A cell culture vessel

Abstract

This application provides a cell culture container, belonging to the technical field of cell culture equipment. The culture container includes a container tank, an air intake and exhaust controller, a gas guiding component, a detector group, a fluorescence detection module, and a computer controller. The computer controller receives the fluorescence intensity signal detected in real time by the fluorescence detection module. After filtering and temperature drift compensation, it calculates the current cell oxygen consumption rate using a preset fluorescence intensity-cell oxygen consumption rate correlation model, and controls the air intake pump according to this rate to achieve on-demand oxygen supply. The correlation model can be a linear proportional model, a piecewise linear model, or a dynamic correction model. Preferably, the fluorescence detection module uses multi-wavelength excitation, which can simultaneously detect the first fluorescence signal characterizing cell activity and the second fluorescence signal characterizing cell death, and stops air intake and alarms when the dead cell signal exceeds a threshold. This invention achieves precise gas control based on cell metabolic activity, improving the success rate of culture.

Description

Technical Field

[0001] This application relates to the field of cell culture equipment technology, and more particularly to a cell culture container. Background Technology

[0002] In the field of biotechnology, current cell culture techniques face significant challenges in accurately simulating the requirements of specific gaseous environments. This challenge is particularly pronounced when it comes to cells that need to be cultured under high pressure and low oxygen conditions. Cells exhibit different growth and metabolic characteristics in different gaseous environments, and creating a suitable high-pressure, low-oxygen environment for cells is no easy task.

[0003] Existing bioreactors typically employ a conventional method to control the gas environment for cell culture. They often regulate the entry of O2, CO2, and other gases by controlling individual gas valves, attempting to achieve the specific conditions required for cell culture. However, these bioreactor devices have significant drawbacks. Their structures are often overly complex, containing numerous components and intricate control systems. This not only increases manufacturing costs and maintenance difficulty but also makes them prone to malfunctions during operation. Furthermore, these devices are bulky and space-consuming, hindering efficient cell culture work within limited laboratory spaces. Summary of the Invention The purpose of this invention is to solve the above-mentioned problems by providing a cell culture container.

[0004] The technical solution of this application is implemented as follows: In a first aspect, this application provides a cell culture container, the culture container including a container, the top of the container having an opening, a top cover being detachably installed at the opening, and the container being used to store cells; An intake and exhaust controller includes an intake pump and an exhaust valve installed on the upper cover. The intake pump is used to introduce gas from the mixed gas tank into the container, and the exhaust valve is used to discharge gas from the container. A gas guiding component, installed on the inner wall of the container, is used to agitate the gas inside the container; A detector array, installed on the inner wall of the container, is used to detect the gas pressure, gas content, and temperature inside the container; A fluorescence detection module is used to detect the fluorescence intensity of the cell culture medium in the container in real time, and the fluorescence intensity is related to cell metabolic activity; A computer controller is electrically connected to the detector group, the gas guiding assembly, the inlet and outlet controller, and the fluorescence detection module. The computer controller is configured as follows: Receive the fluorescence intensity signal output by the fluorescence detection module; Based on the fluorescence intensity signal, the current cell oxygen consumption rate is calculated using a preset fluorescence intensity-cell oxygen consumption rate correlation model. Based on the current cell oxygen consumption rate, the intake volume of the air pump is controlled to adjust the oxygen concentration in the container; the computer controller is configured to calculate the current cell oxygen consumption rate using the fluorescence intensity-cell oxygen consumption rate correlation model in the following manner: The fluorescence intensity signal output by the fluorescence detection module is preprocessed, including filtering and temperature drift compensation, to obtain the compensated fluorescence intensity value F(t); The current cellular oxygen consumption rate OUR(t) is calculated based on a pre-defined correlation model.

[0005] In one implementation, the correlation model is a linear scaling model: ; Wherein α is the comprehensive proportionality coefficient obtained through offline calibration experiments, which include: simultaneously measuring fluorescence intensity F(ti) and actual oxygen consumption rate OUR(ti) at multiple time points, and obtaining the proportionality coefficient α through linear regression fitting.

[0006] In one implementation, the correlation model is a piecewise linear model: ; in The preset fluorescence intensity threshold, , , These are the model parameters obtained by fitting experimental data.

[0007] In one implementation, the correlation model is a dynamic correction model: ; in This is the temperature compensation coefficient. The pH compensation coefficient is calculated in real time based on the temperature and pH value detected by the detector group.

[0008] In one embodiment, the fluorescence detection module includes: a multi-wavelength excitation source and a corresponding multispectral signal receiver; The computer controller is further configured to receive at least two different wavelength fluorescence intensity signals output by the multispectral signal receiver, including a first fluorescence signal characterizing cell activity. and the second fluorescent signal characterizing cell death According to the first fluorescence signal and the second fluorescence signal Calculate the cell health index ; When the second fluorescence signal When the preset threshold is exceeded, the computer controller controls the intake and exhaust controller to stop the intake and issue an alarm signal.

[0009] In one embodiment, the computer controller is equipped with a machine learning module; the computer controller is further configured to: collect historical culture data, including fluorescence intensity, temperature, pH value, oxygen consumption rate and air intake, and train a predictive model; Using the prediction model, based on the fluorescence intensity at the current moment and temperature Predicting the rate of cellular oxygen consumption at future moments ; Based on the cellular oxygen consumption rate at the future time. Adjust the air intake volume of the air intake pump in advance.

