Patent Publication Number: US-2020291341-A1

Title: Pseudo sample creation method and apparatus, and numerical model creation system and method

Description:
TECHNICAL FIELD 
     The present invention relates to a pseudo sample creation method, a pseudo sample creation apparatus, a numerical model creation system, and a numerical model creation method. 
     BACKGROUND ART 
     As a method of grasping the progress status after culturing a sample such as cells, microorganisms, or fungi in a culture solution, a method of measuring a predetermined component (for example, a component derived from the sample or a component as a nutrient of the sample) in the culture solution by optical analysis is known. In the optical analysis, the culture solution including the sample is non-destructively, non-invasively, and instantaneously analyzable. Accordingly, the culture solution after the optical analysis is reusable, that is, culture of a sample in the culture solution after the optical analysis can be further carried out. Therefore, the optical analysis is widely used to grasp the progress status of culturing a sample using a culture solution. In order to measure a predetermined component in a culture solution using optical analysis, it is necessary to create a numerical model in advance. 
     The numerical model is a model representing a correlation between spectral data obtained by optical analysis and the concentration of a predetermined component in a culture solution. 
     In the related art, in order to create a numerical model, it is necessary to carryout the process of actually culturing a sample using a culture solution multiple times. That is, for the culture, a variation in the concentration of the above-described predetermined component is generated during multiple times of the culture processes. Therefore, by using data obtained during multiple times of the culture processes for creating a numerical model, a high-accuracy numerical model can be constructed in consideration of a variation in the concentration of a predetermined component in a culture solution. 
     As described above, in the related art, it is necessary to perform culture processes multiple times to create a numerical model, and massive human labor and time is required. 
     In order to reduce the human labor and time, for example, NPL 1 describes a method of creating a numerical model after adding glucose or the like as a material to be measured to a culture solution of sampled cells to increase the signal intensity of the material to be measured for the numerical model. 
     CITATION LIST 
     Non-Patent Literature 
     NPL 1: Jiang Qiu, et al., “On-line near infrared bioreactor monitoring of cell density and concentrations of glucose and lactate during insect cell cultivation”, Journal of Biotechnology 173, 106-111, 2014 
     SUMMARY OF INVENTION 
     Technical Problem 
     When the method described in NPL 1 is used for a culture solution in which the concentration of a component changes over time, it is necessary to carry out periodic sampling of a culture solution, mixing of a material to be measured, and optical measurement or the like multiple times. Therefore, human labor and time is still required. 
     The present invention has been made under these circumstances, and an object thereof is to reduce required human labor and time such that a high-accuracy numerical model can be created. 
     Solution to Problem 
     The present application includes a plurality of means for solving the above-described problem, and one example thereof is as follows. 
     In order to solve the above-described problems, according to one aspect of the present invention, there is provided a pseudo sample creation method including mixing a pre-culture liquid medium that is not used to culture a sample, a post-culture liquid medium that is used to culture the sample, and an additive with one another to create a pseudo sample. 
     Advantageous Effects of Invention 
     According to one aspect of the present invention, it is possible to reduce required human labor and time to create a high-accuracy numerical model. 
     Objects, configurations, and effects other than those described above will be clarified through the description of the following embodiments. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a diagram illustrating the summary of a pseudo sample creation method according to a first embodiment of the present invention. 
         FIG. 2  is a diagram illustrating the summary of the pseudo sample creation method according to the first embodiment of the invention. 
         FIG. 3  is a flowchart illustrating an example of the pseudo sample creation method. 
         FIG. 4  is a diagram illustrating a configuration example of a numerical model creation system according to a second embodiment of the invention. 
         FIG. 5  is a diagram illustrating a configuration example of a spectrometer. 
         FIG. 6  is a diagram illustrating an example of a shape of an analysis cell. 
         FIG. 7  is a diagram illustrating a configuration example of an analyzer. 
         FIG. 8  is a diagram illustrating an effect of a created pseudo sample. 
         FIG. 9  is a diagram illustrating a configuration example of a pseudo sample creation system according to a third embodiment of the invention. 
