Culture solution changing method, system and medium

Abstract

The invention discloses a culture solution changing method and system and a medium, and the method comprises the following steps: when the solution changing time is up, monitoring a culture container to obtain measurement parameters and preset parameters; according to the measurement parameters and the preset parameters, the liquid changing volume is predicted; determining liquid change time consumption based on the liquid change volume; according to the liquid changing time and the liquid changing time consumption, the liquid changing volume is adjusted, the next liquid changing time is determined, and liquid changing operation is executed according to the adjusted liquid changing volume. According to the method, the measurement parameters and the preset parameters are obtained by monitoring the culture container, so that the volume of the replaced liquid is predicted, and the time consumed for replacing the liquid is accurately determined. By means of the liquid changing time and the liquid changing time consumption, adjustment of the liquid changing volume and determination of the next liquid changing time, the cells can receive nutritional ingredients in time, the cells are better cultured, accurate determination of the next liquid changing time is achieved, and then automation and standardization of the culture process are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biological treatment technology, and in particular to a culture fluid replacement method, system and medium. Background Art

[0002] Currently, cell therapy is a key development area at the forefront of international medicine. Cell culture is a key step in cell therapy. During the cell culture process, the liquid in the culture container needs to be replaced to avoid insufficient liquid concentration. Therefore, it is very important to accurately identify the liquid replacement time in the culture container.

[0003] In the prior art, the pH value of the culture medium is usually monitored, and waste liquid is discharged only when the pH value drops below a certain value due to the influence of cell metabolism, thereby reducing the waste of culture medium. However, the direction of glycolysis reaction of cells in anaerobic and aerobic environments is different. In anaerobic environments, lactic acid produced by glycolysis will have a more significant impact on pH value.

[0004] In addition, when the culture scale is increased, due to the uneven distribution of nutrients and metabolites, the measured results deviate more from the actual values. Therefore, how to accurately identify the time to change the liquid in the container is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] The embodiments of the present invention provide a culture fluid replacement method, system and medium to solve the problem of low accuracy in identifying the time of fluid replacement in a culture container in the prior art.

[0006] A method for replacing a culture medium, comprising:

[0007] When the time for medium change is reached, the culture vessel is monitored to obtain measured parameters and preset parameters;

[0008] Predicting a fluid replacement volume according to the measured parameters and the preset parameters;

[0009] Determining the fluid replacement time based on the fluid replacement volume;

[0010] According to the fluid replacement time and the fluid replacement time consumption, the fluid replacement volume is adjusted and the next fluid replacement time is determined, and the fluid replacement operation is performed according to the adjusted fluid replacement volume.

[0011] A culture fluid replacement system comprises a culture container and a controller connected to the culture container, wherein the controller is used to execute the culture fluid replacement method.

[0012] A computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the culture medium replacement method is implemented.

[0013] The above-mentioned culture fluid replacement method, system and medium, in the culture fluid replacement method of the present invention, when the fluid replacement time is reached, the culture container is monitored to obtain measurement parameters and preset parameters; the fluid replacement volume is predicted based on the measurement parameters and the preset parameters; the fluid replacement time is determined based on the fluid replacement volume; according to the fluid replacement time and the fluid replacement time, the fluid replacement volume is adjusted and the next fluid replacement time is determined, and the fluid replacement operation is performed according to the adjusted fluid replacement volume. The present invention realizes the prediction of the fluid replacement volume by monitoring the culture container to obtain measurement parameters and preset parameters, and then realizes the accurate determination of the fluid replacement time. By adjusting the fluid replacement volume and determining the next fluid replacement time according to the fluid replacement time and the fluid replacement time, the accurate determination of the next fluid replacement time is realized, which allows cells to receive nutrients in a timely manner and better culture cells, thereby realizing the automation and standardization of the culture process. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0015] Figure 1 is a flow chart of a method for replacing culture medium in one embodiment of the present invention;

[0016] Figure 2 is a flow chart of step S20 of a method for replacing culture medium in one embodiment of the present invention;

[0017] Figure 3 is a flow chart of step S30 of the culture medium replacement method in one embodiment of the present invention;

[0018] Figure 4 Schematic diagram of a culture medium replacement system in one embodiment of the present invention. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] In one embodiment, if Figure 1 As shown, a method for replacing culture medium is provided, comprising the following steps:

[0021] S10: When the liquid replacement time is reached, the culture container is monitored to obtain the measured parameters and the preset parameters.

[0022] Understandably, the fluid change time refers to the time point after the cells have been cultured for a period of time, that is, the time point when the glucose in the culture medium is consumed from the initial concentration to the preset fluid change concentration (set according to actual conditions). For example, if the culture starts at 8 o'clock, and the time for the concentration to be consumed to the preset fluid change concentration is 5 hours (obtained by calculation), then the fluid change time is 13 o'clock. After the fluid change is completed, it may be maintained for a certain period of time, and the fluid change operation will be performed at the next fluid change time. Preset parameters refer to known parameters or parameters that can be set in advance, such as the working volume of the culture container, the liquid inlet speed during fluid change, etc. Measurement parameters refer to parameters that need to be measured to be known, such as cell density, drainage speed, etc.