[0010] In one embodiment, the gas guiding assembly includes a first fan and a second fan; The projections of the first fan and the second fan in the vertical direction have a vertical height difference; The projections of the first fan and the second fan in the horizontal direction are both on the same side of the first diameter dividing line of the container, and both are mounted on the inner walls on both sides of the second diameter dividing line of the container. The first diameter boundary line is perpendicular to the second diameter boundary line. The first and second fans are both tilted downwards at 45°. The computer controller controls both the first fan and the second fan to blow air downwards. When the computer controller controls the intake pump to take in air and controls the exhaust valve to exhaust air, the first fan blows air upwards and the second fan blows air downwards.

[0011] In one implementation, based on the current cellular oxygen consumption rate Based on the given numerical range, switch the operating modes of the first and second fans: when When the temperature is below the first threshold, both the first fan and the second fan are controlled to run intermittently or at low speed. when When the airflow is above the first threshold and below the second threshold, both the first and second fans are controlled to blow air downwards. When the intake pump and exhaust valve are working, the first fan is controlled to blow air upwards and the second fan to blow air downwards.

[0012] In one embodiment, the intake and exhaust controller further includes: An intake detection unit, located between the intake pump and the container, is used to detect the intake flow rate. and intake oxygen concentration ; An exhaust detection unit located at the rear end of the exhaust valve (32) is used to detect the exhaust flow rate. And exhaust oxygen ; The computer controller (50) is further configured to: according to the formula Calculation of cellular oxygen consumption rate based on gas balance ; The cell oxygen consumption rate based on gas balance The current cellular oxygen consumption rate calculated based on fluorescence intensity. The system compares the two values; when the deviation exceeds a preset threshold, an alarm signal is issued.

[0013] The advantages or beneficial effects of the above technical solutions include at least the following: Compared to existing technologies that use multiple gas storage tanks to store various gases, this method reduces equipment costs. The computer controller, through data transmitted from the detector components, controls a single mixed gas tank. This allows the computer controller to control the intake pump and exhaust valve, enabling precise pressure control within the tank. Furthermore, pre-mixed gas can be poured into the gas tank and then into the storage tank, eliminating the need for precise control of the gas content in the air. This reduces manufacturing costs, eliminates the need for expensive precision computers, and lowers the cost of controlling gas pressure and content. Attached Figure Description

[0014] The accompanying drawings illustrate exemplary embodiments of the present application and, together with the description thereof, serve to explain the principles of the present application. These drawings are included to provide a further understanding of the present application and are incorporated in and constitute a part of this specification.

[0015] Figure 1 A schematic diagram of the structure of a cell culture container according to an embodiment of the present invention is shown; Figure 2This diagram illustrates the airflow within the container during storage, according to an embodiment of the present invention. Figure 3 This diagram shows a top view of the airflow inside the container in a storage state, according to an embodiment of the present invention. Figure 4 A schematic diagram showing the tilt angles of the first and second fans according to an embodiment of the present invention is provided. Figure 5 This diagram illustrates the airflow within the container during air intake by the intake pump and exhaust by the exhaust valve, according to an embodiment of the present invention. Figure 6 A schematic diagram of the installation of the top cover and the receiving tank according to an embodiment of the present invention is shown; Figure 7 A schematic diagram showing the position of the inclined groove according to an embodiment of the present invention is shown; Figure 8 A schematic diagram of a detector array according to an embodiment of the present invention is shown; Figure 9 A schematic diagram showing the position of the electric heating wire and a cross-sectional schematic diagram of the support portion on the mounting base are shown in an embodiment of the present invention.

[0016] Reference numerals: 10, container; 11, first fan; 12, second fan; 13, first diameter dividing line; 14, second diameter dividing line; 15, fixing rod; 16, thickened part; 161, inclined surface; 17, annular groove; 18, bolt; 20, top cover; 21, inclined groove; 22, annular protrusion; 30, intake and exhaust controller; 31, intake pump; 32, exhaust valve; 40, detector group; 41, mounting base; 411, support part; 42, detection probe; 50, computer controller; 60, mixed gas tank; 70, electric heating wire. Detailed Implementation

[0017] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While some embodiments of this application are shown in the drawings, it should be understood that this application can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of this application. It should be understood that the drawings and embodiments of this application are for illustrative purposes only and are not intended to limit the scope of protection of this application.

[0018] It should be noted that, where there is no conflict, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0019] It should be understood that the term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to". The term "based on" means "at least partially based on". The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments". Definitions of other terms will be given in the following description. It should be noted that the concepts of "first", "second", etc., mentioned in this application are used only to distinguish different devices, modules, or units, and are not intended to limit the order of functions performed by these devices, modules, or units or their interdependencies.

[0020] It should be noted that the terms "one" and "more" used in this application are illustrative rather than restrictive, and those skilled in the art should understand that, unless otherwise expressly indicated in the context, they should be understood as "one or more".

[0021] The names of the messages or information exchanged between multiple devices in the embodiments of this application are for illustrative purposes only and are not intended to limit the scope of these messages or information.

[0022] A cell culture container, comprising the following components: The container 10 has a cylindrical structure, which reduces weld seams and improves sealing. An opening at the top of the container 10 allows the user to place the cell culture dish inside. A top cover 20 is detachably mounted on the top of the container 10 for closing or opening the opening. Specifically, a fixing rod 15 is fixedly connected to the base of the container 10. The end of the fixing rod 15 away from the base is detachably connected to the top cover 20 via bolts 18, ensuring a tight fit of the top cover 20 and preventing air loss from the container 10. The bolts can also be used to tighten the top cover. 18. Remove the cover 20 from the top of the container 10 to access the culture container 10 inside the container 10. As a further improvement, the container 10 has an outwardly extending thickened portion 16 at the opening, and the thickened portion 16 has an outwardly inclined slope 161. The bottom of the cover 20 has a groove 21 that matches the slope 161 to increase the contact area between the cover 20 and the container 10 and further improve the sealing between the cover 20 and the container 10. An annular groove 17 is provided between the thickened portion 16 and the inner wall of the container 10, and the bottom of the cover 20 has an annular protrusion 22 that matches the annular groove 17.

