Cell determination method, screening method, cell separation method, and gel

Abstract

To provide a cell determination method, a screening method, a cell separation method, and a gel that allow determination and separation of metastatic cancer cells with high throughput.SOLUTION: A cell determination method comprises: seeding a plurality of cells on a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer, which is one selected from the group consisting of a natural polymer, a derivative thereof, and an artificial protein, the hydrogel having a modulus of elasticity of 10 to 50 kPa; detecting cells adhered to the hydrogel; and estimating the cells adhered to the hydrogel as metastatic cancer cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cell evaluation method, a screening method, a cell separation method, and a gel. [Background technology]

[0002] Cancer is a disease caused by the proliferation of abnormal cancer cells in the body, and is the leading cause of death among Japanese people. For this reason, methods for quickly and accurately identifying cancer cells are required in the fields of cancer treatment drug development research and cancer diagnosis. Metastatic cancer cells repeatedly invade and metastasize within the body, and the mechanisms of this invasion and metastasis are thought to be related to the morphological changes and motility of cancer cells.

[0003] Research into cancer drug discovery, cancer diagnosis, and elucidation of the mechanisms of metastasis requires a method for isolating cancer cells for characterization. For example, Patent Document 1 reports a method for identifying cancer cells based on their cellular motility on a highly rigid polystyrene culture surface. Non-Patent Document 1 reports a method for identifying cancer cells based on their cellular morphology on a plastic culture surface. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-185888 [Non-patent literature]

[0005] [Non-Patent Document 1] Partin AW, et al., Proc. Natl. Acad. Sci. USA, vol. 86, pp. 1254-1258, 1989 Summary of the Invention [Problem to be solved by the invention]

[0006] The methods disclosed in Patent Document 1 and Non-Patent Document 1 have room for further improvement in terms of isolating metastatic cancer cells with high throughput.

[0007] An object of the present invention is to provide a cell determination method, a screening method, a cell separation method, and a gel that can determine and separate metastatic cancer cells with high throughput. [Means for solving the problem]

[0008] The present invention includes the following aspects. [1] A method for determining cells, comprising seeding a plurality of cells onto a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer that has an elastic modulus of 10 to 50 kPa and is one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins; detecting cells that have adhered to the hydrogel; and inferring that the cells that have adhered to the hydrogel are metastatic cancer cells. [2] The cell evaluation method described in [1], wherein the hydrophilic polymer is gelatin. [3] The cell evaluation method according to [1] or [2], wherein a culture medium is present on the hydrogel. [4] The cell evaluation method according to [3], wherein the medium is Dulbecco's modified Eagle's medium. [5] The cell evaluation method according to any one of [1] to [4], further comprising: photographing an image of a cell adhering to the hydrogel; acquiring at least one of a first variable representing the migration speed of the photographed cell, a second variable representing the angle at which the photographed cell changes direction, and a third variable representing the number of times the photographed cell changes direction; and identifying whether the photographed cell is a metastatic cancer cell based on the acquired one or more variables from among the first variable, the second variable, and the third variable. [6] The cell evaluation method according to any one of [1] to [5], further comprising recovering cells that have not adhered to the hydrogel, seeding the recovered cells on a plastic substrate, and presuming that the cells that have not adhered to the plastic substrate are non-cancer cells. [7] A method for screening a candidate substance, comprising seeding metastatic cancer cells onto a hydrogel having an elastic modulus of 10 to 50 kPa and having a radiation-crosslinked structure of a hydrophilic polymer selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins; confirming that the metastatic cancer cells have adhered to the hydrogel; adding a candidate substance onto the hydrogel; and, if it is detected that there are cells that have not adhered to the hydrogel after adding the candidate substance, determining that the candidate substance has a medicinal effect on metastatic cancer cells. [8] The method for screening a candidate substance according to [7], wherein the hydrophilic polymer is gelatin. [9] The method for screening a candidate substance according to [7] or [8], wherein a culture medium is present on the hydrogel.

[10] The method for screening a candidate substance according to [9], wherein the medium is Dulbecco's modified Eagle's medium.

[11] The method for screening a candidate substance according to any one of [7] to

[10] , further comprising: photographing an image of a cell adhering to the hydrogel; acquiring at least one of a first variable representing the speed of movement of the photographed cell; a second variable representing the angle at which the photographed cell changes direction; and a third variable representing the number of times the photographed cell changes direction; and identifying whether the photographed cell is a metastatic cancer cell based on the acquired one or more variables from among the first variable, the second variable, and the third variable.

[12] The method for screening a candidate substance according to any one of [7] to

[11] , further comprising recovering cells that do not adhere to the hydrogel, seeding the recovered cells on a plastic substrate, and presuming that cells that do not adhere to the plastic substrate are non-cancer cells.

[13] A cell separation method comprising seeding a plurality of cells onto a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer, the hydrogel having an elastic modulus of 10 to 50 kPa and being one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins; separating the cells into those that adhere to the hydrogel and those that do not; and isolating the cells that adhere to the hydrogel as metastatic cancer cells.

[14] The cell separation method described in

[13] , wherein the hydrophilic polymer is gelatin.

[15] The cell separation method according to

[13] or

[14] , wherein a culture medium is present on the hydrogel.

[16] The cell separation method according to

[15] , wherein the medium is Dulbecco's modified Eagle's medium.

[17] The cell separation method according to any one of

[13] to

[16] , further comprising: photographing an image of a cell adhering to the hydrogel; acquiring at least one of a first variable representing the migration speed of the photographed cell, a second variable representing the angle at which the photographed cell changes direction, and a third variable representing the number of times the photographed cell changes direction; and identifying whether the photographed cell is a metastatic cancer cell based on the acquired one or more variables from among the first variable, the second variable, and the third variable.

[18] The cell separation method according to any one of

[13] to

[17] , further comprising recovering cells that do not adhere to the hydrogel, seeding the recovered cells on a plastic substrate, and presuming that the cells that do not adhere to the plastic substrate are non-cancer cells.

[19] A gel for determining whether or not a cell is a metastatic cancer cell, the gel having an elastic modulus of 10 to 50 kPa, and being a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer that is one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins.

[20] The gel for determining whether or not a cancer cell is metastatic, according to

[19] , wherein the hydrophilic polymer is gelatin.

[21] A gel for separating metastatic cancer cells from a plurality of cells, the gel having an elastic modulus of 10 to 50 kPa, and being a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer that is one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins.

[22] The gel for separating metastatic cancer cells according to

[21] , wherein the hydrophilic polymer is gelatin. [Effects of the Invention]

[0009] According to the above aspects, it is possible to provide a cell determination method, a screening method, a cell separation method, and a gel that can determine and separate metastatic cancer cells with high throughput. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram illustrating an example of the configuration of a cell identification device according to an embodiment. [Figure 2] FIG. 1 illustrates an example of a cell centroid according to one embodiment. [Figure 3] FIG. 10 is a diagram illustrating an example of a trajectory generation process according to an embodiment. [Figure 4] FIG. 1 is a diagram illustrating a k-fold cross-validation method according to an embodiment. [Figure 5] 10 is a flowchart illustrating an example of the operation of the cell identification device according to one embodiment. [Figure 6] FIG. 10 is a diagram illustrating a turning angle according to an embodiment. [Figure 7] 1 is a graph showing the cell adhesion rates of Examples 1 and 2, Reference Example 1, and Comparative Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Cell determination method> The cell evaluation method of this embodiment involves seeding a plurality of cells onto a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer that has an elastic modulus of 10 to 50 kPa and is one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins, detecting cells that have adhered to the hydrogel, and inferring that the cells that have adhered to the hydrogel are metastatic cancer cells.

[0012] (hydrogel) The hydrogel used in this embodiment has a radiation-crosslinked structure of a hydrophilic polymer selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins. The hydrogel is a gel for determining whether or not a cell is a metastatic cancer cell. The hydrogel is also a gel for separating metastatic cancer cells.

