Method for enriching mitotic cells, and its application.

Abstract

This invention provides a method for enriching mitotic cells that allows for efficient collection of mitotic cells and better observation of the properties of cell aggregates, as well as enabling the use of such enriched cells. [Solution] A method for enriching mitotic cells is used, which involves first dispersing densely cultured cells, and then enriching the cells while culturing them in suspension to increase the proportion of mitotic cells.

Description

[Technical Field]

[0001] This invention relates to a method for enriching mitotic cells and its applications. [Background technology]

[0002] Chromosome analysis is a method of evaluating the state of cells by revealing the shape and / or number of chromosomes, or the presence and / or copy number changes of specific genes on chromosomes. In particular, information on the shape and / or number of chromosomes that appear during cell division is used to discuss the genetic stability and safety of cell-based therapies and regenerative medicine drugs.

[0003] In chromosome analysis, the cell population to be analyzed is cultured, and if necessary, the cell cycle of the cell population is synchronized using chemicals, etc. Then, the cell population is fixed with a fixative. After that, the fixed cells are dropped onto a glass slide to prepare a slide specimen. After staining the slide specimen, the chromosomes and / or interphase cell nuclei are photographed using a microscope, and the state of the cells is evaluated based on the captured images (see, for example, Patent Documents 1 and 2).

[0004] Specific methods for chromosome analysis include, for example, karyotype analysis to determine the karyotype of a cell (e.g., G-band analysis, Q-band analysis, mFISH analysis), and FISH analysis to analyze changes in the position and / or copy number of specific genes. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Special Publication No. 2023-503614 [Patent Document 2] Patent No. 6542344 [Overview of the project] [Problems that the invention aims to solve]

[0006] However, the conventional techniques described above have the problem that they are inefficient at harvesting mitotic cells from cells cultured in a dense state (e.g., cell aggregates), and that they cannot accurately observe the properties of the cell aggregates.

[0007] For example, in the technique described in Patent Document 1, cell aggregates are separated into single cells and cultured in 2D before chromosome analysis is performed. However, the technique in Patent Document 1 has the problem that because 2D culture takes a relatively long time, the properties of the cells and the cell population change during the 2D culture, making it difficult to observe the properties of the target cell aggregate itself.

[0008] The technique described in Patent Document 2 involves treating cell aggregates with colseminide. Consequently, the technique in Patent Document 2 results in differences in nutritional status and oxygen partial pressure between the outside and inside of the cell aggregate, leading to differences in cell proliferation capacity between the outside and inside of the cell aggregate, and making it impossible to uniformly treat the cell aggregate with colseminide. Therefore, the technique in Patent Document 2 yields a small number of mitotic cells. Furthermore, in the technique in Patent Document 2, the process of separating the cell aggregate into single cells after the nuclear membrane has disappeared in the mitotic phase causes the cells to rupture and be lost. As a result, the technique in Patent Document 2 has the problem of being difficult to efficiently recover the desired mitotic cells.

[0009] One aspect of the present invention aims to realize a method for enriching mitotic cells that can efficiently collect mitotic cells and allow for better observation of the properties of cell aggregates, as well as the utilization of the same. [Means for solving the problem]

[0010] In order to solve the above-mentioned problems, the present inventors conducted extensive research and found that by first dispersing densely cultured cells and then performing a treatment to increase the proportion of mitotic cells while culturing the cells in suspension, it is possible to efficiently collect mitotic cells and to better observe the properties of the cell aggregates, thus completing the present invention. One embodiment of the present invention encompasses the following invention.

[0011] [1] A method for enriching mitotic cells, comprising a step of first dispersing densely cultured cells and then enriching the cells while culturing them in suspension to increase the proportion of mitotic cells.

[0012] [2] The method for enriching mitotic cells according to [1], wherein the densely cultured cells are spheroids, organoids, cell sheets, cell populations formed by bioprinting technology, cell populations formed using scaffolding materials, tissue aggregates, or organs.

[0013] [3] The method for enriching mitotic cells according to [1] or [2], wherein the enrichment step is performed on a culture dish or in a container for temporarily storing cells.

[0014] [4] A method for enriching mitotic cells according to any one of [1] to [3], wherein the enrichment step is performed by administering a mitotic inhibitor to cells in suspension culture.

[0015] [5] The method for enriching mitotic cells according to [4], wherein the mitotic inhibitor is colsemid, colchicine, nocodazole, vinblastine, or vincristine.

[0016] [6] A method for enriching mitotic cells according to any one of [1] to [3], wherein the enrichment step is performed by administering a progression inhibitor that inhibits the progress of interphase of the cell cycle to cells in suspension culture.

[0017] 〔7〕The enrichment method of mitotic cells according to 〔6〕, wherein the progress inhibitor is Amethopterin (methotrexate), 5-fluorodeoxyuridine (FdUrD, or FUdR), thymidine, or BrdU.

[0018] 〔8〕The enrichment method of mitotic cells according to any one of 〔1〕 to 〔3〕, wherein the enrichment step is performed by administering a growth promoter that promotes cell growth to the cells in suspension culture.

[0019] 〔9〕The enrichment method of mitotic cells according to 〔8〕, wherein the growth promoter is a growth factor, cytokine, antibody, peptide, or low molecular weight compound.

[0020] 〔10〕A cell fixation method having a fixation step of fixing mitotic cells obtained by the enrichment method according to any one of 〔1〕 to 〔9〕.

[0021] 〔11〕A chromosome analysis method including an analysis step of analyzing chromosomes of the fixed cells obtained by the fixation method according to 〔10〕.

[0022] 〔12〕The chromosome analysis method according to 〔11〕, wherein in the analysis step, the chromosomes are analyzed by G-band analysis, Q-band analysis, mFISH analysis, or FISH analysis.

Advantages of the Invention

[0023] According to one aspect of the present invention, it is possible to efficiently collect mitotic cells and better observe the properties of cell aggregates, and to provide an enrichment method for mitotic cells and its use.

Brief Description of the Drawings

[0024] [Figure 1] An image showing the result of karyotyping in an embodiment of the present invention. [Figure 2]This image shows a comparison between the amount of immobilized sample obtained in Test 1 and the amount of immobilized sample obtained in Control Test 1 in an embodiment of the present invention. [Modes for carrying out the invention]

[0025] The following describes in detail some examples of embodiments of the present invention, but the present invention is not limited to these. The present invention can be modified in various ways within the scope of the claims. Embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic and patent documents mentioned herein are incorporated herein by reference. Unless otherwise specified herein, "X~Y" representing a numerical range means "X or more and Y or less".