         FIG. 10  is a diagram illustrating a configuration example of a culturing device. 
         FIG. 11  is a diagram illustrating a configuration example of a mixing device. 
         FIG. 12  is a diagram illustrating a configuration example of an analysis cell introducing device. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Hereinafter, a plurality of embodiments of the present invention will be described with reference to the drawings. In all diagrams for describing each of the embodiments, basically, the same members are represented by the same reference numerals, and the description thereof will not be repeated. In addition, in the following embodiments, it goes without saying that the components (including element steps and the like) are not necessarily required, unless expressly stated otherwise and unless they are considered to be clearly required in principle or other reasons. In addition, when referring to “including A”, “composed of A”, “having A”, and “containing A”, it goes without saying that the elements other than the specified one should not be excluded, unless expressly stated the fact that there is only the particular element, or other reasons. Also, in the following embodiment, when referring to the shape, the positional relationship, or other characteristics of the components and the like, elements that substantially approximate or similar to the shape or other characteristics are included unless expressly stated otherwise and unless they are clearly considered not to be so in principle. 
     Summary of Pseudo Sample Creation Method According to First Embodiment of Present Invention 
       FIGS. 1 and 2  are diagrams illustrating the summary of a pseudo sample creation method according to a first embodiment of the present invention. 
     In the pseudo sample creation method according to the first embodiment of the invention, a pre-culture liquid medium  1 , a post-culture liquid medium  2 , and an additive  3  are mixed with one another to create a pseudo sample  4 . 
     &lt;Definitions of Pre-Culture Liquid Medium  1 , Post-Culture Liquid Medium  2 , and Additive  3 &gt; 
     The pre-culture liquid medium  1  is a liquid medium that is not used to culture a sample. The pre-culture liquid medium  1  does not contain saccharides and an amino acid. However, the pre-culture liquid medium  1  may contain a predetermined amount or less of at least one of saccharides and an amino acid. 
     The post-culture liquid medium  2  is a liquid medium that is obtained when culture of a sample such as cells, microorganisms, or fungi ends. The post-culture liquid medium  2  may remove the sample such as cells, microorganisms, or fungi from the liquid medium after culture by filtering or the like. Here, the time when the culture of the sample ends is the time when the concentration of a metabolite such as lactic acid produced by metabolic activity of the sample exceeds a predetermined threshold. The predetermined threshold is a value of the concentration of the metabolite in a state where the culture process is assumed to end and can be freely defined by a user. 
     The additive  3  contains at least one of saccharides, an amino acid, a metabolite, and protein. The additive  3  maybe in any form, for example, a solid such as powder or a tablet or a liquid such as a solution. 
     The pseudo sample  4  is a liquid that is created by mixing the pre-culture liquid medium  1 , the post-culture liquid medium  2 , and the additive  3 . 
     &lt;Tools Used to Create Pseudo Sample  4 &gt; 
     In order to create the pseudo sample  4 , a pipette for weighing each of the pre-culture liquid medium  1 , the post-culture liquid medium  2 , and the additive  3 , a stirring device such as a stirrer or a spatula for mixing the components, and a container for storing the pseudo sample  4  are used. 
     &lt;Method of Creating Pseudo Sample  4 &gt; 
       FIG. 3  is a flowchart illustrating an example of the method of creating the pseudo sample  4 . 
     First, a creator weighs the pre-culture liquid medium  1  by Va [ml] using the pipette (Step S 1 ). Next, the creator weighs the post-culture liquid medium  2  by Vb [ml] using the pipette (Step S 2 ). Next, the creator stores and mixes the weighed pre-culture liquid medium  1  and the weighed post-culture liquid medium  2  with each other in the container (Step S 3 ). 
     At this time, the liquid medium having a volume of Vc (=Va+Vb) [ml] is stored in the container. A proportion of each of the volume Va of the pre-culture liquid medium  1  and the volume Vb of the post-culture liquid medium  2  to the volume Vc may be a value in a range of 0% to 100% (including 0% and 100%). 
