Culture management methods, culture management systems, and programs

Abstract

To provide a technique for supporting cell culture.SOLUTION: A management device in a culture management system uses a first cell count, which is the number of cells in a pre-cultured sample, and a second cell count, which is the number of cells in a post-cultured sample, to determine whether the proliferative potential of the cells included in the sample is below a given reference (S126). When the proliferative potential of the cells is determined below the given reference, the management device calculates the amount of change for each of one or more candidate substances during the culture process (S118). The management device identifies a responsible substance from among the one or more candidate substances on the basis of the amount of change for each of the one or more candidate substances (S128), and then reports the responsible substance (S130).SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] The present invention relates to the management of cell culture. [Background Art]

[0002] Conventionally, various techniques have been proposed for cell culture. For example, as a technique for stable proliferation and growth of human-derived cells (hereinafter referred to as human somatic cells), Patent Document 1 (Japanese Patent Laid-Open No. 7-274951) discloses a technique of adding vitamins to a culture medium, and Patent Document 2 (Japanese Patent Laid-Open No. 2006-254753) discloses a technique of adding growth factors such as FGF (Fibroblast Growth Factor) and EGF (Epidermal Growth Factor) to a culture medium. [Prior Art Documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Laid-Open No. 7-274951 [Patent Document 2] Japanese Patent Laid-Open No. 2006-254753 [Summary of the Invention] [Problem to be Solved by the Invention]

[0004] The conventional techniques as described above are techniques for improving the culture environment of cells in which poor culture (poor proliferation) due to cell death has been observed. There still remains a need for techniques for supporting cell culture, including such techniques.

[0005] The present invention has been made in view of such circumstances, and an object thereof is to provide a technique for supporting cell culture. [Means for Solving the Problem]

[0006] A culture management method according to a certain aspect of the present disclosure is a method for managing the culture of a group of cells contained in a sample, comprising the steps of: determining whether the proliferative capacity of the group of cells contained in the sample after culture is lower than a given standard, using a first cell number, which is the number of cells in the sample before culture, and a second cell number, which is the number of cells after the sample has been cultured; and calculating the amount of change in each of one or more candidate substances in the supernatant of the sample during culture, wherein the one or more candidate substances consist of a group comprising at least one of one or more metabolites and one or more enzymes, and further comprising the step of selecting some of the candidate substances from the one or more candidate substances based on the amount of change of each of the one or more candidate substances in the sample before and after culture, and notifying the selected candidate substances, if it is determined that the proliferative capacity of the group of cells contained in the sample is lower than the given standard.

[0007] A program that conforms to certain aspects of this disclosure is executed by a computer, thereby causing the computer to perform the culture management method described above.

[0008] A culture management system according to a certain aspect of this disclosure comprises a cell culture device for culturing cells contained in a sample, an analysis device for counting the number of cells in the sample, an analysis device for outputting the analysis results of the supernatant of the sample, and a control device, wherein the control device is configured to determine whether the proliferative capacity of the cell population contained in the sample after culturing is lower than a given standard, using a first cell count, which is the number of cells in the sample before culturing, and a second cell count, which is the number of cells after the sample has been cultured, and to calculate the amount of change in each of one or more candidate substances in the supernatant of the sample during culture, wherein the one or more candidate substances consist of a group consisting of at least one of one or more metabolites and one or more enzymes, and if the control device determines that the proliferative capacity of the cell population contained in the sample is lower than the given standard, it is configured to select some of the candidate substances from the one or more candidate substances based on the amount of change in each of the one or more candidate substances in the sample before and after culture, and to notify the user of the selected candidate substances. [Effects of the Invention]

[0009] In accordance with a certain aspect of this disclosure, if the proliferative capacity of a cell population contained in a sample is determined to be lower than a given standard, the causative substance that caused the lower-than-usual proliferative capacity is identified from among the one or more candidate substances based on the change in each of the one or more candidate substances during culture, and the causative substance is notified. This provides a technology to support cell culture. [Brief explanation of the drawing]

[0010] [Figure 1] This is a schematic diagram of the culture management system 1000 according to Embodiment 1 of this disclosure. [Figure 2] This figure shows an example of the change in cell number before and after culturing three different samples. [Figure 3] Figure 2 shows an example of the change in the amount of candidate substance after 6 passages of culture for sample B, relative to the amount before culture. [Figure 4] Figure 2 shows an example of the change in the amount of candidate substance after 8 passages of culture for sample C, relative to the amount before culture. [Figure 5] This figure shows an example of the data structure of a measurement results database. [Figure 6] This diagram illustrates "M0", "M1", "M2", and "ME" in equation (1). [Figure 7] This figure shows an example of the data structure of related tables. [Figure 8] This is a flowchart of the processes performed in the culture management system 1000. [Figure 9] This is a flowchart of the processes performed by the control device 100. [Modes for carrying out the invention]

[0011] The embodiments of this disclosure will be described in detail below with reference to the drawings. In the drawings, the same or corresponding parts are denoted by the same reference numerals, and their descriptions will not be repeated.

[0012] [Embodiment 1] (1) Culture management system FIG. 1 is a schematic configuration diagram of a culture management system 1000 according to Embodiment 1 of the present disclosure. The culture management system 1000 cultures cells contained in a sample and manages information related to said culture. The culture management system 1000 includes a management device 100, a mass spectrometer 200, a subculture device 300, an analysis device 400, and a transfer device 500.

[0013] (1-1) Management device 100 The management device 100 is configured by, for example, a general-purpose computer. In the example of FIG. 1, the management device 100 includes a processor 101, a memory 102, an input / output interface (I / F) 103, and a communication I / F 104.

[0014] The processor 101 executes various programs for the management device 100 to perform various processes. The memory 102 stores programs executed by the processor 101 and various data necessary for execution of the programs. In the culture management system 1000, the input / output I / F 103 causes the processor 101 to communicate with the mass spectrometer 200, the subculture device 300, the analysis device 400, and the transfer device 500. The communication I / F 104 causes the processor 101 to communicate with devices outside the culture management system 1000 via a network.

[0015] The management device 100 further includes a display 110 and an input device 120. The display 110 displays results of arithmetic processing by the processor 101, and the input device 120 (such as a mouse, a keyboard, a touch sensor, etc.) accepts data input operations to the processor 101.

[0016] The management device 100 controls the operation timing of the mass spectrometer 200, the subculture device 300, the analysis device 400, and the transfer device 500. Accordingly, in the culture management system 1000, each device operates in cooperation with other devices.

[0017] (1-2) Mass spectrometer 200 The mass spectrometer 200 is constituted by a mass spectrometer (for example, "Liquid Chromatograph Mass Spectrometer LCMS-8060NX" manufactured by Shimadzu Corporation). In one implementation, the mass spectrometer 200 performs mass spectrometry on a sample and identifies the types and amounts of substances contained in the culture solution or culture supernatant.

