JP2019216708A - Cell-containing container, method for producing cell-containing container, and cell chip - Google Patents

Abstract

To provide cell-containing containers that enable efficient evaluation tests using cells in one container.SOLUTION: A cell-containing container 1 has at least two recesses 3, the recess contains cells, the types of cells are at least two for a container 2, and the shortest distance between the centers of two nearest neighboring recesses is 9.0 mm or less. In a preferable embodiment, the recess contains liquid, the total amount of the liquid with respect to the recess is 10.0 μL or less, and in more preferable embodiment, the filling accuracy of the number of cells contained in the recess is 30% or less.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a cell-containing container, a method for producing the cell-containing container, and a cell chip.

  In recent years, there has been an increasing demand for instruments for performing toxicity or drug efficacy evaluation tests using cells in vitro.

  One of the reasons is that the number of experimental animals is reduced by promoting 3Rs (“Replacement”, “Reduction”, and “Refinement”) in animal experiments, and the method using experimental animals is further reduced. There is a need for alternative methods.

  In addition to the above-mentioned reasons for the promotion of 3Rs in experimental animals, experiments using living cells in vitro have many advantages, such as a reduction in costs for experimental animals and a reduction in test time. Is mentioned.

In order to conduct experiments using living cells in vitro, for example, in order to reproduce the interaction between cells in vitro, a cell culture container in which microwells for accommodating cells are uniformly arranged has been proposed. (For example, see Patent Document 1).
In addition, a plate in which the shape of the well for accommodating the granular material is designed in accordance with the size of the object to be accommodated in order to accommodate only one granular material in each well while securing a necessary liquid amount in the well of the plate. A container has been proposed (for example, see Patent Document 2).

  An object of the present invention is to provide a cell-containing container that can efficiently perform an evaluation test using cells in one container.

  The cell-containing container of the present invention as a means for solving the above problem has at least two concave portions, the concave portions include cells, and the type of the cells is at least two types with respect to the container. Wherein the shortest distance between the centers of two adjacent concave portions is 9.0 mm or less.

  ADVANTAGE OF THE INVENTION According to this invention, the cell containing container which can perform an evaluation test using a cell efficiently in one container can be provided.

FIG. 1 is a graph showing the relationship between the average value x of the number of cells and the coefficient of variation CV. FIG. 2 is a perspective view showing an example of the cell-containing container of the present invention. FIG. 3 is a perspective view showing another example of the inspection device of the present invention. FIG. 4 is a side view of FIG. FIG. 5A is a flowchart showing an example of the method for producing a cell-containing container of the present invention. FIG. 5B is a flowchart showing another example of the method for producing a cell-containing container of the present invention. FIG. 5C is a flowchart showing another example of the method for producing a cell-containing container of the present invention. FIG. 6A is a schematic diagram illustrating an example of a discharge head of the electromagnetic valve type. FIG. 6B is a schematic diagram illustrating an example of a piezo-type ejection head. FIG. 6C is a schematic view of a modified example of the piezo-type ejection head in FIG. 6B. FIG. 7A is a schematic diagram illustrating an example of a voltage applied to the piezoelectric element. FIG. 7B is a schematic diagram illustrating another example of the voltage applied to the piezoelectric element. FIG. 8A is a schematic diagram illustrating an example of a state of a droplet. FIG. 8B is a schematic diagram illustrating an example of a state of a droplet. FIG. 8C is a schematic diagram illustrating an example of a state of a droplet. FIG. 9 is a schematic view showing an example of a dispensing device for sequentially landing droplets in a concave portion. FIG. 10 is a schematic diagram illustrating an example of a droplet forming apparatus. FIG. 11 is a diagram illustrating a hardware block of a control unit of the droplet forming apparatus of FIG. FIG. 12 is a diagram illustrating functional blocks of control means of the droplet forming apparatus of FIG. FIG. 13 is a flowchart illustrating an example of the operation of the droplet forming apparatus. FIG. 14 is a schematic diagram illustrating a modified example of the droplet forming apparatus. FIG. 15 is a schematic diagram illustrating another modified example of the droplet forming apparatus. FIG. 16A is a diagram illustrating a case where a flying droplet includes two fluorescent particles. FIG. 16B is a diagram exemplifying a case where a flying droplet contains two fluorescent particles. FIG. 17 is a diagram illustrating a relationship between the luminance value Li when particles do not overlap with each other and the actually measured luminance value Le. FIG. 18 is a schematic diagram illustrating another modified example of the droplet forming apparatus. FIG. 19 is a schematic diagram illustrating another example of the droplet forming apparatus. FIG. 20 is a schematic diagram illustrating an example of a method of counting cells that have passed through a microchannel. FIG. 21 is a schematic diagram illustrating an example of a method for acquiring an image near the nozzle portion of the ejection head. FIG. 22 is a graph showing the relationship between the probability P (> 2) and the average cell number. FIG. 23 is a graph showing the cell viability in Example 1. FIG. 24 is a graph showing the cell membrane damage rate in Example 1. FIG. 25 is a graph showing the secretion amount of the inflammatory substance in Example 1. FIG. 26A is a diagram illustrating an example of dispensing by a dispenser. FIG. 26B is a diagram illustrating an example of dispensing by a dispenser. FIG. 26C is a diagram illustrating an example of dispensing by a dispenser. FIG. 26D is a diagram illustrating an example of dispensing by a dispenser.

