Method for evaluating cardiomyocytes using Raman scattering

Abstract

Provided are methods for evaluating differentiation into cardiocytes and maturation of the cardiocytes. Method for evaluating cardiocytes using Raman scattering: The Raman spectrum of cardiocytes artificially induced to differentiate from pluripotent stem cells is acquired, the intensity of Raman-scattered light for proteins that include heme b and / or heme c as prosthetic groups is acquired according to the Raman spectrum, and the state of progress of maturation of the cardiocytes is evaluated on the basis of the intensity of the Raman-scattered light. Method for evaluating differentiation into cardiocytes using Raman scattering: Differentiation of pluripotent stem cells into cardiocytes is artificially induced, the Raman spectrum of the cell for which differentiation was induced is acquired, the intensity of Raman-scattered light for heme b and / or heme c is acquired according to the Raman spectrum, and the state of progress of differentiation into cardiocytes is evaluated on the basis of the intensity of the Raman-scattered light.

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating cardiomyocytes using Raman scattering. [Background technology]

[0002] With the progress of research on iPS cells and other pluripotent stem cells, research is being conducted every day with the aim of differentiating pluripotent stem cells into various organ cells and applying this to clinical applications. Research is also being actively conducted on inducing cardiomyocytes from pluripotent stem cells. As a result of this research, it has become possible to artificially create thin sheets of cardiomyocytes.

[0003] In clinical medicine, cardiomyocytes differentiated from pluripotent stem cells through artificial induction are formed into sheets, which are then transplanted into patients. This requires the establishment of methods for evaluating cardiomyocytes during the differentiation process, cardiomyocytes immediately after differentiation, and cardiomyocyte sheets, and strict quality assurance based on these evaluation methods.

[0004] In current clinical medicine, immediately after differentiation, those cardiomyocytes that are expected to grow into cardiomyocytes are formed into sheets, and then the cells in the sheets are allowed to mature.To evaluate the degree of differentiation of cardiomyocytes immediately after differentiation, the cells are fixed, stained with an antibody to TnT2, a cardiomyocyte marker, and the degree of differentiation is evaluated using FACS (Fluorescence Activated Cell Sorting).

[0005] After evaluation, the quality of the cardiomyocytes is analyzed by measuring the strength of the pulsation of the cardiomyocyte sheet as the cardiomyocytes mature and their drug response. Quality analysis involves microscopic observation of fluorescently stained cells, FACS, and quantitative PCR (polymerase chain reaction) of nucleic acids extracted from the cells. All of these analytical methods are invasive to the cardiomyocytes. Other analytical methods also involve invasiveness.

[0006] When using cardiomyocytes immediately after differentiation or cardiomyocyte sheets in clinical medicine, it is difficult to apply the cardiomyocytes themselves, which have been evaluated using the above-mentioned invasive methods, to a living body. Therefore, it is necessary to rely on tests using samples separate from the cells to be applied to the living body. For this reason, a non-invasive method for evaluating cardiomyocytes immediately after differentiation and cardiomyocytes artificially formed into sheets is needed. This also applies when cardiomyocytes in shapes other than sheets are used in clinical medicine.

[0007] The following prior art techniques are known for evaluating cardiomyocytes using Raman spectroscopy.

[0008] Paragraph

[0038] of Patent Document 1 states that the Raman spectrum of the cytoplasm of cardiac myocytes obtained from the subepicardium of a rat heart was measured. The Raman spectrum showed peaks at 751, 1130 and 1582 cm -1 These Raman bands are thought to be specific vibrational modes arising from the porphyrin ring at the center of the heme. Among these heme proteins, the peaks of reduced cytochrome b5 and reduced cytochrome c correspond well to the peaks of the cardiomyocytes mentioned above.

[0009] In Non-Patent Document 1, C2C12 mouse striated muscle cells, which have undergone significant changes in shape due to differentiation, are evaluated using Raman scattering. -1 The Raman scattering image of the cells was obtained using the cytochrome c peak.

[0010] In Non-Patent Document 2, a 570 cm peak was detected in the Raman spectrum of myoglobin extracted from horse heart. -1 A Raman shift of 1000 nm has been observed. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2010 / 103661 [Patent Document 2] Japanese Patent Publication No. 2020-018242 [Patent Document 3] International Publication No. 2020 / 067479 [Patent Document 4] International Publication No. 2013 / 111875 [Patent Document 5] International Publication No. 2016 / 060260 [Patent Document 6] International Publication No. 2018 / 124210

Non-licensed literature

[0012]

Non-patent document 1

[0013]

Non-patent document 2

[0014] [Non-Patent Document 3] Giacomelli, Elisa et al., “Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease”, May 26, 2020, Cell Stem Cell, Volume 26, Issue 6, 862-879, e11, [online], [retrieved on 2021-06-03]. Retrieved from <https: / / doi.org / 10.1016 / j.stem.2020.05.004>

[0015] [Non-Patent Document 4] Anton Mihic, Jiao Li, Yasuo Miyagi, Mark Gagliardi, Shu-Hong Li, Jean Zu, Richard D. Weisel, Gordon Keller, Ren-Ke Li, “The effect of cyclic stretch on maturation and 3D tissue formation of human embryonic stem cell-derived cardiomyocytes”, Biomaterials, Volume 35, Issue 9, 2014, Pages 2798-2808, [online], [retrieved on 2021-06-03]. Retrieved from <https: / / doi.org / 10.1016 / j.biomaterials.2013.12.052>

[0016] [Non-Patent Document 5] Nunes , S. , Miklas , J. , Liu , J. et al. “Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes”, Nat Methods 10, 781–787 (2013), [online], [retrieved on 2021-06-03]. Retrieved from<https: / / doi.org / 10.1038 / nmeth.2524>

[0017]

Outdoor Configuration6

[0018]

