Polyester fiber flow feeding culture device for improving CHO cell density and viability

Abstract

The application discloses a polyester fiber flow feeding culture device for improving CHO cell density and viability, and is characterized in that the device comprises a shell, an adherent culture device in the shell, a feeding and dissolved oxygen device, a liquid outlet device, a main controller and a data transmission unit; the adherent culture device arranged in the shell is provided with a three-dimensional polyester fiber carrier and a layered structure design, so that the mass transfer limitation problem in high-density culture is solved from the perspective of optimizing the mass transfer path, the cell density is increased to 7.5-8*10 8 6 / mL, and meanwhile, an efficient dissolved oxygen and intelligent feeding system is constructed, the dissolved oxygen saturation is stabilized at 30%-60%, the pH value is accurately controlled, and the coupling interference problem of the dissolved oxygen and the pH control is solved.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a polyester fiber fed-batch culture device for improving the density and viability of CHO cells. Background Technology

[0002] CHO cells (Chinese Hamster Ovary cells) are a mammalian cell line derived from the ovaries of Chinese hamsters. They possess the ability to perform complex human-like glycosylation modifications, enabling the expressed products to have conformations and biological activities highly similar to natural proteins. They are widely used in the production of recombinant proteins, monoclonal antibodies, fusion proteins, and vaccine antigens. In actual production, cell culture density and viability are key factors affecting production efficiency and cost. High-density seeding maximizes the utilization of the reactor volume, while high viability ensures a sufficient number of viable cells within the system, thereby improving the production efficiency of the target protein.

[0003] However, existing large-scale culture facilities still exhibit numerous technical bottlenecks, severely restricting the simultaneous improvement of these two key indicators: 1) Traditional planar culture devices (such as culture flasks and multilayer culture plates) mainly rely on a limited culture interface for cell attachment and growth, resulting in low space utilization and making it difficult to exceed 5×10⁻⁶ viable cell density per unit volume. 6 Cells / mL. When cells grow in layers or aggregate locally, the bottom cells are surrounded by the cell layer and boundary layer above, and are in a state of hypoxia and nutrient starvation for a long time. At the same time, lactic acid, ammonia and carbon dioxide produced by cell metabolism cannot diffuse outward in time, leading to the accumulation of metabolic byproducts, accelerating cell apoptosis, reducing protein expression efficiency, and making it difficult to meet the needs of high-density production.

[0004] 2) Currently, most bioreactors still rely on surface aeration or mechanical stirring to achieve gas-liquid mixing. However, as cell culture density increases, the demand for oxygen increases, and the above methods have significant oxygen supply deficiencies, making it difficult to stabilize dissolved oxygen saturation above 40%. While increasing the aeration rate can temporarily increase dissolved oxygen, it will strip dissolved carbon dioxide from the culture medium, disrupting the bicarbonate buffer balance and causing pH changes in the culture medium, making it difficult to achieve synergistic optimization of pH and dissolved oxygen efficiency.

[0005] 3) Most existing CHO cell culture devices are single-loop designs, containing only culture medium circulation pipelines. They lack a way to remove metabolic substances generated during the culture process, resulting in a large amount of metabolic byproducts such as lactic acid and ammonia produced by cells during the exponential growth phase circulating and accumulating with the culture system. This shortens the single cell culture cycle in the reactor and makes it difficult to carry out long-term, high-density continuous culture.

[0006] 4) The automation level is low, requiring frequent manual sampling and testing of key parameters, which is cumbersome and prone to introducing pollution risks. Existing monitoring systems are mostly offline or single-parameter detection, making it difficult to control multiple parameters such as pH, temperature, dissolved oxygen, glucose, and lactic acid during the culture process.

[0007] To address the aforementioned issues, there is an urgent need to develop a fed-batch device that combines mass transfer efficiency and dissolved oxygen efficiency with the removal of metabolic byproducts, in order to overcome the technical bottleneck of high-density and high-viability CHO cell culture in existing technologies and provide a cell culture foundation for the large-scale production of complex bioproducts. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a polyester fiber fed-batch culture device for improving CHO cell density and viability.