[0018] (1-3) Subculture apparatus 300 The subculture apparatus 300 is constituted by a subculture apparatus (for example, "Automatic Subculture System KB-4000" manufactured by Jtech Corporation). In one implementation, the subculture apparatus 300 performs subculturing and cultivation of cells contained in a sample. The subculture apparatus 300 holds one or more types of reagents, and is configured to add (pipette) a specified reagent selected from among the one or more types of reagents to a sample, and configured to collect (sample) culture supernatant from the sample during or after cultivation.

[0019] (1-4) Analysis apparatus 400 The analysis apparatus 400 is constituted by an apparatus that analyzes sample information using techniques such as image processing (for example, "Cell Culture Analysis Apparatus CS-1" manufactured by Shimadzu Corporation). The analysis apparatus 400 is configured to analyze an image of the sample and identify the number of cells contained in the sample.

[0020] (1-5) Conveyance apparatus 500 The conveyance apparatus 500 is configured to move containers among the mass spectrometer 200, the subculture apparatus 300, and the analysis apparatus 400. In one implementation, the conveyance apparatus 500 includes an arm that moves the container, and a motor that moves the arm.

[0021] (2) Culture management (2-1) Candidate substances and causative substances The culture management system 1000 may be configured to identify the substance (causative substance) that caused the cell proliferation ability to fall below a given standard during subculturing, and to manage the culture by adding the identified causative substance to the sample.

[0022] For the purpose of managing the culture, one or more candidate substances are defined as potential causative substances. If the cell proliferation ability falls below a given standard, the culture management system 1000 identifies one or more causative substances from among the one or more candidate substances based on the changes in the amount of each of the one or more candidate substances in the sample before and after culturing. The culture management system 1000 then adds at least one of the identified causative substances to the sample.

[0023] (2-2) Specific examples of changes in cell number and candidate substance amount during culture (2-2-1) Changes in cell number Figure 2 shows an example of the change in cell number before and after culture for three different samples (A, B, and C). Different types of hatching are used for each sample in Figure 2.

[0024] The vertical axis of the graph in Figure 2 represents the ratio of the number of cells after culture to the number of cells seeded in the container. A ratio of "1" means that the number of cells after culture has not changed from the number of seeds. A ratio less than "1" means that the number of cells has decreased, and a ratio greater than "1" means that the number of cells has increased. The horizontal axis represents the number of passages. For example, "4th passage" means that the number of passages performed is 4. The value for "4th passage" represents the number of cells after seeding in the 4th passage.

[0025] The graphs in Figure 2 include the results after 4 to 8 passages for each of Sample A and Sample B.

[0026] In the example in Figure 2, the ratio for sample B after 6 passages of culture is approximately 0.5. That is, in sample B, the number of cells after 6 passages of culture is lower than the number of seeded cells. Also in the example in Figure 2, the ratio for sample C after 8 passages of culture is approximately 0.5. That is, in sample C, the number of cells after 8 passages of culture is lower than the number of seeded cells.

[0027] The culture management system 1000 identifies the causative substance for a sample if the number of cells after culturing falls below the number of cells that were seeded. The identification of the causative substance will be explained in detail below with reference to Figures 3 and 4.

[0028] (2-2-2) Changes in the amount of candidate substances (2-2-2-1) First specific example Figure 3 shows an example of the change in the amount of candidate substance after 6 passages of culture for sample B in Figure 2, relative to the amount before culture. The example in Figure 3 shows indicators representing the amount for each of the four candidate substances (glycine, cysteine, riboflavin, and 4-hydroxyproline).

[0029] The index shown in Figure 3 is calculated using the ratio (Po / Ps) of the peak area (Po) of each substance to the peak area (Ps) of the internal standard substance (1 mM 2-isopropylmalic acid) in the mass spectrometry results of a fixed amount of supernatant. This ratio changes according to the concentration of each substance (amount per unit volume of supernatant). Therefore, this ratio corresponds to the amount of each substance.

[0030] The above index is calculated by subtracting the ratio calculated before culturing from the ratio calculated after culturing for a given number of passages. In other words, the index value is an example of the amount of change during culturing. If the culture medium is changed during culturing, the difference between the amount of candidate substance in the culture medium before culturing and the amount of candidate substance in the supernatant at the time of medium change can be calculated for each medium change, and this difference can be added to the difference between the amount of candidate substance in the culture medium before culturing and the amount of candidate substance in the supernatant after culturing to obtain the index value.

[0031] A value of 0 for the index means that the amount of candidate substance after culturing at a given passage has not changed from the amount at the start of culturing. A positive value for the index means that the amount after culturing at a given passage has increased compared to the amount at the start of culturing. A negative value for the index means that the amount after culturing at a given passage has decreased compared to the amount at the start of culturing.

[0032] The index shown in Figure 3 is calculated by subtracting the ratio calculated above for the culture medium used for six passages from the ratio calculated above for the supernatant after six passages of culture.

[0033] In the example in Figure 3, the index is positive for 4-hydroxyproline, but negative for glycine, cysteine, and riboflavin, respectively.

[0034] In one implementation example, the culture management system 1000 identifies candidate substances whose index values ​​(differences) shown in Figure 3 are negative as causative substances. That is, in the example in Figure 3, the culture management system 1000 identifies glycine, cysteine, and riboflavin as causative substances.

[0035] The culture management system 1000 may add at least one of these three causative substances to the sample. This is expected to increase the cell proliferation capacity in sample B during subsequent cultures.

[0036] (2-2-2-2) Second specific example Figure 4 shows an example of the change in the amount of candidate substance after 8 passages of culture relative to the amount before culture for sample C in Figure 2. As with Figure 3, Figure 4 shows the values ​​of the indicators for each of the four candidate substances.

[0037] In the example in Figure 4, similar to the example in Figure 3, the index is positive for 4-hydroxyproline, but negative for glycine, cysteine, and riboflavin, respectively.

[0038] In one implementation example, the culture management system 1000 identifies glycine, cysteine, and riboflavin as the causative substances, as shown in the example in Figure 4.

[0039] The culture management system 1000 adds at least one of these three causative substances to the sample. This is expected to increase the cell proliferation capacity in sample C during subsequent cultures.

[0040] (2-3) Substances to be added The candidate substance (causative substance) may be a metabolite or an enzyme. If the substance identified as the causative substance is a metabolite, the substance added may be the metabolite itself, or a substance located upstream of the metabolite in the metabolic pathway (refer to Figure 7 for explanation as an "upstream metabolite").

[0041] (3) Database (3-1) Measurement results Figure 5 shows an example of the data structure of the measurement results database. The measurement results database contains measurement results for a sample and is stored, for example, in memory 102. The measurement results database may also be stored in a storage device outside the management device 100, as long as it is accessible to the processor 101.

