Treatment method, cell culture method, medium manufacturing method, substance manufacturing method, medium and program

Abstract

To provide a processing method or the like by which information contributing to the development of culture medium for an objective can be obtained.SOLUTION: A processing method in which a cell is cultured by using each of a plurality of kinds of culture media 41, culture data 51 in which a kind of the culture medium 41, a result of gene expression analysis and a cell characteristic value showing the characteristic of a cell are associated is acquired, and a correlated gene having correlation with the cell characteristic value is extracted on the basis of the culture data 51. Culture is performed by subculture, and the culture data is acquired in a plurality of generations of the subculture.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a treatment method, a cell culture method, a method for producing a medium, a method for producing a substance, a method for culturing a cell, and a method for producing a culture medium. Local and programs. [Background technology]

[0002] Intensive, extensive experiments supported by statistical design of experiments (DoE) are currently the standard method for determining the optimal composition of cell culture media. However, this method is time-consuming and expensive. To improve cost-effectiveness, a method for optimizing the composition of cell culture media based on exometabolome assays has been proposed (Patent Document 1). Using this technique, any number of cellular functions can be optimized via media factors. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2019-193587 Summary of the Invention [Problem to be solved by the invention]

[0004] However, in the method of Patent Document 1, cells are cultured using many different media with different compositions, which requires a huge amount of experimental time and expense to develop an optimized medium.

[0005] In one aspect, the present invention provides a method for processing and the like that can provide information that contributes to the development of a tailored culture medium with a relatively small number of experiments. [Means for solving the problem]

[0006] The treatment method involves culturing cells with uniform characteristics and genetic backgrounds in multiple media containing different types or concentrations of compounds, and determining the type of media and the The genes expressed in the cultured cells and the expression levels of each of the genes were measured. Culture data is obtained that associates the gene expression analysis results with cell characteristic values ​​that indicate cell characteristics, and based on the culture data, The expression level Correlated genes that are correlated with the cell characteristic value are extracted.

[0007] In this processing method, the culture is performed by subculture, and the culture data is obtained for multiple generations of the subculture.

[0008] In this processing method, the gene expression analysis result is a gene profile that associates a plurality of genes with the expression levels of each of the genes.

[0009] In this processing method, the correlated genes are The expression level A positively correlated gene that has a positive correlation with the cell characteristic value, or The expression level The negatively correlated gene has a negative correlation with the cell characteristic value.

[0010] This processing method extracts compounds that affect the expression levels of the extracted correlated genes based on a database that records the relationships between compounds and gene expression levels.

[0011] This processing method extracts compounds that increase the expression level of the correlated gene if the correlated gene has a positive correlation with the cell characteristic value, and extracts compounds that decrease the expression level of the correlated gene if the correlated gene has a negative correlation with the cell characteristic value.

[0012] In this treatment method, the cell characteristic value indicates the proliferation ability or therapeutic effect of the cell.

[0013] The cell culture method involves culturing cells with uniform characteristics and genetic backgrounds in multiple media containing different types or concentrations of compounds, and determining the type of media and The genes expressed in the cultured cells and the expression levels of each of the genes were measured.Culture data correlating the gene expression analysis results with cell characteristic values ​​indicating cell characteristics is recorded, and based on the culture data, The expression level is Correlated genes that correlate with cell characteristic values ​​are extracted, and compounds that affect the expression levels of the extracted correlated genes are extracted based on a database that records the relationship between compounds and gene expression levels.A second medium containing the extracted compounds is prepared, and cells are cultured using the second medium.

[0014] The method for producing the culture medium involves culturing cells with uniform characteristics and genetic backgrounds in a plurality of media containing different types or concentrations of compounds, and then determining the type of medium and the The genes expressed in the cultured cells and the expression levels of each of the genes were measured. Culture data correlating the gene expression analysis results with cell characteristic values ​​indicating cell characteristics is recorded, and based on the culture data, The expression level Correlated genes that correlate with cell characteristic values ​​are extracted, and compounds that affect the expression levels of the extracted correlated genes are extracted based on a database that records the relationship between compounds and gene expression levels, and compounds selected from the extracted compounds are mixed into the base material of the culture medium.

[0015] The method for producing the substance involves culturing cells with uniform characteristics and genetic backgrounds in multiple media containing different types or concentrations of compounds, and then determining the type of media and the The genes expressed in the cultured cells and the expression levels of each of the genes were measured. Culture data correlating the gene expression analysis results with cell characteristic values ​​indicating cell characteristics is recorded, and based on the culture data, The expression level Correlated genes that correlate with cell characteristic values ​​are extracted, and compounds that affect the expression levels of the extracted correlated genes are extracted based on a database that records the relationship between compounds and gene expression levels.A second medium containing the extracted compounds is prepared, cells are cultured using the second medium, and substances secreted from the cultured cells are extracted.

[0016] The culture medium is made by culturing cells with uniform characteristics and genetic backgrounds in multiple media containing different types or concentrations of compounds, and the type of culture medium and The genes expressed in the cultured cells and the expression levels of each of the genes were measured.Culture data correlating the gene expression analysis results with cell characteristic values ​​indicating cell characteristics is recorded, and based on the culture data, The expression level is This medium is prepared by extracting genes that correlate with cell characteristic values, extracting compounds that affect the expression levels of the extracted correlated genes based on a database that records the relationship between compounds and gene expression levels, and mixing a compound selected from the extracted compounds into the base material of the medium.

[0017] The program acquires culture data that correlates the type of culture medium for multiple generations of cells that have been subcultured using multiple types of culture media each containing different types or concentrations of compounds, gene expression analysis results that measure genes expressed in the cultured cells and the expression levels of each of the genes, and cell characteristic values ​​that indicate the characteristics of the cells, and causes a computer to execute a process of extracting correlated genes whose expression levels correlate with the cell characteristic values ​​based on the culture data.

