Method for detecting enzyme activity of CYP enzymes in cells

Abstract

The present invention provides a method for evaluating the intracellular or extracellular activity of CYP enzymes, the method comprising a step for determining the number of molecules of oxidation-type CYP enzymes.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting the activity of CYP metabolic enzymes by utilizing a Stokes Raman scattering signal (hereinafter also referred to as a Raman scattering signal or a Raman signal) that indicates the activity of CYP metabolic enzymes. [Background technology]

[0002] Cytochrome p450 (CYP), a group of enzymes responsible for the metabolism of drugs and toxins, is a hemoprotein expressed in hepatocytes and small intestinal epithelial cells and localized in the endoplasmic reticulum (ER). 57 CYP genes are known in humans. CYPs form a gene superfamily consisting of multiple molecular species (enzyme groups) with different properties, including substrate specificity.

[0003] Traditionally, CYP detection and analysis have relied on spectroscopy using absorbance spectroscopy or electromagnetic paramagnetic resonance, fluorescence immunostaining, luminescence analysis utilizing metabolic reactions of CYPs, and X-ray analysis. However, to increase the detection sensitivity of the target CYPs, CYP proteins must be extracted, purified, or labeled, which is invasive. These tests destroy cellular tissue and can only be applied to a single cell once. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] David B. Hawkes, Gregory W. Adams, Alma L. Burlingame, Paul R. Ortiz de Montellano, James J. De Voss, “Cytochrome P450cin (CYP176A), Isolation, Expression, and Characterization”, Journal of Biological Chemistry, Volume 277, Issue 31, 2002, Pages 27725-27732, [online], [retrieved on 2021-06-04]. Retrieved from<https: / / doi.org / 10.1074 / jbc.M203382200>

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[0005] The inventors have found the following problem. In conventional CYP expression measurement using Raman spectroscopy, CYP proteins have been extracted and purified to improve the detection accuracy of the target CYP. This requires invasive and destructive procedures. Furthermore, conventional techniques using such invasive and destructive procedures make it difficult to measure the enzymatic activity of CYP enzymes in living cells.

[0006] An objective of the present invention is to provide a method for detecting the enzymatic activity of CYP enzymes in cells with high resolution without relying on invasion or labeling. [Means for solving the problem]

[0007] As a result of extensive research into the above-mentioned problems, the present inventors have found that (1) The enzymatic activity of CYP enzymes correlates with the number of oxidized CYP enzyme molecules. (2) By measuring the number of oxidized CYP enzyme molecules in living cells using Raman signals, the enzymatic activity of CYP enzymes in the cells can be evaluated non-invasively; and (3) We discovered that by simultaneously detecting Raman signals from multiple biomolecules in living cells, we can non-invasively and multifacetedly analyze the state of cells (e.g., the degree of drug response, the degree of differentiation / undifferentiation, etc.). Based on this finding, we conducted further research and completed the present invention.

[0008] That is, the present invention is as follows.

[0009] <1> A method for evaluating the enzymatic activity of intracellular or extracellular CYP enzymes, which comprises a step of measuring the number of molecules of oxidized CYP enzymes.

[0010] <2> In the case of evaluating the enzymatic activity of intracellular CYP enzymes, the step of measuring the number of molecules of oxidized CYP enzymes comprises: Irradiating the cells with excitation light and acquiring a Raman spectrum using a photodetector; and extracting Raman scattering signals derived from the CYP enzyme group from the Raman spectrum; <1> The method described.

[0011] <3> The wavenumber of the Raman scattering signal derived from the CYP enzyme group is 300-600, 620-880, 920-1320, or 1320-1660 cm -1 is within the range of <1> or <2> The method described.

[0012] <4> The wave number is 1370 cm -1 or 1636 cm -1 That is, <3> The method described.

[0013] <5> The cells are derived from any one of the liver, small intestine, kidney, and brain. <1> ~ <4> 1. The method according to claim 1 ,

[0014] <6> The cells are derived from the liver. <5> The method described.

[0015] <7> The cells are cells derived from pluripotent stem cells. <1> ~ <4> 1. The method according to claim 1 ,

[0016] <8> The method further comprises a step of observing at least one selected from the group consisting of cell shape, cell size, and intracellular distribution of intracellular components in the region where the number of molecules of the CYP enzyme group is measured. <1> ~ <7> 1. The method according to claim 1 ,

[0017] <9> further extracting Raman scattering signals derived from substances other than the CYP enzyme group; <1> ~ <9> 1. The method according to claim 1 ,

[0018] <10> the substance other than the CYP enzymes is at least one selected from the group consisting of reduced heme b, reduced / oxidized heme c, glycogen, reduced / oxidized cytochrome c, phenylalanine, and lipids; <9> The method described.

[0019] <11> A method for assessing metabolic capacity of hepatic parenchymal cells, comprising the steps of: Irradiating the liver parenchymal cells with excitation light and acquiring a Raman spectrum using a photodetector; and A step of detecting Raman signals of biomolecules related to the metabolic capacity of liver parenchymal cells from the Raman spectrum.

[0020] <12> The biomolecule involved in the metabolism of hepatic parenchymal cells is at least one selected from the group consisting of CYP enzymes, glycogen, cytochrome b5, cytochrome c, lipids, and phenylalanine. <11> The method described.

[0021] In another aspect, the present invention is as follows.

[0022] <1'> A detection method for detecting the enzymatic activity of CYP enzymes by detecting oxidized CYP enzymes.

[0023] <2'> The detection method according to <1'>, which detects the enzymatic activity of the oxidized CYP enzyme group inside and outside the cell using a Raman spectrum containing a Raman scattering signal having a specific wavenumber that indicates the enzymatic activity of the oxidized CYP enzyme group.

[0024] <3'> The detection method according to <1'> or <2'>, wherein the wavenumber of the Raman spectrum is derived from the type b oxidized heme bound to the CYP enzyme group.

[0025] <4'> The specific wavenumber is a wavenumber of 300-600, 620-880, 920-1320, or 1320-1660 cm -1 The detection method according to any one of <1'> to <3'>, wherein the range is:

[0026] <5'> The specific wave number is 1370 cm -1 or 1636 cm -1 The detection method according to <4'>,

[0027] <6'> The detection method according to any one of <1'> to <5'>, wherein the cells are cells having CYP activity.

[0028] <7'> The detection method according to <6'>, wherein the cells having CYP activity are cells derived from any one of the liver, small intestine, kidney, and brain.

[0029] <8'> The detection method according to any one of <1'> to <7'>, wherein the cells are derived from pluripotent stem cells, such as iPS cells and ES cells.

[0030] <9'> The detection method according to any one of <1'> to <8'>, wherein the cells are hepatic parenchymal cells.

[0031] <10'> The detection method according to any one of <1'> to <7'>, wherein the cells are a cell line derived from a human liver tumor or cells differentiated therefrom.

[0032] <11'> The detection method according to any one of <1'> to <10'>, wherein, in addition to detecting the enzymatic activity of the CYP enzyme group, at least one of cell shape, cell size, and intracellular distribution of intracellular components is observed in the region where the enzymatic activity of the CYP enzyme group is detected.

[0033] <12'> The detection method according to any one of <1'> to <11'>, wherein, simultaneously with the detection of the enzymatic activity of the CYP enzyme group, another molecule or its redox state is detected using another Raman scattering signal contained in the Raman spectrum.

[0034] <13'> The detection method according to <12'>, wherein the other Raman scattering signal having a specific wavenumber indicating the other molecule is derived from any one of reduced heme b, reduced / oxidized heme c, glycogen, reduced / oxidized cytochrome c, phenylalanine, and lipid.

[0035] <14'> The wave number of the Raman scattering signal derived from the reduced heme b is 650-680 cm -1 , 900-1000cm -1 , 1300-1373cm -1 , 1490-1500cm -1 or 1570-1590cm -1 The detection method according to <13'>, wherein the range is:

[0036] <15'> The wavenumber of the Raman scattering signal derived from the reduced / oxidized heme c is 590-640 cm -1 ,730-755cm -1 , 1120-1130cm -1 , 1310-1370cm -1 , or 1580-1640cm -1 The detection method according to <13'> or <14'>, wherein the range is:

[0037] <16'> The wave number of the Raman scattering signal derived from the glycogen is 440-580 cm -1 , 840-945cm -1 , 1020-1660cm -1 , or 2900-2940cm -1 The detection method according to any one of <13'> to <15'>, wherein the range is:

[0038] <17'> A step of irradiating the cells with excitation light and acquiring a Raman spectrum using a photodetector; and extracting Raman scattering signals derived from the CYP enzyme group from the Raman spectrum, In the step of extracting the Raman scattering signal, A peak and waveform of a specific wavenumber of the Raman spectrum are identified, and the intersection of a line connecting both ends of the base of the peak and a line drawn from the peak apex perpendicularly to the wavenumber axis is set as the origin, and the height from the origin to the peak apex is calculated as the Raman scattering intensity. The detection method according to any one of <2'> to <16'>.

[0039] <18'> A step of irradiating the cell with excitation light and acquiring a Raman spectrum using a photodetector; and extracting Raman scattering signals derived from the CYP enzyme group from the Raman spectrum, In the step of extracting the Raman scattering signal, A peak and a waveform of a specific wavenumber of the Raman spectrum are identified, and the area of ​​a region surrounded by a line connecting both ends of the base of the peak and the waveform of the Raman spectrum after the noise removal is calculated as the Raman scattering intensity. The detection method according to any one of <2'> to <16'>.

[0040] <19'> The detection method according to <17'> or <18'>, further comprising evaluating at least one of the type, differentiation level, and maturity level of a cell or tissue using Raman scattering signals of glycogen and cytochrome c extracted from the Raman spectrum.

[0041] <20'> A method for detecting the enzymatic activity of purified CYP proteins in an environment simulating an intracellular environment, using Raman spectra having specific wavenumbers that indicate the enzymatic activity of CYP enzymes. [Effects of the Invention]

[0042] According to the present invention, the enzymatic activity of CYP enzymes in cells can be detected with high resolution without invasiveness or labeling. [Brief explanation of the drawings]

[0043] [Figure 1] FIG. 1 is a diagram illustrating Raman spectrum detection. [Figure 2] 10 is a flowchart illustrating a method for extracting a Raman scattering signal. [Figure 3] An example of the measurement results of the Raman spectrum is shown below. [Figure 4] Method 1 for extracting Raman scattering signals is shown. [Figure 5] Method 2 for extracting Raman scattering signals is shown below. [Figure 6] Raman spectra of liver parenchymal cells with CYP3A4 induction. [Figure 7] Graph showing the results of CYP3A4 measurement by luminescence method. [Figure 8] Western blotting results. [Figure 9] Raman spectra of liver parenchymal cells and bile duct epithelial cells. [Figure 10] Raman image constructed based on specific Raman peaks. Wavenumber 600 cm-1: reduced heme c, mainly reduced cytochrome c; wavenumber 675 cm-1: reduced heme b, mainly reduced cytochrome b5; wavenumber 940 cm-1: glycogen; wavenumber 1636 cm-1: oxidized heme b, mainly oxidized CYP enzymes. [Figure 11]Raman image constructed based on specific Raman peaks. Wavenumber 600 cm-1: reduced heme c, mainly reduced cytochrome c; wavenumber 675 cm-1: reduced heme b, mainly reduced cytochrome b5; wavenumber 940 cm-1: glycogen; wavenumber 1000 cm-1: phenylalanine; wavenumber 1370 cm-1: oxidized heme b, mainly oxidized CYP enzymes; wavenumber 1636 cm-1: oxidized heme b, mainly oxidized CYP enzymes. [Figure 12] Immunostaining image of CYP3A4. [Figure 13] Raman images of HepaRG cells with CYP enzymes induced at rifampicin concentrations of 0, 0.04, 0.4, and 4 μM are shown. Wavenumber 1636 cm-1: Oxidized heme b, mainly oxidized CYP enzymes. [Figure 14] Graph showing the activity and Raman signals of CYP enzymes in HepaRG cells. [Figure 15] The detected Raman spectra were averaged for each cell, and the Raman signal at a wavenumber of 1636 cm-1 was plotted as a boxplot. [Figure 16] Graph showing the activity and Raman signals of CYP enzymes in HepaRG cells cultured with different inducers. [Figure 17] The detected Raman spectra were averaged for each cell, and the Raman signal at a wavenumber of 1636 cm-1 was plotted as a boxplot. [Figure 18] Graph showing the activity and Raman signals of CYP enzymes in HepaRG cells in which the expression of CYP enzymes was downregulated using IL-6. [Figure 19] The detected Raman spectra were averaged for each cell, and the Raman signal at a wavenumber of 1636 cm-1 was plotted as a boxplot. [Figure 20] Graph showing the activity of CYP enzymes in HepaRG cells and the change in Raman signal over time. [Figure 21] The detected Raman spectra were averaged for each cell, and the Raman signal at a wavenumber of 1636 cm-1 was plotted as a boxplot. [Figure 22]Graph showing the activity of CYP enzymes in HepaRG cells in which the activity of CYP enzymes was downregulated using Azamulin. [Figure 23] The detected Raman spectra were averaged for each cell, and the Raman signal at a wavenumber of 1636 cm-1 was plotted as a boxplot. [Figure 24] Comparison of Raman image and immunostaining results. Wavenumber 1636 cm-1: Oxidized heme b, mainly oxidized CYP enzymes. [Figure 25] Raman peaks derived from various biomolecules observed in human hepatocytes. [Figure 26] Raman images of various biomolecules. Wavenumber 675 cm-1: reduced heme b, mainly reduced cytochrome b5; wavenumber 940 cm-1: glycogen; wavenumber 1636 cm-1: oxidized heme b, mainly oxidized CYP enzymes; wavenumber 600 cm-1: reduced heme c, mainly reduced cytochrome c; wavenumber 1000 cm-1: phenylalanine; wavenumber 2850 cm-1: lipids. [Figure 27] Graph showing the Raman signal of frozen primary human hepatocytes (PHH) and HepaRG cells. [Figure 28] Image showing the distribution of each molecule within PHH and HepaRG cells, obtained by visualizing the Raman signals corresponding to each spectrum. Wavenumber 600 cm-1: reduced heme c, mainly reduced cytochrome c; wavenumber 675 cm-1: reduced heme b, mainly reduced cytochrome b5; wavenumber 750 cm-1: all cytochromes; wavenumber 1636 cm-1: oxidized heme b, mainly oxidized CYP enzymes; wavenumber 1000 cm-1: phenylalanine, mainly proteins; wavenumber 1157 cm-1: carotenoids; wavenumber 1512 cm-1: carotenoids. [Figure 29] Graph showing the Raman signals of liver-like cells (HLCs) and HepaRG cells. [Figure 30]Image showing the distribution of each molecule within HLC and HepaRG cells, obtained by visualizing the Raman signals corresponding to each spectrum. Wavenumber 600 cm-1: reduced heme c, mainly reduced cytochrome c; wavenumber 675 cm-1: reduced heme b, mainly reduced cytochrome b5; wavenumber 750 cm-1: all cytochromes; wavenumber 1636 cm-1: oxidized heme b, mainly oxidized CYP enzymes; wavenumber 1000 cm-1: phenylalanine. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be appropriately modified and implemented without departing from the spirit of the present invention. All publications cited in this specification, such as prior art documents, public announcements, patent publications, and other patent documents, are incorporated herein by reference.