(Cell-containing container)
The cell-containing container of the present invention has at least two recesses, the recesses include cells, and the type of the cells is at least two types with respect to the container. Has a minimum distance of 9.0 mm or less, and further has other members as necessary.

The present inventors have studied a cell-containing container that can efficiently perform an evaluation test using cells in one container, and have obtained the following knowledge.
For example, in a conventional cell culture container and a plate-shaped container, when a user performs a test, it is necessary to fill the container with cells, and in that case, a predetermined well is filled with a desired number and type of cells. There is a problem that is difficult.
The present inventors have considered that a container having at least two types of cells and having a minimum distance of 9.0 mm or less between the centers of two nearest and adjacent concave portions, for a container having at least two concave portions, That is, it has been found that an evaluation test using cells can be efficiently performed in one container by using a container having a large number of cells and a large number of cells per area.

<Recess>
The concave portion is a section provided in the container, and includes a cell to be described later, and is a portion for disposing other members.
The number of concave portions is at least two, preferably 5 or more, and more preferably 50 or more.

In the cell-containing container of the present invention, the shortest distance between the centers of two nearest and adjacent recesses is 9.0 mm or less, preferably 5.0 mm or less, more preferably 4.5 mm or less, and more preferably 2.25 mm. The following are more preferred. Note that in this specification, the shortest distance between the centers of two nearest neighboring recesses may be referred to as the shortest recess pitch or the shortest pitch.
Here, “closest and adjacent” means that the distance between the center of one concave portion and the center of the other concave portion is the shortest when the distance between the centers of the concave portion and the adjacent concave portion is compared. The center here means the center of gravity of the shape of the opening of the recess.
Further, the shortest distance means the shortest of the lines connecting the two points, that is, the length of the line connecting the two points.

For example, a multi-well plate, a micro-well slide (hereinafter, referred to as a chip) May also be referred to).
Examples of the multi-well plate include a 96-well plate, a 384-well plate, and a 1,536-well plate.
Microwell slides include, for example, 192 well, 768 well, or 3,456 well microwell slides. A microwell slide can also be manufactured by attaching a sheet provided with holes in dimethylpolysiloxane (PDMS) to a substrate having high light transmittance and low autofluorescence.
The number of concave portions is not particularly limited as long as it is at least two, and can be appropriately selected depending on the purpose. For example, 192 or more and 3,456 or less are preferable. When the number of recesses is 192 or more and 3,456 or less, the number of samples that can be handled using one cell-containing container can be increased. Can be performed efficiently.