[0013] In this embodiment, a hydrogel is a material that encapsulates water and hardens into a gel state. For example, it is formed by crosslinking constituent molecules of hydrophilic polymers such as proteins so that they bridge each other. As will be described later, crosslinking by radiation utilizes the high energy inherent in radiation and does not require a so-called crosslinking agent (also known as a thermal crosslinking agent (polymerization initiator) or an ultraviolet crosslinking agent (photopolymerization initiator)). Therefore, a radiation-crosslinked structure that does not contain any crosslinking agent is formed. Such a radiation-crosslinked structure allows the hydrogel state to be maintained even when maintained for a long period of time under cell culture conditions. Furthermore, since the radiation-crosslinked structure does not contain any crosslinking agent, it is not cytotoxic and does not affect the cell culture results.

[0014] Although not particularly limited, radiation crosslinking is a process in which active sites are generated on polymer chains by irradiation, and the polymer chains bond from these sites in an X- or T-shape, forming a three-dimensional mesh structure (also called a network structure). A distinctive feature of radiation crosslinking is that it proceeds at room temperature or below, without the use of additives such as crosslinking agents, and is used to gel materials, improve heat resistance, and impart shape memory properties.

[0015] Furthermore, a hydrophilic polymer refers to a polymer having a hydrophilic group in the molecule. Examples of hydrophilic groups include a hydroxyl group, an amino group, a carboxyl group, an ether group, an acyl group, and a sulfo group. That is, a hydrophilic polymer is a polymer having at least one, preferably two or more, such hydrophilic groups in the molecule.

[0016] Examples of hydrophilic polymers include hydrophilic polymers derived from natural products, such as proteins, peptides, polysaccharides, and nucleic acids, or derivatives thereof. "Naturally derived" means that the polymer can be obtained by extraction or purification from natural products, i.e., earth resources, typically living organisms such as animals, plants, and fungi, and is not limited to being a natural product itself. For example, synthetic proteins artificially synthesized using extracts or purified products from natural products are included in the above-mentioned naturally derived hydrophilic polymers (hereinafter also referred to as "natural polymers"). Note that "synthetic proteins" herein include both proteins synthesized in a cell-based protein synthesis system and proteins synthesized in a cell-free protein synthesis system.

[0017] Specific examples of the naturally occurring hydrophilic polymers include polysaccharides such as dextrin, dextran, chitin, chitosan, agar, agarose, gellan gum, xanthan gum, karaya gum, carrageenan, cellulose, and starch; proteins such as collagen, gelatin, fibrin, albumin, laminin, keratin, ovalbumin, myosin, globulin, and peptides; and nucleic acids such as DNA and RNA.

[0018] A single subtype of naturally occurring hydrophilic polymer may be used as a hydrogel raw material, or multiple different subtypes may be combined to form a hydrogel raw material. For example, collagen is known to have subtypes such as type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen. Therefore, one or more of these subtypes may be used in combination. Type I collagen is preferred because it is the most abundant collagen in the body and can be obtained relatively inexpensively. Type IV collagen is also preferred because it is present in the basement membrane of the skin and can be obtained relatively easily.

[0019] A protein refers to a polymer formed by binding multiple amino acids together via peptide bonds, and is not limited by the number of amino acids that constitute the protein. For example, it includes peptides consisting of two or more amino acids. In this specification, the term "peptide" refers to a polymer consisting of two to 2,000 amino acids.

[0020] Derivatives of hydrophilic polymers derived from natural products are not particularly limited, and examples include derivatives of the naturally derived polymers substituted with lower alkyl groups, lower alkoxyalkyl groups, or hydroxy-lower alkyl groups. Specific examples include natural polymer derivatives selected from the group consisting of lower alkyl group-substituted cellulose derivatives, lower alkoxyalkyl group-substituted cellulose derivatives, hydroxy-lower alkyl group-substituted cellulose derivatives, lower alkoxyalkyl group-substituted chitosan derivatives, lower alkoxyalkyl group-substituted chitin derivatives, lower alkoxyalkyl group-substituted starch derivatives, and lower alkoxyalkyl group-substituted carrageenan derivatives.

[0021] Hydrophilic polymers may also include polymers having artificially designed sequences, such as artificial proteins (including peptides) having artificially designed sequences.

[0022] The hydrogel may be one in which only one of the hydrophilic polymers is cross-linked (bonded) to one another, or two or more hydrophilic polymers are cross-linked (bonded) to one another.

[0023] From the viewpoint of different adhesive properties to metastatic cancer cells, non-metastatic cancer cells, and non-cancer cells, hydrogels containing gelatin as a main component are preferred, and hydrogels consisting of gelatin are more preferred. Examples of gelatin include bovine gelatin and porcine gelatin. Gelatin is purified from type I collagen, which is the most abundant collagen in the body, and is inexpensive and easy to handle.

[0024] The elastic modulus of the hydrogel is 10 to 50 kPa, preferably 20 to 50 kPa. The elastic modulus (hereinafter sometimes referred to as hardness for convenience) is defined by a known method for measuring a stress-strain curve in accordance with JIS K 6272:2003. As an example, the elastic modulus of the hydrogel can be calculated from the stress-strain curve obtained by measuring a hydrogel having a diameter of 35 mm and a thickness of 1.5 mm at 25°C using a creep meter (e.g., Yamaden RE2-33005C, load 2N).

[0025] When the elastic modulus of the hydrogel is 10 to 50 kPa, the adhesion rates of metastatic cancer cells, non-metastatic cancer cells, and non-cancer cells to the hydrogel are different, so that cells can be seeded on the hydrogel and, based on the difference in adhesion, it can be determined whether the cells are metastatic cancer cells, non-metastatic cancer cells, or non-cancer cells.

[0026] The hydrogel may contain 5% by mass or more of water, 10% by mass or more of water, or 30% by mass or more of water relative to the total mass of the hydrogel. The upper limit of the amount of water relative to the total mass of the hydrogel is not particularly limited, and may be, for example, 99% by mass.

[0027] The ratio of the mass of the hydrophilic polymer to the total mass of the hydrogel may be, for example, 1% by mass or more, and preferably 3% by mass or more. The upper limit of the ratio of the mass of the hydrophilic polymer to the total mass of the hydrogel is not particularly limited, and may be, for example, 50% by mass, and preferably 40% by mass.

[0028] The molecular weight of the hydrophilic polymer is not particularly limited, and may be appropriately selected from a range of, for example, 150 to 2,000,000. Typically, a polymer may be appropriately selected from a range of 1,000 to 1,000,000. Note that for polymers with the same composition, the higher the molecular weight, the greater the elastic modulus of the hydrogel tends to be. In this specification, unless otherwise specified, "molecular weight" refers to the weight-average molecular weight. Note that the weight-average molecular weight can be measured using a conventionally known size exclusion chromatogram.

[0029] The hydrogel may be contained in a suitable container, such as a cell culture dish.

[0030] A plurality of cells are seeded on the hydrogel, and then cells adhering to the hydrogel are detected. A culture medium may be present on the hydrogel. Examples of the culture medium include Dulbecco's Modified Eagle Medium (hereinafter sometimes referred to as DMEM), L-15, Roswell Park Memorial Institute (RPMI) 1640 medium, and MEMα, with DMEM being preferred. In particular, when the metastatic cancer cells and non-metastatic cancer cells are breast cancer cells, DMEM is preferred as the culture medium.

[0031] For example, it is preferable to pre-culture the cells before seeding them on the hydrogel. The medium used for pre-culture can be appropriately selected depending on the cell type, and examples include DMEM and L-15. The pre-culture period can be appropriately set, for example, between 3 and 10 days.

[0032] The pre-cultured cells are preferably suspended in the above-mentioned medium and seeded on the hydrogel. After seeding on the hydrogel, the cells are left to stand until they adhere to the hydrogel, and evaluation is preferably performed before the cells completely cover the top surface of the hydrogel (i.e., before they reach confluence). For example, after seeding the cells on the hydrogel, the cells are preferably left to stand for less than 100 hours, preferably 24 to 70 hours, more preferably 30 to 50 hours, and even more preferably 35 to 45 hours. After seeding on the hydrogel, the cells may be cultured.

