Method for calculating corrected spectral intensities from Raman spectra of cell culture media containing glucose

Abstract

To correct variations of a base line in a Raman spectrum of a cell culture medium.SOLUTION: A method for calculating spectral intensity obtained by correcting a Raman spectrum of a cell culture medium containing glucose, includes acquiring the Raman spectrum generated when the cell culture medium is irradiated with excitation light having a single wavelength, extracting spectrum intensity at a first wavenumber and spectrum intensity at a second wavenumber in at least two culture times, and calculating spectrum intensity of glucose at 1127 cm-1 from a segment in which the first wavenumber and the second wavenumber are set so that the segment indicating a relation between the spectrum intensity at the first wavenumber and the spectrum intensity at the second wavenumber indicates a linear change with time according to a change in culture time.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for calculating corrected spectral intensities from Raman spectra of cell culture media containing glucose. [Background technology]

[0002] When quantifying cell culture medium components using Raman spectroscopy, the spectrum may change from measurement to measurement depending on the cell culture medium component. In particular, the baseline of the Raman spectrum may change over time. This is manifested as a shift in the slope and intercept of the baseline of the Raman spectrum, for example, as the concentration of a cell culture medium component decreases or as unwanted components increase.

[0003] Patent Document 1 describes a component analyzer suitable for analyzing components in blood using Raman spectroscopy. Patent Document 2 describes an analyzer suitable for analyzing components contained in blood or interstitial fluid using Raman spectroscopy. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6826463 specification [Patent Document 2] Patent No. 6842322 specification Summary of the Invention [Problem to be solved by the invention]

[0005] If the concentration of a target analyte is calculated from its measurement results without correcting for baseline drift, the calculated concentration obtained from the spectrum acquired by the Raman spectrometer will be inflated or will differ depending on the rise or fall of the baseline. In this case, the reliability of the measurement results may be lost, or the calculated concentration may not be able to keep up with changes over time.

[0006] On the other hand, while conventional waveform analysis methods have a baseline correction function, they use a fixed correction value, and in most cases cannot correct for continuous fluctuations, making it difficult to make the correction follow continuous fluctuations. When analyzing, the user has no choice but to analyze each piece of data manually, and unless this problem is resolved, it is time-consuming and complicated.

[0007] The present invention aims to calculate corrected spectral intensities from Raman spectra of cell culture media containing glucose, and to correct fluctuations in the Raman spectra due to changes over time. [Means for solving the problem]

[0008] In one embodiment of the method according to the present invention, 1. A method for calculating corrected spectral intensities from a Raman spectrum of a cell culture medium containing glucose, comprising: Irradiating the cell culture medium with excitation light of a single wavelength to obtain a Raman spectrum; extracting a spectral intensity at a first wavenumber and a spectral intensity at a second wavenumber for at least two incubation times; the first wavenumber and the second wavenumber are set so that a line segment showing the relationship between the spectral intensity at the first wavenumber and the spectral intensity at the second wavenumber shows a linear change over time due to a change in the culture time, From the line, 1127 cm -1 A method for calculating the spectral intensity of glucose in a [Effects of the Invention]

[0009] According to the present invention, corrected spectral intensities can be calculated from the Raman spectrum of a cell culture medium containing glucose. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a conceptual diagram showing an example of the configuration of an analysis mechanism equipped with an analysis device used in the method of this embodiment. [Figure 2] FIG. 2 is a conceptual diagram showing an example of the configuration of an analytical device used in the method of this embodiment. [Figure 3] FIG. 3 is a diagram showing an example of the change over time in the Raman spectrum of a cell culture medium. [Figure 4] FIG. 4 is an explanatory diagram showing how to obtain the baseline from the spectral intensity at the incubation time Tn. [Figure 5] FIG. 5 is a diagram showing the change over time in the slope of the line segment of the spectral intensity of the Raman spectrum. [Figure 6] FIG. 6 is a diagram showing the change over time in the intercept of the line segment of the spectral intensity of the Raman spectrum. [Figure 7] FIG. 7 shows Raman spectra measured over a period of days in a cell culture medium in which NPCs (Nephron progenitor cells) isolated from mouse fetuses were cultured, and in particular shows the glucose detection position at 1127 cm −1 . [Figure 8] FIG. 8 shows Raman spectra measured over a period of days in a cell culture medium in which NPCs (Nephron progenitor cells) isolated from mouse fetuses were cultured, and in particular shows the detection position of lactic acid at 857 cm −1 . [Figure 9] FIG. 9 is a flowchart showing an example of the method of this embodiment. [Figure 10] FIG. 10 shows the change in the spectral intensity of samples d4 to d8, and explains the change in the Raman spectrum over time. [Figure 11] FIG. 11 shows the results of a regression analysis of the relationship between the slope and the elapsed time (culture time). [Figure 12] FIG. 12 shows the results of a regression analysis of the relationship between the intercept and the elapsed time (culture time). [Figure 13] FIG. 13 is a diagram for explaining calculation of the spectrum intensity of glucose at a certain incubation time. [Figure 14]FIG. 14 is a diagram for explaining calculation of the spectrum intensity after correction for glucose at a certain culture time acquired in FIG. [Figure 15] FIG. 15 shows the results of comparing glucose concentrations with and without correction. [Figure 16] FIG. 16 is a diagram illustrating the calculation of the spectral intensity of lactic acid at a certain incubation time. [Figure 17] FIG. 17 is a diagram for explaining calculation of the corrected spectral intensity of lactic acid at a certain culture time acquired in FIG. [Figure 18] FIG. 18 shows the results of comparing lactate concentrations with and without correction. DETAILED DESCRIPTION OF THE INVENTION

[0011] An example of an embodiment of the present invention will be described below with reference to the accompanying drawings. The embodiment exemplified below is intended to facilitate understanding of the present invention and is not intended to limit the present invention. The present invention can be modified and improved as appropriate without departing from the spirit and scope of the present invention.

[0012] <Analysis mechanism> 1 is a conceptual diagram showing an example of the configuration of an analysis mechanism 10 equipped with an analysis device 26 used in the method of this embodiment. The analysis mechanism 10 is a mechanism for analyzing the spectrum of Raman scattered light generated in a sample 16 when irradiated with excitation light. The analysis mechanism 10 includes a light source 12, a reflector 14, a sample 16, an optical system 18, a monochromator 20, and the analysis device 26 as its main components.

