Perfusion-type cell culture bag and cell perfusion system

Abstract

The present invention relates to a perfusion-type cell culture bag, comprising a bag body, wherein an inner wall of the bag body is provided with a membrane material; one or more perfusion regions are provided in the bag body; each perfusion region is fixedly connected to the inner wall of the bag body; each perfusion region has an internal cavity; a perfusion filter membrane is provided on a partial or the entire surface of the perfusion region; the internal cavity is filled with a spacer mesh for supporting the perfusion region; and a liquid output tube is provided in each perfusion region and is in communication with the outside of the bag body.

Description

A perfusion cell culture bag and cell perfusion system Technical Field

[0001] This invention relates to the field of cell culture technology, and specifically to a cell culture bag and cell perfusion system for cell perfusion culture. Background Technology

[0002] Disposable bioreactors use disposable bags as culture containers, with the stirrer integrated inside. The sealed bags and stirrers are pre-sterilized. In use, the disposable bag is installed in the fixed reactor container, and then the stirrer is connected to the actuator. Due to the significant advantages of mammalian cell perfusion culture technology in terms of product yield, quality, and cost, its application is becoming increasingly widespread, making the development and optimization of perfusion culture processes a current research hotspot in mammalian cell culture technology.

[0003] The most commonly used perfusion methods in the industry are ATF (Automatic Transmission Flow) and TFF (Transfer Flow). ATF works by using a diaphragm pump as a power source, causing the cell culture medium to move alternately within a hollow fiber tube. During this movement, cells are trapped and returned to the bioreactor, while the culture medium permeates through the hollow fibers to the harvest end. TFF also uses hollow fiber tangential flow technology, but the difference is that the TFF system can generate a stronger scouring effect on the surface of the hollow fiber filaments through a higher flow rate, thus reducing membrane clogging. While ATF or TFF systems can achieve perfusion culture, because they rely on a power pump to circulate the culture medium, damage to cells or microcarriers by the pump cannot be avoided, especially adversely affecting shear-sensitive cell cultures. Furthermore, ATF or TFF systems are independent of the bioreactor, making it impossible to control parameters such as temperature, pH, and dissolved oxygen (DO), thus affecting the overall culture environment of the bioreactor. Additionally, the hollow fiber membranes used in perfusion culture are expensive, resulting in high costs. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a perfusion cell culture bag, comprising a bag body with a membrane material on its inner wall, one or more perfusion zones disposed within the bag body, the perfusion zones being fixedly connected to the inner wall of the bag body, each perfusion zone having an internal cavity, and part or all of the surface of the perfusion zone being covered with a perfusion filter membrane; the internal cavity is filled with a diaphragm mesh for supporting the perfusion zones, and an outlet pipe is disposed on the perfusion zones communicating with the outside of the bag body.

[0005] Furthermore, the internal cavity is formed in the following manner:

[0006] The perfusion filter membrane covers a portion of the inner wall of the bag, and the periphery of the perfusion filter membrane 3 is sealed to the membrane material; or,

[0007] Enclosed by a perfusion filter membrane; or,

[0008] The perfusion filter membrane forms the first side, and a membrane made of the same or different material forms the second side, with the edges of the first and second sides bonded together.

[0009] Furthermore, a stirring mechanism is also provided inside the bag.

[0010] Furthermore, the distance h from the lower edge of the irrigation zone to the bottom of the bag is 5%-50% of the total bag height H.

[0011] Furthermore, the irrigation zone covers 1%-80% of the inner wall area of ​​the bag.

[0012] Furthermore, the number of filter membrane layers is at least one.

[0013] Furthermore, the pore size of the perfusion filter membrane is 0.1um-500um.

[0014] Furthermore, the lower part of the outer wall of the bag is also provided with a sampling interface, a liquid outlet, a temperature measurement interface, a pH measurement interface, and a DO measurement interface; the sampling interface is used to sample and detect the cell production status, and the liquid outlet is used to extract the supernatant; the temperature measurement interface, pH measurement interface, and DO measurement interface are respectively used to install sensors to measure the temperature, pH, and DO value of the culture bag.