[0033] Whether or not cells have adhered to the hydrogel can be confirmed, for example, by the following method: The medium on the hydrogel and the cells floating in the medium are collected. If any cells remain on the hydrogel, they are detected as adhered cells.

[0034] Since metastatic cancer cells adhere to the hydrogel of this embodiment, cells adhering to the hydrogel are presumed to be metastatic cancer cells.

[0035] As will be shown in the Examples below, the majority of cells adhering to the hydrogel are metastatic cancer cells, but approximately 20% of the cells adhering to the hydrogel may be non-metastatic cancer cells or non-cancer cells. Therefore, in this specification, "presumed to be metastatic cancer cells" means that the cells adhering to the hydrogel are judged to be highly likely to be metastatic cancer cells.

[0036] Alternatively, multiple cells can be seeded onto the hydrogel, and then time-lapse images of the cells obtained by live cell imaging can be analyzed using image analysis software (e.g., Image J) to analyze migration parameters and detect adherent cells.

[0037] For example, adhering cells can be detected using the cell identification device 1 shown in Figure 1. The cell identification device 1 is a device that distinguishes between metastatic cancer cells, non-metastatic cancer cells, and non-cancerous cells. The cell identification device 1 distinguishes between metastatic cancer cells, non-metastatic cancer cells, and non-cancerous cells based on the motility of the captured cancer cells. Motility refers to, for example, the characteristics of reciprocating movement.

[0038] The cell identification device 1 includes a communication unit 10 , a storage unit 11 , a trajectory generation unit 12 , a display device 13 , a variable acquisition unit 14 , and an identification unit 15 .

[0039] Some or all of the functional units of the cell identification device 1 are realized as software by a processor such as a CPU (Central Processing Unit) executing a program stored in a storage unit having a non-volatile storage medium (non-transitory storage medium). The program may be recorded on a computer-readable storage medium. Examples of computer-readable storage media include portable media such as flexible disks, magneto-optical disks, ROMs (Read Only Memory), and CD-ROMs (Compact Disc Read Only Memory), and non-transitory storage media such as hard disks built into computer systems.

[0040] Some or all of the functional units of the cell identification device 1 may be realized using hardware including electronic circuits (electronic circuits or circuitry) using, for example, an LSI (Large Scale Integration circuit), an ASIC (Application Specific Integrated Circuit), a PLD (Programmable Logic Device), or an FPGA (Field Programmable Gate Array).

[0041] Below, we will explain a cell group containing non-cancer cells and metastatic cancer cells, and also explain an example in which a medium is added to a hydrogel and cells are cultured under appropriate conditions for an appropriate period of time, and their adhesiveness is evaluated.

[0042] When the user inputs a photographing instruction into the computer, a camera (not shown) connected to the microscope photographs cells cultured for an appropriate time in a time-series group of frames, for example, in an environment of 37°C and 5% carbon dioxide. The camera (not shown) photographs each cell for each group of cells at 5-minute intervals for a total of 2 hours. The microscope is capable of detecting the outlines of cells, and may be, for example, a phase-contrast microscope or a microscope using differential interference contrast. The outlines of the cells photographed in the time-series group of frames may be enhanced using image processing such as filtering, which is performed after photographing, as described below.

[0043] Next, the medium on the hydrogel is collected along with the non-adherent cells, seeded onto an empty plastic substrate, and the same medium is added to the gel. This allows for the removal of non-adherent cells while leaving the adherent cells on the gel on the substrate at the time of live cell imaging, allowing for imaging of only the adherent cells. When the user inputs imaging instructions into the computer, a camera (not shown) connected to the microscope performs live cell imaging of the adherent cells, for example, at 5-minute intervals after the addition of medium, for a total of 30 hours.

[0044] According to the program, the communication unit 10 acquires a time series of moving or still image frames from a camera (not shown). The communication unit 10 records the time series frames in the memory unit 11. The memory unit 11 stores the time series frames for each cell group. The presence or absence of cell-substrate adhesion is evaluated based on the migration speed and migration distance from the initial position (Euclidean distance) calculated from the acquired images, and cells adhered to the hydrogel are detected. For example, cells may be distinguished as non-adherent or adherent if the migration speed exceeds a threshold value of 100 μm / h or if the migration distance from the initial position (Euclidean distance) exceeds a threshold value (250 μm) at least once within a unit time (6 h). Visual detection may also be used as criteria for the migration speed, migration distance from the initial position (Euclidean distance), and the presence or absence of cellular pseudopodia. For example, the difference in the speed at which non-adherent and adherent cells cross the field of view (the migration distance from the initial position (Euclidean distance)) and the presence or absence of pseudopodia may be used as criteria for the determination.

[0045] The number of adherent cells and non-adherent cells may be counted by evaluating a combination of the above-mentioned migration speed, the migration distance from the initial position (Euclidean distance), the presence or absence of cell pseudopodia, etc. The cell adhesion rate shown in formula (1.1) may be used as an evaluation index for cell adhesiveness.

[0046]

number

[0047] The display device 13 is a display device such as a liquid crystal display. When the user inputs an instruction to play the acquired moving image or still image file into the computer, the display device 13 displays frames of the moving images or still images recorded in the storage unit 11 in chronological order.

[0048] Furthermore, motility analysis of adhered cells may be performed using the cell identification device 1. For example, an image of cells adhering to a hydrogel may be captured, and at least one of a first variable representing the movement speed of the captured cells, a second variable representing the angle at which the captured cells change direction, and a third variable representing the number of times the captured cells change direction may be obtained, and whether or not the captured cells are metastatic cancer cells may be identified based on the one or more variables obtained from the first variable, the second variable, and the third variable.

[0049] As used herein, "identifying whether or not a cell is a metastatic cancer cell" means performing motility analysis on cells that have adhered to the hydrogel, i.e., cells that are presumed to be metastatic cancer cells, to confirm the likelihood that they are metastatic cancer cells.

[0050] When a user inputs a trajectory generation instruction into the computer, the trajectory generation unit 12 acquires time-series frames for each cell group from the storage unit 11. That is, the trajectory generation unit 12 acquires time-series frames in which adhered cells are photographed from the storage unit 11. The trajectory generation unit 12 generates a trajectory for each photographed cell using predetermined image analysis software (image analysis algorithm).

[0051] When the user inputs an instruction to play the generated trajectory image file into the computer, the display device 13 displays the image of the trajectory generated by the trajectory generating unit 12.

[0052] To obtain images of cell trajectories, a tool using MATRLAB2020a can be used to automatically track the centroid of cells from time-lapse images of cells obtained by imaging adherent cells for a certain period of time (e.g., 30 hours) (see Hiromi Miyoshi, "Diagnosing Cancer Cells through Cell Movement," Japan Society for Medical and Biological Engineering, May 20, 2023). The detailed procedure is shown below.

[0053] A Sobel filter is applied to the whole cell image acquired at time t0 shown in Figure 2(a), and edges including the cell contour are extracted as shown in Figure 2(b). A Sobel filter is a type of first-order differential filter that emphasizes contours. The Sobel filter multiplies the nine pixel values ​​above, below, left, and right of a pixel of interest by kernel coefficients as shown in Equation (1.2) and sums the results. In this example, pixels based on two coefficient matrices for the horizontal and vertical directions are combined to emphasize edges in all directions. In addition, the threshold for binarization after Sobel filter processing can be manually set to an appropriate value depending on the cell type.

[0054]

number

[0055] After edge extraction, an edge expansion process is performed as shown in Figure 2(c). After cells and other objects that touch the boundary of the cropped image are removed, noise is removed by performing a contraction process as shown in Figure 2(d). From the image obtained through the above image processing, the cell outline and centroid are calculated as shown in Figure 2(e). The dot in Figure 2(e) indicates the cell centroid, and the solid line indicates the cell outline. The scale bar is 50 μm. Similar processing is performed on the entire cell image at each time point, and cell motility is evaluated and analyzed from the movement of the centroid over time.