[0023] Specifically, after the culture container is installed, the acquisition device is fixed above the culture container. During the entire process of culturing cells, the viewing angle of the acquisition device remains unchanged, so as to ensure the accuracy of identifying and measuring parameters. When the time for changing the liquid is reached, the culture container is monitored to obtain the measurement parameters and preset parameters. That is, after the culture period has passed and the time for changing the liquid is reached, the controller controls the acquisition device to start measuring the parameters in the culture container after receiving the instruction, thereby obtaining the measurement parameters and retrieving the preset parameters from the database.

[0024] S20: Predicting a fluid replacement volume according to the measurement parameters and the preset parameters.

[0025] It can be understood that the replacement volume refers to the volume of culture medium that needs to be replaced.

[0026] Specifically, after obtaining the measurement parameters and the preset parameters, the volume of the culture fluid that needs to be replaced in the culture container is calculated, that is, the volume of the culture fluid to be replaced is calculated through the measurement parameters and all or part of the parameters in the preset parameters, and the fluid replacement volume can be obtained. Among them, a fluid replacement volume prediction model can be set up, and all parameters can be input into the fluid replacement volume prediction model to predict the fluid replacement volume. Alternatively, a preset fluid replacement volume relationship can be used, and a label can be set at the corresponding position in the relationship, and a parameter label can be added in front of the parameter. In this way, the parameter can be brought into the preset fluid replacement volume relationship through the parameter label for calculation, thereby obtaining the fluid replacement volume.

[0027] S30: Determine the fluid replacement time based on the fluid replacement volume.

[0028] It can be understood that the time required for replacing the culture medium refers to the time required for replacing the culture medium.

[0029] Specifically, the drainage time of the liquid exchange volume is calculated by using some parameters in the measured parameters and some parameters in the preset parameters, and the inlet time of the liquid exchange volume is calculated by using some parameters in the measured parameters and some parameters in the preset parameters, and the drainage time and the inlet time are summed to obtain the liquid exchange time. Among them, the parameters for calculating the inlet time and calculating the drainage time are different except for the liquid exchange volume.

[0030] S40: adjusting the fluid replacement volume and determining the next fluid replacement time according to the fluid replacement time and the fluid replacement time consumption, and performing a fluid replacement operation according to the adjusted fluid replacement volume.

[0031] It can be understood that the next medium change time refers to the time point when the culture medium is replaced next time.

[0032] Specifically, after obtaining the fluid change time, the time threshold is obtained, and the fluid change time and the time threshold are compared. When the fluid change time is less than the time threshold, the fluid change operation is performed according to the fluid change volume and the next fluid change time is determined. When the fluid change time is greater than or equal to the time threshold, the fluid change volume is adjusted so that the fluid change time is less than the time threshold, and the fluid change operation is performed according to the adjusted fluid change volume. Then, according to the fluid change time and the fluid change time, the next fluid change time is determined, that is, the end time point at which the fluid change process ends after the fluid change time is calculated by the fluid change time and the fluid change time. Then, the time consumed by the glucose from the initial concentration to the preset fluid change concentration after the fluid change is calculated, and then, according to the end time point and the consumption time, the next fluid change time can be calculated.

[0033] In the embodiment of the present invention, by monitoring the culture container to obtain the measurement parameters and the preset parameters, the volume of the fluid change is predicted, and then the time consumption of the fluid change is accurately determined. By adjusting the fluid change volume and the time consumption of the fluid change, the next fluid change time can be determined, so that the cells can receive nutrients in time, the cells can be better cultured, and the next fluid change time can be accurately determined, thereby realizing the automation and standardization of the culture process.

[0034] In one embodiment, in step S10, monitoring the culture container to obtain measurement parameters; the measurement parameters include cell density and / or consumption rate, including:

[0035] S101, measuring the cell density of the culture container by using a cell density acquisition device to obtain the cell density.

[0036] Specifically, the cell density of the culture container is measured by the cell density acquisition device. The light source in the cell density acquisition device can be controlled to emit light to the culture container, pass through the transparent protrusion in the culture container, and then receive the light through the imaging lens to collect the cell image. The light source in the cell density acquisition device can also be controlled to emit light to the first reflective device in the culture container, and the light is reflected through the transparent protrusion to the second reflective device by the first reflective device to obtain reflected light. The imaging lens is controlled to receive the reflected light to obtain a cell image, and then the cell image is put into a pre-trained neural network model, and the cells in the cell image are identified and counted by the pre-trained neural network model to obtain the number of cells in the cell image. The cell density can be obtained by performing density calculation based on the number of cells and the parameter information of the cell image.

[0037] or / and

[0038] S102, measuring the consumption rate of the culture container by using a consumption rate acquisition device to obtain the consumption rate.

[0039] It can be understood that the consumption rate refers to the average rate at which cells consume the culture medium per unit time.

