Bioreactor

Abstract

The embodiment of the invention discloses a bioreactor. The bioreactor can comprise a reactor cabin arranged on the swing support, the reactor cabin comprises a shell and a reaction cavity in the shell, and the shell is provided with at least one liquid inlet connector and at least one filter-out connector; the liquid adding mechanism comprises a liquid inlet pipeline, and the liquid inlet pipeline is communicated with the reaction cavity through a liquid inlet connector; the filtering-out mechanism comprises a liquid discharging pipeline, and the liquid discharging pipeline is communicated with the reaction cavity through a filtering-out connector; and the controller is used for controlling the liquid adding mechanism to supplement liquid into the reaction cavity, controlling the filtering mechanism to discharge liquid from the reaction cavity and controlling the swinging amplitude of the swinging support. The method can be used for large-scale perfusion culture of organisms such as cells or microorganisms.

Description

Technical Field

[0001] This application relates to the field of organism culture technology, and more specifically, to a bioreactor. Background Technology

[0002] Currently, the cultivation of living microorganisms such as cells and microorganisms often utilizes swing-type bioreactors, such as the WAVE (wave-type) bioreactor. This type of bioreactor employs a sterile cell culture bag pre-filled with a cell retention membrane, enabling perfusion culture. Perfusion culture involves adding microorganisms or cells along with the culture medium to the reactor, and then continuously removing portions of the conditioned medium while simultaneously injecting fresh medium during the growth of the microorganisms or cells or the formation of products.

[0003] Currently, depending on the size of the WAVE bioreactor, cell culture can be achieved in volumes ranging from 0.1 liters to 25 liters. However, while such WAVE bioreactors are feasible for small-scale laboratory cell culture, they cannot meet the continuous medium-change requirements of perfusion culture processes for medium to large-scale production. Furthermore, the sterilized culture bags are disposable, expensive, and uneconomical, making them unsuitable for large-scale biological culture. Summary of the Invention

[0004] The technical solution provided in this application at least partially solves the above-mentioned technical problems.

[0005] According to one aspect of this application, a bioreactor is provided, comprising: a reactor chamber disposed on a rocking support, the reactor chamber including a shell and a reaction chamber within the shell, the shell having at least one liquid inlet and at least one filtration outlet; a liquid addition mechanism including a liquid inlet pipe connected to the reaction chamber via the liquid inlet; a filtration mechanism including a liquid discharge pipe connected to the reaction chamber via the filtration outlet; and a controller for controlling the liquid addition mechanism to add liquid to the reaction chamber, controlling the filtration mechanism to discharge liquid from the reaction chamber, and controlling the rocking amplitude of the rocking support.

[0006] In an exemplary embodiment, the filtration mechanism further includes a hollow fiber column for retaining substances contained in the liquid discharged from the reaction chamber.

[0007] In an exemplary embodiment, the liquid inlet pipeline includes multiple liquid inlet branches, each liquid inlet branch is connected to a liquid storage container at its inlet end, and each liquid inlet branch is equipped with a pump or flow valve; the controller is used to control the liquid addition mechanism to replenish the reaction chamber by controlling the pump or flow valve; wherein, the liquid storage container stores any one of the following: culture medium or cleaning solution.

[0008] In an exemplary embodiment, the reactor chamber further includes an air inlet; the bioreactor further includes an air inlet mechanism connected to the air inlet, the air inlet mechanism including one or more air inlet branches, each air inlet branch being provided with an air flow valve; the controller is also used to control the opening degree of the air flow valve to deliver one or more gases into the reaction chamber; wherein, the gas includes any one or more of the following: oxygen, carbon dioxide, nitrogen, and air.

[0009] In an exemplary embodiment, the bioreactor further includes a temperature control device; the controller is also used to control the temperature control device to control the temperature of the reactor chamber.

[0010] In an exemplary embodiment, the drain line is equipped with a peristaltic pump; the controller is also used to control the flow rate of liquid discharged from the reaction chamber by controlling the rotational speed of the peristaltic pump.

[0011] In an exemplary embodiment, the reaction chamber is made of 316L stainless steel, or the reaction chamber is a disposable culture bag.

[0012] In an exemplary embodiment, the bioreactor further includes a sensor array and a display unit; wherein the sensor array includes any one or more of the following: the sensors include a temperature sensor, an oxygen content sensor, a flow sensor, and a pH sensor; the display unit is used to display real-time data from the sensor array.