[0056] FIG. 3 is a diagram showing an example of a trajectory generation process. In FIG. 3, cell 20 moves from the position of cell 20-0 to the position of cell 20-25. The trajectory generation unit 12 connects the time-series positions of cell 20 in the time-series frames. In this way, a trajectory of the moved cell 20 is generated. In the following, as an example, the trajectory generation unit 12 generates a trajectory of the moved cell 20 by connecting the time-series positions of cell 20 every 60 minutes.

[0057] 3, during the lapse of 60 minutes from the initial time "t0", cell 20 moves from the position of cell 20-0 to the position of cell 20-12, for example, via the positions of cell 20-1 and cell 20-11. Furthermore, during the lapse of another 60 minutes from the time when cell 20 reaches the position of cell 20-12, cell 20 moves from the position of cell 20-12 to the position of cell 20-24, for example, via the positions of cell 20-13 and cell 20-23.

[0058] When a user inputs a variable acquisition instruction into the computer, the variable acquisition unit 14 derives the movement speed (first variable) of the cell 20 for each trajectory, for example, for 60 minutes. The variable acquisition unit 14 derives the direction change angle (second variable) of the cell 20 for each trajectory, for example, for 60 minutes. For example, in FIG. 3, the angle formed by the line connecting the position of cell 20-0 to the position of cell 20-12 (the movement direction of cell 20 from "time t0" to "time t0+60") and the line connecting the position of cell 20-12 to the position of cell 20-24 (the movement direction of cell 20 from "time t0+60" to "time t0+120") represents the direction change angle "θ" of the cell 20 when the cell 20 moves from the position of cell 20-0 to the position of cell 20-24. Similarly, after 120 minutes have passed since the initial time "t0", the variable acquisition unit 14 derives the direction change angle "θ" of the cell 20 based on the movement direction every 60 minutes when the movement direction of the cell 20 is derived.

[0059] Instead of the variable acquisition unit 14 deriving each direction change angle "θ", each direction change angle "θ" may be derived by a user who measures a trajectory displayed on the display device 13 or the like. Data on each direction change angle "θ" derived by the user who measures the trajectory is stored in the storage unit 11, for example.

[0060] When deriving the direction change angle, the variable acquisition unit 14 selects one threshold from among a plurality of predetermined thresholds as the threshold for the direction change angle. For each selected threshold, the variable acquisition unit 14 derives the number of direction changes (third variable) of the cell 20 in, for example, 1200 minutes. In the following, the variable acquisition unit 14 derives the direction change angle using a predetermined threshold "θ=90°". Furthermore, the variable acquisition unit 14 derives the direction change angle using a predetermined threshold "θ=120°". Furthermore, the variable acquisition unit 14 derives the direction change angle using a predetermined threshold "θ=150°".

[0061] When the variable acquisition unit 14 selects the threshold value for the direction change angle "θ=90°", it derives the total number of times in 60 minutes that the direction change angle "θ" was 90° or greater as the number of direction changes of the cell 20 in 1200 minutes. When the variable acquisition unit 14 selects the threshold value for the direction change angle "θ=120°", it derives the total number of times in 60 minutes that the direction change angle "θ" was 120° or greater as the number of direction changes of the cell 20 in 1200 minutes. When the variable acquisition unit 14 selects the threshold value for the direction change angle "θ=150°", it derives the total number of times in 60 minutes that the direction change angle "θ" was 150° or greater as the number of direction changes of the cell 20 in 1200 minutes.

[0062] In the following, symbols written above letters in mathematical formulas will be written immediately before the letter. For example, the symbol "-" written above the letter "x" in a mathematical formula will be written immediately before the letter as "(-)x".

[0063] The identification unit 15 performs discriminant analysis based on the Mahalanobis distance from the derived variables (first variable, second variable, and third variable), i.e., the motility parameters, to evaluate the motility of each cell. Using the Mahalanobis distance allows data dispersion to be taken into account. Note that when the variance-covariance matrix S is a unit matrix, the Mahalanobis distance becomes the Euclidean distance. A discriminant analysis is used to calculate a discriminant boundary from this Mahalanobis distance, resulting in an (n-1)-dimensional discriminant boundary in n-dimensional space.

[0064] Discriminant analysis (DA) using Mahalanobis distance is performed, for example, as follows. The calculation formula for two-group, two-dimensional discrimination is shown below. First, the mean vector and variance-covariance matrix are obtained for the set of training set parameters shown in equations (1.6) and (1.7). n1 and n2 represent the sample sizes of the training sets of the first and second groups, respectively. The mean vector (-)x i is the variance-covariance matrix S i is calculated using equations (1.10) and (1.11).

[0065]

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[0066]

number

[0067]

number

[0068]

number

[0069]

number

[0070]

number

[0071] At this time, the Mahalanobis distance d i is calculated using equation (1.12), and d2 2 -d1 2 ≧0, parameter x=(ab) tThe cells having the above are identified as group 1.

[0072]

number

[0073] Furthermore, cross-validation may be performed to verify generalization performance. That is, a given dataset may be divided into a training set and a test set, a discrimination boundary may be calculated using the training set, and the discrimination performance for unknown data may be verified by discriminating the test set.

[0074] For example, the generalization performance of a model can be verified using k-fold cross-validation. In k-fold cross-validation, the entire dataset is divided into k parts. A decision boundary is created using k-1 datasets as training datasets, and the remaining datasets are used as test datasets to discriminate from the decision boundary. The accuracy is recorded in the confusion matrix in Table 1, and the discrimination accuracy ACC is calculated using equation (1.13). This is repeated k times, and the generalization performance is evaluated by taking the average of the discrimination accuracy (Figure 4).

[0075]

number

[0076] [Table 1]

[0077] When a user inputs a cell identification command into the computer, the identification unit 15 determines that the cell 20 is a metastatic cancer cell based on one or more acquired variables from the first variable, the second variable, and the third variable. For example, the identification unit 15 determines the mean migration speed and the displacement index (DI) slope, which will be described later, based on the result of pattern matching between the trajectory of the cell 20 and the trajectory of a predetermined metastatic cancer cell.

[0078] Next, an example of the operation of the cell identification device 1 will be described. 5 is a flowchart showing an example of the operation of the cell identification device 1 in the first embodiment. The variable acquisition unit 14 acquires one of a first variable representing the movement speed of the photographed cell 20, a second variable representing the direction change angle of the photographed cell 20, and a third variable representing the number of direction changes of the photographed cell 20 (step S101). The identification unit 15 identifies whether the photographed cell 20 is a cancer cell based on one of these variables. For example, the identification unit 15 identifies whether the cell 20 is a metastatic cancer cell based on the Mahalanobis distance (step S102).

[0079] As described above, the variable acquisition unit 14 acquires one of three types of variables (parameters): the first variable, the second variable, and the third variable. The identification unit 15 identifies whether the photographed cell 20 is a metastatic cancer cell based on the acquired one of these variables. This makes it possible to identify metastatic cancer cells based on their motility.

[0080] Although the above description has been given of an example in which the plurality of cells seeded on the hydrogel includes metastatic cancer cells and non-cancer cells, and metastatic cancer cells are detected and estimated, the present invention is not limited to this. For example, the plurality of cells may include at least one of metastatic cancer cells, non-metastatic cancer cells, and non-cancer cells. An example of a plurality of cells including at least one of metastatic cancer cells, non-metastatic cancer cells, and non-cancer cells is a specimen sampled from a patient.

[0081] Furthermore, metastatic cancer cells and non-metastatic cancer cells may be distinguished based on the adhesiveness of the cells to the hydrogel, and non-metastatic cancer cells and non-cancer cells may be distinguished based on the adhesiveness of the cells to the hydrogel.

[0082] Alternatively, metastatic cancer cells and non-cancer cells may be distinguished based on cell motility, and non-metastatic cancer cells and non-cancer cells may be distinguished based on cell motility. The variable for determining motility can be appropriately set based on the cell type to be distinguished so as to increase the true positive rate or true negative rate.