[0026] [1. Methods for enriching mitotic cells] A method for enriching mitotic cells (in other words, a method for increasing the proportion of mitotic cells) according to one embodiment of the present invention comprises an enrichment step in which densely cultured cells (for example, cell aggregates, etc.) are dispersed in advance, and then the proportion of mitotic cells is increased while the cells are cultured in suspension.

[0027] In a method for enriching mitotic cells according to one embodiment of the present invention, "suspension culture" includes a culture system in which suspension cells are seeded onto a dish or the like, and then the dish or the like is centrifuged together with the suspension cells to forcibly attach the suspension cells to the dish or the like in a short time.

[0028] With the above configuration, since the proportion of mitotic cells is increased while pre-dispersed cells are cultured in suspension, at least one of the following can be achieved: (i) by using suspension culture, which allows for easy handling of cells, a treatment is performed to quickly increase the proportion of mitotic cells before any changes in cell properties occur; (ii) a treatment is performed uniformly on individual cells to increase the proportion of mitotic cells; (iii) mitotic cells, which have become fragile due to the loss of the nuclear membrane from the cell aggregate, can be easily recovered without performing any treatment that could damage the cells, such as dispersing them; (iv) changes in cell properties caused by the cell's anchorage-dependent proliferation can be suppressed; and (v) differences in nutritional status and oxygen partial pressure that occur within the cell aggregate can be eliminated. Therefore, with the above configuration, mitotic cells can be recovered efficiently, and the properties of the cell aggregate can be observed more clearly.

[0029] The above-mentioned cells are not limited to, but include, for example, cells collected from living organisms, stem cells, cancer cells, established cell lines (e.g., HT1080, HepG2, HEK293, etc.), and genetically modified cells thereof. The above-mentioned genetically modified cells can be produced by known methods (for example, by introducing an expression vector containing a desired gene into a desired cell, by disrupting a desired endogenous gene within a desired cell, by exposing a desired cell to a mutagen, by accidentally causing genetic modification during the culture of a desired cell, etc.).

[0030] The stem cells mentioned above are not limited to those mentioned above, and examples include pluripotent stem cells and tissue stem cells.

[0031] The above-mentioned pluripotent stem cells are not limited to, and examples include ES cells (embryonic stem cells), iPS cells (induced pluripotent stem cells), EC cells (embryonal carcinoma cells), EG cells (embryonic germ cells), mGS cells (multipotent germ stem cells), ntES cells (nuclear transfer embryonic stem cells), AgES cells (androgenetic embryonic stem cells), PgES cells (parthenogenetic embryonic stem cells), EpiSC cells (epiblast stem cells), and MUSE cells (Multi-lineage differentiating Stress Enduring cells).

[0032] The above-mentioned tissue stem cells are not limited to, but include, for example, stem cells derived from ectodermal tissues (e.g., neural stem cells, neural crest stem cells, retinal stem cells, corneal stem cells, keratinocyte epidermal stem cells, melanocyte stem cells, mammary gland stem cells, trophoblast stem cells, etc.), stem cells derived from endodermal tissues (e.g., embryonic stem cells, airway stem cells), stem cells derived from mesodermal tissues (e.g., hematopoietic stem cells, mesenchymal stem cells, cardiac stem cells, vascular endothelial progenitor cells, vascular pericytes, skeletal muscle stem cells, adipose stem cells, renal progenitor cells, etc.), and stem cells derived from reproductive tissues (e.g., spermatogonial stem cells, etc.).

[0033] The densely cultured cells described above are not limited to, but may include, for example, spheroids, organoids, cell sheets, cell populations formed by bioprinting technology, cell populations formed using scaffolding materials, tissue aggregates, or organs. With the method for enriching mitotic cells according to one embodiment of the present invention, even when using such densely cultured cells, mitotic cells can be efficiently recovered and the properties of the cell aggregates can be observed more clearly.

[0034] The densely cultured cells described above may be cell aggregates consisting of many cells (for example, more than 100 cells, more than 500 cells, or more than 1000 cells) or few cells (for example, 100 cells or less, 500 cells or less, or 1000 cells or less), but the number of cells contained in the cell aggregate is not limited. If the densely cultured cells described above are cell aggregates consisting of few cells, the adhesion between cells is weak, so the cell aggregate can be easily dispersed, and the proportion of cells in the mitotic phase can be easily increased with mitotic phase inhibitors, progression inhibitors and / or proliferation promoters, etc.

[0035] The spheroids, organoids, cell sheets, cell populations formed by bioprinting technology, and cell populations formed using scaffolding materials can be prepared according to known methods.

[0036] As an example of a method for producing the spheroids described above, for example, the method described in Reference 1 (Yoshitaka Miyamoto, Masashi Ikeuchi, Hirofumi Noguchi, Tohru Yagi and Shuji Hayashi, Spheroid Formation and Evaluation of Hepatic Cells in a Three-Dimensional Culture Device, Cell Medicine, Aug. 2015;8(1-2):47-56) and Reference 2 (Junichi Takahashi, Tomohiro Mizutani, Hady Yuki Sugihara, Sayaka Nagata, Shu Kato, Yui Hiraguri, Sayaka Takeoka, Mao Tsuchiya, Reiko Kuno, Sei Kakinuma, Mamoru Watanabe and Ryuichi Okamoto, Suspension culture in a rotating bioreactor for efficient generation of human intestinal organoids, Cell Reports Methods, Nov. 2022;2(11)) can be cited.

[0037] As an example of a method for producing the above organoids, for example, the method described in Reference 2 and Reference 3 (Aneta Przepiorski, Veronika Sander, Tracy Tran, Jennifer A. Hollywood, Brie Sorrenson, Jen-Hsing Shih, Ernst J. Wolvetang, Andrew P. McMahon, Teresa M. Holm, and Alan J. Davidson, A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells, Stem Cell Reports, Aug. 2018;11(2):470-484) can be cited.