     Next, the creator creates the pseudo sample  4  by adding a predetermined amount of the additive  3  to the liquid medium in which the pre-culture liquid medium  1  and the post-culture liquid medium  2  are mixed with each other and sufficiently mixing the components with one another (Step S 4 ). During mixing, the stirring device such as a stirrer or a spatula may be used, or the container maybe covered with a lid and shaken by the creator. 
     Using the above-described creation method, a plurality of pseudo samples  4  having different mixing ratios among the pre-culture liquid medium  1 , the post-culture liquid medium  2 , and the additive  3  are created. 
     When the additive  3  is insoluble, the additive  3  that remains without being dissolved may be removed from the mixed pseudo sample  4  using a filter or the like. 
     Numerical Model Creation System  20  According to Second Embodiment of Present Invention 
     Next,  FIG. 4  illustrates a configuration example of a numerical model creation system  20  according to a second embodiment of the present invention. 
     The numerical model creation system  20  includes a spectrometer  21 , a reference value measuring device  22 , and an analyzer  23 . 
     The spectrometer  21  performs spectrometry of the pseudo sample  4  created by the creator and outputs spectral data obtained by the spectrometry to the analyzer  23 . 
     The reference value measuring device  22  is an existing device adopting a method other than spectrometry such as an enzyme reaction or chromatography. The reference value measuring device  22  measures the concentration of a predetermined component in the pseudo sample  4  created by the creator and outputs the result to the analyzer  23  as a reference value. 
     The analyzer  23  creates a numerical model representing a correlation between the spectral data and the reference value by multi-variable analysis based on the spectral data and the reference values obtained from the plurality of pseudo samples  4 . 
     Next,  FIG. 5  illustrates a configuration example of the spectrometer  21 . 
     The spectrometer  21  includes a control unit  211 , a light source unit  212 , a light receiving unit  213 , a holding unit  214 , and a fixing tool  215 . An XYZ coordinate system in the spectrometer  21  is as illustrated in the drawing, and the same is applied to the subsequent drawings. 
     The control unit  211  is configured with, for example, a computer including a CPU (Central Processing Unit), a memory, a storage, and a communication interface, and each function of the control unit  211  is implemented by the CPU executing a predetermined program. The control unit  211  controls emission of the light source unit  212  and creates spectral data based on a light receiving signal from the light receiving unit  213 . The controller  211  may transmit the light receiving signal from the light receiving unit  213  to the analyzer  23  without creating the spectral data such that the analyzer  23  creates the spectral data. In addition, the control unit  211  may also function as the analyzer  23  described below. 
     For example, the light source unit  212  emits light having a predetermined wavelength (for example, infrared light, near infrared light, visible light, ultraviolet light, or X-ray) to the pseudo sample  4  that is stored in an analysis cell  201  held by the holding unit  214 . The light receiving unit  213  is configured with, for example, a photomultiplier tube, a Si photodiode, an InGaAs photodiode, or a PbS photoconductive cell. The light receiving unit  213  receives transmitted light emitted from the light source unit  212  and transmitted through the pseudo sample  4 , and outputs a light receiving signal representing a light receiving effect to the control unit  211 . The light receiving unit  213  may be positioned where not only the transmitted light transmitted through the pseudo sample  4  but also light reflected or scattered from the pseudo sample  4  can be received. 
     The holding unit  214  is formed of, for example, a material such as plastic or metal that is not likely deformed. The holding unit  214  is held by and fixed to the analysis cell  201  in which the pseudo sample  4  is stored. 
     The fixing tool  215  maintains a relative positional relationship among the light source unit  212 , the light receiving unit  213 , and the holding unit  214 . 
     Next,  FIG. 6  illustrates an example of a shape of the analysis cell  201 . 
     The analysis cell  201  is a container for storing the pseudo sample  4 , and is formed to have at least one pair of planes facing each other. In the same drawing, an external shape and an internal space of the analysis cell  201  are formed of a rectangular parallelepiped. The analysis cell  201  is formed of a material that allows transmission of light emitted from the light source unit  212 , for example, fused silica or an acrylic resin. 