[0083] The adhesion rate of metastatic cancer cells to the hydrogel of this embodiment is higher than that of non-metastatic cancer cells, so there is a high possibility that cells adhering to the hydrogel are metastatic cancer cells.

[0084] The adhesion rate of metastatic cancer cells to the hydrogel of this embodiment is higher than that of non-cancer cells, and therefore, cells adhering to the hydrogel are likely to be metastatic cancer cells.

[0085] The adhesion rate of non-metastatic cancer cells to the hydrogel of this embodiment is higher than that of non-cancer cells. Therefore, when a cell population consists of non-metastatic cancer cells and non-cancer cells, the cells adhering to the hydrogel are likely to be non-metastatic cancer cells.

[0086] The metastatic cancer cells of this embodiment are not particularly limited, but examples thereof include metastatic breast cancer cells, metastatic lung cancer cells, and metastatic prostate cancer cells.

[0087] The non-metastatic cancer cells of this embodiment are not particularly limited, but examples thereof include non-metastatic breast cancer cells, non-metastatic lung cancer cells, and non-metastatic prostate cancer cells.

[0088] The non-cancerous cells of this embodiment include non-cancerous cells corresponding to the metastatic cancer cells and non-metastatic cancer cells.

[0089] As shown in the examples, non-cancerous breast epithelial cells, MCF-10A, no longer adhere to plastic substrates when pre-cultured in DMEM. Therefore, the following procedure makes it possible to distinguish non-cancerous cells, metastatic cancer cells, and non-metastatic cancer cells from a cell population. First, a cell population containing non-cancerous cells, metastatic cancer cells, and non-metastatic cancer cells is seeded in a container containing hydrogel and DMEM on the hydrogel and cultured. Cells that adhere to the hydrogel are then assumed to be metastatic cancer cells. Cells that do not adhere to the hydrogel are collected and seeded on a plastic substrate. Cells that adhere to the plastic substrate are assumed to be non-metastatic cancer cells, and cells that do not adhere to the plastic substrate are determined to be non-cancerous cells.

[0090] Examples of materials contained in the plastic substrate include polystyrene, acrylic resin, polylactic acid, etc., and polystyrene (polystyrene for tissue culture having a hydrophilized surface) is preferred.

[0091] According to the cell determination method of this embodiment, metastatic cancer cells can be determined with high throughput.

[0092] (Method for manufacturing hydrogel) The hydrogel of the present invention can be produced by irradiating a precursor, which is an aqueous solution containing a hydrophilic polymer and water, with radiation at a dose of 1 to 1,000 kGy. Here, radiation is a concept that includes electromagnetic waves with wavelengths shorter than ultraviolet rays, electron beams, and ion beams, and electromagnetic waves include X-rays and gamma rays. Any known radiation generator for these types of radiation can be used as the radiation irradiator.

[0093] The radiation irradiation is carried out by continuously or intermittently irradiating the precursor with radiation at a dose of 1 to 1000 kGy using a radiation irradiation device. The lower limit of the dose may be 2 kGy or more, 3 kGy or more, 5 kGy or more, 10 kGy or more, 100 kGy or more, or 500 kGy or more, and the upper limit of the dose may be 500 kGy or less, 300 kGy or less, 200 kGy or less, or 100 kGy or less. The upper and lower limit values ​​of the dose may be combined arbitrarily. In one embodiment, radiation irradiation at a dose of 5 to 200 kGy is preferably used.

[0094] Here, the dose refers to the amount of radiation irradiated, and can be measured using a dosimeter commercially available for each type of radiation. In addition, it is desirable to set the irradiation energy in the radiation irradiator to approximately 100 keV to 10 MeV.

[0095] When the precursor is irradiated with radiation, the hydrophilic polymer (e.g., protein) partially cleaves, generating radicals, and a crosslinking reaction occurs, resulting in the formation of a gel that encapsulates water. In other words, a hydrogel is produced. The crosslinking reaction immediately stops when the radiation irradiation is completed. The crosslink density during the reaction depends on the radiation dose, and as a result, the elastic modulus of the gel also depends on the radiation dose.

[0096] The higher the crosslink density of the hydrophilic polymer, the lower the water content. In other words, the higher the crosslink density of the hydrophilic polymer, the higher the content of the hydrophilic polymer in the hydrogel. Therefore, the content of the hydrophilic polymer in the hydrogel also depends on the dose of radiation used.

[0097] During radiation irradiation, it is desirable that the ambient temperature be 4 to 50°C and the dissolved oxygen concentration in the precursor be 0 to 40 mg / L. The ambient temperature during radiation irradiation may be set appropriately depending on the properties of the hydrophilic polymer. For example, when collagen is used, radiation crosslinking can be efficiently achieved by irradiating at a low temperature of about 4 to 25°C, and when gelatin is used, radiation crosslinking can be efficiently achieved by irradiating in a temperature environment of 10 to 30°C.

[0098] The dissolved oxygen in the precursor during radiation irradiation has the effect of capturing radicals. In other words, increasing the dissolved oxygen concentration in the precursor reduces the crosslink density, thereby reducing the elastic modulus of the hydrogel. Specific methods for increasing the dissolved oxygen concentration in the precursor include irradiating the precursor with radiation in an environment with a high oxygen concentration, leaving the precursor in an environment with a high oxygen concentration, or bubbling oxygen into the precursor.

[0099] By lowering the dissolved oxygen concentration in the precursor, the crosslinking density increases, and as a result, the elastic modulus of the hydrogel can be increased.Specific methods for reducing the dissolved oxygen concentration in the precursor include irradiating the precursor with radiation in a nitrogen environment or bubbling nitrogen through the precursor.

[0100] By irradiating a precursor with a low dissolved oxygen concentration under high oxygen concentration, it is possible to change the dissolved oxygen concentration between the surface that comes into contact with oxygen (typically the cell adhesion surface) and other parts. By controlling the oxygen concentration, it is possible to adjust only the surface of the hydrogel used for cell culture (only the surface that comes into contact with the cells to be cultured) to a desired elastic modulus, and to make other parts have a higher elastic modulus than the surface. For example, in the case of a flat hydrogel, it is possible to make the basal part have a high elastic modulus and only the cell adhesion surface have a low elastic modulus.

[0101] <Candidate substance screening method> The screening method for candidate substances of this embodiment includes seeding metastatic cancer cells onto a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer that has an elastic modulus of 10 to 50 kPa and is one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins; confirming that the metastatic cancer cells have adhered to the hydrogel; adding a candidate substance onto the hydrogel; and, if it is detected that there are cells that have not adhered to the hydrogel after adding the candidate substance, determining that the candidate substance has a medicinal effect on the metastatic cancer cells.

[0102] In cancer drug discovery, determining whether a candidate substance has a medicinal effect on cancer cells is generally done based on whether the cancer cells survive or die. Therefore, if a candidate substance causes metastatic cancer cells to change into non-metastatic cancer cells or non-cancerous cells without causing cell death, it cannot be determined that the candidate substance has a medicinal effect on metastatic cancer cells.

[0103] According to the method for screening a candidate substance of this embodiment, metastatic cancer cells can be distinguished from non-metastatic cancer cells or non-cancer cells based on the presence or absence of adhesion to the hydrogel, making it possible to screen for candidate substances that have the medicinal effect of converting metastatic cancer cells into non-metastatic cancer cells or non-cancer cells without causing cell death.

[0104] The screening method for candidate substances of this embodiment is carried out, for example, as follows. First, metastatic cancer cells suspended in a medium are seeded on a hydrogel and cultured. The cells may be pre-cultured in advance. The metastatic cancer cells are cultured on the hydrogel, and the metastatic cancer cells adhere to the hydrogel.

[0105] The candidate substance is then added to the hydrogel. After an appropriate time has passed, cells that have not adhered to the hydrogel are collected. If the collected cells are cultured and proliferate, it is believed that the metastatic cancer cells have transformed into other cells. This suggests that the candidate substance has the medicinal effect of transforming metastatic cancer cells into non-metastatic cancer cells or non-cancerous cells.