[0038] As an example of a method for preparing the above-mentioned cell sheets, for example, the method described in Reference 4 (Nai-Chen Cheng, Yuan-Kun Tu, Ning-Hsu Lee and Tai-Horng Young, Influence of Human Platelet Lysate on Extracellular Matrix Deposition and Cellular Characteristics in Adipose-Derived Stem Cell Sheets, Frontiers in Cell and Developmental Biology, Oct. 2020;8) and Reference 5 (Ayami Hasegawa-Haruki, Koya Obara, Nanako Takaoka, Kyoumi Shirai, Yuko Hamada, Nobuko Arakawa, Ryoichi Aki, Robert M. Hoffman and Yasuyuki Amoh, Hair-follicle associated pluripotent (HAP)-cell sheet implantation enhanced wound healing in diabetic db / db mice, PLOS ONE, Jun. 2024;19(6)) can be cited.

[0039] As an example of a method for producing cell populations formed by the bioprinting technology described above, see, for example, Reference 6 (Tobias Grix, Alicia Ruppelt, Alexander Thomas, Anna-Klara Amler, Benjamin P. Noichl, Roland Lauster and Lutz Kloke, Bioprinting Perfusion-Enabled Liver Equivalents for Advanced Organ-on-a-Chip Applications, Genes(Basel), Mar. 2018;9(4):176), Reference 7 (Paulina Nunez Bernal, Manon Bouwmeester, Jorge Madrid-Wolff, Marc Falandt, Sammy Florczak, Nuria Gines Rodriguez, Yang Li, Gabriel Grobacher, Roos-Anne Samsom, Monique van Wolferen, Luc JW van der Laan, Paul Delrot, Damien Loterie, Jos Malda, Christophe Moser, Bart Spee and Riccardo Levato, Volumetric Bioprinting of Organoids and Optically Tuned Hydrogels to Build Liver-Like Metabolic Biofactories, Advanced Materials, Apr.Examples include the methods described in 2022;34(15)) and reference 8 (Daniela F Duarte Campos, Christopher D Lindsay, Julien G Roth, Bauer L LeSavage, Alexis J Seymour, Brad A Krajina, Ricardo Ribeiro, Pedro F Costa, Andreas Blaeser and Sarah C Heilshorn, Bioprinting Cell- and Spheroid-Laden Protein-Engineered Hydrogels as Tissue-on-Chip Platforms, Frontiers in bioengineering and biotechnology, Apr. 2020;8:374).

[0040] As an example of a method for preparing cell populations formed using the above-mentioned scaffolding material, for example, the method described in Reference 8 and Reference 9 (Meghan M. Capeling, Michael Czerwinski, Sha Huang, Yu-Hwai Tsai, Angeline Wu, Melinda S. Nagy, Benjamin Juliar, Nambirajan Sundaram, Yang Song, Woojin M. Han, Shuichi Takayama, Eben Alsberg, Andres J. Garcia, Michael Helmrath, Andrew J. Putnam and Jason R. Spence, Nonadhesive Alginate Hydrogels Support Growth of Pluripotent Stem Cell-Derived Intestinal Organoids, Stem Cell Reports, Feb. 2019;12(2):381-394) can be cited. These references are incorporated herein by reference.

[0041] The tissue aggregates and organs described above can be tissue aggregates and organs taken from a desired living organism (e.g., a human, or a non-human animal (e.g., a monkey, a cow, a pig, a sheep, a goat, a horse, a dog, a cat, a rabbit, a mouse, a guinea pig, and a rat, etc.)). Furthermore, the tissue aggregates and organs may be (i) tissue-like or organ-like cell aggregates obtained by culturing cells derived from tissues or organs taken from a desired living organism, or (ii) tissue-like or organ-like cell aggregates obtained by culturing desired cells (e.g., cells taken from a living organism, stem cells, cancer cells, established cell lines, or genetically modified cells thereof, etc.).

[0042] The above-mentioned tissue masses and organs are not limited to, but may include, for example, skin, cartilage, hair follicles, fat, bone marrow, kidneys, liver, spleen, esophagus, stomach, duodenum, small intestine, large intestine, rectum, pancreas, heart, blood vessels, lungs, testes, ovaries, prostate, fallopian tubes, uterus, placenta, umbilical cord, amniotic membrane, retina, cornea, lens, muscle, spinal cord, and brain.

[0043] The densely cultured cells described above are dispersed beforehand. The method for dispersing the densely cultured cells is not limited and should be appropriately selected considering the adhesion strength between cells and / or the cells' resistance to the dispersion method. Examples of methods for dispersing densely cultured cells include: (i) dispersing the densely cultured cells by enzymatic treatment (e.g., trypsin, collagenase, dispase, etc.); (ii) dispersing the densely cultured cells by chelating agent treatment (e.g., EDTA, etc.); (iii) dispersing the densely cultured cells by physical treatment (e.g., pipetting, tapping, use of a cell strainer, shaking, etc.); and (iv) any combination of (i) to (iii) above.

[0044] The state of the densely cultured cells after dispersion is not limited. The densely cultured cells may be dispersed to the state of single cells, to the state of cell clumps containing multiple cells (for example, 100 or fewer cells, 500 or fewer cells, or 1000 or fewer cells), or to the state in which single cells and cell clumps are mixed.

[0045] In the enrichment process described above, the proportion of cells in the mitotic phase is increased while the pre-dispersed cells are cultured in suspension.

[0046] In the above suspension culture, it is not necessary for all cells to be in a suspension state; some cells may be in an adherent state (for example, cells adhered to the culture dish, and / or cells adhered to each other). In the above suspension culture, it is preferable that, for example, more than 50%, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of all cells are in a suspension state. In the above suspension culture, for example, if there are 1 × 10 cells in a suspension state... 5 More than one piece / dish, etc., 1 x 10 4 More than one piece / dish, etc., 1 x 10 3 More than one piece / dish, or 1 x 10 2 It is preferable that the number of cells per dish is greater than 10. In the above suspension culture, (i) more than 50%, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the total cells are in a suspension state, and (ii) the number of cells in the suspension state is 1 × 10⁶. 5 More than one per dish, 1 x 10 4 More than one per dish, 1 x 10 3 1 or more per dish, or 1 x 10 2 It may also be a dish or similar. In the above suspension culture, it is preferable that the proportion of cells in a suspension state is large and / or that the number of cells in a suspension state is large. With this configuration, mitotic cells in a suspension state can be easily recovered, thereby reducing damage to mitotic cells and allowing for efficient recovery of mitotic cells.