[0018] This medium is the AOF medium. [Effects of the Invention]

[0019] In one aspect, a treatment method or the like can be provided that allows information contributing to the development of a culture medium suited to a purpose to be obtained with a relatively small number of experiments. In another aspect, a therapeutic drug for a specific disease can be produced from cells produced by the treatment method of the present invention, secretions from the cells, or information obtained by analyzing them. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is an explanatory diagram outlining the procedure for preparing a culture medium suited to a purpose. [Figure 2] FIG. 10 is an explanatory diagram illustrating a specific example of a procedure for creating culture data. [Figure 3] FIG. 2 is an explanatory diagram illustrating the record layout of a culture data DB. [Figure 4] FIG. 2 is an explanatory diagram illustrating the procedure for determining the concentration shown in step S507 of FIG. [Figure 5] FIG. 1 is an explanatory diagram illustrating a configuration of an information processing system. [Figure 6] 10 is a flowchart illustrating the flow of processing of a program. [Figure 7] 10 is a flowchart illustrating the process flow of a subroutine for extracting correlated genes. [Figure 8]FIG. 10 is an explanatory diagram illustrating a specific example of a procedure for creating culture data according to the second embodiment. [Figure 9] FIG. 10 is an explanatory diagram illustrating a record layout of a culture data DB according to the second embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0021] [Embodiment 1] Cell cultures are used for experiments in various research fields, including regenerative medicine, cell engineering, and genetic engineering. Cultured cells are also used in regenerative medicine. Mass-cultured cells are also used to produce proteins and peptides for use in foods and pharmaceuticals. Cell secretions and exosomes obtained from these cells are also used for the above-mentioned purposes.

[0022] In addition, various types of cell culture media are commercially available for various target cells, primarily to improve the proliferation rate of those cells. Some media contain unknown components, such as animal-derived component-containing media, which are basal media supplemented with animal-derived component supplements such as serum.

[0023] According to the findings of the inventors, the components of a culture medium affect the growth rate during cell culture, the properties of the cultured cells, and the characteristics of substances secreted by the cells. Figure 1 is an explanatory diagram outlining the procedure for preparing a culture medium suited to a purpose. First, cells with uniform cell properties and genetic background are seeded into multiple types of culture medium 41 (step S501).

[0024] Figure 1 illustrates three types of media: "medium S," "medium T," and "medium U." In the following explanation, "medium S" may be referred to as medium 41S, "medium T" as medium 41T, and "medium U" as medium 41U. The components contained in each of medium 41, medium 41S, medium 41T, and medium 41U, may be unknown, but it is preferable that the media contain different components.

[0025] When using a medium derived from animal components, there is a risk that proteins on the cell surface may be coated with proteins contained in the medium 41. Therefore, it is desirable that the medium 41 be an AOF (Animal Origin Free) medium or a completely synthetic medium that can reflect the original state of the cell surface.

[0026] 1, the medium 41 contained in a petri dish-like culture vessel is shown as a disk-shaped schematic, but other culture vessels such as a culture flask, a roller bottle, or a multi-well plate may also be used. Note that the petri dish itself is not shown in FIG.

[0027] The cells seeded in each medium 41 are cultured (step S502). Then, the cell characteristic value and gene expression level are measured for each medium 41 (step S503). Here, the cell characteristic value is a characteristic of the cell evaluated in accordance with the purpose of creating the medium. Specific examples of the cell characteristic value will be described later.

[0028] The gene expression level is quantified by the amount of each gene measured by gene expression analysis, which is an analytical method for measuring the types and amounts of base sequences, such as mRNA (messenger ribonucleic acid), miRNA (micro ribonucleic acid), and lncRNA (long noncoding ribonucleic acid), in cultured cells.

[0029] Gene expression analysis is performed using, for example, a next-generation sequencer 16 (see FIG. 5). Gene expression analysis may also be performed using other methods, such as microarrays or real-time PCR (Polymerase Chain Reaction). In the following description, the base sequences detected by gene expression analysis, including non-coding RNAs such as miRNAs and lncRNAs, will be referred to as genes. In the following description, data summarizing the cell characteristic values ​​and gene expression analysis results using each culture medium 41 will be referred to as culture data 51. Examples of culture data 51 will be described later.

[0030] By data analysis of the culture data 51, multiple genes whose expression levels are correlated with the cell characteristic value are extracted (step S504). In the following explanation, the genes extracted in step S504 will be referred to as correlated genes. The data analysis method for step S504 will be described later. In step S504, it is possible to extract both genes whose expression levels are positively correlated with the cell characteristic value and genes whose expression levels are negatively correlated.

[0031] Thereafter, a plurality of compounds that affect the expression of the plurality of correlated genes extracted in step S504 are extracted (step S506) with reference to gene DB (Database) 61. In the following description, the compounds extracted in step S506 will be referred to as influencing compounds.

[0032] Here, gene DB61 is a database that records changes in gene expression levels when various compounds are added to cultured cells. For example, gene DB61 can use the LINCS dataset provided by the National Institutes of Health (NIH) in the LINCS (Library of Integrated Network-Based Cellular Signatures) program. Gene DB61 can also use databases provided by other research institutions or companies, proprietary databases based on in-house experimental results, or a combination of these.

[0033] For each of the influential compounds extracted in the above-mentioned step S506, a concentration that shows an appropriate effect is determined (step S507). A specific example of a method for determining the concentration in step S507 will be described later. The concentration may be determined for only a portion of the influential compounds extracted in step S506.

[0034] Typically, a new medium is prepared by using a basal medium containing a nutrient source for the cells as a base material and mixing each of the influencing compounds to the concentrations determined in step S507 (step S508). As a result, a second medium 42 having the desired function is completed.

[0035] 2 is an explanatory diagram illustrating a specific example of the procedure for creating culture data 51. The following describes an example of creating a second culture medium 42 suitable for obtaining a large number of cells by culture. While FIG. 2 shows "medium S," similar processing is also performed for "medium T" and "medium U," etc.