[0015] Furthermore, the upper part of the outer wall of the bag is also provided with an exhaust port, a feeding port, a surface ventilation port, and a liquid inlet. The exhaust port is used to discharge the culture exhaust gas inside the culture bag, the feeding port is used to add factors and other materials required for the cell culture process to the culture bag, the surface ventilation port is used to introduce sterile air into the bag, and the liquid inlet is used to add culture medium during the culture process.

[0016] Furthermore, a ventilation plate is provided at the bottom of the bag, and the ventilation plate is provided with a full drainage port and a deep ventilation port. The full drainage port and the deep ventilation port are connected to the inside of the bag. The full drainage port is used to collect cell culture products, and the deep ventilation port is used to supply the gas required for cell culture into the bag.

[0017] Furthermore, the liquid outlet pipeline includes a first bag buckle, a first silicone tube, a first right-angle elbow, a second silicone tube, a second right-angle elbow, a third silicone tube, and a double-hook bag buckle connected in sequence.

[0018] The present invention also provides a cell perfusion system, including an external tubing, a drive pump, and a waste bag. The external tubing includes a second bag buckle, a fourth silicone tube, and the aforementioned perfusion cell culture bag. One end of the second bag buckle is connected to the outlet tubing, and the other end is connected to one end of the fourth silicone tube. The other end of the fourth silicone tube is connected to the waste bag. The drive pump is located in the middle of the fourth silicone tube. When the drive pump is running, it continuously squeezes the fourth silicone tube to extract the culture medium or liquid from the waste bag from the perfusion functional area.

[0019] Furthermore, the liquid outlet pipeline includes a first bag buckle, a first silicone tube, a first right-angle elbow, a second silicone tube, a second right-angle elbow, a third silicone tube, and a double-hook bag buckle connected in sequence, with one end of the second bag buckle connected to the double-hook bag buckle.

[0020] In this invention, the traditional culture medium filtration device (hollow fiber tube / membrane) is placed inside the culture bag from the outside of the culture bag, which has the following advantages: (1) Cells or microcarriers are left inside the culture bag through the perfusion filtration membrane. During the operation of the drive pump, the cells or microcarriers will not flow through the drive pump, thereby avoiding the squeezing of the cells or microcarriers, reducing the damage to the cells or microcarriers, and improving the quality of cell culture; (2) What is extracted from the culture bag is the culture medium that has consumed nutrients, and the waste liquid goes directly into the waste liquid tank. The cells or microcarriers are always inside the bag, which is conducive to controlling the stability of the temperature, pH, DO and other parameters of the entire culture environment; (3) The perfusion filtration membrane and the liquid outlet pipeline are part of the culture bag and form an integral whole with the culture bag, without occupying additional space; (4) The perfusion area is fixed. When the stirring direction and / or stirring speed of the stirring paddle are controlled, and / or the swing angle and speed of the culture bag are controlled, the culture system will form a certain speed and / or flow towards the liquid flow, which washes the carriers and cells on the filter membrane and prevents them from accumulating and clogging the filter membrane. Attached Figure Description

[0021] Figure 1 is a structural diagram of a perfusion cell culture bag;

[0022] Figure 2 is a top view of the irrigation area;

[0023] Figure 3 is a cross-sectional view of AA in Figure 1;

[0024] Figure 4 is a cross-sectional view of BB in Figure 1;

[0025] Figure 5 is a schematic diagram of the perfusion system using the perfusion cell culture bag of the present invention.

[0026] Figure 6 is a comparison of the perfusion rates between the swing cell culture bag (2D) and the stirred cell culture bag (3D).

[0027] Figure 7 shows the change in perfusion rate as a function of pump speed during adherent cell perfusion culture. Detailed Implementation

[0028] Referring to Figures 1-2, the perfusion cell culture bag 10 of the present invention includes a bag body 1, a stirring mechanism 2 is provided inside the bag body 1, a perfusion filter membrane 3 is also provided inside the bag body 1, a membrane material 11 is provided in a portion of the inner wall of the bag body 1, the perfusion filter membrane 3 covers the membrane material 11 to form a perfusion zone 4 with an internal cavity, the perfusion zone 4 is filled with a diaphragm mesh 5 for supporting the perfusion zone 4, and an outlet pipe 6 is connected to the membrane material 11 to connect the perfusion zone to the outside.