[0047] The enrichment step described above is preferably performed on a culture dish or in a container for temporarily storing cells (e.g., a tube, etc.). With this configuration, the enrichment step can be carried out according to a simple procedure.

[0048] The culture dish and the container for temporarily storing the cells are not limited, and commercially available ones can be used as appropriate.

[0049] It is preferable that the culture dish and the container for temporarily storing the cells described above are designed to be resistant to cell adhesion. With this configuration, suspension culture can be easily performed. Examples of such culture dishes and containers include: (i) culture dishes and containers whose surfaces in contact with the cells are not coated with cell-adhering substrates (e.g., gelatin, collagen, fibronectin, retronectin, laminin, adhesamine, poly-L-lysine, poly-L-ornithine, vitronectin, Matrigel, etc.); (ii) culture dishes and containers whose materials in contact with the cells are not cell-adhering; and (iii) culture dishes and containers that combine (i) and (ii). Note that reagents that inhibit cell aggregation (anti-clumping agents, etc.) may be used in the form of being added to the culture medium used for suspension culture.

[0050] The enrichment step described above is preferably carried out by administering a mitotic inhibitor to cells in suspension culture. This configuration makes it easy to increase the proportion of cells in the mitotic phase. Administering a mitotic inhibitor to cells in suspension culture can be done, for example, by administering the mitotic inhibitor to the culture medium in which the cells are being cultured.

[0051] The above-mentioned mitotic inhibitor is not limited to any one that increases the proportion of cells in the mitotic phase. The mitotic inhibitor is preferably, for example, colsemid, colchicine, nocodazole, vinblastine, or vincristine. In the enrichment step, it is possible to use one type of mitotic inhibitor, or any combination of multiple types of mitotic inhibitors.

[0052] The enrichment step described above is preferably carried out by administering a cell cycle progression inhibitor to cells in suspension culture that inhibits the progress of interphase (e.g., G1 phase (DNA synthesis preparation phase), S phase (DNA synthesis phase), or G2 phase (mitotic preparation phase)). This configuration makes it easy to increase the proportion of cells in the mitotic phase. Inhibiting the progress of interphase tends to result in a more uniform progression of the cell cycle within the cell population. For example, the proportion of cells in the mitotic phase can be increased by adjusting the timing of the completion of the enrichment step. Administering a cell cycle progression inhibitor to cells in suspension culture can be done, for example, by administering the inhibitor to the culture medium in which the cells are being cultured.

[0053] The above-mentioned progression inhibitor may be administered to densely cultured cells before dispersion. The progression inhibitor causes cells in interphase of the cell cycle to have a nuclear membrane. Therefore, with this configuration, densely cultured cells can be dispersed without causing damage.

[0054] The above-mentioned progression inhibitors are not limited to those that inhibit the progression of the interphase of the cell cycle. Preferably, the progression inhibitors are, for example, amethopterin (methotrexate), 5-fluorodeoxyuridine (FdUrD, or FUdR), thymidine, or BrdU. In the enrichment step, it is possible to use one type of progression inhibitor, or any combination of multiple types of progression inhibitors.

[0055] The enrichment process described above can also be carried out according to the method described in Reference 10: "The AGT Cytogenetics Laboratory Manual (2017)," or more specifically, by any of the following methods (1) to (4): (1) Treatment to release the inhibition of interphase progression of the cell cycle with thymidine or BrdU, after the inhibition of interphase progression by amethopterin (methotrexate); (2) Release treatment of the cell cycle progression inhibition with thymidine or BrdU after inhibiting the progression of interphase with 5-fluorodeoxyuridine (FdUrD or FUdR); (3) Treatment to release the inhibition of interphase progression of the cell cycle, performed after inhibiting interphase progression with an excess amount of thymidine, by 2-deoxicytidine, BrdU, medium change, or simply continuing to culture; (4) Treatment to release the cell cycle progression inhibition by thymidine or medium exchange, which is performed after inhibiting the progression of interphase of the cell cycle with an excess amount of BrdU.

[0056] The enrichment step described above is preferably carried out by administering a growth promoter to cells in suspension culture. This configuration makes it easy to increase the proportion of cells in the mitotic phase. Furthermore, promoting cell proliferation can increase the proportion of cells in the mitotic phase even in cell populations with slow division rates or cell populations that have stopped dividing. Administering a growth promoter to cells in suspension culture can be done, for example, by administering the growth promoter to the culture medium in which the cells are being cultured.

[0057] The above growth promoter may be administered to densely cultured cells before dispersion. Cells in the interphase of the cell cycle will have a nuclear membrane due to the above growth promoter. Therefore, with this configuration, densely cultured cells can be dispersed without causing damage.

[0058] The growth promoter described above is not limited to any agent that promotes cell proliferation. Preferably, the growth promoter is a growth factor, cytokine, antibody, peptide, or small molecule compound. In the enrichment step, it is possible to use one type of growth promoter or any combination of multiple growth promoters.

[0059] The above growth factors may be cell-specific growth factors, such as EGF, IGF, FGF, HGF, TGF, PDGF, VEGF, NGF, EPO, CTGF, Wnt3A, or TPO.

[0060] The above cytokines may be cell-specific cytokines, such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-15, IL-17, M-CSF, G-CSF, GM-CSF, SCF, LIF, Activin A, CD27L, CD30L, etc.

[0061] The antibodies mentioned above may be cell-specific antibodies, such as anti-CD3 antibodies, anti-CD16 antibodies, anti-CD28 antibodies, etc.

[0062] The above peptides may be cell-specific peptides, such as HGF substitute peptides, VEGF substitute peptides, Wnt3a substitute peptides, TPO substitute peptides, etc.

[0063] The low molecular weight compounds mentioned above refer, for example, to compounds with a molecular weight of less than 500. These low molecular weight compounds may be cell-specific compounds and may include, for example, A 83-01, 5-Aza-2'-deoxycytidine (Decitabine), CHIR 99021, SB 431542, Y-27632, etc.

[0064] The enrichment step described above is preferably carried out by administering at least one, at least two, or three substances selected from the group consisting of the mitotic phase inhibitor, the mitotic progress inhibitor, and the growth promoter to cells in suspension culture. Administering two or more substances selected from the group consisting of the mitotic phase inhibitor, the mitotic progress inhibitor, and the growth promoter to cells in suspension culture makes it even easier to increase the proportion of cells in the mitotic phase. As mentioned above, the mitotic progress inhibitor and the growth promoter may also be administered to densely cultured cells before dispersion.