[0009] A fed-batch culture device for improving the density and viability of CHO cells using polyester fibers includes a shell, an adherent culture device inside the shell, a feeding and dissolved oxygen device, a liquid dispensing device, a main controller, and a data transmission unit. The housing is a sealed structure, with a liquid inlet, an air inlet pipe and an exhaust port at the top, and a liquid outlet pipe at the bottom. The adherent culture device is filled inside the shell and includes multiple independently distributed three-dimensional polyester fiber carrier layers. The three-dimensional polyester fiber carrier is a three-dimensional structure formed by accordion folding of polyethylene terephthalate nonwoven fiber layers. The feeding and oxygenation device is divided into two parts: the upper part is the feeding and pre-oxygenation unit, and the lower part is the oxygenation unit. The feeding and pre-oxygenation unit is located above the wall-mounted culture device and includes a sprinkler distributor and a gas enrichment chamber. The sprinkler distributor is connected to the liquid inlet of the shell, and multiple nozzles are evenly distributed on the bottom surface of the sprinkler distributor. The oxygenation unit (i.e., the low-pressure microporous aeration head) is located between the inverted conical guide plate of the liquid outlet device and the lower part of the wall-mounted culture device. The liquid outlet device includes an inverted conical guide plate, which is located at the bottom of the inner cavity of the shell and directly below the wall-adhering culture device, for collecting the culture medium flowing through the wall-adhering culture device; the bottom of the inverted conical guide plate is provided with a liquid outlet interface, which is connected to the liquid outlet pipe through a bottom pipe; The main controller and data transmission unit are used to control and transmit various parameters during the cultivation process.

[0010] In this invention, adherent culture of CHO cells is achieved by setting up an adherent culture device, a feeding and oxygenation device, and a liquid dispensing device inside a sealed shell. The adherent culture device comprises multiple independently distributed three-dimensional polyester fiber carriers, whose accordion-folded three-dimensional structure increases the equivalent volumetric cell density. The feeding and oxygenation device fully integrates the culture medium flowing from the nozzles of a sprinkler dispenser with the oxygen within the device, stabilizing the dissolved oxygen saturation at 40%–60% during the growth phase, thus improving oxygenation efficiency and meeting the oxygen requirements of high-density CHO cell culture.

[0011] In this invention, the shell of the fed-batch culture device for polyester fibers is a sealed cylinder with a diameter of 25-28 cm and a height of 50-60 cm. The top is equipped with a liquid inlet, an air inlet pipe and an exhaust port, and the bottom is equipped with a liquid outlet pipe. It is suitable for small- to medium-scale culture systems of 20-25 L and is applicable to small- to medium-scale culture of CHO cells.

[0012] In this invention, the three-dimensional polyester fiber carrier, referencing the nonwoven polyester fiber tape in patent document CN111235111A, uses polyethylene terephthalate nonwoven fibers. These fibers are folded into a three-dimensional structure using a gusseted method and uniformly arranged in a disc-shaped adherent culture device. This three-dimensional structure provides ample attachment sites for CHO adherent cells, enabling them to adhere and proliferate in the early stages of culture. It also ensures smooth flow of the culture medium within the three-dimensional structure of the carrier, promoting uniform distribution of the culture medium and oxygen within the culture device, and timely removal of metabolic byproducts (such as lactic acid and ammonia).

[0013] Preferably, the inverted conical guide plate is further equipped with a pH sensor, a temperature sensor, a dissolved oxygen sensor, a glucose detector, a lactic acid detector, and an ammonia detector (i.e., Figure 1 (As shown in reference numerals 12-17), the sensors are connected to the main controller, which regulates the aforementioned parameters. Specifically, multiple probe interfaces are provided on the side wall of the inverted conical guide plate. Each sensor probe extends into the liquid collection area inside the inverted conical guide plate to monitor the pH value, temperature, dissolved oxygen saturation, glucose concentration, lactic acid concentration, and ammonia concentration of the culture system in real time, and transmits the data to the main controller 20 for the control and adjustment of the culture process parameters.

