Estimation method, computer program, and estimation system

Abstract

To provide an estimation method or the like capable of easily estimating a calcium addition amount of a solvent suitable for culture.SOLUTION: Acquiring an additive amount of calcium to a first solvent used for swelling dry microcarriers containing calcium alginate and swelling information indicating a swelling state of the dry microcarriers swollen with the first solvent, estimating a recommended additive amount of calcium to the first solvent at which stability of the dry microcarriers after swelling with the first solvent and evaluation of cell culture using the dry microcarriers satisfy a predetermined requirement based on the acquired additive amount of calcium and the swelling information, and outputting information on the estimated recommended additive amount of calcium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an estimation method, a computer program, and an estimation system. [Background technology]

[0002] Efficient mass cultivation of cells and tissues is required in fields such as pharmaceutical production, gene therapy, regenerative medicine, and immunotherapy. Microcarrier culture is known as a mass culture technique for cells and the like. In microcarrier culture, for example, cells, a culture medium, and microcarriers that serve as a scaffold for cell adhesion are introduced into a culture vessel, the culture medium is intermittently stirred to suspend the cells and microcarriers, and the suspended cells descend and come into contact with the microcarriers, thereby adhering to the surface of the microcarriers and growing.

[0003] For example, Patent Document 1 discloses a cell culture carrier characterized by providing a coating layer made of collagen on the surface of beads made of granular calcium alginate. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 5-81 Summary of the Invention [Problem to be solved by the invention]

[0005] In cell culture using microcarriers containing calcium alginate, the amount of calcium added to the solvent used with the microcarriers significantly affects the cell culture conditions. Finding optimal conditions by conducting multiple experiments with different solvent conditions is time-consuming and costly. Therefore, a technology that can easily estimate the amount of calcium added to the solvent suitable for culture is needed.

[0006] An object of one aspect of the present disclosure is to provide an estimation method or the like that can easily estimate the amount of calcium to be added to a medium suitable for culture. [Means for solving the problem]

[0007] An estimation method according to one embodiment of the present disclosure includes acquiring an amount of calcium to be added to a first solvent used to swell dry microcarriers containing calcium alginate and swelling information indicating the swelling state of the dry microcarriers swollen with the first solvent, estimating a recommended amount of calcium to be added to the first solvent that satisfies predetermined requirements for stability of the dry microcarriers after swelling with the first solvent and evaluation of cell culture using the dry microcarriers based on the acquired amount of calcium to be added and swelling information, and outputting information related to the estimated recommended amount of calcium to be added. The processing is performed by a computer.

[0008] An estimation method according to one aspect of the present disclosure involves obtaining the swelling ratio of dry microcarriers containing calcium alginate swollen in each solvent containing different amounts of calcium added, as well as an evaluation of the stability of the dry microcarriers after swelling in the solvent and cell culture using the dry microcarriers, and a computer-implemented process to estimate the swelling ratio range of the dry microcarriers whose stability and cell culture evaluations meet specified requirements based on the obtained swelling ratios for each solvent and the evaluations of stability and cell culture.

[0009] A computer program according to one embodiment of the present disclosure acquires the amount of calcium to be added to a first solvent used to swell dry microcarriers containing calcium alginate and swelling information indicating the swelling state of the dry microcarriers swollen with the first solvent, and based on the acquired amount of calcium to be added and the swelling information, estimates a recommended amount of calcium to be added to the first solvent that satisfies specified requirements for the stability of the dry microcarriers after swelling with the first solvent and for evaluation of cell culture using the dry microcarriers, and outputs information regarding the estimated recommended amount of calcium to be added.

[0010] An estimation system according to one embodiment of the present disclosure includes a processing unit that acquires the amount of calcium to be added to a first solvent used to swell dry microcarriers containing calcium alginate and swelling information indicating the swelling state of the dry microcarriers swollen with the first solvent, and based on the acquired amount of calcium to be added and the swelling information, estimates a recommended amount of calcium to be added to the first solvent that satisfies specified requirements for the stability of the dry microcarriers after swelling with the first solvent and for evaluation of cell culture using the dry microcarriers, and outputs information regarding the estimated recommended amount of calcium to be added. [Effects of the Invention]

[0011] According to the present disclosure, the amount of calcium to be added to a medium suitable for culture can be easily estimated. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram of an estimation system according to an embodiment of the present invention. [Figure 2] FIG. 2 is a block diagram illustrating an example configuration of an estimation device and a terminal device. [Figure 3] FIG. 10 is a diagram illustrating an example of information stored in a model DB. [Figure 4] 10 is a flowchart illustrating an example of a processing procedure for estimating an acceptable range of swelling ratio. [Figure 5]10 is a flowchart showing an example of a processing procedure for estimating a recommended range of calcium addition amounts. [Figure 6] FIG. 10 is a diagram showing an example of a prediction function showing the correlation between the amount of calcium added and the swelling rate for a user culture solution. [Figure 7] 10 is a flowchart showing an example of a processing procedure for estimating a recommended range of calcium addition amounts in the second embodiment. [Figure 8] 11 is a flowchart showing an example of a processing procedure for estimating a recommended range of calcium addition amounts and a recommended range of sodium addition amounts in the third embodiment. [Figure 9] 1 shows phase contrast microscope images of microcarriers after swelling for each amount of calcium added. [Figure 10] Fluorescence microscopy images of cells stained with calcein-AM are shown for each amount of calcium added. [Figure 11] 1 shows a prediction function indicating the correspondence between the amount of calcium added and the swelling ratio in Example 1. [Figure 12] Fluorescence microscopy images of calcein-AM stained cells are shown. [Figure 13] 1 shows a prediction function indicating the correspondence between the amount of calcium added and the swelling ratio in a model case. [Figure 14] 1 shows a prediction function indicating the correspondence between the amount of calcium added and the swelling ratio in Example 2. [Figure 15] 10 shows a prediction function indicating the correspondence between the amount of sodium and calcium added and the swelling ratio in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure will be specifically described with reference to the drawings showing embodiments thereof.

[0014] (First embodiment) FIG. 1 is a schematic diagram of an estimation system 100 according to this embodiment. The estimation system 100 includes an estimation device 1 as a main device. The estimation device 1 is connected to a terminal device 2 via a network N so as to be able to communicate with each other. The network N is a wired or wireless network including a public communication network such as the Internet, a carrier network, etc. The number of terminal devices 2 may be one or three or more. The estimation device 1 and the terminal device 2 are not limited to being separate devices, but may be a single common processing device.

[0015] The estimation system 100 is a system for estimating culture conditions suitable for cell culture using dry microcarriers. In application of the estimation system 100, cell culture is performed using dry microcarriers. The dry microcarriers are used as microcarriers for cell culture. By adding a solvent to the dry microcarriers, the dry microcarriers can absorb water and swell. Cells can be seeded on the swollen microcarriers, and the cells can be attached to the microcarrier surface to grow. In this embodiment, by swelling the dry microcarriers with a solvent to which an additive solution containing a predetermined component has been added, a microcarrier state more suitable for cell culture is achieved. The estimation system 100 estimates the conditions for the amount of a predetermined component added to the solvent as the culture conditions.

[0016] The estimation device 1 is an estimation device capable of various information processing and information transmission and reception, and is, for example, a server computer, a personal computer, a quantum computer, etc. The estimation device 1 is managed, for example, by a provider (e.g., a manufacturer) of dried microcarriers. Based on information received from the terminal device 2, the estimation device 1 estimates suitable culture conditions according to the solvent used by the user who will be culturing the cells.

[0017] The terminal device 2 is an information processing terminal used by a user, such as a personal computer, a smartphone, or a tablet terminal. The terminal device 2 displays the culture conditions received from the estimation device 1 to the user. Examples of users who receive the culture conditions include culturists who use dried microcarriers to perform culture, and providers of dried microcarriers. In this embodiment, the culture conditions are provided to culturists.

[0018] 2 is a block diagram showing an example of the configuration of the estimation device 1 and the terminal device 2. The estimation device 1 includes a processing unit 11, a storage unit 12, and a communication unit 13. The estimation device 1 may be a single computer, or may be a computer system configured with multiple computers and peripheral devices. The estimation device 1 may be a virtual machine whose entity is virtualized, or may be a cloud.

