Continuous cell culture method based on culture mixed model

Abstract

The invention relates to the technical field of cell culture, and particularly discloses a continuous cell culture method based on a culture mixed model, which comprises the following steps: acquiring cell culture environment data; segmenting the environment data according to preset culture time, and calculating statistical characteristics and analysis characteristics of the environment data in each culture time period; performing factor analysis on the statistical characteristics to obtain culture characteristics; carrying out integrated analysis on the analysis features to obtain mixed features; classifying the culture features into a plurality of culture types, and establishing an identification rule based on the culture features of the category center; classifying the mixed features into a plurality of mixed types by using database analysis, and establishing a judgment rule based on the mixed types of professional judgment; combining the recognition rule and the judgment rule to establish a composite judgment rule; and collecting and inputting current cell culture data, and outputting a cell culture mode based on the composite discrimination rule. By means of the culture method, the mixed state of cell culture can be efficiently adjusted, and therefore the success rate of continuous cell culture is increased.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a continuous cell culture method based on a culture mixing model. Background Art

[0002] Continuous culture refers to a culture method in which microbial strains are inoculated in a non-closed culture system, and fresh nutrient solution is continuously added during the culture process to remove inhibitory factors, optimize the growth environment, and continuously collect culture products. Since continuous culture is a culture method developed and implemented based on in-depth research on the growth curve of microorganisms in batch culture, it has significant characteristics and advantages. It can artificially control a certain period in the growth curve to a certain extent according to the purpose of the researcher, shorten or prolong it, or accelerate / reduce the cell growth rate in a certain period, thereby greatly improving the controllability of the microbial culture process.

[0003] Cell culture refers to a technology that allows cells to grow, reproduce and maintain their main structures and functions under artificially created conditions similar to the in vivo environment, such as suitable temperature, pH, and certain nutrition. For example, the Chinese invention patent with patent publication number CN113286650A discloses a continuous cell culture method, which continuously cultures shear-sensitive cells in a bioreactor system with a controlled gas outlet velocity. Specifically, a cell population composed of shear-sensitive cells is cultured in a bioreactor system with at least 25L of culture medium and a gas outlet velocity of up to 20m / s to achieve a steady-state viable cell concentration in the culture medium within the range of 20x106 cells / mL to 15x107 cells / mL. Cell culture is divided into primary culture and subculture according to different cell types, and can be divided into adherent culture and suspension culture according to different cell growth characteristics. Primary culture refers to the process of culturing cells directly separated from tissues in cell culture bottles or culture dishes. When cells continue to grow and multiply, they are closely attached to each other, with small space and high density, insufficient nutrients and accumulation of metabolites, which leads to slow cell growth, deterioration of cell status and even death. Therefore, subculture is needed to avoid this situation. Subculture is the process of digesting and diluting cells and dividing them into two or more culture bottles or culture dishes to allow them to continue to grow. Adherent culture refers to the process in which anchorage-dependent cells adhere to a certain solid surface for growth and reproduction. Suspension culture refers to the process in which certain cells that do not have the characteristics of adherent growth can only grow and reproduce in a flowable liquid culture medium. Among them, during the suspension culture process, the culture fluid needs to be shaken and oscillated.

[0004] Therefore, in the process of continuous suspension culture of cells, it is necessary to carry out continuous oscillation culture of cells. Oscillation culture refers to a culture method in which microbial cells are inoculated into a liquid culture medium and placed on a shaker or oscillator for continuous oscillation. For example, the Chinese invention patent with patent publication number CN104759226A discloses a shaker and a cell suspension culture method that can be placed in a carbon dioxide incubator. The shaker includes a power system, a drive device and an actuator. The power system and the drive device are connected by a power cord that can pass through the mating position of the inner door of the incubator and the sealing gasket of the door frame or through the through hole on the back of the incubator to cross the inside and outside of the incubator. The power cord can separate the drive device and the actuator from the power system inside and outside the incubator, thereby solving the problem of overheating of the heat generated by the power system when the ordinary shaker is placed in a carbon dioxide incubator, resulting in failure of the incubator temperature control and moisture damage to its electronic components.