[0036] First, the same type of cells as in step S501 are seeded in "medium S." Subculture is performed using "medium S." Since the purpose of the culture is to obtain a large number of cells, a value indicating the proliferation ability of the cells is used as the cell characteristic value. Subculture of the cells is performed using medium S. For example, at the end of the second and nth generations of culture, the proliferation ability and gene expression level are measured. Measurements may be performed for all generations of subculture.

[0037] In the following explanation, a value indicating the proliferation ability of cells will be referred to as proliferation ability. Proliferation ability is, for example, PDL (Population Doubling Number: the number of cell divisions). PDL can be calculated based on the ratio of the number of cells seeded in the medium 41 to the number of cells at the end of the culture. The number of cells can be measured using a cell measuring device 17 (see FIG. 5), such as a flow cytometer or an imaging cytometer.

[0038] The proliferation potential may be measured as DT (doubling time). DT can be calculated by analyzing images of cells in culture taken over time using an imaging cytometer. The proliferation potential may be measured based on the amount of cell cycle proteins, such as PCNA (proliferating cell nuclear antigen), expressed by dividing cells. The proliferation potential of the second or subsequent passages may be the proliferation potential of the current generation or the cumulative proliferation potential calculated by accumulating the proliferation potential from the first generation onward.

[0039] FIG. 3 is an explanatory diagram illustrating the record layout of the culture data DB 52. The culture data DB 52 is a database that records the culture data 51. The culture data DB 52 has a passage number field, a medium number field, and a culture result field. The culture result field has a gene expression level field and a proliferation potential field. The gene expression level field has fields corresponding to each gene detected by gene expression analysis, such as a "CNOT10" field and a "SLC39A11" field.

[0040] The number of passages field records the generation of subculture. The medium number field records an identification number assigned to the medium 41 used. Instead of or together with the medium number field, a field may be provided in which the name of the medium 41, such as "medium S" or "medium T," is recorded. The expression level of each gene is recorded in each subfield of each gene expression level field. That is, a horizontal row of the gene expression level field in Figure 3 records a gene profile that associates the genes expressed in cells cultured using the medium recorded in the medium number field with the expression level of each gene.

[0041] For example, when a next-generation sequencer 16 is used for gene expression analysis, the gene expression level can be calculated using formula (1). When formula (1) is used, the unit of gene expression level is RPM (Reads Per Million mapped reads). Gene expression level of gene A = number of reads mapped to gene A / total number of mapped reads (1)

[0042] The proliferation potential field records the proliferation potential of the cells. The proliferation potential field is an example of a field for recording cell characteristic values. The culture data DB 52 has one record for one culture medium of one generation.

[0043] An outline of the method for extracting correlated genes by data analysis of culture data 51 shown in step S504 in Fig. 1 will be described below. In the following explanation, an example will be given in which subculture is performed using J types of culture media, and data evaluating the expression levels of I types of genes in each medium is recorded in culture data DB 52 for K culture generations.

[0044] In the following explanation, the expression level of each gene recorded in the gene expression field of the culture data DB52 is expressed as X ijk X ijk indicates the expression level of the i-th gene in the k-th subculture generation using the j-th medium. The total number of data recorded in the gene expression field is (I x J x K). Furthermore, in the following explanation, the matrix indicating proliferation potential recorded in the proliferation potential field of the culture data DB 52 will be referred to as proliferation potential matrix Q. The proliferation potential matrix Q has J x K elements.

[0045] First, the gene expression level normalized to satisfy all of equations (2) to (4) is calculated.

[0046]

number

[0047] In the following explanation, the normalized gene expression level x ijk A third-order tensor with elements I×J×K is called tensor R. The number of components of tensor R is (I×J×K).

[0048] Next, tensor decomposition is performed on the tensor R as shown in equation (5).

[0049]

number

[0050] Note that the singular value matrix u fi , u gj , u hk are all orthogonal matrices. Tensor decomposition is performed using a known method such as HOSVD (Higher Order Singular Value Decomposition). HOSVD is one method of Tucker decomposition. For tensor decomposition, known methods such as CP decomposition or tensor train decomposition may also be used.

[0051] Next, the singular value matrix u gj The correlation coefficient between the singular value vector corresponding to each row of the matrix Q and the proliferation potential matrix Q is calculated. gj is a singular value matrix corresponding to the type of culture medium 41. For example, Pearson's product-moment correlation coefficient is used as the correlation coefficient. Note that Spearman's rank correlation coefficient, Kendall's rank correlation coefficient, etc. may also be used as the correlation coefficient.

[0052] Then, the singular value vector with the largest absolute value of the calculated correlation coefficient is selected. In the following description, the selected singular value vector is referred to as u Mj Here, the singular value vector u Mj is, for example, the singular value vector u with the largest absolute value of the correlation coefficient. Mj The singular value vectors u with significantly larger absolute values ​​of correlation coefficients than other rowsMj may be selected.

[0053] Then, the singular value matrix u corresponding to the gene type is fi From, u Mj The singular value vector u corresponding to Ni Specifically, first, the elements G(f,M,h) where g = M are extracted from the core tensor G(f,g,h). Among the extracted elements, N corresponding to the largest element G(N,M,h) is selected. The singular value matrix u corresponding to the type of gene is fi From the singular value vector u corresponding to N Ni is extracted.

[0054] where u Ni Based on the null hypothesis that follows a Gaussian distribution, the P value Pi for each gene i is calculated using equation (6). Here, the P value represents the probability that a statistic contrary to the null hypothesis will be observed.

[0055]

number

[0056] Then, Pci, which is the corrected P value corresponding to each gene i, is calculated by multiple comparison correction. Multiple comparison correction is performed, for example, by the Benjamini-Hochberg method. Multiple comparison correction may also be performed by any method such as the HSD (Honestly Significant Difference) test, the Scheffe method, the Dunnett method, or the Games-Howell method.