[0045] The present invention provides a method for evaluating the enzymatic activity of intracellular or extracellular CYP enzymes, which comprises a step of measuring the number of molecules of oxidized CYP enzymes (hereinafter, sometimes referred to as the "evaluation method of the present invention").

[0046] First, the present inventors were the first in the world to discover that "the enzymatic activity of CYP enzymes correlates with the number of molecules of the oxidized form of the CYP enzymes" (specifically, this is demonstrated by the data shown in Example 6 of the present specification). In other words, the present inventors discovered that when CYP enzymes are present in an intracellular or extracellular environment, the enzymatic activity of the CYP enzymes can be evaluated by measuring the "number of molecules" of the oxidized form of the CYP enzymes using an appropriate method.

[0047] In the evaluation method of the present invention, the method used to measure the number of oxidized CYP enzyme molecules in the CYP enzyme group is not particularly limited, and any method known per se may be used. Examples include, but are not limited to, infrared spectroscopy, labeling with fluorescent or luminescent substances, and mass spectrometry. In an embodiment for evaluating the enzymatic activity of intracellular CYP enzymes, Raman spectroscopy, which allows non-destructive analysis, is preferably used. One spectroscopic technique is the identification of the redox state of cytochrome P450 using absorption spectroscopy (Non-Patent Document 1).

[0048] In one embodiment of the evaluation method of the present invention, the enzymatic activity of intracellular CYP enzymes can be evaluated. In this embodiment, Raman spectroscopy can be used in the step of measuring the number of molecules of oxidized CYP enzymes. More specifically, the method for measuring the number of molecules of oxidized CYP enzymes includes the steps of irradiating cells with excitation light and obtaining a Raman spectrum using a photodetector, and extracting Raman scattering signals derived from the CYP enzymes from the Raman spectrum, thereby measuring the number of molecules of oxidized CYP enzymes. In this embodiment, a Raman spectrum containing Raman scattering signals having a specific wavenumber is used to evaluate the enzymatic activity of CYP enzymes in the intracellular environment.

[0049] First, the CYPs to be evaluated by the evaluation method of this embodiment will be described. Here, the CYPs to be evaluated in one aspect of the present invention are CYPs derived from any and all organisms. Unless otherwise specified, the term "CYP enzyme group" used herein refers to CYPs derived from any and all organisms. CYPs are hemoproteins that contain heme as a prosthetic molecule.

[0050] An evaluation method according to one embodiment of the present invention detects a "redox state" derived from a molecular structure specific to CYP molecules. The molecular structure specific to CYP molecules is as follows: There are subtypes (e.g., type a, type b) depending on the porphyrin structure of the heme, and CYP has a type b heme structure. CYPs form a gene superfamily consisting of multiple molecular species (enzymes) with different properties, including substrate specificity. 57 types of CYP genes are known in humans. Of the 57 types of CYP genes, the main CYP enzymes involved in the metabolism of drugs and toxic substances include CYP3A4, CYP1A2, and CYP2B6. In humans, CYP enzymes are mainly expressed in hepatocytes in the liver and intestinal epithelial cells and are localized in the endoplasmic reticulum (ER). In particular, CYP3A4 is most highly expressed in the human liver, accounting for 35% of the entire CYP family (Non-Patent Document 2). In an evaluation method according to one embodiment of the present invention, the total intensity of Raman scattered light at specific Raman shifts is detected for all CYP molecules contained in a CYP enzyme group, without distinguishing between the types of molecules.

[0051] In one embodiment, the "CYP enzymes" whose activity is evaluated by the evaluation method of the present invention are the 57 human CYP enzymes. In another embodiment, the "CYP enzymes" may be CYP enzymes, among the 57 human CYP enzymes, whose expression is induced by an inducer known to induce the expression of CYP enzymes. (Specific examples of such inducers include, but are not limited to, rifampicin, phenytoin, carbamazepine, and dexamethasone.) The inventors have confirmed that the four inducers listed above induce the gene expression of at least one of four CYP enzymes selected from the group consisting of CYP3A4, CYP1A2, CYP2B6, and CYP2C9. The CYP enzyme whose expression is induced by an inducer may be at least one selected from the group consisting of CYP3A4, CYP1A2, CYP2B6, and CYP2C9.

[0052] In one embodiment of the present invention, the "CYP enzyme group" may be any one of the following (1) to (15): (1) Human CYP3A4 (2) Human CYP1A2 (3) Human CYP2B6 (4) Human CYP2C9 (5) Human CYP3A4 and human CYP1A2 (6) Human CYP3A4 and human CYP2B6 (7) Human CYP3A4 and human CYP2C9 (8) Human CYP1A2 and human CYP2B6 (9) Human CYP1A2 and human CYP2C9 (10) Human CYP2B6 and human CYP2C9 (11) Human CYP3A4, human CYP1A2, and human CYP2B6 (12) Human CYP3A4, human CYP1A2, and human CYP2C9 (13) Human CYP3A4, human CYP2B6, and human CYP2C9 (14) Human CYP1A2, human CYP2B6, and human CYP2C9 (15) Human CYP3A4, human CYP1A2, human CYP2B6, and human CYP2C9.

[0053] The evaluation method of the present invention can evaluate the metabolic enzyme activity of not only human CYP3A4 but also CYPs derived from any organism. Heme b is an iron porphyrin complex with an iron atom as the central metal. One highly reactive oxygen atom is bound to the iron atom of heme b. CYPs introduce one oxygen atom into target compounds such as drugs and poisons, oxidizing the compound, thereby increasing the water solubility of the compound and promoting its excretion from the body. Therefore, the iron atom of heme b undergoes a redox reaction to become a trivalent oxidized form (Fe 3+ ) and the divalent reduced form (Fe 2+ In the evaluation method of the present invention, the metabolic enzymatic activity of CYP enzymes is evaluated based on the measurement results of the redox state of CYP enzymes.

[0054] The detection principle used in the evaluation method of this embodiment is Raman spectroscopy. Raman spectroscopy enables non-destructive, stain-free, and non-invasive measurement of CYP enzyme activity. In Raman spectroscopy, Raman scattered light is generated when an object to be measured is irradiated with excitation light. The spectrum obtained from the Raman scattered light has peaks at multiple specific wavenumbers. These peaks are derived from chemical bonds within and between molecules.

[0055] In this embodiment, the "Raman scattering signal having a specific wavenumber" is derived from oxidized heme type b (hereinafter also referred to as oxidized heme b), which is a prosthetic group bound to the CYP enzyme. Specifically, the specific wavenumber is preferably 300-600, 620-880, 920-1320, or 1320-1660 cm -1 More preferably, the wavenumber is in the range of 1360-1380 or 1630-1640 cm -1 Particularly preferred are wave numbers of 1370 or 1636 cm -1 In a specific embodiment, first, cells are irradiated with excitation light, and the resulting Raman scattered light is dispersed by a diffraction grating. Next, the Raman spectrum detected by the detection device is analyzed by, for example, the wavelength range of 1630-1640 cm. -1 A Raman scattering signal (Raman scattering intensity) is obtained at this wavenumber. The fact that a Raman scattering signal is obtained at this wavenumber indicates that the CYP enzymes have enzymatic activity. The bond between the CYP enzyme and heme b is a covalent or non-covalent bond.

[0056] In this embodiment, the cell shape, size, and intracellular distribution of intracellular components may be observed in the region where the CYP enzyme activity is evaluated, along with the evaluation of the CYP enzyme activity. Here, the "region where the CYP enzyme activity is evaluated" refers, for example, to a plane of a predetermined size, X μm wide and Y μm long, containing cells spread on a substrate, as shown in FIG. 1. While FIG. 1 shows a single cell placed in the region, multiple cells may be placed. Observation of the cell shape, size, and intracellular distribution of intracellular components can be performed using, for example, optical measurement methods. Optical measurement methods refer to methods for generating images using bright field ( FIG. 26 ) or fluorescence ( FIG. 12 , FIG. 24 ). The combined use of optical measurement methods allows for the localization of the target cell population and the identification of the target cell population (liver parenchymal cell portion), thereby achieving efficient measurement and highly sensitive signal extraction. Furthermore, individual cell identification (cell outline extraction) is also possible, enabling analysis such as quantification of cell activity. Furthermore, by fluorescently staining the organelles and analyzing the acquired fluorescence and Raman images, it is possible to analyze the distribution of intracellular organelles and CYP enzyme activity.

[0057] In this embodiment, other molecules or their redox states may be detected using other Raman scattering signals contained in the Raman spectrum simultaneously with the evaluation of the enzymatic activity of the CYP enzymes. The Raman scattering signals having specific wavenumbers indicating molecules may be derived from any of reduced / oxidized heme b, reduced / oxidized heme c, glycogen, cytochrome c, phenylalanine, and lipids.

[0058] Other substances to be evaluated using the evaluation method of this embodiment will be described. In this embodiment, in addition to CYPs, cytochrome c and glycogen can be used as indicators of liver metabolic capacity. Metabolism is a reaction that produces the energy necessary for life support and synthesizes necessary polymer compounds, and a thorough understanding of this is necessary for the development of medical technology and drug discovery. Luminescence and fluorescence methods have been widely used to measure cytochrome c and glycogen. In recent years, unstained measurements using Raman spectroscopy have been attracting attention. Raman spectroscopy utilizes the characteristic spontaneous Raman scattering of substances. In particular, in the evaluation of hepatotoxicity and hepatic metabolism, drug metabolism by CYPs, mitochondrial activity or toxicity using cytochrome c as an indicator, and intracellular energy production and storage using glycogen as an indicator are important parameters that are correlated with each other. Non-Patent Document 3 discloses Raman observation of cytochrome c in cells undergoing apoptosis.

[0059] Cytochrome c is a heme c protein present in cells and tissues. It is localized in mitochondria, a type of intracellular organelle, and is involved in apoptosis and energy production. Toxic effects on mitochondria are a cause of drug-induced liver injury, and the quantification and localization of cytochrome c are important factors. Cytochrome c contains type c heme as a prosthetic molecule. It emits a characteristic Raman signal that is different from type b heme contained in CYPs and other proteins. In other words, the Raman scattering signal derived from the oxidized / reduced state of cytochrome c can be used as a biomolecule other than CYP enzymes.

[0060] Furthermore, type b heme proteins such as cytochrome b5 can also be detected. Cytochrome b5 plays an important role in the catalytic cycle of CYP enzymes by providing electrons. Another example is the detection of phenylalanine, which indicates cytoplasmic activity. The intensity of the phenylalanine Raman scattering signal reflects the density of the cytoplasm in the region observed by Raman.

[0061] The specific activity of CYP can be obtained by normalizing (dividing) the Raman scattering signal derived from CYP activity by the Raman scattering signal derived from these molecules. For example, if the Raman scattering signal of cytochrome c is used for normalization, the specific activity of CYP relative to mitochondrial activity can be obtained. If the Raman scattering signal of cytochrome b is used, the specific activity of CYP relative to catalytic activity can be obtained. If normalization is performed by the Raman scattering signal of glycogen, the specific activity of CYP relative to glucose metabolism can be obtained.

[0062] Furthermore, because cytochromes c and b, glycogen, and phenylalanine show specific distributions within cells that are derived from organelles (cytochrome c: distributed in mitochondria, cytochrome b: distributed in the endoplasmic reticulum, phenylalanine: distributed throughout the cytoplasm), it is expected that the relationship between each cellular function that can be measured from these Raman scattering signals and the shape and localization of the organelles can be utilized to quantify the state of the cell and drug response.

[0063] Here, we explain the difference in Raman scattering signals due to differences in the structure of heme. Reduced heme c exhibits a wavenumber of 604 cm. -1 , reduced heme b has a wavenumber of 675 cm -1 The reduced heme structure (common to b and c) has a wavenumber of 750 cm -1 The oxidized heme structure (common to b and c) has a wavenumber of 1638 cm -1 It has been reported that the CYP enzymes with heme structure or CYP3A4 have multiple characteristic Raman scattering signals, such as the wavenumber 1368 cm when they take on an oxidized form (Non-Patent Document 4). -1 , 1371-1373 cm -1 , 1490 cm -1 , 1500 cm -1 , 1570 cm -1 , 1590 cm -1 , 1630 cm -1 , 1640 cm -1 It has been reported that there is a characteristic Raman scattering signal around this region (Non-Patent Documents 5 to 12).