[0005] Oscillation can make the culture medium fully contact with oxygen, increase the supply of dissolved oxygen, and the bacteria cultured by oscillation reproduce uniformly and have high culture efficiency. However, the oscillation time in the continuous cell culture process is relatively long, and the oscillation has a greater impact on the results of continuous cell culture. However, in actual use, due to the single frequency of the oscillation device and the relatively simple setting of the oscillation amplitude, the oscillation effect of the upper and lower cells in the continuous cell culture process is very different, and the degree of contact between cells in different parts of the culture medium and the culture fluid and the degree of contact with oxygen are also quite different, resulting in a low success rate of continuous cell culture. Summary of the invention

[0006] The present invention provides a continuous cell culture method based on a culture mixing model, through which the mixing state of cell culture can be efficiently adjusted, thereby improving the success rate of continuous cell culture.

[0007] The technical solution adopted by the present invention to solve the above technical problems is: a continuous cell culture method based on a culture mixing model, comprising the following steps:

[0008] S1: Acquire cell culture environment data, the environment data includes culture solution status data and mixer status data, the culture solution status data includes culture solution temperature, culture solution pH value, culture solution oxygen content, the mixer status data includes mixing mode, mixing frequency, mixer oxygen content;

[0009] S2: segmenting the environmental data according to the preset cultivation time, and calculating the statistical characteristics and analytical characteristics of the environmental data in each cultivation time period;

[0010] S3: Perform factor analysis on statistical features to obtain training features; at the same time, perform integration analysis on analytical features to obtain mixed features;

[0011] S4: Classify the culture characteristics into multiple culture types and establish identification rules based on the culture characteristics of the category center;

[0012] S5: Use database analysis to classify hybrid features into multiple hybrid types, and establish judgment rules based on professionally judged hybrid types;

[0013] S6: Combine the recognition rules and judgment rules to establish a composite discrimination rule;

[0014] S7: Collect and input current cell culture data, and output the cell culture method based on the composite discrimination rule.

[0015] Furthermore, step S2 specifically includes:

[0016] S211: segmenting the environmental data at time intervals of T2 minutes to obtain a plurality of second culture segments;

[0017] S212: Calculate the mixing mode coefficient θ, the average mixing frequency L1, and the average mixer oxygen content Q1 in each second culture segment as analysis features;

[0018] S221: segmenting the environmental data at time intervals of T1 minutes to obtain a plurality of first culture segments;

[0019] S222: Calculate the average culture solution temperature C1, the standard deviation P1 of the culture solution pH value, and the average culture solution oxygen content H1 in each first culture segment as statistical features;

[0020] The mixing mode coefficient θ is determined by the mixing mode used in the second culture segment.

[0021] Furthermore, T1 and T2 satisfy the following relationship:

[0022]

[0023] Among them, μ represents the mixing frequency coefficient, γ represents the oxygen risk coefficient, and the mixing frequency coefficient is obtained by dividing the average mixing frequency L1 by the set mixing frequency L s The oxygen risk factor is determined by the average mixer oxygen content Q1 and the set mixer oxygen content Q s The deviation between them is determined.

[0024] Furthermore, the cultivation characteristics include a first cultivation characteristic X1 and a second cultivation characteristic X2, and the first cultivation characteristic X1 and the second cultivation characteristic X2 are respectively obtained by analyzing in the following manners:

[0025]

[0026]

[0027] Wherein, C0 represents the set optimal culture solution temperature, P0 represents the maximum set difference of the culture solution pH value, H0 represents the set optimal culture solution oxygen content, δ represents the culture solution temperature factor, ρ represents the culture solution oxygen content factor, represents the culture medium temperature factor, ln() represents the logarithmic function with the constant e as the base, max(C1, C0) represents the maximum value between C1 and C0, and min(P0, P1) represents the minimum value between P0 and P1.