[0013] The light source 12 emits excitation light for irradiating the sample 16. A solid-state laser is preferable as the light source 12 because the shorter the wavelength of this excitation light, the higher the efficiency of Raman scattering and the higher the spatial resolution. The spectrum of Raman scattered light indicates the difference in wavenumber of the Raman scattered light relative to the wavenumber of the excitation light (Raman shift amount). Therefore, it is preferable that the wavelength of the excitation light is a single wavelength. Specific examples of the wavelength of the excitation light are, for example, a single wavelength selected from 514 nm, 532 nm, 633 nm, 670 nm, and 785 nm.

[0014] The reflector 14 reflects the excitation light emitted from the light source 12 and guides it to the sample 16. Note that other members may be used instead of or in addition to the reflector 14, as long as they guide the excitation light emitted from the light source 12 to the sample 16.

[0015] The sample 16 is composed of a cell culture medium and a container (not shown) for containing the cell culture medium. The container for containing the cell culture medium is made of an inert plastic material and has a sealed structure with a vertically elongated internal volume of 25 to 50 ml. The measurement cell used is a rectangular, vertically elongated container made of synthetic quartz glass with a capacity of 1 to 10 ml. In addition to a method in which the sample 16 is placed in such a measurement cell, a flow cell measurement method is also available in which the sample 16 is flowed through a flow-through measurement cell. The container may be a housing made of a chemically inert inorganic material, plastic material, synthetic resin, or metal with excellent corrosion resistance against acids and alkalis. The container has a structure with an inlet and outlet for the sample 16, and a structure with an optical window for incident Raman scattered light and an optical window for receiving Raman scattered light that has passed through the sample 16, or a reflective structure in which Raman scattered light can be incident and received using a single optical element. The container is not limited to these types, and is more preferably structured to allow for the entry and exit of the sample 16, the incidence of Raman scattered light, irradiation of the sample 16, and reception of the Raman scattered light after irradiation.

[0016] <Cell culture medium> Cell culture media are usually composed of, but not limited to, inorganic substances, amino acids, pH indicators, pH buffers, and nutrient sources (roughly divided into three types: carbon sources, mineral sources, and nitrogen sources). The concentrations of components in cell culture media may vary depending on the purpose. In Raman spectroscopy, the concentration of inorganic substances is basically Raman scattering is rarely observed, and only organic matter is Raman scattering Represents.

[0017] <Metabolic production> When cells begin to grow, they consume nutrients such as glucose, and the lactate and ammonium produced within the cells are small molecules that pass through the cell membrane, are released outside the cells, and accumulate in the cell culture medium. The accumulation of waste products such as lactate and ammonium causes a decrease in the pH of the cell culture medium, cytotoxicity, and inhibits cell growth, so it is necessary to monitor the concentrations of lactate and ammonium.

[0018] Before measuring sample 16 with a Raman spectrometer, it is recommended to perform background correction using pure water. It is desirable to use pure water or purified water (distilled water purified with ion exchange resin) with few impurities. Because background measurement significantly affects the soundness of subsequent measurements of sample 16, it is recommended to measure pure water after background measurement and correction, and repeat this process until the baseline is as flat as possible. Furthermore, in case there are any unknown peaks in the cell culture medium that are difficult to identify, and they appear near the main peaks of lactate or glucose, it is also recommended to measure the cell culture medium before cell culture (day 0 of culture) and perform background correction using the acquired spectrum, in addition to background correction using pure water.

[0019] When a high concentration of matrix components is present, an environment with little fluctuation in ambient temperature is desirable. Note that the ambient temperature here is, for example, in the range of 20 to 30°C, and fluctuations in a very short period of time are within ±5°C.

[0020] The optical system 18 guides the Raman scattered light generated in the sample 16 to the monochromator 20. When the sample 16 is irradiated with excitation light, the interaction between the excitation light and the sample 16 generates Raman scattered light of a wavelength different from that of the excitation light, and Rayleigh scattered light of the same wavelength as that of the excitation light. The analyzing mechanism 10 has the optical system 18 on the waveguide to remove this Rayleigh scattered light, and guides the Raman scattered light obtained via the optical system 18 to the monochromator 20.

[0021] The monochromator 20 includes a diffraction grating 22 and a CCD (Charge Coupled Device) detector 24. The diffraction grating 22 separates the light supplied from the sample 16 via the optical system 18 into wavelengths and guides the separated light to the light-receiving surface of the CCD detector 24. The CCD detector 24 has multiple light-receiving elements arranged one-dimensionally or two-dimensionally within its light-receiving surface. The CCD detector 24 accumulates electric charges proportional to the intensity of the Raman scattered light of each wavelength exposed to the light-receiving elements within its light-receiving surface, and outputs Raman spectrum data indicating the intensity distribution of the Raman scattered light by wavelength to an analyzer 26. Instead of the CCD detector 24, a detector using a photodiode, a CMOS (Complementary Metal Oxide Semiconductor), or the like can be used.

[0022] <Analyzer> The analyzer 26 detects components contained in the cell culture medium of the sample 16 based on data provided by the CCD detector 24 of the monochromator 20, and calculates the concentrations of the detected components. The analyzer 26 also notifies the user of the types and concentrations of the components contained in the sample 16 as necessary.

[0023] 2 is a conceptual diagram showing an example of the configuration of an analysis device 26 used in the method of this embodiment. The analysis device 26 includes an input unit 30, a storage unit 32, a control unit 34, and a display unit 36 ​​as its main components.

[0024] The input unit 30 is a section into which various information such as commands and setting values ​​can be input in response to user operations. Specific examples of the input unit 30 include a mouse, a keyboard, a touch panel, a portable semiconductor connector type memory, and a communication interface for sending and receiving information via network communication or serial communication. Examples of semiconductor connector type memory include a USB (Universal Serial Bus) memory and a CF (Compact Flash) memory.

[0025] The storage unit 32 is a part that stores data given from the input unit 30 or the control unit 34 and reads out the stored data. Specific examples of the storage unit 32 include semiconductor memory, magnetic disks, and optical disks.

[0026] The memory unit 32 includes a first storage area (not shown) for storing various programs, a second storage area (not shown) for storing various data such as setting data input from the input unit 30, and a storage area (not shown) for storing programs and data. The first storage area stores an analysis program for executing a process for analyzing the cell culture medium of the sample 16.