The shape, volume, material, color, and the like of the concave portion are not particularly limited, and can be appropriately selected according to the purpose.
The shape of the recess is not particularly limited as long as cells to be described later can be arranged, and can be appropriately selected depending on the purpose. For example, a recess such as a flat bottom, a round bottom, a U bottom, and a V bottom, and a substrate The upper section and the like can be mentioned. Further, the shape of the concave portion used differs depending on the specifications of the inspection apparatus, and a round bottom is generally used in PCR, and a flat bottom is generally used in inspection by optical observation using a microscope or the like.
The volume of the concave portion is not particularly limited and can be appropriately selected depending on the purpose. For example, considering the amount of a reagent used in a general evaluation method, the volume is preferably 0.1 μL or more and 1,000 μL or less. It is preferable that the volume of the solution is small in the evaluation with a rare reagent, so that the volume is more preferably 0.1 μL or more and 10 μL or less.
Examples of the color of the recess include transparent, translucent, colored, and completely shaded. In the case of inspection of an optical system, it is not preferable that interference between adjacent concave portions occurs. Therefore, a container having a transparent bottom surface and colored side surfaces is more preferable.

  The material of the concave portion is not particularly limited as long as it has a low affinity for cells described later, that is, if it has cell non-adhesiveness, and can be appropriately selected depending on the purpose. Materials. Examples of the cell non-adhesive material include the organic materials and inorganic materials described below. These may be used alone or in combination of two or more. Among these, those that easily adsorb the cell adhesive material are preferable. If the material of the container easily adsorbs the cell-adhesive material, the cell-adhesive material can be stably adhered when the cell-adhesive material is discharged into the container at the bottom of the recess.

-Cell non-adhesive material-
The cell non-adhesiveness means that the adhesiveness to at least target cells is lower than that of the cell adhesive material used.
The cell non-adhesion measurement method is not particularly limited and can be appropriately selected depending on the purpose. For example, a needle-shaped AFM probe is inserted into a cell cultured on a container and pulled up. And a method of measuring the load applied to the probe by AFM by peeling the cells from the container, and measuring and evaluating the adhesiveness of the cells to the container. As another method, for example, a method in which pure water or the like is flowed through cells cultured on a container, and the rate of adhesion and detachment of the cells to the container before and after the method is evaluated.

The cell non-adhesive material is not particularly limited and may be appropriately selected depending on the purpose. However, a water-repellent material is preferable. When the cell non-adhesive material is a water-repellent material, it is advantageous in that cells are less likely to adhere.
The cell non-adhesive material is not particularly limited and may be appropriately selected depending on the intended purpose. However, a silicon-containing material is preferable.
The silicon-containing material is not particularly limited and may be appropriately selected depending on the purpose. However, polydimethylsiloxane (PDMS) is preferable from the viewpoint of biocompatibility.

The organic material is not particularly limited and may be appropriately selected depending on the purpose. For example, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetylcellulose), polyimide (PI) , Nylon (Ny), low density polyethylene (LDPE), medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, urethane acrylate and other acrylic materials, cellulose, Silicone-based materials such as polydimethylsiloxane (PDMS) are exemplified.
The inorganic material is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include glass and ceramics.

-Cell adhesive material-
A cell adhesive material having higher cell adhesiveness than the container is further provided on the bottom surface of the concave portion.
The cell adhesive material is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include proteins selected from extracellular matrices.
Examples of proteins selected from the extracellular matrix include fibronectin, laminin, tenascin, vitronectin, RGD (arginine-glycine-aspartic acid) sequence-containing peptide, YIGSR (tyrosine-isoleucine-glycine-serine-arginine) sequence-containing peptide, collagen , Atelocollagen, gelatin and the like. In addition, examples of the protein selected from the extracellular matrix include a mixture of the above proteins, Matrigel, PuraMatrix, and fibrin. Among these, iMatrix 511 (manufactured by Nippi Co., Ltd.) which imitates collagen or a partial structure of laminin used in stem cell culture or the like is preferable. Furthermore, examples of the protein selected from the extracellular matrix include a basic polymer such as polylysine and a basic compound such as aminopropyltriethoxysilane.
As a method of providing the cell adhesive material in the concave portion, there is a method of applying a solution containing the cell adhesive material to the concave portion. In this case, the solution may contain biocompatible particles.