[0064] The Raman scattering signal originating from reduced / oxidized heme b is at 650-680 cm -1 , 900-1000 cm -1 , 1300-1373 cm -1 , 1490-1500 cm -1 or 1570-1590 cm -1 may be within the range (Non-Patent Document 13).

[0065] The wavenumber of the Raman scattering signal originating from reduced / oxidized heme c is 590-640 cm -1 ,730-755 cm -1 , 1120-1130 cm -1 , 1310-1370 cm -1 , or 1580-1640 cm -1 may be in the range of

[0066] Glycogen is a polymer synthesized to temporarily store excess glucose and is known to be synthesized mainly in the liver and skeletal muscle. It is an effective indicator of liver function and glucose metabolism. The wavenumber of the Raman scattering signal derived from glycogen is 440-580 cm -1 , 840-945 cm -1 , 1020-1660 cm -1 (More preferably, 1020-1120 cm -1 ), or 2900-2940 cm -1 may be in the range of

[0067] The wavenumber of the lipid-derived Raman scattering signal is 1450 cm -1 or 2850 cm -1 may be.

[0068] Hereinafter, a method for detecting Raman scattering from intracellular CYP enzymes and other substances, and a method for identifying a Raman scattering signal having a specific wavenumber from an acquired Raman spectrum will be described with reference to FIGS.

[0069] Figure 1 is a diagram explaining the principle of Raman scattering. As shown in Figure 1, when a sample is irradiated with excitation light of wavelength λ0, such as from a laser, scattering occurs in addition to reflection, refraction, and absorption. Of the scattered light of wavelength λ, light with the same wavelength as the incident light is called Rayleigh scattering. On the other hand, light with a reduced frequency compared to the incident light, i.e., whose wavelength is shifted to longer wavelengths, is called Stokes Raman scattering (hereinafter referred to as Raman scattered light). The Raman scattered light generated from the sample is dispersed using a diffraction grating and detected by a detection device such as a CCD image sensor to obtain a Raman spectrum. The Raman scattering signal is then extracted from the obtained Raman spectrum.

[0070] Examples of samples to be irradiated with excitation light include biological tissues (in vivo), cultured cells (in vitro), and fixed biological tissues. Examples of biological tissues include human or animal-derived liver tissue, intestinal tissue (preferably small intestinal tissue), kidney tissue, brain tissue, and cells contained therein. Examples of cultured cells include hepatocytes, small intestinal epithelial cells, bile duct epithelial cells, kidney cells, nerve cells, and glial cells. In one embodiment, the liver cells are hepatic parenchymal cells. In one embodiment, the cultured cells are primary cultured cells or immortalized cells. In one embodiment, the primary cultured cells may be human-derived primary hepatic parenchymal cells (PHH). In another embodiment, the immortalized cells are a human liver tumor-derived cell line. In another embodiment, the cultured cells are cells differentiated therefrom. Examples of human liver tumor-derived cell lines include HepaRG (trademark, HPR116, BIOPREDIC International) cells. Hepatic parenchymal cells differentiated or dedifferentiated from HepaRG cells may also be used. In another embodiment, the cultured cells may be cells differentiated from stem cells. Stem cells include, but are not limited to, ES cells, iPS cells, and somatic stem cells (e.g., neural stem cells, hepatic stem cells, epithelial stem cells, etc.). In one embodiment, the stem cells may be pluripotent stem cells (i.e., ES cells or iPS cells). In one embodiment of the evaluation method of the present invention, the cultured cells may be liver cells (hepatocyte-like cells (HLCs)) obtained by inducing differentiation of human iPS cells.

[0071] Raman scattering of purified CYP enzyme proteins may also be observed. "Purified protein" refers to CYP extracted from cells or synthesized CYP from which contaminants have been completely or partially removed. Synthetic CYP is a genetically recombinant protein prepared microbiologically. "An environment mimicking the intracellular environment" refers to an in vitro reaction system containing intracellular substances other than CYP enzymes.

[0072] Cultured cells are diluted in medium and seeded on a quartz substrate dish for Raman observation. A quartz substrate dish for Raman observation is a plastic dish with a quartz substrate attached to the bottom of the dish. When seeding the cultured cells, a cloning cylinder may be used to seed the cells only on the quartz substrate. As an example, when using HepaRG™ cells, cells cultured at 37°C in a 5% CO2 atmosphere for 3 days may be used as a sample.

[0073] In addition to the above samples, when detecting Raman scattered light extracellularly, the target sample for Raman scattering may be, but is not limited to, a solution sample extracted from tissue that contains impurities, such as microsomes. Note that "extracellular" in this specification means "in vitro" unless otherwise specified, but may also mean "outside the cell (extracellular)" depending on the context.

[0074] The CYP enzymes whose enzymatic activity is evaluated in this embodiment may be induced in cells using an inducer. Rifampicin, a type of CYP enzyme inducer, primarily induces the expression of CYP3A4 among the CYP enzymes. As described above, this inducer can also induce the expression of CYP2B6, CYP2C9, and other enzymes.

[0075] In this embodiment, a Raman microscope (Non-Patent Document 3) may be used to identify a Raman scattering signal from a Raman spectrum having a specific wavenumber. In this microscope, laser light is focused into a linear shape of a predetermined length (X μm, hereinafter also referred to as a line) to excite a Raman signal from the sample. Note that the laser light may also be focused into a point shape to excite a Raman signal.

[0076] For example, a water immersion objective lens may be used for excitation and detection of Raman scattering. The Raman scattered light collected by the objective lens passes through a dichroic mirror and an edge filter that transmits long wavelengths, and is then imaged on the entrance slit of the spectrometer. The Raman scattering signal imaged on the slit is then dispersed by a diffraction grating inside the spectrometer and detected by a CCD image sensor. The CCD image sensor may be, for example, a cooled CCD image sensor.

[0077] Raman scattering signals scattered from various points along the focused line are detected by different pixels on the CCD image sensor. It is preferable to determine the laser irradiation time so that these Raman scattering signals can be measured simultaneously and independently. The laser irradiation time is, for example, 0.001 seconds to 3 minutes, preferably 0.01 seconds to 30 seconds, more preferably 0.1 seconds to 15 seconds, and particularly preferably 1 second to 10 seconds. When focused linearly, each Raman scattering signal is detected simultaneously in a linear region (line region) of a predetermined length where the light is focused. Laser irradiation and detection of Raman scattering signals are repeated while shifting the focusing position in a direction perpendicular to the direction of the focused line. For example, as shown in Figure 1, a Raman image is acquired in a rectangular region of X μm × Y μm on a substrate.

[0078] When the light is focused to a point, a Raman image of any area can be obtained by repeatedly detecting the Raman scattering signal while shifting the focus to any distance in the X and Y directions. This ultimately results in a Raman image of an area of ​​X μm in width and Y μm in height. The X and Y directions can also be reversed.

[0079] The laser light used as excitation light has a wavelength of, for example, 406 nm to 561 nm, and preferably has a wavelength of 532 nm. The inventors focused on resonance Raman scattering. In resonance Raman scattering, molecules are excited in their own absorption bands, allowing the vibrations originating from the absorption bands to be selectively measured due to the resonance effect. Heme b and heme c have an absorption band known as the β / α band near 520 to 560 nm. Therefore, using a laser light with a wavelength of 532 nm as excitation light can significantly increase the Raman scattering light due to the resonance effect. Because the above heme proteins also have an absorption band known as the Soret band near 380 to 460 nm, laser light with wavelengths of 488, 561, 556, 543, 526, 523, 520, 515, 501, 450, or 406 nm may be used to excite Raman scattering. The Raman scattered light detected by the Raman scattering method of this embodiment is due to resonance Raman scattering. In this embodiment, Raman scattered light due to non-resonance Raman scattering may also be detected.

[0080] Figure 2 is a flowchart explaining the method for extracting Raman scattering signals. First, cosmic rays and the offset signal of the CCD image sensor, which are noise contained in the measured Raman scattering signal, are removed. The offset signal is a signal used to adjust the offset of the CCD image sensor. Next, processing is performed to reduce the influence of noise that was not completely removed by the above method. The noise removal method can be either a method using singular value decomposition (SVD) or a method that averages the Raman spectrum for each desired analysis region. The singular value decomposition method can be used to construct a Raman image. Furthermore, the method that averages the Raman spectrum for each desired analysis region can be used to create a dot plot.

[0081] After removing noise contained in the Raman scattering signal, the Raman scattering signal to be analyzed is extracted. Here, the Raman scattering signal to be analyzed is the wavenumber 1636 cm when detecting the enzyme activity of the CYP enzyme group. -1The term "extraction" refers to the Raman scattering signal (Raman scattering intensity) in the target sample. Furthermore, "extraction" of the target Raman scattering signal means defining an arbitrary Raman scattering intensity as the target Raman scattering signal using a predetermined method. The first or second method described below can be used to extract the target Raman scattering signal. The first method will be described using FIGS. 3 and 4, and the second method will be described using FIGS. 3 and 5.

[0082] Figure 3 shows the Raman spectrum after noise removal. As shown in Figure 3, the Raman spectrum (Raman scattering signal) is expressed by a Raman scattering intensity (Raman scattering signal intensity) on the vertical axis and a Raman shift (wave number) on the horizontal axis. The Raman shift (wave number) on the horizontal axis represents the difference in wave number between the incident light from excitation light such as a laser and the Raman scattered light.

[0083] Figure 4 is an enlarged view of the area indicated by the dashed line in Figure 3. This figure shows a method for extracting the Raman scattering signal of an object of analysis using the first method. The first method is suitable when a peak in the Raman scattering intensity of a Raman scattering signal derived from another living organism is present near the Raman scattering signal of the object of analysis.

[0084] In the first method, the difference in intensity between the linearly or nonlinearly approximated background signal intensity and the peak of the Raman scattering intensity is defined as the Raman scattering signal to be analyzed. More specifically, first, a Raman scattering intensity peak at a specific wavenumber and a waveform including the peak are identified. For the Raman scattering intensity peak, the intersection of a line connecting both ends of the peak base, i.e., a straight or curved line, and a line drawn perpendicularly from the peak apex to the wavenumber axis is used as the origin, and the height to the peak apex is calculated as the Raman scattering signal to be analyzed. In this specification, the "line connecting both ends of the peak base" is not particularly limited as long as it can define the region of the peak. The line may be an n-th order curve. If n=1, the line is a straight line, and if n>1, the line is a so-called curve.

[0085] Figure 5 is an enlarged view of the area indicated by the dashed line in Figure 3. This figure shows a method for extracting the Raman scattering signal of the analysis target using the second method. The second method is suitable when the Raman scattering signal of the analysis target has a sufficient signal-to-noise ratio (SN ratio) relative to the background signal and there are no peaks of Raman scattering signal intensity from other living organisms nearby.

[0086] In the second method, first, a peak of Raman scattering intensity at a specific wavenumber and a waveform including the peak are identified. For the Raman scattering intensity peak to be analyzed, the area of ​​the region surrounded by a line connecting both ends of the base of the peak, i.e., a straight line or curve, and the waveform of the Raman scattering signal is calculated, and defined as the Raman scattering signal to be analyzed. In this specification, the "line connecting both ends of the base of the peak" is not particularly limited as long as it can define the region of the peak. The line may be an n-th order curve. If n=1, the line is a straight line, and if n>1, the line is a so-called curve.

[0087] In addition to the first and second methods, it is also possible to extract the Raman scattering signal of the analysis target using another method depending on the required quantitative accuracy. For example, the difference between the intensity of the peak of the Raman scattering intensity of the analysis target and the intensity of the base of the peak is defined as the Raman signal of the analysis target. The base of the peak refers to a point that is convex in the negative y-axis direction near the peak of the Raman scattering intensity of the analysis target.

[0088] By the above method, the wavenumber 1320-1660 cm -1 , preferably 1636 cm -1The Raman scattering signal to be analyzed is defined in the above. If the Raman scattering signal (Raman scattering intensity) increases within the cell, it indicates that the enzymatic activity of the CYP enzyme group within the cell is increased. Using Raman spectroscopy, Raman signals of biomolecules related to metabolic activity can be detected from living cells under a microscope. Raman signals of biomolecules that indicate the state and structure of the cell can also be detected simultaneously. Furthermore, the distribution of each detected biomolecule within the cell can be understood with spatial resolution at the organelle level, for example, with a spatial resolution of 200 nm. Therefore, when examining cellular drug responses in metabolism and energy production, a comprehensive study can be performed in conjunction with the behavior of biomolecules localized in other organelles. 1636 cm -1 Observing the Raman signals of glycogen and cytochrome c can contribute not only to measuring CYP enzyme activity but also to elucidating the mechanisms of their cellular functions. Furthermore, by detecting indicators of liver function such as glucose metabolism and mitochondrial activity / toxicity using the Raman scattering signals of glycogen and cytochrome c extracted from the Raman spectrum, it is possible to evaluate at least one of the following: type of cell or tissue, differentiation level, and maturity level.

[0089] Based on the above facts, the present invention provides a method for replacing hepatocytes, which comprises the following steps: Also provided is a method for assessing metabolic capacity of hepatocytes (hereinafter, sometimes referred to as the "method for assessing metabolic capacity of hepatocytes of the present invention"). Irradiating the liver parenchymal cells with excitation light and acquiring a Raman spectrum using a photodetector; and A step of detecting Raman signals of biomolecules related to the metabolic capacity of liver parenchymal cells from the Raman spectrum.