[0019] The processing unit 11 includes one or more processors such as a central processing unit (CPU), a microprocessing unit (MPU), a graphics processing unit (GPU), etc. The processing unit 11 includes a memory that is a temporary storage medium such as a static random access memory (SRAM) or a dynamic random access memory (DRAM). The processing unit 11 may include functions such as a timer that measures the elapsed time from when a measurement start instruction is given to when a measurement end instruction is given, a counter that counts numbers, and a clock that outputs date and time information. The processing unit 11 may be realized by software, or partly or entirely by hardware such as an application specific integrated circuit (ASIC) or a field programmable gate array (FPGA).

[0020] The storage unit 12 includes a nonvolatile storage device such as a hard disk or a flash memory. The storage unit 12 may be separate from the estimation device 1 and may be one or more external storage devices connected externally. The storage unit 12 stores various computer programs and data referenced by the processing unit 11. The storage unit 12 of this embodiment stores a program 1P for causing a computer to execute processing related to the estimation of culture conditions, and a model DB (Data Base) 121. The model DB 121 is a database that stores information related to cell culture experiments using a specific culture medium that serves as a model. Details of the model DB 121 will be described later.

[0021] A computer program (program product) including program 1P may be provided by a non-transitory recording medium 1A on which the computer program is readably recorded. Recording medium 1A is a portable memory such as a CD-ROM, a USB memory, or an SD (Secure Digital) card. Processing unit 11 reads the desired computer program from recording medium 1A using a reading device (not shown) and stores the read computer program in memory unit 12. Alternatively, the computer program may be provided via communication. Program 1P may be a single computer program or may be composed of multiple computer programs. Program 1P may also be executed on a single computer or may be executed cooperatively by multiple computers.

[0022] The communication unit 13 includes a communication device that realizes communication via the network N. The processing unit 11 transmits and receives data to and from the terminal device 2 through the communication unit 13.

[0023] The configuration of the estimation device 1 is not limited to the above example, and may include, for example, a display unit that displays images, an operation unit that accepts user operations, and the like.

[0024] The terminal device 2 includes a processing unit 21, a storage unit 22, a communication unit 23, a display unit 24, and an operation unit 25.

[0025] The processing unit 21 includes one or more processors such as a CPU, a GPU, or the like. The storage unit 22 includes a non-volatile storage device such as a hard disk or a flash memory. The storage unit 22 stores various computer programs and data referenced by the processing unit 21. The storage unit 22 of this embodiment stores a program 2P for causing a computer to execute processing related to the acquisition of culture conditions. A computer program (program product) including the program 2P may be provided by a non-transitory recording medium 2A on which the computer program is readably recorded, or may be provided via communication.

[0026] The communication unit 23 includes a communication device that realizes communication via the network N. The processing unit 21 transmits and receives data to and from the estimation device 1 via the communication unit 23.

[0027] The display unit 24 includes a display device such as a liquid crystal display, an organic EL (Electro Luminescence) display, etc. The display unit 24 displays the culture conditions received from the estimation device 1 in accordance with instructions from the processing unit 21.

[0028] The operation unit 25 is an interface that accepts user operations. The operation unit 25 includes, for example, a keyboard, a mouse, a touch panel device with a built-in display, a speaker, a microphone, etc. The operation unit 25 accepts operation input from the user and sends a control signal to the processing unit 21 according to the operation content.

[0029] In the present system configured as described above, suitable culture conditions for cell culture using dry microcarriers are estimated.

[0030] The dry microcarriers (cell culture carriers) used in this system contain calcium alginate. More specifically, the dry microcarriers contain alginate cross-linked with calcium ions.

[0031] When used for cell culture, dry microcarriers are swollen in a solvent (e.g., culture medium) and used in the swollen state. In the swollen state of the microcarriers, calcium ions bond across two carboxyl groups (ionic cross-linking), forming a gel that is insoluble in water. To maintain the stability of gelled calcium alginate, the amount of calcium present in the same system as the calcium in the calcium alginate gel is important. If the calcium content is low, the ionic cross-linking will break down, causing deformation and collapse of the gel, making the gelled calcium alginate unstable. On the other hand, if the calcium content is too high, subsequent cell growth may deteriorate. To achieve appropriate cell culture, it is important to appropriately adjust the amount of calcium present in the same system as the calcium in the calcium alginate gel, i.e., the calcium content (calcium ion concentration) of the culture medium that swells the dry microcarriers. The calcium content of the culture medium can be adjusted by changing the amount of calcium added to the culture medium.

[0032] The method of conducting multiple cell culture experiments under different calcium addition conditions to the culture medium and determining optimal conditions based on the results of the cell culture is time-consuming and cost-intensive. Furthermore, it is expected that a variety of culture media will be used in cell culture. Determining optimal conditions for each culture medium with a different composition would require even more time and cost.

[0033] After extensive research, the inventors discovered that there is a correlation between the amount of calcium added to the culture medium and the swelling rate of dried microcarriers caused by the culture medium, and that this correlation between the amount of calcium added and the swelling rate can be used to determine the optimal amount of calcium added from actual measured swelling rate data.

[0034] The swelling ratio of dry microcarriers refers to the degree of swelling of dry microcarriers. In this specification, the swelling ratio is expressed by the following formula: Swelling rate (%) = (average particle size of swollen microcarriers) ÷ (average particle size of dry microcarriers) × 100 In the formula, the average particle size of dry microcarriers is the average value of the equivalent sphere diameters of dry microcarriers, and the average particle size of swollen microcarriers is the average value of the equivalent sphere diameters of the dry microcarriers after swelling with a solvent (culture medium). The number of microcarriers used to calculate the average particle size is not particularly limited, but can be, for example, 80 or more.

[0035] As used herein, "calcium addition amount (mmol / L)" refers to the amount of calcium (mmol) added per 1 L of solvent (e.g., culture medium, swelling solution, etc.). "Sodium addition amount (mmol / L)" refers to the amount of sodium (mmol) added per 1 L of solvent.

[0036] An overview of the flow of cell culture in this embodiment will be described. As shown in Figure 1, when culturing cells, a culture kit containing dry microcarriers and an additive solution is provided to a user by a provider of dry microcarriers. The user also prepares a culture solution to be used for culturing (hereinafter also referred to as user culture solution) and cells. The user can select any culture solution and cells depending on the purpose of culturing. The user culture solution is an example of a first solvent used to swell the dry microcarriers. Alternatively, a culture kit containing cells may be provided to the user.

[0037] The dry microcarriers used in this system are dry microcarriers for cell culture and contain calcium alginate as described above. It is preferable that the dry microcarriers further contain a cell adhesive substance to enhance cell adhesion. Examples of cell adhesive substances include, but are not limited to, scaffold proteins such as gelatin, collagen, laminin, elastin, proteoglycan, fibronectin, and vitronectin, peptides or domains of sequences that exhibit their activity (e.g., RGD peptide, laminin E8, etc.), and serum. Among these, gelatin is preferred from an economical standpoint. Known dry microcarriers can be used as such dry microcarriers.

[0038] The additive liquid to be added to the culture medium is an aqueous solution containing calcium. The additive liquid is used to adjust the calcium ion concentration of the culture medium. The additive liquid is not particularly limited, but an example thereof is an aqueous calcium chloride solution.

[0039] As the culture medium, a known culture medium for cell culture can be used. The culture medium may already contain calcium (before the addition of the additive solution). In this specification, the amount of calcium added to the culture medium (solvent) means the amount of calcium added by subsequently adding the additive solution to the culture medium. In other words, the amount of calcium added to the culture medium does not include the amount of calcium already contained in the culture medium.

[0040] Adherent cells are preferably used as cells for use with microcarriers so that they can be cultured on the microcarrier surface. Examples of such cells include hepatocytes (liver parenchymal cells), Kupffer cells, endothelial cells (e.g., vascular endothelial cells and corneal endothelial cells), fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermal cells (e.g., epidermal keratinocytes), epithelial cells (e.g., tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, and corneal epithelial cells), mammary gland cells, pericytes, myoblasts, myotubes, satellite cells, muscle cells (e.g., smooth muscle cells and cardiac myocytes), kidney cells, pancreatic islet cells, nerve cells (e.g., peripheral nerve cells and optic nerve cells), chondrocytes, and bone cells. These cells may be primary cells directly collected from tissues or organs, or may be cells that have been passaged for several generations. Furthermore, these cells may be undifferentiated cells such as embryonic stem cells and iPS cells, somatic stem cells such as mesenchymal stem cells that have the ability to differentiate, unipotent stem cells such as vascular endothelial progenitor cells that have the ability to differentiate into a single type of cell, or cells that have completed differentiation. Cells used with microcarriers may also include CHO cells, 293 cells, 3T3 cells, Vero cells, MRC5 cells, HeLa cells, hybridoma cells, and other cells widely used as cell substrates for the production of biopharmaceuticals and viral vectors, as well as established cell lines derived from these cells. Furthermore, the cells used for culturing may be a single type of cell, or two or more types of cells may be co-cultured.