The biocompatible particles are not particularly limited as long as they have an affinity for a living body such as a cell, and can be appropriately selected. Examples thereof include gelatin particles and collagen particles. These may be used alone or in combination of two or more.
When the biocompatible particles are gelatin particles, gelatin, which is a raw material of the gelatin particles, is not particularly limited and can be appropriately selected depending on the purpose. Manufactured by a company).
The particulate gelatin particles can improve the adhesion of the cells to the container, and can be disposed at a desired position without being decomposed into cells for a long period of time as compared with non-particle gelatin. it can. Therefore, gelatin particles have the advantage of improving cell adhesion and being used as a cell nutrient for a long period of time.

  Further, the biocompatible particles are preferably cross-linked by a cross-linking agent in the structure.

  The crosslinking agent is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include aldehydes such as glutaraldehyde and formaldehyde; ethylene propylene diglycidyl ether, glycerol polyglycidyl ether, diglycerol polyglycidyl ether, and sorbitol poly. Glycidyl ethers such as glycidyl ether and ethylene glycol diglycidyl ether; hexamethylene diisocyanate, α-tolidine isocyanate, tolylene diisocyanate, naphthylene-1,5-diisocyanate, 4,4-diphenylmethane diisocyanate, triphenylmethane-4,4,4 -Isocyanates such as triisocyanate; calcium gluconate, (1S, 2R, 6S) -2-hydroxy-9- (hydroxymethyl) -3- Kisabishikuro [4.3.0] non-4,8-diene-5-carboxylate (genipin), a combination of a polyphenol and an oxidizing agent such as horseradish peroxidase, and the like compounds having a succinimide group. These may be used alone or in combination of two or more. Of these, aldehydes are preferred, and glutaraldehyde is more preferred.

The content of the crosslinking agent is preferably 1% by mass or more and 20% by mass or less, more preferably 2% by mass or more and 10% by mass or less based on the total amount of the raw material of the biocompatible particles.
The content of the biocompatible particles is preferably from 0.5% by mass to 10% by mass, more preferably from 1% by mass to 5% by mass, based on the total amount of the solution containing the cell adhesive material.

-Preparation example of sample solution containing cell adhesive material-
Biocompatible particles are dispersed at a concentration of 0.5% by mass in pure water obtained using a pure water production apparatus (trade name: GSH-2000, manufactured by ADVANTEC). The sample liquid can be prepared by stirring the 20-mm rotor at 200 rpm for about one day using a stirrer with a liquid volume for measurement of 5 mL.

-Measurement conditions-
・ Solvent: water (refractive index: 1.3314, viscosity at 25 ° C .: 0.884 mPa · s (cP), optimal light amount adjustment is appropriately set by an ND filter)
・ Measurement probe: thickening probe ・ Measurement routine: measurement: 25 ° C. for 180 seconds → measurement: 25 ° C. for 600 seconds (when the main body side is changed to 35 ° C., the liquid temperature gradually changes from 25 ° C. to 35 ° C. Particles in the meantime) Monitor diameter change) → Measurement: 180 seconds at 35 ° C

  The recess preferably further contains a liquid.

−Liquid−
The liquid is not particularly limited as long as it can be used as a dispersion medium for dispersing cells when used in the production of the cell-containing container of the present invention described below, and can be appropriately selected depending on the purpose. And phosphate buffered saline.
Further, in addition to the liquid, a cell culture medium (sometimes referred to as a cell culture solution), a wetting agent, a dispersant, a pH adjuster, and the like may be added.