     On an upper surface of the analysis cell  201 , an introduction port  2011  for introducing the pseudo sample  4  is provided. In addition, on a lower surface of the analysis cell  201 , a discharge port (not illustrated) for discharging the pseudo sample  4  may be provided. 
     In addition, the entirety of the analysis cell  201  is put into a box or the like to block external light, and an opening for allowing the light emitted from the light source unit  212  to be incident into the box and an opening for allowing the transmitted light to exit may be provided. 
     The analysis cell  201  is reusable, and the pseudo sample  4  may be replaced multiple times for spectrometry. In addition, a plurality of the same analysis cells  201  may be prepared such that the analysis cell  201  to be used can be replaced at any timing. When the analysis cell  201  is reused, it is necessary to replace the pseudo sample  4 . However, labor required to replace the analysis cell  201  can be reduced. When a plurality of the same analysis cells  201  are used, a decrease in analysis accuracy caused by carry-over can be prevented. 
     Next, an example of a spectral data creation process by the spectrometer  21  will be described. 
     In the spectrometer  21 , background measurement is performed. In the background measurement, for example, a light receiving signal representing a state where the empty analysis cell  201  not storing the pseudo sample  4  is held by the holding unit  214  is acquired, or a light receiving signal representing a state where the analysis cell  201  is not held by the holding unit  214  is acquired. By subtracting the result of the background measurement from a light receiving signal representing a state where the analysis cell  201  storing the pseudo sample  4  is held by the holding unit  214 , true spectral data of the pseudo sample  4  can be obtained. 
     First, a measurer (that may be the same as or different from the creator of the pseudo sample  4 ) cleans the analysis cell  201  using pure water, a cleaning solution (for example, an organic solvent), or the pseudo sample  4 . When the analysis cell  201  is not used, cleaning is not necessarily performed. During cleaning, the cleaning solution or the like may be introduced from or may be discharged to the introduction port  2011  of the analysis cell  201 . 
     After cleaning the analysis cell  201 , the measurer introduces the pseudo sample  4  from the introduction port  2011  of the analysis cell  201 . After the introduction, the analysis cell  201  is held by and fixed to the holding unit  214 . 
     Next, the light source unit  212  starts emitting light having a predetermined wavelength to the analysis cell  201  storing the pseudo sample  4  in accordance with the control from the control unit  211 . The light receiving unit  213  starts receiving light transmitted through the analysis cell  201  in accordance with a control signal from the control unit  211 , and outputs a light receiving signal representing a light receiving effect to the control unit  211 . The control unit  211  creates spectral data based on the light receiving signal. Next, the measurer disposes the pseudo sample  4  stored in the analysis cell  201  and ends a single spectrometry operation. 
     By repeating the above-described spectrometry by at least the number of the pseudo samples  4 , the spectral data creation process by the spectrometer  21  ends. 
     Next,  FIG. 7  illustrates a configuration example of functional blocks configuring the analyzer  23 . 
     As in the control unit  211  of the above-described spectrometer  21 , the analyzer  23  is configured with a computer, and each of the functions of the analyzer  23  is implemented by a CPU executing a predetermined program. The analyzer  23  may also function as the control unit  211 . The analyzer  23  includes a pre-processing unit  231 , a calculating unit  232 , and a storage unit  233 . 
     The pre-processing unit  231  performs a predetermined pre-processing (for example, four arithmetic operation, differential, integral, or normalization) on the spectral data and the reference values obtained from the pseudo samples  4  in order to convert the spectral data and the reference value into a data format suitable for multi-variable analysis in the calculating unit  232  described below, and outputs the results to the calculating unit  232 . The pre-processing unit  231  is not necessarily provided. 
     The calculating unit  232  creates a numerical model representing a correlation between the spectral data and the reference value by performing multi-variable analysis on the pre-processed spectral data and the pre-processed reference values obtained from the plurality of pseudo samples  4 . 
     As a method of the multi-variable analysis, for example, single regression analysis, multiple regression analysis, quantification theory type I, quantification theory type II, quantification theory type III, discriminant analysis, logistic regression analysis, principal component analysis, partial least squares regression (PLS regression), factor analysis, cluster analysis, correspondence analysis, multidimensional scaling, conjoint analysis, support vector machine, decision tree, Random forest, naive bayes, neural network, or deep learning can be assumed. However, multi-variable analysis other than the above-described examples may be adopted. 
