Method for manufacturing cell chips and method for cell analysis

Abstract

When a cell suspension is dispensed from a liquid dispensing head, proteins leak from the cells during the dispensing process. As a result, proteins contaminate not only droplets containing cells but also droplets without cells, making accurate measurement of individual cells impossible. [Solution] A method for manufacturing cell chips, comprising a fixation step of immersing cells in a cell fixative, a placement step of dispensing a cell suspension containing the fixed cells and liquid from an inkjet-type dispensing unit toward a dispensing medium such as a membrane to place droplets of the cell suspension on the dispensing medium, and a staining step of staining cell-derived components, characterized in that the fixation step, placement step, and staining step are carried out in that order.

Description

[Technical Field]

[0001] The present invention relates to a method for manufacturing cell chips and a method for cell analysis. [Background technology]

[0002] Dot blotting is a known method for evaluating cells by detecting whether or not they express a particular protein. In dot blotting, multiple cells are lysed to form a single sample, and this lysate is placed on an extrusion medium such as a polyvinylidene fluoride membrane (hereinafter also called a PVDF membrane). After drying, an antigen-antibody reaction is performed. For example, if the antibody is fluorescently labeled, a device that images the fluorescence can be used to detect whether or not the protein specifically recognized by the antibody was present in multiple cells.

[0003] In recent years, inkjet printers have also come into use in the field of biotechnology. Specifically, this method involves filling an inkjet-type liquid ejection head with a cell suspension instead of ink, and ejecting it onto a medium such as a petri dish instead of paper. If the cell suspension can be ejected from the liquid ejection head using this method and cells can be arranged one by one on the medium, dot blotting can be performed on a single-cell basis. That is, by arranging cells one by one on the medium, drying them, performing an antigen-antibody reaction, and imaging the fluorescence, the presence or absence of proteins specifically recognized by the antibody can be quantitatively analyzed on a cell-by-cell basis. Patent Document 1 describes using an inkjet printer to arrange growth medium and cells on a substrate so that they overlap. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2005-130859 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, our investigations have revealed that when droplets containing cells are ejected using an inkjet printer as described in Patent Document 1, proteins leak out from the cells during the ejection process. As a result, proteins become mixed not only in droplets containing cells but also in droplets without cells on the ejection medium, potentially making it impossible to accurately perform quantitative measurements of proteins at the single-cell level.

[0006] Therefore, an object of the present invention is to provide a method for manufacturing a cell chip that can place a cell-derived component equivalent to that of a single cell on a dispensing medium and can accurately measure the amount of cell-derived component for each individual cell. Another object of the present invention is to provide a method for cell analysis using a cell chip manufactured by this method. [Means for solving the problem]

[0007] The present invention provides a method for manufacturing a cell chip in which cell-derived components are arranged on a dispensing medium, comprising: a fixation step of immersing cells in a cell fixative; a placement step of dispensing droplets of a cell suspension containing the cells and liquid that have undergone the fixation step from a discharge port provided in an inkjet type discharge unit toward the dispensing medium, thereby arranging the droplets on the dispensing medium; and a staining step of staining the cell-derived components contained in the droplets arranged on the dispensing medium in the placement step.

[0008] Furthermore, the present invention provides a cell analysis method characterized by having a detection step for detecting a signal from the stained cell-derived components in a cell chip manufactured by the above method. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a method for manufacturing a cell chip capable of arranging cell-derived components for one cell on a discharged medium and accurately measuring the amount of cell-derived components for each cell. Further, according to the present invention, it is possible to provide a cell analysis method using the cell chip manufactured by this method.

Brief Description of the Drawings

[0010] [Figure 1] It is a schematic diagram of a cell placement device according to an embodiment of the present invention. [Figure 2] It is a view of a liquid discharge head according to an embodiment of the present invention. Fig. 2(a) is a schematic perspective view of the liquid discharge head. Fig. 2(b) is a schematic cross-sectional view along line A-A of Fig. 2(a). Fig. 2(c) is an enlarged view of the central portion of Fig. 2(b). Fig. 2(d) is a schematic view of the discharge port when viewed from the lower side of Fig. 2(a). [Figure 3] It is a process diagram for manufacturing a cell chip. [Figure 4] It is a diagram schematically showing the state when a cell suspension is placed on a discharged medium. Fig. 4(a) is a diagram schematically showing the state when a cell suspension that has undergone a fixation process is placed on a discharged medium. Fig. 4(b) is a diagram schematically showing the state when a cell suspension that has not undergone a fixation process is placed on a discharged medium. [Figure 5] In Examples 2 and 3, it is an image of a cell chip when a cell suspension is placed by changing the droplet placement interval in the placement process and the cell-derived components are stained.

Embodiments for Carrying Out the Invention

[0011] Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. In the present invention, physical property values are values at room temperature (25°C) unless otherwise specified.

[0012] (Cell suspension) In the present invention, the cells include eukaryotic cells and prokaryotic cells without limitation, but are preferably mammalian cells. Mammals include, without limitation, humans, mice, rats, rabbits, hamsters, guinea pigs, monkeys, cats, dogs, and all other mammals. Also, the types of cells include, without limitation, somatic cells, germ cells, blood cells, nerve cells, cancer cells, stem cells, and the like. Further, the cells include primary cultured cells, cultured cells, established cells, cancerous cells, chimeric cells, transformed cells, and the like.

[0013] In the present invention, a cell chip is manufactured by placing a cell suspension on a discharged medium and staining it. Therefore, when using adherent cultured cells in the present invention, as a preparatory work, it is necessary to use cells detached from a culture dish using trypsin or the like. Also, when using cells contained in blood in the present invention, red blood cells are specifically lysed with a hemolytic agent, and the remaining cells are used. Further, when using cells in a tissue in the present invention, the extracellular matrix is lysed using a collagenase solution or the like, and the cells isolated from the tissue are used. If necessary, centrifuge to remove the supernatant, add a liquid for dispersing the cells, mix well, and obtain a cell suspension. Particularly, when observing a mammalian cell suspension under a phase contrast microscope, particles with a diameter of about 10 μm and substantially spherical and independent of each other can be seen.

[0014] (Cell fixing solution) In this invention, a cell fixative is used to crosslink or denature the proteins that make up cells. Therefore, the cell fixative in this invention is a liquid that crosslinks or denatures the proteins that make up cells. As the cell fixative in this invention, a cell fixative used when performing immunocytochemistry can be used. In immunocytochemistry, cells are immersed in the cell fixative before immersing them in the cell permeate. This is to prevent cell-derived components from leaking out of the cells, even if the cell permeate creates holes in the cell membrane, that is, removes the lipids that make up the cell membrane. According to this invention, when a cell suspension is ejected from an inkjet-type ejector after immersing cells in the cell fixative, it is possible to prevent cell-derived components from leaking out of the cells due to the impact during ejection. In other words, it has been found that the cell fixative is also effective in preventing the leakage of cell-derived components when ejecting a cell suspension from an inkjet-type ejector.

[0015] Specifically, the solution used to crosslink proteins contains compounds such as formaldehyde, glutaraldehyde, and dimethyl suberuiminate. By dissolving formaldehyde in water, methylene glycol, which has hydroxyl groups at both ends, can be obtained. Since these hydroxyl groups undergo a dehydration reaction with amino groups, methylene glycol plays a role in crosslinking cell-derived components that have amino groups. When cells are immersed in a solution containing compounds such as formaldehyde, glutaraldehyde, and dimethyl suberuiminate, even if the cells are subjected to force when the cell suspension is ejected from the inkjet-type ejector unit, cell-derived components are less likely to leak from the cells into the liquid that disperses them.

[0016] Specifically, the liquids that denature proteins are those containing organic solvents such as ethanol and methanol. These organic solvents are thought to penetrate the hydrogen bonds of proteins, disrupting their three-dimensional structure and reducing their solubility in water. Therefore, even when cells are subjected to force as the cell suspension is ejected from the inkjet-type ejector unit, proteins are less likely to leak from the cells into the liquid dispersing them.