  The volume of the liquid is not particularly limited and can be appropriately selected depending on the purpose. For example, it is preferable that the total amount of the liquid with respect to the concave portion constituting the cell-containing container is 10.0 μL or less. When the total amount of the liquid with respect to the recesses constituting the cell-containing container is 10.0 μL or less, the amount of reagents (cells, drugs, and the like) used in one test can be reduced.

  The method for measuring the volume of the liquid is not particularly limited and can be appropriately selected depending on the purpose.For example, the weight is measured by a microbalance before and after the liquid is applied, and the liquid is measured by ultrasonic scanning of the liquid surface after the liquid is applied. Surface detection (for example, device name: LABCYTE (registered trademark), manufactured by Kiko Tech Co., Ltd.) and the like.

-Cells-
Cells are not particularly limited and can be appropriately selected depending on the purpose.

In the cell-containing container of the present invention, there are at least two types of cells for the container constituting the cell-containing container. In other words, the type of cells arranged in the cell-containing container means that there are at least two types of cells contained in the container, that is, the cell-containing container.
For example, when disposing two types of cells (cell A and cell B) in a container having 96 concave portions, 48 positions may be disposed as cell A, and the remaining 48 positions may be disposed as cell B. The cells B may be arranged in all the 96 concave portions. Thus, how the cells are arranged in the concave portion of the container can be appropriately selected.
Here, the type of cells means not only different types of cells such as, for example, neurons and muscle cells, but also, for example, neurons a obtained from one mouse A and cells obtained from another mouse B. Like the obtained nerve cell b, cells having the same type of cell but obtained from different sources are also regarded as different cell types. Also, in the case where cells differentiated using pluripotent stem cells are used, if the donor from which the pluripotent stem cells have been obtained is different, it is regarded as a different cell type.

  The cells are not particularly limited and can be appropriately selected depending on the purpose. For example, the cells can be used for all cells regardless of eukaryotic cells, prokaryotic cells, multicellular biological cells, and unicellular biological cells. . In addition, as a cell, a living cell is preferable.

  Eukaryotic cells are not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include animal cells, insect cells, plant cells, fungi, algae, and protozoa. These may be used alone or in combination of two or more. Among these, animal cells and fungi are preferred.

  The adhesive cells may be primary cells directly collected from tissues or organs, or may be primary cells directly collected from tissues or organs that have been passaged for several generations, and can be appropriately selected depending on the purpose. For example, differentiated cells, undifferentiated cells and the like can be mentioned.

  The differentiated cells are not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include hepatocytes, which are parenchymal cells of the liver; stellate cells; Kupffer cells; vascular endothelial cells; Endothelial cells such as cells; fibroblasts; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells such as epidermal keratinocytes; tracheal epithelial cells; gastrointestinal epithelial cells; cervical epithelial cells; Epithelial cells such as breast cells; pericytes; muscle cells such as smooth muscle cells and cardiomyocytes; renal cells; pancreatic Langerhans islet cells; nerve cells such as peripheral nerve cells, optic nerve cells; chondrocytes; bone cells; And differentiated cells derived from ES cells.

  The undifferentiated cells are not particularly limited and can be appropriately selected depending on the purpose.Examples include undifferentiated cells, such as embryonic stem cells, pluripotent stem cells such as mesenchymal stem cells having pluripotency, Examples include unipotent stem cells such as vascular endothelial progenitor cells having unipotency, induced pluripotent stem (iPS) cells, Embronic Stem (ES) cells, and stem cells obtained from the human body.

The fungus is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include mold and yeast. These may be used alone or in combination of two or more. Among them, yeast is preferable because it can regulate the cell cycle and can use haploid.
The cell cycle means a process in which when cells increase, cell division occurs, and the cells (daughter cells) generated by cell division become cells that undergo cell division again (mother cells) to produce new daughter cells.