Direct Environment 7

[0019] [Non-Patent Document 8] Feyen, Dries A.M. et al., “Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes”, Cell Reports, Volume 32, Issue 3, 107925, [online], [retrieved on 2021-06-03]. Retrieved from <https: / / doi.org / 10.1016 / j.celrep.2020.107925>

[0020] [Non-Patent Document 9] Antje Ebert, Amit U. Joshi, Sandra Andorf, Yuanyuan Dai, Shrivatsan Sampathkumar, Haodong Chen, Yingxin Li, Priyanka Garg, Karl Toischer, Gerd Hasenfuss, Daria Mochly-Rosen, Joseph C. Wu, “Proteasome-Dependent Regulation of Distinct Metabolic States During Long-Term Culture of Human iPSC-Derived Cardiomyocytes”, Circulation Research. 2019;125:90-103, 20 May 2019, [online], [retrieved on 2021-06-03]. Retrieved from<https: / / doi.org / 10.1161 / CIRCRESAHA.118.313973> Summary of the Invention [Problem to be solved by the invention]

[0021] The present invention provides methods for assessing differentiation into cardiomyocytes and maturation of cardiomyocytes. [Means for solving the problem]

[0022] <1> A method for evaluating cardiomyocytes using Raman scattering, comprising: Raman spectra were obtained from cardiomyocytes artificially induced to differentiate from pluripotent stem cells. The intensity of Raman scattered light of a protein containing at least one of heme b and heme c as a prosthetic group is obtained from the Raman spectrum; evaluating the progress of maturation of cardiomyocytes based on the intensity of the Raman scattered light; method. <2> the protein containing at least one of heme b and heme c as a prosthetic group is reduced cytochrome c, The intensity of the Raman scattered light of reduced cytochrome c was obtained as the Raman scattered light, and the intensity peak was at a wavenumber of 743 cm -1 From 755cm -1 is calculated based on the intensity of Raman scattered light at the Raman shift of <1> The method described below. <3> the protein containing at least one of heme b and heme c as a prosthetic group is oxymyoglobin; The intensity of the Raman scattered light of oxymyoglobin is obtained, and the intensity is at least 565 cm -1 From 575cm -1 is calculated based on the intensity of Raman scattered light at the Raman shift of <1> The method described below. <4> Utilizing the Raman spectrum excited by light with a wavelength of 532 nm, <1> ~ <3> A method according to any one of the preceding claims. <5> Further, the intensity of Raman scattered light of lipids is obtained from the Raman spectrum. Evaluating the maturation of cardiomyocytes based on the intensity of Raman scattered light from lipids. <1> ~ <4> A method according to any one of the preceding claims. <6> The pluripotent stem cells are human cells. <1> ~ <5> A method according to any one of the preceding claims. <7> The cardiomyocytes are gathered together to form sheet-like tissue fragments. <6> The method described below. <8> The cardiomyocytes gather around the core material to form a cell mass. <6> The method described below. <9> The cardiomyocytes form spheroids together with human cardiac fibroblasts, human cardiac endothelial cells, and human mesenchymal cells; or The cardiomyocytes form spheroids together with human cardiac fibroblasts and human cardiac endothelial cells. <6> The method described below. <10> The cardiomyocytes form cardiac organoids. <6> The method described below. <11> Prior to acquiring the Raman spectrum, the method further comprises promoting maturation of the cardiomyocytes by at least one of the following: providing a mechanical stimulus to the cardiomyocytes; providing an electrical stimulus to the cardiomyocytes; introducing a gene into the cardiomyocytes; Co-culturing precardiomyocytes with other cells; and adding an oxidizing substrate, compound, or factor to the medium in which the cardiomyocytes are cultured; <6> The method described below. <12> acquiring the Raman spectrum, acquiring the intensity of the Raman scattered light, and evaluating the progress of maturation of the same cardiomyocytes over time; <6> ~ <11> A method according to any one of the preceding claims. <13> further comprising culturing the cardiomyocytes in the presence of a drug before acquiring the Raman spectrum; and evaluating the effect of the drug on the maturation of cardiomyocytes by evaluating the progress of maturation of the cardiomyocytes. <6> ~ <12> A method according to any one of the preceding claims. <14> the pluripotent stem cells are any of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and embryonic germ cells (EGCs); The embryonic stem cells (ESCs) include somatic cell-derived embryonic stem cells (ntESCs), <1> ~ <13> A method according to any one of the preceding claims. <15> The peak of the excitation light when acquiring the Raman spectrum is in the range of 400 nm to 600 nm. <1> ~ <14> A method according to any one of the preceding claims. <16> When acquiring the Raman spectrum, the temperature of the cardiomyocytes is lowered to 4°C or less. <1> ~ <15> A method according to any one of the preceding claims. <17> A method for evaluating differentiation into cardiomyocytes using Raman scattering, comprising: Pluripotent stem cells are artificially induced to differentiate into cardiomyocytes, obtaining a Raman spectrum of the cells induced to differentiate; The intensity of Raman scattered light from at least one of heme b and heme c is obtained from the Raman spectrum; evaluating the progress of differentiation of the cells into cardiomyocytes based on the intensity of the Raman scattered light; method. [Effects of the Invention]

[0023] The present invention provides methods for assessing differentiation into cardiomyocytes and maturation of cardiomyocytes. [Brief explanation of the drawings]

[0024] [Figure 1] Evaluation method flow [Figure 2] Schematic diagram of Raman spectroscopy for cardiomyocyte sheets [Figure 3] Raman spectrum [Figure 4] Raman scattering intensity distribution on cardiomyocyte sheet [Figure 5] Time change in the intensity of Raman scattered light with a Raman shift of 570 cm-1 [Figure 6] Time variation of the intensity of Raman scattered light with a Raman shift of 748 cm-1 [Figure 7] Myoglobin gene expression level [Figure 8] Cytochrome c gene expression level [Figure 9] Fluorescent staining of cardiomyocyte sheet [Figure 10] Evaluation of the degree of differentiation using Raman spectroscopy [Figure 11] Schematic diagram of Raman spectroscopy for cell aggregates [Figure 12] Schematic diagram of Raman spectroscopy on a well plate [Figure 13] Schematic of Raman spectroscopy in a microchannel DETAILED DESCRIPTION OF THE INVENTION

[0025] 1 shows a flow chart of a method for evaluating cardiomyocytes using Raman scattering according to one embodiment of the present invention. The evaluation items are the progress of differentiation and maturation of cardiomyocytes. In step S11, a Raman spectrum of the cardiomyocytes is acquired. Details of the acquisition of the Raman spectrum are described below.