[0017] The cell fixative is preferably a solution that crosslinks the proteins that make up the cells. In other words, it is preferable that the cell fixative has the effect of crosslinking cell-derived components with each other. In particular, it is especially preferable that the cell fixative contains at least one compound selected from the group consisting of formaldehyde, glutaraldehyde, and dimethyl suberuiminate. One reason why this cell fixative is preferred is that ethanol and methanol, which are solutions that denature proteins, also have the effect of removing lipids that make up the cell membrane, depending on the type of cell membrane. If cells are fixed using a solution that crosslinks proteins, the cell membrane is maintained, and even if the cells are subjected to force when the cell suspension is ejected from the inkjet ejector, there is little risk of proteins leaking from the cells into the liquid that disperses the cells. A second reason why this cell fixative is preferred is that organic solvents such as ethanol and methanol have the effect of lowering surface tension, making it difficult to stably eject the cell suspension from the inkjet ejector. Even if the cell fixative is removed by centrifugation, it is difficult to remove it completely. On the other hand, the protein crosslinking solution has little effect on lowering surface tension, so the surface tension of the cell dispersion solution does not decrease, allowing the cell suspension to be stably ejected from the inkjet-type ejector unit.

[0018] Furthermore, when cellular proteins cross-link or denature, nucleic acids, lipids, and polysaccharides surrounding the proteins are more easily retained within the cell. Therefore, even if cells are subjected to force after being immersed in a cell fixative, not only proteins but also cell-derived components including nucleic acids, lipids, and polysaccharides are less likely to leak out of the cells.

[0019] (Inkjet ejection unit) After immersion in a cell fixative, droplets of a cell suspension containing cells and a cell-dispersing liquid are ejected using an inkjet method and placed on the ejection medium. The method for ejecting droplets using an inkjet method involves a liquid ejection head and a cell placement device that drives the liquid ejection head. An example of the cell placement device and liquid ejection head is shown below.

[0020] Figure 1 shows an example of a cell placement device that drives a liquid ejection head. The cell placement device 1 includes a holder 3 on which an inkjet-type liquid ejection head 2 can be mounted, a drive motor 4, and a drive belt 5 connected to the drive motor 4 to move the holder 3 to a predetermined location. The drive motor 4 allows the liquid ejection head 2 to be moved to any location within the transportable area. The liquid ejection head 2, filled with liquid (e.g., cell suspension), is mounted on the holder 3. Then, droplets consisting of the cell suspension filled in the liquid ejection head 2 are ejected from the liquid ejection head 2 toward the medium to be ejected 10 on the stage 9. The stage 9 is provided with a drive motor 11, which allows the stage 9 to be moved to any location within the transportable area. For example, the drive motor 4 can move the liquid ejection head 2 in the left-right direction in Figure 1, and the drive motor 11 can move the stage 9 in the depth direction in Figure 1.

[0021] Furthermore, by bringing the liquid dispensing head 2 into contact with the suction recovery mechanism 6 and driving the suction motor 7, any liquid remaining in the liquid dispensing head 2 can be discharged. The liquid generated by suction and discharge is discharged outside the system through the waste liquid tube 8. The suction recovery mechanism 6 can also be used as a cap to prevent the liquid dispensing head 2 from drying out when it is not dispensing a cell suspension.

[0022] Figure 2(a) is a perspective view showing one embodiment of the liquid dispensing head 2. The liquid dispensing head 2 has a pressure element substrate 12, a space 13 that can hold the liquid to be dispensed, and an electrical connection part 14 that sends power and signals from the cell placement device 1 to the pressure element substrate 12.

[0023] Figure 2(b) is a cross-sectional view of the pressure element substrate 12 along line AA in Figure 2(a). Figure 2(c) is an enlarged view of a part of the cross-sectional view in Figure 2(b). The pressure element substrate 12 consists of a discharge port 15 for discharging liquid, a flow path 16 for supplying liquid to the discharge port 15, a discharge energy generating element 17 that generates energy for discharging liquid, and an electronic circuit element (not shown) that controls the discharge energy generating element 17. A resistance heating heater (heating element) can be used as the discharge energy generating element 17. By applying a voltage, the heater is heated, causing the liquid near the heater to foam on the order of microseconds, resulting in a thermal inkjet type liquid discharge head 2 that can produce a mechanical effect. The flow path 16 is connected to a liquid supply port 18 and a space 13 that can hold the liquid to be discharged. By applying a voltage to the discharge energy generating element 17, a cell suspension is discharged from the discharge port 15. The area around the discharge port 15 is composed of a water-repellent layer 19, which makes it difficult for the discharged cell suspension to adhere.

[0024] Figure 2(d) is a view of a liquid dispensing head 2 suitable for use in the present invention, as seen from the side of the pressure element substrate 12. The liquid dispensing head 2 may have multiple dispensing ports 15 as shown in Figure 2(d), or it may have only one dispensing port 15. Having multiple dispensing ports 15 allows for the dispensing of a larger amount of cell suspension in a shorter time. For example, the dispensing ports 15 are arranged at intervals of 300 dpi (84.6 μm) in the longitudinal direction (Y direction). In Figure 2(d), the rows of dispensing ports 15 in the longitudinal direction are arranged in two rows with a liquid supply port 18 in between, and the combined spacing of the two rows of dispensing ports 15 is 600 dpi (42.3 μm), arranged in a staggered pattern. When cell suspension is dispensed from all dispensing ports 15, the cell suspension can be distributed on a membrane or glass slide at intervals of 600 dpi. Note that the numerical values ​​for the spacing of the dispensing ports described above are just examples and are not limited thereto.

[0025] When liquid is ejected from the ejection unit of an inkjet system, droplets of 200 pl or less are generally ejected from the ejection port at a speed of 0.5 m / s or more and 30 m / s or less. When a cell suspension is ejected from the ejection unit of an inkjet system, the cell suspension that has passed through the channel 16 is ejected from the ejection port 15 by the force of the ejection energy generating element 17. The cells are subjected to force as they move from the channel 16 to the ejection port 15. When the diameter of the ejection port is 20 μm or more and 50 μm or less, and the speed of the cell suspension coming out of the ejection port is 0.5 m / s or more and 30 m / s or less, it has been confirmed that when the cell suspension is ejected from the ejection unit, holes are created in the cell membrane of cells that have not undergone a fixation process. When holes are created in the cell membrane, cell-derived components leak into the liquid that disperses the cells.

[0026] In this example, an electrothermal conversion element (heating element) is used as the discharge energy generation element, but it is also possible to configure the system to discharge liquid from the discharge port using an electromechanical conversion element such as a piezoelectric element. In this case as well, it is possible to discharge a cell suspension, similar to the case where an electrothermal conversion element is used.

[0027] (Discharged medium) The ejected medium refers to a membrane or slide glass onto which the cell suspension ejected from the ejection unit of an inkjet printer lands. The ejected medium used in this embodiment is not particularly limited, but a membrane or slide glass with a surface modified to facilitate protein adsorption, commonly used in dot blotting, Western blotting, and immunocytochemistry, is selected. Specifically, the material of the ejected medium is preferably at least one selected from the group consisting of polyvinylidene fluoride (PVDF), nitrocellulose, and glass.

[0028] Furthermore, PVDF and nitrocellulose membranes readily adsorb cell-derived components due to hydrophobic interactions. Additionally, if the glass slide is coated with aminosilane, the amino groups of the aminosilane ionically bond with the carboxyl groups of the cell-derived components, thus facilitating their adsorption. It is advisable to select the discharge medium according to the cell-derived components to be detected.

[0029] (staining) By staining cell-derived components using a staining solution that can specifically stain them, and then quantifying the degree of staining, it is possible to measure what types of cell-derived components and in what quantities were present in the cells placed on the exudation medium. For example, when staining proteins among cell-derived components, and specifically actin among proteins, phalloidin and actin antibodies can be cited as substances that can specifically recognize actin. Furthermore, if the phalloidin or actin antibody is pre-labeled with fluorescence, the amount of actin can be measured by measuring its fluorescence intensity.

[0030] For fluorescent labeling, fluorescent substances with various wavelengths can be selected. In addition to fluorescent labeling, horseradish peroxidase labeling and alkaline phosphatase labeling can also be used. By combining multiple labeling methods, it is possible to detect multiple types of cell-derived components simultaneously. Furthermore, as staining solutions, trypan blue aqueous solution, which stains proteins blue-violet, and Hoechst aqueous solution, which specifically stains the nucleus, can also be used.