[0027] The storage unit 32 stores data of a line segment that indicates the relationship between the spectral intensity at a first wave number and the spectral intensity at a second wave number.

[0028] The first wavenumber and the second wavenumber are set so that the change in the slope and the change in the intercept of the line segment showing the relationship between the spectral intensity at the first wavenumber and the spectral intensity at the second wavenumber show a linear change over time with the change in the incubation time. -1 , 600cm -1 , 400cm -1 The second wave number can be, for example, 2700 cm -1 , 2600cm -1 , 2500cm -1In this case, the first wavenumber and the second wavenumber are set so that the line segment showing the relationship between the spectral intensity at the first wavenumber and the spectral intensity at the second wavenumber changes linearly with the change in culture time, that is, changes linearly with the passage of culture time. In the present application, the first wavenumber is set to 600 cm -1 or 400cm -1 The second wave number is 2600 cm -1 or 2500cm -1 Although the explanation for the case where is omitted, it is known that the same results can be obtained even in these cases.

[0029] The first wave number and the second wave number are 800 cm -1 , 2700cm -1 In this case, the storage unit 32 stores, for example, 800 cm in 96 hours (4 days). -1 and 2700 cm -1 Spectral intensity at 800cm -1 and 2700 cm -1 The data of the first line showing the relationship between the -1 and 2700 cm -1 Spectral intensity at 800cm -1 and 2700 cm -1 and data of the second line segment indicating the relationship between the first line segment and the second line segment.

[0030] Figure 3 shows an example of the change over time in the Raman spectrum of a cell culture medium. As shown in Figure 3, it was found that the baseline of the spectrum fluctuated over time. -1 and 2700cm -1 In addition, under the conditions of cell culture medium measurement, RamanIt was found that no spectrum was detected. In the present application, as described above, the baseline fluctuation is read by expressing a line between two points as a linear equation, and expressing the slope and intercept as a relational expression of elapsed time, so that the spectral intensity can be obtained. In the figure, d0 means the sample on day 0 of culture, d2 means the sample on day 2 of culture, d4 means the sample on day 4 of culture, d6 means the sample on day 6 of culture, d8 means the sample on day 8 of culture, and d8frozen means the sample thawed from the frozen state on day 8 of culture (the same applies below).

[0031] FIG. 4 is an explanatory diagram showing how to determine the spectral intensity C. Here, the first and second wave numbers are 800 cm -1 , 2700cm -1 When this is the case, the spectral intensity C is expressed by the following formula (1) using the incubation time T and the wave number ν (nu) to be measured. C=a×ν+b Equation (1) a: wave number 800cm -1 and 2,700 cm -1 The slope of the line connecting the spectrometer outputs at b: wave number 800cm -1 and 2,700 cm -1 Intercept of the line connecting the spectrometer outputs at

[0032] Wave number 800cm -1 and 2700 cm -1 From equation (1), the line connecting The relational expression for the spectral intensity at incubation time T1 is: C1=a1×ν+b1 The relational expression for the spectral intensity at incubation time T2 is: C2 = a2 × ν + b2 … Culture time T n The relation between the spectral intensity in: C n =a n ×ν+b n By calculating as follows, a line segment showing the relationship between the spectral intensity and the wave number can be obtained.

[0033] However, since the above slope a depends on the incubation time T, the following relational equation (2) between the slope a and the incubation time T is used: a=c×T+d Formula (2) is calculated and substituted into equation (1). In addition, since the intercept b depends on the incubation time T, the following relation between the intercept b and the incubation time T is given by Equation (3): b=e×T+f Equation (3) is calculated and substituted into equation (1).

[0034] Figure 5 shows the time-dependent change in the slope of the line segments of the spectral intensity of the Raman spectrum. n Changes in the incubation time T1, T2, T n From this, the slope c and intercept d of equation (2) for the slope a and the incubation time T can be calculated (see equation (2)).

[0035] Figure 6 shows the time-dependent change in the intercepts of the line segments of the spectral intensity of the Raman spectrum. n Changes in the incubation time T1, T2, T n From this, the slope e and intercept f of equation (3) with intercept b and incubation time T can be calculated (see equation (3)).

[0036] In the above case, the storage unit 32 stores the slopes a1, a2, . . . a n and incubation times T1, T2, T n The data of equation (2) showing the relationship between b1, b2...b n and incubation times T1, T2, T n The data of the formula (3) showing the relationship between the above is stored.

[0037] The wave number ν for measuring glucose is 1127 cm -1 The wave number ν of lactic acid to be measured is 857 cm -1 The reason for this is that both are wavenumbers of the main spectrum, the detected spectral intensity is strong, and they are easy to distinguish.

[0038] Figure 7 shows the Raman spectra of the cell culture medium in which NPCs (Nephron progenitor cells) isolated from mouse embryos were cultured over a period of several days. In particular, the glucose detection position at 1127 cm -1 In Figure 7, measurements were taken of the cell culture medium over the course of several days (glucose measurements were taken using pure water and background correction was performed). Figure 8 shows the Raman spectra of the measurement results over the course of several days of a cell culture medium in which NPCs (Nephron progenitor cells) isolated from mouse fetuses were cultured, and in particular, the 857 cm peak, where lactate was detected. -1 FIG.

[0039] To create a calibration curve, a solution with the composition shown in Table 1 was prepared with the aim of simulating a solution with cell culture medium components as close as possible. The composition was limited to the minimum necessary components to create a calibration curve, and phenol red, which is contained in cell culture medium, was not added.

[0040] The standard substances used to create the calibration curve are glucose or lactic acid alone, or a mixture of two or more of these. As a pH buffer, use 10x D-PBS(-), Code No. 048-29805, manufactured by Fujifilm Wako Pure Chemical Industries, or prepare a phosphate-buffered saline-based simulation solution according to Table 1. Preparation method Based on the SDS from Fujifilm Wako Pure Chemical Industries, prepare the following: KCl: 2000.0 mg / L, NaCl: 80,000.0 mg / L, KH2PO4: 2000.0 mg / L, Na2HPO4: 11,500.0 mg / L. The 10x concentration means that the solution must be diluted 10 times for use. Obtain the above-mentioned reagents and use distilled, purified, and filtered ultrapure water to prepare 1 L of the above 10x concentrated solution of phosphate buffer solution, which can then be diluted to use as a stock solution. · Adjust the mixing ratio of KH2PO4 and NaHPO4 to adjust the pH to 6.5-7.5. ·NaCl and KCl are kept constant at 137mM and 2.7mM, respectively. Using the above solution as a base, glucose concentrations of 50, 75, 150, 200, 300, 500, and 750 mg / dl, and lactic acid concentrations of 9, 45, 90, 180, 450, and 900 mg / dl were added to a pH buffer-based solution, and pure water was added to bring the total volume to 100 ml to create a mixed solution with the desired concentration.