[0042] As shown in Figure 5, the measurement results database associates the identification number (measurement ID) corresponding to the measurement performed by the mass spectrometer 200 with the identification number (container ID) of the cell culture vessel, passage number, cell number, seeded cell number, and the respective changes in multiple candidate substances. In Figure 5, the measurement ID is represented as M1, M2, ..., etc., the container ID is represented as V1, V2, ..., etc., the cell number is represented as N1, N2, ..., etc., the seeded cell number is represented as P0, P1, ..., etc., and the changes in candidate substances are represented as QA1, QA2, ... QB1, QB2, ..., etc.

[0043] The passage number indicates the number of times a cell has been passed through. A passage number of 0 means that the first culture has been performed. For example, in the example in Figure 5, the sample with measurement ID "M1" shows a change in cell number from P0 to N1 during the first culture. In this sample's culture, the change in glycine, one example of a candidate substance, is QA1.

[0044] A passage number of 1 means that the culture is performed after one passage. For example, in the example in Figure 5, the sample with measurement ID "M4" shows a change in cell number from P1 to N4 after one passage. In the culture of this sample, the change in glycine, one example of a candidate substance, is QA4.

[0045] The change in candidate substances represents the change in the amount of candidate substances during culture. If no culture medium is changed during culture, the change in candidate substances is derived by subtracting the amount of candidate substances in the supernatant after culture from the amount of candidate substances in the culture medium (supernatant) before culture. If a culture medium is changed during culture, the change in candidate substances is derived using the amounts of candidate substances before and after culture, as well as the amounts of candidate substances before and after the culture medium change. For example, if the same culture medium used at the start of culture is used for the culture medium change, and the amount of candidate substances in that culture medium is M0, the change in candidate substances is derived according to the following equation (1).

[0046] [Change in candidate substance] = (M1-M0) + (M2-M0) + ... + (ME-M0) (1) Figure 6 is a diagram illustrating "M0", "M1", "M2", and "ME" in equation (1). In Figure 6, "M0" represents the amount of candidate substance in the culture medium used to start the culture and in the culture medium used for the medium change. "M1" represents the amount of candidate substance in the supernatant at the time of the first medium change (immediately before the medium change). "M2" represents the amount of candidate substance in the supernatant at the time of the second medium change (immediately before the medium change). "ME" represents the amount of candidate substance in the supernatant at the end of the culture.

[0047] The "M1-M0" terms represent the change in candidate substances before and after the first culture medium change. The "M2-M0" terms represent the change in candidate substances before and after the second culture medium change. In other words, the change in candidate substances shown in equation (1) is derived as the sum of the changes in candidate substances before and after each culture medium change.

[0048] In the example in Figure 5, "hexokinase" is shown as an example of an enzyme in the candidate substance.

[0049] (3-2) Related Tables (Upstream Metabolites) Figure 7 shows an example of the data structure of the association table. The association table associates a metabolite, which is an example of a candidate substance, with other metabolites located upstream of it in the metabolic pathway (upstream metabolites). The association table is prepared in advance by the administrator of the culture management system 1000, for example. The association table is stored in memory 102, for example, but may also be stored in a storage device outside the management device 100, as long as it is a storage device accessible by the processor 101.

[0050] The upstream metabolites for each candidate substance registered in the related table are described below. Note that in the culture management system 1000, the upstream metabolites for each candidate substance in Figure 7 are not limited to those shown in Figure 7. Substances other than those shown in Figure 7 may be selected as upstream metabolites, taking into account various requirements such as ease of availability and ease of direct addition to the sample.

[0051] (3-2-1) Glycine The example in Figure 7 includes four substances (threonine, L-alothreonine, sarcosine, and 2-amino-3-oxobutyric acid) as upstream metabolites registered for the candidate substance "glycine." These four substances are located upstream of glycine in the "glycine, serine, and seleonine metabolic pathway," assuming that metabolism proceeds in the direction of synthesizing pyruvate, which is used in the citric acid cycle. The "glycine, serine, and seleonine metabolic pathway" is provided, for example, at Kyoto University's KEGG (Kyoto Encyclopedia of Genes and Genomes) (https: / / www.genome.jp / kegg-bin / show_pathway?map00260).

[0052] (3-2-2) Cysteine The example in Figure 7 includes three substances (acetyl-L-serine, L-cystathionine, and S-sulfo-L-cysteine) as upstream metabolites registered for the candidate substance "cysteine." These three substances are located upstream of cysteine ​​when metabolism is assumed to proceed toward the synthesis of pyruvate in the "cysteine ​​and methionine metabolic pathway." The "cysteine ​​and methionine metabolic pathway" is provided, for example, at KEGG at Kyoto University (https: / / www.genome.jp / kegg-bin / show_pathway?amc00270).

[0053] (3-2-3) Riboflavin The example in Figure 7 includes three substances (6,7-dimethyl-8-ribityrrumazine, GTP (guanosine triphosphate), and ribulose-5-phosphate) as upstream metabolites registered for the candidate substance "riboflavin." These three substances are located upstream of riboflavin, for example, in the "riboflavin metabolic pathway," assuming that the synthesis of FAD (flavin adenine dinucleotide), which is used in the reaction when fumarate is synthesized from succinate in the citric acid cycle, proceeds. The "riboflavin metabolic pathway" is provided, for example, at KEGG at Kyoto University (https: / / www.genome.jp / kegg-bin / show_pathway?map00740).

[0054] (3-2-4)4-hydroxyproline The example in Figure 7 includes three substances (4-oxoproline, proline, and cis-4-hydroxy-D-proline) as upstream metabolites registered for the candidate substance "4-hydroxyproline." These three substances are located upstream of 4-hydroxyproline, for example, in the "arginine and proline metabolic pathway," assuming that metabolism proceeds toward the synthesis of pyruvate. The "arginine and proline metabolic pathway" is provided, for example, at KEGG at Kyoto University (https: / / www.genome.jp / kegg-bin / show_pathway?hsa00330).

[0055] (4) Processing flow Figure 8 is a flowchart of the process performed in the culture management system 1000. This process begins with the culture sample set in the culture management system 1000. In one implementation example, the control performed for this process in each of the control device 100, mass spectrometer 200, subculture device 300, analysis device 400, and transport device 500 is achieved by the processor contained in each device executing a given program. The details of this process are described below.

[0056] In step S100, the analysis device 400 counts the number of cells in a container holding cells to be used for culture. At this time, the container is set in the analysis device 400 by the transport device 500. The analysis device 400 transmits the count value from step S100 to the control device 100. In the control device 100, the processor 101 registers the received count value as the original number of cells in the memory 102.

[0057] In step S102, the user determines the number of cells to seed in each container and inputs the determined number of cells along with the container ID into the control device 100. The processor 101 of the control device 100 registers the number of cells in each container that has been entered into the measurement results database (Figure 5). More specifically, in the measurement results database, the processor 101 assigns a measurement ID to each container ID entered by the user, and further registers the number of cells entered in association with each container ID as the "number of seeded cells" (P0, etc.). The user may also input the number of passages associated with each container ID into the control device 100. The processor 101 may also register the number of passages entered in association with each container ID in the measurement results database.