[0065] The enrichment step described above is preferably carried out by sorting cells in suspension culture to separate cells in the mitotic phase (for example, separating cells in the mitotic phase according to cell type). This configuration makes it easy to increase the proportion of cells in the mitotic phase.

[0066] More specifically, in the enrichment step described above, (i) cells in suspension culture may be sorted to separate mitotic cells, or (ii) at least one, at least two, or three agents selected from the group consisting of the mitotic inhibitor, the mitotic progression inhibitor, and the growth promoter may be administered to cells in suspension culture, and then the cells in suspension culture may be sorted to separate mitotic cells. In the case of (ii) above, a larger number of mitotic cells can be obtained.

[0067] The sorting described above can be performed using a commercially available cell sorter, following the protocol provided with the cell sorter.

[0068] In the enrichment process described above, the timing for initiating the treatment to increase the proportion of mitotic cells is not particularly limited. For example, the timing for initiating the treatment to increase the proportion of mitotic cells may be within 3 hours, 2 hours, 1 hour, 30 minutes, or 15 minutes after dispersing the densely cultured cells. The earlier the timing for initiating the treatment to increase the proportion of mitotic cells relative to the cell division cycle, the more mitotic cells that better reflect the properties of the cell aggregate can be obtained.

[0069] In the enrichment process described above, the length of time from the start of the process to increase the proportion of cells in the mitotic phase to the end of the process is not particularly limited.

[0070] In the enrichment process described above, when administering a mitotic inhibitor to cells in suspension culture, the length of time from the start of the process to increase the proportion of mitotic cells to the end of the process to increase the proportion of mitotic cells may be, for example, more than 0 hours and within 8 hours.

[0071] In the enrichment process described above, when the progress inhibitor is administered to cells in suspension culture, the length of time from the start of the process to increase the proportion of mitotic cells to the end of the process to increase the proportion of mitotic cells may be, for example, 8 hours or more.

[0072] In the enrichment step described above, when a growth promoter is administered to cells in suspension culture, the length of time from the start of the process to increase the proportion of cells in the mitotic phase to the end of the process to increase the proportion of cells in the mitotic phase may be, for example, the length of time from the start to the end of the enrichment step described above.

[0073] [2. Method of cell fixation] A cell fixation method according to one embodiment of the present invention includes a fixation step of fixing mitotic cells obtained by an enrichment method according to one embodiment of the present invention.

[0074] With the above configuration, it is possible to efficiently prepare a sample in which mitotic cells that better reflect the properties of the cell aggregate are immobilized, and in which a sufficient number of mitotic cells are immobilized.

[0075] The above fixation step may include, for example, a contact step of bringing mitotic cells into contact with a fixative. This contact step can fix the mitotic cells. The above fixation step can be carried out, for example, by replacing the solution surrounding the mitotic cells with a fixative (fixative solution).

[0076] The above-mentioned immobilizing agents are not limited to those mentioned above, and examples include alcohol-based fixatives (e.g., Carnoy), formaldehyde-based fixatives, and glutaraldehyde-based fixatives.

[0077] The lower limit of the contact time (the length of time during which the mitotic cells are in contact with the immobilizing agent) is not limited and may be, for example, 1 second or more, 1 minute or more, 5 minutes or more, or 10 minutes or more. The upper limit of the contact time (the length of time during which the mitotic cells are in contact with the immobilizing agent) is not limited and may be, for example, 1 minute or less, 5 minutes or less, 10 minutes or less, or 1 hour or less. With this configuration, the mitotic cells can be sufficiently immobilized.

[0078] The above immobilization step may include, for example, a hypotonic treatment step in which mitotic cells are brought into contact with a hypotonic solution before the above contact step. This hypotonic treatment step makes it easier to spread the chromosomes onto a glass slide.

[0079] The above hypotonic solution is not limited to this, and examples include KCl solutions (e.g., 0.05M to 0.075M KCl solutions).

[0080] The duration of the hypotonic treatment process described above (the length of time the mitotic cells are in contact with the hypotonic solution) is not limited and may be, for example, 1 second to 60 minutes, 1 minute to 60 minutes, 1 minute to 45 minutes, 1 minute to 30 minutes, or 5 minutes to 30 minutes. Mitotic cells are fragile because their nuclear membrane has disappeared. Therefore, shortening the duration of the hypotonic treatment process can reduce the damage inflicted on the cells.

[0081] [3. Chromosome analysis method] A chromosome analysis method according to one embodiment of the present invention includes an analysis step of analyzing the chromosomes of fixed cells obtained by a fixation method according to one embodiment of the present invention.

[0082] According to the fixation method of one embodiment of the present invention, a sample can be efficiently prepared in which mitotic cells that better reflect the properties of the cell aggregate are fixed, and in which a sufficient number of mitotic cells are fixed. Furthermore, in the chromosome analysis method of one embodiment of the present invention, since the chromosomes are analyzed using the sample, the chromosomes of cells forming the cell aggregate can be analyzed efficiently and accurately.

[0083] In the above analysis process, for example, the chromosomes can be analyzed by G-band analysis, Q-band analysis, mFISH analysis, or FISH analysis. With this configuration, the chromosomes of cells forming a cell aggregate can be easily analyzed.

[0084] These analytical methods are commonly used in the field. The G-band analysis described above can be performed, for example, according to the method described in Reference 11 (Erik McIntire, Kimberly Leonhard, Seth Taapken and Anna Lisa Larson, G-Banded Karyotyping of Human Pluripotent Stem Cell Cultures, Methods in Molecular Biology, 2021;2239, 251-268).

[0085] The above Q-band analysis can be performed, for example, according to the method described in Reference 12 (Huaigeng Xu, Bo Wang, Miyuki Ono, Akihiro Kagita, Kaho Fujii, Noriko Sasakawa, Tatsuki Ueda, Peter Gee, Misato Nishikawa, Masaki Nomura, Fumiyo Kitaoka, Tomoko Takahashi, Keisuke Okita, Yoshinori Yoshida, Shin Kaneko, Akitsu Hotta, Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility, Cell Stem Cell, Apr. 2019;24(4), 566-578).