[0031] (Fixed process) The fixation process involves immersing cells in a cell fixative. When the cell suspension is dispensed from the nozzle of the inkjet-type ejector unit (liquid ejection head), the cells are subjected to force within the ejector unit. In cells that have not undergone the fixation process, proteins may leak from the cells into the liquid in which they are dispersed. In contrast, in cells that have undergone the fixation process, proteins are less likely to leak from the cells into the liquid in which they are dispersed when the cell suspension is dispensed from the ejector unit. Furthermore, the proteins are cross-linked or denatured in the cell fixative, making it less likely for cellular components, including nucleic acids, lipids, and polysaccharides surrounding the proteins, to leak from the cells.

[0032] An example of the fixation process is described below. After centrifuging the cell suspension prepared in the preparation step, the supernatant is removed. Cell fixative is added to the precipitated cells and mixed well to form a cell suspension (cell suspension in cell fixative). This cell suspension is incubated for a certain period of time. For example, if a 10% formaldehyde aqueous solution is used as the cell fixative, it is incubated at room temperature for 15 minutes.

[0033] After incubation, the cell suspension may be centrifuged to remove the supernatant, which is the cell fixative. For example, one method involves incubating in a 10% formaldehyde aqueous solution at room temperature for 15 minutes, then adding phosphate buffer (PBS) and mixing well, followed by centrifuging at 90G for 10 minutes to remove the supernatant. When dispensing liquid from the ejection unit of an inkjet system, there are limitations on the physical properties of the liquid in order to ensure stable dispensing. However, if the cell fixative is removed after incubation, the range of choices for the cell fixative expands. However, removing it as completely as possible requires repeated centrifugation, which is time-consuming. Therefore, it is preferable to use a cell fixative that does not have the effect of reducing surface tension.

[0034] If the supernatant is removed, the cells can be redispersed in a commonly used cell-dispersing solution to obtain a cell suspension. Examples of solutions for redispersion include buffers such as physiological saline, PBS, and Tris, as well as cell culture media such as D-MEM (Dulbecco's Modified Eagle Medium), IMDM (Iscove's Modified Dulbecco's Medium), HBSS (Hanks' Balanced Salt Solutions), EMEM (Earle's minimal essential medium), and RPMI (Roswell Park Memorial Institute Medium) 1640.

[0035] Since we want to adsorb cell-derived components onto the discharge medium, it is preferable that the liquid used to disperse the cells in the cell suspension does not contain substances similar to cell-derived components, such as proteins. Furthermore, it is preferable that the liquid used to disperse the cells in the cell suspension contains only inorganic components, such as PBS. It is preferable that the cell-derived components contain proteins. The types of proteins contained in cell-derived components include proteins that exist inside the cell, such as actin, and proteins that exist on the cell surface, such as CD14. The phenomenon of protein leakage was particularly observed with proteins that exist inside the cell. It is thought that proteins on the cell surface, being held by the cell membrane, did not detach from the cell membrane and leak into the liquid that disperses the cells, even if the cell was subjected to force in the discharge area. On the other hand, it is thought that proteins inside the cell were more likely to leak into the liquid that disperses the cells when the cell was subjected to force and a hole was created in the cell membrane. Cells that have undergone the fixation process can prevent leakage of intracellular proteins. Proteins found inside cells include, but are not limited to, actin, glyceraldehyde-3-phosphate dehydrogenase, and extracellular signal-regulated kinase.

[0036] (Placement process) The placement process involves dispensing droplets of a cell suspension containing cells and liquid, which have undergone the fixation process, from an ejection port provided in the inkjet-type ejection unit toward the ejection medium, thereby placing the droplets onto the ejection medium. The cell suspension, which has undergone the fixation process, is filled into a space in the ejection unit (liquid ejection head) that can hold liquid, and the ejection unit is attached to the holder. Then, by driving the drive motor 4 to move the liquid ejection head 2 in the left-right direction in Figure 1, or by driving the drive motor 11 to move the stage 9 in the depth direction in Figure 1, the ejection energy generating element 17 is driven, causing droplets of the cell suspension to be ejected from the ejection port 15 and land on the ejection medium 10.

[0037] Droplet volume refers to the volume of liquid dispensed in one pass from a single nozzle 15 of the inkjet dispensing unit. The dispensing unit may be a liquid dispensing head 2 with one nozzle 15, or a liquid dispensing head 2 with multiple nozzles 15. By dispensing multiple times from the nozzle 15 into a petri dish or the like, the weight of the droplet dispensed in one pass can be calculated by dividing the change in weight of the petri dish or the liquid dispensing head by the number of passes. Furthermore, the volume of the dispensed droplet (droplet volume) can be calculated from the specific gravity of the liquid. In this case, a liquid that disperses cells (e.g., PBS) is used as the liquid to be dispensed. Preferably, the cells contained in the cell suspension dispensed as droplets in the placement step are cells that have undergone a fixation step and have had the cell fixative removed by centrifugation.

[0038] In the present invention, it is preferable to adjust the cell concentration (number concentration) in the cell suspension so that the probability of one cell or zero cells being contained in one droplet is higher than the probability of two cells being contained in one droplet discharged from the discharge port, and then fill the discharge port with the suspension. As will be described later in the staining process, it is relatively easy to distinguish whether or not there were cells in a single location on the discharge medium where a droplet is placed. On the other hand, it is difficult to distinguish whether there was one cell or two or more cells. Therefore, it is preferable to adjust the cell number concentration in the cell suspension so that the probability of two cells being contained in one droplet is 0.1 or less, and more preferably, the probability of two cells being contained in one droplet is 0.05 or less. The cell concentration should be adjusted according to the amount of droplet discharged from the discharge port and the size of the cells (volume of one cell).

[0039] The following describes the objective of placing one cell in a single location on the discharged medium. The relationship between the probability of a cell being contained in a single discharged droplet and the volume of the droplet was calculated using the following formula: where n is the cell number concentration and V is the volume of one cell. c In that case, the volume concentration N of the cell is, N=nV c ...(1) It can be expressed as follows: Let m be the maximum number of cells contained in one droplet, and V be the volume of one droplet. d So, m = V d / V c ···(2) can be expressed as follows. Assuming there are m boxes per droplet and the probability that a cell enters a single box is N, the probability P k that k cells enter a single droplet is [Number] Substituting equations (1) and (2) into equation (3), [Number] we get the following. If the number concentration n of the cells in the cell suspension is adjusted such that P2 < P1 + P0, the probability that a droplet contains 1 or fewer cells is higher than the probability that a droplet contains 2 cells. Since the diameter of the cells in the cell suspension is about 10 μm, the volume V c of a single cell can be estimated to be 1 pl. When the volume of the droplet is 22 pl and the cell concentration is 1×10 7 cells / ml, calculating the probabilities P0, P1, and P2 that 0, 1, and 2 cells enter a single droplet respectively, we get P0 = 0.80, P1 = 0.18, and P2 = 0.02. That is, in this case, the probability that 2 cells enter a single droplet is sufficiently low, and the probability that a single droplet contains 0 or 1 cell is sufficiently high. Comparing the probabilities that a single droplet contains 0 or 1 cell, among the droplets placed on the ejected medium, a liquid containing 1 cell will be placed at approximately 1 out of 5 locations.

[0040] In this invention, it is preferable to arrange droplets with spacing between them so that multiple droplets do not overlap on the dispensing medium. If droplets overlap before they dry on the dispensing medium, cells move within the connected droplets on the medium, staining the cell-derived components, and then the stained points on the dispensing medium appear randomly distributed. When imaging and quantifying cell-derived components using image processing, randomly stained points make it difficult to distinguish them from noise, and also difficult to determine which parts are cell-derived components from a single cell, making it difficult to quantify the cell-derived components. In contrast, when droplets are regularly arranged with spacing on the dispensing medium, the cells are also regularly arranged. In this case, when cell-derived components are stained and observed, an image is obtained in which stained points are regularly distributed on the dispensing medium. When there are regularly stained points, it is easier to quantify the cell-derived components using image processing.

[0041] Whether droplets overlap or not on the dispensing medium depends on the droplet's spread at the moment of impact and its spread on the medium until drying. The former is difficult to measure and is judged by whether or not the cell-derived components are regularly arranged on the dispensing medium. The latter, on the other hand, can be measured. The way droplets spread on the dispensing medium differs depending on the type of liquid used to disperse the cells, the type of dispensing medium, and the amount of droplets. It is advisable to change the spacing between droplets depending on the type of liquid used to disperse the cells, the type of dispensing medium, and the amount of droplets.