[0058] In step S104, the mass spectrometer 200 identifies the amount of each candidate substance by analyzing the culture medium used for cultivation. At this time, the container containing the culture medium is set in the mass spectrometer 200 by the transport device 500. The mass spectrometer 200 aspirates a certain amount from the culture medium, performs the analysis, and transmits the analysis results to the control device 100. The control device 100 registers the analysis results transmitted in step S102 in memory 102, associating them with the container ID transmitted in the same manner.

[0059] In step S106, the subculture apparatus 300 seeds the number of cells determined in step S102 into each container. At this time, each container is also filled with the culture medium analyzed in step S104. Cell seeding and / or filling of the culture medium may be performed by the user.

[0060] In step S108, the subculture device 300 determines whether the conditions for ending culture of the sample have been met. An example of a condition for ending culture is that a predetermined culture time has elapsed since the start of culture. However, the conditions for ending culture can be set as appropriate. The subculture device 300 proceeds to step S110 (NO in step S108) until it determines that the conditions for ending culture have been met (YES in step S108), and then proceeds to step S116.

[0061] In step S110, the subculture device 300 determines whether or not to perform a sample culture medium change. One example of a condition for performing a culture medium change is that a predetermined culture time has elapsed since the start of culture. However, the conditions for performing a culture medium change may be set as appropriate. The subculture device 300 continues the culture by returning control to step S108 (NO in step 110) until it determines that the conditions for performing a culture medium change have been met, and then proceeds to step S112.

[0062] In step S112, the mass spectrometer 200 identifies the container ID of the sample and, similar to how the culture medium was analyzed in step S104, analyzes the supernatant of the sample to determine the amount of each candidate substance. At this time, the supernatant is collected by the subculture device 300, and the container containing the collected supernatant is set in the mass spectrometer 200 by the transport device 500. During collection, the subculture device 300 transmits the container ID of the container to be collected to the control device 100. The mass spectrometer 200 aspirates a certain amount of supernatant from the sample and performs the analysis. The mass spectrometer 200 transmits the container ID and the amount of each identified candidate substance to the control device 100. In the control device 100, the processor 101 registers the transmitted amount of each candidate substance in memory 102, associating it with the transmitted container ID. At this time, the amount of each candidate substance is registered as the amount immediately before the culture medium change.

[0063] In step S114, the subculture device 300 discards the culture medium in the culture vessel and adds new culture medium to perform a culture medium exchange.

[0064] In step S115, the mass spectrometer 200 identifies the amount of each candidate substance by analyzing the supernatant of the sample, similar to how it analyzed the culture medium in step S104, and transmits this information to the control device 100. The control device 100 registers the transmitted amount of each candidate substance in the memory 102. At this time, the amount of each candidate substance is registered as the amount immediately after the culture medium change. If the same culture medium used in step S104 is used in the culture medium change, the analysis of the culture medium may be omitted in step S115, and the same amount registered in step S104 may be registered.

[0065] In step S116, the mass spectrometer 200 identifies the container ID of the sample and, similar to how the culture medium was analyzed in step S104, analyzes the supernatant of the sample to determine the amount of each candidate substance. At this time, the supernatant is collected by the subculture device 300, and the container containing the collected supernatant is set in the mass spectrometer 200 by the transport device 500. During collection, the subculture device 300 transmits the container ID of the container to be collected to the control device 100. The mass spectrometer 200 aspirates a certain amount of supernatant from the sample and performs analysis. The mass spectrometer 200 transmits the container ID and the amount of each identified candidate substance to the control device 100. In the control device 100, the processor 101 registers the transmitted amount of each candidate substance in memory 102, associating it with the transmitted container ID. At this time, the amount of each candidate substance is registered as the amount at the end of culture.

[0066] In step S118, the control device 100 calculates the change in each candidate substance for each sample, as described with reference to equation (1).

[0067] In step S120, the subculture device 300 determines whether the subculture completion conditions for the sample have been met. An example of a subculture completion condition is reaching the number of subgenerations determined at the start of the culture. However, the subculture completion conditions may be set as appropriate. The subculture device 300 proceeds to step S122 until it determines that the subculture completion conditions have been met (NO in step S120), and when it determines that the subculture completion conditions have been met (YES in step S120), it terminates the subculture of the sample. The culture vessel containing the sample after subculture completion is transported out of the culture management system 1000 by the transport device 500. The control device 100 may, in response to an external instruction, have the transport device 500 transport the culture vessel out of the culture management system 1000. The external instruction may be input to the control device 100 via the input device 120, or from an external device via the communication I / F 104.

[0068] In step S122, the subculture device 300 removes cells that have lost their ability to proliferate from the sample. Note that cells that have lost their ability to proliferate are not limited to cells that have completely lost their ability to proliferate, but also include cells that are judged to have low proliferative capacity according to predetermined conditions.

[0069] In one implementation, the subculture device 300 has the function of identifying cells that have lost their ability to proliferate and the function of removing some cells from the sample. In one implementation, the function of identifying cells that have lost their ability to proliferate is realized by a camera that takes pictures of the sample and a computer that analyzes the images taken by the camera. The computer identifies features (for example, color and shape) that have been pre-registered as characteristics of cells that have lost their ability to proliferate in the captured images. In one implementation, the function of removing some cells from the sample is realized by a suction device and an arm that moves the suction device. The computer causes the suction device to aspirate the portion of the sample in which the identified features have been identified, and then discharges that portion into a container other than the container from which the removal will take place.

[0070] In step S124, the analysis device 400 counts the number of cells in the sample. At this time, the container containing the sample is set in the analysis device 400 by the transport device 500. The analysis device 400 transmits the count value to the control device 100. In the control device 100, the processor 101 registers the received count value as the number of cells (N1, etc.) in the measurement result database (Figure 5).

[0071] In step S126, the control device 100 determines whether the proliferative capacity of the cells contained in the sample is lower than a given standard.

[0072] In one implementation example, one example of a given criterion is that the ratio (C1 / C2) of the number of cells counted in step S124 (C1) to the number of seeded cells registered in step S106 (C2) is 1. In this example, if the ratio is less than 1, it is determined that the cell proliferation capacity is lower than the given criterion, and if the ratio is 1 or greater, it is determined that the cell proliferation capacity is not lower than the given criterion.

[0073] In step S126, the control device 100 calculates the above ratio. If the calculated ratio is less than 1, it determines that the proliferative capacity of the cells in the sample is lower than the given standard (YES in step S126), and proceeds to step S128. On the other hand, if the calculated ratio is 1 or greater, the control device 100 determines that the proliferative capacity of the cells in the sample is not lower than the given standard (NO in step S126), and the control returns to step S102, thereby performing subculturing.

[0074] The aforementioned "given criteria" may be set as appropriate depending on the circumstances under which the culture management system 1000 is applied.