[0013] In an exemplary embodiment, the liquid addition mechanism is used to add culture medium to the reaction chamber, the culture medium including one or more culture substances; the filtration mechanism further includes a return pipeline for returning the filtered substances or culture medium to the reaction chamber.

[0014] In an exemplary embodiment, the temperature control device includes a heating plate.

[0015] The bioreactor provided according to the embodiments of this application can replace the culture medium in the reaction chamber in stages or continuously during the cell or microbial culture process, thereby realizing perfusion culture.

[0016] Furthermore, the bioreactor provided according to the embodiments of this application uses a hollow fiber column to retain cells in a tangential flow manner, which can effectively avoid clogging and adverse effects on cell growth compared to cell retention through a pre-placed cell retention membrane in a culture bag. Attached Figure Description

[0017] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments, with reference to the accompanying drawings:

[0018] Figure 1 A schematic diagram of the composition of a bioreactor according to Embodiment 1 of this application is shown;

[0019] Figure 2 A schematic diagram of the composition of a bioreactor according to Embodiment 2 of this application is shown.

[0020] Figure 3 A schematic diagram of the structure of a bioreactor according to Embodiment 2 of this application is shown;

[0021] Figure 4 A logic diagram of the controller of a bioreactor according to Embodiment 2 of this application is shown. Detailed Implementation

[0022] To better understand this application, various aspects of this application will be described in more detail with reference to the accompanying drawings. It should be understood that these detailed descriptions are merely illustrative of exemplary embodiments of this application and are not intended to limit the scope of this application in any way. Throughout the specification, the same reference numerals refer to the same elements. The expression "and / or" includes any and all combinations of one or more of the associated listed items.

[0023] It should also be understood that expressions such as "comprising," "including," "having," "containing," and / or "comprising" are open-ended rather than closed-ended expressions in this specification, indicating the presence of the stated features, elements, and / or components, but not excluding the presence of one or more other features, elements, components, and / or combinations thereof. Furthermore, when expressions such as "at least one of..." appear after a list of listed features, they modify the entire list of features, not just individual elements in the list. Additionally, when describing embodiments of this application, the word "may" is used to mean "one or more embodiments of this application." And the term "exemplary" is intended to refer to examples or illustrations.

[0024] Unless otherwise specified, all terms used herein (including engineering and technical terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should also be understood that, unless expressly stated herein, terms defined in common dictionaries shall be interpreted as having the meaning consistent with their meaning in the context of the relevant art, and not as having an idealized or overly formalized meaning.

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. Any one or more embodiments or elements thereof described herein, or arrangements or combinations of some or all embodiments or elements thereof, are merely exemplary and are not intended to limit the scope of this application.

[0026] As mentioned above, perfusion culture, as an important process in cell culture, provides a stable environment conducive to cell culture by continuously removing metabolic byproducts while continuously infusing new culture medium during cell growth and product formation.

[0027] Current perfusion culture devices mainly include stirred bioreactors and wave-type bioreactors. Compared to traditional stirred bioreactors, wave-type bioreactors use a shaking table to agitate the culture medium in disposable culture bags, increasing the contact area between gas and liquid, and promoting nutrient transfer and oxygen exchange efficiency. This gentle and efficient mixing method avoids the high shear force damage to cells caused by the stirring impeller, while also significantly improving the oxygen mass transfer coefficient, thus increasing cell density and yield.

[0028] However, the cell retention membrane built into the disposable culture bag can easily become clogged during the cell culture cycle, causing the entire perfusion culture to fail and thus affecting the culture cycle and culture cost.

[0029] The bioreactor provided in this application can at least solve or partially solve at least one of the problems existing in the aforementioned existing perfusion culture devices, or other problems. The features, principles, and other aspects of this application will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0030] Example 1

[0031] Figure 1 A bioreactor 100 according to one embodiment of this application is shown. This bioreactor 100 can be used, for example, for cell or microbial culture. For simplicity, embodiments of this application will be described below using cell culture as an example. However, it should be understood that this application is not limited to cell culture, but can also be used for the culture of organisms such as microorganisms.

[0032] Combination Figure 1 As shown, the bioreactor 100 may include a reactor chamber 10, a liquid addition mechanism 30, a filtration mechanism 40, and a controller (not shown in the figure).