     The storage unit  233  is used as a work area, for examples, stores data in the process of calculation by the pre-processing unit  231  or the calculating unit  232 . 
     Next, an example of a numerical model creation process by the analyzer  23  will be described. 
     In the analyzer  23 , first, the pre-processing unit  231  performs a predetermined pre-processing on the spectral data obtained from the spectrometer  21  and the reference values obtained from the reference value measuring device  22 , and outputs the results to the calculating unit  232 . Next, the calculating unit  232  creates a numerical model by performing multi-variable analysis on the pre-treated spectral data and the pre-treated reference values. The created numerical model is output to the outside of the analyzer  23  and is used for actual spectrometry (for example, estimation of the concentration of a predetermined component in a culture solution). 
     &lt;Evaluation of Numerical Model Created by Numerical Model Creation System  20 &gt; 
     Next, the evaluation of the numerical model created by the numerical model creation system  20  will be described based on examples. 
     First, creation of a numerical model using a method of the related art will be described. 
     First, CHO (Chinese Hamster Ovary) cells were cultured eight times. Hereinafter, the eight culture processes will be referred to as A, B, C, D, E, F, G, and H, respectively. In the culture solution used, glucose and L-glutamine were added to a Dulbecco&#39;s modified eagle pre-culture liquid medium with low glucose (1.0 [g/ 1 ]), and the concentration of glucose was adjusted to be 5.5 to 6.5 [g/l] to perform culture. 
     The eight (A to H) culture processes were performed using 125 [ml] of a pre-culture liquid medium in an incubator at an air temperature of 37.5° C. in an environment where the concentration ratio of CO 2  to air was 5%. 
     In four culture processes (A to D) among the eight (A to H) culture processes, five samples were obtained at a frequency of once per day to perform the spectrometry and the reference value measurement. A numerical model was created using spectral data and reference values obtained from each of a plurality of samples. 
     Specifically, first, a numerical model was created using data (spectral data and reference values) of five samples obtained each of the four culture processes (A, B, C, D). In this case, four numerical models were created. 
     Next, a numerical model was created using each of all the data (spectral data and reference values) obtained from all the combinations (AB, AC, AD, BC, BD, and CD) of two culture processes among the four culture processes (A to D). In this case, six numerical models were created. 
     Next, a numerical model was created using each of all the data (spectral data and reference values) obtained from all the combinations (ABC, ABD, ACD, and BCD) of three culture processes among the four culture processes (A to D). In this case, four numerical models were created. 
     Further, a numerical model was created using each of all the data (spectral data and a reference value) obtained from all the combination (ABCD) of four culture processes among the four culture processes (A to D). In this case, one numerical model was created. 
     On the other hand, in the pseudo sample creation method according to the first embodiment, a culture solution after completion (eight days after culture) of a single culture process (for example, A) among the four culture processes (A to D) was adopted as the post-culture liquid medium  2 , and the pseudo sample  4  was created. As the pre-culture liquid medium  1 , a Dulbecco&#39;s modified eagle medium with low glucose (1.0 [g/l]) was adopted. As the additive  3 , glucose was adopted. 
     The pre-culture liquid medium  1 , the post-culture liquid medium  2 , and the additive  3  were mixed with one another in random fractions to prepare 66 types of pseudo samples  4  having different mixing ratios. The 66 types of pseudo samples  4  were sequentially input to the numerical model creation system  20  ( FIG. 4 ) to create a numerical model. As the multi-variable analysis in the analyzer  23 , PLS regression was adopted. 
     Spectral data and reference values were acquired from a culture solution after completion (eight days after culture) of the remaining four culture processes (E to H) among the eight (A to H) culture processes and were used to verify the accuracy of the numerical model. Specifically, the spectral data was used to estimate the concentration of glucose using the numerical model. The reference value was used as a true value of the concentration of glucose. 
     Next,  FIG. 8  is a diagram illustrating an example of a numerical sample created using the pseudo sample creation method according to the first embodiment. 