[0041] [Table 1]

[0042] The control unit 34 controls the analyzer 26 based on the program stored in the first storage area of ​​the storage unit 32 and the data stored in the second storage area of ​​the storage unit 32.

[0043] When the control unit 34 reads out the analysis program stored in the first storage area of ​​the memory unit 32, it loads the read analysis program into the load area of ​​the memory unit 32. In this case, the control unit 34 functions as an acquisition unit 40, an extraction unit 42, a component identification unit 44, a calculation unit 46, and a notification unit 48, and executes the analysis process for analyzing the cell culture medium. A specific example of the control unit 34 is a chip component having a CPU (Central Processing Unit), ROM (Read Only Memory), and RAM (Random Access Memory).

[0044] The display unit 36 ​​is a part that displays information indicated in data given from the control unit 34. Specific examples of the display unit 36 ​​include a liquid crystal display, a plasma display, and an EL (Electro Luminescence) display.

[0045] The acquisition unit 40 acquires a Raman spectrum that shows the intensity distribution for each wavenumber of Raman scattered light generated when a sample, which is a cell culture medium containing a desired component, is irradiated with excitation light of a single wavelength. Specifically, the acquisition unit 40 drives the light source 12 by appropriately adjusting the intensity of the excitation light to be emitted from the light source 12, and acquires Raman spectrum data output from the CCD detector 24 of the monochromator 20 via the light source 12, reflector 14, sample 16, and optical system 18 in this order.

[0046] The extracting unit 42 converts the wavelengths of the Raman spectrum acquired by the acquiring unit 40 into Raman shift amounts to extract the spectral intensity. Specifically, each wavelength in the Raman spectrum is converted into a wavenumber, which is the number of waves contained in a unit length. Each converted wavenumber is then converted into a Raman shift amount, which is the difference from the wavenumber of the excitation light emitted from the light source 12. Each converted wavenumber is then converted into a Raman shift amount, which is the difference from the wavenumber of the excitation light emitted from the light source 12. This results in a Raman spectrum with intensity on the vertical axis and wavenumber on the horizontal axis. It is known that substances have unique vibrational energy depending on their molecular structure and crystalline structure, and the Raman shift amount, which is the difference between the wavenumber (frequency) of the Raman scattered light and the wavenumber (frequency) of the incident light, has a unique waveform that reflects the molecular structure and crystalline structure of the substance.

[0047] The extracting unit 42 extracts the spectral intensity at the first wavenumber and the spectral intensity at the second wavenumber for at least two culture times. The culture time can be, for example, one of 96 hours (4 days), 120 hours (5 days), 144 hours (6 days), 168 hours (7 days), and 192 hours (8 days), but is not limited to these. For example, the 800 cm at 96 hours (4 days) -1 and 2700 cm -1 and the spectral intensity at 800 cm after 120 hours (5 days). -1 and 2700 cm -1 The spectral intensities at and can be extracted.

[0048] In analytical instruments that perform spectral detection, particularly liquid chromatography, ion chromatography, and gas chromatography, quantitative calculations are performed by determining the peak height or the area of ​​the peak waveform. This is because it is assumed that the peaks of the object being measured are clearly separated or that the peaks are normally distributed. In Raman spectroscopy, the peaks of the detected spectrum are not necessarily normally distributed, so quantitative calculations are generally and preferably performed using the peak height of the spectrum.

[0049] It may be possible to calculate the area of ​​a Raman spectrum if it is a single, simple spectrum or peak. However, in the presence of multiple matrices, there may be adjacent peaks, and even if the area is calculated, errors may occur depending on the method of peak division. For this reason, Raman spectra are generally processed using the peak height of the spectrum. This differs from separation analysis, which divides the detected spectrum and peaks, as is done in waveform processing for liquid chromatography and ion chromatography, when analyzing Raman spectroscopy data. This takes advantage of the fact that detected peaks appear at different wavenumbers due to differences in molecular structure and crystalline structure.

[0050] The component identification unit 44 searches for and identifies components of the cell culture medium based on the Raman spectrum extracted by the extraction unit 42 and the wave numbers specific to peaks that appear in the Raman spectrum depending on the components of the cell culture medium.

[0051] The calculation unit 46 calculates the concentration of the component corresponding to the peak based on the spectral intensity of the peak detected by the component identification unit 44. That is, the calculation unit 46 reads out calibration curve data for the component and wavenumber corresponding to the peak detected by the component identification unit 44 from the second storage area, and calculates the concentration (mg / dl) of the component corresponding to the detected peak from the spectral intensity of the peak using the calibration curve.

[0052] In this embodiment, the calculation unit 46 calculates the slopes a1, a2, . . . a nChanges in the incubation time T1, T2, T n From the above, the equation (2) of the slope a and the incubation time T is calculated, and the slope c and intercept d of the equation (2) are calculated. n Changes in the incubation time T1, T2, T n From the above, the equation (3) of the slope b and the incubation time T is calculated, and the slope e and intercept f of the equation (3) are calculated. Furthermore, the calculation unit 46 calculates the slope a at the desired incubation time T, the intercept b at the desired incubation time T, and the wave number for glucose, 1127 cm -1 or 857 cm, which is the wavenumber for lactic acid -1 From this, the spectral intensity C of glucose or lactate at the desired incubation time T is calculated (see equation (1) above).

[0053] The component discriminating unit 44 recognizes the wave number of a certain component read from the second storage area, and determines the wave number within ±6 cm based on that wave number. -1 In this case, the peak is searched for within ±6 cm of the above wave number. -1 If even one peak is detected within this range, it means that the component corresponding to that peak is contained in the cell culture medium. In this application, for ease of understanding, only wave numbers of certain numerical values ​​are shown as wave numbers, but these wave numbers are within ±6 cm. -1 For example, 800cm -1 is 800±6cm -1 means 857cm -1 is 857±6cm -1 means 1127cm -1 is 1127±6cm -1 means 2700cm -1 is 2700±6cm -1 means.