[0041] The user conducts a swelling experiment in which dry microcarriers are swelled using the prepared user culture solution, and obtains swelling information indicating the swelling state of the dry microcarriers. More specifically, the user obtains two or more points of swelling information for dry microcarriers swollen in the user culture solution while changing the amount of calcium added to the user culture solution. The amount of calcium added to the user culture solution may be set arbitrarily by the user, or may be selected appropriately from a pre-set candidate calcium amount or candidate calcium amount range. The amount of calcium added in the swelling experiment may be zero. In other words, the swelling experiment may be conducted using a user culture solution to which no additive liquid has been added.

[0042] The swelling information includes, for example, the swelling ratio of the dry microcarriers. The swelling information may be any information that can be used to calculate the swelling ratio, and may be, for example, images (e.g., microscopic images, phase contrast images, etc.) of the dry microcarriers observed by a microscope before and after swelling. The acquired swelling information corresponding to each amount of calcium added is transmitted from the terminal device 2 to the estimation device 1.

[0043] The estimation device 1 estimates a recommended amount of calcium to be added to a user's culture solution when swelling dry microcarriers with the user's culture solution, based on swelling information related to the user's culture solution. In this embodiment, the estimation device 1 estimates a recommended range of calcium addition amounts defined by upper and lower limit values ​​of the recommended addition amount. The lower limit value of the recommended range of calcium addition amounts may be zero. In other words, the recommended amount of calcium addition may include zero. The recommended range of calcium addition amounts is estimated taking into account a predetermined acceptable range of swelling ratios of dry microcarriers. The acceptable range of swelling ratios corresponds to a swelling ratio range within which the stability of microcarriers in cell culture and the evaluation of cell culture meet specified requirements.

[0044] Microcarrier stability is information indicating the stability of microcarriers swollen in a culture medium (swollen dry microcarriers). Stability can be determined based on the quality of the microcarrier shape (approximately circular shape) and the presence or absence of collapse after a predetermined culture period when cell culture is performed using microcarriers swollen in a culture medium.

[0045] The cell culture evaluation is information indicating the evaluation of cell culture using microcarriers swollen with culture medium. The cell culture evaluation includes, for example, fluorescence observation results and cell proliferation rate. The fluorescence observation results are the results of evaluating the cell proliferation state by staining live cells attached to microcarriers with a fluorescent substance (e.g., calcein-AM) after a predetermined culture period and observing them under a fluorescence microscope. The cell proliferation rate is the ratio of the number of live cells after a predetermined culture period to the initial number of live cells seeded (number of live cells / initial number of live cells seeded). The cell culture evaluation may also use the amount of antibody production, the amount of cytokine secretion, the amount of exosome production, etc.

[0046] The acceptable range of swelling ratio used to estimate the recommended range of calcium addition is determined based on the results of actual cell culture experiments using the above-mentioned dry microcarriers and a model specific culture medium (hereinafter also referred to as model culture medium). Details of the method for estimating the acceptable range of swelling ratio will be described later. The model DB 121 stores various information obtained from cell culture experiments using dry microcarriers swollen in model culture medium. The model culture medium is an example of a second solvent.

[0047] 3 is a diagram showing an example of the content of information stored in the model DB 121. The model DB 121 stores data on a plurality of experiments in which the amount of calcium added to a model culture solution (the amount of calcium-containing additive solution added) was varied. The model DB 121 stores records that use, for example, an experiment number as a key to identify the experiment, linking together information such as the ratio of the amount of additive solution added to the model culture solution (additive solution / model culture solution), the amount of calcium added to the model culture solution (mmol / L), the swelling rate (%) of dried microcarriers, stability data indicating the stability of the microcarriers, and cell evaluation data indicating the evaluation of the cell culture.

[0048] In the example shown in FIG. 3, the cell evaluation data includes the results of fluorescence microscope observation and the proliferation rate of cells. The results of stability and fluorescence microscope observation are each evaluated in two stages: good, represented by a circle (◯), and poor, represented by a cross (×). If no measurement has been performed, information indicating that measurement is not possible (e.g., "-") is recorded. The evaluations of stability and fluorescence microscope observation may be classified into three or more stages, or may be quantitative information. Note that the contents stored in the model DB 121 are not limited to the example shown in FIG. 3. The model DB 121 may store, for example, microscope images, the average particle diameter of the microcarriers, the number of viable cells, etc.

[0049] The estimated range of the recommended amount of calcium to be added is provided to the user via the terminal device 2. The user prepares a culture solution by adding an additive solution so that the amount of calcium to be added falls within the recommended range. The user then uses the resulting culture solution to swell dried microcarriers, seeding the target cells onto the microcarriers and proceeding with the culture.

[0050] The cell culture procedure is not limited to the above example. For example, the dry microcarriers and the additive solution may be provided to the user at different times. The dry microcarriers may be provided to the user first, and a swelling experiment may be performed using the user's culture solution under conditions of zero calcium addition, i.e., no additive solution added. After identifying the preferred range based on the results of the swelling experiment, the additive solution may be provided to the user. The swelling experiment may also be performed by someone other than the culturist, for example, the provider of the dry microcarriers.

[0051] 4 is a flowchart showing an example of a processing procedure for estimating an acceptable range of a swelling ratio. The following processing is executed by the processing unit 11 in accordance with a program 1P stored in the storage unit 12 of the estimation device 1.

[0052] The processing unit 11 of the estimation device 1 acquires multiple pieces of experimental data related to cell culture experiments conducted using model culture solutions (step S11). The experimental data may be acquired, for example, by communication with an external device or by receiving input from an operator. The cell culture experiments are conducted using dry microcarriers containing calcium alginate and model culture solutions under multiple conditions in which the amount of calcium added to the model culture solutions is different. The experimental data include, for example, the amount of additive solution added, the amount of calcium added, the swelling rate, stability data, cell evaluation data, etc., for each condition.

[0053] The processing unit 11 receives requirements for cell culture using dry microcarriers (step S12). The processing unit 11 receives the requirements based on, for example, an operation by a provider of the dry microcarriers. The requirements for cell culture are information for determining an acceptable range for the swelling ratio, and specifically include requirements regarding the stability of the microcarriers and requirements regarding the evaluation of the cell culture.

[0054] Based on the acquired experimental data for the multiple amounts of calcium added, the processing unit 11 estimates an acceptable range of swelling ratios for which the evaluation of microcarrier stability and cell culture satisfies the requirements acquired in step S12 (step S13). More specifically, by estimating the range of calcium added amounts for which the evaluation of stability and cell culture satisfies the requirements, the processing unit 11 determines the range of swelling ratios corresponding to the range of calcium added amounts. Typically, the lower limit of the amount of calcium added depends on the results of the stability evaluation, and the upper limit depends on the results of the cell culture evaluation.

[0055] The above requirement may be, for example, that the stability of the microcarrier is equal to or greater than a predetermined threshold, and that the evaluation of the cell culture is equal to or greater than a predetermined threshold. The threshold may be a classification threshold. In this embodiment, as an example, an acceptable range of swelling ratio is specified corresponding to a range of calcium addition amounts such that the microcarrier has good stability, good fluorescence microscopic observation, and a proliferation rate of 1.1 times or greater.

[0056] Assume that six experimental data points, as shown in Figure 3, are obtained from a cell culture experiment. In Figure 3, the calcium addition amount increases gradually from b1 to b6. In this case, the experiment IDs that satisfy the above requirements are ID2 to ID5, so the lower limit and upper limit of the calcium addition amount that satisfy the above requirements are b2 and b5, respectively. The minimum value (e.g., c5) and the maximum value (e.g., c2) of the swelling ratios c2, c3, c4, and c5 corresponding to the calcium addition amounts b2 to b5 can be set as the lower and upper limits of the acceptable range of swelling ratios, respectively, to set the acceptable range of swelling ratios c5 to c2.

[0057] The processing unit 11 stores the acquired experimental data and the specified acceptable range of the swelling ratio in the storage unit 12 (step S14), and ends the series of processes.

[0058] In the above process, the estimation device 1 may receive requirements for cell culture from a culturist via the terminal device 2. The requirements for cell culture may be set for each culturist or each type of user culture solution for which the recommended addition amount range is to be estimated. In addition, the acceptable range of the swelling ratio may be set for each type of cell based on experimental data for each type of cell.