[0033] The reactor chamber 10 may include a shell and a reaction chamber located within the shell, which may be a sealed shell. The reaction chamber may be made of materials that are easy to clean and sterilize, such as glass or 316L stainless steel, so that it can be repeatedly cleaned and sterilized before use. The reactor chamber 10 may be mounted on a swing support 20. In an exemplary embodiment, a controller may be used to control the swing amplitude of the swing support 20 to provide an optimal culture or growth environment for cells or microorganisms in the reactor chamber. Exemplarily, the swing support 20 may be provided with a swing mechanism, and the controller may control the swing mechanism through a drive mechanism to drive the swing support 20 to swing back and forth within a certain angle range. The controller may also control the swing amplitude and swing frequency of the swing mechanism to prevent cell precipitation or aggregation in the reaction chamber and to ensure uniform cell distribution in the reaction chamber.

[0034] In an exemplary embodiment, the reactor chamber 10 has at least one liquid inlet on its shell. The liquid filling mechanism 30 may include a liquid inlet line connected to the reaction chamber of the reactor chamber 10 via the liquid inlet. The liquid filling mechanism 30 may also include a pump or a flow valve.

[0035] In an exemplary embodiment, the reactor chamber 10 has at least one filter port connected to the filter mechanism 40. The filter mechanism 40 includes a drain pipe and a hollow fiber column 41. The drain pipe is connected to the reaction chamber of the reactor chamber 10 through the filter port. The hollow fiber column 41 is used to trap substances contained in the liquid discharged from the reaction chamber. The hollow fiber column 41 uses a tangential flow method, which is less likely to cause cell blockage, and it is easy to disassemble and replace during cell culture.

[0036] The membrane pore size and membrane surface area of ​​the hollow fiber column 41 can be flexibly set according to different requirements of the target product. In an exemplary embodiment, the selectable fiber pore size is, for example, 0.1 μm to 0.65 μm.

[0037] By using hollow fiber columns 41, cells or microorganisms of the target size can be effectively retained, while the culture medium containing metabolic waste is discharged from the bioreactor, thus retaining cells or microorganisms within the bioreactor. The bioreactor provided according to the embodiments of this application utilizes hollow fiber columns to filter the culture medium during cell culture, meeting the continuous and large-scale filtration needs of medium- to large-scale production without harming the cultured living cells. Furthermore, compared to disposable cell culture bags, it eliminates the need for pre-installed retention membranes in the cell culture bags, saving costs, and also avoids the clogging problems easily caused by retention membrane filtration.

[0038] In an exemplary embodiment, both the liquid addition mechanism 30 and the filtration mechanism 40 are electrically connected to the controller. The controller can control the liquid addition mechanism 30 to add or replenish liquid to the reaction chamber. For example, it can control the pump or flow valve in the liquid addition mechanism 30 to deliver or replenish culture medium (which may include one or more culture substances), buffer solution, etc., to the reaction chamber. At the same time, it can also control the filtration mechanism 40 to drain liquid from the reaction chamber. For example, by draining a certain amount of culture medium from the reaction chamber into the filtration mechanism 40, waste liquid containing metabolic waste can be filtered out.

[0039] The bioreactor provided in this application is suitable for cell suspension and the culture of various microorganisms. It allows for the phased or continuous replacement of the culture medium in the reaction chamber during cell or microbial culture, meaning that a portion of the waste liquid can be discharged while new culture medium is added, thus achieving perfusion culture. The reactor chamber includes a sealed shell, along with sterile interfaces and tubing, ensuring that the cell culture process is not contaminated by the external environment.

[0040] Furthermore, existing methods of cell retention using pre-placed cell retention membranes in culture bags often result in cell accumulation on the membrane surface due to the perpendicular flow direction of the liquid. This can easily cause blockage and negatively impact cell growth, sometimes necessitating the transfer of the entire culture to a new culture bag. In contrast, the bioreactor provided in this application uses a hollow fiber column with tangential flow to retain cells. Compared to cell retention using pre-placed cell retention membranes in culture bags, this method effectively avoids blockage and adverse effects on cell growth.

[0041] Example 2

[0042] Figure 2 A bioreactor 200 according to another embodiment of this application is shown, which can be used for cell or microbial culture. The bioreactor 200 may include a reactor chamber 10, a liquid addition mechanism 30, a filtration mechanism 40, a heating plate 50, and a controller (not shown). The reactor chamber 10 is located on a rocking support 20.