[0040] Specifically, the nutrient concentration in the culture container is collected by the consumption rate acquisition device, and the preset liquid exchange concentration in the culture container at the liquid exchange time is collected by the consumption rate acquisition device. Then, the time consumed from the nutrient concentration to the preset liquid exchange concentration is measured to obtain the consumption time. Then, the ratio of the difference between the nutrient concentration and the preset liquid exchange concentration and the consumption time is calculated to obtain the consumption rate. That is, in this embodiment, the precise calculation of the measurement parameters is achieved through different measuring devices, thereby improving the precision and accuracy of subsequent time calculations.

[0041] In one embodiment, in step S101, the cell density acquisition device includes a light source and an imaging lens, and a transparent protrusion is provided on one side of the culture container; the cell density acquisition device cooperates with the transparent protrusion.

[0042] That is, measuring the cell density of the culture container by a cell density acquisition device to obtain the cell density includes:

[0043] The light source is controlled to emit light through the imaging lens of the transparent protrusion to obtain a cell image.

[0044] It can be understood that the light source of the cell density acquisition device emits light, which can be directly irradiated to the imaging lens through the transparent protrusion, and then the cell image is acquired through the imaging lens. The cell image is an image formed after the light irradiates the cells in the transparent protrusion.

[0045] The cells in the cell image are identified and counted using a pre-trained neural network model to obtain the number of cells in the cell image.

[0046] The cell density is obtained according to the cell number and the parameter information of the cell image.

[0047] It can be understood that the pre-trained neural network model can adopt the network structure of the YOLO series, for example, the network structure of yolox, etc.

[0048] Specifically, the neural network model of pre-training is retrieved, and all cell images are input into the neural network model of pre-training, the cells in the cell image are identified and counted by the neural network model of pre-training, that is, the number of cells in the cell image is identified and counted by the yolox network completed by training, so as to calculate the cell density, and the processing mode of the decoupled head (Decoupled head) can be applied using the yolox network structure, the speed of detection is accelerated, and the detection performance is also more accurate. Or, first, all cell images are identified by the recognition module with clarity, and the clarity of the cell image can also be identified by the Tenengrad gradient method, the Laplacia gradient method or the variance method, so as to obtain the clarity value, and filter out the cell image with the maximum clarity value. Wherein, the process of identifying by the yolox network is not described in detail. Next, the cell images selected are counted by the counting module, that is, the cell images are first grayed to obtain the corresponding gray images, then the gray images are segmented, and the gray images are supplemented by morphological processing, that is, the cell edges are first expanded and enlarged, and then the corrosion is reversed to determine the position of the cell nucleus, and then the connected cells are segmented. The segmented image is processed by Gaussian filtering, so that the image becomes blurred, the cell nucleus is darker than the cell edge, and the position of the cell nucleus is highlighted. And the blurred image is sharpened to obtain the contour position information of the cell and remove the edge. Finally, the detected cells are numbered and counted after median filtering, and the number of cells in the cell image can be obtained. According to the number of cells and the parameter information of the cell image, that is, the area of ​​the cell image is calculated by the parameter information of the cell image, the cell data is divided by the area, and the units are converted at the same time to obtain the cell density. That is, in this embodiment, the shooting of cells in the culture container is realized, and the clarity of the cell image is improved. And the recognition of the number of cells is realized, and then the calculation of the cell density is realized, and the accuracy of recognition is improved.

[0049] In another embodiment, in step S101, measuring the cell density of the culture container by a cell density acquisition device to obtain the cell density includes:

[0050] S1011, controlling the light source to emit light to the first reflective device and reflect the light through the transparent protrusion to the second reflective device to obtain reflected light.

[0051] S1012, controlling the imaging lens to receive reflected light to obtain a cell image.

[0052] S1013, identifying and counting cells in the cell image using a pre-trained neural network model to obtain the number of cells in the cell image.

[0053] S1014, obtaining the cell density according to the cell quantity and the parameter information of the cell image.

[0054] Wherein, the cell density acquisition device comprises at least two reflective devices, a light source and an imaging lens, and a transparent protrusion is provided on one side of the culture container; the cell density acquisition device cooperates with the transparent protrusion.

[0055] Understandably, the reflected light refers to the light reflected by the first reflective device and the second reflective device. The cell image refers to the image of the cells in the culture container taken by the imaging lens, which can be one or more images. The cell density refers to the number of cells per unit volume. The parameter information refers to the length and width of the cell image.

[0056] Specifically, when measuring the cell density of a culture container by using a cell density acquisition device, the light source is first controlled to emit light into the culture container, and the emitted light passes through the transparent protrusion through the reflection of the first reflector and falls on the second reflector. Then, the light is reflected out of the culture container by the reflection of the second reflector. An imaging lens is provided on the reflection path of the second reflector, and the imaging lens is controlled to receive the reflected light, that is, the light reflected out of the culture container is received by the imaging lens, thereby obtaining at least one cell image.