[0023] The intake and exhaust controller 30 includes an intake pump 31 and an exhaust valve 32 mounted on the top of the cover 20. The intake pump 31 is also detachably connected to a mixed gas tank 60, which contains a mixed gas of 2% O2, 5% CO2 and 93% N. The gas in the mixed gas tank 60 can enter the receiving tank 10 through the intake pump 31. When the mixed gas is filled into the receiving tank 10, it will increase the gas pressure in the receiving tube, forming a pressure chamber. In addition, the gas in the receiving tank 10 can be discharged through the exhaust valve 32.

[0024] Meanwhile, a HEPA filtration system is installed between the air pump 31 and the gas tank. The mixed gas passes through the HEPA filtration system to filter out impurities and bacteria in the gas, creating a relatively sterile environment in the container tank 10. A gas guiding assembly is installed on the inner wall of the receiving tank 10. The gas guiding assembly specifically includes the following components: a first fan 11 and a second fan 12; as shown below. Figure 2 As shown, the projections of the first fan 11 and the second fan 12 in the vertical direction have a vertical height difference, wherein the height of the first fan 11 is greater than that of the second fan 12; the projections of the first fan 11 and the second fan 12 in the horizontal direction are both located on the same side of the first diameter dividing line 13 of the receiving tank 10, and both are installed on the inner walls on both sides of the second diameter dividing line 14 of the receiving tank 10, as shown. Figure 3 As shown, the first fan 11 blows the air at the top of the container 10 downwards, and the air flows downwards along the inner wall of the cylindrical container 10. At this time, the second fan 12 located below continues to blow the drawn air towards the bottom of the container 10, causing the air to form a vortex inside the container 10. (Refer to...) Figure 2 This increases the airflow within the container 10, thereby increasing the air mixing within the container 10; wherein, the first diameter dividing line 13 and the second diameter dividing line 14 are perpendicular to each other; and the orientation of the first fan 11 and the second fan 12 is both tilted downward at 45°. The culture vessel further includes: a detector assembly 40, installed on the inner wall of the container 10, for detecting data such as gas pressure, gas content and temperature inside the container 10; and an electric heating wire 70, installed at the bottom of the container 10, for heating the air inside the container 10.

[0025] The culture vessel further includes a fluorescence detection module for real-time detection of the fluorescence intensity of the cell culture medium within the container 10, the fluorescence intensity being correlated with cell metabolic activity. The culture container further includes a computer controller 50, which is electrically connected to the detector assembly 40, the gas guiding assembly, the inlet and outlet controller 30, and the fluorescence detection module. The culture container further includes an electric heating wire 70, which is installed at the bottom of the container 10. The computer controller 50 is also electrically connected to the electric heating wire 70 installed at the bottom of the container 10, and the computer controller 50 further controls the operation of the electric heating wire 70.

[0026] The computer controller 50 receives data from the detector group 40, such as air composition, air pressure, and temperature data inside the container 10. Based on this data, the computer controller 50 controls the intake and exhaust controller 30 and the gas guiding component to operate. Specifically, when the mixed gas in the mixed gas tank 60 is introduced into the container 10, it changes the concentration and pressure of O2 and CO2 in the container 10. At the same time, the detector group 40 senses O2 and CO2 and uploads the data to the computer controller 50. Similarly, while the computer controller controls the heating wire 70 to raise the temperature, the detector group 40 uploads the temperature signal to the computer controller 50. After processing the information, the computer controller 50 adjusts the intake pump 31 and the heating wire 70 to dynamically balance the environment inside the container 10, creating suitable conditions for cell culture at the bottom of the container 10.

[0027] More specifically: Computer controller 50 is configured as follows: Receive the fluorescence intensity signal output by the fluorescence detection module; and calculate the current cell oxygen consumption rate based on the fluorescence intensity signal using a preset fluorescence intensity-cell oxygen consumption rate correlation model. Based on the current cellular oxygen consumption rate, the intake volume of the air pump 31 is controlled to adjust the oxygen concentration within the container 10; the computer controller 50 is configured to calculate the current cellular oxygen consumption rate using a fluorescence intensity-cellular oxygen consumption rate correlation model in the following manner: The fluorescence intensity signal output by the fluorescence detection module is preprocessed, including filtering and temperature drift compensation, to obtain the compensated fluorescence intensity value F(t); The current cellular oxygen consumption rate OUR(t) is calculated based on a pre-defined correlation model. In the first embodiment, the correlation model is a linear scaling model: ; Wherein α is the comprehensive proportionality coefficient obtained through offline calibration experiments, which include: simultaneously measuring fluorescence intensity F(ti) and actual oxygen consumption rate OUR(ti) at multiple time points, and obtaining the proportionality coefficient α through linear regression fitting.

[0028] In the second embodiment, the correlation model is a piecewise linear model: ; in The preset fluorescence intensity threshold, , , These are the model parameters obtained by fitting experimental data.

[0029] In the third embodiment, the correlation model is a dynamic correction model: ; in This is the temperature compensation coefficient. This is the pH compensation coefficient, which is calculated in real time based on the temperature and pH value detected by detector group 40.