     In  FIG. 8 , the horizontal axis represents the number of culture solutions used for creating a numerical sample. The number of culture solutions used to create a numerical sample in the method of the related art is one to four, and the number of culture solutions used to create a numerical sample in the pseudo sample creation method according to the first embodiment is one. In  FIG. 8 , the vertical axis represents an error index RMSEP [g/l] between a concentration of glucose estimated using a numerical sample and a true value of the concentration of glucose. As the value of RMSEP decreases, the accuracy of the numerical model increases. When the value of RMSEP is 0.6 [g/l] or lower, the accuracy of the numerical model can be considered to be high. 
     In  FIG. 8 , white circles represent values of RMSEP corresponding to the method of the related art, black triangles represent average values of RMSEP corresponding to the method of the related art, and a black square represents a value of RMSEP corresponding to the pseudo sample creation method according to the first embodiment. 
     It can be seen that in the method of the related art, as the number of culture solutions used to create a numerical model increases, the number of data increases; therefore, the high accuracy of the numerical model can be realized. 
     On the other hand, it can be seen that, in the pseudo sample creation method according to the first embodiment, the number of culture solutions (post-culture liquid medium  2 ) used to create a numerical model was one, but  66  types of pseudo samples  4  having different mixing ratios between the pre-culture liquid medium  1  and the additive  3  were created; therefore, a high-accuracy numerical model in which RMSEP is 0.57 [g/l] was able to be created. 
     The reason why a high-accuracy numerical model can be created using the pseudo sample  4  created by the pseudo sample creation method according to the first embodiment can be described by assuming that a numerical model is configured with two factors including a principal factor and an inhibiting factor. 
     That is, the principal factor is a factor that determines the true component concentration and, here, corresponds to single spectra of glucose. The inhibiting factor is a factor that provides error to the component concentration determined by the principal factor and, here, corresponds to lactic acid or the like. The pseudo sample  4  created by changing mixing ratios among the pre-culture liquid medium  1 , the post-culture liquid medium  2 , and the additive  3  succeeds to remove a concentration correlation between the principal factor and the inhibiting factor such that the principal factor and the inhibiting factor can be clearly distinguished from each other. Therefore, it is presumed that a high-accuracy numerical model can be created. 
     Pseudo Sample Measurement System  100  According to Third Embodiment of Present Invention 
     In the pseudo sample creation method according to the first embodiment, the pseudo sample is manually created. However, the pseudo sample may be automatically created. 
     Next,  FIG. 9  illustrates a configuration example of a pseudo sample measurement system  100  according to a third embodiment of the present invention. 
     The pseudo sample measurement system  100  automatically executes a process of creating the pseudo sample  4  and introducing the pseudo sample  4  into the analysis cell without manually executing the process. The pseudo sample measurement system  100  includes a culturing device  110 , a mixing device  120 , and an analysis cell introducing device  130 . 
     The culturing device  110  creates the post-culture liquid medium  2  and outputs the post-culture liquid medium  2  to the mixing device  120 . The mixing device  120  mixes the pre-culture liquid medium  1  and the additive  3  with the post-culture liquid medium  2  output from the culturing device  110  to create the pseudo sample  4 . The analysis cell introducing device  130  introduces the pseudo sample  4  created by the mixing device  120  to the analysis cell  201 . 
     Next,  FIG. 10  illustrates a configuration example of the culturing device  110 . 
     The culturing device  110  includes a culture tank  1100 , a first pump  1105 , an optical analysis cell  1106 , and a second pump  1108 . 
     A stirring blade  1101  is provided in the culture tank  1100 . In addition, a first outlet  1102  and an inlet  1103  are provided in the culture tank  1100 . The first outlet  1102  and the inlet  1103  are connected to each other through a first flow path  1104 . On the first flow path  1104 , the first pump  1105  and the optical analysis cell  1106  are provided. 
     Further, a second outlet  1107  is provided in the culture tank  1100 . The second outlet  1107  is connected to the mixing device  120  through a second flow path  1109 . On the second flow path  1109 , the second pump  1108  is provided. 