[0014] Preferably, the wall-adherent culture device comprises 3 to 5 independent three-dimensional polyester fiber carrier layers, each layer having a thickness of 8 to 10 cm. Each three-dimensional polyester fiber carrier layer is provided with a support grid on both the top and bottom, with a grid aperture of 6 to 8 mm, to prevent deformation of the three-dimensional polyester fiber carrier and ensure smooth flow of the culture medium.

[0015] Preferably, the shower head distributor is annular, with 24 to 48 nozzles evenly distributed on its bottom surface. The nozzles have an orifice diameter of 200 to 500 μm. A micro peristaltic pump is connected to the shower head distributor to control the liquid flow rate at 50 to 200 mL / min.

[0016] In this invention, based on the cell density and dissolved oxygen data provided by the data transmission unit, the main controller dynamically and adaptively adjusts the liquid flow rate of the micro peristaltic pump, so that the feed solution flows evenly into the wall-mounted culture device through the nozzle of the shower distributor; by controlling the liquid flow rate, the culture medium can have a greater contact with the oxygen inside the entire device, thereby improving the dissolved oxygen efficiency.

[0017] Preferably, the gas enrichment chamber is located above the shower head distributor, the volume of the gas enrichment chamber is 5~7.5L, the working pressure is 0.01~0.03 MPa, and a gas flow meter is installed inside it to monitor the gas flow rate entering the device.

[0018] More preferably, the gas enrichment chamber is provided with a baffle plate with an inclination angle of 30~60°. The gas forms a rotating uniform flow through the baffle plate and is guided to the gas phase space below the shower distributor. It then comes into gas-liquid contact with the culture liquid falling from the nozzle of the shower distributor, improving the mixing effect of the gas and the culture liquid and achieving the first stage of pre-dissolved oxygen.

[0019] In this invention, by regulating the pressure inside the gas enrichment chamber, combined with adjusting the tilt angle of the baffle and the liquid flow rate in the shower distributor, the gas and culture medium are fully mixed before entering the adherent culture device, thus avoiding fluctuations in oxygen content during CHO cell culture due to uneven mixing.

[0020] Preferably, the dissolved oxygen unit is located at the bottom of the shell, directly below the wall-mounted culture device, and adopts a low-pressure microporous aeration head structure with a working pressure of 0.03~0.08 MPa, generating uniform microbubbles with a diameter of 100~500 μm. The presence of microbubbles can increase the gas-liquid contact area, improve oxygen transfer efficiency, and at the same time reduce the damage to CHO cells caused by local fluid disturbance and shear force generated by large bubbles during their rise and collapse.

[0021] In this invention, the culture solution and gas flowing out through the shower head distributor complete the initial oxygenation process. The mixed culture solution flows into the wall-mounted culture device, while the microbubbles generated by the low-pressure microporous aeration head at the bottom pass through the wall-mounted culture device during their ascent and come into contact with the mixed culture solution to complete the secondary oxygenation process. Ultimately, the dissolved oxygen saturation in the device is stabilized at 40%~60%, which is 2~3 times higher than the traditional single oxygenation method.

[0022] More preferably, a gas-liquid separation physical defoaming component is provided inside the shell, below the shower head distributor and above the adherent culture device. This defoaming component is composed of a porous intercepting mesh made of polytetrafluoroethylene (PTFE), with a mesh size of 0.5~2 mm and a porosity of 70%~90%. In this invention, the culture medium can flow into the adherent culture area below through the intercepting mesh. The low surface area of ​​the PTFE material makes it difficult for liquid films or bubbles to adhere to the mesh surface. Microbubbles rising from the bottom and local microbubbles formed during the shower spraying process are eliminated when passing through the intercepting mesh, achieving gas-liquid separation and physical defoaming. This reduces the accumulation of local bubbles in the culture system, effectively preventing bubble rupture from impacting the adherent cells, and significantly improving the stability and survival rate of CHO cells during high-density culture.