[0059] 5 is a flowchart showing an example of a processing procedure for estimating the recommended calcium addition amount range. The following processing is executed by the processing unit 11 in accordance with a program 1P stored in the storage unit 12 of the estimation device 1, and by the processing unit 21 in accordance with a program 2P stored in the storage unit 22 of the terminal device 2.

[0060] The processing unit 21 of the terminal device 2 acquires a plurality of pieces of data relating to a swelling experiment using a user culture solution, in which the amount of calcium added to the user culture solution corresponds to the swelling rate of the dry microcarriers relative to the amount of calcium added (step S21). The user varies the amount of calcium added to the user culture solution and measures the swelling rate of the dry microcarriers swollen in the user culture solution at multiple points to generate a plurality of pieces of experimental data. The processing unit 21 acquires the amount of calcium added and the swelling rate by, for example, accepting input from the user.

[0061] The processing unit 21 associates the acquired multiple amounts of calcium added with the swelling ratio of the dry microcarriers for each amount of calcium added, and transmits them to the estimation device 1 (step S22). The data such as the amount of calcium added may be associated with identification information for identifying the terminal device 2 or the user.

[0062] The processing unit 11 of the estimation device 1 receives a plurality of amounts of calcium to be added and the swelling ratio of the dry microcarriers corresponding to each amount of calcium to be added, in association with each other (step S23). Note that the estimation device 1 may also receive the amount of additive liquid added to the user's culture solution, and calculate the amount of calcium to be added in the user's culture solution based on the received amount of additive liquid.

[0063] The processing unit 11 derives correlation information indicating the correlation between the amount of calcium added and the swelling rate for the user culture solution based on the received multiple amounts of calcium added and the swelling rate (step S24).

[0064] In this embodiment, the correlation information used is a prediction model (prediction function) that has been machine-learned to determine the correlation between the amount of calcium added to a user's culture solution and the swelling rate of dried microcarriers caused by the user's culture solution. The machine learning algorithm is not particularly limited, but Gaussian process regression may be used, for example, and is preferably an algorithm using a kernel function. Examples of kernel functions include the RBF (Radial Basis Function) kernel, Matern kernel, ExpSineSquared kernel, and DotProduct kernel, with the Matern kernel being preferred. When using the Matern kernel, it is preferable to set the number of differentiable fractions to 0. Furthermore, in order to take into account the influence of measurement errors, adding a White kernel is preferable, as this can further improve prediction accuracy.

[0065] Furthermore, the processing unit 11 calculates the predictive distribution of output data for new input data. In a prediction model using a Gaussian process, Bayesian estimation is used, and therefore the function estimated in Gaussian process regression is obtained as a distribution of functions rather than a single function. In calculating the predictive distribution using Bayesian estimation, it is preferable to use a Gaussian distribution (normal distribution) with a mean of 0 and a variance of 1 as the prior distribution. The numerical value of the confidence interval used to derive the predictive distribution can be set appropriately. The method for calculating the predictive distribution is not limited to Bayesian estimation using a Gaussian process, and other methods may also be used.

[0066] The prediction model may be any model that can predict the swelling ratio relative to the amount of added calcium. The prediction model may be a model based on other machine learning algorithms such as linear regression, neural networks, support vector machines, and decision trees. The prediction model is not limited to machine learning models and may be a rule-based model.

[0067] Figure 6 is a diagram showing an example of a prediction function that shows the correlation between the amount of calcium added to a user's culture solution and the swelling rate. The horizontal axis of the graph shown in Figure 6 is the amount of calcium added to the culture solution (mmol / L), and the vertical axis is the swelling rate (%). In Figure 6, dots represent actual measured values ​​from swelling experiments, the solid line represents the prediction function, and the band-like area above and below the solid line represents the standard deviation in the prediction function.

[0068] Returning to FIG. 5, the processing unit 11 estimates the range of calcium addition amounts corresponding to the acceptable range of swelling rates based on the derived correlation information (prediction model) between the amount of calcium addition and the swelling rate for the user's culture solution and a predetermined acceptable range of swelling rates (step S25). The range of calcium addition amounts estimated in step S25 corresponds to the recommended range of calcium addition amounts for the user's culture solution. When a predicted distribution of the prediction model has been obtained, the processing unit 11 preferably specifies the upper and lower limits of the recommended range of calcium addition amounts, taking into account the confidence interval of the predicted distribution, as shown in FIG. 6. Note that when the acceptable range of swelling rates is set for each cell type, the processing unit 11 may read out the acceptable range of swelling rates corresponding to the cell type used by the user.

[0069] The processing unit 11 transmits the estimated recommended range of calcium addition amount to the terminal device 2 (step S26). The processing unit 11 may transmit the estimation result to the terminal device 2 corresponding to the identification information associated with the calcium addition amount and swelling rate acquired in step S23.

[0070] The processing unit 21 of the terminal device 2 receives the recommended range of calcium addition amounts for the user's culture solution (step S27). The processing unit 21 displays the received recommended range of calcium addition amounts on the display unit 24 (step S28), and ends the series of processes.

[0071] In the above-described processing, the estimation device 1 may acquire images of the dry microcarriers before and after swelling from the terminal device 2, instead of or in addition to the swelling ratio of the dry microcarriers. The images of the dry microcarriers are preferably associated with the imaging magnification, imaging conditions, scale bar, etc., used when the images were captured. When images are acquired, the estimation device 1 determines the average particle diameter of the dry microcarriers before and after swelling based on each acquired image, and calculates the swelling ratio using the determined average particle diameters before and after swelling.

[0072] The estimation result output by the estimation device 1 is not limited to the recommended calcium addition range, but may also be a recommended addition range of an additive solution containing calcium. The estimation device 1 calculates a recommended addition range of the additive solution corresponding to the recommended calcium addition range based on the estimated recommended calcium addition range and the additive solution composition. The estimation result may also be one or more recommended calcium addition amounts corresponding to the recommended calcium addition range. The estimation device 1 can identify one or more addition amounts within the estimated recommended calcium addition range, for example, according to a predetermined rule.

[0073] If there is no recommended amount of calcium to be added that corresponds to the acceptable range of swelling ratio, the estimation device 1 may generate, as an estimation result, information indicating that the user's culture solution is unsuitable for culture, or may generate information regarding components other than calcium to be added to the culture solution. Examples of components other than calcium include chelating agents. Adding a chelating agent to the culture solution captures calcium in the culture solution, thereby reducing the amount of calcium that reacts with the calcium alginate gel. Therefore, even if there is no recommended amount of calcium to be added that corresponds to the acceptable range of swelling ratio, suitable culture conditions can be presented. Specific examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), glycol ether diaminetetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), and nitrilotriacetic acid (NTA), with EDTA being preferred. When chelating agent addition information is generated as the estimation result, a new additive containing the chelating agent may be provided to the user in correspondence with the output of the estimation result.

[0074] The processing entity for each process in the above-described flowchart is not limited. Some or all of the processes executed by the estimation device 1 may be executed by, for example, the terminal device 2. When the terminal device 2 executes the process for deriving the recommended range of calcium addition amount, the estimation device 1 may transmit data indicating the acceptable range of the swelling ratio to the terminal device 2 in advance.

[0075] The estimation device 1 may acquire the swelling information without going through the terminal device 2. For example, the estimation device 1 may acquire the swelling information by accepting an input operation of the swelling information from a provider of dried microcarriers. The estimation results may also be output to a device other than the terminal device 2. For example, the estimation device 1 may output the estimation results via a display unit of its own device, or may output the estimation results to another computer, a specified printing device, or the like.

[0076] The estimation device 1 may also perform processing that takes into account the influence of measurement error when estimating the acceptable range of the swelling ratio. For example, the estimation device 1 performs processing similar to step S24 of FIG. 5 based on experimental data on the model culture solution to obtain a prediction model and a prediction distribution that indicate the correlation between the amount of calcium added and the swelling ratio for the model culture solution. Taking into account the confidence interval of the prediction distribution, the estimation device 1 estimates the acceptable range of the swelling ratio based on the obtained prediction model and the range of the amount of calcium added that corresponds to the acceptable range of the swelling ratio estimated in step S13. In this case, the upper and lower limits of the swelling ratio are set to correspond to the lower and upper limits of the amount of calcium added, taking into account the width of the predetermined confidence interval. By taking into account the measurement error, an acceptable range of the swelling ratio that is more suitable for estimation can be set.