[0075] In step S128, the control device 100 identifies the causative substance using the change in each candidate substance identified in step S118, as described above in "(2-2-2) Change in the amount of candidate substance".

[0076] In step S130, the control device 100 notifies the user of the causative substance identified in step S128.

[0077] The notification in step S130 may be a notification to a predetermined person. The "predetermined person" is, for example, the person (requester) who requested the administrator of the culture management system 1000 to provide information about the sample. The memory 102 of the control device 100 may contain information (for example, an email address) that specifies how to notify the requester, and the control device 100 notifies the requester of the causative substance by sending an email describing the identified causative substance to that email address.

[0078] The notification in step S130 may also be provided by displaying the causative substance on the display 110.

[0079] In the notification in step S130, the causative substance may be displayed along with the passage number of the culture in question (generation information in the subculture). For example, as shown in Figure 3, the display of the amount of each candidate substance may include information identifying the sample (sample B) and generation information (6th passage). Furthermore, the display may represent the causative substance among one or more candidate substances in a different manner from the other candidate substances (for example, by a different color).

[0080] In step S132, the control device 100 determines whether or not it has received instructions to add the causative substance to the sample. In one implementation example, after notifying the user of the causative substance in step S130, the control device 100 receives instructions from the user regarding whether or not to add the causative substance to the sample for a certain period of time. If the control device 100 determines that it has received instructions to add the causative substance within the aforementioned period of time (YES in step S132), it proceeds to step S134; otherwise (NO in step S132), it returns control to step S102.

[0081] In step S134, the control device 100 instructs the subculture device 300 to add the causative substance. The subculture device 300 adds the causative substance identified in step S120 to the culture medium. In one example, the subculture device 300 includes a sample rack containing one or more candidate substances (causative substances) and a pipetting device. The subculture device 300 causes the pipetting device to add the causative substance contained in the sample rack to the culture medium. An upstream metabolite may be added instead of, or together with, the causative substance. The amount of causative substance (and / or upstream metabolite) added may be predetermined or set according to the change in the causative substance. More specifically, the amount added may be set to be larger the greater the decrease in the causative substance.

[0082] Priority may be set in advance for one or more candidate substances. If two or more types of causative substances are identified in step S130, the subculture device 300 may add only a predetermined number of causative substances to the sample, starting with those with the highest priority among the two or more identified causative substances.

[0083] If the identified causative substance is a metabolite, the subculture device 300 may add an upstream metabolite (Figure 7) corresponding to the metabolite to the sample in place of the metabolite, or in addition to the metabolite. The above priority order may be set for one or more candidate substances and one or more upstream metabolites. The subculture device 300 may add only a predetermined number of substances from the identified one or more causative substances and their upstream metabolites, starting with those with the highest priority, to the sample.

[0084] Subsequently, control is returned to step S102. In the process shown in Figure 8, if the proliferative capacity of the cells contained in the container is lower than a given standard, the causative substance is identified. In the process shown in Figure 8, the number of seeded cells determined in step S102 is an example of the "first cell number." On the other hand, the number of cells counted after culturing in step S124 is an example of the "second cell number."

[0085] In the process shown in Figure 8, step S102 identifies and registers the number of cells at the start of each generation of subculture, and step S124 identifies and registers the number of cells at the end of each generation of subculture.

[0086] Furthermore, the number of cells in the sample may be obtained during the culture of each generation. For example, after the analysis of the supernatant in step S112 and before the exchange of the culture medium in step S114, the number of cells in the sample may be determined by image analysis of the sample. In this case, if the number of cells has decreased since the start of culture, the processor 101 may use the analysis results in step S112 to identify the causative substance and notify the user. The user may add the notified causative substance to the sample and input the type and amount of the added causative substance into the control device 100. The user may also instruct the control device 100 to add the causative substance to the sample. The processor 101 of the control device 100 may register the input amount of the causative substance in the measurement result database.

[0087] After the analysis of the supernatant in step S112 and before the exchange of the culture medium in step S114, the user may determine whether there are any abnormalities in the cell shape based on the image analysis of the sample. The processor 101 may use the analysis results in step S112 to identify the causative substance and notify the user. If the user determines that there are abnormalities in the cell shape, they may add the notified causative substance to the sample and input the type and amount of the added causative substance into the control device 100. The processor 101 of the control device 100 may register the input amount of the causative substance in the measurement results database.

[0088] Furthermore, in the process shown in Figure 8, the control device 100 may omit the control in step S128 and, in step S130, notify of candidate substances whose quantity has changed significantly since the start of culture. In this case, "significant change in quantity" can mean either a large decrease in quantity or a large increase in quantity. In one implementation example, when the control device 100 determines in step S126 that the growth capacity is low, it may select candidate substances whose quantity at that time has changed from the quantity at the start of culture within a given range, and notify of the name of the selected candidate substance in step S130. The "given range" can be appropriately set depending on the circumstances under which the culture management system 1000 is implemented. The control device 100 may also notify of the change in the quantity of each candidate substance from the quantity at the start of culture in step S130. In step S130, the control device 100 may notify of candidate substances with large changes (and their changes) along with the notification of the causative substances as described above.

[0089] [Embodiment 2] (1) Overview In Embodiment 2, the processes that were performed by the device in the culture management system 1000 of Embodiment 1 (culturing, subculturing, supernatant analysis, and cell counting) are performed manually.

[0090] In Embodiment 2, the number of cells and the amount of each candidate substance in the sample are input to the control device 100 (Figure 1). The number of cells and the amount of each candidate substance may be input by an operator via the input device 120, or they may be input from an external device via a communication interface through a network.

[0091] The control device 100 uses the cell count to determine whether the cell proliferation capacity is lower than a given standard. If it determines that the cell proliferation capacity is lower than a given standard, the control device 100 uses the results of the supernatant analysis to identify the causative substance. The following describes the processes performed by the control device 100.

[0092] (2) Processing flow Figure 9 is a flowchart of the process performed in the control device 100. The process in Figure 9 is performed for each sample, for example, by the processor 101 executing a given program. The number of cells and the amount of candidate substance used are for the sample in question.

[0093] In step S200, the control device 100 acquires the cell count value before culture. The cell count value before culture refers to the value for samples with passage number 0 that have not been cultured at all, or samples collected during the passage process performed during subculturing.

[0094] In step S202, the control device 100 obtains a count value of the number of cells after a given number of passages. The "given number of passages" is specified, for example, by the user of the control device 100.

[0095] In step S204, the control device 100 determines whether the proliferation capacity of the cells after the given number of subculturing cycles is lower than a given standard. The determination in step S204 may be the same as the determination in step S114 (Figure 8). That is, in one example, an example of a given standard is that the ratio (CA / CB) of the cell count value (CA) obtained in step S202 to the cell count value (CB) obtained in step S200 is 1. In this example, if the ratio is less than 1, it is determined that the proliferation capacity of the cells is lower than a given standard, and if the ratio is 1 or greater, it is determined that the proliferation capacity of the cells is not lower than a given standard.