[0090] In the method of the present invention for evaluating the metabolic capacity of hepatocytes, the "biomolecule associated with the metabolic capacity of hepatocytes" is not particularly limited as long as it is a biomolecule associated with the metabolic capacity of hepatocytes, but may be, for example, at least one selected from the group consisting of CYP enzymes, glycogen, cytochrome b5, cytochrome c, lipids, and phenylalanine. Known wavenumbers may be used for the Raman scattering signal capable of detecting these biomolecules. Specific examples include, but are not limited to, the wavenumbers used in the following examples. [Example]

[0091] The above embodiments will be described in more detail with reference to Examples 1 to 7. Experimental conditions common to these Examples are described in (1) to (8) below. In the following Examples, CYP3A4 and other CYP enzymes in cells derived from human liver tumors are detected. As described above, the detection method of the present invention utilizes the redox state derived from the molecular structure characteristic of CYP enzymes. Furthermore, as described above, the redox state of CYP enzymes is related to the metabolic mechanism of CYP enzymes. Therefore, the data obtained in the following Examples for CYP3A4 demonstrate that the enzyme activity of metabolism by CYPs derived from all kinds of organisms, not just human CYP3A4, can be detected by the detection method of the present invention.

[0092] (1) Seeding and culturing of hepatocytes In the examples, cultured cells were used. More specifically, frozen HepaRG cells (HPR116, BIOPREDIC International) differentiated into hepatocytes and bile duct epithelial cells were used. The frozen HepaRG cells were thawed in a 37°C water bath. The thawed HepaRG cells were then diluted with culture medium (MIL600, ADD670, BIOPREDIC International). The diluted HepaRG cells were then seeded onto a quartz substrate dish for Raman observation (SF-S-D12, Fine Plus International, Japan).

[0093] Seeding will be explained in more detail. The "quartz substrate dish for Raman observation" used for seeding is a plastic dish with a quartz substrate 0.15 mm thick and 12 mm in diameter attached to the bottom inside the dish. In order to seed HepaRG cells only on the quartz substrate of the dish, a cylindrical cloning cylinder (outer diameter 10 mm, inner diameter: 8 mm, 1980005, Hilgenberg GmbH) was first placed on the quartz substrate. 1.2 x 10 cells were placed inside the cloning cylinder. 5 The cloning cylinder was placed on a quartz substrate. In other words, the cloning cylinder was not glued or fixed to the quartz substrate.

[0094] Twenty-four hours after seeding, HepaRG cells adhered to the quartz substrate. Once the seeded HepaRG cells had adhered to the quartz substrate, the cloning cylinder was removed from the quartz substrate. When the cloning cylinder was removed, the medium was replaced with fresh medium (MIL600, ADD670, BIOPREDIC International). HepaRG cells were then cultured in the new medium. The culture was continued at 37°C in a 5% CO2 atmosphere for 3 days.

[0095] (2) Induction of CYP enzymes expressed in hepatocytes or inhibition of CYP3A Next, the induction or inhibition of CYP enzymes was carried out using HepaRG cells cultured as described in (1) above.

[0096] The induction of CYP enzymes will now be described. First, the medium for the HepaRG cells described in (1) above was replaced with a medium containing an inducer for CYP enzymes. The HepaRG cells were cultured in a medium containing the inducer described below at 37°C in a 5% CO atmosphere for 48 hours to induce CYP enzymes.

[0097] The media used for induction of CYP enzymes were MIL600, ADD650, and BIOPREDIC International. One of the following four inducers for CYP enzymes was added to the medium: Rifampicin (189-01001), Fujifilm Wako, Japan; Phenytoin (16612082), Fujifilm Wako, Japan; Dexamethasone (04718863), Fujifilm Wako, Japan; and Carbamazepine (034-23701), Fujifilm Wako, Japan. The concentrations of each inducer in the medium used in each example are as described in each example. These inducers promote transcription of CYP genes.

[0098] Control cells without induction of CYP enzymes (hereafter referred to as the uninduced cell group) were also cultured. For the uninduced cell group, the medium of the HepaRG cell group (1) was replaced with medium (MIL600, ADD650, BIOPREDIC International) that did not contain the above-mentioned inducers. The cells were cultured at 37°C in a 5% CO2 atmosphere for 48 hours.

[0099] Next, we will explain the inhibition of CYP enzymes. The HepaRG cell population used for the inhibition of CYP3A, a type of CYP enzyme, is a cell population in which the above-mentioned CYP enzymes were induced. Specifically, the medium of the HepaRG cell population (1) was replaced with a medium containing rifampicin, one of the above-mentioned inducers, and the cells were cultured at 37°C in a 5% CO2 atmosphere for 48 hours to induce the CYP enzymes and obtain a cell population. This cell population was used for the inhibition of CYP3A.

[0100] The media used for inhibiting CYP enzymes were MIL600, ADD650, BIOPREDIC International. The inhibitor added to the media was azamulin (18748, Cay Chemical). The azamulin concentration in the media used in each example is as described in the respective examples. The media in which CYP enzymes were induced was replaced with media containing the inhibitor azamulin. CYP3A, a type of CYP enzyme, was inhibited by culturing HepaRG cells in azamulin-containing media at 37°C in a 5% CO2 atmosphere for 5 minutes.

[0101] (3) Detection of CYP3A4 activity in cell populations using a luminescence method To confirm the enzymatic activity of CYP3A4, a CYP enzyme expressed in hepatocytes, in the cell populations observed with Raman imaging, a luminescence assay (hereinafter referred to as the luminescence assay) was conducted in parallel with the Raman imaging. The cell populations used in the luminescence assay were isolated from the cultured cells used for the Raman imaging described in (5). That is, the cells used were cultured under the same conditions and at the same time as the cultured cells used for the Raman imaging. The CYP luminescence assay was performed using a commercially available luminescence assay kit (P450-Glo-CYP3A4-Assay-and-Screening-System) according to the provided protocol.

[0102] The luciferin-IPA substrate is converted to luciferin by CYP3A4, which then becomes a substrate for luciferase, producing luminescence proportional to CYP3A4 activity. The cell culture medium was replaced with substrate-containing medium containing 3 μM of the luciferin-IPA substrate (V9002, Promega, Germany) and incubated at 37°C under 5% CO2 for 1 hour. The luciferin-IPA substrate is specific for CYP3A4. The luciferin-IPA substrate, a precursor to luciferin, is converted to luciferin by the catalytic action of the CYP3A4 enzyme. After incubation, the substrate-containing medium was collected and 50 μl aliquots were dispensed into a 96-well white flat-bottom plate (Corning). 50 μl of luciferase was added dropwise to the substrate-containing medium, and luminescence was generated via the luciferin-luciferase reaction. Luminescence was detected using a microplate reader (Synergy HTX, BioTek). The CYP3A4 activity detected by this luminescence method is shown in Figures 7, 14, 16, 18, 20, and 22.

[0103] (4) Detection of CYPs in cell populations using Western blotting Western blotting was used to detect CYP3A4 protein in cells with CYP enzyme induction. For Western blotting, cells were cultured using the method described above (2) with rifampicin as an inducer. CYP-induced cells were washed twice with chilled PBS and lysed in a cell lysis solution. The cells were lysed in RIPA buffer containing a protease inhibitor cocktail (1-100 dilution, Cat. No. P8340, Sigma-Aldrich) for 30 minutes on ice. The resulting cell lysate was collected using a cell scraper. The collected cell lysate was centrifuged (12,000 g, 20 min) at 4°C, and the supernatant was collected to remove any precipitated cell debris.

[0104] The protein concentration in the cell lysate supernatant was measured using a BCA protein assay kit (Cat. No. 23227, ThermoFisher Scientific). The cell lysate was loaded onto the wells of 12% SDS-PAGE gels (Cat. No. 4568043, BioRad) and subjected to electrophoresis. After SDS-PAGE, a low-fluorescence polyvinylidene fluoride (PVDF) membrane was attached to the gel and transferred. After transfer, the membrane was blocked with TBST (0.1% Tween-20 in TBS) containing 5% ECL blocking agent (Cat. No. RPN2125, GE Healthcare) for 1 hour at room temperature with agitation. The membrane was then washed twice with TBST.

[0105] Next, primary and secondary antibody reactions were performed. The blocked membrane was immersed in blocking buffer containing primary antibodies: mouse anti-CYP3A4 (1:2000), rabbit anti-cytochrome b5 (1:1000), and rabbit anti-β-actin (1:1000 dilution, Cat. No. 4970, Cell Signaling Technology) overnight. Next, the membrane was immersed in blocking buffer containing secondary antibodies: horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:10000) for 1 hour at room temperature. The membrane was then immersed in TBST buffer and washed three times.

[0106] CYP3A4 protein was detected using an ECL detection system (RPN2232, GE Healthcare) and a ChemiDOC MP imaging system (Bio-Rad).

[0107] (5) Raman observation of cell populations using Raman spectroscopy The cell populations cultured by the above method (1) or (2) were quantitatively measured for Raman signals using Raman spectroscopy, and Raman images were constructed. Here, "observation" in this specification includes both detecting the Raman scattering signals of the cell populations and obtaining and observing Raman images of the cell populations. Note that, in some examples, the cell populations to be observed may be obtained through a separate process after culturing by method (1). Details will be explained in each example.

[0108] The culture medium was removed from the cultured cells, and the cells were washed twice with PBS. An observation solution (Live Cell Imaging Solution (A14291DJ, Thermo Fisher Scientific)) was added to the washed cells. The cells thus treated were subjected to Raman observation using the Raman microscope (Non-Patent Document 3) described with reference to Figure 1 (Figures 6, 9-11, 13-21, 23-26).

[0109] A 40x water-immersion objective lens (CFI Apochromat Lambda S 40XC WI) equipped on the Raman microscope was used to excite the Raman scattering light of the cell group and detect the Raman scattering signal. The excitation light for the Raman scattering light of the cell group was irradiated using the water-immersion objective lens by focusing a 532 nm laser beam into a straight line of a specified length (hereinafter referred to as a line).

[0110] Next, we will explain the process for detecting Raman scattered light from cell clusters excited by excitation light. Raman scattered light collected by a water-immersion objective passes through a dichroic mirror and an edge filter that transmits long wavelengths, before being focused on the entrance slit of the spectrometer. The Raman scattered signal focused on the slit is then dispersed by a diffraction grating inside the spectrometer and detected by a cooled CCD image sensor (PIXIS 400B, Princeton Instruments) used as a photodetector. Raman signals scattered from various points along the focused line can be measured simultaneously and independently at different pixels on the CCD. The laser irradiation time was 5 seconds. Each Raman signal was simultaneously detected over a region of approximately 133.3 μm along the focused line. This laser irradiation and Raman signal detection were repeated by shifting the focusing position perpendicular to the focused line by a scanning pitch of Z nm. Specifically, the laser was scanned over a rectangular area of ​​133.3 μm × Y μm to detect the Raman scattering signals of the cells. The laser energy density on the surface of the cells during the laser scanning was 3 mW / μm. 2 The value of Y, which corresponds to the width of the observation area, and the value of the scanning pitch Z in the direction parallel to Y vary depending on the example. Two types of diffraction gratings with different ruling numbers were used for detecting Raman signals. Here, "detection" of Raman scattering signals includes both quantitative measurement of Raman scattering signals and construction of Raman images. A diffraction grating with 1200 ruling numbers (1200 L / mm, BLZ 500 nm) was used for quantitative measurement of Raman scattering signals. On the other hand, a diffraction grating with 600 ruling numbers (600 L / mm, BLZ 500 nm) was used for construction of Raman images. In this study, different diffraction gratings were used depending on the purpose of each experiment, but they do not necessarily have to be used in the above-mentioned manner. In other examples, diffraction gratings with 600 and 1200 ruling numbers can be used for quantitative measurement of Raman scattering signals and construction of Raman images, respectively.

[0111] (6) Noise reduction of Raman scattering signals The Raman scattering signals measured as Raman spectra were analyzed using the calculation software Matlab (Math Works) according to the method described in Non-Patent Document 14. After removing cosmic rays and photodetector offset signals contained in the measured Raman spectra, noise was removed using singular value decomposition (SVD). A signal intensity distribution at an arbitrary Raman shift (wavenumber) was then constructed, and a Raman image was acquired. For quantitative measurement of Raman signals, noise removal using SVD was not performed; instead, data from which cosmic rays and camera offset had been removed was used. If necessary, the average of clear spectra (hereinafter referred to as the average spectrum) was calculated from multiple Raman spectra. The multiple Raman spectra may be Raman spectra from the area where the target cells are concentrated, or from adjacent areas.

[0112] (7) Extraction of Raman scattering signals and construction of Raman images A clear spectrum was obtained by removing noise from the acquired Raman spectrum using the method described in (6) above. The Raman scattering signal to be analyzed was extracted from the obtained spectrum. The extraction method varies depending on the circumstances, such as the intensity of the target Raman scattering signal and whether Raman scattering signals from other biomolecules are present nearby. In this example, the target Raman scattering signal was extracted from the spectrum and used for quantification using the two methods described below.

[0113] First, the peaks of the Raman scattering signal to be analyzed, which are thought to be derived from the CYP metabolic enzymes, and the waveforms containing the peaks were identified from the noise-processed spectrum. In one method, the area enclosed by the line or curve connecting the ends of the peak base and the waveform of the Raman scattering signal was defined as the Raman scattering signal intensity, and the Raman scattering signal intensity was calculated. In another method, the intersection of the line or curve connecting the ends of the peak base and a line drawn perpendicularly from the peak apex to the wavenumber axis was used as the origin, and the height to the peak apex was defined as the Raman scattering signal intensity, and the Raman scattering signal intensity was calculated. To create each boxplot in this example, the signal intensity of the Raman scattering signal extracted using the method described with reference to Figure 4 was used. Using these two methods, the correlation between the Raman scattering signal to be analyzed and CYP activity was investigated.

[0114] (8) Immunohistochemical localization of CYP3A4 and cytochrome b5 in cell populations Immunostaining was performed to detect a wavenumber of 1636 cm -1 We confirmed that the Raman signal originated from the CYP enzyme group. Specifically, we performed immunostaining of CYP3A4 and cytochrome b5 protein, which is a type b oxidized heme protein like the CYP enzyme group. Like the CYP enzyme group, cytochrome b5 is an enzyme involved in drug metabolism. The cell group used for immunostaining was the cell group after Raman observation.