[0026] In one embodiment of the present invention, the cardiomyocytes for which the Raman spectrum is to be acquired are those in the process of being artificially induced to differentiate from pluripotent stem cells, or those that have already been induced to differentiate. In one embodiment, the cardiomyocytes are those in the process of being induced to differentiate from pluripotent stem cells while artificially culturing the pluripotent stem cells, or those that have already been induced to differentiate. In one embodiment, the pluripotent stem cells are human cells.

[0027] Pluripotent stem cells are cells that can differentiate into almost all cells that make up the body. There are several types of pluripotent stem cells. Pluripotent stem cells that have been established to date include embryonic stem cells (ESCs), embryonic germ cells (EGs), and induced pluripotent stem cells (iPSCs). Pluripotent stem cells to which the evaluation method of the present invention can be applied include, but are not limited to, embryonic stem cells (ESCs), embryonic germ cells (EGs), and induced pluripotent stem cells (iPSCs). Embryonic stem cells (ESCs) include not only common embryonic stem cells but also somatic cell-derived embryonic stem cells (ntESCs, nuclear transfer embryonic stem cells).

[0028] FIG. 2 shows a schematic diagram of Raman spectroscopy of a cell sheet 20. In this embodiment, the evaluation method will be described using a cardiomyocyte cell sheet as an example, but the morphology of cardiomyocytes is not limited to this. In embodiments different from that shown in FIG. 2, cardiomyocytes may take the form of a cell sheet, a cell cluster, or individual cells dispersed without bonding to each other. The cell sheet 20 may be replaced with, for example, a group of cells arranged on a substrate before forming a sheet.

[0029] In Figure 2, excitation light 22 is focused into a line parallel to the y-axis through an objective lens (not shown). The focused excitation light 22 is scanned over the cell sheet 20. In the figure, scanning is performed in the +x direction. Scanning causes Raman scattering over a relatively wide area on the cell sheet, for example, a 100 µm square.

[0030] In one embodiment of the present invention shown in Figure 2, the excitation light 22 preferably has a peak at a wavelength of 400 nm to 600 nm, more preferably at a wavelength of 406 nm to 561 nm, and particularly preferably at a wavelength of 532 nm. In one embodiment, the excitation light 22 is laser light. The wavelength of the excitation light 22 is also suitable for excitation light used in various other Raman spectroscopy methods described below.

[0031] In one embodiment of the present invention shown in FIG. 2 , the excitation light 22 applies strong heat to the cell sheet 20. Cardiomyocytes in the cell sheet 20 may be damaged by the heat of the excitation light 22. When scanning the cell sheet 20 with the excitation light 22, the cell sheet 20 is cooled to a temperature lower than the temperature at which the cell sheet 20 was cultured, e.g., 37°C. For example, the temperature of the cell sheet 20 is lowered to 4°C or lower. This prevents damage to the cardiomyocytes. Lowering the temperature of the cell sheet 20 also reduces the pulsation frequency of the cardiomyocytes. This prevents sample misalignment when scanning the cell sheet 20 with the excitation light 22. Lowering the temperature to a temperature lower than 4°C further sharpens the peak of the Raman spectrum acquired from the cell sheet 20. This improves wavenumber resolution and facilitates spectral separation. In one embodiment, the temperature of the cell sheet 20 is lowered to the limit temperature at which the cells do not freeze.

[0032] In Figure 2, the spectrum of Raman scattered light 23 generated within the cell sheet 20 is detected and recorded. The spectrum is detected by passing the Raman scattered light through a spectrometer or bandpass filter and then detecting it with a two-dimensional photodetector. This allows a planar image of the Raman spectrum on the cell sheet 20 to be obtained. In other words, the Raman spectrum is observed under a microscope.

[0033] The cytoplasm of cells that have just completed differentiation into cardiomyocytes differs from the cytoplasm of cells before differentiation and has a composition unique to cardiomyocytes. Cells that have just completed differentiation into cardiomyocytes have gene and protein expression levels that differ from those of cells before differentiation. Furthermore, cells that have just completed differentiation into cardiomyocytes have myocardial function. Therefore, cells that have just completed differentiation into cardiomyocytes exhibit a Raman shift that differs from that of cells before differentiation. It is meaningful to perform microscopic observation of the Raman spectrum of cells in the process of being induced to differentiate from pluripotent stem cells.

[0034] On the other hand, cells that have just completed differentiation into cardiomyocytes are immature as cardiomyocytes. Immature cardiomyocytes and mature cardiomyocytes differ in gene expression levels, protein expression levels, and myocardial function levels. Cells whose maturation has been accelerated before Raman spectra acquisition exhibit a different Raman shift from immature cells.

[0035] This evaluation method evaluates the degree of differentiation from pre-differentiated cells to cardiomyocytes, which has been or is thought to be induced by physical, biological, and chemical methods. Cell differentiation induction may be performed as follows, following the methods for producing cardiomyocytes described in Patent Document 2 and for producing multilayered myocardial tissue cultures described in Patent Document 3. First, embryoid bodies are cultured as spheroids. Next, while the embryoid bodies are cultured in three-dimensional suspension, they are induced to differentiate into cardiomyocytes.

[0036] Furthermore, what is evaluated by this evaluation method is the degree of maturation of cardiomyocytes that has been or is thought to have been promoted by physical, biological, and chemical methods. Cardiomyocytes are cells that have already been induced to differentiate.