The yeast is not particularly limited and may be appropriately selected depending on the intended purpose. For example, yeast that is synchronously cultured in the G0 / G1 phase and fixed in the G1 phase is preferable.
Further, as the yeast, for example, a Bar-1 deficient yeast having an increased sensitivity of a pheromone (sex hormone) that controls the cell cycle to the G1 phase is preferable. When the yeast is a Bar-1 deficient yeast, the abundance of yeast whose cell cycle cannot be controlled can be reduced, and the number of cells to be arranged can be easily controlled.

  Prokaryotic cells are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include eubacteria and archaebacteria. These may be used alone or in combination of two or more.

The cell may be a cell that can emit light when receiving light. If the cells are capable of emitting light when receiving light, the number of cells can be controlled with high precision and landed in the recess.
Receiving light means receiving light.
An optical sensor is a lens that collects visible light that can be seen by the human eye and light that is longer in the near-infrared, short-wave infrared, or thermal infrared region using a lens, and is used as a target cell. Means a passive sensor that acquires the shape and the like as image data.

--- Cells that can emit light when receiving light--
The cell that can emit light when receiving light is not particularly limited as long as it can emit light when receiving light, and can be appropriately selected according to the purpose. Cells that express the protein, cells that are labeled with a fluorescently labeled antibody, and the like are included.
The site of staining with a fluorescent dye, the site of expressing a fluorescent protein, or the site of labeling with a fluorescently-labeled antibody in a cell is not particularly limited, and includes a whole cell, a cell nucleus, a cell membrane and the like.

--- Fluorescent dye ---
Examples of the fluorescent dye include fluoresceins, azos, rhodamines, coumarins, pyrenes, and cyanines. These may be used alone or in combination of two or more. Among these, fluoresceins, azos, and rhodamines are preferable, and eosin, Evans blue, trypan blue, rhodamine 6G, rhodamine B, and rhodamine 123 are more preferable.

  Commercially available products can be used as the fluorescent dye. Examples of the commercially available products include EosinY (manufactured by Wako Pure Chemical Industries, Ltd.), trade name: Evans Blue (manufactured by Wako Pure Chemical Industries, Ltd.), and commercial products. Name: Trypan Blue (manufactured by Wako Pure Chemical Industries, Ltd.), Trade name: Rhodamine 6G (manufactured by Wako Pure Chemical Industries, Ltd.), trade name: Rhodamine B (manufactured by Wako Pure Chemical Industries, Ltd.), trade name: Rhodamine 123 ( Wako Pure Chemical Industries, Ltd.).

--- Fluorescent protein ---
Examples of fluorescent proteins include, for example, Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, TurboGFP, AcGFP, TagGFP, Azami-Green, EGFP, EGFP, EGFP, EFP2 , YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, KusabiraOrange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRedRomTrmRobRomRomRomRomArmRomRonArmRomRomArmRomRomArmRomBrm d, mCherry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, and the like KikumeGR. These may be used alone or in combination of two or more.

--- Fluorescently labeled antibody--
The fluorescently labeled antibody is not particularly limited as long as it is fluorescently labeled, and can be appropriately selected depending on the purpose. Examples thereof include CD4-FITC and CD8-PE. These may be used alone or in combination of two or more.

  The volume average particle diameter of the cells in the free state is preferably 30 μm or less, more preferably 15 μm or less, and particularly preferably 10 μm or less. When the volume average particle diameter is 30 μm or less, it can be suitably used for a droplet discharging means such as an ink jet method or a cell sorter.

The volume average particle size of the cells can be measured, for example, by the following measurement method.
10 μL of the prepared stained yeast dispersion is taken out, placed on a PMMA plastic slide, and the volume average particle diameter can be measured by using an automatic cell counter (trade name: Counts Automated Cell Counter, manufactured by Invitrogen). In addition, the number of cells can be determined by the same measurement method.

  Here, the number of cells included in each recess has a variation that occurs when the cells are filled into the recess, that is, the filling accuracy.