[0096] If the control device 100 determines that the cell proliferation capacity is lower than a given standard (YES in step S204), it proceeds to step S206. On the other hand, if the control device 100 determines that the cell proliferation capacity is not lower than a given standard (NO in step S204), it proceeds to step S220.

[0097] In step S206, the control device 100 obtains the amount of each candidate substance in the culture medium used for cultivation.

[0098] In step S208, the control device 100 determines whether or not the culture medium was changed during the culture. This determination in step 208 is made, for example, by an operator of the control device 100. In one implementation example, after step S206, the operator inputs whether or not the culture medium was changed via a communication interface through the network using the input device 120 or an external device.

[0099] The decision in step S208 may be based on a predetermined plan. In one implementation example, before performing the culture operation, the worker registers the work plan for the culture in memory 102 via a communication I / F through the network from the input device 120 or an external device, and in step S208, it is determined whether the culture medium exchange was performed according to the registered work plan.

[0100] If the control device 100 determines that the culture medium has been changed (YES in step 208), it proceeds to step S210. On the other hand, if the control device 100 determines that the culture medium has not been changed (NO in step S208), it proceeds to step S212.

[0101] In step S210, the control device 100 acquires the amount of each candidate substance in the culture supernatant during a medium exchange. If multiple medium exchanges have been performed, the amount of each candidate substance in the culture supernatant is acquired for each medium exchange.

[0102] In step S212, the control device 100 obtains the amount of each candidate substance after culturing for the given number of passages.

[0103] In step S214, the control device 100 calculates the difference between the amount obtained in step S206 and the amount obtained in step S212 for each candidate substance. The difference may be calculated in the same way as in step S118, as described above in "(2-2-2) Change in the amount of candidate substance". Alternatively, if the amount of candidate substance in the culture supernatant during medium exchange is obtained in step S210, the change in the amount of candidate substance may be calculated by adding the difference between the amount obtained in step S206 and the amount obtained in step S212 to the difference between the amount obtained in step S206 and the amount obtained in step S210.

[0104] In step S216, the control device 100 identifies the causative substance based on the difference for each candidate substance calculated in step S214. The causative substance may be identified as described above in "(2-2-2) Change in the amount of candidate substance," similar to step S130.

[0105] In step S218, the control device 100 displays the causative substance identified in step S216 on the display 110. In addition to the causative substance, or instead of the causative substance, the control device 100 may display upstream metabolites (Figure 7) for the causative substance. After that, the control device 100 terminates the process shown in Figure 9.

[0106] In step S220, the control device 100 displays the result of the determination in step S204, i.e., that the cell proliferation capacity is not lower than the given standard, and terminates the process shown in Figure 9.

[0107] In Embodiment 2 described above, when a sample is subcultured for a given number of passages, the control device 100 determines whether the cell proliferation capacity of the sample is lower than a given standard, using the number of cells before subculturing and the number of cells after a given number of cultures.

[0108] Furthermore, the control device 100 identifies the causative substance using the amount of each candidate substance before passage and the amount after a given number of passages, and displays the causative substance on the display 110. This allows the user to recognize the causative substance. In addition, the user can expect to improve the cell proliferation ability in the sample by adding the causative substance to the sample. Moreover, when culturing the same cells as in the sample, the user can expect to avoid a situation where the cell proliferation ability is reduced in the new culture by increasing the amount of the causative substance in the culture medium.

[0109] In addition, in step S218, the control device 100 may, in the same manner as in step S130 in Figure 8, display candidate substances that have changed significantly since the start of the culture, either instead of displaying the causative substance or, along with displaying the causative substance.

[0110] [Differentiation] (1) Method of analyzing the supernatant The candidate substance amount is an example of the results of the supernatant analysis. The supernatant analysis method used to identify the candidate substance amount is not limited to mass spectrometry. Instead of, or in addition to, mass spectrometry, analysis using liquid chromatography and / or analysis according to the ELISA (Enzyme-Linked Immuno Sorbent Assay) method may be used. In this case, the mass spectrometer 200 in the culture management system 1000 may be appropriately modified according to the analytical method adopted.

[0111] (2) Criteria for determining proliferative capacity The criteria for determining low proliferative capacity (criteria in S128 or S204) are not limited to a ratio of 1 for the number of cells after culture at each passage to the number of cells before culture. This criterion may be set as appropriate by the person implementing the culture management system.

[0112] Another example of a criterion is that the ratio of the number of cells after the current culture to the number of cells after the previous passage is 1. In this case, for example, if the number of cells after the 4th passage is less than the number of cells after the 3rd passage, the cell proliferation capacity in the 4th passage sample is judged to be lower than the given criterion. On the other hand, if the number of cells after the 4th passage is greater than or equal to the number of cells after the 3rd passage, the cell proliferation capacity in the 4th passage sample is judged to be not lower than the given criterion.

[0113] Another example of a criterion is that the increase in cell number from the previous culture to the current culture is the same as the increase in cell number from the culture before last to the previous culture. In this case, for example, if the number of cells after 4 passages is 1.5 times the number of cells after 3 passages, and the number of cells after 3 passages is 2.0 times the number of cells after 2 passages, the former increase (1.5 times) is smaller than the latter increase (2.0 times), so the cell proliferation capacity in the sample after 4 passages is judged to be lower than the given criterion. On the other hand, if the former increase is greater than or equal to the latter increase, the cell proliferation capacity in the sample after 4 passages is judged to be not lower than the given criterion.

[0114] Another example of a criterion may be the relative value of the ratio of the number of cells after culture to the number of cells before culture, for each passage. For example, in the example in Figure 2, the ratio of the number of cells after culture for sample A at passage 4 to the number of cells before culture is approximately 3.8, while for sample B it is approximately 1.2. In other words, the ratio for sample B is less than half of the ratio for sample A. In this case, after 4 passages of culture, sample B is judged to have lower cell proliferation capacity than the given criterion. On the other hand, in the example in Figure 2, the ratio of the number of cells after culture for sample A at passage 7 to the number of cells before culture is approximately 2.2, while for sample B it is approximately 2.0. In other words, the ratios for samples A and B are similar. In this case, the proliferation capacity of samples A and B is similar, and after 7 passages of culture, the cell proliferation capacity of sample B is judged not to be lower than the given criterion. In one implementation, if the ratio of "a certain sample" is less than 0.8 times the ratio of "another sample," the cells of "another sample" are judged to have high proliferative capacity, and the cells of "a certain sample" are judged to have low proliferative capacity. In another implementation, if the ratio of "a certain sample" is between 0.8 times and 1.25 times the ratio of "another sample," the cells of "another sample" and "a certain sample" are judged to have similar proliferative capacity. Here, the multiple samples used for comparison (in the above example, sample A and sample B) are selected on the premise that there are commonalities in the culture conditions of each sample. Commonalities include, for example, the cell type to be cultured, the cell line, the culture medium used, the cells or reagents used in the sample, the number of cells seeded at the start of culture, the number of cells seeded per unit area of ​​the culture vessel, or the number of cells at the start of culture.