[0115] Immediately after Raman imaging, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cell membranes were permeabilized with a 0.1% Triton-X-100 solution diluted in phosphate-buffered saline (PBS). After washing, cells were blocked with 4% bovine serum albumin (A2153, Sigma-Aldrich) for 1 hour at room temperature to suppress nonspecific staining. Primary antibodies, anti-CYP3A4 (1:1000 dilution, SAB5300118, Sigma-Aldrich) and rabbit anti-human cytochrome b5 (1:500 dilution, ab69801, Abcam) in 1% BSA blocking buffer were added dropwise and incubated overnight at 4°C. The next day, the cells were washed three times with PBS and then incubated with blocking buffer containing Alexa Fluor 488 goat anti-mouse antibody (10 μg / ml, A11001, Invitrogen) and Alexa Fluor 594 goat anti-rabbit antibody (10 μg / ml, A11012, Invitrogen) as secondary antibodies for 1 hour at room temperature. After washing three times with PBS, the cells were stained. For staining, 1 M DAPI (D1306, Invitrogen) was added and incubated at room temperature to stain the cell nuclei. Finally, the cells were washed twice with PBS and stored at 4°C in the dark until fluorescence observation. Fluorescence observation was performed using a laser scanning confocal microscope (CLSM, Nikon, Japan) at the same area as the Raman observation. The area for Raman observation was previously labeled with a permanent marker. Based on this labeling, the areas for Raman and fluorescence observation were determined. Fluorescence images obtained by the above method are shown in FIGS. Examples 1 to 7 will be described in detail below with reference to FIGS.

[0116] Example 1 <Detection of Raman spectra resulting from induction of CYP enzymes in human hepatocytes (Figure 6)>

[0117] In this example, we used rifampicin, an inducer of CYP enzymes, to induce the expression of CYP enzymes in HepaRG cells and attempted to detect Raman scattering signals resulting from the induction of CYP enzymes. Rifampicin primarily induces the expression of CYP3A4 among CYP enzymes. This inducer also induces the expression of CYP2B6, CYP2C8, CYP2C9, and CYP2C19 (Non-Patent Document 15). Upon rifampicin induction, changes in the expression levels of the entire CYP enzymes, including the above CYPs, are dominated by increases or decreases in the expression level of CYP3A4. This is because rifampicin primarily induces the expression of CYP3A4 among CYP enzymes, and CYP3A4 accounts for one-third of all CYP enzymes expressed in the liver (Non-Patent Document 2).

[0118] In this example, Raman observation, luminescence testing, and Western blotting were performed on cultured cells in parallel. The luminescence testing was performed to confirm the enzymatic activity of CYP enzymes, particularly CYP3A4, in the cell populations to be observed with Raman spectroscopy. Western blotting was performed to detect CYP3A4 protein in the cell populations.

[0119] First, human hepatocytes (HepaRG cells) were seeded, cultured, and CYP enzyme induction was performed according to the procedures described in (1) and (2) above. A CYP enzyme inducer (Rifampicin) was added at a concentration of 4 μM. As a control for the CYP enzyme-induced cell population, an uninduced cell population was also cultured using medium (MIL600, ADD650, BIOPREDIC International) that did not contain a CYP enzyme inducer.

[0120] After six days of culture, HepaRG cells aggregated into hepatic parenchymal cells and biliary epithelial cells, forming separate cell populations. On the quartz substrate on which the cells were seeded, the regions where hepatic parenchymal cells and biliary epithelial cells formed cell populations could be identified by bright-field microscopy. It is known that CYP enzymes are expressed only in hepatic parenchymal cells. Therefore, the target of Raman observation in this example was hepatic parenchymal cells.

[0121] To perform Raman observation and luminescence testing in parallel, a cell population was separated from a CYP-induced liver parenchymal cell population for Raman observation and luminescence testing. A cell population of liver parenchymal cells without CYP enzyme induction was also separated for these purposes.

[0122] For Raman observation, laser scanning was performed on a rectangular area of ​​133.3 x 84 μm at a scanning pitch of 333 nm according to the procedure described in (5) above. The acquired Raman spectrum was analyzed according to the procedures described in (5) to (7) above. In parallel with the Raman observation, a luminescence test was carried out according to the procedure described in (3) above. The luminescence test was used to measure the activity of CYP3A4 expressed in the cell group in which the CYP enzyme group was induced. Furthermore, Western blotting was performed according to the procedure described in (4) to detect CYP3A4 protein.

[0123] First, the results of the Raman observation will be described with reference to FIG. Figure 6 shows the Raman spectra of liver parenchymal cells in which CYP enzymes were induced. When observing cell populations with or without CYP enzyme induction, the average spectrum of the area where liver parenchymal cells congregated was calculated and plotted. As shown in Figure 6, the light gray line (Induced) represents the spectrum of the liver parenchymal cell population in which CYP enzymes were induced by rifampicin. The dark gray line (Control) represents the spectrum of the liver parenchymal cell population in which CYP enzymes were not induced.

[0124] From the average spectrum in Figure 6, the wavenumbers 1370 and 1636 cm -1 The peaks and waveforms of wave numbers 1370 and 1636 cm -1 The signal intensities of liver parenchymal cells in which CYP enzymes were induced and those in which CYP enzymes were not induced were compared. The liver parenchymal cells in which CYP enzymes were induced by rifampicin showed higher signal intensities at wavenumbers of 1370 and 1636 cm compared to the cells in which CYP enzymes were not induced. -1 It was confirmed that the Raman signal increased.

[0125] Next, the results of luminescence spectroscopy and Western blotting, which were performed in parallel with Raman observation, will be explained with reference to Figures 7 and 8. Luminescence spectroscopy and Western blotting confirmed that CYP3A4, a type of CYP enzyme, was induced in the cultured liver parenchymal cell population.

[0126] The bar graph in FIG. 7 shows that the CYP activity was increased in the cell group in which CYP3A4 was induced using Rifampicin, compared to the cell group in which CYP3A4 was not induced.

[0127] In the Western blotting photograph in Figure 8, no bands are observed in the cell group without CYP3A4 induction, whereas the expression level of CYP enzymes is increased in the cell group in which CYP3A4 was induced using rifampicin.

[0128] Raman observation revealed that the wave numbers were 1370 and 1636 cm -1 It was confirmed that the Raman signals at wavenumbers 1370 and 1636 cm were increased by the induction of CYP enzymes with rifampicin. -1 In addition to the previously known Raman signals of reduced heme (wavenumbers 600, 675, 750 cm -1 ) (Non-Patent Document 4), and a Raman signal (wavenumber 940 cm) indicating glycogen related to glucose metabolism in hepatocytes. -1 ) (Non-Patent Document 16), and the Raman scattering signal (wavenumber 1000 cm) indicating phenylalanine, an essential amino acid abundant in the cytoplasm.-1 ) peaks and waveforms were identified.

[0129] <Intracellular distribution of Raman signals resulting from induction of CYP enzymes in human hepatocytes (Figure 9)>

[0130] Next, we investigated the intracellular distribution of Raman signals resulting from the induction of CYP enzymes, and attempted to identify the biomolecules represented by the detected Raman signals. Using the Raman data measured according to the procedure described in (5) above, we extracted the Raman signals representing each biomolecule according to the methods described in (6) and (7) above, and obtained the intracellular signal intensity distribution.

[0131] Here, the wave number is 1370 cm -1 and 1636 cm -1 It is known that the Raman signals around this region are derived from type b and type c oxidized heme (Non-Patent Document 4). The CYP enzymes induced by rifampicin are type b heme proteins (Non-Patent Document 12). Therefore, the Raman observation results mentioned above are derived from the wavenumbers 1370 and 1636 cm. -1 This is in good agreement with the expectation that the Raman signals at 1370 and 1636 cm are due to CYP enzymes with type b heme as a prosthetic group. As mentioned above, the molecules of these CYP enzymes are mainly CYP3A4. -1 The Raman signal of is detected only in liver parenchymal cells. This result is consistent with the fact that CYP enzymes are expressed only in liver parenchymal cells, and not in bile duct epithelial cells. A detailed explanation will be given below with reference to Figures 9 and 10.

[0132] Figure 9 is a graph showing the Raman spectra of liver parenchymal cells and bile duct epithelial cells. The black line shows the average Raman spectrum within a specified region of the liver parenchymal cells. The gray line shows the average Raman spectrum within a specified region of the bile duct epithelial cells. The vertical axis of the graph represents the Raman scattering intensity (Raman scattering signal intensity), and the horizontal axis represents the Raman shift (wavenumber). As shown in the graph in Figure 9, at a wavenumber of 1636 cm -1The Raman signal at wavenumber 1370 cm can be confirmed only in hepatic parenchymal cells (black line) and not in bile duct epithelial cells (gray line). -1 The Raman signal at wavenumber 1636 cm -1 The Raman signal shows a similar trend to that of

[0133] The specified region where the average Raman spectra of the liver parenchymal cell group and the bile duct epithelial cell group were acquired will be explained using FIG. 10. FIG. 10 is a Raman image constructed based on specific Raman peaks. The leftmost photograph in FIG. 10 is a bright field photograph. The area above the white line drawn in the bright field photograph indicates liver parenchymal cells, and the area below (inside) the white line indicates bile duct epithelial cells. In the second photograph from the left, the specified region of the liver parenchymal cell group indicated by the black line in FIG. 9 is indicated by the upper open square, and the specified region of the bile duct epithelial cell group indicated by the gray line in FIG. 9 is indicated by the lower open square. As mentioned above, FIG. 9 is a graph showing the average Raman spectrum within the specified region. Furthermore, FIG. 10 shows the average Raman spectrum at wavenumbers of 600, 675, 940, and 1636 cm. -1 Although not shown in Figure 10, the Raman images of the 1370 cm wavenumber are shown. -1 The Raman image also shows the wavenumber of 1636 cm -1 The Raman image shows the same tendency as the one in Fig.

[0134] From these facts and experimental results, the wave numbers 1370 and 1636 cm -1 It is suggested that this may be due to CYP enzymes. It can also be used to distinguish between liver parenchymal cells and bile duct epithelial cells. In addition to the Raman signals at the above wavenumbers, the 675 cm -1 The Raman signal can be utilized.

[0135] Wavenumbers 1370 and 1636 cm -1To distinguish whether the Raman signal in this figure originates from type c or type b heme, we focused on the differences in the intracellular distribution of type c and type b heme. It has been reported that CYP enzymes, which are type b heme, are primarily present in the endoplasmic reticulum (Non-Patent Document 17). On the other hand, type c heme is known to be primarily distributed on the mitochondrial membrane and involved in energy metabolism. It has been reported that when observing intracellular structures, the distribution of type c heme coincides with the surface shape of mitochondria (Non-Patent Document 3). Raman images were created using the Raman signals representing the various reduced hemes described above, and the intensity distribution of the Raman signals in hepatocytes with CYP enzymes induced was compared with that in uninduced cells (Figure 11).

[0136] As shown in Figure 11, glycogen (940 cm -1 ) and CYP enzymes (1370, 1636 cm -1 ) indicates biomolecules resulting from the metabolic function of the liver. Other Raman peaks (600, 675 cm -1 ) indicates a heme protein that is different from the CYP enzyme family. -1 The Raman signal of 675 cm corresponds to reduced cytochrome c. -1 The Raman signal at 750 cm indicates reduced cytochrome b. The Raman image constructed using the Raman signals at each wavenumber shows the concentration distribution of the molecules resulting from each Raman signal. -1 The Raman signal at 600 cm comes from reduced cytochrome c and cytochrome b. -1 and 675 cm -1The Raman image of reduced cytochrome c shows the distribution of mitochondria, as cytochrome c is primarily distributed in the mitochondria, while the Raman image of reduced cytochrome b shows the distribution of ER (endoplasmic reticulum), as cytochrome b is primarily distributed in the ER.

[0137] The Raman signal of reduced hemoprotein and phenylalanine (1000 cm -1 In the Raman signal representing glycogen, no change in signal intensity was observed due to the induction of CYP enzymes with Rifampicin. On the other hand, in the Raman signal representing glycogen, it was confirmed that the signal intensity decreased due to the induction of CYP enzymes with Rifampicin compared to cells without CYP enzyme induction.

[0138] As shown in Figure 11, the wavenumber 600 cm -1 The Raman signal intensity distribution within the cell was localized to fine structures such as mitochondria. -1 The intracellular intensity distribution of the Raman signal from 1370 and 1636 cm in Fig. 11 was spread throughout the cytoplasm. The endoplasmic reticulum, where the CYP enzymes, which are type b heme proteins, are distributed, has a finer mesh structure than mitochondria (Non-Patent Document 18). Therefore, it is thought that the spatial resolution of the microscope used in this experiment (approximately 260 nm) is not enough to observe a clear image in which the distribution of the Raman signal matches the structure. Therefore, the Raman signal intensity distribution at 1370 and 1636 cm in Fig. 11 was not enough. -1 The intensity distribution of the Raman signal is consistent with the observation that the endoplasmic reticulum is distributed throughout the cytoplasm.

[0139] wave number 675 cm -1The Raman signal is obtained when cytochrome b is in the reduced state (Non-Patent Document 19). Cytochrome b is a type b hemoprotein like the CYP enzyme group, and its signal distribution is at wavenumbers 1370 and 1636 cm -1 and shows the cytoplasm similar to the distribution of the Raman signal. Therefore, it can be said that the Raman signals at wavenumbers 1370 and 1636 cm -1 are type b hemoproteins distributed in the endoplasmic reticulum.

[0140] In addition to hemoproteins, the intensity distribution of the Raman signal at wavenumber 940 cm -1 derived from glycogen related to glucose metabolism in hepatocytes was also confirmed. The results are shown in Fig. 11. It was confirmed that the Raman signal derived from glycogen, like the CYP enzyme group, is distributed only in the liver parenchymal cells with metabolic ability.