[0054] The notification unit 48 notifies the user of the types of cell culture components detectable by the component identification unit 44 and the concentrations of the cell culture components detected by the component identification unit 44 and calculated by the calculation unit 46. For example, the types of cell culture components and the concentrations of the cell culture components calculated by the calculation unit 46 are displayed on the display screen of the display unit 36 ​​in a predetermined display format. Instead of or in addition to displaying the information on the display unit 36, the types of cell culture components and the concentrations of the cell culture components calculated by the calculation unit 46 may be output as audio from a speaker.

[0055] The control unit 34 functions by the acquisition unit 40, extraction unit 42, component identification unit 44, calculation unit 46, and notification unit 48, and executes the process of analyzing the components of the cell culture medium.

[0056] In Raman spectroscopy, the sample 16 can be directly placed in a container and measured without pre-treatment. Raman scattering In cell culture media where the composition of the solution is known in advance, the high concentration of matrix Raman It is preferable to know what is or is not detected as a spectrum.

[0057] <Baseline correction> Furthermore, by using the following formula (4), fluctuations in the slope and intercept of the baseline can be corrected, and the measurement target component of the cell culture medium can be quantified by Raman spectroscopy. Corrected density=A×(C-D)+B Equation (4) A: The slope of the calibration curve obtained from the relationship between the concentration of the standard substance and the spectral intensity in an aqueous system. B: Intercept of the calibration curve obtained from the relationship between the concentration of the standard substance and the spectral intensity in an aqueous system C: Corrected spectral intensity D: Spectral intensity used as the basis for calculating peak height

[0058] 9 is a flowchart showing an example of the method of this embodiment. In the method 50 of this embodiment, first, a Raman spectrum generated by irradiating a cell culture medium containing glucose and lactic acid with excitation light of a single wavelength is acquired (step 52).

[0059] Next, the first wave number, 800 cm -1 The spectral intensity of the second wavenumber, 2700 cm -1 The spectral intensities at T1, T2, T n The extract is then extracted after incubation for the following period (step 54).

[0060] Here, the first wave number is 800 cm -1 Spectral intensity at the second wavenumber, 2700 cm -1 The line segment showing the relationship between the spectral intensity at the first wavenumber, 800 cm, shows a linear change over time with the change in incubation time. -1 and the second wavenumber, 2700 cm -1 is set.

[0061] Next, from the line above, 1127cm -1 The spectral intensity of glucose at 857 cm -1 The spectral intensity of lactate in the region is calculated (step 56).

[0062] After step 56, there is an additional 1127 cm -1 From the spectral intensity of glucose at 1127cm -1 The glucose concentration can be calculated using a calibration curve of the glucose concentration and the spectral intensity at 857 cm. -1 From the spectral intensity of lactic acid at 857cm -1 The concentration of lactic acid can be calculated using a calibration curve of the concentration of lactic acid and the spectral intensity.

[0063] When actually measuring cell culture media using a Raman spectrometer, it is preferable to perform in-line measurement, as shown below, that is, to incorporate the spectrometer into a culture system in which culture is actually being carried out. (1) For example, the start time of Raman spectroscopic measurement is set to culture time 0. (2) By operating the Raman spectrometer so that it automatically measures at preset intervals, the measurement start time can also be obtained as data. For example, if measurements are taken every hour for half a day, 12 pieces of data will be accumulated. If the measurement start time is 9:00 AM, 12 hours of data will have been accumulated by 9:00 PM, resulting in 12 hours of incubation time and 12 pieces of data. A program can be constructed in advance to automatically determine the relational equation for baseline fluctuations using, for example, 3 to 12 of the 12 pieces of data obtained as the incubation time for 12 hours. In this case, it is preferable to automatically update the coefficients and intercepts of the automatically determined relational equation. (3) The obtained relational expression (correction expression) can be applied to subsequent interval measurements.

[0064] The present invention can be used by acquiring and correcting the incubation time in this manner. Note that the present invention is also applicable to manual analysis, as is actually done in laboratories.

[0065] In the following explanation, for ease of understanding, an example of the action of each component that is the target of each method is explained.

[0066] Furthermore, when determining each of the following equations or line segments, it has been confirmed that they also exhibit linearity in mixed solutions containing 100 to 300 mg / dl of glucose and 0 to 400 mg / dl of lactic acid.

[0067] Example 1 An example in which the corrected spectral intensity of glucose is calculated from the Raman spectrum will be described below, although the present invention is not limited to this example.

[0068] <About cell culture media samples> The results of experiments using cell culture media are shown below. Table 2 shows the types and main components of the cell culture media.

[0069] [Table 2]

[0070] A glucose-containing cell culture medium sample (see Table 2, using the StemFit AK02N described above) was irradiated with a single wavelength laser light of 785 nm to obtain the Raman spectrum of the cell culture medium sample. The Raman analyzer used was a Marqmetrix Model AIO-M73. The blood gas analyzer (blood gas meter) used was a Radiometer Model ABL800 BASIC analyzer.

[0071] Table 3 shows the 800 cm -1 and 2700 cm -1 10 shows the results of extracting the spectral intensities of samples d4 to d8. Fig. 10 shows the changes in the spectral intensities of samples d4 to d8, and explains how the spectra change over time.

[0072] [Table 3]

[0073] As a result of the measurements, it was confirmed that the slope and intercept of the baseline of the spectrum changed with time.

[0074] Table 4 shows the spectral intensities and the values ​​from 800 to 2700 cm for d4 to d8. -1 The change in the value was calculated as a straight line, and the slope and intercept were calculated and shown below.

[0075] [Table 4]

[0076] Fig. 11 is a graph showing the results of a regression analysis of the relationship between the slope and the elapsed time (culture time). Fig. 12 is a graph showing the results of a regression analysis of the relationship between the intercept and the elapsed time (culture time).