[0042] For example, if the cell dispersion solution is PBS, the discharge medium is a PVDF membrane, and the droplet volume is 22 pl, then arranging the droplets at 300 dpi intervals will result in a regular arrangement of cell-derived components. On a PVDF membrane, PBS droplets spread to a diameter of 27 μm, so arranging them at 300 dpi intervals results in a distance of 84.6 μm from the center of one droplet to the center of the next, ensuring sufficient distance between droplets. It is likely that the droplets were sufficiently far apart at the moment of impact.

[0043] To ensure that droplets do not connect with each other on the discharge medium, it is preferable to use the following method for arranging droplets with sufficient spacing between them. Specifically, in the arrangement step, it is preferable to arrange multiple droplets on the recording medium without overlapping by at least one method selected from the group consisting of discharging droplets from different discharge ports among the multiple discharge ports of the discharge unit, discharging droplets by moving the discharge unit, and discharging droplets by moving the discharge medium. An example of a method of discharging droplets by moving the discharge unit is to move the liquid discharge head 2 in the left-right direction in Figure 1 by driving the drive motor 4 to discharge droplets. An example of a method of discharging droplets by moving the discharge medium is to move the stage 9 in the depth direction in Figure 1 by driving the drive motor 11 to discharge droplets.

[0044] As shown in Figure 2(d), we will explain how to arrange droplets using a liquid dispensing head as an example, in which the dispensing ports 15 are arranged at intervals of 300 dpi (84.6 μm) in the Y direction. When droplets are dispensed from all of the dispensing ports 15 in the Y direction, the droplets are arranged on the dispensed medium at intervals of 300 dpi (84.6 μm). If it is desired to arrange the droplets on the dispensed medium at intervals of 150 dpi (169.2 μm), one can alternately select dispensing ports and non-dispensing ports from among the dispensing ports 15, apply a voltage to the dispensing energy generating element 17 corresponding to the dispensing ports, and dispense the droplets.

[0045] Furthermore, if the distance the liquid discharge head 2 is moved by the drive motor 4 between the first and second discharges at a certain discharge port 15 is 84.6 μm, then the droplets discharged from that discharge port 15 will be placed on the discharge medium at intervals of 300 dpi (84.6 μm). To place the droplets on the discharge medium at intervals of 150 dpi (169.2 μm), one can either alternately select the discharge port, such as a discharge port, a non-discharge port, and a discharge port, to double the time between the first and second discharges, or double the speed at which the liquid discharge head 2 moves to double the distance traveled.

[0046] In this invention, it is preferable to adjust the cell concentration of the cell suspension so that the probability of a droplet containing one or fewer cells is higher than the probability of a droplet containing two cells, and then fill the dispensing unit with the suspension. In this case, as described above, the droplets dispensed from the dispensing unit will consist of droplets containing cells and droplets that do not contain cells. If droplets containing cells and droplets that do not contain cells connect on the dispensing medium, the amount of liquid dispersing each cell will increase. After the water in the cell suspension placed on the dispensing medium evaporates, cell-derived components and components that were dissolved in the liquid dispersing the cells remain. If the amount of liquid dispersing the cells increases, the amount of dissolved components increases, and these components inhibit the adsorption of cell-derived components to the dispensing medium, which can result in the cell-derived components becoming more easily detached during the staining process. Therefore, in order to retain cell-derived components on the dispensing medium, it is preferable to place droplets with spacing between them so that they do not overlap on the dispensing medium.

[0047] In the present invention, the volume of a single droplet discharged from the nozzle at one time is preferably 10 pl or more and 50 pl or less. If the volume of a single droplet discharged from the nozzle at one time is less than 10 pl, the discharge of the cell suspension may be unstable, or the cell suspension may not be discharged at all due to cell clogging of the nozzle. On the other hand, if the volume of the droplet (liquid volume) is too large, proteins may easily detach from the discharge medium. In cells that have undergone the fixation process, the functional groups of the proteins are reduced, and the ability to adsorb to the discharge medium is weakened. Therefore, if the volume of the droplet is large, liquid will enter between the protein and the discharge medium, leading to more factors that inhibit the ability of the protein to adsorb to the discharge medium. Then, when the discharge medium on which the cells are placed is immersed in the staining solution in order to stain the protein, the protein will easily detach from the discharge medium. For this reason, the volume of the droplet is preferably 50 pl or less. Further details will be described later in the examples.

[0048] In this invention, it is preferable to dry the droplets placed on the dispensing medium. This allows cell-derived components to be adsorbed onto the dispensing medium. For example, if droplets of a cell suspension placed on the dispensing medium are left at room temperature for a few seconds, water will evaporate from the droplets. Once evaporation is complete, components that were dissolved in the liquid dispersing the cells and cell-derived components will remain on the dispensing medium. At this time, the cell-derived components will be adsorbed onto the dispensing medium. When approximately 20 pl of PBS was placed on a PVDF membrane and observed with an automatic micro-contact angle meter, both the diameter and height of the droplets decreased over time, and it was determined that the water had evaporated after 1.5 to 2 seconds. Alternatively, the water may be actively removed to dry the material. For example, this can be done by suctioning water from the opposite side of the PVDF membrane on which the cell suspension is placed. In this case, the cell-derived components remain on the PVDF membrane. This method allows for more efficient and reliable drying than leaving the material at room temperature.

[0049] As mentioned above, in cells that have undergone a fixation process, the functional groups of the cell-derived components are reduced, resulting in a weaker ability to adsorb to the dispensing medium. However, in this invention, since an inkjet-type dispensing unit is used, droplets of a cell suspension consisting of cells and a very small amount of liquid dispersing the cells can be placed on the dispensing medium. In this case, the amount of components dissolved in the liquid dispersing the cells is extremely small, and there are few components that inhibit the adsorption of cell-derived components to the dispensing medium.

[0050] (dying process) The staining step is a process of staining cell-derived components contained in droplets placed on the dispensing medium in the placement step. In this staining step, it is preferable to stain the cell-derived components with a staining solution. It is preferable that the staining solution contains an antibody. Furthermore, it is preferable that the cell-derived components are proteins present inside cells, and that the staining solution contains an antibody that specifically recognizes proteins. An example of a staining step using an antibody as the staining solution, with proteins among the cell-derived components as the target of staining, will be described. First, in order to prevent nonspecific adsorption of the antibody, the dispensing medium on which the cells are placed is immersed in a blocking solution. Next, the dispensing medium is immersed in a solution containing the antibody. After washing off any unwanted antibodies, the antibody-derived signal is detected. If the antibody is fluorescently labeled, when the dispensing medium is observed with a fluorescence microscope, fluorescent spots will be visible. These spots are the locations where proteins that the antibody specifically recognizes are placed. According to the present invention, since proteins do not leak from the cells into the liquid that disperses the cells during dispensing, the amount of protein per cell can be measured by measuring the brightness derived from the antibody on the dispensing medium.

[0051] In addition to measuring the brightness of each bright spot, brightness may also be measured after double staining. For example, one method involves staining the nucleus with Hoechst and the protein with a fluorescein (FITC)-labeled antibody. Cell suspension was placed on the dispensing medium under the condition that one in five droplets of cell suspension contained a cell, and the dispensing medium was immersed in a staining solution containing Hoechst and a FITC-labeled antibody. After washing, observation with a fluorescence microscope revealed that nuclei stained with Hoechst were observed at a rate of one in five locations where the cell suspension was placed. According to this invention, since proteins and nuclei do not leak out of the cells when cells are dispensed from the dispensing unit, the amount of protein per cell can be measured by accumulating the brightness of FITC at the locations where nuclei are placed in the droplets. Furthermore, if the brightness of FITC at the locations where nuclei are placed in the droplets is below the detection limit, it can be quantified that the amount of that protein per cell was zero. In other words, the amount of protein per cell can be measured more accurately, including cells that did not express that protein.

[0052] As an example of double staining, we have given the example of staining the nucleus with Hoechst, but other methods include staining proteins that are known to be expressed in a certain amount in all cells, such as actin. For example, one method is to stain actin with a FITC-labeled antibody and the protein to be measured with a rhodamine-labeled antibody. The cell suspension is placed on the dispensing medium under the condition that one in five droplets of the cell suspension contains a cell, and the dispensing medium is stained with the FITC-labeled antibody and the rhodamine-labeled antibody. According to this invention, since no protein leaks out of the cells when cells are dispensed from the dispensing part, the amount of protein per cell can be measured by quantifying the brightness of rhodamine at the droplet placement location where the FITC bright spot is located.