[0077] 5, the estimation device 1 may derive an optimal value for the recommended amount of calcium to be added from the obtained range of recommended amounts of calcium to be added. For example, the user may conduct a cell culture experiment using a user culture solution to which calcium has been added so that the amount falls within the range of recommended amounts of calcium to be added displayed in step S28, and collect experimental data. The experimental data may include the amount of calcium to be added in the user culture solution, and an evaluation of the stability of the microcarriers and the cell culture under the conditions of the amount of calcium to be added. The estimation device 1 acquires the experimental data via the terminal device 2.

[0078] The estimation device 1 optimizes the calcium addition amount based on the obtained experimental data, for example, to maximize the proliferation rate of cell culture. The estimation device 1 may optimize the calcium addition amount using a known optimization algorithm. Examples of optimization algorithms include random search, grid search, simulated annealing, genetic algorithms, and Bayesian optimization. Among these, Bayesian optimization is preferred from the perspective of reducing the amount of newly required data. When using Bayesian optimization, a kernel function is preferred as the Gaussian process model function. Examples of kernel functions include the RBF kernel, Matern kernel, ExpSineSquared kernel, and DotProduct kernel. The estimation device 1 outputs the calculated optimal calcium addition amount to the terminal device 2. This eliminates the need to determine a specific calcium addition amount from within the recommended calcium addition amount range, allowing cell culture to be carried out efficiently under more appropriate conditions.

[0079] According to this embodiment, by acquiring experimental data for a model culture solution in advance, it is possible to easily estimate the amount of calcium to be added that is appropriate for various user culture solutions with different compositions. Using the correlation between the amount of calcium to be added in the culture solution and the swelling rate of dry microcarriers, it is possible to efficiently and accurately identify a suitable range of the amount of calcium to be added. This eliminates the need to perform actual cell culture using the user culture solution, and allows the suitable range of the amount of calcium to be estimated using the swelling rate of dry microcarriers, which is relatively easy to measure. This makes it easier to optimize the amount of calcium to be added, and reduces the time and cost required for optimizing the amount of calcium to be added.

[0080] By optimizing the amount of calcium added to the user's culture solution, it becomes unnecessary to analyze the detailed components of the user's culture solution, making optimization easier. By configuring the culturer to provide swelling information, it becomes unnecessary to provide information about the user's culture solution to the estimation device 1, improving the confidentiality of the user's culture solution.

[0081] By considering the stability of the dry microcarriers and the evaluation of cell culture, it is possible to estimate the appropriate range of calcium addition that will not cause the dry microcarriers to become unstable and will achieve suitable cell growth, allowing culture to proceed with a high success rate.

[0082] Using a kernel function allows for accurate prediction of the swelling rate relative to the amount of calcium added. Using the Matern kernel, where X is the amount of calcium added and Y is the swelling rate, prediction accuracy can be improved in cases where Y decays significantly near X = 0 and tends to decay more slowly as the value of X increases. Calculating the predictive distribution through Bayesian estimation using a Gaussian process allows for accurate estimation of the recommended range of calcium addition amounts, taking into account confidence intervals.

[0083] (Second embodiment) In the second embodiment, the details of the method for generating correlation information related to the user culture solution are different from those in the first embodiment. The following mainly describes the differences from the first embodiment, and the same reference numerals are used to designate the same components as in the first embodiment, and detailed descriptions thereof will be omitted.

[0084] Fig. 7 is a flowchart showing an example of a processing procedure for estimating the recommended range of calcium addition amounts in the second embodiment. In Fig. 7, steps that perform the same processing as the steps shown in Fig. 5 are assigned the same step numbers as in Fig. 5.

[0085] The processing unit 21 of the terminal device 2 and the processing unit 11 of the estimation device 1 execute the same processes as steps S21 to S23 in FIG. 5. While the following describes an example in which data on calcium addition amounts and swelling rates for multiple points related to a user culture solution is used, the data on calcium addition amounts and swelling rates used to generate the correlation information in the second embodiment may be at least one point. In the second embodiment, in steps S21 to S23, one or more calcium addition amounts and swelling rates are acquired, transmitted, and received. The calcium addition amount used in the swelling experiment is preferably selected so as to match one of the calcium addition amounts in the experimental data for the model culture solution stored in the model DB 121 in advance. If matching experimental data for the model culture solution is not available, new experimental data for the model culture solution related to the calcium addition amount matching the calcium addition amount used in the swelling experiment may be generated. The experimental data for the model culture solution may be actual measurements or predicted values ​​(e.g., values ​​obtained from a prediction model showing the correlation between the calcium addition amount and swelling rate for the model culture solution described in the first embodiment).

[0086] The processing unit 11 of the estimation device 1 calculates a correction coefficient for correcting the swelling rate of the model culture solution based on a comparison between the swelling rate of the user culture solution and the swelling rate of the model culture solution at the same calcium addition amount (step S31). The processing unit 11, for example, calculates the difference between the swelling rate of the model culture solution and the swelling rate of the user culture solution for each calcium addition amount for which data was obtained, and sets the average value of the calculated differences as the correction coefficient. The processing unit 11 may also determine a function formula for correcting the swelling rate based on the comparison of the swelling rates. The processing unit 11 may also calculate the correction coefficient based on a comparison between the swelling rate of the user culture solution and the swelling rate of the model culture solution for approximately the same or similar calcium addition amounts.

[0087] The processing unit 11 uses the calculated correction coefficients to correct the swelling ratios corresponding to the amounts of calcium added for the model culture solution stored in the model DB 121 (step S32). The processing unit 11 regards each swelling ratio obtained by correction as a predicted swelling ratio for the user culture solution, and associates it with each amount of calcium added for the model culture solution or the user culture solution.

[0088] The processing unit 11 executes the same process as step S24 in FIG. 5, and derives correlation information indicating the correlation between the calcium addition amount and the predicted swelling rate for the user culture solution based on each calcium addition amount and the predicted swelling rate associated with each calcium addition amount (step S33).

[0089] Thereafter, the processing unit 11 of the estimation device 1 and the processing unit 21 of the terminal device 2 execute the same processes as steps S25 to S28 in Fig. 5. In step S25, the processing unit 11 estimates the range of the amount of calcium to be added that corresponds to the acceptable range of the swelling rate, based on the derived prediction model of the amount of calcium to be added and the predicted swelling rate for the user culture solution and a predetermined acceptable range of the swelling rate. The acceptable range of the swelling rate may be obtained by processing that takes into account the influence of the measurement error described above.

[0090] According to this embodiment, estimation is possible using swelling information from at least one point, which reduces the burden of swelling experiments.

[0091] (Third embodiment) The third embodiment differs from the first embodiment in that the amount of sodium added to the solvent is estimated in addition to the amount of calcium added. The following mainly describes the differences from the first embodiment, and the same reference numerals are used to designate the same components as the first embodiment, and detailed descriptions thereof will be omitted.

[0092] In the third embodiment, a swelling liquid different from the culture medium used for cell culture is used as a solvent. The swelling liquid is another example of the first solvent used to swell the first solvent-based dry microcarriers. Calcium and sodium are added to the swelling liquid. By using a liquid containing sodium in addition to calcium to swell the dry microcarriers, the calcium alginate portion of the dry microcarriers, which is difficult to swell, is loosened to a degree that prevents ion exchange reaction with calcium ionically cross-linked to alginic acid by sodium ions, allowing the calcium alginate as a whole to swell satisfactorily. The dry microcarriers are swelled with such a swelling liquid, and after swelling of the dry microcarriers, the swelling liquid is removed. Cell culture can then be carried out using the desired culture medium.

[0093] The swelling liquid may contain at least water, and may contain at least one of calcium and sodium in advance.

[0094] 8 is a flowchart showing an example of a process procedure for estimating the recommended range of calcium and the recommended range of sodium addition amounts in the third embodiment. The recommended range of sodium addition amount is defined by the upper and lower limits of the recommended amount of sodium to be added to the swelling liquid.

[0095] The processing unit 21 of the terminal device 2 acquires a plurality of amounts of calcium and sodium added in a swelling liquid for a swelling experiment using the swelling liquid, and the swelling ratio of the dry microcarriers for each amount of calcium and sodium added, in association with each other (step S41). The processing unit 21 then associates the acquired plurality of amounts of calcium and sodium added with the swelling ratio of the dry microcarriers for each amount of calcium and sodium added, and transmits them to the estimation device 1 (step S42). The swelling experiment may be performed under conditions of zero sodium addition, i.e., using a swelling liquid with no added sodium.