[0141] By detecting the changes in the intensity distributions of the Raman signals showing various hemoproteins and the Raman signal at wavenumber 940 cm -1 molecular information regarding the maturity and drug response of liver parenchymal cells can be measured.

[0142] <Comparison of Raman observation results and immunostaining results of human hepatocytes with CYP enzyme group induction (Fig. 12)>

[0143] To confirm that the Raman signals at wavenumbers 1370 and 1636 cm -1 are derived from the CYP enzyme group and not from cytochrome b5, a comparison was made between the Raman observation results and the immunostaining results of human hepatocytes in which the CYP enzyme group was induced using Rifampicin. CYP3A4 and cytochrome b5 were immunostained and fluorescence observation was performed. As described above, CYP3A4 has the highest expression level among the CYP enzyme group induced by Rifampicin. On the other hand, cytochrome b5 is a type b hemoprotein other than the CYP enzyme group known to be present in the endoplasmic reticulum. Fig. 12 is a fluorescence image obtained by the method in (8) above.

[0144] The distribution of cytochrome b5 and CYP shown in Figure 12 and the Raman signal of reduced hemoprotein type b shown in Figure 11 (wavenumber 675 cm -1 ) and the Raman signals of type b oxidized heme protein (1370, 1636 cm -1 ), both show the entire cytoplasm. Cytochrome b5 has also been reported to be distributed in the endoplasmic reticulum, similar to the CYP enzymes. The inventors' Raman observation results are consistent with previous research reports on cytochrome b5. Therefore, the wavenumbers at 1370 and 1636 cm -1 It can be confirmed that the Raman signal at 1370 and 1636 cm is not derived from cytochrome b5. -1 The Raman signal at wavenumber 675 cm increases in cells induced with Rifampicin. -1 There was no significant difference in the intensity of the Raman signal at 675 cm between induced and uninduced cells. -1 The Raman signal from the aforementioned study is already known to indicate reduced cytochrome b. The immunostaining results also showed a similar trend. The fluorescence intensity of the CYP3A4 label increased only in cells in which the CYP enzymes were induced. There was no significant difference in the fluorescence intensity of the cytochrome b5 label between the induced and control groups. Therefore, the Raman signals at wavenumbers 1370 and 1636 cm originating from the oxidized type b hemoprotein were confirmed. -1 It can be said that the Raman signal of is mainly derived from CYP enzymes rather than cytochrome b5.

[0145] The wavenumbers 1370 and 1636 cm shown in Figure 11 -1In the Raman image and the fluorescence image of CYP3A4 shown in FIG. 12, in some cells, the intensity distribution of the Raman signal does not match the intensity distribution of the fluorescence. This difference in distribution is considered to be derived from CYP molecules other than CYP3A4. This is because only CYP3A4 can be specifically detected in the fluorescence image, while all CYP molecules are detected in the Raman image. As described above, Rifampicin used as an inducer in this experiment has been reported to induce CYP2B6, CYP2C8, CYP2C9, and CYP2C19 (Non-Patent Document 15) as molecules included in the CYP enzyme group other than CYP3A4.

[0146] <Example 2> <Detection of Inducer Concentration Dependence of CYP Activity>

[0147] In this example, an attempt was made to detect the inducer concentration dependence of CYP activity by Raman observation. As described above, the inducer (Rifampicin) induces the expression of CYP3A4 in human hepatocytes (HepaRG cells). Therefore, different concentrations of the inducer (Rifampicin) were used to induce CYP3A4 in human hepatocytes (HepaRG cells). Seeding, culturing, and induction of hepatocytes were performed according to the procedures (1) and (2) above. The concentrations of the inducer (Rifampicin) were added at 0, 0.04, 0.4, and 4 μM. In a part of the induced cells, as a control experiment for Raman observation, the activity of CYP3A4 expressed in the cells was detected using the luminescence method according to the procedure shown in (3) above. Raman observation was performed on other cells according to the procedure shown in (5) above. At three locations in the culture dish, a rectangular region with a length of 133.3 μm and a width of 84 μm was irradiated with a laser at a scanning pitch of 1 μm to obtain Raman spectra. Noise removal was performed on the obtained Raman spectra according to the methods described in (6) and (7) above.

[0148] In the above example, the Raman signals at wavenumbers 1370 and 1636 cm -1 were presumed to be derived from the CYP enzyme group and to correlate with the CYP enzyme group activity. Therefore, as shown in FIG. 13, at a wavenumber of 1636 cm -1The Raman image of a rectangular area was reconstructed using the distribution of the Raman signal of (oxidized type b heme protein) at a wavenumber of 1636 cm. -1 The Raman signal is uniformly distributed throughout the cytoplasm. Figure 13 shows an 84 x 84 μm region cut out from the acquired Raman image.

[0149] Although not specifically shown, the wavenumber is 1370 cm -1 The change in the distribution of the Raman signal and the change in the intensity of the Raman signal at the wavenumber 1636 cm -1 The results showed a similar trend to those of the previous study.

[0150] Figures 14 and 15 show the calculated average spectrum of the liver parenchymal cell region. -1 The intersection of a line connecting both ends of the peak base with a line drawn perpendicularly from the peak of the peak waveform to the wavenumber axis was taken as the origin, and the height to the peak apex was extracted as the signal intensity.

[0151] In Figure 14, the light grey bars show the activity of CYP3A4 detected using the luminescence method, while the dark grey bars show the activity at 1636 cm wavenumber extracted from Raman spectra averaged over a region of liver parenchymal cells. -1 The Raman signal of 1636 cm for each added concentration of Rifampicin is shown. -1 The Raman signal intensity of 1636 cm, which indicates the CYP enzyme group, was compared with the results of the CYP3A4 activity measured using the luminescence method. -1 It was confirmed that the Raman signal intensity of 1636 cm and the activity of CYP3A4 increased. -1 The Raman signal of CYP3A4 is positively correlated with the activity of CYP3A4.

[0152] 15 is a box plot showing the variation in CYP enzyme activity among cells. Each plot was obtained by extracting the wavenumber at 1636 cm from the average spectrum of each cell according to the method described above. -1This image shows the extracted Raman signal from the CYP enzymes. It can be seen that even under constant Rifampicin administration conditions, the induced activity of CYP enzymes varies from cell to cell. Detection of CYP enzyme activity using Raman signals indicates that not all cells in a culture dish will increase their CYP activity uniformly. It was also confirmed that the median value of the plot for each Rifampicin administration concentration condition increases with the Rifampicin administration concentration. This increase in the median value was statistically significant. It also corresponds well to an increase in CYP3A4 activity.

[0153] Example 3 <Detection of CYP activity in hepatocytes after addition of various inducers>

[0154] In this example, we attempted to observe the difference in the response of CYP enzyme activity to different inducers. We used four inducers (rifampicin, phenytoin, dexamethasone, and carbamazepine) known to induce CYP expression in human hepatocytes (HepaRG cells). CYP expression in human hepatocytes (HepaRG cells) was induced using these inducers, and CYP enzyme activity was detected using Raman imaging. These inducers activate the transcription factor PXR (pregnant X receptor: PXR). Activated PXR translocates from the cytoplasm to the nucleus, where it forms a heterodimer with retinoid X receptor (retinoid X receptor: RXR) and binds to promoter sequences, promoting target gene transcription (Non-Patent Documents 20-22), thereby promoting the expression of CYP enzymes. The transcription of CYP3A4 is primarily promoted by PXR.

[0155] Human hepatocytes (HepaRG cells) were seeded, cultured, and induced according to the procedures described in (1) and (2) above. Four inducers (rifampicin, phenytoin, dexamethasone, and carbamazepine) were added at concentrations of 4, 100, 100, and 100 μM, respectively. As a control, uninduced hepatocytes cultured in inducer-free medium (MIL600, ADD650, BIOPREDIC International) were also prepared. Cells were then isolated from these cell populations for luminescence and Raman observation. A portion of the induced human hepatocytes (HepaRG cells) was used for luminescence as a control for Raman observation, while the remaining human hepatocytes (HepaRG cells) were used for Raman observation. Luminescence and Raman observation were performed according to the procedures described in (3) and (5), respectively. CYP3A4 activity in human hepatocytes (HepaRG cells) was measured using the above procedures. As mentioned above, 1636 cm -1 The Raman signal at 1636 cm is expected to correlate well with CYP activity. -1 The Raman signal was extracted and compared with the detection results of CYP3A4 using the luminescence method.

[0156] 16 and 17 show the results of measuring the activity of CYP enzymes when induced using Rifampicin, Phenytoin, Carbamazepine, and Dexamethasone as inducers. The activity of CYP3A4 induced by various drugs was measured at a wavenumber of 1636 cm. -1 The Raman signal of 1636 cm, which is the Raman peak of the CYP enzymes, was measured after averaging the Raman spectra detected from the liver parenchymal cell region. Figure 16 shows the activity of CYP3A4 (light gray) and the Raman signal (dark gray) detected using the luminescence method. The black bar graph in Figure 16 shows the Raman peak of the CYP enzymes at wavenumber 1636 cm, which is the Raman peak of the CYP enzymes, after averaging the Raman spectra detected from the liver parenchymal cell region. -1 The light gray bars show the CYP activity detected by the luminescence method. The signal was extracted at a wavenumber of 1636 cm. -1At the peak including [the relevant content], with the intersection of the straight line connecting both ends of the peak base and the straight line perpendicularly dropped from the peak of the peak waveform to the wavenumber axis as the origin, the height to the peak apex was implemented as the signal intensity. Also, Fig. 17 is a box-and-whisker plot showing the variation in CYP enzyme group activity for each cell. Each individual plot is obtained by extracting the Raman signal at a wavenumber of 1636 cm from the average spectrum for each cell in the same manner as described above. -1 Similar to the results in Fig. 15, even under a certain culture condition, the induced CYP enzyme group activity varies for each cell. Also, the cells in the culture dish do not uniformly increase their CYP activity. Further, the increase and decrease in the median value of the plots for each culture condition well match the variation in the activity of CYP3A4 detected by the luminescence method. The increase in the median value of the plots was statistically significant.

[0157] Although not particularly shown, the change in the distribution of the Raman signal at a wavenumber of 1370 cm -1 and the change in the intensity of the Raman signal also showed a tendency equivalent to those at a wavenumber of 1636 cm -1 .

[0158] <Example 4> <Detection of Suppression of CYP Activity by IL-6 (Interleukin-6)>

[0159] In this example, an attempt was made to observe the suppression of CYP activity. For the suppression of CYP activity, Interleukin-6 (IL-6), which is known to suppress the activity of the CYP enzyme group in human hepatocytes (HepaRG cells), was used. After suppressing, i.e., down-regulating, the activity of the CYP enzyme group in human hepatocytes (HepaRG cells), an attempt was made to detect the activity of the CYP enzyme group by Raman observation. IL6 is a primary mediator of the acute-phase response and is one of the cytokines that play a central role in multiple chronic inflammatory diseases. When cells are exposed to inflammatory or infectious stress, the release of IL6 occurs. IL6 is known to suppress the expression of CYP3A4 mRNA in HepaRG cells, which are a hepatoma cell line (Non-Patent Document 23). As a result of the suppression of mRNA expression, the expression of CYP3A4 protein is suppressed.

[0160] To downregulate CYP enzyme activity using IL-6, hepatocytes were seeded and cultured according to the procedure described above (1). Before IL-6 administration, all CYP enzymes were induced, i.e., nonspecific induction of CYP enzymes was performed. Nonspecific induction of CYP enzymes was performed by culturing HepaRG cells in medium (MIL600, ADD620, BIOPREDIC International) containing 2% DMSO for 2 days. Subsequently, the cells were cultured for 48 hours in medium (MIL600, ADD650, BIOPREDIC International) containing 2 ng / ml IL-6 (206-IL-010 / CF, R&D Systems). Control hepatocytes were also cultured for 48 hours in medium without IL-6 (MIL600, ADD650, BIOPREDIC International). Cells were then isolated from these cell populations for luminescence and Raman imaging.

[0161] A portion of the induced and IL-6-administered human hepatocytes (HepaRG cells) was used for the luminescence method as a control experiment for Raman observation, and the remaining human hepatocytes (HepaRG cells) were used for Raman observation. The luminescence method and Raman observation were carried out according to the procedures described above in (3) and (5), respectively, to measure the activity of CYP3A4 in the human hepatocytes (HepaRG cells). Analysis of the Raman observation results revealed that the 1636 cm peak, which was expected to correlate well with CYP activity in (6) and (7) above, was -1 The Raman signal of CYP3A4 was extracted and compared with the results of CYP3A4 using the luminescence method.

[0162] Figures 18 and 19 show the results of Raman signal detection of the downregulation of CYP activity caused by IL-6 administration. The luminescence test also detected a decrease in CYP3A4 activity, which was in good agreement with the Raman measurement results. The bar graph in Figure 18 shows the CYP3A4 activity (light gray) detected using the luminescence method and the Raman signal (dark gray). The dark gray bar graph in Figure 18 shows the Raman peak of the CYP enzymes at wavenumber 1636 cm, after averaging the Raman spectra from the liver parenchymal cell region. -1 The Raman signal intensity is plotted, and the light gray bars indicate CYP activity detected by luminescence. The signal was extracted at a wavenumber of 1636 cm. -1 The signal intensity was measured by taking the intersection of a line connecting both ends of the peak base with a line drawn perpendicularly from the peak of the peak waveform to the wavenumber axis as the origin, and measuring the height to the peak apex.

[0163] 19 is a boxplot showing the variation in CYP enzyme activity for each cell. Each plot is calculated from the average spectrum for each cell at a wavenumber of 1636 cm as described above. -1 This is an extracted Raman signal from the CYP3A4 enzymes. As with the results in Figure 15, CYP enzyme activity differs from cell to cell, even under certain culture conditions. Furthermore, the CYP activity of all cells in the culture dish does not decrease uniformly. Furthermore, the decrease in the median value of the plot under each culture condition (with or without administration of a CYP enzyme expression inhibitor) coincides well with the fluctuations in CYP3A4 activity detected by luminescence. The observed decrease in the mean value of the plot was statistically significant.