[0053] According to the present invention, the cells are dried during the drying process, and their shape is disrupted. When the shape is disrupted, the staining solution can more easily reach the components that were present inside the cells. In general immunohistochemistry, cells are immersed in an infiltration solution before staining, but in the present invention, it is not always necessary to immerse the cells in an infiltration solution. Furthermore, it is easier to remove excess staining solution. These effects are particularly useful when the molecular weight of the staining solution is high. Antibodies can be cited as an example of a staining solution with a high molecular weight.

[0054] (Cell chip) In this invention, as shown in Figure 3, a cell chip is manufactured by performing a fixation step, a placement step, and a staining step in that order. In the fixation step, cell-derived components are prevented from leaking out of the cells; in the placement step, cells are placed one by one at intervals; and in the staining step, cell-derived components are stained for each individual cell. By using a cell chip prepared according to this invention, the cell-derived components of each cell can be accurately measured. Measuring the cell-derived components of each cell allows for the analysis of experimental samples and the diagnosis of diseases.

[0055] Figure 4 shows the process of dispensing a cell suspension from multiple (five locations in the figure) outlets and sequentially placing droplets 20 from the left column to the right column in the figure. As shown in Figure 4(a), when cells 21 that have undergone the fixation process are dispensed from the dispensing unit in the placement process and droplets 20 are placed on the dispensing medium 10, cell-derived components 23 do not leak from the cells into the cell-dispersing liquid, and the amount of cell-derived components 23 from one cell can be contained in a single droplet 22 placed on the dispensing medium 10. Therefore, after drying, the amount of cell-derived components 23 from one cell can be retained in a very small area on the dispensing medium. For this reason, when stained in the staining process, the amount of cell-derived components can be measured for each cell with high sensitivity.

[0056] On the other hand, as shown in Figure 4(b), when cells 24 that have not undergone the fixation process are dispensed from the dispensing unit in the placement process and droplets 20 are placed on the dispensing medium 10, the cells are subjected to force within the dispensing unit, causing cell-derived components 23 to leak into the liquid that disperses the cells, and cell-derived components 23 also mix with droplets 20 that do not contain cells (nuclei 25). Therefore, droplets 22 that do not contain cells (nuclei 25) but do contain cell-derived components 23 are placed on the dispensing medium 10 after drying. In this state, even if staining is performed, it is not possible to accurately measure the amount of cell-derived components 23 for each cell.

[0057] In general immunohistochemistry, the steps are performed in the order of arrangement, fixation, and staining. In the arrangement step, live cells are seeded onto a glass slide using a pipette or similar tool and incubated until the cells adhere to the slide. Once the cells have gone through the fixation step, they die and become less likely to adhere to the slide. Therefore, it is necessary to perform the arrangement step followed by the fixation step.

[0058] On the other hand, in this invention, a cell suspension is placed using an inkjet-type ejection unit, and since the amount of liquid used to disperse each cell is very small, even cells that have undergone a fixation process (dead cells) can be adsorbed onto the ejection medium. Therefore, the fixation process and the placement process can be carried out in that order.

[0059] In conventional immunohistochemistry, cells are thinly seeded on a glass slide so that they do not overlap. However, when observing a glass slide with adhered cells, the cells are arranged irregularly, and there are areas where cells are adjacent to each other, making it difficult to distinguish where one cell ends and another begins. In contrast, in this invention, the cell suspension is placed using an inkjet-type ejector, so the cells are arranged regularly, making it easy to distinguish where one cell ends and another begins.

[0060] In typical cell chips, to separate cells individually, a plate-like structure is created with walls to separate the cells and recesses for individual cells, or a sea-island structure consisting of areas where cells adhere easily and areas where they do not. Incubation is then required until the cells adhere. Therefore, as with general immunohistochemistry, the process must be carried out in the order of arrangement, fixation, and staining.

[0061] Furthermore, some common cell chips have a wall separating cells and through-holes at the bottom of the recesses separated by the wall. The size of the recess separated by the wall is approximately the same as that of a single cell, and liquids such as cell suspensions and staining solutions are introduced and removed through the through-holes. However, in such a structure, it is necessary to preserve the shape of the cells so that the cells and cell-derived components do not flow out through the through-holes. In particular, when staining cell-derived components present inside cells, the staining solution diffuses into the cells and specifically binds to the cell-derived components. Then, excess staining solution is removed by washing. At this time, the excess staining solution diffuses inside the cells and dissolves in the washing solution. In addition, although staining solution can be introduced and removed through the through-holes, it is difficult to remove the staining solution that is trapped between the wall, the bottom of the recess, and the cells.

[0062] On the other hand, in the cell chip of the present invention, a cell suspension is ejected using an inkjet method, and droplets of the cell suspension are placed on the ejection medium. Therefore, since the amount of liquid used to disperse each cell is very small, cell-derived components can be adsorbed onto the ejection medium. In addition, the cells do not need to maintain their shape during the staining process, making it easier for cell-derived components and the staining solution to come into contact, and excess staining solution can be easily removed. Furthermore, in the present invention, the cell suspension is ejected using an inkjet method, and the cells are placed at a distance from each other on the ejection medium, so there is no need for walls to separate the cells on the ejection medium. In addition, no staining solution remains between the walls and the cells, making it easy to remove excess staining solution. For these reasons, compared to cell chips using plates with through-holes, the cell chip of the present invention can stain cell-derived components with high precision and has a higher signal-to-noise ratio.

[0063] In typical cell analysis using a cell sorter, the process is carried out in the following order: fixation, staining, and placement. Specifically, cells are fixed by immersion in a fixative, then immersed in a cell permeable solution to create pores in the cell membrane. Next, the cells are immersed in a staining solution. In particular, when staining cell-derived components present within the cells, the staining solution diffuses into the cells and specifically binds to these components. Excess staining solution is then washed away. The excess staining solution diffuses within the cells and dissolves in the washing solution. Finally, the cells flow one by one through the narrow channels in the cell sorter, and the degree to which each cell is stained is analyzed during the process. Alternatively, the cells can be placed in a 96-well plate or similar.

[0064] On the other hand, the present invention is characterized by performing the staining step after the arrangement step. Since the cells are arranged one by one in the arrangement step, the cells do not need to maintain their shape in the staining step. Cell-derived components and the staining solution come into contact more easily, and excess staining solution is easily removed. In other words, compared to cell analysis methods that are performed in the order of fixation step, staining step, and arrangement step, the present invention, which is performed in the order of fixation step, arrangement step, and staining step, can stain cell-derived components with high precision and has a higher signal-to-noise ratio.

[0065] This invention is characterized by performing the fixation step, placement step, and staining step in that order. As mentioned earlier, when the fixation step is omitted and the process is performed in the order of placement step and staining step, cell-derived components leak out during dispensing, making it impossible to accurately measure the amount of cell-derived components. Furthermore, for comparison, when the process is performed in the order of placement step, fixation step, and staining step, cell-derived components were detected even in areas where no cells were placed.

[0066] As described above, the process is carried out in the order of fixation, placement, and staining. In the placement step, a cell chip in which droplets of cell suspension are placed using an inkjet method allows for accurate and precise measurement of cell-derived components one cell at a time.

[0067] (Cell analysis method) The cell analysis method of this disclosure includes a detection step in which a signal from stained cell-derived components is detected in a cell chip manufactured by the above method. Furthermore, it is preferable to use an imaging device to detect the signal in the detection step. It is also preferable to include an integration step in which the detected signal is integrated using an imaging device in a predetermined region including the location where droplets are placed on the cell chip. For example, a cell chip having stained cell-derived components can be imaged using an imaging device, the obtained signal can be used as image data, and the cell-derived components can be measured by performing image analysis on the image data. As an image analysis method, first, components that are expressed in a certain amount in the nucleus and in all cells are image-analyzed to identify the location where the cell-derived components are placed. Then, a predetermined region including the location where the cell-derived components are placed is determined, and the signal from the staining solution that specifically stains the cell component to be detected is integrated. In general image analysis, it is necessary to determine the threshold for whether the cell-derived components are stained or not. It is also necessary to determine whether it is noise such as debris. According to the present invention, since the cell-derived components equivalent to one cell are regularly arranged in a very small predetermined area, there is no need to arbitrarily determine the threshold, noise can be judged uniformly, and the amount of cell-derived components can be quantified. In other words, cell-derived components can be measured more accurately.