[0023] In this invention, air or oxygen is introduced into the device via a gas mixing unit. The input gas is guided by a baffle plate in the gas enrichment chamber to form a rotating, uniform flow. This, combined with the culture medium flowing out from the sprinkler distributor, ensures thorough mixing of the gas and culture medium, achieving the initial oxygenation process. Microbubbles are generated through low-pressure aeration at the bottom, rising through the carrier layer of the wall-mounted culture device and contacting the falling droplets of the mixed culture medium, completing the secondary oxygenation process. This entire feeding and oxygenation device improves upon the problems of low oxygenation efficiency, difficult control, and uneven nutrient distribution found in traditional devices. Simultaneously, the special three-dimensional carrier layer structure protects the activity of CHO cells, enabling high-density culture and improving product expression efficiency.

[0024] Preferably, the outlet pipe of the liquid outlet device is connected to a bypass circulation pipe via a three-way valve. The bypass circulation pipe is equipped with an adsorption unit to adsorb metabolic byproducts from the flowing culture medium. The adsorption unit is located at the bottom of the device and is connected to the outlet pipe of the culture chamber. The three-way valve is used to switch the fluid path. When adsorption treatment is required, the bypass circulation pipe is opened, allowing the culture medium to flow through the adsorption unit; when adsorption treatment is not required, the bypass circulation pipe is closed, and the culture medium flows along the outlet pipe or the circulation pipe. The adsorption unit can reduce the accumulation level of metabolic byproducts such as lactic acid and ammonia generated during the culture process, thereby helping to maintain the stability of the culture system.

[0025] The present invention also provides a method for culturing CHO cells, wherein the culturing method is performed in the above-described apparatus.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: In this invention, a three-dimensional polyester fiber carrier and layered structure design are proposed by using an adherent culture device set inside the shell. This addresses the problem of limited mass transfer in high-density culture from the perspective of optimizing the mass transfer pathway, thereby increasing cell density to 7.5~8×10⁻⁶. 8cells / mL; meanwhile, an efficient dissolved oxygen and intelligent feeding system is constructed, which can stabilize the dissolved oxygen saturation at 40% - 60% during the growth stage and at 30% - 40% during the production stage, and achieve precise regulation of the pH value, solving the coupling interference problem of dissolved oxygen and pH regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 FIG. is a schematic structural diagram of a polyester fiber fed-batch culture device for improving the density and viability of CHO cells according to the present invention, wherein, 1 - housing; 2 - liquid inlet interface; 3 - air inlet pipe; 4 - gas enrichment chamber; 5 - spoiler; 6 - shower distributor; 7 - gas-liquid separation type physical defoaming component; 8 - adherent culture device unit containing three-dimensional polyester fiber carriers (wherein, the three-dimensional polyester fiber carriers are arranged in an accordion-fold manner and each unit structure is replaceable); 9 - low-pressure microporous aeration head; 10 - inverted conical deflector; 11 - adsorption unit; 12 - pH sensor; 13 - temperature sensor; 14 - dissolved oxygen sensor; 15 - glucose detector; 16 - lactate detector; 17 - ammonia detector; 18a - micro peristaltic pump a; 18b - micro peristaltic pump b; 18c - micro peristaltic pump c; 19a - solenoid valve controller a; 19b - solenoid valve controller b; 19c - solenoid valve controller c; 20 - main controller (wired between + and -); 21 - liquid outlet pipe; 22 - exhaust interface (equipped with a sterile filtration component).

[0028] Figure 2 FIG. is a schematic diagram of an adherent culture device unit of the present invention, wherein 23 is a support grid.

[0029] Figure 3 FIG. is a statistical chart of the cell density and cell viability of CHO cells in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The following combines examples to further elaborate on the present invention in detail, but the embodiments of the present invention are not limited to the following examples.