[0043] The liquid inlet pipeline of the liquid addition mechanism 30 may include multiple liquid inlet branches 31, and the inlet end of each liquid inlet branch 31 may be connected to different liquid storage containers, such as culture medium, washing solution, or buffer solution. In an exemplary embodiment, the liquid addition mechanism 30 is used to replenish culture medium into the reaction chamber, and the culture medium may include one or more culture substances required for cell growth. Each liquid inlet branch 31 is equipped with a liquid replenishment peristaltic pump or flow valve 32, which can be connected to a controller. The controller can control the type and flow rate of liquid replenished into the reaction chamber by controlling the liquid replenishment peristaltic pump or flow valve 32.

[0044] The filtration mechanism 40 may include a reflux filtration structure consisting of a drain pipe 42, a hollow fiber column 41, a return pipe 44, and a peristaltic pump 45. This structure draws a certain amount of culture medium from the reaction chamber, filters it through the hollow fiber column 41, and then returns a portion of the culture medium containing cells or other culture materials to the reaction chamber via the return pipe 44, while the remaining portion without cells or other culture materials is discharged. The controller can control the flow rate or velocity of the liquid drawn from the reaction chamber by controlling the rotational speed of the peristaltic pump 45.

[0045] In an exemplary embodiment, the filtration mechanism 40 may further include a pump 43 for periodically pumping cleaning fluid into the hollow fiber column 41 to rinse the fiber column and prevent cells from adhering inside the fiber column.

[0046] Combination Figure 2 and Figure 3 As shown, in an exemplary embodiment, the bioreactor 200 further includes an air intake mechanism 70, which may include one or more air intake branches, each equipped with an air intake flow control valve (also referred to herein as a flow valve). The air intake mechanism 70 is connected to the air intake interface 60 of the reactor chamber. As an exemplary embodiment, a controller can control the opening degree of each flow valve to deliver one or more gases into the reaction chamber, such as any one or more of the following gases: oxygen, carbon dioxide, nitrogen, and air.

[0047] In an exemplary embodiment, the bioreactor 200 may further include a temperature control device 50, which may include, but is not limited to, a heating plate. A controller can control the temperature control device 50 to control the temperature of the reactor chamber 10.

[0048] As an exemplary embodiment, the bioreactor 200 may further include a sensor group, which may include any one or more of the following sensors: a temperature sensor, an oxygen content sensor, a flow sensor, and a pH sensor. For example, the temperature sensor can monitor the temperature of the reaction chamber in real time, the flow sensor can monitor the flow rate or flow of the flow valve, and the oxygen content sensor and pH sensor can detect the dissolved oxygen content and pH of the culture medium in the reactor chamber.

[0049] As an exemplary embodiment, the bioreactor 200 also includes a display unit 80, which can be used to display data such as real-time temperature, flow rate of various liquids or gases, frequency and amplitude of swaying, dissolved oxygen and pH during culture, etc., collected by the sensor, or to display parameter settings related to cell culture.

[0050] The bioreactor according to this embodiment can control various growth conditions and environmental parameters required during cell culture in real time, thereby improving the reliability of the cell culture process.

[0051] Figure 4 A logical schematic diagram of a controller for a bioreactor provided according to an embodiment of this application is shown. For the sake of brevity, descriptions of structures similar to those in Embodiments 1 and 2 described above will be omitted in this embodiment.

[0052] Combination Figure 4 As shown, the bioreactor mainly includes a reactor chamber, a liquid addition mechanism, a filtration mechanism, an air inlet mechanism, and a controller.

[0053] As an exemplary embodiment, in addition to the reusable 316L stainless steel reaction chamber, the reactor chamber in this embodiment can also use disposable culture bags, such as microbial culture bags or cell culture bags.

[0054] The microbial culture bags or cell culture bags of this embodiment can be made of multi-layered biocompatible pharmaceutical-grade materials, with high mechanical strength and multiple interfaces such as liquid inlet, liquid outlet, and air inlet, making them compatible with various inoculation and feeding operations. They have good airtightness, which can largely prevent cell transfer between different containers during culture, reducing the risk of contamination, and require no cleaning or sterilization before use, simplifying operation. Furthermore, the cell culture bags are available in different sizes to suit the culture of various cell types and different production scales.

[0055] It should be noted that the microbial culture bag or cell culture bag of this application does not require a pre-placed cell retention membrane, which greatly reduces the processing difficulty and production cost, and is conducive to improving the convenience and economy of large-scale mass production.

[0056] According to the bioreactor provided in this application, by placing a culture bag containing culture medium and product inside the reactor chamber, and controlling the temperature of the heating plate, the swing amplitude of the swing support, and the gas flow rate through a controller, precise control of the temperature and gas content inside the reactor chamber can be achieved, so that the cultured cells or microorganisms have better cell viability and cell state.