     It is desirable that the first flow path  1104  and the second flow path  1109  have high heat resistance, pressure resistance, and mechanical strength, have easiness of cleaning, sterilization, and the like, and are non-invasive to materials in the culture tank  1100 . The optical analysis cell  1106  is formed of a material that allows transmission of light, for example, fused silica or an acrylic resin. 
     In the culturing device  110 , in a state where the first outlet  1102  of the culture tank  1100  is filled with the culture solution up to the upstream side and the sample is put into the culture tank  1100 , the first outlet  1102  and the inlet  1103  are opened, the second outlet  1107  is closed, and the first pump  1105  is driven. As a result, the culture solution flows out from the first outlet  1102 , passes through the first flow path  1104 , and returns to the culture tank  1100  through the inlet  1103 . By performing optical analysis on the optical analysis cell  1106  on the first flow path  1104 , the progress status of culture of the sample in the culture solution can be checked. In the culturing device  110 , the optical analysis can be performed on the optical analysis cell  1106  without exposing the culture solution to external air. Therefore, incorporation of contaminants can be prevented during the optical analysis. 
     When it is verified from the optical analysis that the progress status of culture is desirable, the first outlet  1102  and the inlet  1103  are closed, the first pump  1105  is stopped, the second outlet  1107  is opened, and the second pump  1108  is driven. As a result, the culture solution (post-culture liquid medium  2 ) flows out from the second outlet  1107 , passes through the second flow path  1109 , and is output to the mixing device  120 . 
     In the culturing device  110 , the culture solution in which the progress status of culture is desirable can be obtained by using one culture tank  1100  without using a plurality of culture tanks  1100 . 
     Next,  FIG. 11  illustrates a configuration example of the mixing device  120 . The mixing device  120  corresponds to the pseudo sample creation apparatus according to the present invention. 
     The mixing device  120  includes a first container  1201 , a second container  1202 , a third container  1203 , a pump  1204 , a flow path  1205 , a fourth container  1206 , a fifth container  1207 , a shaker  1209 , and a weighing and mixing unit  1210 . 
     It is desirable that the first container  1201 , the second container  1202 , the third container  1203 , the flow path  1205 , the fourth container  1206 , and the fifth container  1207  have high heat resistance, pressure resistance, and mechanical strength, have easiness of cleaning, sterilization, and the like, and are non-invasive to materials in the culture tank  1100 . 
     In the first container  1201  and the fourth container  1206 , a predetermined amount of the additive  3  is stored in advance. In the third container  1203 , the pre-culture liquid medium  1  is stored. The post-culture liquid medium  2  is introduced from the culturing device  110  into the first container  1201  and the second container  1202 . The pre-culture liquid medium  1  is introduced from the third container  1203  into the fourth container  1206  and the fifth container  1207 . 
     The shaker  1209  shakes the first container  1201  and the fourth container  1206 . The weighing and mixing unit  1210  creates the pseudo sample  4  by weighing, acquiring, and mixing the culture solution (the pre-culture liquid medium  1 , the pre-culture liquid medium  1  with which the additive  3  is mixed, the post-culture liquid medium  2 , and the post-culture liquid medium  2  with which the additive  3  is mixed) introduced from the first container  1201 , the second container  1202 , the fourth container  1206 , and the fifth container  1207 . 
     In the mixing device  120 , the post-culture liquid medium  2  is introduced from the culturing device  110  into the first container  1201  and the second container  1202 . In addition, the pump  1204  is driven such that the pre-culture liquid medium  1  stored in the third container  1203  is introduced into the fourth container  1206  and the fifth container  1207  through the flow path  1205 . 
     Next, the shaker  1209  shakes the first container  1201  that stores the post-culture liquid medium  2  and the fourth container  1206  that stores the pre-culture liquid medium  1  to be mixed with the additive  3  stored in advance. Next, the weighing and mixing unit  1210  creates a plurality of pseudo samples  4  having different mixing ratios by weighing, acquiring and mixing the culture solutions from the first container  1201 , the second container  1202 , the fourth container  1206 , and the fifth container  1207 . 