[0029] The distance h from the lower edge of the irrigation zone 4 to the bottom of the bag body is 5%-50% of the total height H of the bag body. The irrigation zone covers 1%-80% of the inner wall area of ​​the bag body 1, and the distance D between the irrigation filter membrane 3 and the inner wall of the bag body 1 is 0-300mm.

[0030] In the above embodiment, the perfusion filter membrane 3 covers part of the membrane material 11 on the inner wall of the bag body 1, and the periphery of the perfusion filter membrane 3 is welded to the membrane material 11 to form the perfusion zone 4. Alternatively, the perfusion zone 4 can be directly formed from the filter membrane 3, that is, the filter membrane 3 can be made into a flat bag, which is then placed inside the bag body 1 and welded to the inner wall of the bag body 1; or the filter membrane 3 and the membrane material can be first formed into a flat bag (one side is the filter membrane, and the other side is a membrane material that is the same as or different from the inner wall of the bag body 1), and then this flat bag can be placed inside the bag body 1 and welded to the inner wall of the bag body 1. The advantage of this is that the perfusion zone can be prepared in advance and then welded to the bag body 1. Preferably, one side of the perfusion zone 4 has the same material as the membrane material of the inner wall of the bag body 1, which facilitates welding.

[0031] Because a portion of the bag body 1 has a fixed infusion zone 4, when the liquid inside the bag body 1 is stirred, the stirring paddle will not touch the filter membrane on the infusion zone 4, thus preventing damage to the infusion zone 4.

[0032] It is important to note that the connection between the irrigation zone 4 and the bag body 1 requires careful consideration of the influence of the stirring paddle. In existing technologies, the bag body 1 is typically 2D in shape, formed by welding two membrane materials around their perimeters. During use, the swaying of the bag body has minimal impact on the irrigation zone. However, in this invention, because a stirring paddle is installed inside the bag, the entire bag body is 3D. During stirring, it is crucial to ensure that the irrigation zone does not drift or undergo elastic deformation to avoid contact with the stirring paddle. Therefore, the irrigation zone must be welded to the bag body 1 to achieve a fixed connection between the irrigation zone and the bag body.

[0033] Referring to Figures 1-4, a ventilation plate 7 is also provided at the bottom of the bag body 1. The ventilation plate 7 is provided with a full drainage port 71 and a deep ventilation port 72. The full drainage port 71 and the deep ventilation port 72 are connected to the inside of the bag body. The full drainage port 71 is used to collect cell culture products, and the deep ventilation port 72 is used to supply the gas required for cell culture into the bag body.

[0034] The lower part of the outer wall of the bag body 1 is also provided with a sampling interface 12, a liquid outlet 13, a temperature measurement interface 14, a pH measurement interface 15, and a DO measurement interface 16. The sampling interface 12 is used to sample and detect the cell production status, and the liquid outlet 13 is used to discharge the supernatant; the temperature measurement interface 14, pH measurement interface 15, and DO measurement interface 16 are respectively used to install corresponding sensors to measure the temperature, pH, and DO value of the culture bag.

[0035] The upper part of the outer wall of the bag body 1 is also provided with an exhaust port 21, a feeding port 22, a surface ventilation port 23, and a liquid inlet 24. The exhaust port 21 is used to discharge the culture exhaust gas in the culture bag, the feeding port 22 is used to add factors and other materials required for the cell culture process to the culture bag, the surface ventilation port 23 is used to introduce sterile air into the bag, and the liquid inlet 24 is used to add culture medium during the culture process.

[0036] Referring to Figure 5, which is a schematic diagram of the perfusion system using the perfusion cell culture bag of the present invention, the perfusion system includes a perfusion cell culture bag 10, an external tubing 30, a drive pump 40, and a waste liquid bag 50. Inside the culture bag 10, the aforementioned outlet tubing 6 includes, in sequence, a first bag buckle 61, a first silicone tube 62, a first right-angle elbow 63, a second silicone tube 64, a second right-angle elbow 65, a third silicone tube 66, and a double-barbed bag buckle 67. The outlet tubing 6 is located inside the culture bag, allowing for arbitrary placement of the double-barbed bag buckle 67 and the filter membrane, and enhancing the sealing between the external connection and the inside of the outlet tubing. Since the inside of the culture bag is a sterile environment, this eliminates the sealing problems associated with direct external connection to the inside of the culture bag.