[0028] Furthermore, the analysis feature includes a first analysis feature Y1 and a second analysis feature Y2, and the first analysis feature Y1 and the second analysis feature Y2 are respectively obtained by analyzing in the following manners:

[0029]

[0030]

[0031] Among them, L0 represents the set optimal mixing frequency, Q0 represents the set optimal mixer oxygen content, α represents the mixing frequency factor, β represents the mixer oxygen content factor, represents the logarithmic function with L0 as the base, represents the logarithmic function with Q0 as the base, sin() represents the sine function, is the θ square of Y1.

[0032] Further, in step S4, the first culture feature X1 and the second culture feature X2 are classified into N different culture types through a two-dimensional coordinate axis, and the difference in the number of culture features between every two culture types is less than 20; wherein N∈(4, 16).

[0033] Further, in step S4, the culture feature F at the center is selected from each culture type. ij , F ij Included and F ij represents the culture characteristics at the center of the i×j-th culture type, and They represent the first culture characteristic value and the second culture characteristic value at the center of the i×j-th culture type, and the following identification rules are established:

[0034]

[0035]

[0036] in, represents the first culture characteristic value of the input, Represents the second culture characteristic value of the input.

[0037] Furthermore, in step S5, the first analysis feature Y1 and the second analysis feature Y2 are input into the established database, and the mixed features are classified into M different mixed types by the value Z output by the database.

[0038] Furthermore, in step S5, one of the M mixing types is selected, and the value Z output by the corresponding database is determined according to the selected mixing type, and the following judgment rule is established:

[0039]

[0040] in, represents the first analyzed eigenvalue of the input, represents the value of the input second analysis feature, q represents the first analysis feature value adjustment factor, and p represents the second analysis feature value adjustment factor.

[0041] Furthermore, in step S6, the judgment rule is used as the first operation rule, and after the judgment rule can be operated, the identification rule is started to be operated, thereby establishing a composite judgment rule.

[0042] Compared with the prior art, the advantages of the present invention are: by analyzing the detection data of the mixing mode, mixing frequency, and oxygen content of the mixer in the mixer for successful continuous cell culture, as well as the detection data of the culture medium temperature, culture medium pH value, and culture medium oxygen content, a feasible culture mixing model is established, thereby improving the reproducibility and success rate of continuous cell culture; moreover, the method of the present application also establishes a variety of culture types and mixing types, increases the mixing mode and frequency, and culture mode, which can effectively improve the quality of continuous cell culture, and at the same time reduce the reliance on manual identification and manual adjustment during the culture process, thereby reducing the labor cost of culture. DETAILED DESCRIPTION

[0043] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below.

[0044] This embodiment provides a continuous cell culture method based on a culture mixing model, and the specific steps are as follows:

[0045] S1: Obtain cell culture environment data, the environment data includes culture solution status data and mixer status data, the culture solution status data includes culture solution temperature, culture solution pH value, culture solution oxygen content, the mixer status data includes mixing mode, mixing frequency, mixer oxygen content.

[0046] The cell culture environment data obtained is mainly detected from the test of successful continuous cell culture, and the obtained cell culture environment data includes environmental data of at least 5 groups of cells of the same type. The method of the present application mainly detects the state of the culture solution and the state of the mixer during the continuous cell culture process, thereby obtaining the data of the overall culture environment during the continuous cell culture process.

[0047] S2: segmenting the environmental data according to the preset cultivation time, and calculating the statistical characteristics and analytical characteristics of the environmental data in each cultivation time period;

[0048] The environmental data is segmented at time intervals of T2 minutes to obtain multiple second culture segments; the mixing mode coefficient θ, the average mixing frequency L1, and the average mixer oxygen content Q1 in each second culture segment are calculated as analysis features; the environmental data is segmented at time intervals of T1 minutes to obtain multiple first culture segments; the average culture solution temperature C1, the standard deviation of the culture solution pH value P1, and the average culture solution oxygen content H1 in each first culture segment are calculated as statistical features.