[0077] The linear equation from d4 to d8 was calculated from the above equation (2), and the slope of this linear equation was used as the relational equation between wavenumber and incubation time, resulting in the following equation (5): a=(-0.0157×T-2.91) Equation (5)

[0078] Similarly, the linear equation for the intercepts from d4 to d8 was calculated from the above equation (3), and when the intercept of this linear equation was used as a relational equation for the culture time, the following equation (6) was obtained: b=(43.7×T+8509) Equation (6)

[0079] From the above equations (1), (5), and (6), the following equation (7) is obtained: Corrected spectral intensity = (-0.0157 × T - 2.91) × ν + (43.7 × T + 8509) Equation (7)

[0080] The calibration curve, which was previously obtained by measuring a standard solution to calculate the glucose concentration, was expressed by the following formula (8), which was previously obtained. When creating the calibration curve, a solution was prepared that was as close as possible to the cell culture medium components, with a phosphate-buffered saline base containing 137 mM NaCl and 27 mM KCl, respectively, and glucose concentrations of 50, 75, 150, 200, 300, 500, and 750 mg / dl. The calibration curve was calculated from the spectral intensity obtained by measurement using the simulated cell culture medium to which glucose had been added and the weighed concentrations of the added glucose of 50, 75, 150, 200, 300, 500, and 750 mg / dl. Glucose concentration (mg / dl) = 0.3018 × spectral intensity + 0.9079 Equation (8)

[0081] Table 5 shows the characteristic wavenumber of glucose, 1127 cm -1 The spectral intensity and height before correction are shown below. The spectral intensity, height, and calculated concentration of the baseline to be calculated are shown below.

[0082] [Table 5]

[0083] A method for calculating the spectral intensity of glucose will now be described. FIG. 13 is a diagram illustrating the calculation of the spectral intensity of glucose at a certain incubation time. FIG. 14 is a diagram illustrating the calculation of the corrected spectral intensity of glucose at a certain incubation time obtained in FIG. 13. In FIG. 13, (A) represents the spectral intensity before correction in Table 5, (B) represents the spectral intensity of the baseline used to calculate the pre-correction height in Table 5, and (C) represents the height obtained by subtracting (B) from (A) in Table 5. In FIG. 14, (D) represents the spectral intensity after correction in Table 6, (E) represents the spectral intensity of the baseline used to calculate the post-correction height in Table 6, and (F) represents the corrected height obtained by subtracting (E) from (D) in Table 6 (corrected glucose spectral intensity). In FIGS. 13 and 14, the spectral intensity of glucose is the sum of the spectral intensity of glucose and the spectral intensity of the baseline.

[0084] Even if the baseline fluctuates, the spectral intensity of (D) is also composed of the ratio of (B) / (A), and the corrected baseline spectral intensity (E) is calculated. That is, (E) = (D) × (I) / (A) The corrected baseline spectral intensity (O) is calculated as follows:

[0085] The corrected glucose spectral intensity (F) can be obtained by subtracting the calculated (E) from (D). The calculated glucose concentration can be obtained from the spectral intensity (F).

[0086] To calculate the spectral intensity of glucose, the corrected spectral intensity (d) was calculated using ν=1127 from equation (7) as follows: as ν:1127 d4: Corrected spectral intensity = (-0.0157 × 96 - 2.91) × 1127 + (43.7 × 96 + 8509) = 7724.9 d5: Corrected spectral intensity = (-0.0157 × 120 - 2.91) × 1127 + (43.7 × 120 + 8509) = 8349

[0087] Similarly, calculations were performed for d6, d7, and d8. From the corrected spectral intensities obtained, the characteristic wavenumber of glucose, 1127 cm -1 The corrected spectral intensity of glucose was calculated, and the calculated concentration of glucose was then calculated.

[0088] [Table 6]

[0089] Table 7 shows the results of comparing glucose concentrations with and without correction. Figure 15 shows the results of comparing glucose concentrations with and without correction. Note that the blood gas measurement results are used here as a comparison target for relative evaluation with the concentration detected by the Raman spectrometer and obtained by calculation.

[0090] [Table 7]

[0091] From Table 7 and Figure 15, it is clear that the corrected values ​​are relatively closer to the blood gas meter values ​​compared to the uncorrected values.

[0092] <Example 2> Hereinafter, an example will be described in which baseline correction was performed from the peak of the Raman spectrum for lactic acid. Note that the cell culture medium sample used is the same as that used for glucose, so its description will be omitted.

[0093] A 785 nm single-wavelength laser was irradiated onto a cell culture medium sample containing lactic acid (using the StemFit AK02N described above) from Table 2, and the Raman spectrum of the cell culture medium sample was obtained. The analyzer used was a Marqmetrix Model AIO-M73. The blood gas analyzer (blood gas meter) used was a Radiometer Model ABL800 BASIC analyzer.

[0094] The calibration curve, which was obtained in advance by measuring a standard solution in order to calculate the lactic acid concentration, is the following equation (9), which was obtained in advance. When creating the calibration curve, a solution was simulated as close as possible to the cell culture medium components, with a phosphate-buffered saline base containing 137 mM NaCl and 27 mM KCl, respectively, and lactic acid concentrations of 9, 45, 90, 180, 450, and 900 mg / dl. The calibration curve was calculated from the spectral intensity obtained by measurement using the simulated cell culture medium to which lactic acid had been added and the weighed concentrations of the added lactic acid of 9, 45, 90, 180, 450, and 900 mg / dl. Lactic acid concentration (mM) = 0.0192 × spectral intensity - 3.8465 Equation (9)

[0095] Table 8 shows the characteristic wave number of lactic acid, 857 cm -1 The spectral intensity and height before correction are shown below. The spectral intensity, height, and calculated concentration of the baseline to be calculated are shown below.

[0096] [Table 8]

[0097] The following equation is obtained from the above equations (1), (2), and (3): Corrected spectral intensity = (-0.0157 × T - 2.91) × ν + (43.7 × T + 8509) Equation (10) was used to calculate the following: as ν:857 d4: Corrected spectral intensity = (-0.0157 × 96 - 2.911) × 857 + (43.7 × 96 + 8509) = 8917.8 d5: Corrected spectral intensity = (-0.0157 × 120 - 2.911) × 857 + (43.7 × 120 + 8509) = 9643.7 Similarly, the concentrations of d6, d7, and d8 were calculated. From the corrected spectral intensities, the wavenumber specific to lactic acid, 857 cm -1 The spectral intensity of lactic acid after correction was calculated, and the calculated concentration of lactic acid was then calculated.