[0057] Then, genes whose corrected P value Pci is equal to or less than a predetermined threshold are extracted. The extracted genes are the aforementioned correlated genes. Note that correlated genes with a positive correlation coefficient are positively correlated genes that have a positive correlation with proliferation potential, and correlated genes with a negative correlation coefficient are negatively correlated genes that have a negative correlation with proliferation potential.

[0058] Figure 4 is an explanatory diagram illustrating the concentration determination procedure shown in step S507 of Figure 1. A basal medium selected according to the type of cell, the purpose of culture, etc. is used as a base medium to prepare a plurality of concentration-adjusted media 49 by mixing the influence compounds extracted in step S506 of Figure 1. Table 1 shows the concentrations of the influence compounds. The "M" in the concentration indicates the molar concentration.

[0059] [Table 1]

[0060] D1 is a so-called control containing only the basal medium. The concentrations of the influencing compounds in D2 to D5 are all examples. The same type of cells as in step S501 are seeded in each of a plurality of concentration-adjusted media 49 (step S521).

[0061] First, cells seeded in each concentration-adjusted medium 49 are cultured (step S522). Then, the proliferation ability of the cells is measured for each concentration-adjusted medium 49 (step S523). An example of a graph showing the relationship between concentration and proliferation is shown at the bottom of FIG. 4. The proliferation ability is highest when the concentration of the influencing compound is D3. Therefore, the concentration of this influencing compound is determined to be D3.

[0062] Here, the experiment described using Figure 4 may be repeated using a concentration-adjusted medium 49 in which the concentration is finely divided around the concentration determined to have high proliferation potential, and an appropriate concentration of the influential compound may be determined with high precision. Note that if the experiment described using Figure 4 does not show a significant effect compared to the control, the influential compound is not used in the second medium 42.

[0063] The above-described procedure allows the determination of an appropriate concentration for each of multiple influence compounds. Therefore, a medium containing multiple influence compounds at appropriate concentrations can be determined. In the experiment described using FIG. 4, multiple influence compounds may be contained in one medium 41. The appropriate concentration can be determined by taking into account the effects of interactions between multiple influence compounds.

[0064] 5 is an explanatory diagram illustrating the configuration of an information processing system 10. The information processing system 10 includes an information processing device 20 and a server 30 connected via a network. The information processing system 10 may also include a culture device 15, a next-generation sequencer 16, and a cell measuring device 17.

[0065] The information processing device 20 includes a control unit 21, a main memory device 22, an auxiliary memory device 23, a communication unit 24, a reading unit 29, and a bus. The control unit 21 is an arithmetic and control device that executes the program of this embodiment. The control unit 21 uses one or more central processing units (CPUs), graphics processing units (GPUs), multi-core CPUs, or the like. The control unit 21 is connected to each hardware unit that constitutes the information processing device 20 via the bus.

[0066] The main memory device 22 is a storage device such as an SRAM (Static Random Access Memory), a DRAM (Dynamic Random Access Memory), or a flash memory. The main memory device 22 temporarily stores information required during processing performed by the control unit 21 and programs currently being executed by the control unit 21.

[0067] The auxiliary storage device 23 is a storage device such as an SRAM, a flash memory, a hard disk, or a magnetic tape. The auxiliary storage device 23 stores a culture data DB 52, programs to be executed by the control unit 21, and various data required for executing the programs. The communication unit 24 is an interface for communication between the information processing device 20 and a network.

[0068] The information processing device 20 is a general-purpose personal computer, a tablet, a smartphone, or the like. The information processing device 20 may be a computer built into the culture device 15 or the next-generation sequencer 16. The information processing device 20 may be a mainframe computer or a virtual machine running on a mainframe computer. The information processing device 20 may be configured with hardware such as multiple personal computers or mainframe computers that perform distributed processing. The information processing device 20 may be configured with a cloud computing system or a quantum computer.

[0069] The control unit 21 may read the program 97 recorded on the portable recording medium 96 via the reading unit 29 and store it in the auxiliary storage device 23. The control unit 21 may read the program 97 stored in a semiconductor memory 98, such as a flash memory, implemented in the information processing device 20. Furthermore, the control unit 21 may download the program 97 from another server computer (not shown) connected via the communication unit 24 and a network (not shown) and store it in the auxiliary storage device 23.

[0070] The server 30 includes a control unit 31, a main memory device 32, an auxiliary memory device 33, a communication unit 34, and a bus. The control unit 31 uses one or more CPUs, GPUs, multi-core CPUs, etc. The control unit 31 is connected to each hardware unit constituting the server 30 via the bus.

[0071] The main memory device 32 is a memory device such as an SRAM, a DRAM, a flash memory, etc. The main memory device 32 temporarily stores information required during processing performed by the control unit 31 and programs being executed by the control unit 31.

[0072] The auxiliary storage device 33 is a storage device such as an SRAM, flash memory, hard disk, or magnetic tape. The auxiliary storage device 33 stores the gene DB 61, the programs to be executed by the control unit 31, and various data required for executing the programs. The communication unit 34 is an interface for communication between the server 30 and the network. The server 30 is, for example, a server for providing the LINCS program.

[0073] The culture device 15, next-generation sequencer 16, and cell measuring device 17 may be so-called stand-alone devices that are not connected to a network. When stand-alone devices are used, the culture technician performs the subculture work and also measures the gene expression level and proliferation ability using the next-generation sequencer 16 and cell measuring device 17. The culture technician then records the measurement results in the culture data DB 52.

[0074] The control unit 21 of the information processing device 20 may acquire the gene expression level from the next-generation sequencer 16 via the network and automatically record it in the culture data DB 52. The control unit 21 may acquire the proliferation potential from the cell measuring device 17 via the network and automatically record it in the culture data DB 52.