[0164] Although not specifically shown, the wavenumber is 1370 cm -1 The change in the distribution of the Raman signal and the change in the intensity of the Raman signal at the wavenumber 1636 cm -1 The results showed a similar trend to those of the previous study.

[0165] <Example 5> <Time-course Raman observation of changes in CYP activity following hepatocyte seeding>

[0166] In this example, we attempted to use Raman spectroscopy to detect changes in CYP enzyme activity over time in human hepatocytes (HepaRG cells) cultured for several days. First, frozen human hepatocytes (HepaRG cells) (HPR116, BIOPREDIC International) were thawed. HepaRG cells are known to exhibit high CYP enzyme activity after seeding, which then declines over the next 48 hours.

[0167] Human hepatocytes (HepaRG cells) were seeded and cultured according to the procedure described in (1) above. Cells were separated from these cell populations for luminescence and Raman observation. CYP enzyme activity was detected by luminescence and Raman observation 6, 24, and 48 hours after seeding the HepaRG cells.

[0168] A portion of the prepared human hepatocytes (HepaRG cells) was used for the luminescence method as a control experiment, and the remaining human hepatocytes (HepaRG cells) were used for Raman observation. The luminescence method and Raman observation were carried out according to the procedures described in (3) and (5) above, respectively, to measure the activity of CYP3A4 in the human hepatocytes (HepaRG cells). In the Raman observation of this example, different cell groups seeded and cultured at the same timing were observed for each time point. Analysis of the Raman observation results revealed that the 1636 cm peak, which was expected to correlate well with CYP activity in (6) and (7) above, was observed. -1 The Raman signal of CYP3A4 was extracted and compared with the results of CYP3A4 using the luminescence method.

[0169] Figures 20 and 21 show the results of detecting the time course of CYP activity in HepaRG cells by luminescence and Raman observation, respectively. The time course of the CYP enzyme activity after seeding of HepaRG cells was measured at a wavenumber of 1636 cm. -1 The dark gray bar in Figure 20 shows the Raman signal of the CYP enzyme group at wavenumber 1636 cm, which was obtained after averaging the Raman spectrum from the liver parenchymal cell region. -1The Raman signal intensity is plotted, and the light gray bar graph indicates the CYP activity detected by the luminescence method. The signal extraction was performed by using, as the origin, the intersection of the straight line connecting both ends of the peak base at the peak including the wavenumber 1636 cm -1 and the straight line dropped vertically from the peak apex of the peak waveform to the wavenumber axis, and taking the height up to the peak apex as the signal intensity.

[0170] Also, FIG. 21 is a box-and-whisker plot showing the variation in the CYP enzyme group activity for each cell. Each individual plot was obtained by extracting the Raman signal at the wavenumber 1636 cm -1 from the average spectrum for each cell in the same manner as described above. Similar to the results in FIG. 15, even under certain culture conditions, the CYP enzyme group activity varies for each cell. Also, the cells in the culture dish do not all uniformly lose CYP activity. Further, the decrease in the median value of the plots at each culture condition (difference in time points) well matches the variation in the activity of CYP3A4 detected by the luminescence method. The decrease in the median value of the plots was statistically significant.

[0171] From the results of Examples 2 to 5, it was confirmed that there is a positive correlation between the Raman signal at the wavenumber 1636 cm -1 indicating the oxidized CYP enzyme group and the activity of CYP3A4 detected by the luminescence method. From this, it can be said that the activity of the CYP enzyme group can be measured using the Raman signal at the wavenumber 1636 cm -1 .

[0172] Although not particularly shown, the change in the distribution and the change in the intensity of the Raman signal at the wavenumber 1370 cm -1 also show a tendency equivalent to that at the wavenumber 1636 cm -1 . Therefore, the Raman signal at the wavenumber 1370 cm -1 can also be utilized for measuring the CYP enzyme activity.

[0173] <Example 6> <Inhibition of CYP Activity by Azamulin>

[0174] In this example, we attempted to detect the inhibition of CYP enzyme activity in human hepatocytes (HepaRG cells) by Raman observation. Azamulin is known as a mechanism-based inhibitor (MBI) that specifically acts on CYP3A (including CYP3A4) (Non-Patent Document 24). Therefore, azamulin inhibits the activity of CYP3As in human hepatocytes (HepaRG cells).

[0175] Azamulin, metabolized by the catalytic action of CYP3As, produces highly reactive metabolites. These metabolites irreversibly form covalent bonds with CYP3As, including CYP3A4, inhibiting the catalytic action (metabolic ability) of CYP3As. When CYP3As are inhibited, drugs are not metabolized in the body, resulting in elevated blood drug concentrations. This results in altered therapeutic efficacy and an increased risk of serious side effects. Therefore, detecting a decrease in CYP3As activity (drug metabolic ability) caused by inhibitors is important in evaluating CYP3As activity.

[0176] Hepatocyte seeding, culture, induction, and inhibition were performed according to the procedures described in (1) and (2) above. The inducer (Rifampicin) and inhibitor (Azamulin) were added to the culture medium at concentrations of 4 μM and 10 μM, respectively. As a control, human hepatocytes (HepaRG cells) cultured in medium (MIL600, ADD650, BIOPREDIC International) without the inhibitor (Azamulin) after induction were also prepared. Cells were isolated from these cell groups for luminescence and Raman observation. A portion of the prepared human hepatocytes (HepaRG cells) was used for luminescence as a control experiment for Raman observation, while the remaining human hepatocytes (HepaRG cells) were used for Raman observation. Luminescence and Raman observation were performed according to the procedures described in (3) and (5), respectively, to measure CYP3A4 activity in human hepatocytes (HepaRG cells). In the analysis of the Raman observation results, the peak at 1636 cm , which was expected to correlate well with CYP activity in (6) and (7) above, was observed. -1 The Raman signal of CYP3A4 was extracted and compared with the results of CYP3A4 using the luminescence method.

[0177] Figures 22 and 23 show the activity of CYP enzymes detected by luminescence and Raman observation, respectively. In Figure 22, CYP3A4 activity was measured by luminescence with and without CYP3A4 inhibition by azamulin. As mentioned above, CYP was induced with rifampicin before administering azamulin. The luminescence detection results in Figure 22 confirmed that 82% of all CYP3A4 activity was lost after 5 minutes of CYP enzyme inhibition by azamulin.

[0178] Figure 23 shows the relationship between the presence or absence of CYP3A4 inhibition by azamulin and the wavenumber 1636 cm -1 The Raman scattering signal is extracted at a wavenumber of 1636 cm. -1 The signal intensity was measured by taking the point of intersection of a line connecting both ends of the base of the peak and a line drawn perpendicular to the wavenumber axis from the peak of the peak waveform as the origin, and measuring the height to the peak apex. Before administering azamulin, CYP was induced with rifampicin. Figure 23 shows that administration of azamulin resulted in a peak at a wavenumber of 1636 cm. -1 The individual plots show a decrease in the Raman signal at 1636 cm from the Raman spectrum averaged over each cell. -1 The Raman signal at wavenumber 1636 cm -1 The Raman signal at 1636 cm varies among individual cells, indicating differences in CYP enzyme activity among cells. The mean value of the plot is reduced in human hepatocytes (HepaRG cells) treated with azamulin compared to cells induced with rifampicin but not treated with azamulin. This indicates that the wavenumber at 1636 cm -1 By observing the Raman signal, it can be said that the inhibition of CYP enzyme activity was detected. It was also confirmed that the decrease in the median value of the plot was statistically significant.

[0179] Based on the prior art and previous experimental results, the signal of oxidized heme b (wavenumber 1636 cm -1) correlates with the CYP enzyme activity of the CYP enzymes in cells. More specifically, by considering the following (1) to (7), it can be understood that the enzyme activity of the CYP enzymes clearly correlates with the number of oxidized CYP enzyme molecules: (1) As a basic premise, CYP3A4 accounts for the majority (approximately 30%) of CYPs present in the human liver. (Pharmacology & Therapeutics 138 (2013) 103-141. Cytochrome P450 enzymes in drug metabolism: Regulation of gene expression, enzyme activities, and impact of genetic variation) (2) When rifampicin was used as a CYP enzyme inducer, the 1636cm -1 The increase in the signal from CYP3A4 shows a high correlation with the results of evaluation using existing CYP3A4 enzyme activity evaluation methods (Figures 13, 14, and 15). (3) Even when CYP inducers other than rifampicin were used, the 1636 cm -1 The increase in the signal from CYP3A4 shows a high correlation with the results of evaluation using existing CYP3A4 enzyme activity evaluation methods (Figures 16 and 17). (4) The tendency of IL-6 to inhibit CYP activity was also evaluated using existing methods for evaluating CYP enzyme activity. -1 The evaluation results based on the signals show a high correlation (Figures 18 and 19). (5) Even without induction of CYP enzymes, the time course of the decline in CYP activity was comparable to the results of evaluation using existing methods for evaluating CYP enzyme activity. -1 The evaluation results based on the signals show a high correlation (Figures 20 and 21). (6) In the experimental system where Azamulin, a competitive inhibitor of CYP3A4, was added, the decrease in CYP enzyme activity was 1636 cm , which was different from the results of the evaluation using existing CYP enzyme activity evaluation methods. -1The evaluation results based on the signals show a high correlation (Figures 22 and 23). Considering the above (1) to (6), the enzymatic activity of CYP enzymes has a very strong correlation with the molecular amount of oxidized CYP enzymes, indicating that the enzymatic activity of CYP enzymes can be measured by measuring the molecular amount of oxidized CYP enzymes. Furthermore, the correlation between the amount of oxidized CYP molecules and the enzymatic activity of the CYP enzymes can be explained by the theoretical interpretation shown in (7). (7) The reaction cycle of CYP enzymes involves repeated structural changes: oxidized CYP → reduced CYP → oxidized CYP → reduced CYP. Addition of a competitive inhibitor does not change the amount of CYP enzyme molecules, but the reaction cycle stops at the reduced CYP stage, resulting in a decrease in the amount of oxidized CYP molecules.

[0180] Raman observation was performed on human hepatocytes (HepaRG cells) in which CYP enzymes were inhibited with azamulin. -1 It was confirmed that the Raman signal at wavenumber 1636 cm -1 The Raman signal at 1636 cm is mainly due to the heme containing iron (Fe) in the oxidized heme protein. -1 The decrease in the Raman signal of 1636 cm is thought to be due to the following three reasons: 1) A decrease in the number of hemoprotein molecules containing heme; 2) The hemoprotein changes from an oxidized state to a reduced state; and 3) The molecular structure changes, resulting in a decrease in the Raman signal at a wavenumber of 1636 cm. -1 The molecular structure that exhibits the Raman signal is reduced or eliminated.

[0181] In this example, the possibility of cause 1) is considered low. This is because it is considered that the number of hemoproteins does not change when CYP enzyme activity is inhibited for 5 minutes using azamulin. Therefore, it is considered that causes 2) and 3) affect the Raman observation results shown in Figure 23. To confirm the influence of cause 2), the wavenumber 675 cm, which indicates reduced hemoprotein type b, was used. -1The Raman signal intensity distribution of 1636 cm indicates the oxidized heme protein type b. -1 The Raman signal intensity distribution (Raman image) was compared with that of the CYP enzymes and cytochrome b5, which are type b hemoproteins contained in HepaRG cells, to confirm the intracellular distribution of hemoprotein molecules. Furthermore, to confirm the intracellular distribution of hemoprotein molecules, immunostaining was performed for the CYP enzymes and cytochrome b5, which are type b hemoproteins contained in HepaRG cells. The results are shown in Figure 24. Figure 24 includes Raman images obtained by the method (5) above and fluorescence images obtained by the method (8) above. If the oxidized CYP enzymes were converted to a reduced state by the administration of azamulin, as shown in cause 2), then the intracellular wavenumber of 675 cm -1 However, as can be seen from the Raman observation results in Figure 24, the Raman signal at wavenumber 675 cm -1 From the above results, the remaining reason 3) is the wavenumber 1636 cm -1 This is thought to be the true cause of the decrease in the Raman signal of CYP3A4. In other words, administration of Azamulin changes the molecular structure of CYP3A4, resulting in a decrease in the wavenumber of 1636 cm, which reflects the enzyme activity. -1 It can be seen that the molecular structure showing the Raman signal of

[0182] Although not specifically shown, the wavenumber is 1370 cm -1 The change in the distribution of the Raman signal and the change in the intensity of the Raman signal at the wavenumber 1636 cm -1 The results showed a similar trend to those of the previous study.

[0183] Example 7 <Multifaceted measurement of liver function using Raman scattering signals>

[0184] In this example, the wavenumbers 1370-1636 cm , which indicate CYP metabolic activity, -1 The Raman scattering signal group was obtained by adding other Raman signals to the Raman signal of 940 cm. -1 The Raman signal at 600 cm originates from glycogen, which is related to sugar metabolism. -1 is derived from cytochrome c, which is involved in energy production. Wavenumber 2850 cm -1The Raman signals from these are related to the metabolism of lipids in the liver. By detecting these Raman scattering signals collectively, we attempted to measure the cellular functions of human liver cells (HepaRG cells) from multiple angles.

[0185] Hepatocytes were seeded, cultured, and induced according to the methods described in (1) and (2) above. However, the timing of medium change, CYP induction, and CYP measurement differed from those described in (1) and (2) above. In this example, the medium was replaced with medium (MIL600, ADD670, BIOPREDIC International) 1 and 4 days after seeding. CYP induction was performed on days 6 and 7 after seeding by replacing the medium with medium containing 4 μM rifampicin (MIL600, ADD650, BIOPREDIC International). Control cells were cultured in medium (MIL600, ADD650, BIOPREDIC International) without the inhibitor (azamulin). A portion of the prepared human hepatocytes (HepaRG cells) was used for luminescence as a control experiment for Raman imaging, and the remaining human hepatocytes (HepaRG cells) were used for Raman imaging. Luminescence and Raman observations were performed on days 1 and 8 after seeding according to the procedures described in (3) and (5) above, respectively, to measure the activity of CYP3A4 in human hepatocytes (HepaRG cells).