[0106] The hydrogel used is the same as that described above in (Hydrogel), and therefore a detailed description thereof will be omitted. Furthermore, the metastatic cancer cells and the culture medium used are the same as those described above, and therefore a detailed description thereof will be omitted.

[0107] Candidate substances include the above-mentioned organic synthetic compounds, proteins, peptides, antibodies, bacterial metabolites, etc. that are suspected to have medicinal effects on cancer cells.

[0108] According to the method for screening a candidate substance of this embodiment, it is possible to screen for a candidate substance that has a novel pharmacological effect on metastatic cancer cells.

[0109] <Cell separation method> The cell separation method of this embodiment involves seeding a plurality of cells onto a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer that has an elastic modulus of 10 to 50 kPa and is one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins, separating the cells into those that adhere to the hydrogel and those that do not, and isolating the cells that adhere to the hydrogel as metastatic cancer cells.

[0110] In research into cancer drug discovery, cancer diagnosis, and elucidation of the mechanisms of metastasis, it is important to isolate cancer cells for characterization. The cell separation method of this embodiment can separate metastatic cancer cells from non-metastatic cancer cells or non-cancer cells based on the presence or absence of adhesion to the hydrogel.

[0111] The cell separation method of this embodiment is carried out, for example, as follows. First, a plurality of cells suspended in a culture medium are seeded on a hydrogel and cultured. The cells may be pre-cultured in advance. The cells are cultured on the hydrogel, and metastatic cancer cells adhere to the hydrogel.

[0112] The cells that do not adhere to the hydrogel are then collected, and the remaining cells that adhere to the hydrogel are isolated as metastatic cancer cells.

[0113] Furthermore, non-cancerous breast epithelial cells, MCF-10A, no longer adhere to plastic substrates when pre-cultured in DMEM, as shown in the Examples. Therefore, non-cancerous cells, metastatic cancer cells, and non-metastatic cancer cells can be separated from a cell population by the following procedure. First, a cell population containing non-cancerous cells, metastatic cancer cells, and non-metastatic cancer cells is seeded in a container containing hydrogel and DMEM on the hydrogel and cultured. Subsequently, cells that do not adhere to the hydrogel are collected, and cells that adhere to the hydrogel are collected as metastatic cancer cells. Cells that do not adhere to the hydrogel are seeded on a plastic substrate. Subsequently, cells that do not adhere to the plastic substrate are collected as non-cancerous cells, and cells that adhere to the plastic substrate are collected as non-metastatic cancer cells.

[0114] The hydrogel used is the same as that described above in (Hydrogel), and therefore a detailed description thereof will be omitted. Furthermore, the metastatic cancer cells, non-metastatic cancer cells, non-cancer cells, and culture media used are the same as those described above, and therefore a detailed description thereof will be omitted.

[0115] According to the cell separation method of this embodiment, metastatic cancer cells can be separated with high throughput. [Example]

[0116] The present invention will be explained in more detail below with reference to examples, but it should be understood that the present invention is not limited to these examples.

[0117] <Preparation of gel> The gels used in the following examples were prepared according to the description in Oyama, TG et al., Collagen hydrogels with controllable combined cues of elasticity and topography to regulate cellular processes, Biomedical Materials, 16(4), 2021.

[0118] Specifically, gels were prepared as follows: Japanese Pharmacopoeia gelatin (GLS250, manufactured by Nitta Gelatin Co., Ltd.) was dissolved in ultrapure water at 50°C to obtain aqueous gelatin solutions containing 10% and 15% by mass of the total mixture. 1.5 mL of this aqueous gelatin solution was poured into a 35 mm cell culture dish and allowed to stand overnight at 20°C to form a physical gel.

[0119] Next, the dishes were irradiated with gamma rays at a dose rate of 4 kGy / h or 5 kGy / h in a deoxygenated state at the Cobalt-60 gamma ray irradiation facility of the Takasaki Advanced Quantum Science and Technology Research Institute. After irradiation, 2 mL of phosphate-buffered saline (PBS(-) solution) was poured into each dish and washed at 37°C for 2 hours to obtain swollen gels with adjusted elastic modulus.

[0120] The elastic modulus of the resulting gel was calculated from the stress-strain curve obtained using a creep meter (Yamaden Co., Ltd., RE2-33005C, load 2N). The swollen gel was immersed in culture medium at 37°C for 1 hour, and the absorbed PBS(-) was replaced before use in cell culture. The combination of the gelatin aqueous solution concentration and gamma ray irradiation dose to obtain a gel with an elastic modulus of 25 kPa used in Example 1 below was 10 wt% and 8 kGy. The combination of the gelatin aqueous solution concentration and gamma ray irradiation dose to obtain a gel with an elastic modulus of 50 kPa used in Example 2 was 10 wt% and 10 kGy. The combination of the gelatin aqueous solution concentration and gamma ray irradiation dose to obtain a gel with an elastic modulus of 160 kPa used in Example 3 was 15 wt% and 20 kGy.

[0121] <Reagents> The culture media used were Dulbecco's Modified Eagle's Medium-low glucose (Sigma, D6046, DMEM), MEGM Mammary Epithelial Cell Growth Medium Bullet Kit (Lonza, CC-3150, MEGM), and Leibovitz's L-15 Medium with L-Glutamine, Phenol Red, and Sodium Pyruvate (Fujifilm Wako Pure Chemical Industries, Ltd., 128-06075, L-15). DMEM was prepared by adding 10% Fetal Bovine Serum (Cosmobio, P200302, hereafter referred to as FBS) and 1% Penicillin-Streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd., 168-23191, diluted 100-fold, containing 100 units / mL penicillin and 0.1 mg / mL streptomycin, hereafter referred to as P / S). L-15 was prepared by adding 15% FBS and 1% Penicillin-Streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd., 168-23191, diluted 100-fold, containing 100 units / mL penicillin and 0.1 mg streptomycin, P / S). MEGM was prepared by adding MEGM BulletKit (Lonza, CC-3150) and 100 ng / mL Cholera Toxin solution for cell biology (Fujifilm Wako Pure Chemical Industries, Ltd., 030-20621).

[0122] <Pre-culturing cells before gel seeding> The cancer cell models used were metastatic breast cancer cells MDA-MB-231 (European Collection of Authenticated Cell Cultures (ECACC)) and non-metastatic breast cancer cells MCF-7 (Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank)), and the non-cancer cell model used was mammary epithelial cells MCF-10A (American Type Culture Collection (ATCC)). The trypsin-EDTA solution, culture medium, and observation medium used for pre-culture of each cell type are shown in Table 2. In Table 2, 0.25T / E is trypsin-EDTA solution (0.25%, Fujifilm Wako Pure Chemical Industries, Ltd., 201-16945), and 0.05T / E is trypsin-EDTA solution (0.05%, Fujifilm Wako Pure Chemical Industries, Ltd., 202-16931).

[0123] (1) Metastatic breast cancer cell line MDA-MB-231 The cells were cultured in an incubator (As One, i-CUBE (HOT & COOL), FCI-280) at 37°C. Subculture was performed when the cells in a 60 mm dish (Corning, 353002, FALCON®) reached 80-90% confluence. After removing the medium from the dish with an aspirator, 1 mL of 0.05% trypsin-EDTA solution was added and the cells were detached from the dish by incubation at 37°C and 5% CO2 for 5 minutes. The trypsin / EDTA reaction was then stopped by adding L-15. The cells were then precipitated by centrifugation at 300 rpm for 3 minutes (Kubota, 2410), recovered by adding L-15, and seeded. The cells were then transferred to an incubator and cultured at 37°C.