     Next,  FIG. 12  illustrates a configuration example of the analysis cell introducing device  130 . 
     The analysis cell introducing device  130  includes a sampling unit  1301 , a control valve  1302 , and a control unit  1303 . 
     The sampling unit  1301  introduces the pseudo sample  4  input from the mixing device  120  into the analysis cell  201  in accordance with the control from the control unit  1303 . 
     Although not illustrated in the drawing, the analysis cell  201  illustrated in  FIG. 12  is held by the holding unit  214  of the spectrometer ( FIG. 5 ), and spectrometry can be performed immediately after the pseudo sample  4  is introduced. 
     The control valve  1302  is provided in a waste liquid tube  2013  that is connected to a discharge port  2021  provided on the lower surface of the analysis cell  201 . The control valve  1302  is, for example, a solenoid valve and is opened in accordance with control from the control unit  1303  such that the pseudo sample  4  stored in the analysis cell  201  is discharged. 
     It is desirable that the waste liquid tube  2013  has high heat resistance, pressure resistance, and mechanical strength, is deformable, has easiness of cleaning, sterilization, and the like, and is formed of a material such as rubber, silicon, or Tygon. 
     The control unit  1303  is implemented by, for example, a computer including a CPU, a memory, a communication interface, and a storage. The control unit  1303  controls the sampling unit  1301  and the control valve  1302 . 
     In the analysis cell introducing device  130 , first, the control valve  1302  is closed by the control unit  1303 . Next, the sampling unit  1301  introduces the pseudo sample  4  input from the mixing device  120  into the analysis cell  201  through the introduction port  2011  in accordance with the control from the control unit  1303 . In a state where the pseudo sample  4  is stored in the analysis cell  201 , spectrometry or the like is performed. After the analysis ends, the control valve  1302  is opened by the control unit  1303 . As a result, the pseudo sample  4  is discharged from the analysis cell  201  through the waste liquid tube  2013 . The above-described operation is repeated by the number of the pseudo samples  4 . 
     In the above-described pseudo sample measurement system  100 , the series of processes including the creation of the post-culture liquid medium  2 , the creation of the pseudo sample  4 , the introduction of the pseudo sample  4  into the analysis cell  201 , the optical analysis of the pseudo sample  4 , and the replacement of the pseudo sample  4  in the analysis cell  201  can be automatically executed without being manually executed. 
     The present invention is not limited to the embodiments and the modification examples and includes various modification examples. For example, the respective embodiments have been described in detail in order to easily describe the invention, and the present invention is not necessarily to include all the components described above. In addition, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment. Further the configuration of one embodiment can be added to the configuration of another embodiment. In addition, addition, deletion, and replacement of another configuration can be made for a part of the configuration each of the embodiments. 
     REFERENCE SIGNS LIST 
       1 : pre-culture liquid medium 
       2 : post-culture liquid medium 
       3 : additive 
       4 : pseudo sample 
       20 : numerical model creation system 
       21 : spectrometer 
       22 : reference value measuring device 
       23 : analyzer 
       100 : pseudo sample measurement system 
       110 : culturing device 
       120 : mixing device 
       130 : analysis cell introducing device 
       201 : analysis cell 
       211 : controller 
       212 : light source unit 
       213 : light receiving unit 
       214 : holding unit 
       215 : fixing tool 
       231 : pre-processing unit 
       232 : calculating unit 
       233 : storage unit 
       1100 : culture tank 
       1101 : stirring blade 
       1102 : first outlet 
       1103 : inlet 
       1104 : first flow path 
       1105 : first pump 
       1106 : optical analysis cell 
       1107 : second outlet 
       1108 : second pump 
       1109 : second flow path 
       1201 : first container 
       1202 : second container 
       1203 : third container 
       1204 : pump 
       1205 : flow path 
       1206 : fourth container 
       1207 : fifth container 
       1209 : shaker 
       1210 : weighing and mixing unit 
       1301 : sampling unit 
       1302 : control valve 
       1303 : control unit 
       2011 : introduction port 
       2013 : waste liquid tube 
       2021 : discharge port