[0049] The relationship between T1 and T2 is as follows:

[0050]

[0051] Among them, μ represents the mixing frequency coefficient, γ represents the oxygen risk coefficient, and the mixing frequency coefficient is obtained by dividing the average mixing frequency L1 by the set mixing frequency L s The oxygen risk factor is determined by the average mixer oxygen content Q1 and the set mixer oxygen content Q s The mixing mode coefficient θ is determined by the mixing mode used in the second culture segment.

[0052] In step S2, the obtained data is first segmented according to time, and then analyzed according to time periods. The obtained culture fluid state data is segmented according to the time interval of T2 minutes, T2∈[5,10], and the obtained mixer state data is segmented according to the time interval of T1 minutes, and the above relationship is satisfied between T1 and T2. Limiting the relationship between T1 and T2 can obtain a more reasonable segmentation of the mixer state data. Through the comprehensive judgment between the average mixing frequency L1, the average mixer oxygen content Q1 and the mixing mode coefficient θ, the relationship between the mixing mode and the mixing frequency can be balanced. At the same time, the oxygen content in the mixer improves the division of T1 as a whole.

[0053] In step S2, the analysis features are limited to the mixing mode coefficient θ, the average mixing frequency L1, and the average mixer oxygen content Q1 in each second culture segment. The mixing modes mainly include stirring and oscillation. The stirring types include central rotation and stirring along the axis. The oscillation types mainly include horizontal oscillation, swing oscillation, variable amplitude oscillation, up and down oscillation, and three-dimensional space oscillation. Different mixing mode coefficients θ are assigned according to different mixing modes. When taking the average value of the mixing frequency and the mixer oxygen content, it is also necessary to reduce the noise of the data and remove unreasonable values.

[0054] In step S2, the statistical features are also limited to the average culture solution temperature C1, the standard deviation P1 of the culture solution pH value, and the average culture solution oxygen content H1 in each first culture segment. Since the culture solution temperature and the culture solution oxygen content change slowly, the average value can be used to reflect them. When taking the average value of the culture solution temperature and the culture solution oxygen content, it is also necessary to reduce the noise of the data and remove unreasonable values. Since the culture solution pH value changes quickly and is relatively sensitive, it is reflected in the form of standard deviation.

[0055] S3: Perform factor analysis on the statistical features to obtain the cultivation features; the cultivation features include the first cultivation feature X1 and the second cultivation feature X2, and the first cultivation feature X1 and the second cultivation feature X2 are respectively obtained by the following analysis methods:

[0056]

[0057]

[0058] Wherein, C0 represents the set optimal culture solution temperature, P0 represents the maximum set difference of the culture solution pH value, H0 represents the set optimal culture solution oxygen content, δ represents the culture solution temperature factor, ρ represents the culture solution oxygen content factor, represents the culture medium temperature factor, ln() represents the logarithmic function with the constant e as the base, max(C1, C0) represents the maximum value between C1 and C0, and min(P0, P1) represents the minimum value between P0 and P1.

[0059] At the same time, the analysis features are integrated and analyzed to obtain mixed features; the analysis features include a first analysis feature Y1 and a second analysis feature Y2, and the first analysis feature Y1 and the second analysis feature Y2 are respectively obtained by the following analysis methods:

[0060]

[0061]

[0062] Among them, L0 represents the set optimal mixing frequency, Q0 represents the set optimal mixer oxygen content, α represents the mixing frequency factor, β represents the mixer oxygen content factor, represents the logarithmic function with L0 as the base, represents the logarithmic function with Q0 as the base, sin() represents the sine function, is the θ square of Y1.