[0068] When arranging a plurality of droplets on the ejected medium such that the droplets are spaced apart from each other so as not to overlap, it is preferable that the distance between the droplets is more than twice the resolution of the imaging device. According to the present invention, the cell-derived components for one cell are regularly arranged in a predetermined region with an extremely small amount. If such a regular arrangement is more than twice the resolution of the imaging device, the cell-derived components for one cell can be sufficiently detected. Conversely, widening the arrangement interval can reduce the resolution of the imaging device and shorten the imaging time. As the imaging device, a known one can be used. Examples will be shown below to explain the present invention in more detail.

Example

[0069] <Example 1, Comparative Example 1> (Effect of preventing leakage of cell-derived components by performing the fixing step) It was examined whether cell-derived components leaked out when cells that had not undergone the fixing step were ejected from an inkjet-type ejection unit, and whether leakage of cell-derived components could be prevented when the fixing step was performed. In Comparative Example 1, cells were ejected from a liquid ejection head without undergoing the fixing step, and in Example 1, cells that had undergone the fixing step were ejected from a liquid ejection head. Both in Example 1 and Comparative Example 1, staining was performed using an actin antibody that can specifically stain actin, which is a protein present inside the cells.

[0070] First, as a preparatory work, mouse macrophage RAW264.7 cells were cultured in D-MEM supplemented with 10% serum and 1% MEM Non-Essential Acid Soution until they reached 80% confluence. The cells were detached from the culture dish using trypsin. After centrifugation, the supernatant was removed, and this was used as the target cells to be analyzed in Example 1 and Comparative Example 1 (as well as Examples 2 - 13).

[0071] Next, as the fixing step, in Example 1, 1.6×10 6100 μl of 10% formaldehyde aqueous solution (Reagent A from Abcam's Cell Fixation & Permeabilization Kit), which is the cell fixative, was added to each target cell and mixed thoroughly. The mixture was incubated at room temperature for 15 minutes. 5 ml of PBS was then added and mixed thoroughly. The mixture was then centrifuged at 90 G for 10 minutes using a centrifuge (Kubota Shoji, S300TR), and the supernatant containing the cell fixative was removed. In contrast, this fixation step was not performed in Comparative Example 1. The cells from both the fixed cells (Example 1) and the unfixed cells (Comparative Example 1) were measured. 6 Add 200 μl of PBS to each and mix well, then 8 × 10 6 Cell suspensions were prepared at concentrations of cells / ml.

[0072] Next, as a placement step, the cell suspensions of Example 1 and Comparative Example 1 were respectively filled into liquid ejection heads (droplet volume 22 pl, nozzle diameter 23.4 μm) that can be mounted on an inkjet printer (Canon, G1310). In addition, a PVDF membrane (Merck Holdings, Immobilon-FL, registered trademark), which is the ejection medium, was cut into 3 x 3 cm pieces and attached to A4 size inkjet paper with tape, and set in the inkjet printer. Then, the liquid ejection heads filled with the cell suspensions of Example 1 and Comparative Example 1 were mounted in the inkjet printers, and droplets of the respective cell suspensions were ejected toward the PVDF membranes at 75 dpi intervals, arranging multiple droplets on the ejection medium so that they did not overlap. Each PVDF membrane with the cell suspension droplets was dried at room temperature for at least 1 hour and stored at 4°C.

[0073] Next, as a staining step, each PVDF membrane prepared in the above preparation step was immersed in 4 ml of blocking solution (Merck Holdings, Immobilon blok-FL, registered trademark) and shaken at room temperature for 1 hour. Subsequently, the PVDF membranes were immersed in Tris Buffer Saline with Tween 20 solution (hereinafter referred to as TBS-T) and shaken for 10 minutes, and the excess blocking solution was washed off. This washing was performed three times. Subsequently, the washed PVDF membranes were immersed in 4 ml of primary antibody solution prepared by diluting actin antibody (Abcam, ab219733) 200-fold with Can Get Signal Solution 1 (Toyobo, registered trademark) and shaken at room temperature for 2 hours. Then, the PVDF membranes immersed in the primary antibody solution were immersed in TBS-T and shaken for 10 minutes, and the excess primary antibody solution was washed off. This washing was performed three times. Next, the PVDF film after washing was treated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 680) (Life Technologies Japan, A21109, registered trademark) was immersed in 4 ml of secondary antibody solution diluted 5000-fold with immunoassay reagent (Toyobo, Can Get Signal Solution 2, registered trademark), and shaken at room temperature for 1 hour. Then, the PVDF membrane immersed in the secondary antibody solution was immersed in TBS-T and shaken for 10 minutes to wash off the excess secondary antibody solution. This washing was performed three times. Subsequently, the washed PVDF membrane was immersed in 4 ml of a solution of 1 mg / ml Hoechst aqueous solution diluted 1500-fold with PBS, and shaken at room temperature for 10 minutes. Then, the PVDF membrane immersed in the diluted Hoechst aqueous solution was immersed in PBS and shaken for 3 minutes to wash off the excess Hoechst aqueous solution. Thus, in Example 1, a cell chip was obtained in which the fixation, arrangement, and staining steps were performed in this order, while in Comparative Example 1, a cell chip was obtained in which the arrangement and staining steps were performed in this order without performing the fixation step.

[0074] Next, cell analysis was performed on the cell chips obtained in Example 1 and Comparative Example 1. Specifically, bright spots of antibodies that specifically recognized actin were observed using a near-infrared fluorescence imaging device (LI-COR, Odyssey CLx, excitation 685 nm, fluorescence 710-730 nm, resolution 21 μm, registered trademark), and bright spots of nuclei intercalated with Hoechst were observed using a fluorescence microscope (KEYENCE, BZ-X, DAPI filter), and images were taken of each.

[0075] Next, an image was formed from the signals based on the light reception of the bright spots. From the acquired image, the bright spots of antibodies that specifically recognized actin were quantified. For quantification, the image analysis software Fiji-ImageJ (manufactured by the National Institutes of Health, hereinafter referred to as Fiji) was used. First, background subtraction was performed on the image of the bright spots of the intercalated nuclei of Hoechst. Using Fiji's Subtract Background function, the Rolling Ball Radius was set to 20 pixels and the subtraction process was performed. Binarization was performed to extract the bright spots. Using Fiji's Threshold function, binarization was performed so that the top 0.9% of the pixel value distribution of the entire image was extracted. After binarization, the position information of the bright spots was obtained using Fiji's Analyze Particles function. When using the Analyze Particles function, the conditions for identifying bright spots were set so that bright spots with a size of 26 pixels or more and 280 pixels or less, and a circularity of 0.5 to 1.0 were treated as bright spots. Based on the above, we obtained the positional information of the bright spot where Hoechst and the nucleus interposed, i.e., the positional information of the cell injected by the liquid discharge head.

[0076] Next, we proceeded with image analysis of the bright spots of antibodies that specifically recognized actin. We used images of the antibody bright spots observed in the same field of view as the image used to identify the nuclear positions. As with the nuclear position information identification, we performed background light subtraction. Then, using the macro function, we calculated the sum of the antibody brightness values ​​for rectangular areas with sides of 40 μm, centered on the position of each bright spot of the nucleus.

[0077] In Example 1, a liquid dispensing head with a droplet volume of 22 pl was used, with 8 × 10 6 A cell suspension at a concentration of cells / ml was filled and dispensed onto a PVDF membrane. Calculations showed that the probability of a droplet containing one or zero cells on the PVDF membrane was higher than the probability of a droplet containing two cells. Furthermore, based on the probability of one or zero cells, it was estimated that approximately one in five droplets on the PVDF membrane would contain one cell. Indeed, observation of the intercalated nuclear nuclei revealed that nuclei were observed at approximately one in five locations. Additionally, there was one nuclear nucleus at each location where a nucleus was observed. Therefore, by calculating the cell concentration at which the probability of a droplet containing one or zero cells was higher than the probability of a droplet containing two cells, and then adjusting the cell suspension to a concentration below that level and dispensing it from the liquid dispensing head, it was possible to place one cell per droplet on the PVDF membrane.