[0086] The above mFISH analysis is described, for example, in Reference 13 (Archana Bhartiya, Ian Robinson, Mohammed Yusuf, and Stanley W. Botchway, Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation, Front. Mol. Biosci., Mar 2021:8) and Reference 14 (M'kacher R, Colicchio B, Borie C, Junker). S, Marquet V, Heidingsfelder L, Soehnlen K, Najar W, Hempel WM, Oudrhiri N, Wilhelm-Murer N, Miguet M, Arnoux M, Ferrapie C, Kerbrat W, Plesch A, Dieterlen A, Girinsky T, Voisin P, Deschenes G, Tabet AC, Yardin C, Bennaceur-Griscelli A, Fenech M, Carde P, This can be done according to the method described in Jeandidier E. Telomere and Centromere Staining Followed by M-FISH Improves Diagnosis of Chromosomal Instability and Its Clinical Utility. Genes (Basel), Apr. 2020;27, 11(5), 475).

[0087] The above FISH analysis can be performed, for example, according to the method described in Reference 14. These references are incorporated herein by reference.

[0088] According to an embodiment of the present invention, it is possible to efficiently collect cells in the mitotic phase and to better observe the properties of cell aggregates, and to provide a method for enriching cells in the mitotic phase and its use. An embodiment of the present invention may also contribute to the achievement of, for example, Goal 3, "Good health and well-being for all", of the Sustainable Development Goals (SDGs) proposed by the United Nations.

Example

[0089] <Experimental series I> <1. Regarding various treatments> <1-1. Cells> In the following tests, HT1080 was used as the cells.

[0090] <1-2. Three-dimensional (3D) culture> The cells were seeded on a cell non-adhesive culture dish (IWAKI, 1010-060, 60 mm) at 5×10 4 cells / 5 mL medium / dish or 4×10 4 cells / 4 mL medium / dish. The medium was added every 2 - 4 days, and the cells were passaged every 7 - 12 days. As a result, densely cultured cells (cell aggregates) were produced.

[0091] The composition of the basic medium used for 3D culture was as follows, and a medium obtained by adding Pen Strep (gibco, 15140-122) to 1% of the basic medium was used (3D Tumoresphere Medium XF (1% P.S.)).

Table 1

[0092] For 2D culture, we used a culture medium (MEM(10%FBS, 1%PS1%NEAA)) consisting of MEM (Gibco, 11095-080) supplemented with 10% FBS (Gibco, 10437-028 or 10270-106), 1% Pen Strep (Gibco, 15140-122), and 1% MEM NEAA (Gibco, 11140-050).

[0093] <1-4. Single-cell conversion process> Cell aggregates or cells were converted into single cells according to the following single-cell conversion process (1) or single-cell conversion process (2).

[0094] Single-cell formation treatment (1): When forming cell aggregates created by 3D culture into single cells and placing them in a suspension state, the cell aggregates were treated with an enzyme (trypsin) and a chelating agent (EDTA), and then pipetting was performed to form single cells.

[0095] Single-cell formation treatment (2): When cells adhering to the surface of a culture dish by 2D culture are formed into single cells and suspended, the cells are detached using an enzyme (trypsin) and a chelating agent (EDTA), and then formed into single cells by pipetting.

[0096] <1-5. Treatment with mitotic inhibitors> Cell aggregates, single cells in suspension (suspension culture), or cells adhered to the surface of a culture dish or container by 2D culture were treated with a mitotic inhibitor for a desired time according to the following mitotic inhibitor treatments (1), (2), or (3). Colsemid was used as the mitotic inhibitor.

[0097] Treatment with mitotic inhibitors (1): When treating cell aggregates formed by 3D culture with mitotic inhibitors, the treatment was carried out by adding the desired concentration of mitotic inhibitor to the culture medium containing the cell aggregates and maintaining the culture for the desired time.

[0098] Treatment with mitotic inhibitors (2): When treating cells that have been separated into single cells and are in a suspension state (suspension culture) with mitotic inhibitors, the treatment was carried out by adding the desired concentration of mitotic inhibitor to the culture medium containing the cells and maintaining it for the desired time.

[0099] Treatment with mitotic inhibitors (3): When treating cells adhered to the surface of a culture dish by 2D culture with mitotic inhibitors, the treatment was carried out by adding the desired concentration of mitotic inhibitor to the culture medium containing the cells and maintaining it for the desired time.

[0100] <1-6. Hypotonic Treatment> After a series of treatments (3D culture, 2D culture, single-cell treatment, and mitotic inhibitor treatment), the cells obtained were added to a hypotonic solution (0.075 M KCl) and maintained for a desired time to perform hypotonic treatment.

[0101] <1-7. Fixation Process> After hypotonic treatment, the cells obtained were fixed by adding an alcohol-based fixative (Carnoy's).

[0102] <2. Examination of the timing of procedures to increase the proportion of cells in the mitotic phase> (Test 1) First, cell aggregates were prepared according to "1-2. Three-dimensional (3D) culture" above. Next, the cell aggregates were separated into single cells and placed in a suspension state (suspension culture) according to "Single-cell treatment (1)" above. Immediately after starting the suspension culture, the single-cell cultured cells were treated with a mitotic inhibitor according to "Mitotic inhibitor treatment (2)" above. At this time, the colsemid concentration in the culture medium was set to 0.5 μg / mL and the treatment time was set to 3 hours. Next, the cells were subjected to hypotonic treatment according to "1-6. Hypotonic treatment" above. At this time, the treatment time was set to 5 minutes. Next, the cells were immobilized according to "1-7. Immobilization treatment" above. In this way, a sample of cells in the mitotic phase was obtained. After spreading the obtained sample onto a glass slide, Hoechst staining was performed and the number of mitotic cells on the glass slide was counted.

[0103] (Controlled study 1) First, cell aggregates were prepared according to "1-2. 3D Culture" above. The obtained cell aggregates were separated into single cells according to "Single Cell Formation Treatment (1)" above. Using the obtained cells, cells adhered to the surface of a culture dish were prepared according to "1-3. 2D Culture" above. These cells were cultured on the surface of the culture dish for approximately 22 hours. Next, the cells adhered to the surface of the culture dish were treated with a mitotic inhibitor according to "Mitotic Phase Inhibitor Treatment (3)" above. At this time, the colsemid concentration in the culture medium was set to 0.5 μg / mL and the treatment time was set to 3 hours. Next, the cells adhered to the surface of the culture dish were separated into single cells and suspended according to "Single Cell Formation Treatment (2)" above. Next, the cells were subjected to hypotonic treatment according to "1-6. Hypotonic Treatment" above. At this time, the treatment time was set to 5 minutes. Next, the cells were immobilized according to "1-7. Immobilization Treatment" above. As described above, we obtained a sample in which cells in the mitotic phase were immobilized.