[0075] The culture device 15, next-generation sequencer 16, and cell measuring device 17 may cooperate to perform the subculture, measurement of gene expression levels, and measurement of proliferation ability described with reference to Figure 2, and record the data in the culture data DB 52. A robot (not shown) may operate the culture device 15, next-generation sequencer 16, and cell measuring device 17. The culture device 15, next-generation sequencer 16, and cell measuring device 17 may be configured as an integrated device.

[0076] Fig. 6 is a flowchart illustrating the flow of program processing. The program in Fig. 6 is a program that executes the process of extracting correlated genes described in step S504 of Fig. 1 and the process of extracting influential compounds described in step S506.

[0077] The control unit 21 of the information processing device 20 acquires the culture data 51 from the culture data DB 52 (step S601). The control unit 21 starts a subroutine for extracting correlated genes (step S604). The subroutine for extracting correlated genes is a subroutine for extracting correlated genes based on the culture data DB 52. The processing flow of the subroutine for extracting correlated genes will be described later.

[0078] The control unit 21 transmits the positively correlated genes from the correlated genes extracted in step S604 to the server 30 (step S605). The control unit 31 of the server 30 receives the positively correlated genes (step S701). The control unit 31 extracts influencing compounds that increase the expression levels of the correlated genes received from the gene DB 61 (step S702). The control unit 31 transmits the extracted influencing compounds (step S703).

[0079] The control unit 21 of the information processing device 20 receives the influencing compound (step S607). The influencing compound received in step S607 is a compound that increases the expression level of a gene that has a positive correlation with proliferation potential. It is expected that proliferation potential will be increased by using a medium containing a large amount of this compound.

[0080] The control unit 21 transmits negatively correlated genes from among the correlated genes extracted in step S604 to the server 30 (step S608). The control unit 31 of the server 30 receives the negatively correlated genes (step S704). The control unit 31 extracts influencing compounds that decrease the expression levels of the received correlated genes from the gene DB 61 (step S705). The control unit 31 transmits the extracted influencing compounds (step S706).

[0081] The control unit 21 of the information processing device 20 receives the influencing compound (step S609). The influencing compound received in step S609 is a compound that decreases the expression level of a gene that has a negative correlation with proliferation potential. It is expected that proliferation potential will increase by reducing the content of this compound or by using a medium from which this compound has been removed.

[0082] The control unit 21 displays the influencing compounds received in step S607 and the influencing compounds received in step S609 (step S610), and then ends the process.

[0083] 7 is a flowchart illustrating the process flow of the correlated gene extraction subroutine, which extracts correlated genes based on the culture data DB 52.

[0084] The control unit 21 normalizes the gene expression levels included in the culture data based on equations (2) to (4) (step S631). The control unit 21 constructs the normalized gene expression data into tensor R, which is a third-order tensor having three modes: type of medium, gene expression level, and passage number (step S632). The control unit 21 performs tensor decomposition shown in equation (5) and decomposes tensor R constructed in step S632 into a core tensor G and three singular value matrices (step S633).

[0085] The control unit 21 constructs a proliferation potential matrix Q corresponding to the proliferation potential field of the culture data DB 52 (step S634). gi The control unit 21 calculates the correlation coefficient between each singular value vector constituting the matrix Q and the proliferation potential matrix Q (step S635). Mj is extracted (step S636).

[0086] The control unit 21 extracts the singular value vector u extracted in step S636 from the singular value vector G(f, g, h). Mj The control unit 21 selects the largest element G(N, M, h) from among the elements of the extracted core tensor G (step S637). The control unit 21 extracts the singular value matrix u corresponding to the type of gene. fi From the singular value vector u corresponding to N Ni is extracted (step S638).

[0087] The control unit 21 calculates a P value for each gene based on equation (6) (step S639). The control unit 21 calculates a corrected P value for each gene (step S640). The control unit 21 extracts correlated genes whose corrected P values ​​are equal to or less than a predetermined threshold (step S641). Thereafter, the control unit 21 ends the process. [Experimental Example] This example describes the development of a second culture medium 42 that improves the proliferation potential of mesenchymal stem cells collected from human adipose tissue. Eight types of culture medium 41 commercially available for the proliferation and culture of mesenchymal stem cells were used. Mesenchymal stem cells were seeded into each of the eight types of culture medium and subcultured for nine passages. The proliferation potential of the cultured cells at the third and ninth passages was measured using an automated cell counter (FACSCOPE B, manufactured by Curiosis) and the gene expression level was measured using a next-generation sequencer (Ion Proton® Semiconductor Sequencer, manufactured by ThermoFisher Scientific®), creating culture data 51. Proliferation potential was evaluated using PDL.

[0088] Based on the culture data 51, eight elements for the mode of medium type, 20,812 elements for the mode of gene expression level, and two elements for the number of subculture generations were recorded in the culture data DB 52. Table 2 shows the correlation coefficients calculated by the control unit 21 in step S635 of the program for calculating correlated genes, which was described using Figure 7.

[0089] [Table 2]

[0090] Based on the correlation coefficients shown in Table 2, the control unit 21 calculates the singular value vector u corresponding to the culture medium number 1 in step S636. 1j and the singular value vector u corresponding to medium number 3 3j Let us denote the singular value vector u gj Extracted from.

[0091] Furthermore, the control unit 21 extracted elements corresponding to g=1 and g=3 from the core tensor G(f, g, h). The control unit 21 selected the element with the largest absolute value from the extracted elements. The control unit 21 then calculated the singular value matrix u corresponding to the gene type. fi From the singular value vector u corresponding to the selected element Ni was extracted.

[0092] The control unit 21 calculates the singular value vector u Ni Based on equations (6) and (7), the P value and corrected P value corresponding to each gene i were calculated. The control unit 21 extracted 141 genes whose corrected P values ​​were equal to or less than the threshold value of 0.01. The genes extracted by the control unit 21 consisted of 109 positively correlated genes having a positive correlation with proliferation potential and 32 negatively correlated genes having a negative correlation with proliferation potential.

