with nitrogen, air, and carbon dioxide in the mixing container 11, providing the environment and nutrients required for cell growth. The mixed culture medium is transported to the interior of the reaction container 21 through the third interface 211 at the bottom of the reaction container 21 by the first peristaltic pump. The reacted culture medium flows back to the mixing container 11 from the fourth interface 212 at the top of the reaction container 21 and is mixed with nitrogen, air, and carbon dioxide again. During the culture process, the cell growth can be observed under a microscope.

[0054] During cell culture, for example, a control mechanism can be used to control the pH value in the mixer 1 and the bioreactor 2 to 7.5±0.02, the DO value to 40%±10%, and the temperature to 37±0.2°C. The flow rate of the mixer 1 can be 50 mL / min.

[0055] Harvesting cells: Close the fourth interface 212 on the upper portion of the reaction container 21, switch the third interface 211 on the lower portion of the reaction container 21 to connect to the waste liquid bag, reverse the first peristaltic pump, and recover the culture supernatant into the waste liquid bag. Close the third interface 211 on the lower portion of the reaction container 21, open the fourth interface 212 on the upper portion of the reaction container 21, use the second peristaltic pump to inject the enzymatic solution into the reaction container 21, and use the rotating mechanism 5 to drive the reaction container 21 to flip and shake. After standing for a period of time (for example, about 1 minute to 5 minutes), open the third interface 211 on the lower portion of the reaction container 21, reverse the first peristaltic pump, and harvest all the cell suspension into the harvest bag.

[0056] As described above, the present application further provides a cultivation method, which uses the cultivation system 100 described in the above embodiment. The cultivation method includes the following steps:

[0057] After being mixed in the mixer 1, the culture medium flows into the reaction container 21 through the second interface 112 and the third interface 211, flows along the first direction and through at least part of the through hole 221 of the culture plate 22, flows to the fourth interface 212, and flows back to the mixer 1 through the first interface 11.

[0058] This achieves sufficient material exchange between culture materials and cells, improves the culture effect, makes the cell growth rate faster, saves culture materials, and allows the cells to maintain their normal natural growth form.

[0059] In one or more embodiments of the culture method, as described above, the cell harvesting step can be as follows: disconnecting the bioreactor 2 from the mixer 1, closing one of the third and fourth interfaces 211 and 212 of the bioreactor (e.g., the fourth interface 212), and draining the culture medium from the other interface (e.g., the third interface 211); then, the other interface (e.g., the third interface 211) is closed, the one interface (e.g., the fourth interface 212) is opened, and an enzymatic solution is injected; after treatment, the one interface (e.g., the fourth interface 212) is closed, the other interface (e.g., the third interface 211) is opened, and the cell suspension is drained. This achieves a relatively simple and easy-to-operate cell harvesting step, further improving cell production efficiency.

[0060] In summary, the beneficial effects of the cell culture system, bioreactor, and culture method described above include, but are not limited to, a structure in which the cell culture system is connected to the mixer and the bioreactor, in particular, the third interface can be connected to the second interface of the mixer, and the fourth interface can be connected to the first interface of the mixer, thereby providing perfusion culture in the bioreactor. In addition, a structure in which culture plates with through holes are stacked in the bioreactor not only provides more locations for cell growth to attach, but also further optimizes the flow effect of the culture medium and the structure of the through holes, thereby further enhancing the effect of perfusion culture of cells in the bioreactor, achieving sufficient material exchange between the culture material and the cells in the bioreactor, improving the culture effect, making the cell growth rate faster, saving culture material, and allowing the cells to maintain a normal natural growth morphology.