[0037] The external tubing 30 includes a second bag buckle 31 and a fourth silicone tube 32. One end of the second bag buckle 31 is connected to the double-barbed bag buckle 67, and the other end is connected to one end of the fourth silicone tube 32. The other end of the fourth silicone tube 32 is connected to the waste bag 50. The drive pump 40 is located in the middle of the fourth silicone tube 32. When the drive pump 40 is running, it continuously squeezes the fourth silicone tube 32 to extract the culture medium from the perfusion functional area. Cells or microcarriers are trapped by the filter membrane and do not enter the perfusion tubing. They are ultimately collected through the cell culture bag 10. The drive pump 40 is located in the middle of the fourth silicone tube 32. When the drive pump 40 is running, it continuously squeezes the fourth silicone tube 32 to extract the liquid from the waste bag through the perfusion functional area. The liquid at a certain flow rate backwashes the filter membrane to prevent cells or carriers from accumulating on the filter membrane and preventing blockage. The drive pump 40 is controlled by a control program to run continuously at a certain speed, so that the culture medium is extracted from the culture bag at a specified rate. At the same time, fresh culture medium and gas are added to the culture bag through other culture bag interfaces, thereby realizing the cell perfusion culture process.

[0038] In this invention, the traditional culture medium filtration device (hollow fiber tube / membrane) is placed inside the culture bag from the outside of the culture bag, which has the following advantages: (1) Cells or microcarriers are left inside the culture bag through the perfusion filter membrane 3. During the operation of the drive pump, the cells or microcarriers will not flow through the drive pump, thereby avoiding the squeezing of the cells or microcarriers, thus reducing the damage to the cells or microcarriers and improving the quality of cell culture; (2) What is extracted from the culture bag is the culture medium that has consumed nutrients, and the waste liquid goes directly into the waste liquid tank. The cells or microcarriers are always inside the bag, which is conducive to controlling the stability of the temperature, pH, DO and other parameters of the entire culture environment; (3) The perfusion filter membrane 3 and the liquid outlet pipe 6 are themselves part of the culture bag, forming an integral whole with the culture bag, without occupying additional space; (4) The perfusion area 4 is fixed. When the stirring direction and / or stirring speed of the stirring paddle are controlled, and / or the swing angle and speed of the culture bag are controlled, the culture system will form a certain speed and / or flow towards the liquid flow, which washes the carriers and cells on the filter membrane and prevents them from accumulating and clogging the filter membrane.

[0039] Filter membranes separate fluid components through their pore size and material. The choice of pore size depends on the filtration purpose; for example, 0.1-10µm pores are considered microfiltration membranes, capable of removing microparticles, colloids, and bacteria. In the field of cell culture, cell retention requires consideration of cell size. Cell size varies greatly depending on the cell type. Prokaryotic cells have a simple structure, averaging 1-10µm; eukaryotic cells have a complex structure, averaging 3-30µm. The smallest known cell is mycoplasma, with a diameter of only 0.1-0.3µm. Furthermore, with the rise of tissue engineering, in vitro cell culture has evolved to allow for suspension-scale culture on microcarriers, with microcarrier sizes generally ranging from 20-500µm. With technological advancements, filtration membrane materials are becoming increasingly innovative. These materials include, but are not limited to, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), modified polyethersulfone (PES), mixed cellulose, nylon, polypropylene, glass fiber, and quartz. When selecting a filtration material, the following aspects must be considered: 1) The properties and chemical compatibility of the filtered fluid: Different fluid properties and chemical environments require different materials to ensure filtration efficiency and membrane stability. 2) Membrane area: A suitable membrane area should be selected based on the treatment requirements to optimize efficiency and cost. 3) Special membrane requirements: Such as leaching, adsorption, flux, and color; membrane materials meeting specific requirements should be selected based on the specific application scenario.