[0031] All raw materials used in the present invention are commercially available. The three-dimensional polyester fiber carrier of the present invention adopts the non-woven polyester fiber strip material in the patent document with the publication number of CN111235111A.

[0032] Such as Figure 1As shown, the fed-batch culture device for polyester fibers of the present invention includes a shell 1, a wall-adhering culture device 8 inside the shell, a feeding and oxygenation device, a liquid outlet device, a circulation pipeline, a main controller 20, and a data transmission unit. The shell 1 is a cylindrical, sealed structure with a liquid inlet 2, an air inlet 3, and an exhaust port 22 equipped with a sterile filter assembly at the top. A miniature peristaltic pump 18c is connected to the pipe of the liquid inlet 2 for precise control of feeding and delivery. The wall-adhering culture device 8 (e.g., Figure 2 The three-dimensional polyester fiber carrier (as shown) is filled inside the shell 1 and contains multiple layers of independently distributed three-dimensional polyester fiber carriers. Each layer of the three-dimensional polyester fiber carrier is provided with a support grid 23 on the top and bottom to prevent deformation of the three-dimensional polyester fiber carrier and ensure smooth flow of the culture medium. The feeding and oxygenation device is divided into two parts: the upper part is the feeding and pre-oxygenation unit, and the lower part is the oxygenation unit. The feeding and pre-oxygenation unit is located above the wall-adhering culture device 8 and includes a gas enrichment chamber 4 and a sprinkler distributor 6. The sprinkler distributor 6 is connected to the liquid inlet 2 at the top of the shell 1, and a nozzle is opened on the bottom surface of the sprinkler distributor 6. The oxygenation unit (i.e., the low-pressure microporous aeration head 9) is set at the bottom of the shell 1, between the liquid outlet device (inverted conical guide plate 10) and the bottom of the wall-adhering culture device 8. The liquid outlet device includes a liquid outlet pipe 21 and an inverted conical guide plate 10. The inverted conical guide plate 10 is set below the wall-adhering culture device 8 and is used to collect the culture medium flowing through the wall-adhering culture device 8. The sidewall of the inverted conical flow guide plate 10 is equipped with a pH sensor 12, a temperature sensor 13, a dissolved oxygen sensor 14, a glucose detector 15, a lactic acid detector 16, and an ammonia detector 17 to monitor the pH value, temperature, dissolved oxygen saturation, glucose concentration, lactic acid concentration, and ammonia concentration of the culture system in real time, and transmit the data to the main controller 20. The bottom of the inverted conical guide plate 10 is provided with a liquid outlet. The liquid outlet is connected to a flow path switching assembly consisting of multiple solenoid valves via a pipeline, which is used to switch different fluid circuits according to the culture state. The flow path switching assembly includes solenoid valve controllers 19a, 19b, and 19c, with each solenoid valve controlling a corresponding functional branch. The bypass of solenoid valve controller 19a is connected to an adsorption unit 11, which is used to adsorb and remove metabolic byproducts in the culture medium. Solenoid valve controller 19b is connected to an external circulation pipeline, and a micro peristaltic pump 18b is installed on the return pipeline to pump the culture medium back to the top inlet port 2, realizing the recycling of the culture medium. Solenoid valve controller 19c is connected to the micro peristaltic pump 18a and the final liquid outlet pipe 21 to complete the discharge of the culture medium. The main controller 20 is electrically connected to each sensor, the micro peristaltic pump, and the solenoid valve controllers to realize the linkage control of multiple parameters and data transmission.

[0033] In the feeding and pre-oxygenation unit, the gas enrichment chamber 4 is located above the shower distributor 6. The gas enrichment chamber 4 is equipped with a baffle 5. The gas input from the air inlet pipe 3 forms a rotating uniform flow through the baffle 5 and is guided to the gas phase space below the shower distributor 6. It then comes into gas-liquid contact with the culture liquid falling from the nozzle of the shower distributor, improving the mixing effect of the gas and the culture liquid and achieving the first stage of pre-oxygenation.