[0104] (Controlled study 2) First, cell aggregates were prepared according to "1-2. Three-dimensional (3D) culture" above. Next, the cell aggregates were treated with a mitotic inhibitor according to "Mitotic inhibitor treatment (1)" above. At this time, the colsemid concentration in the culture medium was set to 0.5 μg / mL and the treatment time was set to 3 hours. Next, the cell aggregates were separated into single cells and suspended according to "Single-cell treatment (1)" above. Next, the cells were subjected to hypotonic treatment according to "1-6. Hypotonic treatment" above. At this time, the treatment time was set to 5 minutes. Next, the cells were immobilized according to "1-7. Immobilization treatment" above. In this way, a sample of cells in the mitotic phase was obtained. After spreading the obtained sample onto a glass slide, Hoechst staining was performed and the number of mitotic cells on the glass slide was counted.

[0105] (Controlled study 3) First, cells were prepared by adhering them to the surface of a culture dish according to "1-3. 2D Culture" above. These cells did not undergo "1-2. 3D Culture" above. Next, the cells adhering to the surface of the culture dish were treated with a mitotic inhibitor according to "3. Mitotic Inhibitor Treatment" above. At this time, the colsemid concentration in the culture medium was set to 0.05 μg / mL, and the treatment time was 4.5 hours. Next, the cells adhering to the surface of the culture dish were separated into single cells and suspended according to "2. Single Cell Formation Treatment" above. Next, the cells were subjected to hypotonic treatment according to "1-6. Hypotonic Treatment" above. At this time, the treatment time was 15 minutes. Next, the cells were immobilized according to "1-7. Immobilization Treatment" above. In this way, a sample of cells in the mitotic phase was obtained.

[0106] (Controlled study 4) First, cell aggregates were prepared according to "1-2. Three-dimensional (3D) culture" above. Next, the cell aggregates were treated with a mitotic inhibitor according to "Mitotic inhibitor treatment (1)" above. At this time, the colsemid concentration in the culture medium was set to 0.05 μg / mL and the treatment time was set to 4.5 hours. Next, the cell aggregates were separated into single cells and suspended according to "Single-cell treatment (1)" above. Next, the cells were subjected to hypotonic treatment according to "1-6. Hypotonic treatment" above. At this time, the treatment time was set to 15 minutes. Next, the cells were immobilized according to "1-7. Immobilization treatment" above. In this way, a sample of cells in the mitotic phase was obtained.

[0107] (Test results) In Experiment 1 described above, a sufficient number of mitotic cells were immobilized in the sample. Furthermore, because no two-dimensional (2D) culture or other processing was performed in Experiment 1, the time from "Single-cell formation (1)" to "1-7. Immobilization" was very short (approximately 4 hours). This indicates that the sample obtained by immobilizing mitotic cells better reflects the properties of the cell aggregate. The average number of mitotic cells per slide was 115.3 (n=3).

[0108] On the other hand, in the aforementioned control study 2, it was not possible to efficiently obtain samples with fixed mitotic cells (the average number of mitotic cells per slide was 68.7 (n=3)). This revealed that treating cell aggregates with mitotic inhibitors, as in conventional techniques, does not efficiently yield the desired effect of the mitotic inhibitor.

[0109] Furthermore, in control experiment 1, 2D culture required a long time (approximately 22 hours). This indicates that the sample obtained by fixing mitotic cells does not reflect the properties of the cell aggregate.

[0110] Furthermore, in Control Experiment 3, even when the treatment time with mitotic inhibitors was extended to increase the cell number during mitosis in cells maintained in 2D culture, a sufficient number of immobilized mitotic cells could be obtained. However, since Control Experiment 3 does not go through the "1-2. 3D culture" process described above, it is not possible to observe the properties of cell aggregates in Control Experiment 3.

[0111] On the other hand, in control experiment 4, under the same conditions as control experiment 3, the cells ruptured during single-cell separation, making it impossible to obtain a fixed sample. This indicates that it is difficult to optimize the conditions for increasing the number of mitotic cells in the cell aggregate state.

[0112] <3. Chromosome Analysis> First, cell aggregates were prepared according to "1-2. Three-dimensional (3D) culture" described above. Next, the cell aggregates were separated into single cells and placed in a suspension state (suspension culture) according to "Single-cell treatment (1)" described above. Immediately after starting the suspension culture, the single-cell cultured cells were treated with a mitotic inhibitor according to "Mitotic inhibitor treatment (2)" described above. At this time, the colsemid concentration in the culture medium was set to 0.5 μg / mL and the treatment time was set to 3 hours. Next, the cells were subjected to hypotonic treatment according to "1-6. Hypotonic treatment" described above. At this time, the treatment time was set to 5 minutes. Next, the cells were immobilized according to "1-7. Immobilization treatment" described above. In this way, a sample of cells in the mitotic phase was obtained.

[0113] The obtained samples were spread onto glass slides, stained with Hoechst stain, and then subjected to chromosome analysis (Q-band analysis) by karyotyping.

[0114] The test results are shown in Figure 1. Figure 1A shows the unfolded chromosomes of one cell on a glass slide, and Figure 1B shows the result of calliotyping (caliogram) of Figure 1A. More specifically, the arrows in Figure 1B indicate the locations of structural abnormalities in the chromosomes of the cell. From Figures 1A and 1B, it is clear that chromosome analysis can be performed more effectively using samples prepared by immobilizing mitotic cells obtained by the mitotic cell enrichment method of the present invention.

[0115] <Experiment Series II> <1. Regarding various processes> <1-1.Cell> In the following experiments, human subcutaneous adipose-derived mesenchymal stem cells (hereinafter referred to as hADSCs) were used as the cells.

[0116] <1-2. Forming cells into sheets> Place the cells on a cell-adhering culture dish (Thermo Fisher Scientific, 130184, 6-well plate) in a 5 × 10⁶ arrangement. 5 Cells were seeded in 2 mL of culture medium per well (Day 0 of culture). The culture medium was changed on Day 1 of culture. From Day 2 onwards, multiple cells overlapped (cell sheets) were formed.