[0030] In practical applications, the type of correlation model also varies depending on the type of cell. The selection of the correlation model—whether it is a linear proportional model, a piecewise linear model, or a dynamic correction model—is determined based on the following methods: 1. In some immortalized cell lines (such as CHO cells and HEK293 cells) during the exponential growth phase, their main energy source is glycolysis or aerobic oxidation, and their metabolic pattern is relatively constant. The fluorescence signal and oxygen consumption rate can maintain a good linear relationship; no complex compensation is required. At this time, the correlation model is suitable for the linear proportional model.

[0031] 2. When the cells are adherent cells or stem cells with a distinct growth cycle, such as mesenchymal stem cells, these cells have low metabolism and weak fluorescence signal immediately after seeding, resulting in a low proportion coefficient. Smaller; cells are proliferating rapidly, oxygen consumption is increasing dramatically, fluorescence signal is strong, and the proportionality coefficient is low. The ratio is relatively large; as cells fuse, contact inhibition occurs, and metabolism transitions to a maintenance state, the relationship between fluorescence signal and oxygen consumption may change again, and the proportionality coefficient may change. Unlike before, and in biopharmaceutical process development, if a production line's process for producing a specific protein is mature, the behavioral patterns of cells at various time points are predictable. Thresholds can be preset based on historical data. , With the corresponding parameters, the piecewise model can capture this phased change well and is suitable for piecewise linear models.

[0032] 3. Due to fluorescence signal Not only are they affected by cellular metabolism, but they are also significantly interfered with by the physical environment (temperature, pH). Interference is corrected before metabolic calculations are performed; this is crucial in high-precision physiological studies or drug screening scenarios, such as studying the immediate effects of a drug on mitochondrial metabolism. In such cases, if a 0.5°C temperature fluctuation causes a 5% shift in the fluorescence signal, while the drug effect only changes the signal by 10%, failing to correct for temperature will lead to completely erroneous conclusions about drug efficacy. The dynamic correction model can isolate environmental interference and restore the true changes in cellular metabolism. Furthermore, temperature and pH fluctuate briefly each time the culture vessel is opened. In these scenarios, the detector array 40 detects these fluctuations in real time, and the computer controller utilizes… and These two coefficients correct fluorescence readings in real time, ensuring the calculated oxygen consumption rate. It always reflects the actual needs of the cells, rather than disturbances in the physical environment.

[0033] Based on a further improvement to the above structure, the computer controller 50 is further configured to receive at least two different wavelength fluorescence intensity signals output by a multispectral signal receiver, including a first fluorescence signal characterizing cell activity. and the second fluorescent signal characterizing cell death According to the first fluorescence signal Second fluorescence signal Calculate the cell health index ; Among them, the first fluorescence signal Representing cell viability, this typically involves detecting the functional state of mitochondria in living cells, the level of intracellular reducing power (such as NADH), or the activity of enzymes specific to living cells. Mitochondrial membrane potential probes (such as TMRM and JC-1) are used; only cells with normal mitochondrial membrane potential (i.e., viable cells with energy metabolism) will accumulate these probes and emit fluorescence; the second fluorescence signal... Cell death is a fluorescent signal associated with the loss of cell membrane integrity (i.e., cell death). It utilizes the increased permeability of the cell membrane of dead cells to allow dyes that normally cannot enter living cells to enter the cell interior. After binding with DNA or other cell nuclear materials, it emits light. It is detected using cell membrane-impermeable nucleic acid dyes (such as propidium iodide, PI). These dyes can only enter dead cells with lost membrane integrity and emit light after binding with DNA. Therefore, its intensity reflects the number of dead cells in the culture system.

[0034] When the second fluorescence signal When the preset threshold is exceeded, the computer controller 50 controls the intake and exhaust controller 30 to stop the intake and issue an alarm signal.

[0035] Specifically, the fluorescence detection module includes a multi-wavelength excitation source and a corresponding multispectral signal receiver. Two fluorescent probes are added to the culture medium simultaneously: the first is a cell viability probe, specifically the TMRM (mitochondrial membrane potential) which emits the first fluorescence signal. The second type is a cell death probe, specifically propidium iodide (PI), which can only penetrate the cell membrane of dead cells and bind to DNA, emitting a second fluorescent signal. Among them, TMRM is tetramethylrhodamine methyl ester, a cationic fluorescent dye that can pass through living cell membranes and selectively accumulate in the normal mitochondrial matrix depending on mitochondrial membrane potential. Computer controller 50 simultaneously receives and And calculate the cell health index: During normal cultivation, the computer controller 50 mainly relies on... Calculate the oxygen consumption rate and control the gas supply. When monitored... When the temperature suddenly rises and exceeds the preset safety threshold, it indicates that the culture system may be contaminated or there may be large-scale cell death. At this time, the computer controller 50 immediately issues an instruction to stop the air intake pump 31 and close the exhaust valve 32.

[0036] Based on the above structure, the computer controller 50 controls both the first fan 11 and the second fan 12 to blow air downwards, such as Figure 2 As shown, to ensure airflow within the container 10 and uniform gas distribution, when the computer controller 50 controls the air intake pump 31 to intake air and controls the exhaust valve 32 to exhaust air, as... Figure 5 As shown, the first fan 11 blows air upwards, assisting the air in the container 10 to be blown out from the exhaust port of the exhaust valve 32, and the second fan 12 blows air downwards, assisting the mixed gas entering from the air intake pump 31 to flow from the top to the bottom of the container 10, so that the mixed gas is quickly and evenly distributed in the container 10.