[0115] [Pattern] Those skilled in the art will understand that the above-described exemplary embodiments are specific examples of the following embodiments.

[0116] (Section 1) A culture management method according to one embodiment is a method for managing the culture of a group of cells contained in a sample, comprising the steps of: determining whether the proliferative capacity of the group of cells contained in the sample after culture is lower than a given standard, using a first cell number, which is the number of cells in the sample before culture, and a second cell number, which is the number of cells after the sample has been cultured; and calculating the amount of change in each of one or more candidate substances in the supernatant of the sample during culture, wherein the one or more candidate substances consist of a group comprising at least one of one or more metabolites and one or more enzymes, and if it is determined that the proliferative capacity of the group of cells contained in the sample is lower than the given standard, the method may further comprise the steps of: selecting some of the candidate substances from among the one or more candidate substances based on the amount of change of each of the one or more candidate substances before and after culture; and notifying the selected some of the candidate substances.

[0117] According to the culture management method described in paragraph 1, if the cell proliferation capacity in the sample is judged to be low compared to a given standard, a substance selected based on the change before and after culture is notified. This provides information to support cell culture.

[0118] (Section 2) In the culture management method described in Section 1, the selection step may include selecting candidate substances whose change before and after culture exceeds a given range.

[0119] According to the culture management method described in Section 2, when the proliferation capacity of the cell population is judged to be lower than a given standard, the user can identify candidate substances that showed a large change before and after culturing.

[0120] (Clause 3) In the culture management method described in paragraph 1 or 2, the selection step may include identifying, as some candidate substances, the causative substances that cause the growth capacity to be judged to be lower than the given standard.

[0121] According to the culture management method described in Section 3, users can expect to improve the proliferation capacity of cells in a given sample by adding the causative substance to the culture medium. Furthermore, when culturing the same cells as in the sample, users can expect to avoid a situation where the proliferation capacity of cells is reduced in the new culture by increasing the amount of the causative substance in the culture medium.

[0122] (Clause 4) In the culture management method described in any one of paragraphs 1 to 3, the step of making a determination may include determining that the proliferative capacity is lower than a given standard when the ratio of the number of second cells to the number of first cells falls below a given value.

[0123] According to the culture management method described in Section 4, the ability to proliferate can be easily determined. (Clause 5) In the culture management method described in any one of paragraphs 1 to 4, the notification step may include notifying a substance located upstream of the causative substance in the metabolic pathway.

[0124] According to the culture management method described in Section 5, even if the causative substance is a difficult-to-obtain substance or a substance that is difficult to add directly to the sample, the user can add a substance located upstream of the causative substance in the metabolic pathway to the sample.

[0125] (Clause 6) The culture management method described in any one of paragraphs 1 to 5 further comprises the step of performing a culture medium exchange of the sample, and the step of calculating the amount of change may include deriving the amount of change for each of the one or more candidate substances using the amount in the supernatant at the start of culture, the amount in the exchanged culture medium, and the amount in the supernatant at the end of culture.

[0126] According to the culture management method described in Section 6, cell culture management can be performed even when the culture medium is changed.

[0127] (Section 7) The culture control method described in any one of Sections 1 to 6 may further include the step of adding the causative substance to the sample.

[0128] According to the culture management method described in paragraph 7, the management of cell culture can be carried out comprehensively, along with the response taken when it is determined that the cell's proliferative capacity has fallen below a given standard.

[0129] (Clause 8) The culture control method described in any one of paragraphs 1 to 6 may further comprise the step of adding at least one of the causative substance and a substance located upstream of the causative substance in the metabolic pathway to the sample.

[0130] According to the culture management method described in paragraph 8, the management of cell culture can be carried out comprehensively, along with the response taken when it is determined that the cell's proliferative capacity has fallen below a given standard.

[0131] (Section 9) The culture management method described in any one of Sections 1 to 8 may further include a step of removing cells from the sample that have lost their ability to proliferate.

[0132] According to the culture management method described in paragraph 9, the management of cell culture can be carried out comprehensively, including the removal of cells that have lost their proliferative capacity from the sample. (Clause 10) In the culture management method described in any one of paragraphs 1 to 9, the culture includes any generation of culture in subculturing, and the step of notifying the causative substance may include outputting the causative substance together with the generation information in subculturing.

[0133] According to the culture management method described in Section 10, in subculturing, information regarding the culture of each generation is provided along with information about that generation.

[0134] (Clause 11) A program according to one embodiment is executed by a computer, causing the computer to carry out the culture management method described in any one of paragraphs 1 to 5.

[0135] According to the program described in paragraph 11, the management of cell culture can be performed by computer.

[0136] (Clause 12) A culture management system according to one embodiment comprises a cell culture device for culturing cells contained in a sample, an analysis device for counting the number of cells in the sample, an analysis device for outputting the analysis results of the supernatant of the sample, and a control device, wherein the control device is configured to determine whether the proliferation capacity of the cell population contained in the sample after culturing is lower than a given standard, using a first cell count, which is the number of cells in the sample before culturing, and a second cell count, which is the number of cells after the sample has been cultured, and to calculate the change in each of one or more candidate substances in the supernatant of the sample during culture, wherein one or more candidate substances consist of a group consisting of at least one of one or more metabolites and one or more enzymes, and the control device may be configured to select some of the candidate substances from among the one or more candidate substances based on the change in each of the one or more candidate substances in the sample before and after culture, and notify the user of some of the candidate substances, when it is determined that the proliferation capacity of the cell population contained in the sample is lower than a given standard.

[0137] According to the culture management system described in Section 12, if the cell proliferation capacity in a sample is determined to be low compared to a given standard, a substance selected based on the change before and after culture is notified. This provides information to support cell culture.

[0138] (Clause 13) In the culture management system described in paragraph 12, selecting some candidate substances may include selecting some candidate substances whose change before and after culture exceeds a given range.

[0139] According to the culture management system described in Section 13, when the proliferation capacity of a cell population is determined to be lower than a given standard, the user can identify candidate substances that showed a large change before and after culturing.

[0140] (Clause 14) In the culture management system described in paragraph 12 or 13, the selection of some candidate substances may include identifying some candidate substances that are the cause of the growth capacity being judged to be lower than the given criteria.

[0141] According to the culture management system described in Section 14, users can expect to improve the proliferation capacity of cells in a given sample by adding the causative substance to the culture medium. Furthermore, when culturing the same cells as in the sample, users can expect to avoid a situation where the proliferation capacity of cells is reduced in the new culture by increasing the amount of the causative substance in the culture medium.

[0142] (Clause 15) In the culture management system described in any of Clauses 12 to 14, the control device may determine that the proliferative capacity is lower than a given standard when the ratio of the number of second cells to the number of first cells falls below a given value.

[0143] According to the culture management system described in Section 15, the determination of proliferation ability can be made easily.