[0057] Further, the neural network model of pre-training is retrieved, and all cell images are input into the neural network model of pre-training, the cells in the cell image are identified and counted by the neural network model of pre-training, that is, the number of cells in the cell image is identified and counted by the yolox network completed by training, so as to calculate the cell density, and the processing mode of decoupled head (Decoupled head) can be applied using yolox network structure, the speed of detection is accelerated, and the detection performance is also more accurate. Or, first, all cell images are identified by definition by recognition module, that is, the definition of cell image can be identified by Tenengrad gradient method, Laplacia gradient method or variance method, so as to obtain definition value, and filter out the cell image with maximum definition value. Then, the cell image filtered out is counted by counting module, that is, first graying is carried out to cell image, corresponding gray image is obtained, then, gray image is segmented, and gray image is supplemented by morphological processing mode, that is, first expansion expands cell edge, then corrosion is negated, to determine the position of nucleus, and then connected cells are segmented. The segmented image is subjected to Gaussian filtering so that the image becomes blurred, the color of the cell nucleus is darker than that of the cell edge, and the position of the cell nucleus is highlighted. The blurred image is sharpened to obtain the cell contour position information and remove the edge. Finally, the detected cells are numbered and counted after median filtering to obtain the number of cells in the cell image. According to the number of cells and the parameter information of the cell image, that is, the area of ​​the cell image is calculated by the parameter information of the cell image, the cell data is divided by the area, and the units are converted at the same time to obtain the cell density. That is, in this embodiment, the shooting of cells in the culture container is realized, and the clarity of the cell image is improved. The recognition of the number of cells is realized, and then the calculation of the cell density is realized, and the accuracy of recognition is improved.

[0058] In one embodiment, if Figure 2 As shown, in step S20, the liquid replacement volume is predicted based on the measured parameters and the preset parameters; the measured parameters include cell density and consumption rate; the preset parameters include device parameters and peripheral parameters, including:

[0059] S201, predicting the expansion multiple according to the cell type in the culture container, the nutrient concentration in the peripheral parameters and the consumption rate.

[0060] Understandably, the expansion multiple refers to the ratio of the number of expanded cells to the initial number of cells after a period of culture. Cell types include T cells, K562 tumor cells, etc. The cell expansion model can be a collection of historical cultures of different cell types under different nutrient concentrations, through which a functional relationship can be fitted or a deep learning model can be obtained by training. Through simulation prediction, the expansion multiple can be predicted. Among them, the nutrient concentration refers to the concentration of the culture component required for the cells to be cultured. For example, T cells need the glucose component in the culture medium for incubation, and the nutrient concentration here is the glucose component concentration in the culture medium.

[0061] Specifically, after obtaining the measurement parameters and the preset parameters, the expansion multiple is predicted according to the cell type in the culture container, the nutrient concentration and the consumption rate in the peripheral parameters, that is, a preset cell expansion model is obtained, and the cell type in the culture container, the nutrient concentration and the consumption rate in the peripheral parameters are input into the preset cell expansion model, and the cell expansion multiple is predicted by the preset cell expansion model, that is, the preset cell expansion model calculates the cell expansion multiple through the absorption of culture components by the cells of the cell type in the culture container, resulting in changes in nutrient concentration and the consumption rate of the cells, thereby obtaining the expansion multiple.

[0062] In another embodiment, the expansion multiple is predicted by identifying and counting the number of cells before and after the culture, that is, the number of cells at the beginning is identified and counted by a measuring device, that is, the first number of cells obtained by the above steps S1011 to S1014, or the first number of cells obtained by the method in another embodiment. When the time for changing the medium is reached, the current number of cells is identified and counted by a measuring device, that is, the second number of cells obtained by the above steps S1011 to S1014, or the second number of cells obtained by the method in another embodiment, and the ratio of the second number of cells to the first number of cells is calculated and rounded to obtain the expansion multiple.

[0063] S202, obtaining the culture volume, the first liquid replacement ratio and the second liquid replacement ratio in the equipment parameters.

[0064] Understandably, the culture volume refers to the working volume of the culture container. The working volume is known based on the volume of the container. The first liquid replacement ratio and the second liquid replacement ratio are respectively the maximum liquid replacement ratio and the minimum liquid replacement ratio.

[0065] Specifically, when the liquid change time is reached, the culture volume, the first liquid change ratio and the second liquid change ratio in the equipment parameters are obtained, that is, the culture volume is the working volume when culturing cells, which can be set through specific experiments. The upper and lower limits of the first liquid change ratio and the second liquid change ratio are obtained based on the experience of manual control or the experimental results of large-scale culture. For example, after a large number of experiments, the upper and lower limits of the first liquid change ratio are 40% to 50%, and the upper and lower limits of the first liquid change ratio are 10% to 20%. Therefore, it can be directly obtained when in use, and when adjusting the ratio later, it can be adjusted according to experience.

[0066] S203, inputting the expansion multiple, the cell density, the culture volume, the first fluid replacement ratio and the second fluid replacement ratio into a fluid replacement volume prediction model to predict the fluid replacement volume.