[0093] 109 positively correlated genes were submitted to the L1000 database service site, and approximately 300 influential compounds that increase the expression levels of the genes were received. 32 negatively correlated genes were submitted to the L1000 database service site, and approximately 300 influential compounds that decrease the expression levels of the genes were received. The L1000 database is an example of the database provided by the aforementioned LINCS program.

[0094] The compounds that increased the expression levels of the 109 positively correlated genes included compounds that normally suppress cell proliferation, such as anticancer drugs and antibacterial agents. A person skilled in the art would not consider using such compounds in the second culture medium 42, which is intended to obtain a large number of cells. A literature search for these compounds revealed no reports of their effect on promoting cell proliferation.

[0095] For these compounds, which normally inhibit cell proliferation, we investigated the relationship between the concentration of each compound and the proliferation rate, as explained using Figure 4, and confirmed that all of them had the effect of promoting cell proliferation. In particular, for certain anticancer drugs, the greatest proliferation-promoting effect was obtained at a concentration of 100 nM. To confirm this, we cultured cells for 20 days using medium containing the compound at a concentration of 100 nM, and found that the proliferation ability increased by approximately 20% compared to the control basal medium.

[0096] A medium can be manufactured by adding to the medium an influence compound that increases the expression level of each positively correlated gene or an influence compound that decreases the expression level of each negatively correlated gene. Furthermore, instead of adding a single influence compound, a combination of influence compounds may be selected and added to produce the most effective combination. In this case, the type and concentration of the influence compound to be added are appropriately selected. Furthermore, these combinations may be selected using AI (artificial intelligence) from a database of correlations between compounds or literature information. Here, the second medium 42 was developed by selecting multiple influence compounds and determining the appropriate concentration for each. This concludes the description of this example.

[0097] According to this embodiment, in addition to the mesenchymal stem cells described in the examples, a second culture medium 42 can be provided that increases the proliferation ability of any cells, such as pluripotent stem cells such as embryonic stem cells and iPS cells (induced pluripotent stem cells), multipotent stem cells such as tissue stem cells and tissue stem cells, and somatic cells such as epidermal cells of the skin.

[0098] The medium 41 is not limited to those commercially available for human cells. For example, media for microbial culture, such as bacterial culture, fungal culture, and algal culture, can also be used as long as the target cells are not killed.

[0099] The target cells are not limited to human cells, and a culture medium for cells of any organism for which a gene DB61 similar to the LINCS dataset is available can be developed based on the method of this embodiment.

[0100] According to this embodiment, as illustrated in the experimental examples, a new medium can be developed by adding compounds to the composition that are not thought of by those skilled in the art, but which actually have an effect. By developing such a new medium and using it for culture, the target cells can be cultured efficiently.

[0101] [Variation 1-1] In this modified example, subculture is not performed, and second culture medium 42 is prepared based on one generation's worth of culture data 51. That is, the number of passages field is not necessary in culture data DB 52 described with reference to FIG.

[0102] In step S632 of the flowchart described with reference to Figure 7, control unit 21 organizes the normalized gene expression data into a second-order tensor, i.e., a matrix, having two modes: medium type and gene expression. In the following step S633, control unit 21 decomposes the second-order tensor organized in step S632 using a known method such as nonnegative matrix factorization or singular value decomposition. The subsequent processing is the same as in embodiment 1.

[0103] [Variation 1-2] This section describes a modified example for developing a medium with high cell viability. Instead of proliferation potential, cell viability at the end of culture is used as a cell characteristic value. The viability can be calculated as the ratio of the number of surviving cells at the end of culture to the total number of cells.

[0104] [Variation 1-3] This section describes a modified example of a culture medium for increasing cell secretions. Secretions include, for example, exosomes, which are extracellular vesicles, as well as functional proteins such as hormones and RNA contained within the exosomes. These secretions play an important role in intercellular signaling and are expected to be used, for example, as pharmaceuticals. In this modified example, the amount of secretions contained in the culture medium is used as a cell characteristic value.

[0105] The total amount of secreted substances or the amount of a specific secreted substance may be used as the cell characteristic value. The cell characteristic value is preferably determined appropriately depending on the use of the medium.

[0106] [Variation 1-4] This modification describes the development of a culture medium that suppresses the generation of morphologically abnormal cells. In this modification, the normal cell rate is used as the cell characteristic value. The normal cell rate is the ratio of morphologically normal cells to the total number of cells. The normal cell rate can be measured, for example, by counting the number of morphologically normal cells and the number of morphologically abnormal cells using an imaging cytometer.

[0107] Imaging cytometers can quantify visual characteristics of cells, such as average cell size or cell size variability, and these visual values ​​may be used as cell property values.

[0108] Multiple cell characteristic values ​​may be evaluated in a single experiment. By using an influencing compound that increases the expression level of a correlated gene common to multiple cell characteristic values, a second medium 42 that simultaneously exerts multiple effects can be developed. By using an influencing compound common to multiple cell characteristic values, a second medium 42 that simultaneously exerts multiple effects can also be developed. Here, the influencing compounds can be combined by weighting them according to the symptoms of, for example, concomitant diseases.

[0109] [Embodiment 2] This embodiment relates to the development of a medium for culturing mesenchymal stem cells that is highly effective in treating diabetic nephropathy. Explanation of parts common to the first embodiment will be omitted.

[0110] Fig. 8 is an explanatory diagram illustrating a specific example of the procedure for creating culture data 51 in embodiment 2. Fig. 8 shows a procedure that is performed instead of the procedure using Fig. 2. Fig. 8 shows "medium S," but similar processing is also performed for "medium T" and "medium U," etc.

[0111] Mesenchymal stem cells are seeded in "medium S." Subculture is performed using "medium S." For example, at the end of the second and nth culture generations, cell characteristic values ​​and gene expression levels are measured. In this embodiment, the cell characteristic value is a value indicating the therapeutic effect when the cultured mesenchymal stem cells are administered to a patient with diabetic nephropathy, specifically, the patient's fasting blood glucose level.