[0037] Based on the above structure, the detector assembly 40 includes: a mounting base 41, which is installed on the side wall of the container 10, with one side flush with the inner wall of the container 10 and the other side extending out of the container 10. The side extending out of the container 10 is connected to the computer controller 50 via a wire; and a detection probe 42, which is installed on the side of the mounting base 41 that extends into the container 10, so that the detection probe 42 can detect the inside of the container 10. The detection probe 42 includes a temperature sensor, a pressure sensor, a gas content sensor, etc., for detecting various data inside the container 10. Further improvements to the above structure: A support portion 411 extends from the side of the mounting base 41 near the detection probe 42, and a groove corresponding to the extended support portion 411 is provided on the inner wall of the container 10 to increase the contact area between the support portion 411 and the side wall of the container 10, making the mounting base 41 more stable on the side wall of the container 10 and less likely to fall off.

[0038] Many studies have attempted to simulate mechanical stress using bioreactors, with the key being how to transfer pressure to cartilage. Early bioreactors employed direct contact pressurization (compression loading), but this method easily leads to cartilage damage. In contrast, hydrostatic pressure can generate more matrix (such as GAG, collagen I and II) and improve mechanical properties. Later, pressurization was achieved by squeezing the liquid (pressure range approximately 5-10 MPa), but this differs from the hydrostatic pressure of a human knee joint when standing (approximately 5 MPa). In recent years, gas pressurization (pressure range approximately 10-100 kPa) has yielded results showing that low hydrostatic pressure can promote cartilage regeneration, which is more consistent with clinical research. However, previous bioreactors did not consider the issue of hypoxic culture. The partial pressure of oxygen in the human knee joint cavity is 50±9 mmHg, while the partial pressure of oxygen in ordinary culture containers is 140 mmHg, far exceeding the oxygen partial pressure within the joint cavity. The cartilage tissue culture device of this application achieves hypoxic conditions simultaneously through gas pressurization; current cell culture technologies face challenges in simulating the requirements of specific gas environments, especially for cell cultures requiring high-pressure hypoxic conditions. Existing bioreactors typically achieve culture conditions by controlling multiple gas valves to allow the entry of O2, CO2, and other gases separately. However, these devices are often too complex and bulky. In this application, the gases from multiple gas tanks are mixed into one gas tank, and then the mixed gas in the mixed gas tank is introduced into the cell culture container of this application by controlling the gas valve, which reduces the burden on the equipment. Furthermore, the computer controller 50 controls the individual mixed gas tank by controlling the various data transmitted back by the detector group 40, which facilitates the computer controller 50 to control the air intake pump 31 and the exhaust valve 32 to achieve precise pressure control within the container 10.

[0039] Based on the further improvement of the above structure, the computer controller 50 is equipped with a machine learning module; the computer controller 50 is further configured to: collect historical culture data, including fluorescence intensity, temperature, pH value, oxygen consumption rate and air intake, and train a prediction model. Using a predictive model, based on the fluorescence intensity at the current moment and temperature Predicting the rate of cellular oxygen consumption at future moments ; Based on the cellular oxygen consumption rate at future moments Adjust the intake volume of the intake pump 31 in advance.

[0040] Computer controller 50 controls both the first fan 11 and the second fan 12 to blow air downwards; When the computer controller 50 controls the intake pump 31 to take in air and controls the exhaust valve 32 to exhaust air, the first fan 11 blows air upward and the second fan 12 blows air downward.

[0041] During the initial stage of cultivation, historical cultivation data, including fluorescence intensity, are collected systematically. ,temperature pH value, and real-time calculated oxygen consumption rate The training dataset is composed of data such as air intake volume. This data is then used to train a recurrent neural network model that can learn the temporal dynamics of cell metabolism.

[0042] Once formal culture begins, the computer controller 50 uses a trained prediction model to determine the fluorescence intensity at the current moment. and temperature Predicting the future Cellular oxygen consumption rate at a specific time (30 minutes later) The controller adjusts the intake volume of the intake pump 31 in advance based on this predicted value. For example, when it is predicted that the cells will enter the exponential growth phase and oxygen consumption will surge in 1 hour, the system increases the basic oxygen supply in advance to achieve feedforward control and effectively avoid the lag defect of traditional feedback control.

[0043] Based on the current cellular oxygen consumption rate Given the current value range, switch the operating mode of the first fan 11 and the second fan 12: when When the temperature is below the first threshold, both the first fan 11 and the second fan 12 are controlled to run intermittently or at low speed. when When the airflow is above the first threshold and below the second threshold, both the first fan 11 and the second fan 12 are controlled to blow air downwards. When the intake pump 31 and exhaust valve 32 are working, the first fan 11 is controlled to blow air upwards and the second fan 12 is controlled to blow air downwards.

[0044] Further improvements to the above technical solution further link fan control with cellular metabolic state. The computer controller 50 calculates the current cellular oxygen consumption rate... Within the given numerical range, the operating modes of the first fan 11 and the second fan 12 are dynamically switched: rest mode, growth mode, and air exchange mode, respectively. In resting mode: when When the temperature drops below the first threshold (e.g., when cells are newly seeded or under cryogenic storage), it indicates that cell metabolism is weak and the cells are sensitive to airflow shear force. At this time, the first fan 11 and the second fan 12 are controlled to operate intermittently, specifically for 1 minute every 10 minutes, or at a very low speed, to maintain only weak convection to prevent local hypoxia and minimize mechanical damage to the cells.

[0045] In the growth pattern: when When the temperature is above the first threshold and below the second threshold (when the cell is in the normal proliferation phase), both the first fan 11 and the second fan 12 are controlled to continuously blow downwards at a medium speed to ensure the uniform distribution of gas and nutrients and meet the needs of cell growth.