[0124] (2) Non-metastatic breast cancer cell line MCF-7 Cells were cultured in an incubator (Thermo Fisher Scientific, STERI-CYCLE i160, 41981653) at 37°C and 5% CO2. Subculture was performed when cells in a 60 mm dish reached 80-90% confluence. After removing the medium from the dish with an aspirator, 1 mL of 0.25% trypsin-EDTA solution was added and cells were detached from the dish by centrifugation at 37°C and 5% CO2 for 2 minutes. DMEM was then added to stop the trypsin / EDTA reaction. The cells were then pelleted by centrifugation at 300 rpm for 3 minutes (Kubota, 2410). After collection and seeding, the cells were transferred to an incubator and cultured at 37°C and 5% CO2.

[0125] (3) Non-cancerous cells mammary epithelial cells MCF-10A Cells were cultured in an incubator (Thermo Fisher Scientific, STERI-CYCLE i160, 41981653) at 37°C and 5% CO2. Subculture was performed when cells in a 60 mm dish reached 80-90% confluence. After removing the medium from the dish with an aspirator, 1 mL of 0.05% trypsin-EDTA solution was added and cells were detached from the dish by 20 minutes at 37°C and 5% CO2. The trypsin / EDTA reaction was then stopped by adding MEGM. The cells were then pelleted by centrifugation at 130 rpm for 10 minutes (Thermo Fisher Scientific, Sorvall ST 8, 75007202). MEGM was added, recovered, and seeded. The cells were then transferred to an incubator and cultured at 37°C and 5% CO2.

[0126] [Table 2]

[0127] <Evaluation, Observation> (1) Live cell imaging The cells that had been pre-cultured for 7 days were treated with trypsin / EDTA solution and washed by centrifugation in the same manner as in the pre-culture using each of the observation media shown in Table 1, and a cell suspension was prepared using the observation medium.

[0128] 2 × 10 cells were plated on each of the 25, 50, and 160 kPa gels and 35 mm polystyrene dishes containing 1 mL of observation medium warmed to 37 °C. 3 cells / cm 2 The cells were seeded in a 1000 ml / min solution and cultured for 40 hours at 37°C for L-15 observation medium, or at 37°C and 5% CO2 for other media. Live cell imaging was then performed at 5-minute intervals for a total of 2 hours using an incubation monitoring system (Olympus, CM20) at 37°C for L-15 observation medium, or at 37°C and 5% CO2 for other media.

[0129] Next, the medium on the gel was removed along with the non-adherent cells and seeded onto an empty 35 mm polystyrene dish. 1 mL of the same medium was added to the gel. This process was repeated twice. This allowed for the removal of non-adherent cells while leaving the adherent cells on the gel on the substrate at the time of live cell imaging, enabling imaging of only the adherent cells. Live cell imaging of the adherent cells was performed at 5-minute intervals for a total of 30 hours after the addition of medium.

[0130] (2) Adhesiveness analysis The time-lapse images (i.e., time-series frames) of cells obtained by 2-hour live cell imaging were visually evaluated for the presence or absence of cell-substrate adhesion based on cell migration speed, migration distance, cell pseudopodia, etc. using the image analysis software ImageJ, and the numbers of adherent and non-adherent cells were counted. The cell adhesion rate shown in the above formula (1.1) was used as an index to evaluate cell adhesiveness.

[0131] (3) Motility analysis (3-1) Cell tracking To quantitatively evaluate cell motility from time-lapse images of cells obtained by 30 hours of live cell imaging, MATRLAB2020a was used to automatically track the cell centroid as described above. Similar processing was performed on whole-cell images at each time point, and cell motility was evaluated from the movement of the centroid over time. Cells whose migration speed exceeded a threshold (100 μm / h) or whose migration distance from the initial position (Euclidean distance) exceeded a threshold (250 μm) at least once within a unit time (6 h) were determined to be non-adherent cells and were excluded from motility analysis.

[0132] (3-2) Motion parameters Cell motility was analyzed from cell centroids obtained using an automated tracking tool. Parameters related to migration speed and direction were obtained from cell centroids every 5 minutes. The Mann-Whitney U test was used to compare two average values. Thirty hours of live-cell imaging data was used for motility analysis, but tracking times varied depending on the cell type. Therefore, calculation of motility parameters was limited to cells that could be tracked for 300 minutes or more.

[0133] (3-2-1)Mean Migration Speed The displacement of the cell centroid was obtained at an imaging interval of 5 minutes, and the average velocity within the tracking time was calculated.

[0134] (3-2-2) Displacement Index (DI) Slope The time change of the displacement index was calculated using the following equations (1.3) to (1.5).

[0135]

number

[0136]

number

[0137]

number

[0138] x(t) and y(t) are the centroid coordinates of the cell at time t, τ is the lag time, and <> is the time average. The maximum lag time was set to half the tracking time. DI was plotted against the lag time τ, and the slope was calculated from a logarithmic DI-lag time graph. The parameters used for the DI slope were the average DI slope over the entire lag time (Average DI Slope), the slope between the minimum (5 min) and maximum (half the tracking time) points of the lag time (DI Slope of Start-End), and the average DI slope up to a lag time of 50 min (DI Slope in 50 min).

[0139] (3-2-3) Frequency of Turns The number of times a cell turned in a certain angular range was calculated. Normalization was performed using the tracking time for each cell, and the values ​​were standardized to a value of 30 hours of tracking. The turning angle θ was defined as shown in Figure 6. (x i ,y i ) at time t i The regions used were 0°-30°, 30°-60°, 60°-90°, 90°-120°, 120°-150°, and 150°-180°. Note that if there is no movement of the cell centroid (i.e., x i =x i+1 ) was not counted in any angle.

[0140] (3-2-4) Average migration distance between turns within a certain angle range The average distance traveled by cells before they made a change in direction within a certain angular range was calculated. The angular ranges used were 0°-30°, 30°-60°, 60°-90°, 90°-120°, 120°-150°, and 150°-180°. Note that if the number of changes in the angular range of interest was zero, it was set to 0 μm.

[0141] (3-2-5) Sum of Turn Angles The sum of the cell turning angles for each imaging interval was calculated and normalized using the tracking time for each cell to be consistent for 30 hours of tracking.

[0142] (3-2-6)Stop Time The average time it took for a cell to travel a distance greater than the threshold was defined as the stasis time, which was determined by using the average migration distance per imaging interval as the threshold.

[0143] (3-2-7) Total Length Migrated The total distance traveled by the cells was calculated for each imaging interval. The total distance traveled here is not the Euclidean distance between the initial position and the final position.

[0144] (3-2-8) Time Between Turns The average time it took for cells to change direction within a certain angular range was calculated. The following regions were used: 0°-30°, 30°-60°, 60°-90°, 90°-120°, 120°-150°, and 150°-180°. Note that if the number of changes within the angular range of interest was zero, the time was set to 0 minutes.

[0145] (4) Cancer cell differentiation As described above, discriminant analysis was performed using the calculated motility parameters based on the Mahalanobis distance to evaluate differences in motility between cell types at each substrate elasticity. Discriminant analysis was then performed using this Mahalanobis distance to calculate a discriminant boundary, resulting in an n-1-dimensional discriminant boundary in n-dimensional space. In this example, cross-validation was performed to verify generalization performance. Specifically, the given dataset was divided into a training set and a test set. The discriminant boundary was calculated using the training set, and the test set was discriminated to verify the discrimination performance against unknown data.

[0146] (4-1) Cancer cell discrimination analysis After performing discriminant analysis (DA) using Mahalanobis distance, the generalization performance of the model was verified using k-fold cross-validation. In this example, k=5 k-fold cross-validation was used.

[0147] <Examples 1 and 2> In Example 1, a gel with an elastic modulus of 25 kPa was used, and the cell adhesion rates of metastatic breast cancer cells MDA-MB-231, non-metastatic breast cancer cells MCF-7, and non-cancerous breast epithelial cells MCF-10A were evaluated by the method described in (2) Adhesion Analysis using the observation medium in Table 2. In Example 2, the same procedure as in Example 1 was performed, except that a gel with an elastic modulus of 50 kPa was used instead of the gel with an elastic modulus of 25 kPa, and the cell adhesion rate was evaluated.