[0040] Example 1

[0041] The perfusion rate of the stirred perfusion culture bag (3D) and the rocking cell culture bag (2D) was tested to verify the perfusion effect of the perfusion zone. The stirred perfusion culture bag (3D) described in Example 1 is composed of four welded membrane materials. The stirring paddle is located in the center of the bag and is driven by a stirring shaft to continuously stir and achieve the exchange of substances and gases in the culture. The perfusion zone is fixed to the inner wall of the bag and does not move during the stirring process. In contrast, the rocking cell culture bag (2D) is composed of two welded membrane materials and is placed horizontally on a shaker. Its perfusion zone is connected to the culture suspension through tubing, and the exchange of substances and gases in the culture is achieved through the continuous rocking of the bag.

[0042] Test Result 1: The stirred perfusion culture bag (3D) was continuously stirred for 12 days. The perfusion zone and tubing were firmly fixed, the filter membrane was undamaged, and no culture particles adhered to the surface. The rocking cell culture bag (2D), after continuous rocking for 12 days, showed that the perfusion zone swayed with the liquid surface and could not be completely submerged in the culture medium. The small and unstable surface area in contact with the liquid resulted in a slow perfusion rate. Furthermore, some test bags showed damaged filter membranes, and some test bags had a layer of culture adhering to the filter area.

[0043] Test Result 2: The stirred perfusion culture bag (3D) used a 5-micron pore size filter membrane to perfuse cultures with particles ranging from 50 to 500 microns. Adjusting the peristaltic pump speed (which affects the negative pressure in the perfusion zone), as shown in Figure 7, gradually adjusting the pump speed to 8 rpm resulted in a maximum perfusion rate of 3.5 ml / min, achieving a perfusion flow rate of 2 VVD / day, meeting the needs of cell culture. Further increasing the pump speed (increasing the negative pressure in the perfusion zone) resulted in a slow decrease in perfusion rate; even at a pump speed of 15 rpm, a perfusion rate of 2 ml / min was maintained. The 2D swing-type cell culture bag uses a 5-micron pore size filter membrane for perfusion of cultures with particles ranging from 50 to 500 microns. Adjusting the peristaltic pump speed (affecting the negative pressure in the perfusion zone) is shown in Figure 6, where the perfusion rate of the 2D perfusion bag varies with the peristaltic pump speed. When using a swing-type mixing method to prevent culture from clogging the filter membrane, gradually adjusting the peristaltic pump speed to 7 rpm resulted in a maximum perfusion rate of 3 ml / min. Compared to the stirred perfusion bag, both the pump speed and the perfusion rate were lower, indicating clogging. Further increasing the pump speed (increasing the negative pressure in the perfusion zone) rapidly reduced the perfusion rate. When the pump speed reached 15 rpm, the perfusion rate was 0, indicating complete clogging of the perfusion zone.

[0044] Example 2

[0045] The perfusion culture bag was used to culture suspension cells such as CHO. The filter membrane in the perfusion zone of the perfusion culture bag has a pore size of 1.2 micrometers. During stirring, the cells move at high speed in the culture system, overcoming the negative pressure of the retention device, thus accelerating the perfusion rate. When stirring stops, the cells cannot settle quickly, and the perfusion zone, located in the cell suspension, is prone to clogging under the negative pressure, leading to a slower perfusion rate. Therefore, the perfusion rate is faster during stirring than when stirring is stopped.

[0046] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A perfusion cell culture bag, comprising a bag body (1), wherein the inner wall of the bag body (1) has a membrane material (11), characterized in that, The bag body (1) is provided with one or more irrigation zones (4), the irrigation zones (4) are fixedly connected to the inner wall of the bag body (1), the irrigation zones (4) have an internal cavity, and part or all of the surface of the irrigation zones is covered with an irrigation filter membrane (3); the internal cavity is filled with a diaphragm mesh (5) for supporting the irrigation zones (4), and an outlet pipe (6) is provided on the irrigation zones (4) to communicate with the outside of the bag body (1).