[0078] Next, when we observed the bright spots of the antibody that specifically recognized actin, we found that the bright spots of the antibody were located in the same places as the bright spots of the nucleus. From this, we can conclude that actin did not leak from the cells into the cell dispersion liquid during the process of dispensing the cells from the liquid dispensing head. Furthermore, since the nucleus and actin of one cell were detected at a rate of one in five locations where the droplets were placed, we can conclude that the cell-derived components were sufficiently adsorbed onto the dispensing medium during drying, and that the cell-derived components were not detached from the dispensing medium during the staining process.

[0079] On the other hand, in Comparative Example 1, antibody spots were observed even in areas where there were no nuclear spots. These antibody spots coincided with the locations where the cell suspension was placed. In other words, it indicated that actin was present in droplets that did not contain cells. From this, it can be interpreted that in cells that had not undergone the fixation process, actin leaked out of the cells into the liquid dispersing solution due to the force exerted during dispensing within the liquid dispensing head.

[0080] Finally, actin was quantified using image analysis. The areas where nuclei were stained with Hoechst were identified as cell locations, and the fluorescence intensity derived from the antibody was accumulated uniformly within a 40 μm square area centered on each nucleus location. Although the resolution of the imaging device was 21 μm, the droplet spacing was 75 dpi (338.4 μm), which is more than twice the resolution, allowing for sufficient analysis of the fluorescence intensity derived from the antibody. As a result, the signal-to-noise ratio (SNR) was higher in Example 1 compared to Comparative Example 1. This is thought to be because, since there was no actin leakage, the amount of actin equivalent to one cell was present in the area where the nucleus was located. In other words, it can be said that the amount of actin equivalent to one cell could be accurately quantified after the fixation process.

[0081] <Examples 2-3> (Droplet spacing and ease of component analysis for each individual cell) In Examples 2 and 3, the fixation and placement steps were carried out in the same manner as in Example 1, except that the droplet spacing was changed. In the staining step, proteins were stained on the PVDF membrane. Specifically, in Example 1, the droplet spacing was 75 dpi, in Example 2 it was 300 dpi, and in Example 3 it was 600 dpi, and the droplets were dispensed onto the PVDF membrane. In addition, an aqueous trypan blue solution was used as the solution to stain the proteins in the staining step. Specifically, in the staining step, the PVDF membrane was immersed in the aqueous trypan blue solution for 1 minute, then the excess trypan blue was washed off with PBS, and the PVDF membrane was observed under a microscope.

[0082] Figure 5 shows the results of microscopic observation. In Example 2, which is set to 300 dpi, the dots stained with trypan blue are arranged regularly. On the other hand, in Example 3, which is set to 600 dpi, trypan blue stained dots are visible, but they are arranged randomly. Since the trypan blue stained dots originate from a single cell, it is possible to analyze each cell individually in both Example 2 and Example 3. However, in Example 3, the trypan blue stained dots are arranged randomly, so when performing image analysis, it is necessary to determine where to set the threshold in order to distinguish each dot by looking at the image. In Example 2, this determination is not necessary, and it is possible to quantify the cell-derived components from a single cell more accurately.

[0083] In Examples 2 and 3, similar to Example 1, the cell dispersion solution was PBS, the discharge medium was a PVDF membrane, and the amount of liquid discharged from the liquid discharge head was 22 pl, resulting in droplets with a diameter of 27 μm on the PVDF membrane. Therefore, when droplets were placed at 300 dpi intervals, the distance from the center of one droplet to the center of the next was 84.6 μm, and when droplets were placed at 600 dpi intervals, the distance from the center of one droplet to the center of the next was 42.3 μm. The random distribution of cell-derived components when droplets were placed at 600 dpi intervals is thought to be due to the droplets overlapping at the moment of impact and the movement of cells within the droplets.

[0084] <Examples 4-8> (Droplet volume and dispensing stability) In Examples 4-8, as shown in Table 1 below, we confirmed whether the cell suspension could be dispensed stably by dispensing the cell suspension from dispensing nozzles with different liquid volumes (droplet volumes) per dispensing.

[0085] The cells, which had undergone the fixation process in the same manner as in Example 1, were redispersed in D-MEM, resulting in 2 × 10⁻⁶ cells. 6Cell suspensions with a concentration of 10 cells / ml were prepared. The prepared cell suspensions were loaded into liquid ejection heads with different droplet volumes that could be mounted on a Canon inkjet printer G1310. A culture dish was placed under the liquid ejection head, and the liquid ejected into the culture dish was mixed with trypan blue aqueous solution in a 1:1 ratio to stain the cells. The cell concentration was then measured using a cell counter (Thermo Fisher Scientific, Countess II FL, registered trademark). When cells clogged the liquid ejection head, the cell concentration increased to 2 × 10⁶. 6 Since the cell concentration was lower than cells / ml, we determined whether stable dispensing was possible by measuring how much the cell concentration had decreased.

[0086] The results are summarized in Table 1. The droplet volume from each liquid dispensing head is as shown in the table. In Examples 5-8, the cell concentration in the cell suspension remained almost unchanged before and after dispensing, and stable dispensing was determined. In Example 4, the cell concentration in the cell suspension was 80% or less before and after dispensing, and it was determined to be somewhat unstable.

[0087] [Table 1] Criterion A: There is almost no change in cell concentration in the cell suspension before and after dispensing. B: Change in cell concentration in cell suspension before and after dispensing is 80% or less.

[0088] For example, if the cell suspension is 10 6 There are several, and from those, 10 4 When analyzing individual cells one by one, it is not necessary to place all cells in the cell suspension onto a PVDF membrane. However, it is preferable for the cells to be stably dispensed from the liquid dispensing head. There is an error of about ±5% in the dispensing volume. Including this error, a droplet volume of 10 pl or more is preferable.

[0089] <Examples 9-13> (Droplet volume and detachment of cell-derived components) In Examples 9-13, as shown in Table 2 below, we confirmed whether proteins could be stained on the PVDF membrane by dispensing cell suspensions from dispensing nozzles with different droplet volumes toward the PVDF membrane and comparing the results.

[0090] In the same manner as in Example 1, the cells that have undergone the fixation process are redispersed in PBS, and 8 × 10 6 Cell suspensions with a concentration of cells / ml were prepared. The prepared cell suspensions were loaded into liquid ejection heads with different droplet volumes, which are compatible with a Canon inkjet printer G1310. Then, as in Example 1, each was ejected onto a PVDF membrane at 75 dpi, and the PVDF membranes on which the cell suspension droplets were placed were dried for more than one hour and stored at 4°C to complete the placement process.

[0091] In the staining process, a trypan blue aqueous solution was used to stain proteins blue-violet. Trypan blue was used instead of antibodies to allow for a simple observation of whether the proteins would detach from the PVDF membrane during the immersion in the staining solution. Each PVDF membrane was immersed in the trypan blue aqueous solution for 1 minute. Afterward, the excess trypan blue was washed off with PBS, and the PVDF membrane was observed under a microscope.

[0092] The results are summarized in Table 2. Microscopic observation revealed bluish-purple dots. These dots were approximately the size of a single cell. In Example 13, bluish-purple dots were observed, but some bluish-purple dots were also seen in areas where no droplets were placed. This can be interpreted as cells detaching from the PVDF membrane and adhering to other locations during the immersion process in the trypan blue aqueous solution or PBS.

[0093] When the water in the solution that disperses (suspends) the cells evaporates, the proteins that make up the cells adsorb to the PVDF membrane. If the droplet volume is large, the amount of solution dispersing each cell increases, and so does the amount of precipitate that remains after the water evaporates. These precipitates inhibit the adsorption of proteins to the PVDF membrane, so it is thought that the proteins are more likely to detach from the PVDF membrane during the staining process. In Example 13, not all cell-derived components (proteins) detached from the PVDF membrane, but some detachment was observed. Since there is an error of about ±5% in the droplet volume during dispensing, it is preferable that the droplet volume be 50 pl or less, including this error.