[0148] <Reference example 1> In Reference Example 1, the same procedure as in Example 1 was carried out except that a gel having an elastic modulus of 160 kPa was used instead of the gel having an elastic modulus of 25 kPa, and the cell adhesion rate was evaluated. <Comparative Example 1> In Comparative Example 1, the same procedure as in Example 1 was carried out except that a polystyrene dish (manufactured by VIOLAMO, TCPS, VTC-D35N) was used instead of the gel, and the cell adhesion rate was evaluated.

[0149] The experiments in Examples 1 and 2, Reference Example 1, and Comparative Example 1 were each carried out twice, and the average value was taken as the cell adhesion rate. The results are shown in FIG.

[0150] In Comparative Example 1, the adhesion rate was 90% or higher for all cells and culture media, whereas in Examples 1 and 2 and Reference Example 1, it was found that the adhesion rate varied depending on the cells. In particular, when DMEM was used as the culture medium, it was found that the adhesion rate was high for metastatic cancer cells and low for non-cancer cells.

[0151] For example, when metastatic cancer cells and non-cancer cells are mixed in a 1:1 ratio, if the gel of Example 1 and DMEM are used to remove non-adherent cells and recover adherent cells, approximately 95% of the recovered cells are metastatic cancer cells. This suggests that metastatic cancer cells can be isolated with a high probability.

[0152] Furthermore, it was found that the adhesion rates of metastatic cancer cells and non-metastatic cancer cells were significantly different when DMEM was used as the medium in Examples 1 and 2. This suggests that when metastatic cancer cells and non-metastatic cancer cells are mixed, the gels of Examples 1 and 2 can be used to remove non-adhered cells and recover adhered cells, thereby enabling the separation of metastatic cancer cells.

[0153] For example, when metastatic cancer cells and non-metastatic cancer cells are mixed in a 1:1 ratio, if the gel of Example 1 and DMEM are used to remove non-adherent cells and recover adherent cells, approximately 75% of the recovered cells are metastatic cancer cells. This suggests that metastatic cancer cells can be isolated with a high probability.

[0154] Furthermore, it was found that the adhesion rates of non-metastatic cancer cells and non-cancer cells were significantly different when DMEM was used as the medium in Examples 1 and 2. This suggests that when non-metastatic cancer cells and non-cancer cells are mixed, the gels of Examples 1 and 2 can be used to remove the non-adhered cells and recover the adhered cells, thereby enabling the separation of non-metastatic cancer cells.

[0155] For example, when metastatic cancer cells and non-metastatic cancer cells are mixed in a 1:1 ratio, if the gel of Example 1 and DMEM are used to remove non-adherent cells and recover adherent cells, approximately 75% of the recovered cells are metastatic cancer cells. This suggests that metastatic cancer cells can be isolated with a high probability.

[0156] From the results of Example 1, when the medium is DMEM and metastatic cancer cells and non-metastatic cancer cells are mixed in a 1:1 ratio, it is estimated that approximately 75% of the adherent cells obtained by the above method are metastatic cancer cells. To further confirm this from the motility of the adherent cells, motility analysis was performed using the above method. As a result, cancer cell discrimination was performed based on two variables, mean migration speed and time change in displacement index (Avg. DI slope), and it was found that approximately 75% of metastatic cancer cells and approximately 25% of non-metastatic cancer cells could be distinguished with 72.3% accuracy.

[0157] Furthermore, when metastatic and non-metastatic cancer cells were mixed in a 1:1 ratio in DMEM culture medium, approximately 88% of the non-adherent cells were estimated to be non-metastatic. To further confirm this, motility analysis was performed using the method described above to determine the motility of adherent cells. The results showed that cancer cells could be distinguished with 74.0% accuracy from approximately 88% of non-metastatic cancer cells and approximately 12% of metastatic cancer cells based on two variables: the frequency of turns under 60° and the time change in the displacement index (DI slope in 50 min).

[0158] From the results of Example 1, when the medium is DMEM and non-metastatic cancer cells and non-cancer cells are mixed in a 1:1 ratio, it is estimated that approximately 90% of the adherent cells obtained by the above method are non-metastatic cancer cells. To further confirm this from the motility of the adherent cells, motility analysis was performed using the above method. As a result, cancer cell discrimination was performed based on a single variable, the average time it took to turn 120° or more (Time Between Turns Over 120°), and it was found that approximately 90% of non-metastatic cancer cells and approximately 10% of non-cancer cells could be distinguished with 76.4% accuracy.

[0159] Furthermore, it was found that non-cancerous breast epithelial cells MCF-10A become non-adherent to polystyrene dishes (VIOLAMO, TCPS, VTC-D35N) when pre-cultured in DMEM medium. Therefore, when non-metastatic cancer cells and non-cancerous cells are mixed in a 1:1 ratio in DMEM medium, the adherent cells can be recovered as non-metastatic cancer cells and the non-adherent cells can be recovered as non-cancerous cells by collecting the adherent cells using the above method and seeding them on a polystyrene dish. [Explanation of symbols]

[0160] 1... cell identification device, 10... communication unit, 11... memory unit, 12... trajectory generation unit, 13... display device, 14... variable acquisition unit, 15... identification unit, 20... cell

Claims

1. seeding a plurality of cells onto a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer, the hydrogel having an elastic modulus of 10 to 50 kPa and being one selected from the group consisting of naturally-derived polymers, derivatives of naturally-derived polymers, and artificial proteins; detecting cells adhering to the hydrogel; A cell assessment method comprising inferring that cells adhering to the hydrogel are metastatic cancer cells.

2. The cell evaluation method according to claim 1 , wherein the hydrophilic polymer is gelatin.

3. The cell evaluation method according to claim 1 or 2, wherein a culture medium is present on the hydrogel.

4. The cell evaluation method according to claim 3 , wherein the medium is Dulbecco's modified Eagle's medium.

5. Furthermore, an image of the cells adhering to the hydrogel is taken, acquiring at least one of a first variable representing a moving speed of the photographed cell, a second variable representing a direction change angle of the photographed cell, and a third variable representing a number of direction changes of the photographed cell; The cell evaluation method according to claim 1 or 2, further comprising identifying whether the photographed cell is a metastatic cancer cell based on one or more acquired variables from among the first variable, the second variable, and the third variable.

6. Furthermore, cells that have not adhered to the hydrogel are collected, seeding the collected cells on a plastic substrate; The cell evaluation method according to claim 1 or 2, further comprising presuming that cells that do not adhere to the plastic substrate are non-cancer cells.

7. seeding metastatic cancer cells onto a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer, the hydrogel having an elastic modulus of 10 to 50 kPa and being one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins; confirming that the metastatic cancer cells are adhered to the hydrogel; Adding a candidate substance onto the hydrogel; A method for screening a candidate substance, comprising determining that the candidate substance has medicinal effect against metastatic cancer cells when it is detected that there are cells that have not adhered to the hydrogel after adding the candidate substance.

8. seeding a plurality of cells onto a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer, the hydrogel having an elastic modulus of 10 to 50 kPa and being one selected from the group consisting of naturally-derived polymers, derivatives of naturally-derived polymers, and artificial proteins; The cells are separated into those adhered to the hydrogel and those not adhered to the hydrogel, A cell separation method comprising separating cells adhering to the hydrogel as metastatic cancer cells.

9. A gel for determining whether a cell is a metastatic cancer cell, The gel has an elastic modulus of 10 to 50 kPa; The gel is a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer that is one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins, and is used to determine whether or not the cells are metastatic cancer cells.

10. The gel for determining whether a cell is a metastatic cancer cell according to claim 9 , wherein the hydrophilic polymer is gelatin.

11. 1. A gel for separating metastatic cancer cells from a plurality of cells, comprising: The gel has an elastic modulus of 10 to 50 kPa; The gel for separating metastatic cancer cells is a hydrogel having a radiation-crosslinked structure of a hydrophilic polymer, which is one selected from the group consisting of naturally occurring polymers, derivatives of naturally occurring polymers, and artificial proteins.

12. The gel for separating metastatic cancer cells according to claim 11, wherein the hydrophilic polymer is gelatin.

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