[0096] The processing unit 11 of the estimation device 1 receives a plurality of calcium and sodium addition amounts and the swelling ratios of the dry microcarriers corresponding to the respective calcium and sodium addition amounts in association with each other (step S43). The processing unit 11 derives correlation information indicating the correlation between the calcium and sodium addition amounts for the swelling solution and the swelling ratio based on the received plurality of calcium and sodium addition amounts and the swelling ratios (step S44). In step S44, the processing unit 11 executes a process similar to step S24 of Fig. 5 to derive a prediction function using the calcium and sodium addition amounts as explanatory variables and the swelling ratio as a response variable.

[0097] The processing unit 11 estimates the range of calcium and sodium addition amounts corresponding to the acceptable range of swelling rate based on the derived correlation information between the amounts of calcium and sodium addition and the swelling rate for the swelling solution and a predetermined acceptable range of swelling rate (step S45). The range of calcium and sodium addition amounts estimated in step S45 corresponds to the recommended range of calcium and sodium addition amounts, respectively. In step S45, the recommended range of sodium addition amounts may be set in advance taking into account the effect of osmotic pressure on cells. The recommended range of sodium addition amounts may be, for example, 150 mmol / L to 160 mmol / L. The processing unit 11 can estimate the recommended range of calcium and sodium addition amounts corresponding to the acceptable range of swelling rate and the recommended range of sodium addition amounts.

[0098] The processing unit 11 transmits the recommended range of calcium and the recommended range of sodium to be added to the swelling liquid to the terminal device 2 (step S46).

[0099] The processing unit 21 of the terminal device 2 receives the recommended range of calcium and sodium to be added to the swelling liquid (step S47), and displays them on the display unit 24 (step S48), thereby completing the series of processes.

[0100] The target for estimating the recommended ranges of calcium and sodium addition amounts is not limited to the swelling solution, but may be a culture solution. The processing unit 11 may identify one or more recommended amounts of calcium and sodium addition according to the recommended ranges of calcium and sodium addition amounts.

[0101] According to this embodiment, it is possible to estimate the optimum range of the amount of sodium to be added in addition to the amount of calcium to be added, which increases the flexibility of the solvent used to swell the dry microcarriers. Instead of a culture medium containing expensive ingredients such as amino acids, vitamins, and proteins, a swelling solution composed of inexpensive ingredients such as sodium and calcium can be used, which improves economy. [Example]

[0102] Examples of the present invention will be specifically described below, but it is not intended that the present invention be limited to these examples.

[0103] [Example 1] <Setting the acceptable range for swelling ratio in a model case using a model culture medium> α-MEM (Nacalai Tesque, 21444-05) was prepared and adjusted to 10% FBS (GIBCO, 26140-079) and 20 ng / mL basic fibroblast growth factor (b-FGF) (hereafter referred to as basal culture medium). Furthermore, 500 mM calcium chloride solution (CaCl2 water) was prepared and added to the basal culture medium in 1 / 800, 1 / 400, 1 / 200, 1 / 100, 1 / 50, 1 / 25, 1 / 20, 1 / 15, 1 / 12.5, 1 / 6.25, and 1 / 3.125 volumes to prepare 11 culture medium conditions. Basal culture medium without calcium chloride solution was also prepared.

[0104] For each of the prepared culture solutions, swelling ratio measurements were performed using dried microcarriers containing calcium alginate and gelatin as a cell adhesive substance, obtained according to the method described in Japanese Patent No. 7281116, and a 24-well plate (Sumitomo Bakelite Co., Ltd., MS-90240) treated to reduce cell adsorption. Specifically, 2 mg of the dried microcarriers and 1 mL of the culture solution prepared under one of the conditions were placed in one well of the 24-well plate, and the plate was incubated at 37°C and 5% CO2 for at least 2 hours to allow the dried microcarriers to swell (this swelling was performed for all conditions).

[0105] The mean particle size was then determined from the phase contrast images of the swollen microcarriers under each condition, with the average equivalent sphere diameter of 80 or more swollen microcarriers measured. The value obtained from this measurement was taken as the mean particle size of the swollen microcarriers. The dried microcarriers were then placed in a 24-well plate (Sumitomo Bakelite Co., Ltd., MS-90240), and the mean equivalent sphere diameter of 80 or more dried microcarriers measured from the phase contrast images. The value obtained from this measurement was taken as the mean particle size of the dried microcarriers. The mean particle size of the dried microcarriers was 91 μm. From these data, the swelling ratio for each condition was calculated using the above-mentioned formula. When measuring each microcarrier, the bottom area of ​​the container and the amount of each microcarrier placed were adjusted to ensure that the microcarriers could be observed without overlapping.

[0106] After calculating the swelling rate, 5.6 × 10 human adipose-derived stem cells (manufacturer: Lonza Co., Ltd., PT-5006) were added to each well. 4 After seeding with cells, the total culture medium volume per well was adjusted to 2 mL, and the cells were cultured for 7 days at 37°C and a CO2 concentration of 5%. On days 1, 2, 3, 6, and 7 of culture, the suspension containing the cells and microcarriers in the well was gently pipetted using a 1 mL pipette.

[0107] After 7 days of culture, the microcarriers were observed under phase contrast and their state was confirmed, and stability was evaluated according to the following criteria. Figure 9 shows phase contrast microscope images of the microcarriers after swelling for each amount of calcium added. The measurement results are shown in Table 1. (Evaluation criteria) ○: Approximately circular and not collapsed ×: Not roughly circular, partially collapsed

[0108] For #2 to #12 in Table 1, for which the microcarrier condition was rated as "good," live cells were stained with calcein-AM solution (Nacalai Tesque, 19177-14) and photographed under a fluorescence microscope to evaluate the cells according to the following criteria: Figure 10 shows fluorescence microscope images of calcein-AM stained cells for each amount of calcium added. (Evaluation criteria) ○: Cell adhesion was observed, and cell proliferation was confirmed to cover the microcarrier surface. ×: Cell adhesion is observed, but cell proliferation is not confirmed to cover the microcarrier surface.

[0109] For unstained wells #2 to #12, the culture supernatant was removed from each well, and the microcarriers containing the attached cells were washed with 1 mL of PBS. The PBS supernatant was then removed again, and 0.5 mL of TrypLE (GIBCO, 12563-029) was added. The microcarriers were dissolved at 37°C for 5 minutes, and the cells were recovered. The recovered cells were added to a total volume of 1 mL of basal culture medium to prepare a cell suspension. The viable cell count for this cell suspension was determined using a NucleoCounter NC-202 (ChemoMetec, 970-2020) under the conditions: Count & Viability. The initial seeding number of viable cells was 5.6 x 10 4 The proliferation rate was calculated for each cell.

[0110] 9 and 10, it was found that when the swelling ratio was in the range of 214 to 259%, the microcarriers maintained their approximately circular shape, were stable without collapse, and the cells proliferated to such an extent that they covered the microcarrier surface, achieving a proliferation rate of more than 1x the initial seeding number of live cells, enabling favorable cell proliferation. Based on this, the upper limit of the acceptable range for the swelling ratio was determined to be 259%, and the lower limit to be 214%.

[0111] [Table 1]

[0112] <Measurement of swelling rate of any culture medium> A different culture medium from that used in the model case was used: PRIME-XV MSC XSFM MDF1 (Fujifilm Wako Pure Chemical Industries, Ltd., 991333-1L). Furthermore, a 500 mM calcium chloride solution was prepared. Two culture medium conditions were prepared: one without the solution and one with 1 / 100 the amount added to the basal culture medium.

[0113] For each culture medium, dried microcarriers containing calcium alginate and gelatin as a cell adhesive, prepared according to the method described in Japanese Patent Publication No. 7281116, and a 24-well plate (Sumitomo Bakelite Co., Ltd., MS-90240) treated for low cell adsorption were used to prepare for swelling ratio measurements. (This swelling was performed for all conditions.) Then, the mean particle size was determined from the phase contrast images for each condition, and the swelling ratio was calculated, in the same manner as for setting the acceptable range for swelling ratio. The measurement results are shown in Table 2.

[0114] [Table 2]

[0115] <Estimating the optimal range of calcium content> Based on Table 2, a prediction function was estimated using a posterior distribution with the explanatory variable X representing the amount of calcium added to the culture medium (mmol / L) and the response variable Y representing the swelling ratio (%). The kernel function Matern was selected as the model for the prediction function (the number of differentiable functions was set to 0). To account for the influence of measurement error, a White kernel was added to the function. Furthermore, the prediction distribution was calculated by Bayesian estimation using a Gaussian process with a confidence interval set to 95%. A Gaussian distribution (normal distribution) with a mean of 0 and a variance of 1 was selected as the prior distribution used for Bayesian estimation. Figure 11 shows the prediction function indicating the correspondence between the amount of calcium added and the swelling ratio in Example 1. The horizontal axis of the graph in Figure 11 represents the amount of calcium added to the culture medium (mmol / L), and the vertical axis represents the swelling ratio (%). In Figure 11, dots represent actual measurements, the solid line represents the prediction function, and the bands above and below the solid line represent the 95% confidence interval.