[0063] In step S3, the first culture feature X1 and the second culture feature X2 are used to represent the culture feature, the first culture feature X1 is used to reflect the temperature of the culture solution, and the second culture feature X2 is used to reflect the overall condition of the culture solution. In step S3, the first analysis feature Y1 and the second analysis feature Y2 are also used to represent the mixing feature, the first analysis feature Y1 is used to reflect the comprehensive condition of the mixing frequency and the oxygen content of the mixer, and the second analysis feature Y2 is used to represent the overall condition of the mixer.

[0064] S4: Classify the culture characteristics into multiple culture types, and establish identification rules based on the culture characteristics at the center of the category: classify the first culture characteristic X1 and the second culture characteristic X2 into N different culture types through the two-dimensional coordinate axis, and the difference in the number of culture characteristics in each two culture types is less than 20; where N∈(4,16), select the culture characteristic F at the center from each culture type ij , F ij Included and F ij represents the culture characteristics at the center of the i×j-th culture type, and They represent the first culture characteristic value and the second culture characteristic value at the center of the i×j-th culture type, and the following identification rules are established:

[0065]

[0066]

[0067] in, represents the first culture characteristic value of the input, Represents the second culture characteristic value of the input.

[0068] Step S4 is to obtain the summarized culture types by classifying the culture characteristics, so as to facilitate rapid adjustment of the culture methods in the subsequent steps.

[0069] S5: Use database analysis to classify the mixed features into multiple mixed types, and establish judgment rules based on the mixed types judged by professionals: input the first analysis feature Y1 and the second analysis feature Y2 into the established database, classify the mixed features into M different mixed types through the numerical value Z output by the database, select one of the M mixed types, and determine the numerical value Z output by the database corresponding to the selected mixed type, and establish the following judgment rules:

[0070]

[0071] in, represents the first analyzed eigenvalue of the input, represents the value of the input second analysis feature, q represents the first analysis feature value adjustment factor, and p represents the second analysis feature value adjustment factor.

[0072] In step S5, a database is established using currently known cell culture mixing methods, and a value is assigned according to the effect of the mixing method in the database, that is, the value Z output by the database, and then the mixing characteristics are classified into M different mixing types according to the value Z output by the database.

[0073] S6: Establish an operation order for the judgment rule and the identification rule: take the judgment rule as the first operation rule, and when the judgment rule can be operated, start to operate the identification rule, so as to establish a composite judgment rule.

[0074] S7: Collect and input current cell culture data, and output the cell culture method based on the composite discrimination rule.

[0075] In step S7, according to the established operation rules, the collected current cell culture data is input, and the judgment rules are first used to determine whether the current operating mixer state meets the rules. When the mixer state meets the operating conditions, the culture fluid state is judged, and finally the judged cell culture method is output.

[0076] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A continuous cell culture method based on a culture mixing model, characterized in that: The steps include: S1: Acquire cell culture environment data, wherein the environment data includes culture solution state data and mixer state data, wherein the culture solution state data includes culture solution temperature, culture solution pH value, and culture solution oxygen content, and the mixer state data includes mixing mode, mixing frequency, and mixer oxygen content; S2: segmenting the environmental data according to preset cultivation time, and calculating statistical characteristics and analytical characteristics of the environmental data in each cultivation time period; S3: performing factor analysis on the statistical features to obtain the cultivation features; and at the same time, performing integration analysis on the analysis features to obtain mixed features; S4: classifying the culture characteristics into multiple culture types, and establishing identification rules based on the culture characteristics of the category center; S5: using database analysis to classify the mixing characteristics into multiple mixing types, and establishing judgment rules based on professionally judged mixing types; S6: Combining the identification rule and the judgment rule to establish a composite judgment rule; S7: collecting and inputting current cell culture data, and outputting a cell culture method based on the composite discrimination rule.