[0098] [Table 9]

[0099] A method for calculating the spectral intensity of lactic acid will be described. FIG. 16 is a diagram illustrating the calculation of the spectral intensity of lactic acid at a certain incubation time. FIG. 17 is a diagram illustrating the calculation of the corrected spectral intensity of lactic acid at a certain incubation time obtained in FIG. 16. In FIG. 16, (A) represents the spectral intensity before correction in Table 8, (B) represents the spectral intensity of the baseline used to calculate the height before correction in Table 8, and (C) represents the height obtained by subtracting (B) from (A) in Table 8. In FIG. 17, (D) represents the spectral intensity after correction in Table 9, (E) represents the spectral intensity of the baseline used to calculate the height after correction in Table 9, and (F) represents the corrected height obtained by subtracting (E) from (D) in Table 9 (corrected lactic acid spectral intensity). In FIGS. 16 and 17, the spectral intensity of lactic acid is the sum of the spectral intensity of lactic acid and the spectral intensity of the baseline.

[0100] Even if the baseline fluctuates, the spectral intensity of (D) is also composed of the ratio of (B) / (A), and the corrected baseline spectral intensity (E) is calculated. That is, (E) = (D) × (I) / (A) The corrected baseline spectral intensity (O) is calculated as follows:

[0101] The corrected spectral intensity of lactic acid (F) can be calculated by subtracting the calculated (E) from (D). The calculated concentration of lactic acid can be calculated from the spectral intensity of (F).

[0102] Table 10 shows the results of comparing lactate concentrations with and without correction. Figure 18 shows the results of comparing lactate concentrations with and without correction.

[0103] [Table 10]

[0104] From Table 10 and Figure 18, it is clear that the corrected values ​​are relatively closer to the blood gas meter values ​​compared to the uncorrected values.

[0105] <Modification> In the above examples, a specific cell culture medium was used, however, the present invention can of course be used with other cell culture media.

[0106] In the above example, 800 cm -1 and 2700 cm -1 However, the present invention can also extract spectral intensities at other wavenumbers, for example, 800 cm -1 , 600cm -1 , 400cm -1 and one selected from the group consisting of 2700 cm -1 , 2600cm -1 , 2500cm -1 The spectral intensity can be detected by one selected from the group consisting of: The spectral intensity may be measured by any combination of these.

[0107] In the above examples, the time-dependent changes were measured for a specific number of days (or hours), but the present invention can measure the time-dependent changes in the spectrum for other number of days (or hours).

[0108] The above configuration is based on the following findings made by the present inventors: [1] It was found that the line segment showing the relationship between the spectral intensity at two wavenumbers in the Raman spectrum of cell culture media containing glucose or lactate showed a linear change over time as the incubation time changed. [2] The baseline of the Raman spectrum of the cell culture medium, which changes over time, was considered to be a straight line, and the "slope" and "intercept" were calculated. A time-dependent relationship was found as the culture time changed. [3] We found that the baseline change over time can be corrected from the "slope" and "intercept" of the baseline, which shows time dependence, rather than calculating the spectral intensity. [4] The baseline change over time in culture was 1127 cm -1 It was found that the change in the baseline, plotted as a line segment showing time dependence, affects the glucose concentration in the blood. It was also found that the Raman spectral intensity of glucose can be corrected from the "slope" and "intercept" of the baseline change, and that the glucose concentration can be calculated from the corrected Raman spectral intensity of glucose. [5] The baseline change over time in culture was 857 cm -1 It was found that the Raman spectral intensity of lactic acid can be corrected from the "slope" and "intercept" of the baseline change, which is plotted as a line segment showing time dependence, and the concentration of lactic acid can be calculated from the corrected Raman spectral intensity of lactic acid.

[0109] The first embodiment of the present invention is 1. A method for calculating corrected spectral intensities from a Raman spectrum of a cell culture medium containing glucose, comprising: Irradiating the cell culture medium with excitation light of a single wavelength to obtain a Raman spectrum; extracting a spectral intensity at a first wavenumber and a spectral intensity at a second wavenumber for at least two incubation times; the first wavenumber and the second wavenumber are set so that a line segment showing the relationship between the spectral intensity at the first wavenumber and the spectral intensity at the second wavenumber shows a linear change over time due to a change in the culture time, From the line, 1127 cm -1 A method for calculating the spectral intensity of glucose in a

[0110] This allows for the calculation of corrected spectral intensities from the Raman spectrum of a glucose-containing cell culture medium, thereby correcting for variations in the Raman spectrum over time. By correcting for variations in the spectrum from one measurement to the next and correcting for baseline variations, more accurate glucose spectral intensities can be calculated. This reduces the discrepancy between the measured spectrum and the actual spectrum. For example, a relational equation can be calculated in advance using statistical processing software, and the results of the equation representing the variations can be automatically input to track continuous changes in the calculated concentration. Alternatively, a least-squares algorithm can be incorporated into the program in advance to continuously calculate the relational equation. To track continuous changes, as described above, it is necessary to use the least-squares method in real time.

[0111] In a second embodiment of the present invention, the first wave number is 800 cm -1 , 600cm -1 , 400cm -1 The compound is one selected from the group consisting of:

[0112] In this application, the analytes to be measured are glucose and lactic acid. Although the analytes not to be measured are also contained in the cell culture medium components, no substances are detected at the relevant wavenumbers. Therefore, other analytes that are not to be measured under the conditions of cell culture medium measurement containing glucose are not detected. Raman The analysis can be performed without detecting the spectrum. Raman Since no spectrum is detected, the spectral intensity of glucose can be calculated without being affected by the spectral intensities of other components.

[0113] In a third embodiment of the present invention, the second wave number is 2700 cm -1 , 2600cm -1 , 2500cm -1The compound is one selected from the group consisting of:

[0114] In this application, the analytes targeted for measurement are glucose and lactic acid. Although analytes not targeted for measurement are also contained in the cell culture medium components, no substances are detected at the relevant wavenumbers. Therefore, other analytes are not detected under the conditions of cell culture medium measurement. Raman The analysis can be performed without detecting the spectrum. Raman Since no spectrum is detected, the spectral intensity of glucose can be calculated without being affected by the spectral intensities of other components.