[0034] Inside the housing 1, below the shower distributor 6 and above the wall-mounted culture device 8, there is a gas-liquid separation physical defoaming component 7, which is used to physically break up the foam and separate the gas and liquid during the culture process.

[0035] Example 1 (1) Cell resuscitation and seed culture preparation: Remove frozen cells from the liquid nitrogen tank and thaw them quickly in a 37°C water bath. After complete thawing, transfer the cell suspension to a 15 mL sterile centrifuge tube, add 5 mL of preheated serum-free, protein-free, and chemically defined basal medium, and gently mix by pipetting. Centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 5 mL of medium. Transfer the resuspended cells to a 50 mL shake flask and add basal medium to a total volume of 20 mL. Incubate the shake flask at 37°C, 100 rpm, and 70%–90% humidity. Sample and measure cell density every 24 hours, and add medium (Shanghai OPMA Biotechnology, AltairCHO® Medium, C673017) as needed to maintain a cell density of 3–6 × 10⁻⁶. 6 Cells / mL. Through stepwise scale-up, the culture system was successively transferred to 100 mL, 500 mL, and 1000 mL shake flasks for further amplification. After 5-6 days of culture, approximately 500 mL of seed culture was obtained, with a cell density of approximately 3.0 × 10⁻⁶ cells / mL. 6 per mL.

[0036] (2) Inoculation and culture in a 25 L reactor Two vials of seed culture were inoculated into a 25 L reactor, with an initial working volume of 2 L and a cell density of 1–1.5 × 10⁻⁶ cells / mL. 6 Cells / mL. Culture conditions were set as follows: temperature 37℃, dissolved oxygen saturation 30%~40%, pH 6.8~7.2. Cells were sampled daily for counting, and basal medium (Shanghai OPMA Biotechnology, AltairCHO® Medium, C673017) was gradually added until the total culture volume reached 25 L.

[0037] (3) Polyester fiber fed-batch culture device for amplification culture Once the cell density in the 25 L reactor reaches 1.0 × 10⁻⁶ 7 After [number] cells / mL, connect as follows: Figure 1The aforementioned fed-batch polyester fiber culture device introduces cell slurry into the device through an inlet, allowing cell growth to occur in an adherent culture unit within the device. This adherent culture unit comprises four independently distributed three-dimensional polyester fiber carriers. The culture conditions are maintained at: temperature 37°C, pH 6.8–7.2, and dissolved oxygen saturation 40%.