[0067] Specifically, the expansion multiple, cell density, culture volume, first fluid exchange ratio and second fluid exchange ratio are input into the fluid exchange volume prediction model, and the fluid exchange volume is predicted by the fluid exchange volume prediction model to predict the fluid exchange volume. Among them, the neural network model is trained by sample data and sample labels, that is, historical measurement parameters and historical prediction parameters are used as inputs, and historical fluid exchange volumes are used as outputs, so that the neural network model continuously learns the relationship between historical measurement parameters, historical prediction parameters and historical fluid exchange volumes, so that the neural network model converges to obtain a fluid exchange volume prediction model. That is, in this embodiment, the calculation of the cell expansion multiple is realized, and the prediction of the fluid exchange volume is realized, thereby improving the precision and accuracy of fluid exchange volume recognition.

[0068] In one embodiment, in step S203, the expansion multiple, the cell density, the culture volume, the first fluid replacement ratio and the second fluid replacement ratio are input into a fluid replacement volume prediction model to predict the fluid replacement volume, including:

[0069] The fluid replacement volume prediction model includes:

[0070] L=((P1-P2)×V / (E-1))×(D-1)+P2×V;

[0071] Wherein, L refers to the volume of fluid replacement;

[0072] P1 refers to the first fluid change ratio;

[0073] P2 refers to the second fluid change ratio;

[0074] V refers to the culture volume;

[0075] E refers to the amplification factor;

[0076] D refers to cell density.

[0077] Understandably, the first liquid replacement ratio refers to the maximum liquid replacement ratio, such as 50%, etc. The second liquid replacement ratio refers to the minimum liquid replacement ratio, such as 10%, etc. The culture volume is the working volume of the culture container, which is generally provided by the merchant and is a preset parameter. The expansion multiple is the multiple of the cell density or cell data growth during the culture period, for example, 5 times, etc. Cell density refers to the number of cells per unit volume (which can be per square centimeter or per square millimeter).

[0078] Specifically, the expansion multiple, cell density, culture volume, first fluid replacement ratio and second fluid replacement ratio are input into the fluid replacement volume prediction model, and the fluid replacement volume is calculated by the functional relationship learned by the fluid replacement volume prediction model during the training process, that is, all the input parameters are substituted into the corresponding positions in the functional relationship to obtain the fluid replacement volume. That is, in this embodiment, the accurate calculation of the fluid replacement volume is achieved, thereby improving the accuracy of identifying the culture fluid replacement volume.

[0079] In one embodiment, if Figure 3 As shown, in the step S30, the liquid replacement time is determined based on the liquid replacement volume; the preset parameters include the liquid inlet speed, the first maximum liquid discharge speed and the second maximum liquid discharge speed, including:

[0080] S301, determining a drainage speed according to the cell density, the first maximum drainage speed, and the second maximum drainage speed.

[0081] S302, determining a drainage time based on the fluid replacement volume and the fluid drainage speed, and determining a fluid inlet time based on the fluid replacement volume and the fluid inlet speed.

[0082] S303, summing the liquid inlet time and the liquid discharge time to obtain the liquid replacement time.

[0083] Understandably, the drainage rate refers to the rate at which liquid is discharged during the liquid exchange process. The drainage duration refers to the time consumed to discharge the volume of the liquid exchange at the drainage rate. The inlet rate refers to the speed at which the culture medium enters the culture container during the liquid exchange process. The inlet duration refers to the time consumed to start transporting the volume of the liquid exchange into the culture container at the inlet speed. The liquid exchange time refers to the time required for the entire liquid exchange process, that is, the time consumed for one drainage and liquid inlet. The drainage rate refers to the linear decrease relationship between the cell density from 1E6 / mL (per million / ml) to 1E7 / mL (per ten million / ml), that is, the drainage rate = (-(F1+F2) / 9)×D+(10*F1-F2 / 9). Among them, F1 refers to the maximum drainage rate (mL / min) of the cell retention membrane at a cell density of 1E6 / mL. F2 refers to the maximum drainage rate (mL / min) of the cell retention membrane at a cell density of 1E7 / mL. The first maximum drainage rate and the second maximum drainage rate refer to F1 and F2, respectively.

[0084] Specifically, after obtaining the liquid exchange volume, the liquid discharge speed is determined according to the cell density, the first maximum liquid discharge speed and the second maximum liquid discharge speed, that is, the speed of discharging waste liquid is predicted by the trained speed prediction model, that is, the cell density, the first maximum liquid discharge speed and the second maximum liquid discharge speed are input into the speed prediction model, and the speed of discharging waste liquid is calculated by the invisible function relationship learned in the speed prediction model training process, that is, the parameters are substituted into the relationship to obtain the liquid discharge speed. Then, the liquid discharge time is determined based on the liquid exchange volume and the liquid discharge speed, that is, the liquid exchange volume is divided by the liquid discharge speed, and the result is multiplied by one thousand (unit conversion), and the liquid discharge time can be calculated. The liquid inlet time is determined based on the liquid exchange volume and the liquid inlet speed, that is, the liquid exchange volume is divided by the liquid inlet speed, and the result is multiplied by one thousand, and the liquid inlet time can be calculated. Among them, the unit of the liquid discharge speed is milliliter, and the unit of the liquid exchange volume is liter, and unit conversion is required. Furthermore, the liquid filling time and the liquid discharge time are summed to obtain the liquid replacement time, that is, the liquid filling time and the liquid discharge time are summed to obtain the liquid replacement time consumed by the entire liquid replacement process. In another embodiment, the liquid filling process and the liquid discharge process can be performed simultaneously, and the liquid filling time and the liquid discharge time are compared, and the larger time is determined as the liquid replacement time. That is, in this embodiment, the calculation of the liquid discharge speed and the determination of the liquid discharge time and the liquid filling time are realized. In addition, the calculation of the liquid replacement time consumed by the entire liquid replacement process is realized, ensuring the automation and standardization of the culture process.