[0037] Physical methods include, but are not limited to, mechanical and electrical stimulation of immature cardiomyocytes. Biological methods include, but are not limited to, gene transfer to immature cardiomyocytes and co-culture with other cells that are not cardiomyocytes. Chemical methods include the addition of oxidative substrates and other compounds to the medium in which immature cardiomyocytes are cultured. In another aspect, chemical methods include the addition of oxidative substrates and other factors to the medium in which immature cardiomyocytes are cultured.

[0038] Another example of a chemical method involves culturing pluripotent stem cells in a medium containing a WNT signaling activator, followed by culturing them in a medium containing a WNT signaling inhibitor. Examples of WNT signaling activators include GSK3β inhibitors such as BIO and CHIR99021. Examples of WNT signaling inhibitors include low-molecular-weight compounds or proteins, such as KY02111, IWP-2, IWR-1, XAV939, DKK1, and IGFBP.

[0039] Proteins that have heme as a prosthetic group include cytochromes and myoglobin.

[0040] Cytochromes containing heme b are called cytochrome b, and cytochromes containing heme c are called cytochrome c. Cytochrome b and cytochrome c each have an oxidized state and a reduced state.

[0041] <Structural formula of heme b>

[0042] [ka]

[0043] <Structural formula of heme c>

[0044] [ka]

[0045] Myoglobin is a hemoprotein containing heme b as a prosthetic group. Myoglobin is further classified into reduced myoglobin, oxymyoglobin (oxygenated myoglobin), and other myoglobins.

[0046] Returning to Figure 1, in step S12, the Raman shift, i.e., the intensity of Raman scattered light at a Raman shift of a predetermined wavenumber, is obtained from the Raman spectrum. Here, the Raman shift of the predetermined wavenumber is specific to at least one of reduced cytochrome c and oxymyoglobin (oxygenated myoglobin).

[0047] In one embodiment of the present invention, the intensity of Raman scattered light from at least reduced cytochrome c is measured. The intensity of Raman scattered light is measured at wavenumbers of 743-755 cm. -1 Raman shift, preferably at wavenumber 748 cm -1 The Raman shift is calculated based on the intensity of the Raman scattered light at the Raman shift of 1000 nm.

[0048] In one embodiment of the present invention, the intensity of Raman scattered light from at least oxymyoglobin is measured. The intensity of Raman scattered light is measured at a wavenumber of 565-575 cm. -1 Raman shift, preferably at a wavenumber of 570 cm -1 The Raman shift is calculated based on the intensity of the Raman scattered light at the Raman shift of 1000 nm.

[0049] Using Raman spectroscopy, the Raman scattered light intensity of multiple types of molecules, including the above-mentioned reduced cytochrome c and oxymyoglobin, can be obtained in a single measurement of the same cardiomyocyte. As an example, the Raman scattered light intensity of lipids can be obtained. This is useful for cross-sectional analysis of multiple events in the same cell. In contrast, conventional fluorescence analysis and pulsation diagnosis performed on cardiomyocytes cannot evaluate the correlation between multiple biomolecules within the same cardiomyocyte. Cross-sectional analysis of multiple events in the same cell using Raman spectroscopy is useful for analyzing drug effects, drug toxicity, and drug metabolism.

[0050] In step S13 shown in FIG. 1, the intensity of the Raman scattered light acquired in step S12 is compared with the data. The data here includes a correlation between the intensity of the Raman scattered light and the degree of maturation of the cardiomyocytes. "Maturation" here refers to the process of immature cardiomyocytes progressing to mature cardiomyocytes. The data may be acquired in advance, or may be acquired from positive and negative control cardiomyocytes simultaneously with Raman spectroscopy of the target cardiomyocytes. The degree of maturation of the subject cardiomyocytes is evaluated by comparing the acquired Raman intensity with the data.

[0051] As described above, cardiomyocytes confirmed to be mature are transplanted into a living body or applied to drug discovery evaluation. Drug discovery evaluation includes, but is not limited to, analysis of drug efficacy, drug toxicity, and drug metabolism. Here, before applying the cardiomyocytes to a living body, the temperature of the cardiomyocytes is kept lower than when the cardiomyocytes were cultured. Application of the cardiomyocytes to a living body includes, but is not limited to, transplantation of the cardiomyocytes into a living body. Preferably, the temperature of the cardiomyocytes is kept at 4°C. This prevents damage to the cardiomyocytes before transplantation into a living body, even after evaluation of the progress of maturation of the cardiomyocytes.

[0052] In another embodiment, in step S13 shown in FIG. 1, the intensity of the Raman scattered light acquired in step S12 is compared with the data. Here, the data includes a correlation between the intensity of the Raman scattered light and the degree of differentiation of the cells. Differentiation here refers to the process from pre-differentiation cells, such as pluripotent stem cells, to immature cardiomyocytes. The data may be acquired in advance, or may be acquired from positive and negative control cells simultaneously with Raman spectroscopy of the target cells. The degree of differentiation of the test cells is evaluated by comparing the acquired Raman intensity with the data. Cardiomyocytes whose differentiation has been confirmed as described above can also be further induced to obtain mature cardiomyocytes.

[0053] <Experimental Example>

[0054] The following describes embodiments of the present invention with reference to actual experimental examples. The following description is not intended to limit the scope of the present invention, but is merely a sample to deepen understanding of the technical concept of the present invention.

[0055] Cardiomyocyte cell sheets were prepared as follows, following Patent Document 4. First, human iPS cell line 253G1 (cell number HPS0002, RIKEN Cell Materials Research Laboratory -CELL BANK-, Japan) was induced to differentiate into cardiomyocytes (CMs) on a culture dish. To induce differentiation, pluripotent stem cells were first cultured in a medium containing a WNT signal activator. Next, the cells were cultured in a medium containing a WNT signal inhibitor. One week after differentiation induction, pulsation was confirmed in the cells. Two weeks after differentiation induction, the cells were harvested.