[0186] Figure 25 shows the relationship between liver parenchymal cells and various Raman scattering signals. In Figure 25, the light gray dashed line shows the Raman spectrum of HepaRG cells measured on Day 1, the light gray solid line shows the Raman spectrum measured on Day 8, and the dark gray solid line shows the Raman spectrum of cells in which CYP enzymes were induced with 4 μM Rifampicin from Day 6, measured on Day 8. As shown in Figure 25, the wavenumber 1636 cm originating from CYP enzymes is -1 It can be seen that the Raman signal at 940 cm originating from glycogen increases depending on the number of days the liver parenchymal cells are cultured or due to the induction of CYP enzymes. -1The Raman signal of cytochrome b5 and cytochrome c increases with the number of days of culture of liver parenchymal cells. However, the Raman signal decreases with the administration of rifampicin. Rifampicin has the effect of promoting the decomposition of glycogen, and this example demonstrated that this phenomenon can be observed without labeling by using a Raman microscope. -1 The Raman signal intensity per unit cell of the lipid-containing sample did not change. -1 The Raman signal of was slightly reduced by the induction of CYP.

[0187] The intracellular distribution of each of the above Raman signals is shown in Figure 26. Figure 26 shows the Raman data measured according to the procedure described in (5) above, and the Raman signals representing each biomolecule were extracted according to the methods described in (6) and (7) above, and the signal intensity distribution within the cell was obtained. The Raman scattering signals were extracted at a wavenumber of 1636 cm. -1 The signal intensity was measured by measuring the height to the peak apex, with the origin being the intersection of a line connecting both ends of the peak base with a line drawn perpendicularly from the apex of the peak waveform to the wavenumber axis. -1 The Raman signals of are mainly expressed in liver parenchymal cells and are hardly observed in bile duct epithelial cells. The Raman signals of cytochrome c, which is related to energy production, phenylalanine, an essential amino acid, and lipids are observed at wavenumbers of 600, 1000, and 2850 cm. -1 The Raman signal can be seen throughout the entire cell within the field of view. The distribution of each signal intensity corresponds to the mitochondria where cytochrome c is present, the entire cytoplasm where phenylalanine is present, and the lipid range where lipids are present.

[0188] Using this method, we were able to detect Raman signals from biomolecules related to metabolic activity from living cells under a microscope. We were also able to simultaneously detect Raman signals from biomolecules that represent the state and structure of the cell. The intracellular distribution of each detected biomolecule can be grasped with spatial resolution at the organelle level. Therefore, when examining cellular drug responses in metabolism and energy production, we can comprehensively examine the behavior of biomolecules localized in other organelles. -1 and 940 cm -1 We believe that observing the Raman signals will contribute not only to measuring CYP enzyme activity and glucose metabolism, but also to elucidating the mechanisms of these cellular functions.

[0189] Although not specifically shown, the wavenumber is 1370 cm -1 The change in the distribution of the Raman signal and the change in the intensity of the Raman signal at the wavenumber 1636 cm -1 The results showed a similar trend to those of the previous study.

[0190] Example 8 Visualization of CYP activity in primary human hepatocytes Frozen primary human hepatocytes (PHH, Lot No. HC2-50), OptiThaw Hepatocyte Isolation Kit (K8000), OptiCulture Media Kit (K8300M), and OptiPlate Hepatocyte Media (K8200) were purchased from Sekisui XenoTech, LLC. PHH were thawed using the OptiThaw Hepatocyte Isolation Kit and cultured on a 35 mm quartz-bottomed dish (SF-S-D12; Fine Plus International) for Raman observation. Specifically, the quartz bottom of the 35 mm dish was pre-coated with collagen, and a 15 mm inner diameter silicone ring was placed on the quartz. 3.2 × 10 cells were placed inside the ring. 4 The cells were seeded. After the PHHs adhered to the culture surface (7 hours after seeding), Raman measurements were performed. HepaRG (a cell line derived from human hepatocytes) at the same density served as a control.

[0191] Figure 27 shows the Raman spectra of PHH and HepaRG cells. Both cells show a peak at 1636 cm, which indicates CYP activity. -1 Figure 28 shows cell images of PHH and HepaRG cells, visualizing the Raman signals corresponding to each spectrum. -1 Intracellular CYP activity was observed in both PHH and HepaRG cells. These results demonstrate that CYP activity can be detected and its intracellular distribution visualized not only in HepaRG but also in primary hepatocytes.

[0192] Example 9 Visualization of CYP activity in hepatic-like cells (HLCs) differentiated from human induced pluripotent stem cells (hiPSCs)

[0193] hiPSCs were seeded onto a 35 mm dish with a quartz bottom pretreated with Matrigel (BD Science). 5The cell culture area was defined by a 15 mm inner diameter silicone ring. Stepwise differentiation into definitive endoderm cells, hepatoblast-like cells, and hepatocyte-like cells (HLCs) was performed over a 25-day period without intervening passaging. hiPS cells were cultured in RPMI 1640 medium (Sigma-Aldrich) containing 100 ng / ml Activin A (R&D Systems), 1x Gluta-MAX (Thermo Fisher Scientific), and 1x B27 Supplement Minus Vitamin A (Thermo Fisher Scientific) for 4 days to induce differentiation into definitive endoderm cells. To induce hepatoblast-like cell differentiation, the medium was then replaced with RPMI 1640 medium containing 20 ng / ml BMP4 (R&D Systems), 20 ng / ml FGF4 (R&D Systems), 1x GlutaMAX, and 1x B27 Supplement Minus Vitamin A, and cultured for 5 days. The cells differentiated into hepatoblast-like cells were then cultured for 5 days in RPMI1640 medium supplemented with 20 ng / ml HGF, 1x GlutaMAX, 1x B27 Supplement Minus Vitamin A, and then cultured in Hepatocyte Culture Medium Bullet Kit supplemented with 20 ng / ml oncostatin M (Osm). TM The induction of HLC was completed by culturing the cells in HCM (Lonza) for 11 days, after which the HLC cells were observed using a Raman microscope.

[0194] Figure 29 shows the Raman spectra of HepaRG cells in which CYP was induced with rifampicin, HepaRG cells without the addition of an inducer, and HLCs. -1 Figure 30 shows images of cells in which the Raman signals corresponding to each spectrum are visualized. -1Intracellular CYP activity was observed in all of the hepatocytes. These results demonstrate that it is possible to visualize the intracellular distribution of CYPs and their metabolites not only in HepaRG but also in various other hepatocytes.

[0195] <Application example>

[0196] The advantages of the method for detecting the enzymatic activity of CYP enzymes in cells according to the above embodiment are explained below, along with various applications of the method. CYP activity is an indicator of metabolic capacity, which is one of liver functions. Therefore, this method enables rapid and precise (at the organelle level) metabolic capacity diagnosis, thereby improving the efficiency of diagnosis in clinical settings and drug response evaluation in drug discovery. Furthermore, in the development of regenerative medicine technology, the method can be applied to evaluate the metabolic capacity of iPS-derived hepatocytes and constructed three-dimensional tissues.

[0197] Many of the prior art methods for detecting CYP activity are "destructive" or "invasive" tests that destroy cell tissue in a single test using mass spectrometry or colorimetric probes. In contrast, this embodiment enables minimally invasive, label-free detection of CYP activity with organelle-level resolution. Using this technology, CYP activity and its distribution in three-dimensional liver tissue can be rapidly visualized without destroying the tissue.

[0198] The inventors have found that there is a correlation between the redox state of CYP metabolic enzyme molecules and CYP activity. In the above embodiment, the activity of CYP metabolic enzymes is measured by detecting changes in spontaneous Raman scattering light caused by the state of the molecules. The inventors have found that the activity of CYP metabolic enzymes is correlated with the redox state of CYP metabolic enzyme molecules at 1370 cm -1 , 1636 cm -1It has been discovered that the Raman signal derived from oxidized heme b, which contains CYP, correlates with CYP activity. In the above embodiment, the Raman signal is used to measure CYP activity in hepatocytes. In a further application of the above embodiment, the activity of purified CYP proteins and CYPs expressed in other organ tissues is measured. Furthermore, in the above embodiment, the Raman signals of other factors related to the metabolic function of cells or tissues, such as reduced / oxidized heme b, reduced / oxidized heme c, and glycogen, are simultaneously detected while measuring CYP activity, thereby further evaluating the type, differentiation, and maturity of cells or tissues.

[0199] CYP metabolic enzymes are hemoproteins expressed in hepatocytes and small intestinal epithelial cells and localized in the endoplasmic reticulum (ER). They are known as a group of enzymes involved in detoxification and drug metabolism. Because CYP metabolic enzyme activity plays a key role in drug metabolism, it is used as an indicator of hepatic metabolic activity measurement in drug discovery and development. Therefore, there is a strong demand in the fields of drug discovery, regenerative medicine, and drug discovery for a method to detect CYP metabolic enzyme activity without destroying cellular tissue. The inventors have succeeded in non-destructively detecting the activity of CYP metabolic enzymes by utilizing Raman scattering signals. Furthermore, the inventors have discovered a Raman signal that correlates not only with enzyme quantity but also with enzyme activity.

[0200] While Raman signals correlated with the redox state and amount of CYP metabolic enzymes in vitro have been known in prior art, there have been no reports of their measurement within cells or tissues. In contrast, the above-described embodiment detects CYP enzyme activity in addition to the amount of CYP enzymes nondestructively and without staining within cells or tissues. This enables faster and more quantitative CYP activity measurement and measurement of changes in CYP activity over time within the same sample. Furthermore, the above-described embodiment utilizes Raman signals to simultaneously detect Raman signals derived from other metabolic indicators. This allows for the acquisition of the distribution of biomolecules resulting from the metabolic state of cells or tissues. Therefore, the above-described embodiment can also be applied to the evaluation of the differentiation and maturity of cells or tissues. In particular, in hepatic tissue, it is possible to evaluate metabolism related to drug efficacy and toxicity and the regional specificity of metabolic function.

[0201] In the field of regenerative medicine, it is known that the number of passages and culture methods can significantly affect the quality of human ES / iPS cells, in particular. Therefore, there is a need for standardization of cell quality control techniques. Cell quality control is also necessary in the process of producing hepatocytes from ES / iPS cells. In one aspect, quality control is performed by evaluating the metabolic capacity of iPS-derived hepatocytes and constructed three-dimensional tissues. In one aspect, metabolic capacity is evaluated using the method for detecting the enzymatic activity of CYP enzymes in cells, as described in the above-described embodiment.

[0202] In the field of drug discovery, there is a demand for non-destructive, time-dependent evaluation of drug efficacy and toxicity (ADMET evaluation) using cell tissues. In one aspect, such evaluation of drug efficacy and toxicity is carried out by the method for detecting the enzymatic activity of CYP enzymes in cells according to the above-described embodiment.

[0203] In the field of diagnostics, there is a demand for effective indicators that can identify abnormal areas in cells or tissues to be diagnosed, as well as technologies that can rapidly detect and measure these indicators. For example, tissue diagnosis during and after surgery in cancer resection requires advanced judgment by a pathologist. In the above embodiment, imaging of metabolic biomolecules, including CYP activity, is performed nondestructively and without staining. In one aspect, this imaging is used to evaluate the efficacy and toxicity of cells or tissues, evaluate their quality, and perform rapid tissue diagnosis.

[0204] The present invention is not limited to the above-described embodiment, and can be modified as appropriate within the scope of the invention. [Industrial Applicability]

[0205] As described above, the present invention can be extremely useful in the fields of regenerative medicine technology, drug discovery technology, and diagnostic technology.

[0206] This application is based on patent application No. 2021-104309 filed in Japan (filing date: June 23, 2021), the contents of which are incorporated in their entirety into this specification.

Claims

1. A method for evaluating intracellular or extracellular enzymatic activity of a CYP enzyme, comprising a step of measuring the number of molecules of an oxidized CYP enzyme, comprising: The method of claim 1, wherein the greater the number of oxidized CYP enzyme molecules, the higher the CYP enzyme activity of said CYP enzyme.

2. In the case of evaluating the enzymatic activity of an intracellular CYP enzyme, the step of measuring the number of molecules of an oxidized CYP enzyme comprises: Irradiating the cells with excitation light and acquiring a Raman spectrum using a photodetector; and The method of claim 1 , further comprising the step of extracting a Raman scattering signal from the Raman spectrum originating from a CYP enzyme.

3. The wavenumber of the Raman scattering signal derived from the CYP enzyme is 300-600, 620-880, 920-1320, or 1320-1660 cm -1 The method of claim 2, wherein the concentration is within the range of

4. The wave number is 1370 cm -1 Or 1636cm -1 The method according to claim 3, wherein

5. The method according to any one of claims 1 to 4, wherein the cells are derived from any one of the liver, the small intestine, the kidney, and the brain.

6. The method of claim 5 , wherein the cells are derived from the liver.

7. The method according to any one of claims 1 to 4, wherein the cell is a cell derived from a pluripotent stem cell.

8. The method according to any one of claims 1 to 4, further comprising a step of observing at least one selected from the group consisting of cell shape, cell size, and intracellular distribution of intracellular components in the region where the number of CYP enzyme molecules is measured.

9. The method according to any one of claims 1 to 4, further comprising the step of extracting a Raman scattering signal from a substance other than a CYP enzyme.

10. The method according to claim 9, wherein the substance other than the CYP enzyme is at least one selected from the group consisting of reduced heme b, reduced / oxidized heme c, glycogen, reduced / oxidized cytochrome c, phenylalanine, and lipids.

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