[0085] In one embodiment, in step S40, adjusting the fluid replacement volume and determining the next fluid replacement time according to the fluid replacement time and the fluid replacement time consumption includes:

[0086] S401, when the fluid replacement time is greater than or equal to a time threshold, the first fluid replacement ratio and the second fluid replacement ratio are proportionally adjusted to obtain a new fluid replacement volume.

[0087] S402, determining a new liquid replacement time according to the liquid discharge speed, the liquid inlet speed and the new liquid replacement volume.

[0088] S403, determining the next liquid change time according to the liquid change time and the new liquid change time.

[0089] It is understandable that the time threshold refers to the time consumed by the entire liquid replacement process set in advance, which can be set according to actual conditions. Generally, 24 hours, that is, 1440 minutes, is the time threshold.

[0090] Specifically, after obtaining the fluid exchange time, a preset time threshold is obtained, and the fluid exchange time is compared with the time threshold. When the fluid exchange time is less than the time threshold, it indicates that the entire fluid exchange process can be completed within the time threshold, and the culture medium is exchanged, and the next fluid exchange time is determined. When the fluid exchange time is greater than or equal to the time threshold, it indicates that the entire fluid exchange process cannot be completed within the time threshold, and the first fluid exchange ratio and the second fluid exchange ratio are proportionally adjusted, that is, the difference between the first fluid exchange ratio and the second fluid exchange ratio is made smaller by proportional adjustment, so that the adjusted first fluid exchange ratio and the second fluid exchange ratio, as well as the expansion multiple, cell density, and culture volume are input into the fluid exchange volume prediction model to predict the new fluid exchange volume. Further, according to the drainage speed, the inlet speed, and the new fluid exchange volume, the new fluid exchange time is determined, that is, the new drainage time is first calculated by the new fluid exchange volume and the drainage speed, and the new inlet time is calculated by the new fluid exchange volume and the inlet speed, and the new drainage time and the new inlet time are summed to obtain the new fluid exchange time. Then, the next liquid change time is determined according to the liquid change time and the new liquid change time consumption, that is, after the new liquid change time consumption at the time point of the liquid change time, and then the time consumption for the concentration to be consumed to the preset liquid change concentration, the next liquid change time can be determined. That is, in this embodiment, the judgment of whether the liquid change can be completed in one time within the time threshold is realized, the calculation of the new liquid change volume is realized, and then the calculation of the new liquid change time consumption is realized, ensuring that the determination of whether the liquid change can be completed in one time within the time threshold is ensured.

[0091] In one embodiment, after step S401, that is, after the first liquid replacement ratio and the second liquid replacement ratio are proportionally adjusted to obtain a new liquid replacement volume, the preset parameters further include a preset liquid replacement concentration; including:

[0092] S501, determining a fluid replacement time according to the nutrient concentration, the preset fluid replacement concentration, the culture volume, the cell density and the consumption rate.

[0093] S502: Determine a single liquid replacement volume according to the liquid replacement time and the liquid drainage speed.

[0094] S503, calculating the number of fluid changes according to the new fluid change volume and the single fluid change volume.

[0095] Understandably, the fluid replacement time refers to the time point when the cells change from the nutrient concentration to the preset fluid replacement concentration at the consumption rate. The preset fluid replacement concentration is the fluid replacement concentration set in advance. After experiments, it was found that the intersection point of cell proliferation speed and concentration change is within the concentration range of 100 to 150 mmol (millimolar), so the preset fluid replacement concentration is set to 100 mmol.

[0096] Specifically, since the cell proliferation rate will significantly reduce nutrition after the nutrient concentration is lower than 100mmol, the liquid should be replaced before the nutrient concentration is lower than 100mmol. Therefore, when the total volume of the inlet and outlet liquid is large, the inlet and outlet liquid demand within 24 hours is divided into multiple cycles. First, the time when the nutrient concentration drops to 150mmol is calculated by nutrient concentration, culture volume, cell density, consumption rate and concentration of 150mmol, that is, t1 = (V×g0-150mmol) / (D×V×Cg)×60, where g0 is the nutrient concentration; V refers to the culture volume; D refers to the cell density; Cg is the consumption rate, which is the number of millimoles of molecules consumed per 24h; in this way, the first time can be obtained. Then, calculate the time required for the concentration to drop from 150mmol to the preset concentration of 100mmol, that is, t2 = (V×150mmol-100mmol) / (D×V×Cg)×60. In this way, the second time can be obtained. The first time and the second time are summed to obtain the concentration consumption time. The liquid change time can be determined from the time of starting the culture through the concentration consumption time. According to the liquid change time and the drainage speed, the single liquid change volume is determined, that is, the upper limit of the single maximum liquid change volume is calculated by the second time and the drainage speed, and the single liquid change volume can be obtained. Among them, round down to the order of 100mL. The drainage speed changes according to the cell density. The maximum drainage speed is calculated to obtain the single liquid change volume. The number of fluid changes is calculated based on the new fluid change volume and the single fluid change volume, where the unit of the new fluid change volume is liter, and the unit of the single fluid change volume is milliliter. The units are converted, that is, the fluid change volume is divided by the single fluid change volume, multiplied by one thousand and rounded (that is, when the final number does not meet 100mL, add one to the number of times) to get the number of fluid changes.