[0056] A portion of the collected cells was stained with a fluorescent antibody against TnT2, a myocardial marker (SC-20025; Santa Cruz Biotechnology, Dallas, TX, USA). After staining, the efficiency of differentiation induction was confirmed by FACS. As a control, cells in the middle of differentiation were collected and analyzed by FACS in the same way.

[0057] Two weeks after differentiation induction, the cells were further trained. After staining, the maturation state was confirmed by FACS and Raman spectroscopy. As a control, cells that had not been trained were similarly analyzed by FACS and Raman spectroscopy. Figures 3 to 9 show the observation results regarding cardiomyocyte maturation. Figure 10 shows the observation results regarding differentiation leading to cardiomyocytes.

[0058] A portion of the prepared cardiomyocyte sheet was subjected to training, in which the maturation of cardiomyocytes was physically promoted by mechanical or electrical stimulation.

[0059] During the training, the cardiomyocyte sheets that were not trained were stored without any treatment while being continuously cultured in an incubator at 37°C.

[0060] Figure 3 shows Raman spectra acquired within a specific region on a cell sheet. The horizontal axis represents the Raman shift in wavenumber [cm -1 The vertical axis represents the intensity of Raman scattered light, i.e., Raman intensity [au]. The two curves represent the Raman spectrum of a cardiomyocyte sheet that had undergone maturation through two weeks of training (w / training) and the Raman spectrum of a cardiomyocyte sheet that had not undergone maturation through training (w / o training).

[0061] The Raman spectrum was acquired as explained using Figure 2. Raman peaks appear in the Raman spectrum. These peaks represent an increase in the intensity of Raman scattered light at that Raman shift. At the Raman shifts expressed as the following wavenumbers, the Raman intensity increased after maturation through training.

[0062] 570 cm -1 A Raman shift due to oxymyoglobin is observed around the wavenumber of 1. See the description in Non-Patent Document 2.

[0063] 595-605 cm -1 , 635-645 cm -1 , 685-695 cm -1 , 743-755 cm -1 , 915-925 cm -1 , 1120-1130 cm -1 , 1310-1320 cm -1 and 1580-1590 cm -1 The peaks around the wavenumber of 1000 indicate an increase in the Raman signal due to maturation through training. These peaks are thought to be Raman shifts resulting from oxidized and reduced cytochrome b and oxidized and reduced cytochrome c.

[0064] Figure 4 shows the intensity distribution of Raman scattered light at a specific wavenumber of Raman shifts on a cell sheet, known as a Raman image. The top row shows a cardiomyocyte sheet that has undergone maturation through training (w / Training). The bottom row shows a cardiomyocyte sheet that has not undergone maturation through training (Control, i.e., w / o Training).

[0065] In each column, from left to right, 570 cm -1 Raman image of the Raman shift around the wavenumber of 642 cm -1 (640 cm -1 ) Raman shift around the wavenumber, 687 cm -1 Raman image of the Raman shift around the wavenumber of 748 cm -1 Raman image of the Raman shift around the wavenumber of 2910 cm -1 Raman image of the Raman shift around the wavenumber of 2965 cm -1 Raman image of the Raman shift around the wavenumber of Shows.

[0066] Figure 5 shows the 570cm -1 The graph shows the time change in Raman intensity, or the signal intensity of the Raman shift near the wavenumber of 1000. The cardiomyocyte cell sheets used for observation were distributed among four culture dishes. Culture and training began on the culture dishes (w / Training). Each culture dish was observed 2 days (Day 2), 6 days (Day 6), 10 days (Day 10), and 14 days (Day 14) after the start of training. Control cultured cells (w / o Training) were distributed in the same way and observed in the same way after culturing for the same period.

[0067] The Raman intensity from the Raman image was calculated as follows. First, arithmetic operations were performed on the signal intensity of the Raman shift at the above wavenumbers. Arithmetic operations were performed as follows: In the Raman spectrum, the area enclosed by the lines connecting the apex of any Raman peak and the two bases on either side of that peak was calculated, and this was used as the signal intensity. This calculation involves adding the peak signal and subtracting the background signal. The above areas were calculated for Raman peaks at other wavenumbers that have a positive correlation with the signal intensity before and after training. Furthermore, peak signals at different wavenumbers, for example, peaks originating from the same molecule but with different vibrational modes, may be added to enhance the peak signal. Furthermore, trained and untrained cells may be compared based on the number of cells or cell concentration. To quantitatively evaluate the comparison, a Raman signal representing molecular vibrations that do not change the Raman intensity signal amount before and after training, such as the Raman signal of phenylalanine vibration, was used as the comparison standard. The Raman signal intensity was normalized by multiplication or division. The above calculations were performed for each coordinate on the measured cardiomyocyte sheet to obtain a Raman image.

[0068] Next, the Raman intensity in a specified area on the cardiomyocyte sheet is calculated by averaging the Raman signals in a spatial coordinate system that adds the intensity of the Raman signals to the plane of the Raman image. The process of obtaining the average in the spatial coordinate system is performed as follows. First, coordinates having Raman signals are extracted on the plane of the Raman image acquired as described above. The Raman image here consists of a signal intensity distribution of Raman peaks. Extraction is performed by setting a threshold for the Raman peak at an arbitrary signal intensity. The average intensity per pixel is calculated from the extraction. Alternatively, the average Raman intensity of all coordinates within a specified area on the plane of the acquired Raman image is calculated. The average Raman intensity of all coordinates on the plane of the acquired Raman image may also be calculated.

[0069] 570 cm, indicating the presence of oxymyoglobin -1The Raman intensity of the Raman shift around the wavenumber of 1 increased with time in both w / Training and Control (w / o training). The intensity further increased with maturation due to training.