[0046] In ventilation mode: when a need for additional gas is detected (i.e., when intake pump 31 and exhaust valve 32 are operating), regardless of the current... Regardless of the value, priority is given to gas purging, specifically by having the first fan move upwards and the second fan move downwards, to complete gas replacement as quickly as possible and prevent cells from being in an abnormal gas environment for a long time.

[0047] When only gas mixing is required and no air exchange is needed (both intake pump 31 and exhaust valve 32 are closed), the computer controller 50 controls both the first fan 11 and the second fan 12 to blow air downwards, forming a top-to-bottom circulating vortex within the container 10. Figure 5 As shown.

[0048] When gas replacement is required (air intake pump 31 intakes, exhaust valve 32 exhausts), computer controller 50 controls the first fan 11 to blow air upwards, assisting in pushing the stale gas at the top towards the exhaust valve 32; simultaneously, it controls the second fan 12 to blow air downwards, assisting in quickly guiding the newly introduced fresh mixed gas to the cell culture area at the bottom, achieving rapid gas purging. Figure 6 As shown.

[0049] Based on further improvements to the above technical solution, the intake and exhaust controller 30 also includes: An intake detection unit, specifically located on the pipeline between the intake pump 31 and the container 10, is used to detect the intake air flow rate. and intake oxygen concentration The unit includes an intake mass flow controller for accurately measuring the intake flow rate. and intake oxygen concentration sensor An exhaust detection unit located at the rear end of the exhaust valve (32) is used to detect the exhaust flow rate. And exhaust oxygen ; The computer controller (50) is further configured as follows: according to the formula Calculation of cellular oxygen consumption rate based on gas balance ;in, The unit is mL O2 / min Cellular oxygen consumption rate based on gas balance Compared with the current cellular oxygen consumption rate calculated based on fluorescence intensity The system compares the two values; when the deviation exceeds a preset threshold, an alarm signal is issued.

[0050] The specific formula for calculating the deviation between the two is: ; when When the threshold of 20% is exceeded, it indicates a significant discrepancy between the calculation results of the fluorescence model and the experimental results based on gas balance. Possible causes include: contamination of the optical window of the fluorescence detection module; quenching or concentration changes of the fluorescent probe; unexpected changes in cell metabolic state; and other sensor malfunctions.

[0051] At this point, the computer controller 50 immediately triggers an alarm signal. The alarm can be triggered in one or more of the following ways: displaying a warning message (such as "Fluorescence-gas deviation exceeds limits, please check the fluorescence detection module") on the human-machine interface of the culture vessel; activating an audible and visual alarm; or sending an alarm notification to the administrator's linked mobile app, SMS, or email address via wired or wireless network.

[0052] Based on a further improvement of the above technical solution: the computer controller 50 can also calculate the carbon dioxide production rate. To obtain respiratory quotient ; ; ; in, The carbon dioxide concentration in the intake air is measured by an intake carbon dioxide concentration sensor installed on the pipeline between the intake pump 31 and the container tank 10. To measure the carbon dioxide concentration in the exhaust gas, it can be obtained by measuring the exhaust carbon dioxide concentration sensor installed on the exhaust gas pipeline of exhaust valve 32. The computer controller 50 can also calculate the respiratory quotient. This value infers the current metabolic substrate type of the cell and provides culture suggestions to the user. For example: when A value consistently above 0.95 suggests that "cells primarily use carbohydrates as metabolic substrates, and glucose consumption may be accelerated." when When the concentration remains below 0.75, it suggests that "the cells may be shifting to fatty acid metabolism, and it is recommended to check the glucose concentration in the culture medium". when When drastic fluctuations occur, it indicates "unstable metabolic state; it is recommended to check the culture conditions."

[0053] These prompts can be displayed along with alarm messages or provided to users as routine operational data.

[0054] Although the core of this embodiment is verification and alarm, the computer controller 50 can also adjust the control strategy appropriately according to the deviation. For example: When the deviation is small (e.g., less than 10%), the fluorescence model can be considered basically reliable, and the system can continue to operate accordingly. As a basis for intake control; When the deviation exceeds 10% but does not reach the alarm threshold, the system can automatically reduce the level of interference. The trust weight should be adjusted, and the range of intake adjustment should be appropriately narrowed to avoid over-adjustment due to model errors. When the deviation exceeds the alarm threshold, in addition to issuing an alarm, the system can temporarily switch to pure time control or manual control mode until the user confirms and troubleshoots the fault.

[0055] Through the above approach, this embodiment achieves independent verification of the fluorescence model calculation results, significantly improving the system's reliability and data credibility. At the same time, it provides users with timely and accurate anomaly warnings, which helps ensure the smooth progress of cell culture experiments.

[0056] Embodiments of the present invention also provide a cell culture method, based on the cell culture container described above, the method comprising the following steps: S1. Preparation for cultivation: S1.1, Cell seeding: The cells to be cultured are seeded into the culture container within the containment vessel 10, and culture medium containing a fluorescent probe is added. The fluorescence intensity of the fluorescent probe is related to the cell's metabolic activity and can be selected from oxygen-sensitive fluorescent probes, NADH autofluorescence monitoring probes, or mitochondrial membrane potential-sensitive probes.

[0057] S1.2 Model parameter pre-calibration: Based on cell type and culture purpose, select and pre-calibrate fluorescence intensity-oxygen consumption rate correlation models. Model types include: Linear scaling model: Suitable for metabolically stable cell lines; Piecewise linear model: Calculation is performed piecewise based on fluorescence intensity thresholds, and it is suitable for cells with distinct growth cycles; Dynamic correction model: Suitable for high-precision culture in environments that are sensitive to environmental conditions.