[0061] Although the present invention is disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Any person skilled in the art may make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, any modifications, equivalent variations, and modifications made to the above embodiments in accordance with the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A cell culture system, characterized in that, Comprising: A mixer for mixing substances required for cell culture, the mixer including a mixing container and a mixing driving member, the mixing container providing a sealed mixing space, communicating with the outside through a first interface and a second interface, and the mixing driving member being capable of driving the substances to be mixed to move and mix; A bioreactor including a reaction container and a plurality of culture plates, the reaction container providing a closed space, the plurality of culture plates being disposed within the closed space; the closed space communicating with the outside through a third interface and a fourth interface, the third interface being capable of communicating with the second interface of the mixer, and the fourth interface being capable of communicating with the first interface of the mixer to provide perfusion culture within the bioreactor; defining the direction from the third interface to the fourth interface as a first direction, and a plurality of the culture plates being adjacently distributed in the first direction, at least some of the culture plates having through holes that penetrate the thickness of the culture plates in the first direction; Preferably, in the direction from the center to the periphery of the culture plate, there are multiple circles of the through holes, and each circle has a plurality of the through holes; More preferably, the bioreactor is a disposable consumable.

2. The cell culture system according to claim 1, characterized in that, The interval between adjacent ones of the culture plates is 0.5 mm to 10 mm; Preferably, the interval between adjacent ones of the culture plates is 0.5 mm to 2 mm.

3. The cell culture system according to claim 1, characterized in that, Adjacent ones of the plurality of culture plates are separated by a support member; alternatively, a mounting shaft is provided within the closed space, and adjacent ones of the plurality of culture plates are fixedly disposed adjacent to the mounting shaft.

4. The cell culture system according to claim 1, wherein The reaction container further includes a pressure interface for communicating with a pressure source; Preferably, the pressure source is a filtering mechanism or a pressurizing mechanism.

5. The cell culture system according to claim 1, wherein It further includes an observation window and an observation device, the observation window providing a visible surface for at least a part of the closed space, and the observation device being optically communicated with the visible surface; Preferably, the observation device further includes a moving mechanism.

6. The cell culture system according to claim 1, wherein The bioreactor further includes a heat exchange structure disposed outside the reaction container.

7. The cell culture system according to claim 1, characterized in that, The reaction container is connected to the plurality of culture plates separately or integrally.

8. The cell culture system according to claim 1, wherein It further includes a rotating mechanism capable of driving the bioreactor to rotate about an axis.

9. The cell culture system according to claim 1, wherein The mixing container includes a mixing tank body, the mixing driving member is disposed inside the mixing tank body and includes a stirring paddle or a magnetic stirrer; alternatively, the mixing container includes a mixing bag, the mixing driving member is disposed outside the mixing bag and connected thereto, and the mixing driving member includes an orbital shaker.

10. A bioreactor, characterized in that, Comprising: A reaction container and a plurality of culture plates, the reaction container providing a closed space, the plurality of culture plates being disposed within the closed space; the closed space communicating with the outside through a third interface and a fourth interface, the third interface being disposed below the reaction container, and the fourth interface being disposed above the reaction container; defining the direction from the third interface to the fourth interface as a first direction, and a plurality of the culture plates being adjacently distributed in the first direction, at least some of the culture plates having through holes that penetrate the thickness of the culture plates in the first direction; Preferably, the bioreactor is used for the cell culture system according to any one of claims 1-9; More preferably, the bioreactor is a disposable consumable; Even more preferably, the reaction vessel is integrally manufactured with the culture dish.

11. A biological culture method, using the cell culture system according to any one of claims 1-9, the culture method comprising: After being mixed in the mixer, the culture medium flows into the reaction vessel through the second interface and the third interface, flows along the first direction and through the through holes of at least a part of the culture dish, flows to the fourth interface, and flows back to the mixer through the first interface.

12. The biological culture method according to claim 11, characterized in that, It further includes disconnecting the connection between the bioreactor and the mixer, closing one of the third interface and the fourth interface of the bioreactor, and discharging the culture medium through the other interface; then closing the other interface, opening the one interface, injecting the enzyme solution, closing the one interface after treatment, opening the other interface, and discharging the cell suspension.

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