[0057] In a specific application example, the method for achieving cell perfusion culture using any of the above-mentioned bioreactors may include the following steps:

[0058] Step a: The seed cells and culture medium are delivered to the reaction chamber of the reactor compartment via the liquid dispensing mechanism.

[0059] Step b: Control the swing amplitude of the swing support so that the cells and culture medium in the reaction chamber form a uniform suspension.

[0060] Step c involves supplying at least one gas into the reaction chamber via an air intake mechanism, and controlling the temperature of the reactor chamber via a temperature sensor and a temperature control device (e.g., a heating plate).

[0061] Step d: According to preset parameters and conditions, a certain amount of culture medium in the reaction chamber is discharged to the filtration mechanism, and the hollow fiber column is used to retain the cells and other substances contained in the culture medium.

[0062] In some implementations, the flow rate of liquid discharged from the reaction chamber can be controlled by setting the rotational speed of the peristaltic pump.

[0063] Step e involves continuously injecting new culture medium into the reaction chamber using a peristaltic pump.

[0064] In some implementations, the rotation speed of the replenishment peristaltic pump can be controlled by monitoring the flow rate of the discharged culture medium, thereby achieving a balance between the discharge and replenishment amounts.

[0065] Continuous perfusion culture of cells can be achieved through steps a through e described above. It should be noted that there is no fixed order between steps c, d, and e. Depending on the growth conditions of different cells and actual production needs, one or more of these steps may be omitted.

[0066] The above description is merely an illustration of the embodiments of this application and the technical principles employed. Those skilled in the art should understand that the scope of protection involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the technical concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A bioreactor, comprising: A reactor chamber is mounted on a rocking support. The reactor chamber includes a shell and a reaction chamber inside the shell. The shell is provided with at least one liquid inlet and at least one filter outlet. A liquid addition mechanism includes a liquid inlet pipe, which is connected to the reaction chamber through the liquid inlet interface; The filtration mechanism includes a drain pipe, which is connected to the reaction chamber through the filtration port; as well as The controller is used to control the liquid adding mechanism to replenish the reaction chamber, control the liquid discharging mechanism to drain the reaction chamber, and control the swing amplitude of the swing support.

2. The bioreactor according to claim 1, wherein, The filtration mechanism also includes a hollow fiber column, which is used to retain substances contained in the liquid discharged from the reaction chamber.

3. The bioreactor according to claim 1, wherein, The liquid inlet pipeline includes multiple liquid inlet branches, each of which is connected to a liquid storage container at its inlet end, and each of which is equipped with a pump or flow valve. The controller is used to control the liquid adding mechanism to replenish the reaction chamber by controlling the pump or the flow valve; The storage container contains any one of the following: culture medium or cleaning solution.

4. The bioreactor according to claim 1, wherein, The reactor chamber also has an air inlet; The bioreactor also includes an air intake mechanism connected to the air intake interface, the air intake mechanism including one or more air intake branches, each of the air intake branches being provided with an airflow valve; The controller is also used to control the opening degree of the gas flow valve to deliver one or more gases into the reaction chamber; The gas includes any one or more of the following: oxygen, carbon dioxide, nitrogen, and air.

5. The bioreactor according to claim 1, wherein, The bioreactor also includes a temperature control device; The controller is also used to control the temperature control device to control the temperature of the reactor chamber.

6. The bioreactor according to claim 1, wherein, The drainage pipeline is equipped with a peristaltic pump; The controller is also used to control the flow rate of liquid discharged from the reaction chamber by controlling the rotational speed of the peristaltic pump.

7. The bioreactor according to any one of claims 1-6, wherein, The reaction chamber is made of 316L stainless steel, or the reaction chamber is a disposable culture bag.

8. The bioreactor according to claim 7, wherein, The bioreactor also includes a sensor array and a display unit. The sensor group includes any one or more of the following: temperature sensor, oxygen content sensor, flow sensor, and pH sensor; The display unit is used to display real-time data from the sensor group.

9. The bioreactor according to claim 2, wherein, The liquid addition mechanism is used to add culture medium to the reaction chamber, and the culture medium includes one or more culture substances; The filtration mechanism also includes a return pipeline for returning the filtered material or culture medium to the reaction chamber.

10. The bioreactor according to claim 5, wherein, The temperature control device includes a heating plate.

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