[0070] Figure 6 shows the 748 cm -1 The graph shows the time course of the Raman scattering intensity at 748cm, which indicates the presence of reduced hemes c and b, especially reduced cytochrome c and cytochrome b. -1 The Raman intensity of the Raman shift around the wavenumber of 10 increased with time in both w / Training and control (w / o training), reaching a peak around day 10. The intensity further increased with maturation due to training.

[0071] Figure 7 shows the expression level of myoglobin gene in cardiomyocyte sheets. Figure 8 shows the expression level of cytochrome c gene. Controls w / o Training-1 and w / o Training-2 represent independent trials using cardiomyocyte sheets that had not undergone maturation through training. w / Training-1 and w / Training-2 represent independent trials using cardiomyocyte sheets that had undergone maturation through training.

[0072] Gene expression levels were measured using the whole gene analysis method as follows: Total RNA was extracted from cardiomyocyte sheets using Trizol reagent (Life Technologies). cDNA sequencing analysis was performed by acquiring RNA-Seq data using the Illumina Hiseq 4000 platform (Illumina). The vertical axis of the graphs in Figures 7 and 8, "Normalized FPKM," represents the following: First, by looking at the read count data obtained by RNA-Seq, we can see that the transcript amount directly represents the gene expression level. The read count data obtained from RNA-Seq data is corrected by the total number of reads, and then normalized for transcript length. The data obtained is called FPKM (RPKM for single-end reads). FPKM stands for "fragments per kilobase of exon per million reads mapped."

[0073] The gene expression of cytochrome c and myoglobin in cardiomyocytes increased in w / Training-1 and w / Training-2.

[0074] 9 shows fluorescent staining images of cardiomyocyte sheets. "w / Training" indicates a cardiomyocyte sheet that has undergone maturation through training. "w / o Training" indicates a cardiomyocyte sheet that has not undergone maturation through training.

[0075] Fluorescent staining of cardiomyocytes and image acquisition were performed as follows: Cardiomyocytes were fixed with 4% paraformaldehyde for 30 minutes at room temperature. They were then permeabilized with 0.5% (v / v) Triton X-100 in D-PBS for 1 hour. Next, the cardiomyocytes were treated with a blocking solution (0.1% (v / v) Tween-20, 5% (v / v) normal donkey serum, 3% (v / v) bovine serum albumin, 5% (v / v) normal goat serum, D-PBS) for 16 hours at 4°C. The cardiomyocytes were then incubated with anti-troponin antibody as the primary antibody. The cells were then washed with PBS and incubated with a secondary antibody diluted 1:300 in blocking buffer for 1 hour at room temperature. Finally, the cells were incubated with 4'-6-Diamidino-2-phenylindole (DAPI; 300 nM; Wako Pure Chemical Industries, Ltd.) for 30 minutes at room temperature to stain the cell nuclei.

[0076] The bright areas in each image in Figure 9 indicate the localization of cytochrome c. With training, cytochrome c in cardiomyocytes increased.

[0077] The gene expression of cytochrome c and myoglobin and the increase in the abundance of cytochrome c molecules shown in FIGS. 7 to 9 coincide with the increase in the intensity of Raman scattered light.

[0078] The above experimental example demonstrates that Raman spectroscopy can be used to non-invasively evaluate and diagnose the maturity of cardiomyocytes. The Raman spectroscopy in the above experimental example detected Raman scattering reflecting the molecular vibrations of the molecules themselves within cardiomyocytes. The molecular composition of immature cardiomyocytes was analyzed non-invasively.

[0079] As mentioned in the background art, it has not been possible to non-invasively diagnose the degree of maturation of cardiomyocytes. In contrast, the method using Raman spectroscopy is non-invasive. Therefore, it is possible to observe the maturation process of the same cardiomyocytes over time, from the early stages of maturation. Therefore, the method using Raman spectroscopy is useful for studying the maturation of cardiomyocytes. Furthermore, the method using Raman spectroscopy can evaluate the quality of cardiomyocytes to be transplanted without labeling the cardiomyocytes.

[0080] Next, we will show an example of applying Raman spectroscopy to the measurement of differentiation leading to cardiomyocytes. Figure 10 shows an example of evaluating the degree of differentiation using Raman spectroscopy. iPS cells were differentiated into cardiomyocytes. Three types of cells with different degrees of differentiation were evaluated using Raman spectroscopy. The results of evaluating the degree of differentiation for each cell group using FACS are shown in the left column. If the FACS evaluation of the degree of differentiation exceeds 80%, the cell group is determined to have differentiated from its pre-differentiation state into cardiomyocytes. Furthermore, the Raman spectroscopy intensity of each cell group was measured. The average spectrum of the measured area is shown in the right column.

[0081] As shown in Figure 10, a positive correlation was observed between the degree of differentiation determined from the FACS measurement results and the magnitude of specific Raman peaks derived from cytochrome C. The Raman shifts of these Raman peaks were 750, 1125, and 1585 cm -1 These results demonstrate that the degree of differentiation from pre-differentiated cells to cardiomyocytes can be measured noninvasively based on the increase in the Raman signal.

[0082] The above experimental example demonstrates that Raman spectroscopy can be used to non-invasively evaluate and diagnose the degree of differentiation from pre-differentiated cells to cardiomyocytes. The Raman spectroscopy in the above experimental example detected Raman scattering that reflects the molecular vibrations of the molecules themselves within the cells. The molecular composition of pre-differentiated cells was analyzed non-invasively.

[0083] Raman spectroscopy is non-invasive, allowing for the time-dependent observation of the same cells during their differentiation into cardiomyocytes. Therefore, Raman spectroscopy is useful for studying the differentiation process leading to cardiomyocytes. Raman spectroscopy can also be used to assess the quality of cells to be transplanted, such as pluripotent stem cells, without labeling the cells.

[0084] Next, aspects of the present invention other than the cardiomyocyte evaluation method introduced in the above experimental example will be described.