[0112] 9 is an explanatory diagram illustrating the record layout of the culture data DB 52 of embodiment 2. The culture data DB 52 is a database that records the culture data 51. The culture data DB 52 has a passage number field, a medium number field, and a culture result field. The culture result field has a gene expression level field and a fasting blood glucose level field. The gene expression level field has fields corresponding to each gene detected by gene expression analysis, such as an "HGF" field and an "AGER" field.

[0113] The number of passages field records the generation of subculture. The medium number field records an identification number assigned to the medium 41 used. Instead of or together with the medium number field, a field may be provided in which the name of the medium 41, such as "medium S" or "medium T," is recorded. Each subfield of the gene expression level field records the expression level of each gene. That is, a horizontal row of the gene expression level field in Figure 9 records a gene profile that associates the genes expressed in cells cultured using the medium recorded in the medium number field with the expression level of each gene.

[0114] The fasting blood glucose level field records the fasting blood glucose level, which is a value indicating the therapeutic effect of a patient administered with cultured cells. The fasting blood glucose level field is an example of a field for recording cell characteristic values. The culture data DB 52 has one record for one culture medium of one generation.

[0115] Returning to Figure 8, the explanation continues. Gene expression levels and fasting blood glucose levels are recorded in the culture data DB 52 and analyzed using the same procedure as in the first embodiment. Specifically, first, correlated genes that are correlated with fasting blood glucose levels are extracted. Then, influencing compounds that affect the expression of the extracted correlated genes are extracted from the L1000 database. The concentration of the influencing compounds is determined by an experiment similar to the procedure described using Figure 4. The second culture medium 42 is completed by compounding the influencing compounds to contain the determined concentration.

[0116] The disease of the patient to whom the cultured cells are administered is not limited to diabetic nephropathy. The cell characteristic value is not limited to the patient's fasting blood glucose level. Any combination of a disease and a value indicating the therapeutic effect for that disease can be used as appropriate. Specific examples will be described later in the modified examples.

[0117] The cells used are not limited to mesenchymal stem cells. For example, stem cells derived from umbilical cord or bone marrow, IPS (Induced Pluripotent Stem) cells, fibroblasts, or nerve cells may also be used. Cell characteristic values ​​for each of multiple types of cells may be measured to select a combination of cells and culture media with a high therapeutic effect.

[0118] [Variation 2-1] This modification relates to the development of a medium for culturing mesenchymal stem cells that is highly effective in improving a patient's blood insulin level. The patient's blood insulin level is one of the values ​​related to the therapeutic effect of diabetes. Explanation of parts common to the second embodiment will be omitted.

[0119] In this modified example, the cell characteristic value is the blood insulin level when cultured mesenchymal stem cells are administered to a patient. The culture data DB 52 used in this modified example has a blood insulin level field instead of a fasting blood glucose level field. This modified example will be explained using Figure 8. As in the second embodiment, mesenchymal stem cells are seeded in a culture medium 41 such as "culture medium S." Subculture is performed using "culture medium S." For example, at the end of the second and nth culture generations, blood insulin levels and gene expression levels are measured.

[0120] The blood insulin level and gene expression level are recorded in the culture data DB 52. Thereafter, as in the second embodiment, the correlated genes are extracted, the influencing compounds are extracted, and the concentrations of the influencing compounds are determined in sequence. By compounding the influencing compounds at the determined concentrations, the second culture medium 42 suitable for culturing cells that have the effect of improving blood insulin levels is completed.

[0121] [Variation 2-2] This modification relates to the development of a medium for culturing mesenchymal stem cells that is highly effective in improving a patient's urinary albumin level. The patient's urinary albumin level is one of the values ​​related to the therapeutic effect of diseases such as diabetic nephropathy. Explanation of parts common to the second embodiment will be omitted.

[0122] This modified example will be described using Figure 8. As in the second embodiment, mesenchymal stem cells are seeded in a culture medium 41 such as "culture medium S." Subculture is performed using "culture medium S." For example, at the end of the second and nth culture generations, cell characteristic values ​​and gene expression levels are measured. In this modified example, the cell characteristic value is the urinary albumin level when the cultured mesenchymal stem cells are administered to a patient.

[0123] In this modified example, the cell characteristic value is the urinary albumin level when cultured mesenchymal stem cells are administered to a patient. The culture data DB 52 used in this modified example has a urinary albumin level field instead of a fasting blood glucose level field. This modified example will be explained using Figure 8. As in the second embodiment, mesenchymal stem cells are seeded in a culture medium 41 such as "culture medium S." Subculture is performed using "culture medium S." For example, at the end of the second and nth culture generations, the urinary albumin level and gene expression level are measured.

[0124] The urinary albumin level and gene expression level are recorded in the culture data DB 52. Thereafter, as in the second embodiment, the correlated genes are extracted, the influential compounds are extracted, and the concentrations of the influential compounds are determined in sequence. By compounding the influential compounds at the determined concentrations, the second culture medium 42 suitable for culturing cells that has the effect of improving the urinary albumin level is completed.

[0125] [Variation 2-3] The supernatant may be administered to a patient instead of or together with the cultured cells. The supernatant is produced by removing the cells from the medium and then removing impurities by a process such as centrifugation. The supernatant contains secretions from the cells. A value indicating the therapeutic effect on a patient to whom the supernatant is administered can be used as the cell characteristic value.

[0126] The secreted product, such as a specific protein or RNA purified from the culture medium, may be administered to a patient, and the cell characteristic value may be used to indicate the therapeutic effect of the patient to whom the secreted product is administered.

[0127] The cells cultured using the second culture medium 42, the supernatant, and the secretions may be provided to medical institutions as medicines.

[0128] The cells seeded in the culture medium 41 may be cells collected from a specific patient. A customized second culture medium 42 can be developed that is suitable for culturing cells from the specific patient for customized treatments such as regenerative medicine.