[0058] Among them, the proportional coefficient α, the piecewise parameter, and the compensation coefficient , Obtained through offline calibration experiments or real-time calculations.

[0059] S1.3 Initial Environment Setup: Close the top cover 20 and start the computer controller 50. Set the target oxygen concentration range, temperature, and pressure parameters according to the cultivation requirements. The computer controller 50 controls the air intake pump 31 to introduce gas into the mixed gas tank 60 until the data fed back by the detector group 40 reaches the set value.

[0060] S2. Dynamic regulation during the cultivation process: S2.1 Real-time data acquisition: The computer controller 50 receives the following data in real time: Fluorescence signal: The raw fluorescence intensity signal from the fluorescence detection module; Environmental parameters: temperature (T), pH, air pressure, and oxygen concentration from detector group 40.

[0061] S2.2 Signal Preprocessing: Perform the following on the original fluorescence signal: Filtering: A low-pass filter algorithm is used to remove high-frequency noise; Temperature drift compensation: based on real-time temperature The fluorescence signal is corrected using a preset compensation coefficient to obtain the compensated fluorescence intensity value. .

[0062] S2.3 Calculation of oxygen consumption rate: Calculate the current cellular oxygen consumption rate based on the pre-defined correlation model. : If a linear proportional model is used: ; If a piecewise linear model is used: according to The corresponding linear formula is called within the given interval; If a dynamic correction model is used: .

[0063] S2.4 Gas supply regulation based on oxygen consumption rate: The computer controller 50 will calculate Compare with the preset target oxygen consumption rate range: when When the oxygen level exceeds the upper limit of the target range, it indicates that the cell metabolism is vigorous and the oxygen consumption is increased. The controller increases the air intake of the air pump 31 to increase the oxygen concentration in the container 10. when When the temperature drops below the lower limit of the target range, it indicates that cell metabolism has slowed down and oxygen consumption has decreased. The controller then reduces the intake volume of the intake pump 31 to reduce the oxygen supply. when When within the target range, maintain the current air intake or stop air intake.

[0064] S2.5 Airflow disturbance regulation based on oxygen consumption rate: Computer controller 50 according to The operating mode of the gas guiding component is dynamically switched depending on the numerical range it is in. Resting mode: When Below the first threshold, cell metabolism is weak and the cells are sensitive to airflow shear force. The first fan 11 and the second fan 12 are controlled to operate intermittently or at low speed to maintain only weak convection and avoid causing mechanical damage to the cells.

[0065] Growth pattern: when When the temperature is above the first threshold and below the second threshold, the cells are in the normal proliferation phase. The first fan 11 and the second fan 12 are both controlled to blow air downwards at a medium speed to ensure a uniform distribution of gas and nutrients.

[0066] Ventilation mode: When gas replenishment is required (i.e., when intake pump 31 and exhaust valve 32 are working), regardless of the current... Regardless of the value, the scavenging mode is executed first—controlling the first fan 11 to blow upward (to help expel stale gas) and the second fan 12 to blow downward (to help introduce fresh gas), so as to complete the gas replacement as quickly as possible and reduce the exposure time of cells in abnormal gas environments.

[0067] S3. Storage and Maintenance: S3.1 Enter storage mode When cell culture reaches the target density, or when it is necessary to pause the culture for storage, the operator can enter "storage mode" via computer controller 50.

[0068] Step 3.2 Metabolic Inhibition and Maintenance In storage mode, the computer controller 50 performs the following operations: Lowering the temperature: Depending on the cell type, the temperature inside the container 10 is lowered to a suitable storage temperature (such as 4°C or room temperature) to inhibit cell metabolism; Maintain basic oxygen supply: Continuously monitor fluorescence signals and oxygen consumption rate Only maintain a low flow rate of oxygen at a baseline level to prevent cell death due to hypoxia, while avoiding unnecessary energy consumption; Intermittent fan operation: The first fan 11 and the second fan 12 are switched to an intermittent operation mode with extremely low speeds, and are only briefly activated when a local oxygen gradient is detected to maintain basic gas homogeneity.

[0069] S3.3, Awakening and Recovery Culture: When it is necessary to resume culture from storage, the operator sets the "resume culture" mode. The computer controller 50 gradually raises the temperature to the culture temperature and adjusts it according to real-time monitoring data. The changes gradually increase the air intake and fan speed, allowing the cells to smoothly return to a normal metabolic state.

[0070] S4. End of cultivation: S4.1, Termination of culture: When the preset culture endpoint is reached or the user manually terminates the process, the computer controller 50 performs the following operations: Close the intake pump 31 and the exhaust valve 32; Switch the first fan 11 and the second fan 12 to low speed or turn them off; Record and save all data throughout the entire culture process (fluorescence intensity, oxygen consumption rate, temperature, pH, air intake, etc.).

[0071] S4.2 Cell Harvesting and Equipment Cleaning: Open the top cover 20 and remove the culture container for cell harvesting. Then clean and sterilize the inside of the container 10 and all accessible parts for future use.

[0072] In the description of this application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0073] Those skilled in the art should understand that the above embodiments are merely for illustrative purposes and are not intended to limit the scope of this application. Those skilled in the art can make other changes or modifications based on the above disclosure, and these changes or modifications still fall within the scope of this application.