[Filling accuracy]
In the present invention, the filling accuracy refers to a relative value (percentage,%) of a variation in the number of cells filled in each recess, which occurs when cells are filled in the recesses. That is, the filling accuracy means a value representing the coefficient of variation of the number of cells filled in the concave portion in percentage (%). The variation coefficient is a value obtained by dividing the standard deviation σ by the average value x, and is represented by the following equation (1).

  Since the variation coefficient can relatively represent the magnitude of the variation in consideration of the size of the group, it is possible to compare the variation of two groups having different average values.

  When the variation coefficient (CV value) for each average value x is calculated from Expression 1, it is as shown in Table 1 and FIG. In addition, based on the graph shown in FIG. 1, the variation coefficient (the accuracy of the number of ejected cells: the total number of cells contained in the droplets ejected from the inkjet head and arranged in the concave portion) is calculated from the average value x. Can be.

As a method of calculating the cell ejection accuracy (coefficient of variation), for example, the number of cells contained in each recess of the cell-containing container is counted, an average value x and a standard deviation s are calculated, and the obtained standard deviation s is averaged. A method of calculating by dividing by the value x is exemplified.
In addition, as a method for calculating the cell ejection accuracy (coefficient of variation), it can be estimated based on “uncertainty” representing a variation in a measurement result caused by a device, an operation, or the like used for measurement.

"Uncertainty" refers to "parameters that characterize the variability of values that can be reasonably linked to measurands associated with the results of measurements" and ISO / IEC Guide 99: 2007 [International metrology measurement terms-basic and general concepts and Related terms (VIM)].
Here, “a value that can be reasonably linked to a measured quantity” means a candidate of a true value of the measured quantity. That is, the uncertainty means information on the variation in the measurement result due to the operation, equipment, and the like related to the manufacture of the measurement target. The greater the uncertainty, the greater the variability expected as a measurement result.
The uncertainty may be, for example, a standard deviation obtained from a result of performing a measurement for calculating a variation in an operation and a device related to manufacturing, and a value in which a true value is included with a predetermined probability or more. May be set to a value that is half of the confidence level expressed as the range of
As a method of calculating the uncertainty, there is a method of calculating the uncertainty based on the guideline for calculating the uncertainty based on the guideline which can be calculated based on the Guide to the Expression of Uncertainty in Measurement (GUM: ISO / IEC Guide 98-3) and the Japan Accreditation Board Note 10 test. .
As a method of calculating the uncertainty, for example, a type A evaluation method using statistics such as measured values and information on uncertainty obtained from a calibration certificate, a manufacturer's specification, published information, etc. are used. Two methods of the type B evaluation method can be applied.
The uncertainty can be expressed with the same confidence level by converting all uncertainties obtained from factors such as operation and measurement into standard uncertainties. Standard uncertainty refers to the variation in the average value obtained from the measured values.

  As an example of a method of calculating the uncertainty, for example, factors causing the uncertainty are extracted, and the uncertainty (standard deviation) of each factor is calculated. Further, the calculated uncertainties of the respective factors are combined by the sum of squares method to calculate a combined standard uncertainty. Since the sum-of-squares method is used in the calculation of the combined standard uncertainty, among the factors that cause the uncertainty, those that have sufficiently small uncertainties can be ignored. As the uncertainty, a coefficient of variation (CV value) obtained by dividing the synthetic standard uncertainty by an expected value may be used.

Factors that cause uncertainty include, for example, counting and dispensing the number of cells in a cell suspension containing cells. And the frequency at which the placed cells are placed at appropriate positions in the recess.
The means for arranging the cells in the concave portion includes, for example, in the case of an ink jet method described later, the number of cells contained in the liquid droplet when the cell suspension is ejected by the ink jet method to form a liquid droplet, the cell Examples include the dispersibility of the suspension.