[0129] [Embodiment 3] This embodiment relates to the development of a medium for culturing mesenchymal stem cells that are highly effective in improving symptoms of various diseases in pharmacological tests using disease model animals, such as drug-induced or genetically modified mice. Mesenchymal stem cells that are highly effective in disease model animals are highly likely to be highly effective in actual patients. Examples of cell characteristic values ​​include (1) pathological markers in the blood of the model animals, (2) tissue sections prepared and scored for the severity of disease from a pathological perspective, and (3) quantitative values ​​of disease markers obtained by extracting protein and / or RNA from tissue and performing Western blotting and / or qPCR (quantitative polymerase chain reaction). Explanations of parts common to the second embodiment will be omitted.

[0130] The technical features (constituent elements) described in each embodiment can be combined with each other, and by combining them, new technical features can be formed. The embodiments disclosed herein are illustrative in all respects and should not be considered as limiting. The scope of the present invention is defined by the claims, not by the above meaning, and is intended to include all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0131] 10 Information Processing Systems 15 Culture device 16 Next-generation sequencer 17 Cell measurement device 20 Information processing equipment 21 Control section 22 Main storage 23 Auxiliary storage device 24 Communications Department 29 Reading unit 30 servers 31 Control Unit 32 Main storage 33 Auxiliary storage device 34 Communications Department 41 Culture medium 42 Second medium 49 Concentration-adjusted medium 51 Culture Data 52 Culture Data DB 61 Gene DB 96 Portable recording media 97 Programs 98 Semiconductor Memory

Claims

1. Cells with uniform characteristics and genetic backgrounds are cultured in multiple media containing different types or concentrations of compounds, acquiring culture data that associates the type of medium, gene expression analysis results obtained by measuring genes expressed in the cultured cells and the expression levels of each of the genes, with cell characteristic values ​​that indicate cell characteristics; extracting correlated genes whose expression levels correlate with the cell characteristic values ​​based on the culture data; Processing method.

2. The culture is carried out by subculture, The culture data is obtained at multiple generations of the subculture. The processing method according to claim 1 .

3. The gene expression analysis result is a gene profile that associates a plurality of genes with the expression levels of each of the genes. The processing method according to claim 1 or 2.

4. The correlated gene is a positively correlated gene whose expression level is positively correlated with the cell characteristic value, or a negatively correlated gene whose expression level is negatively correlated with the cell characteristic value. The processing method according to any one of claims 1 to 3.

5. Compounds that affect the expression levels of the extracted correlated genes are extracted based on a database that records the relationships between compounds and gene expression levels. The processing method according to any one of claims 1 to 4.

6. If the correlated gene has a positive correlation with the cell characteristic value, extracting a compound that increases the expression level of the correlated gene; If the correlated gene has a negative correlation with the cell characteristic value, a compound that decreases the expression level of the correlated gene is extracted. The processing method according to claim 5.

7. The cell characteristic value indicates the proliferation ability or therapeutic effect of the cell. The processing method according to any one of claims 1 to 6.

8. Cells with uniform characteristics and genetic backgrounds are cultured in multiple media containing different types or concentrations of compounds, recording culture data that associates the type of medium, gene expression analysis results obtained by measuring genes expressed in the cultured cells and the expression levels of each of the genes, with cell characteristic values ​​that indicate cell characteristics; extracting correlated genes whose expression levels correlate with the cell characteristic values ​​based on the culture data; extracting compounds that affect the expression levels of the extracted correlated genes based on a database that records the relationships between compounds and gene expression levels; preparing a second medium containing the extracted compound; Cultivating cells using the second medium Cell culture method.

9. Cells with uniform characteristics and genetic backgrounds are cultured in multiple media containing different types or concentrations of compounds, recording culture data that associates the type of medium, gene expression analysis results obtained by measuring genes expressed in the cultured cells and the expression levels of each of the genes, with cell characteristic values ​​that indicate cell characteristics; extracting correlated genes whose expression levels correlate with cell characteristic values ​​based on the culture data; extracting compounds that affect the expression levels of the extracted correlated genes based on a database that records the relationships between compounds and gene expression levels; A compound selected from the extracted compounds is mixed into the medium base. Method for producing culture medium.

10. Cells with uniform characteristics and genetic backgrounds are cultured in multiple media containing different types or concentrations of compounds, recording culture data that associates the type of medium, gene expression analysis results obtained by measuring genes expressed in the cultured cells and the expression levels of each of the genes, with cell characteristic values ​​that indicate cell characteristics; extracting correlated genes whose expression levels correlate with cell characteristic values ​​based on the culture data; extracting compounds that affect the expression levels of the extracted correlated genes based on a database that records the relationships between compounds and gene expression levels; preparing a second medium containing the extracted compound; Cultivating cells using the second medium; Extracting secreted substances from cultured cells Methods for producing substances.

11. Cells with uniform characteristics and genetic backgrounds are cultured in multiple media containing different types or concentrations of compounds, recording culture data that associates the type of medium, gene expression analysis results obtained by measuring genes expressed in the cultured cells and the expression levels of each of the genes, with cell characteristic values ​​that indicate cell characteristics; extracting correlated genes whose expression levels correlate with the cell characteristic values ​​based on the culture data; extracting compounds that affect the expression levels of the extracted correlated genes based on a database that records the relationships between compounds and gene expression levels; A compound selected from the extracted compounds was mixed into the medium base. Culture medium.

12. Cells with uniform characteristics and genetic background are subcultured using a plurality of types of media each containing a compound of a different type or concentration, and the types of media used for multiple generations of the cells are obtained, and gene expression analysis results are obtained by measuring genes expressed in the cultured cells and the expression levels of each gene, and culture data is obtained that correlates cell characteristic values ​​that indicate the characteristics of the cells; extracting correlated genes whose expression levels correlate with the cell characteristic values ​​based on the culture data; Have the computer perform the process program.

13. The medium is an AOF medium. The program according to claim 12.

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