[0038] When the cell density reaches 4.5 × 10 8 At a cell density of 100 cells / mL, the main controller 20 controls the temperature control device to lower the culture temperature to 34°C. Simultaneously, based on data from the glucose detector 15, the main controller 20 controls the micro-peristaltic pump 18c to feed optimized production culture medium (Shanghai Aopumai Biotechnology, AltairCHO® Feed Plus, P226220) through the inlet pipe 2. The culture medium is dispersed by the top sprinkler dispenser 6 and then evenly sprayed onto the surface of the three-dimensional polyester fibers in the adherent culture device 8 below. Waste liquid is discharged through the inverted conical guide plate 10 and the outlet device, maintaining the cell density at 7.5~8×10⁻⁶. 8 Cells / mL. During adherent fed-batch culture, ammonia and lactate concentrations were monitored using an ammonia detector 17 and a lactate detector 16. The main controller 20 controlled solenoid valves 19a, 19b, and 19c to switch the liquid flow path based on the detection results. The liquid underwent cyclic adsorption treatment via adsorption unit 11, and the treated liquid was then returned to the device, thus maintaining an ammonia concentration <2 mmol / L and a lactate concentration <5 mmol / L. Culture was terminated when cell viability fell below 80%. Throughout the culture process, the main controller 20 adjusted the air inlet pipe 3 and the low-pressure microporous aeration head 9 based on the dissolved oxygen sensor 14 to maintain dissolved oxygen saturation at 30%–40% during the production phase; the pH was maintained at 6.8–7.2 using the pH sensor 12. The device operated continuously for 20 days from reactor inoculation. Specific cell density and viability were as follows: Figure 3 As shown.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fed-batch culture device using polyester fibers to improve the density and viability of CHO cells, characterized in that, It includes a shell, an internal wall-adhering culture device, a feeding and dissolved oxygen device, a liquid discharge device, a main controller, and a data transmission unit; The housing is a sealed structure, with a liquid inlet, an air inlet pipe and an exhaust port at the top, and a liquid outlet pipe at the bottom. The adherent culture device is filled inside the shell and includes multiple independently distributed three-dimensional polyester fiber carrier layers. The three-dimensional polyester fiber carrier is a three-dimensional structure formed by accordion folding of polyethylene terephthalate nonwoven fiber layers. The feeding and oxygenation device is divided into two parts: the upper part is the feeding and pre-oxygenation unit, and the lower part is the oxygenation unit. The feeding and pre-oxygenation unit is located above the wall-mounted culture device and includes a sprinkler distributor and a gas enrichment chamber. The sprinkler distributor is connected to the liquid inlet of the shell, and its bottom surface is evenly distributed with nozzles. The oxygenation unit adopts a low-pressure microporous aeration head and is located between the inverted conical guide plate of the liquid outlet device and the lower part of the wall-mounted culture device. The liquid outlet device includes an inverted conical guide plate, which is located at the bottom of the inner cavity of the shell and directly below the wall-adhering culture device, for collecting the culture medium flowing through the wall-adhering culture device; the bottom of the inverted conical guide plate is provided with a liquid outlet interface, which is connected to the liquid outlet pipe through a bottom pipe; The main controller and data transmission unit are used to control and transmit various parameters during the cultivation process.

2. The polyester fiber fed-batch culture device for improving CHO cell density and viability according to claim 1, characterized in that, The wall-adherent culture device comprises 3 to 5 independent three-dimensional polyester fiber carrier layers, each layer having a height of 8 to 10 cm. Each three-dimensional polyester fiber carrier layer is provided with supporting grids on the top and bottom, with grid apertures of 6 to 8 mm.

3. The polyester fiber fed-batch culture device for improving CHO cell density and viability according to claim 1, characterized in that, The shower head distributor is ring-shaped, with 24 to 48 nozzles evenly distributed on the bottom surface. The nozzles have an orifice diameter of 200 to 500 μm. A micro peristaltic pump is connected to the shower head distributor to control the liquid flow rate at 50 to 200 mL / min.

4. The polyester fiber fed-batch culture device for improving CHO cell density and viability according to claim 1, characterized in that, The gas enrichment chamber is located above the shower head distributor. The volume of the gas enrichment chamber is 5~7.5 L, and the working pressure is 0.01~0.03 MPa. It is equipped with a gas flow meter to control the gas flow rate entering the device.

5. The polyester fiber fed-batch culture device for improving CHO cell density and viability according to claim 4, characterized in that, The gas enrichment chamber is equipped with a baffle plate with an inclination angle of 30 to 60 degrees.

6. The polyester fiber fed-batch culture device for improving CHO cell density and viability according to claim 1, characterized in that, In the dissolved oxygen unit, the low-pressure microporous aerator has an air supply pressure of 0.03~0.08 MPa, generating microbubbles with a diameter of 100~500 μm.

7. The polyester fiber fed-batch culture device for improving CHO cell density and viability according to claim 1, characterized in that, Inside the housing, below the shower distributor and above the wall-mounted culture device, there is also a gas-liquid separation physical defoaming component; the liquid outlet pipe of the liquid outlet device is connected to a bypass circulation pipe through a three-way valve, and the bypass circulation pipe is equipped with an adsorption unit for adsorbing metabolic by-products of the flowing culture medium.

8. A method for culturing CHO cells, characterized in that, The culture method described herein is carried out in the polyester fiber fed-batch culture apparatus for improving CHO cell density and viability as described in any one of claims 1 to 7.

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