2. The perfusion cell culture bag according to claim 1, characterized in that, The internal cavity is formed in the following manner: The perfusion filter membrane (3) covers a portion of the membrane material (11) on the inner wall of the bag body (1), and the periphery of the perfusion filter membrane (3) is sealed and connected to the membrane material (11); or, The perfusion filter membrane (3) is used to form a ring; or, The perfusion filter membrane (3) forms the first side, and a membrane of the same or different material as the membrane material (11) forms the second side, with the edges of the first and second sides adhering together.

3. The perfusion cell culture bag according to claim 1, characterized in that, The bag body (1) is also equipped with a stirring mechanism (2).

4. The perfusion cell culture bag according to any one of claims 1-3, characterized in that, The distance h from the lower edge of the irrigation zone (4) to the bottom of the bag is 5%-50% of the total bag height H.

5. The perfusion cell culture bag according to claim 4, characterized in that, The irrigation zone (4) covers 1%-80% of the inner wall area of ​​the bag (1).

6. The perfusion cell culture bag according to any one of claims 1-5, characterized in that, The filter membrane (3) has at least one layer.

7. The perfusion cell culture bag according to any one of claims 1-5, characterized in that, The pore size of the perfusion filter membrane (3) is 0.1um-500um.

8. The perfusion cell culture bag according to any one of claims 1-3, characterized in that, The lower part of the outer wall of the bag (1) is also provided with a sampling interface (12), a liquid outlet (13), a temperature measurement interface (14), a pH measurement interface (15), and a DO measurement interface (16); the sampling interface (12) is used to sample and detect the cell production status, and the liquid outlet (13) is used to extract the supernatant; the temperature measurement interface (14), pH measurement interface (15), and DO measurement interface (16) are respectively used to install sensors to measure the temperature, pH and DO value of the culture bag.

9. The perfusion cell culture bag according to any one of claims 1-3, characterized in that, The upper part of the outer wall of the bag (1) is also provided with an exhaust port (21), a feeding port (22), a surface ventilation port (23), and a liquid inlet (24). The exhaust port (21) is used to discharge the culture exhaust gas in the culture bag, the feeding port (22) is used to add factors and other materials required for the cell culture process to the culture bag, the surface ventilation port (23) is used to introduce sterile air into the bag, and the liquid inlet (24) is used to add culture medium during the culture process.

10. The perfusion cell culture bag according to any one of claims 1-3, characterized in that, The bottom of the bag (1) is also provided with a ventilation plate (7), which is provided with a full drainage port (71) and a deep ventilation port (72). The full drainage port (71) and the deep ventilation port (72) are connected to the inside of the bag. The full drainage port (71) is used to collect cell culture products, and the deep ventilation port (72) is used to supply the gas required for cell culture into the bag.

11. The perfusion cell culture bag according to any one of claims 1-3, characterized in that, The liquid outlet pipeline (6) includes a first bag buckle (61), a first silicone tube (62), a first right-angle elbow (63), a second silicone tube (64), a second right-angle elbow (65), a third silicone tube (66), and a double hook bag buckle (67) connected in sequence.

12. A cell perfusion system, comprising an external tubing (30), a drive pump (40), and a waste bag (50), wherein the external tubing (30) includes a second bag buckle (31) and a fourth silicone tube (32), characterized in that, It also includes the perfusion cell culture bag according to any one of claims 1-11, wherein one end of the second bag buckle (31) is connected to the liquid outlet pipe (6), and the other end is connected to one end of the fourth silicone tube (32), the other end of the fourth silicone tube (32) is connected to the waste bag (50), and the drive pump (40) is located in the middle of the fourth silicone tube (32). When the drive pump (40) is running, it continuously squeezes the fourth silicone tube (32) to extract the culture medium or waste liquid from the perfusion functional area.

13. The system according to claim 12, characterized in that, The liquid outlet pipeline (6) includes a first bag buckle (61), a first silicone tube (62), a first right-angle elbow (63), a second silicone tube (64), a second right-angle elbow (65), a third silicone tube (66), and a double hook bag buckle (67) connected in sequence. One end of the second bag buckle (31) is connected to the double hook bag buckle (67).

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