[0097] S504, determining a target fluid replacement time according to the fluid discharge speed, the fluid inlet speed, the single fluid replacement volume, and the fluid replacement times.

[0098] S505, determining the next liquid change time according to the liquid change time and the target liquid change time, so as to complete the liquid change operation of a single liquid change volume for the number of liquid changes before the next liquid change time.

[0099] It can be understood that the target fluid exchange time refers to the sum of the times of multiple fluid exchanges.

[0100] Specifically, after obtaining the number of fluid changes, the target fluid change time is determined based on the fluid discharge speed, fluid inlet speed, single fluid change volume, and fluid change number. That is, the single fluid change volume is divided by the fluid inlet speed and multiplied by one thousand (unit conversion) to obtain the single fluid inlet time. The single fluid inlet time is multiplied by the number of fluid changes to obtain the target fluid inlet time. The single fluid change volume is divided by the fluid discharge speed and multiplied by one thousand (unit conversion) to obtain the single fluid discharge time. The single fluid discharge time is multiplied by the number of fluid changes to obtain the target fluid discharge time. The target fluid inlet time and the target fluid discharge time are summed to obtain the target fluid change time. Furthermore, according to the fluid exchange time and the target fluid exchange time, the next fluid exchange time is determined, that is, the concentration consumption time after the fluid exchange is calculated, that is, the concentration consumption time is calculated by the fluid exchange volume and the fluid exchange concentration. The nutrient concentration after the fluid exchange is first determined, and then the concentration consumption time is calculated by the current cell density (i.e., the density after amplification by the amplification multiple) and the consumption rate, and the concentration consumption time can be obtained. Then, after the fluid exchange time, the next fluid exchange time is calculated by the target fluid exchange time and the concentration consumption time, so that the fluid exchange operation of a single fluid exchange volume of the number of fluid exchanges is completed before the next fluid exchange time. That is, in this embodiment, the entire inlet and outlet fluid cycle is divided into multiple single cycles, which avoids the phenomenon of malnutrition of cells when the concentration is low, and ensures that the cells are adequately nourished during the entire fluid exchange process.

[0101] It should be understood that the order of execution of the steps in the above embodiment does not necessarily mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiment of the present invention.

[0102] A culture fluid replacement system comprises a culture container and a controller connected to the culture container, wherein the controller is used to execute the culture fluid replacement method.

[0103] The specific definition of the culture medium replacement system, controller and its various units and modules can be found in the above definition of the culture medium replacement method, which will not be repeated here. Each module in the above controller can be implemented in whole or in part by software, hardware and their combination. It is understandable that, if Figure 4As shown, the controller includes a processor, a memory, a network interface and a database connected by a system bus. Each module of the controller can be embedded in or independent of the processor in hardware form, or can be stored in the memory in software form, so that the processor can call and execute the operations corresponding to the above modules. Among them, the processor is used to provide computing and control capabilities. The memory includes a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system, a computer program and a database. The internal memory provides an environment for the operation of the operating system and the computer program in the non-volatile storage medium. The database is used to store the data used in the culture fluid replacement method in the above embodiment. The network interface is used to communicate with an external terminal through a network connection. When the computer program is executed by the processor, a culture fluid replacement method is implemented.

[0104] In one embodiment, a computer-readable storage medium is provided, wherein the computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the culture medium replacement method described above is implemented.

[0105] Those skilled in the art can understand that all or part of the processes in the above-mentioned embodiment methods can be completed by instructing the relevant hardware through a computer program, and the computer program can be stored in a non-volatile computer-readable storage medium. When the computer program is executed, it can include the processes of the embodiments of the above-mentioned methods. Among them, any reference to memory, storage, database or other media used in the embodiments provided in this application can include non-volatile and / or volatile memory. Non-volatile memory can include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM) or flash memory. Volatile memory can include random access memory (RAM) or external cache memory. As an illustration and not limitation, RAM is available in many forms, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDRSDRAM), enhanced SDRAM (ESDRAM), synchronous link (Synchlink) DRAM (SLDRAM), memory bus (Rambus) direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM).

[0106] Those skilled in the art can clearly understand that for the convenience and simplicity of description, only the division of the above-mentioned functional units and modules is used as an example. In actual applications, the above-mentioned functions can be distributed and completed by different functional units and modules as needed, that is, the internal structure of the device can be divided into different functional units or modules to complete all or part of the functions described above.