[0085] <Examples of methods to promote maturity>

[0086] In one embodiment of the present invention, immature cardiomyocytes are subjected to artificial treatment to promote their maturation. Examples of artificial treatments include, but are not limited to, physical methods, such as mechanical and electrical stimulation; biological methods, such as gene transfer into cardiomyocytes or co-culture with other cells; and chemical methods, such as the addition of oxidative substrates and other compounds or factors to the cardiomyocyte culture medium. The evaluation method of the present invention is then applied to the cardiomyocytes to evaluate changes in gene expression levels, protein levels, and the progress of myocardial function maturation in the cardiomyocytes. Depending on the cardiomyocyte morphology, i.e., whether the cardiomyocytes are cell sheets, cell clusters, or dispersed cells, appropriate artificial treatments are selected.

[0087] In one embodiment of the present invention, cardiomyocytes are sheet-like tissue flakes. In one embodiment, as shown in Patent Document 5, cardiomyocytes are gathered on an oriented scaffold to form a sheet-like tissue flake. The oriented scaffold allows cardiomyocytes to mimic the myocardial structure in vivo. By culturing the cardiomyocytes constituting the sheet-like tissue flakes over time, the gene expression levels, protein expression levels, and myocardial function levels of the cardiomyocytes mature. In another embodiment, cardiomyocytes are allowed to form sheet-like tissue flakes without using an oriented scaffold. This promotes the maturation of cardiomyocytes.

[0088] In one embodiment of the present invention, cardiomyocytes gather three-dimensionally to form cell clusters. In one embodiment, as shown in Patent Document 6, cardiomyocytes gather around a core material to form a torus-shaped cell cluster. The cardiomyocytes constituting the torus-shaped cell cluster are cultured over time to mature. In another embodiment, cardiomyocytes gather around a core material to form a cell cluster having a shape other than a torus shape. This promotes the maturation of cardiomyocytes.

[0089] In another embodiment of the cardiomyocytes forming cell clusters, the cardiomyocytes form spheroids. In one embodiment, as shown in Non-Patent Document 3, the cardiomyocytes form spheroids together with cardiac interstitial cells. In one embodiment, the cardiac interstitial cells are cardiac fibroblasts and cardiac endothelial cells. By co-culturing the cardiomyocytes with these interstitial cells, the cardiomyocytes mature. In one embodiment, by further co-culturing the cardiomyocytes and these interstitial cells with mesenchymal cells, the cardiomyocytes mature.

[0090] In other embodiments of the cardiomyocytes forming cell masses, the cardiomyocytes form cardiac organoids.

[0091] In one aspect of the present invention, cardiomyocytes are cultured under specific culture conditions before Raman spectroscopy is acquired. The culture conditions include, for example, the presence or absence of physical and chemical treatments, as well as the duration, rest periods, and frequency of such treatments.

[0092] In one embodiment of the present invention, the maturation of cardiomyocytes is promoted by mechanical stimulation. In one embodiment, cardiomyocytes are repeatedly stretched and contracted. As shown in Non-Patent Document 4, mechanical stimulation is applied to cardiomyocytes using Stage Flexer (trademark) supplied by FLEXCELL.

[0093] In one embodiment of the present invention, the maturation of cardiomyocytes is promoted by electrical stimulation, and in one embodiment, electrical stimulation is applied to cardiomyocytes using electrodes, as described in Non-Patent Documents 5 and 6.

[0094] In one embodiment of the present invention, the maturation of cardiomyocytes is promoted by gene transfer into cardiomyocytes. An example of gene transfer is the transfer of CDH2 (Non-Patent Document 7).

[0095] In one embodiment of the present invention, cardiomyocyte maturation is promoted by culturing cardiomyocytes in a medium supplemented with an oxidative substrate, such as a lipid. In one embodiment, palmitate is used as the oxidative substrate, as described in Non-Patent Document 8.

[0096] In one embodiment of the present invention, cardiomyocytes are matured by culturing them for a long period of time as described in Non-Patent Document 9 before acquiring a Raman spectrum.

[0097] In one aspect of the present invention, cardiomyocytes are cultured in the presence of a drug before Raman spectra are acquired. The progress of maturation of the cardiomyocytes is evaluated. Based on the above, the effect of the drug on the maturation of the cardiomyocytes is evaluated.

[0098] <Example of Raman spectroscopy>

[0099] In one embodiment of the present invention, a Raman spectroscopy method suitable for various cardiomyocyte morphologies, i.e., whether the cardiomyocytes are a cell sheet, a cell mass, or dispersed cells, is selected. A Raman spectroscopy method using line illumination excitation light according to one embodiment of the present invention has been described with reference to Figure 2. Raman spectroscopy according to other embodiments of the present invention will be described below.

[0100] Figure 11 shows a schematic diagram of Raman spectroscopy of a cell cluster 30. This method is a so-called side-illumination microscopic analysis. Because the cell cluster 30 has a three-dimensional structure, it is thicker than a monolayer cell sheet or dispersed cells. One example of the cell cluster 30 is a spheroid.

[0101] The excitation light 32 shown in Figure 11 is a Bessel beam. The Bessel beam is focused on one or a limited number of cells in the cell mass 30. Raman scattered light 33 is generated from the cells that have received the excitation light 32. By focusing the Bessel beam, an increase in the Raman scattered light generated from other cells and the culture solution 31 is prevented. Therefore, there is little Raman scattered light generated in the background of the cells to be measured by Raman spectroscopy.

[0102] As shown in Figure 11, a convex lens 34 converges the diffusing Raman scattered light 33. The converged Raman scattered light passes through a spectrometer 35. The diffraction grating of the spectrometer 35 is placed parallel to the optical axis of the excitation light 32. The spectrometer 35 resolves the Raman scattered light 33 into wavenumbers. In this way, Raman spectroscopy is performed.