2. The continuous cell culture method according to claim 1, characterized in that The step S2 specifically includes: S211: segmenting the environmental data at time intervals of T2 minutes to obtain a plurality of second culture segments; S212: Calculate the mixing mode coefficient θ, the average mixing frequency L1, and the average mixer oxygen content Q1 in each second culture segment as the analysis features; S221: Segmenting the environmental data at time intervals of T1 minutes to obtain a plurality of first culture segments; S222: Calculate the average culture solution temperature C1, the standard deviation P1 of the culture solution pH value, and the average culture solution oxygen content H1 in each first culture segment as the statistical features; The mixing mode coefficient θ is determined by the mixing mode used in the second culture segment.

3. The continuous cell culture method according to claim 2, characterized in that: The following relationship is satisfied between T1 and T2: Wherein, μ represents the mixing frequency coefficient, γ represents the oxygen risk coefficient, and the mixing frequency coefficient is obtained by combining the average mixing frequency L1 with the set mixing frequency L s The oxygen risk factor is determined by the deviation between the average mixer oxygen content Q1 and the set mixer oxygen content Q s The deviation between them is determined.

4. The continuous cell culture method according to claim 1, characterized in that The culture characteristics include a first culture characteristic X1 and a second culture characteristic X2, and the first culture characteristic X1 and the second culture characteristic X2 are respectively obtained by analyzing in the following manners: Wherein, C0 represents the set optimal culture solution temperature, P0 represents the maximum set difference of the culture solution pH value, H0 represents the set optimal culture solution oxygen content, δ represents the culture solution temperature factor, ρ represents the culture solution oxygen content factor, represents the culture medium temperature factor, ln() represents the logarithmic function with the constant e as the base, max(C1, C0) represents the maximum value between C1 and C0, and min(P0, P1) represents the minimum value between P0 and P1.

5. The continuous cell culture method according to claim 2, characterized in that: The analysis features include a first analysis feature Y1 and a second analysis feature Y2, wherein the first analysis feature Y1 and the second analysis feature Y2 are respectively obtained by analyzing in the following manners: Among them, L0 represents the set optimal mixing frequency, Q0 represents the set optimal mixer oxygen content, α represents the mixing frequency factor, β represents the mixer oxygen content factor, represents the logarithmic function with L0 as the base, represents the logarithmic function with Q0 as the base, sin() represents the sine function, is the θ square of Y1.

6. The continuous cell culture method according to claim 4, characterized in that: In the step S4, the first culture feature X1 and the second culture feature X2 are classified into N different culture types through a two-dimensional coordinate axis, and the difference in the number of the culture features between every two culture types is less than 20; wherein N∈(4, 16).

7. The continuous cell culture method according to claim 6, characterized in that In step S4, the culture feature F at the center is selected from each culture type. ij , F ij Included and F ij represents the culture characteristics at the center of the i×j-th culture type, and They represent the first culture characteristic value and the second culture characteristic value at the center of the i×j-th culture type, and the following identification rules are established: in, represents the first culture characteristic value of the input, Represents the second culture characteristic value of the input.

8. The continuous cell culture method according to claim 5, characterized in that In the step S5, the first analysis feature Y1 and the second analysis feature Y2 are input into an established database, and the mixed features are classified into M different mixed types according to the value Z output by the database.

9. The continuous cell culture method according to claim 8, characterized in that In step S5, one of the mixed types is selected from the M mixed types, and the value Z output by the database corresponding to the selected mixed type is determined, and the following judgment rule is established: in, represents the first analyzed eigenvalue of the input, represents the value of the input second analysis feature, q represents the first analysis feature value adjustment factor, and p represents the second analysis feature value adjustment factor.

10. The continuous cell culture method according to claim 1, characterized in that: In the step S6, the judgment rule is used as the first operation rule. After the judgment rule can be operated, the identification rule is started to be operated, so as to establish the composite judgment rule.

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