[0116] Considering the confidence interval, X corresponding to the set acceptable range of swelling rate (214% to 259%) is 1.318 mmol / L to 5.548 mmol / L (the hatched area in Figure 11). Therefore, it was estimated that the recommended range of calcium addition as an optimal condition is 1.318 mmol / L to 5.548 mmol / L.

[0117] <Cultivation under estimated optimal conditions> The basal culture medium used in the above estimation was PRIME-XV MSC XSFM MDF1 (Fujifilm Wako Pure Chemical Industries, Ltd., 991333-1L). Furthermore, a 500 mM calcium chloride solution was prepared and added to the basal culture medium at a volume of 1 / 200. The calcium content of the resulting culture medium was 2.5 mmol / L, which is within the estimated recommended range.

[0118] Using the above culture medium, 80 mg of dry microcarriers containing calcium alginate and gelatin as a cell adhesive material, obtained according to the method described in Japanese Patent No. 7281116, were swelled, and 6 × 10 human adipose-derived stem cells (manufacturer: Lonza Co., Ltd., PT-5006) were cultured. 5The cells were placed in a spinner flask (Corning, 3152) with the cells, and the culture medium was adjusted to 40 mL (cell seeding concentration: 15,000 cells / mL). The culture was cultured at 37°C and 5% CO2 with intermittent agitation for 1 day and continuous agitation for 3 days (total of 4 days of culture). After culture, the culture was sampled and the viable cells were counted using a NucleoCounter NC-202 (ChemoMetec, 970-2020) and Lysis1 Reagent (ChemoMetec, 910-0010) under Microcarriers conditions. The viable cell count was 242,500 cells / mL, indicating proliferation of approximately 16.2 times the cell seeding concentration.

[0119] Furthermore, a sample of culture medium taken separately from the above counts was stained for live cells using calcein-AM solution (Nacalai Tesque, 19177-14) and photographed under a fluorescence microscope. It was found that the microcarriers maintained a roughly circular shape, were stable without collapse, and had proliferated to the point where they covered the microcarrier surface. Figure 12 shows a fluorescence microscope image of cells stained with calcein-AM. These results demonstrate that by using the estimated recommended dosage range, dry microcarriers containing calcium alginate can be maintained without becoming unstable and favorable cell proliferation can be achieved.

[0120] [Example 2] <Setting the acceptable range for swelling ratio taking into account measurement error> Based on Table 1, and in the same manner as in Example 1, a prediction function was estimated for a model case using a posterior distribution in which the explanatory variable X was the amount of calcium added to the culture medium (mmol / L) and the response variable Y was the swelling ratio (%), and a prediction distribution was obtained. Figure 13 shows a prediction function indicating the correspondence between the amount of calcium added and the swelling ratio for the model case. The horizontal axis of the graph shown in Figure 13 is the amount of calcium added to the culture medium (mmol / L), and the vertical axis is the swelling ratio (%). In Figure 13, dots indicate actual measurements, the solid line indicates the prediction function, and the band-like area above and below the solid line indicates the 95% confidence interval.

[0121] The Y corresponding to the acceptance range of X (upper limit: 33.33 mmol / L, lower limit: 0.625 mmol / L) corresponding to the swelling rate determined in the setting of the acceptance range of the swelling rate in the model case was 218% to 256% (hatched area in Figure 13) taking into account the confidence interval. Therefore, the above range was adopted as the acceptance range taking into account the measurement error.

[0122] <Estimating the optimal range of calcium content> As shown in Table 3, δ was calculated using some of the model case swelling rates (A) shown in Table 1 (calcium content = 0 mmol / L, 5 mmol / L) and the swelling rates (B) of the optional culture medium shown in Table 2, and the average value of these δ was used as a correction coefficient. The correction coefficient was -8%. The predicted swelling rate (B') was calculated by adding the correction coefficient -8 to each of (A).

[0123] [Table 3]

[0124] Based on Table 3, a prediction function was estimated using a posterior distribution in which the explanatory variable X was the amount of calcium added to the culture medium (mmol / L) and the response variable Y was the predicted swelling ratio (%), as in Example 1, to determine the predicted distribution. FIG. 14 shows a prediction function indicating the correspondence between the amount of calcium added and the swelling ratio in Example 2. The horizontal axis of the graph shown in FIG. 14 represents the amount of calcium added to the culture medium (mmol / L), and the vertical axis represents the predicted swelling ratio (%). In FIG. 14, dots represent actual measurements, the solid line represents the prediction function, and the band-like area above and below the solid line represents the 95% confidence interval.

[0125] The X corresponding to the acceptable range of the swelling rate (218% to 256%), which was set taking into account measurement error, was 0.536 mmol / L to 7.376 mmol / L (the hatched area in Figure 14) taking into account the confidence interval. Therefore, the recommended range of calcium to be added to the culture solution under optimal conditions was estimated to be 0.536 mmol / L to 7.376 mmol / L. It was found that a wider range of recommended addition amounts could be estimated than the recommended addition range of 1.318 mmol / L to 5.548 mmol / L predicted in Example 1.

[0126] [Example 3] <Measurement of swelling ratio of swelling liquid> Swelling solutions were prepared by adding calcium chloride and sodium chloride to water according to the compositions shown in Table 4. For each swelling solution, dry microcarriers containing calcium alginate and gelatin as a cell adhesive substance, obtained according to the method described in Japanese Patent Publication No. 7281116, and a 24-well plate (Sumitomo Bakelite Co., Ltd., MS-90240) treated to reduce cell adsorption were used. Preparation for swelling ratio measurement was carried out in the same manner as in Example 1, and the dry microcarriers were allowed to swell (this swelling was carried out under all conditions). Then, as in Example 1, the average particle size was determined from the phase contrast images under each condition, and the swelling ratio was calculated. The measurement results are shown in Table 4.

[0127] [Table 4]

[0128] Based on Table 4, a prediction function was estimated using a posterior distribution in which explanatory variable X1 was the amount of sodium added to water (mmol / L), explanatory variable X2 was the amount of calcium added to water (mmol / L), and objective variable Y was the swelling ratio (%), as in Example 1, to obtain a predicted distribution. Figure 15 shows a prediction function indicating the correspondence between the amount of sodium added and the amount of calcium added and the swelling ratio in Example 3. The horizontal axis of the graph shown in Figure 15 represents the amount of sodium added to water (mmol / L), and the vertical axis represents the amount of calcium added to water (mmol / L), with the swelling ratio (%) represented by color. In Figure 15, dots represent actual measured values.

[0129] The amount of calcium added was determined in advance to be 2.5 mmol / L. Using the predicted swelling ratio of 242% corresponding to this calcium addition amount from Table 3, X1 and X2 corresponding to the predicted swelling ratio of 242% were calculated, taking into account the confidence interval. In Figure 15, the shaded area within the dashed line represents the calculated results of X1 and X2. To minimize damage to cells due to osmotic pressure, it is preferable to select the 150 mmol / L to 160 mmol / L range for X1 within the above range. For example, if saline (9 g / L sodium chloride solution) to which calcium chloride is added is used as the swelling solution, X1 = 154.00 mmol / L, as shown in Figure 15, and X2 will be 2.83 mmol / L to 8.12 mmol / L or 10.00 mmol / L to 10.48 mmol / L. Therefore, it was found that the recommended range of sodium to be added to the swelling liquid (water) under suitable conditions is 150 mmol / L to 160 mmol / L, and the recommended range of calcium to be added is 2.83 mmol / L to 8.12 mmol / L and 10.00 mmol / L to 10.48 mmol / L.

[0130] Thus, it was demonstrated that the acceptable range of swelling ratio can be used to estimate the amounts of sodium and calcium to be added to a medium suitable for cell culture.