[0094] [Table 2] Criteria A: No peeling B: Some peeling present

[0095] <Reference example 1> (penetrant) In general immunohistochemistry, cells are immersed in a fixative before staining. In particular, when using a fixative that cross-links proteins, the cells are sometimes immersed in a lipid-removing infiltration solution after immersion in the fixative to create pores in the cell membrane and facilitate antibody penetration. In this invention, we verified whether it is necessary to immerse the cells in an infiltration solution after immersion in the fixative solution before the staining process using the following method.

[0096] Cells were fixed using formaldehyde, which has a protein cross-linking effect, in the same manner as in Example 1. The cells that had undergone the fixation process were redispersed in PBS and 8 × 10⁻⁶ cells were collected. 6 Cell suspensions with a concentration of cells / ml were prepared. The prepared cell suspensions were loaded into liquid ejection heads with different droplet volumes, which are compatible with a Canon inkjet printer G1310. Then, as in Example 1, each was ejected onto a PVDF membrane at 75 dpi, and the PVDF membranes on which the cell suspension droplets were placed were dried for more than one hour and stored at 4°C to complete the placement process.

[0097] The PVDF membrane, after the placement process, was immersed in 5 ml of PBS to allow it to absorb water. Next, 100 μl of permeation solution (Reagent B from Abcam's Cell Fixation & Permeabilization Kit) was spread over the PVDF membrane and incubated at room temperature for 15 minutes. After that, it was washed three times with 5 ml of PBS. Then, as in Example 1, the PVDF membrane was stained, photographed, and actin was quantified by image analysis.

[0098] The amount of actin per cell did not differ significantly between Example 1 and Reference Example 1. In this invention, the step of immersion in the permeate before staining was not necessary. Compared to general immunohistochemistry, this invention includes a placement step, which replaces the immersion in the permeate step. It is thought that the placement step disrupts the shape of the cells, making it easier for the antibody to approach the protein. Therefore, the fixation solution in this invention only needs to have the effect of crosslinking proteins, and the effect of removing lipids that constitute the cell membrane is not essential. As an effect of this invention, it can be said that if cells are placed using an inkjet printer, the step of immersing cells in the permeate becomes unnecessary.

[0099] The present invention includes the following configuration. (Composition 1) A method for manufacturing a cell chip in which cell-derived components are arranged on a dispensing medium, The fixation process involves immersing cells in a cell fixative solution, A placement step involves discharging droplets of the cell suspension containing the cells and liquid, which have undergone the fixation step, from an ejection port provided in an inkjet ejection unit toward the ejection medium, thereby arranging the droplets on the ejection medium. A method characterized by comprising a staining step of staining cell-derived components contained in the droplets placed on the discharge medium in the placement step. (Configuration 2) The method of configuration 1, wherein in the arrangement step, the droplets placed on the discharge medium are dried. (Composition 3) In the placement step, the method according to Configuration 1 or 2, wherein a plurality of the droplets are placed on the ejected medium so as not to overlap each other. (Configuration 4) In the placement step, at least one method selected from the group consisting of a method of ejecting the droplets from different ejection ports among a plurality of ejection ports included in the ejection unit, a method of ejecting the droplets by moving the ejection unit, and a method of ejecting the droplets by moving the ejected medium, and placing a plurality of the droplets on the ejected medium so as not to overlap each other, the method according to Configuration 3. (Configuration 5) In the placement step, the method according to any one of Configurations 1 to 4, wherein the number concentration of the cells in the cell suspension is adjusted so that the probability that one droplet ejected from the ejection port contains one or zero cells is higher than the probability that one droplet ejected from the ejection port contains two cells. (Configuration 6) In the placement step, when the number concentration of the cells in the cell suspension is n, the volume of one cell is V c , and the volume of one droplet is V d , the method according to Configuration 5, wherein the number concentration n of the cells in the cell suspension is adjusted so that P2 < P1 + P0, where P is the probability that one droplet contains k cells, according to the formula for calculating P k for calculating

Equation

[0100] 1. Cell placement device 2. Liquid dispensing head 3.Holding body 4. Drive motor 5. Drive belt 6.Suction recovery mechanism 7. Suction motor 8. Waste liquid tube 9. Stage 10.Medium to be ejected 11. Drive motor 12. Pressure element substrate 13. Space to hold liquid 14. Electrical connection 15.Discharge port 16. Flow Channel 17. Discharge energy generating element 18.Liquid supply port 19. Water-repellent layer 20.Droplets 21. Cells after the fixation process 22.Droplets 23. Cell-derived components 24. Cells that have not undergone the fixation process 25.Nuclear

Claims

1. A method for manufacturing a cell chip in which cell-derived components are arranged on a dispensing medium, The fixation process involves immersing cells in a cell fixative solution, A placement step involves discharging droplets of the cell suspension containing the cells and liquid, which have undergone the fixation step, from an ejection port provided in an inkjet ejection unit toward the ejection medium, thereby arranging the droplets on the ejection medium. A method characterized by comprising a staining step of staining cell-derived components contained in the droplets placed on the discharge medium in the placement step.

2. The method according to claim 1, wherein in the arrangement step, the droplets arranged on the discharge medium are dried.

3. The method according to claim 1, wherein in the arrangement step, a plurality of the liquid droplets are arranged on the discharge medium so that they do not overlap with each other.

4. The method according to claim 3, wherein in the arrangement step, the plurality of droplets are arranged on the medium to be discharged so as not to overlap each other by at least one method selected from the group consisting of a method of discharging droplets from different discharge ports among a plurality of discharge ports of the discharge unit, a method of discharging droplets by moving the discharge unit, and a method of discharging droplets by moving the medium to be discharged.

5. The method according to claim 1, wherein in the arrangement step, the number concentration of cells in the cell suspension is adjusted such that the probability of one or zero cells being contained in one droplet discharged from the discharge port is higher than the probability of two cells being contained in one droplet discharged from the discharge port.

6. In the above arrangement step, the number concentration of the cells in the cell suspension is n, and the volume of one cell is V. ｃ , and the volume of one droplet is V ｄ In this case, the probability P of a single droplet containing k cells is... ｋ Formula for calculating [Math 1] From, P ２ <P １ +P ０ The method according to claim 5, wherein the number concentration n of the cells in the cell suspension is adjusted to such a result.

7. The method according to claim 5, wherein in the arrangement step, the number concentration of cells in the cell suspension is adjusted so that the probability of two cells being contained in one droplet discharged from the discharge port is 0.1 or less.

8. The method according to claim 5, wherein in the arrangement step, the number concentration of cells in the cell suspension is adjusted so that the probability of two cells being contained in one droplet discharged from the discharge port is 0.05 or less.

9. The method according to claim 1, wherein the cell-derived component includes a protein.

10. The method according to claim 9, wherein the protein is a protein present inside a cell.

11. The method according to claim 1, wherein in the staining step, the cell-derived component is stained with a staining solution.

12. The method according to claim 11, wherein the staining solution contains an antibody.

13. The method according to claim 11, wherein the cell-derived component is a protein present inside a cell, and the staining solution contains an antibody that specifically recognizes the protein.

14. The method according to claim 1, wherein the volume of the droplet discharged from the discharge unit is 10 pl or more and 50 pl or less.

15. The method according to claim 1, wherein the cells contained in the cell suspension discharged as droplets in the arrangement step are obtained by centrifuging the cells that have undergone the fixation step and removing the cell fixative.

16. The method according to claim 1, wherein the material of the discharged medium is at least one selected from the group consisting of polyvinylidene fluoride, nitrocellulose, and glass.

17. The method according to claim 1, wherein the cell fixative comprises at least one compound selected from the group consisting of formaldehyde, glutaraldehyde, dimethyl sveriminoate, methanol, and ethanol.

18. The method according to claim 1, wherein the cell fixative has the effect of crosslinking the cell-derived components.

19. The method according to claim 1, wherein the cell fixative comprises at least one compound selected from the group consisting of formaldehyde, glutaraldehyde, and dimethyl sveluiminate.

20. A cell analysis method characterized by comprising a detection step of detecting a signal from the stained cell-derived component in a cell chip manufactured by the method according to any one of claims 1 to 19.

21. The cell analysis method according to claim 20, wherein the signal is detected using an imaging device in the detection step.

22. The cell analysis method according to claim 21, further comprising an integration step of integrating the signal detected using the imaging device in a predetermined region including the location where the droplet is placed on the cell chip.

23. The cell analysis method according to claim 21, wherein in the cell chip, a plurality of the droplets are arranged at intervals of more than twice the resolution of the imaging device.

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