[0103] Figure 12 shows a schematic diagram of Raman spectroscopy on a well plate 41. This technique is known as multi-focus spectroscopy. This technique is suitable for evaluating a large number of samples, each conditioned by multiple parameters, by Raman observation. One example of this is drug response screening. It is also suitable for spectroscopic analysis of relatively large cardiomyocyte sheets, e.g., the size of the well bottom.

[0104] As shown in Figure 12, cardiomyocyte cell sheets 40a-40c and other cardiomyocyte cell sheets are placed one by one in each well of a well plate. A multi-focus Raman spectrometer generates multiple excitation lights 42a-42c and other excitation lights, each with a focus. Each excitation light is irradiated onto each cardiomyocyte cell sheet. Conditions for promoting maturation of the cardiomyocyte cell sheet differ for each well. Raman scattered light 43 is simultaneously obtained within the plate from each cardiomyocyte cell sheet whose maturation has been promoted under different conditions. Raman spectra are obtained from this Raman scattered light 43 for each well.

[0105] Figure 13 is a schematic diagram of Raman spectroscopy in a microchannel 51 called a flow cell. This method is suitable for Raman spectroscopy of cells that are dispersed among themselves or relatively small cell clusters that are dispersed among themselves. A sheet of excitation light 52 is irradiated onto cells 50 in the microchannel. The cells 50 are dispersed one by one. Raman scattered light 53 is obtained from the cells 50 that have received the excitation light 52. A convex lens 54 converges the Raman scattered light 53.

[0106] 13, cells 50 are further sorted downstream of microchannel 51. This increases the concentration of cells 50 that have or do not have a predetermined intensity of Raman scattered light at a predetermined Raman shift. Such sorting can improve the quality of cells 50.

[0107] This application claims priority based on Japanese Patent Application No. 2022-005252, filed on January 17, 2022, the disclosure of which is incorporated herein in its entirety. [Explanation of symbols]

[0108] 20 cell sheet, 22 excitation light, 23 Raman scattered light, 30 cell mass, 31 culture medium, 32 excitation light, 33 Raman scattered light, 34 convex lens, 35 Raman scattered light, 40a-40c cardiomyocyte cell sheet, 41 well plate, 42a-42c excitation light, 43 Raman scattered light, 50 cell, 51 microchannel, 52 excitation light, 53 Raman scattered light, 54 convex lens, S11-13 steps

Claims

1. A method for evaluating cardiomyocytes using Raman scattering, comprising: Raman spectra were obtained from cardiomyocytes artificially induced to differentiate from pluripotent stem cells. obtaining the intensity of Raman scattered light from a protein containing at least one of heme b and heme c as a prosthetic group from the Raman spectrum; evaluating the progress of maturation of cardiomyocytes based on the intensity of the Raman scattered light; method.

2. the protein containing at least one of heme b and heme c as a prosthetic group is reduced cytochrome c, The intensity of the Raman scattered light of reduced cytochrome c was obtained as the Raman scattered light, and the intensity peak was at a wavenumber of 743 cm -1 From 755cm -1 is calculated based on the intensity of Raman scattered light at the Raman shift of The method of claim 1.

3. the protein containing at least one of heme b and heme c as a prosthetic group is oxymyoglobin; The intensity of the Raman scattered light of oxymyoglobin is obtained, and the intensity is at least 565 cm -1 From 575cm -1 is calculated based on the intensity of Raman scattered light at the Raman shift of The method of claim 1.

4. 2. The method of claim 1, wherein the Raman spectrum is excited with light having a wavelength of 532 nm.

5. Further, the intensity of Raman scattered light of lipids is obtained from the Raman spectrum. Evaluating the maturation of cardiomyocytes based on the intensity of Raman scattered light from lipids. The method of claim 1.

6. The pluripotent stem cells are human cells. The method of claim 1.

7. The cardiomyocytes are gathered together to form sheet-like tissue fragments. The method of claim 6.

8. The cardiomyocytes gather around the core material to form a cell mass. The method of claim 6.

9. The cardiomyocytes form spheroids together with human cardiac fibroblasts, human cardiac endothelial cells, and human mesenchymal cells; or The cardiomyocytes form spheroids together with human cardiac fibroblasts and human cardiac endothelial cells. The method of claim 6.

10. The cardiomyocytes form cardiac organoids. The method of claim 6.

11. The method further includes, prior to acquiring the Raman spectrum, promoting maturation of the cardiomyocytes by at least one of the following: providing a mechanical stimulus to the cardiomyocytes; providing an electrical stimulus to the cardiomyocytes; introducing a gene into the cardiomyocytes; Co-culturing precardiomyocytes with other cells; and adding an oxidizing substrate, compound, or factor to the medium in which the cardiomyocytes are cultured; The method of claim 6.

12. acquiring the Raman spectrum, acquiring the intensity of the Raman scattered light, and evaluating the progress of maturation of the same cardiomyocytes over time; The method of claim 6.

13. further comprising culturing the cardiomyocytes in the presence of a drug before acquiring the Raman spectrum; and evaluating the effect of the drug on the maturation of cardiomyocytes by evaluating the progress of maturation of the cardiomyocytes. The method of claim 6.

14. the pluripotent stem cells are any of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and embryonic germ cells (EGCs); The embryonic stem cells (ESCs) include somatic cell-derived embryonic stem cells (ntESCs); The method of claim 1.

15. The peak of the excitation light when acquiring the Raman spectrum is in the range of 400 nm to 600 nm. The method of claim 1.

16. When acquiring the Raman spectrum, the temperature of the cardiomyocytes is lowered to 4°C or less. The method of claim 1.

17. A method for evaluating differentiation into cardiomyocytes using Raman scattering, comprising: Pluripotent stem cells are artificially induced to differentiate into cardiomyocytes, obtaining a Raman spectrum of the cells induced to differentiate; The intensity of Raman scattered light from at least one of heme b and heme c is obtained from the Raman spectrum; evaluating the progress of differentiation of the cells into cardiomyocytes based on the intensity of the Raman scattered light; method.

Images