Claims

1. A cell culture container, characterized in that, The culture container includes: A container (10) having an opening at the top and a cover (20) detachably attached to the opening, the container (10) being used to store cells; An intake and exhaust controller (30) includes an intake pump (31) and an exhaust valve (32) mounted on the upper cover (20). The intake pump (31) is used to introduce gas from the mixed gas tank (60) into the container (10), and the exhaust valve (32) is used to discharge gas from the container (10). A gas guiding component is installed on the inner wall of the container (10) to agitate the gas inside the container (10); A detector assembly (40) is installed on the inner wall of the container (10) to detect the gas pressure, gas content and temperature inside the container (10); A fluorescence detection module is used to detect the fluorescence intensity of the cell culture medium in the container (10) in real time, wherein the fluorescence intensity is related to cell metabolic activity; A computer controller (50) is electrically connected to the detector group (40), the gas guiding assembly, the inlet and outlet controller (30), and the fluorescence detection module. The computer controller (50) is configured as follows: Receive the fluorescence intensity signal output by the fluorescence detection module; Based on the fluorescence intensity signal, the current cell oxygen consumption rate is calculated using a preset fluorescence intensity-cell oxygen consumption rate correlation model. Based on the current cell oxygen consumption rate, the intake volume of the air pump (31) is controlled to adjust the oxygen concentration in the container (10); the computer controller (50) is configured to calculate the current cell oxygen consumption rate using the fluorescence intensity-cell oxygen consumption rate correlation model in the following manner: The fluorescence intensity signal output by the fluorescence detection module is preprocessed, including filtering and temperature drift compensation, to obtain the compensated fluorescence intensity value F(t); The current cellular oxygen consumption rate OUR(t) is calculated based on a pre-defined correlation model.

2. The cell culture container according to claim 1, characterized in that: The correlation model is a linear proportional model: ； Where α is the comprehensive proportionality coefficient obtained through offline calibration experiments, the offline calibration experiments including: synchronously measuring fluorescence intensity F(t) at multiple time points. i The proportionality coefficient α is obtained by fitting the actual oxygen consumption rate OUR(ti) and the actual oxygen consumption rate OUR(ti) through linear regression.

3. The cell culture container according to claim 1, characterized in that: The correlation model is a piecewise linear model: ； in The preset fluorescence intensity threshold, , , These are the model parameters obtained by fitting experimental data.

4. The cell culture container according to claim 1, characterized in that: The correlation model is a dynamic correction model: ; in This is the temperature compensation coefficient. The pH compensation coefficient is calculated in real time based on the temperature and pH value detected by the detector group (40).

5. The cell culture vessel according to any one of claims 1 to 4, characterized in that: The fluorescence detection module includes: a multi-wavelength excitation source and a corresponding multispectral signal receiver; The computer controller (50) is further configured to receive at least two different wavelength fluorescence intensity signals output by the multispectral signal receiver, including a first fluorescence signal characterizing cell activity. and the second fluorescent signal characterizing cell death According to the first fluorescence signal and the second fluorescence signal Calculate the cell health index ; When the second fluorescence signal When the preset threshold is exceeded, the computer controller (50) controls the intake and exhaust controller (30) to stop the intake and issue an alarm signal.

6. The cell culture container according to claim 5, characterized in that: The computer controller (50) is equipped with a machine learning module; the computer controller (50) is further configured to: collect historical culture data, including fluorescence intensity, temperature, pH value, oxygen consumption rate and air intake, and train a prediction model; Using the prediction model, based on the fluorescence intensity at the current moment and temperature Predicting the rate of cellular oxygen consumption at future moments ; Based on the cellular oxygen consumption rate at the future time. Adjust the air intake volume of the air intake pump (31) in advance.

7. The cell culture container according to claim 6, characterized in that: The gas guiding assembly includes a first fan (11) and a second fan (12); The projections of the first fan (11) and the second fan (12) in the vertical direction have a vertical height difference; The projections of the first fan (11) and the second fan (12) in the horizontal direction are both on the same side of the first diameter dividing line (13) of the container (10), and both are mounted on the inner walls on both sides of the second diameter dividing line (14) of the container (10). The first diameter dividing line (13) and the second diameter dividing line (14) are perpendicular to each other; The first fan (11) and the second fan (12) are both tilted downwards at 45°; The computer controller (50) controls both the first fan (11) and the second fan (12) to blow air downwards; When the computer controller (50) controls the intake pump (31) to take in air and controls the exhaust valve (32) to exhaust air, the first fan (11) blows air upward and the second fan (12) blows air downward.

8. The cell culture container according to claim 7, characterized in that: Based on the current cellular oxygen consumption rate Based on the given numerical range, switch the operating modes of the first fan (11) and the second fan (12): when When the temperature is below the first threshold, the first fan (11) and the second fan (12) are controlled to run intermittently or at low speed. when When the value is higher than the first threshold and lower than the second threshold, both the first fan (11) and the second fan (12) are controlled to blow air downwards; When the intake pump (31) and exhaust valve (32) are working, the first fan (11) is controlled to blow air upward and the second fan (12) blow air downward.

9. The cell culture container according to claim 7, characterized in that: The intake and exhaust controller (30) also includes: An air intake detection unit is installed between the air intake pump (31) and the container (10) for detecting the air intake flow rate. and intake oxygen concentration ; An exhaust detection unit located at the rear end of the exhaust valve (32) is used to detect the exhaust flow rate. And exhaust oxygen ; The computer controller (50) is further configured to: according to the formula Calculation of cellular oxygen consumption rate based on gas balance ; The cell oxygen consumption rate based on gas balance The current cellular oxygen consumption rate calculated based on fluorescence intensity. The system compares the two values; when the deviation exceeds a preset threshold, an alarm signal is issued.

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