[0117] For sheet formation of cells, a medium for MSCs (Vitality, DSGM401) was used.

[0118] <1-3. Single-cell conversion process> To separate sheet-like cell aggregates into single cells and create a suspended state, the cell aggregates were treated with Trypsin (0.25%) and phenol red (Gibco, 15050065), followed by pipetting to separate them into single cells.

[0119] <1-4. Treatment with mitotic inhibitors> Sheet-like cell aggregates or single cells suspended in suspension (suspension culture) were treated with a mitotic inhibitor for a desired time according to either the following mitotic inhibitor treatment (1) or mitotic inhibitor treatment (2). Colsemid was used as the mitotic inhibitor.

[0120] Treatment with mitotic inhibitors (1): When treating sheet-like cell aggregates with mitotic inhibitors, the treatment was carried out by adding the desired concentration of mitotic inhibitor to the culture medium containing the cell aggregates and maintaining the medium for the desired time.

[0121] Treatment with mitotic inhibitors (2): When treating cells that have been separated into single cells and are in a suspension state (suspension culture) with mitotic inhibitors, the treatment was carried out by adding the desired concentration of mitotic inhibitor to the culture medium containing the cells and maintaining it for the desired time.

[0122] <1-5. Hypotonic Treatment> After a series of treatments (cell sheeting, single-cell separation, and mitotic phase inhibitor treatment), the resulting cells were added to a hypotonic solution (0.075 M KCl) and maintained for a desired time to perform hypotonic treatment.

[0123] <1-6. Immobilization Process> After hypotonic treatment, the cells obtained were fixed by adding an alcohol-based fixative (Carnoy's).

[0124] <2. Examination of the timing of procedures to increase the proportion of cells in the mitotic phase> (Test 1) First, cell aggregates were prepared according to "1-2. Cell Sheet Formation" above. Next, the cell aggregates were separated into single cells and placed in a suspension state (suspension culture) according to "1-3. Single Cell Formation Treatment" above. Immediately after starting the suspension culture, the single cells in suspension culture were treated with a mitotic inhibitor according to "Mitotic Inhibitor Treatment (2)" above. At this time, the colsemid concentration in the culture medium was set to 0.8 μg / mL, the treatment time was set to 1 hour, and shaking culture was performed. Next, the cells were subjected to hypotonic treatment according to "1-5. Hypotonic Treatment" above. At this time, the treatment time was set to 1 minute. Next, the cells were immobilized according to "1-6. Immobilization Treatment" above. In this way, a sample of cells in the mitotic phase was obtained.

[0125] (Controlled study 1) First, cells were prepared by attaching them to the surface of a culture dish according to "1-2. Cell Sheet Formation" above. Next, the cells attached to the surface of the culture dish were treated with a mitotic inhibitor according to "Mitotic Inhibitor Treatment (1)" above. At this time, the colsemid concentration in the culture medium was set to 0.8 μg / mL and the treatment time was set to 1 hour. Next, the cells attached to the surface of the culture dish were separated into single cells and suspended according to "1-3. Single Cell Formation Treatment" above. Next, the cells were subjected to hypotonic treatment according to "1-5. Hypotonic Treatment" above. At this time, the treatment time was set to 1 minute. Next, the cells were immobilized according to "1-6. Immobilization Treatment" above. In this way, a sample of cells in the mitotic phase was obtained.

[0126] (Test results) The amount of immobilized sample in Experiment 1 described above was compared with the amount of immobilized sample in Control Experiment 1 (see Figure 2). As shown on the left side of Figure 2, in Control Experiment 1, an amount of sample was obtained that filled the space from the bottom of the tube up to a height of 1.7 mm. On the other hand, as shown on the right side of Figure 2, in Experiment 1, an amount of sample was obtained that filled the space from the bottom of the tube up to a height of 2.1 mm. This indicates that in Control Experiment 1, cells ruptured due to the single-cell treatment performed after treatment with the mitotic inhibitor, while in Experiment 1, cell rupture was reduced. [Industrial applicability]

[0127] The present invention can be used for chromosome analysis of cells. Furthermore, the present invention can be used in the development of pharmaceuticals (e.g., cell-based therapies, regenerative medicine drugs), and more specifically, in tests to evaluate the genetic stability and / or safety of pharmaceuticals.

Claims

1. A method for enriching mitotic cells, comprising a step of first dispersing densely cultured cells and then enriching the cells while culturing them in suspension to increase the proportion of mitotic cells.

2. The method for enriching mitotic cells according to claim 1, wherein the densely cultured cells are spheroids, organoids, cell sheets, cell populations formed by bioprinting technology, cell populations formed using scaffolding materials, tissue aggregates, or organs.

3. The method for enriching mitotic cells according to claim 1, wherein the enrichment step described above is performed on a culture dish or in a container for temporarily storing cells.

4. The method for enriching mitotic cells according to claim 1, wherein the enrichment step is performed by administering a mitotic inhibitor to cells in suspension culture.

5. The method for enriching mitotic cells according to claim 4, wherein the mitotic inhibitor is colsemid, colchicine, nocodazole, vinblastine, or vincristine.

6. The method for enriching mitotic cells according to claim 1, wherein the enrichment step is performed by administering a progression inhibitor that inhibits the progress of interphase of the cell cycle to cells in suspension culture.

7. The method for enriching mitotic cells according to claim 6, wherein the progression inhibitor is amethopterin, 5-fluorodeoxyridine, thymidine, or BrdU.

8. The method for enriching mitotic cells according to claim 1, wherein the enrichment step is performed by administering a growth promoter that promotes cell proliferation to cells in suspension culture.

9. The method for enriching mitotic cells according to claim 8, wherein the growth promoter is a growth factor, cytokine, antibody, peptide, or low molecular weight compound.

10. A method for fixing cells, comprising a fixation step of fixing mitotic cells obtained by an enrichment method according to any one of claims 1 to 9.

11. A chromosome analysis method comprising an analysis step of analyzing the chromosomes of fixed cells obtained by the fixation method described in claim 10.

12. The chromosome analysis method according to claim 11, wherein in the above analysis step, the chromosome is analyzed by G-band analysis, Q-band analysis, mFISH analysis, or FISH analysis.

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