[0131] The following additional notes are provided regarding the above-described embodiments. (Appendix 1) Acquire the amount of calcium added to a first solvent used to swell dry microcarriers containing calcium alginate and swelling information indicating the swelling state of the dry microcarriers swollen with the first solvent; Based on the acquired amount of calcium to be added and the swelling information, a recommended amount of calcium to be added to the first solvent is estimated so that the stability of the dry microcarriers after swelling with the first solvent and evaluation of cell culture using the dry microcarriers satisfy predetermined requirements; Output information about the estimated recommended amount of calcium to be added A method of estimating that the processing is performed by a computer. (Appendix 2) Deriving correlation information between the amount of calcium added and the swelling rate of the dry microcarriers according to the amount of calcium added and the swelling rate of the first solvent; Based on the derived correlation information regarding the first solvent, the stability of the dry microcarriers after swelling with a second solvent, and the swelling ratio range of the dry microcarriers that satisfies predetermined requirements in evaluation of cell culture using the dry microcarriers, a recommended amount of calcium to be added is estimated so that the swelling ratio regarding the first solvent falls within the swelling ratio range. Estimation method described in Appendix 1. (Appendix 3) The swelling ratio range is set based on the swelling ratio of the dry microcarriers swollen with each second solvent containing different amounts of calcium, the stability of each dry microcarrier after swelling with each second solvent, and evaluation of cell culture using each dry microcarrier. Estimation method described in Appendix 2. (Appendix 4) For each of the first solvents having different amounts of added calcium, the amount of added calcium and a swelling ratio corresponding to the swelling information for each amount of added calcium are obtained; The correlation information is derived based on the acquired multiple amounts of calcium added and swelling ratios. The estimation method described in Appendix 2 or Appendix 3. (Appendix 5) For each of the second solvents having different amounts of added calcium, the amount of added calcium and a swelling ratio corresponding to the swelling information for each amount of added calcium are obtained; correcting the acquired swelling ratio for the second solvent based on a comparison between the swelling ratio for the first solvent and the swelling ratio for the second solvent; The correlation information is derived based on the amount of calcium added and the corrected swelling ratio for the second solvent. 1. An estimation method according to any one of Supplementary Note 2 to Supplementary Note 4. (Appendix 6) Gaussian process regression is used to derive the correlation information. 1. An estimation method according to any one of Appendix 2 to Appendix 5. (Appendix 7) estimating a range of recommended amounts of calcium to be added to the first solvent based on the amount of calcium added and the swelling information; A recommended amount of calcium to be added is determined by an optimization algorithm using the estimated amount of calcium to be added within the range of the recommended amount of calcium to be added and an evaluation of cell culture using the dried microcarriers swollen with the first solvent to which the amount of calcium to be added has been applied. 1. An estimation method according to any one of appendices 1 to 6. (Appendix 8) Further obtaining the amount of sodium added to the first solvent; Based on the acquired amount of sodium added, amount of calcium added, and swelling information, a recommended amount of calcium added and a recommended amount of sodium added to the first solvent are estimated, which will satisfy predetermined requirements for the stability of the dry microcarriers after swelling and for evaluation of cell culture using the dry microcarriers. 1. An estimation method according to any one of Appendix 1 to Appendix 7. (Appendix 9) Estimate the recommended amount of calcium to be added to achieve a cell growth rate of 1.1 times or more as an evaluation of the cell culture. 10. The estimation method according to any one of Appendix 1 to Appendix 8.

[0132] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The technical features described in each embodiment can be combined with each other, and the scope of the present invention is intended to include all modifications within the scope of the claims and the scope equivalent to the claims. The sequences shown in each embodiment are not limited, and the order of each process may be changed within a range consistent with the present invention, and multiple processes may be executed in parallel. The entity that performs each process is not limited, and the process of each device may be executed by another device within a range consistent with the present invention.

[0133] The matters described in each embodiment can be combined with each other. In addition, the independent claims and dependent claims described in the claims can be combined with each other in any combination, regardless of the reference format. Furthermore, although the claims use a format in which a claim references two or more other claims (multiple claim format), this is not limited to this format. A multiple claim (multi-multi claim) that references at least one other multiple claim may also be used. [Explanation of symbols]

[0134] 100 Estimation System 1 Estimation device 11 Processing section 12 Storage section 13 Communications Department 1P Program 1A Recording Media 2. Terminal Device 21 Processing section 22 Memory section 23 Communications Department 24 Display 25 Control section 2P Program 2A Recording Media

Claims

1. Acquire the amount of calcium added to a first solvent used to swell dry microcarriers containing calcium alginate and swelling information indicating the swelling state of the dry microcarriers swollen with the first solvent; Based on the acquired amount of calcium to be added and the swelling information, a recommended amount of calcium to be added to the first solvent is estimated so that the stability of the dry microcarriers after swelling with the first solvent and evaluation of cell culture using the dry microcarriers satisfy predetermined requirements; Output information about the estimated recommended amount of calcium to be added A method of estimating that processing is performed by a computer.

2. Deriving correlation information between the amount of calcium added and the swelling rate of the dry microcarriers according to the amount of calcium added and the swelling rate of the first solvent; Based on the derived correlation information regarding the first solvent, the stability of the dry microcarriers after swelling with a second solvent, and the swelling ratio range of the dry microcarriers that satisfies predetermined requirements in evaluation of cell culture using the dry microcarriers, a recommended amount of calcium to be added is estimated so that the swelling ratio in the first solvent falls within the swelling ratio range. The estimation method according to claim 1 .

3. The swelling ratio range is set based on the swelling ratio of the dry microcarriers swollen with each second solvent containing different amounts of calcium, the stability of each dry microcarrier after swelling with each second solvent, and evaluation of cell culture using each dry microcarrier. The estimation method according to claim 2 .

4. For each of the first solvents having different amounts of added calcium, the amount of added calcium and a swelling ratio corresponding to the swelling information for each amount of added calcium are acquired; The correlation information is derived based on the acquired multiple amounts of calcium added and swelling ratios. The estimation method according to claim 2 .

5. For each of the second solvents having different amounts of added calcium, the amount of added calcium and a swelling ratio corresponding to the swelling information for each amount of added calcium are acquired; correcting the acquired swelling ratio for the second solvent based on a comparison between the swelling ratio for the first solvent and the swelling ratio for the second solvent; The correlation information is derived based on the amount of calcium added and the corrected swelling ratio for the second solvent. The estimation method according to claim 2 .

6. Gaussian process regression is used to derive the correlation information. The estimation method according to claim 2 .

7. estimating a range of recommended amounts of calcium to be added to the first solvent based on the amount of calcium added and the swelling information; A recommended amount of calcium to be added is determined by an optimization algorithm using the estimated amount of calcium to be added within the range of the recommended amount of calcium to be added and an evaluation of cell culture using the dried microcarriers swollen with the first solvent to which the amount of calcium to be added has been applied. The estimation method according to claim 1 or 2.

8. Further obtaining the amount of sodium added to the first solvent; Based on the acquired amount of sodium added, amount of calcium added, and swelling information, a recommended amount of calcium added and a recommended amount of sodium added to the first solvent are estimated, which will satisfy predetermined requirements for the stability of the dry microcarriers after swelling and for evaluation of cell culture using the dry microcarriers. The estimation method according to claim 1 or 2.

9. The recommended amount of calcium to be added is estimated to achieve a cell growth rate of 1.1 times or more as an evaluation of the cell culture. The estimation method according to claim 1 or 2.

10. For each solvent containing different amounts of calcium, the swelling ratio of the dry microcarriers containing calcium alginate swollen in the solvent, the stability of the dry microcarriers after swelling in the solvent, and cell culture using the dry microcarriers were evaluated. Based on the swelling ratios for each solvent obtained and the evaluation of stability and cell culture, a swelling ratio range for the dry microcarriers that satisfies predetermined requirements for the evaluation of stability and cell culture is estimated. A method of estimating that processing is performed by a computer.

11. Acquire the amount of calcium added to a first solvent used to swell dry microcarriers containing calcium alginate and swelling information indicating the swelling state of the dry microcarriers swollen with the first solvent; Based on the acquired amount of calcium to be added and the swelling information, a recommended amount of calcium to be added to the first solvent is estimated so that the stability of the dry microcarriers after swelling with the first solvent and evaluation of cell culture using the dry microcarriers satisfy predetermined requirements; Output information about the estimated recommended amount of calcium to be added A computer program that causes a computer to perform a process.

12. Acquire the amount of calcium added to a first solvent used to swell dry microcarriers containing calcium alginate and swelling information indicating the swelling state of the dry microcarriers swollen with the first solvent; Based on the acquired amount of calcium to be added and the swelling information, a recommended amount of calcium to be added to the first solvent is estimated so that the stability of the dry microcarriers after swelling with the first solvent and evaluation of cell culture using the dry microcarriers satisfy predetermined requirements; Output information about the estimated recommended amount of calcium to be added A processing unit for executing processing is provided. Estimation system.

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