[0144] (Section 16) In the culture management system described in any one of Sections 12 to 15, the control device may further notify of substances located upstream of the causative substance in the metabolic pathway.

[0145] According to the culture management system described in Section 16, even if the causative substance is a difficult-to-obtain substance or a substance that is difficult to add directly to the sample, the user can add a substance located upstream of the causative substance in the metabolic pathway to the sample.

[0146] (Section 17) In the culture management system described in any one of Sections 12 to 16, the cell culture device may add the causative substance to the culture medium.

[0147] According to the culture management system described in paragraph 17, cell subculturing and culture, cell counting, and culture management can be carried out comprehensively, along with the response taken when it is determined that the cell proliferation capacity has fallen below a given standard.

[0148] (Section 18) In any of the culture management systems described in Sections 12 to 16, the cell culture device may add at least one of the causative substance and a substance located upstream of the causative substance in the metabolic pathway to the sample.

[0149] According to the culture management system described in paragraph 18, cell subculturing and culture, cell counting, and culture management can be carried out comprehensively, along with the response taken when it is determined that the cell proliferation capacity has fallen below a given standard.

[0150] (Section 19) In the culture management system described in any one of Sections 12 to 18, the cell culture device may remove cells from the sample that have lost their ability to proliferate.

[0151] According to the culture management system described in Section 19, cell subculturing and culture, cell counting, and culture management can be carried out comprehensively, along with the removal of cells that have lost their reproductive capacity from the sample.

[0152] (Section 20) In the culture management system described in any one of Sections 12 to 19, the culture includes any generation of culture in subculturing, and the control device may notify the causative substance along with the generation information in subculturing.

[0153] According to the culture management system described in Section 18, in subculturing, information regarding the culture of each generation is provided along with information about that generation.

[0154] The embodiments disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of this disclosure is indicated by the claims rather than by the description of the embodiments above, and all modifications within the meaning and scope equivalent to the claims are intended to be included. Furthermore, each technology in the embodiments is intended to be practiced individually or, as far as possible, in combination with other technologies in the embodiments. [Explanation of Symbols]

[0155] 100 control devices, 101 processors, 102 memory, 110 displays, 120 input devices, 200 mass spectrometers, 300 subculture devices, 400 analysis devices, 500 transport devices, 1000 culture management systems.

Claims

1. A method for managing the culture of cell populations contained in a sample, A step of determining whether the proliferative capacity of the cell population contained in the cultured sample is lower than a given standard, using a first cell count, which is the number of cells in the sample before culture, and a second cell count, which is the number of cells after the sample has been cultured. The process includes the step of calculating the amount of change in each of the one or more candidate substances in the supernatant of the sample during cultivation. The one or more candidate substances described above are composed of a group consisting of at least one of one or more metabolites and one or more enzymes. If the proliferation ability of the cell population contained in the sample is determined to be lower than the given standard, the step of selecting some candidate substances from the one or more candidate substances based on the amount of change of each of the one or more candidate substances in the sample before and after culture, The further step includes notifying the aforementioned candidate substances, A culture management method comprising the selection step of selecting candidate substances whose change before and after culture exceeds a given range.

2. The culture management method according to claim 1, wherein the selection step includes identifying, as some of the candidate substances, the causative substances that cause the growth capacity to be judged to be lower than the given standard.

3. The culture management method according to claim 1 or 2, wherein the step of making the determination includes determining that the proliferative capacity is lower than the given standard when the ratio of the number of second cells to the number of first cells is lower than a given value.

4. The culture management method according to claim 3, wherein the notification step includes notifying a substance located upstream of the causative substance in the metabolic pathway.

5. The step further comprises performing a culture medium exchange for the aforementioned sample, The culture management method according to claim 1 or 2, wherein the step of calculating the amount of change includes deriving the amount of change for each of the one or more candidate substances using the amount in the supernatant at the start of culture, the amount in the replaced medium, and the amount in the supernatant at the end of culture.

6. The culture management method according to claim 3, further comprising the step of adding the causative substance to the sample.

7. The culture management method according to claim 3, further comprising the step of adding at least one of the causative substance and a substance located upstream of the causative substance in the metabolic pathway to the sample.

8. The culture management method according to claim 1 or 2, further comprising the step of identifying cells that have lost their ability to proliferate from the aforementioned cell population, and removing such cells from the sample by aspirating them with an aspiration device and then discharging them into a container other than the sample container.

9. The culture includes any generation of culture in a subculture, The culture management method according to claim 3, wherein the step of notifying the causative substance includes outputting the causative substance together with generation information in subculturing.

10. A program that, when executed by a computer, causes the computer to perform the culture management method described in claim 1 or claim 2.

11. A cell culture device for culturing cells contained in the sample, An analytical device for counting the number of cells in the aforementioned sample, An analytical device that outputs the analysis results of the supernatant of the aforementioned sample, A transport device for moving the sample from the cell culture apparatus to the analyzer and the analytical apparatus, Equipped with a management device, The aforementioned control device is Using the first cell count, which is the number of cells in the sample before culturing, and the second cell count, which is the number of cells after culturing the sample, it is determined whether the proliferative capacity of the cell population contained in the cultured sample is lower than a given standard. The system is configured to calculate the amount of change in each of the one or more candidate substances in the supernatant of the aforementioned sample during cultivation. The one or more candidate substances described above are composed of a group consisting of at least one of one or more metabolites and one or more enzymes. The aforementioned control device is If the proliferation ability of the cell population contained in the sample is determined to be lower than the given standard, some candidate substances are selected from the one or more candidate substances based on the amount of change of each of the one or more candidate substances in the sample before and after culture. It is configured to notify the aforementioned selection of candidate substances, A culture management system in which the selection of some candidate substances includes selecting candidate substances whose change before and after culture exceeds a given range.

12. The culture management system according to claim 11, wherein the selection of the aforementioned candidate substances includes identifying, as the aforementioned candidate substances, the causative substances that cause the growth ability to be judged to be lower than the given standard.

13. The culture management system according to claim 11 or 12, wherein the control device determines that the proliferation capacity is lower than a given standard when the ratio of the second cell number to the first cell number is lower than a given value.

14. The culture management system according to claim 13, wherein the control device further notifies of a substance located upstream of the causative substance in the metabolic pathway.

15. The cell culture device adds the causative substance to the sample, according to claim 13.

16. The cell culture device adds at least one of the causative substance and a substance located upstream of the causative substance in the metabolic pathway to the sample, according to claim 13.

17. The cell culture apparatus described above, Equipped with an additional suction device, A culture management system according to claim 11 or claim 12, comprising identifying cells that have lost their ability to proliferate from the aforementioned cell population, removing such cells from the sample by aspirating them with the aspiration device and then discharging them into a container other than the sample container.

18. The culture includes any generation of culture in a subculture, The culture management system according to claim 13, wherein the control device notifies the causative substance along with generation information in subculturing.

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