[0115] In a fourth embodiment of the present invention, the 1127 cm -1 From the spectral intensity of glucose at 1127cm -1 The glucose concentration is calculated using a calibration curve showing the relationship between the glucose concentration and the spectral intensity in the sample.

[0116] This allows for more accurate calculation of the glucose concentration by correcting for fluctuations in the Raman spectrum of the cell culture medium over time, i.e., the glucose concentration can be calculated with the background or baseline effect corrected in the Raman spectrum of the cell culture medium.

[0117] A fifth embodiment of the present invention is Irradiating the cell culture medium with excitation light of a single wavelength to obtain a Raman spectrum; extracting a spectral intensity at a first wavenumber and a spectral intensity at a second wavenumber for at least two incubation times; the first wavenumber and the second wavenumber are set so that a line segment showing the relationship between the spectral intensity at the first wavenumber and the spectral intensity at the second wavenumber shows a linear change over time due to a change in the culture time, From the line, 857 cm -1 A method for calculating the spectral intensity of lactic acid in a

[0118] This allows for the calculation of corrected spectral intensities from the Raman spectrum of a cell culture medium containing lactate, and for correction of variations due to changes in the Raman spectrum over time. By correcting for variations in each spectral measurement and correcting for baseline variations, more accurate lactate spectral intensities can be calculated. This reduces the discrepancy between the measured spectrum and the actual spectrum. For example, a relational equation can be calculated in advance using statistical processing software, and the results of the equation showing the variations can be automatically input to track continuous changes in the calculated concentration. Alternatively, a least-squares algorithm can be incorporated into the program in advance to continuously calculate the relational equation. To track continuous changes, as described above, it is necessary to use the least-squares method in real time.

[0119] In a sixth embodiment of the present invention, the first wave number is 800 cm -1 , 600cm -1 , 400cm -1 The compound is one selected from the group consisting of:

[0120] In this application, the analytes to be measured are glucose and lactic acid. Although the analytes not to be measured are also contained in the cell culture medium components, no substances are detected at the relevant wavenumbers. Therefore, other analytes that are not to be measured under the conditions of cell culture medium measurement containing lactic acid are not detected. Raman The analysis can be performed without detecting the spectrum. Raman Since no spectrum is detected, the spectral intensity of lactic acid can be calculated without being affected by the spectral intensities of other components.

[0121] In a seventh embodiment of the present invention, the second wave number is 2700 cm -1 , 2600cm -1 , 2500cm -1 The compound is one selected from the group consisting of:

[0122] In this application, the analytes to be measured are glucose and lactic acid. Although the analytes not to be measured are also contained in the cell culture medium components, no substances are detected at the relevant wavenumbers. Therefore, other analytes that are not to be measured under the conditions of cell culture medium measurement containing lactic acid are not detected. Raman The analysis can be performed without detecting the spectrum. Raman Since no spectrum is detected, the spectral intensity of lactic acid can be calculated without being affected by the spectral intensities of other components.

[0123] In the eighth embodiment of the present invention, the 857 cm -1 From the spectral intensity of lactic acid at 857cm -1 The concentration of lactic acid is calculated using a calibration curve showing the relationship between the concentration of lactic acid and the spectral intensity in the sample.

[0124] This allows for the calculation of corrected spectral intensities from the Raman spectrum of the cell culture medium containing lactic acid, and allows for more accurate calculation of the concentration of lactic acid by correcting for fluctuations in the Raman spectrum due to changes over time. That is, the lactate concentration can be calculated by correcting for the influence of the background or baseline in the Raman spectrum of the cell culture medium containing lactic acid. [Explanation of symbols]

[0125] 10 Analytical mechanism 12 light source 14 Reflector 16 samples 18 Optical system 20 Monochromator 22 Diffraction Grating 24 CCD detectors 26 Analyzer 30 Input section 32 Storage section 34 Control Unit 36 Display section 40 Acquisition Department 42 Extraction part 44 Component Identification Unit 46 Calculation section 48 Notification Department

Claims

1. 1. A method for calculating corrected spectral intensities from a Raman spectrum of a cell culture medium containing glucose, comprising: Irradiating the cell culture medium with excitation light of a single wavelength to obtain a Raman spectrum; extracting a spectral intensity at a first wavenumber and a spectral intensity at a second wavenumber for at least two incubation times; the first wave number and the second wave number are set so that a line segment showing the relationship between the spectral intensity at the first wave number and the spectral intensity at the second wave number shows a linear change over time due to a change in the culture time, From the line, 1127 cm -1 1. A method for calculating a spectral intensity of glucose at The first wave number is 800±6 cm -1 , 600±6 cm -1 , 400±6 cm -1 is one selected from the group consisting of The second wave number is 2700±6 cm -1 , 2600±6 cm -1 , 2500±6 cm -1 is one selected from the group consisting of The method, wherein the first wavenumber and the second wavenumber are free of other Raman spectral detection under cell culture medium measurement conditions.

2. Furthermore, the above 1127 cm -1 From the spectral intensity of glucose at 1127 cm -1 The method according to claim 1 , wherein the glucose concentration is calculated using a calibration curve showing the relationship between the glucose concentration and the spectral intensity in a sample.

3. Irradiating the cell culture medium with excitation light of a single wavelength to obtain a Raman spectrum; extracting a spectral intensity at a first wavenumber and a spectral intensity at a second wavenumber for at least two incubation times; a third wavenumber and a fourth wavenumber are set so that a line segment showing the relationship between the spectral intensity at the first wavenumber and the spectral intensity at the second wavenumber shows a linear change over time due to a change in the culture time; From the line, 857 cm -1 The method according to claim 1 or 2, wherein the spectral intensity of lactic acid is calculated.

4. The third wave number is 800±6 cm -1 , 600±6 cm -1 , 400±6 cm -1 4. The method of claim 3, wherein the ion exchange reaction is one selected from the group consisting of:

5. The fourth wave number is 2700±6 cm -1 , 2600±6 cm -1 , 2500±6 cm -1 5. The method of claim 3 or 4, wherein the hydroxyl group is one selected from the group consisting of:

6. Furthermore, the above 857 cm -1 From the spectral intensity of lactic acid at 857 cm -1 The method according to any one of claims 3 to 5, wherein the concentration of lactic acid is calculated using a calibration curve showing the relationship between the concentration of lactic acid and the spectral intensity in a sample.

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