[0107] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included in the protection scope of the present invention.

Claims

1. A method for replacing culture medium, characterized in that: include: When the time for medium change is reached, the culture vessel is monitored to obtain measured parameters and preset parameters; Predicting a fluid replacement volume according to the measured parameters and the preset parameters; Determining the fluid replacement time based on the fluid replacement volume; According to the fluid replacement time and the fluid replacement time consumption, the fluid replacement volume is adjusted and the next fluid replacement time is determined, and the fluid replacement operation is performed according to the adjusted fluid replacement volume.

2. The method for replacing culture medium according to claim 1, characterized in that: The measurement parameters include cell density and consumption rate; the preset parameters include device parameters and peripheral parameters; The step of predicting the fluid replacement volume according to the measured parameters and the preset parameters includes: Predicting the expansion multiple according to the cell type in the culture container, the nutrient concentration in the peripheral parameters and the consumption rate; Obtaining the culture volume, the first liquid replacement ratio, and the second liquid replacement ratio in the equipment parameters; The expansion multiple, the cell density, the culture volume, the first fluid replacement ratio and the second fluid replacement ratio are input into a fluid replacement volume prediction model to predict the fluid replacement volume.

3. The method for replacing culture medium according to claim 2, characterized in that: The step of inputting the expansion multiple, the cell density, the culture volume, the first fluid replacement ratio and the second fluid replacement ratio into a fluid replacement volume prediction model to predict the fluid replacement volume comprises: The fluid replacement volume prediction model includes: L=((P1-P2)×V / (E-1))×(D-1)+P2×V; Wherein, L refers to the volume of fluid replacement; P1 refers to the first fluid change ratio; P2 refers to the second fluid change ratio; V refers to the culture volume; E refers to the amplification factor; D refers to cell density.

4. The method for replacing culture medium according to claim 1, characterized in that: The measurement parameters include cell density and / or consumption rate; The monitoring of the culture container to obtain the measured parameters comprises: Measuring the cell density of the culture container by using a cell density acquisition device to obtain the cell density; or / and The consumption rate is measured on the culture container by a consumption rate acquisition device to obtain the consumption rate.

5. The method for replacing culture medium according to claim 2, characterized in that: The preset parameters include a liquid inlet speed, a first maximum liquid discharge speed, and a second maximum liquid discharge speed; The step of determining the fluid replacement time based on the fluid replacement volume includes: determining a drainage speed according to the cell density, the first maximum drainage speed, and the second maximum drainage speed; Determining a drainage time based on the fluid replacement volume and the fluid removal speed, and determining a fluid inlet time based on the fluid replacement volume and the fluid inlet speed; The liquid replacement time is obtained by summing the liquid inlet time and the liquid discharge time.

6. The method for replacing culture medium according to claim 5, characterized in that: The adjusting the liquid replacement volume and determining the next liquid replacement time according to the liquid replacement time and the liquid replacement time consumption comprises: When the fluid replacement time is greater than or equal to the time threshold, the first fluid replacement ratio and the second fluid replacement ratio are proportionally adjusted to obtain a new fluid replacement volume; Determining a new liquid replacement time according to the liquid discharge speed, the liquid inlet speed and the new liquid replacement volume; The next liquid change time is determined according to the liquid change time and the new liquid change time.

7. The method for replacing culture medium according to claim 6, characterized in that: The preset parameters also include preset liquid replacement concentration; After the first liquid replacement ratio and the second liquid replacement ratio are adjusted to obtain a new liquid replacement volume, the method further includes: Determining a liquid replacement time according to the nutrient concentration, the preset liquid replacement concentration, the culture volume, the cell density, and the consumption rate; Determine a single liquid replacement volume according to the liquid replacement time and the liquid drainage speed; Calculate the number of fluid changes according to the new fluid change volume and the single fluid change volume; Determining a target fluid replacement time according to the fluid discharge speed, the fluid inlet speed, the single fluid replacement volume, and the fluid replacement times; The next liquid change time is determined according to the liquid change time and the target liquid change time, so that the liquid change operation of a single liquid change volume with the number of liquid changes is completed before the next liquid change time.

8. The method for replacing culture medium according to claim 4, characterized in that: The cell density acquisition device comprises a light source and an imaging lens, and a transparent protrusion is provided on one side of the culture container; the cell density acquisition device cooperates with the transparent protrusion; Measuring the cell density of the culture container by a cell density acquisition device to obtain the cell density comprises: Controlling the light source to emit light through the transparent protrusion to the imaging lens to obtain a cell image; Identifying and counting cells in the cell image using a pre-trained neural network model to obtain the number of cells in the cell image; The cell density is obtained according to the cell number and the parameter information of the cell image.

9. A culture medium replacement system, characterized in that: It comprises a culture container and a controller connected to the culture container, and the controller is used to execute the culture medium replacement method according to any one of claims 1 to 8.

10. A computer-readable storage medium storing a computer program, characterized in that: When the computer program is executed by a processor, the culture medium replacement method according to any one of claims 1 to 8 is implemented.

Images