The cells arranged in the recess with a certain ejection accuracy adhere to the bottom of the recess, and the cells change their form while interacting with each other. Normally, it is known that the cell's original function is expressed by leaving it in an environment close to the body for a certain period of time before starting the test process, and a culture process for 24 hours or longer is necessary become. In particular, when the pluripotent stem cells in the course of differentiation are ejected, it is desirable to start the test step after the completion of the differentiation, and a culture period of about 3 days to 1 month may be required.
In other words, the filling accuracy, which is the variation in the number of cells present in the recess during the inspection process, depends on the variation in the adhesive function of each cell, the variation in the distance between the cells, the variation in the interaction with the substrate, and the environmental factors during culture. It is clear that the value is larger than the ejection accuracy due to the influence of variations and the like.

  The filling accuracy of the number of cells contained in the concave portion of the cell-containing container of the present invention is preferably 30% or less, and more preferably 15% or less. If the filling accuracy is 30% or less, a wide range of tests from tests where the number of cells contained in the cell-containing container has little effect on the results to tests where the number of cells contained in the cell-containing container is required to be strict Can be applied to

<Other components>
The other members are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include an identification unit, a storage unit, a cover member that covers a plurality of recesses, and a cover sheet.

<< identification means >>
The identification means is provided in the cell-containing container of the present invention, and is a means for identifying the cell-containing container.
It is preferable that the identification unit is at least one of an identification unit and an identification display.
The identification unit is not particularly limited and may be appropriately selected depending on the purpose. Examples of the identification unit include a memory, an IC chip, a barcode, a QR code (registered trademark), a Radio Frequency Identifier (RFID), color classification, and printing. No. Among these, when mass-producing cell-containing containers, RFID that can be associated by wireless communication is preferable. In addition, even when the cell-containing container is inserted into the analyzer, the RFID is preferable because it can be associated by wireless communication.

  It is preferable that the identification display is at least one of a character, a symbol, a graphic, and a color. Among these, numbers are particularly preferred. It is preferable that the identification display has a lower creation cost than the identification unit, does not require a reading device for reading information of the identification unit, and can be visually identified.

The position where the identification means is provided is not particularly limited and can be appropriately selected according to the purpose. Is preferred.
The number of identification means is not particularly limited and can be appropriately selected depending on the purpose.

As a method of writing the identification information in the identification unit, for example, a manual input, a method using a writing device, and the like can be given.
As a method of writing the identification information on the container, for example, a method of directly printing the identification information on the container, a method of attaching a seal on which the identification information is printed to the container, and the like can be given.

Reading of the identification information of the identification unit can be performed by a reading mechanism built in the analyzer when the container is mounted on the analyzer. Also, a reading device outside the analyzer can be used.
Reading of the identification display can be carried out visually and by a reading mechanism incorporated in the analyzer when the container is mounted on the analyzer. Also, a reading device outside the analyzer can be used.

<< storage means >>
The storage means is means for storing information on the cell-containing container and information on cells contained in the concave portion in a portion other than the measurement region of the cell-containing container of the present invention. The measurement region of the container is a concave portion (well) portion capable of holding the object to be measured (when the container has a plurality of concave portions, also includes between the concave portions).

  The information on the cell-containing container means information on members constituting the cell-containing container, and includes, for example, the type of the container, the type of the liquid applied to the recess, the type of the cell adhesive material, the measurement date and time, and the like. No.

The information on the cells contained in the recess includes, for example, cell type, cell differentiation history, cell origin, manufacturing company, manufacturing lot number, analysis result (activity value, emission intensity, etc.), and filling of the recess with cells. Results of counting the number of cells in the droplet generated when performing the measurement, the number of cells in the concave portion (the known number counted), the survival rate of the cells in the concave portion, and the position of the concave portion in which the cells are included among the plurality of concave portions. Information, the accuracy of filling the cells in the cell-containing container, and the information on the certainty (or uncertainty) of the number of cells (known number).
Regarding the counting result of the number of cells in the droplet generated when the concave portion is filled with the cells, and the number of cells (the known number counted), for example, observation from the bottom of the concave portion immediately after being placed, or a description will be given later. It can be measured by a droplet discharge counting device.

  Examples of the storage unit include a memory, a hard disk drive, a solid state drive, and an IC chip. These may be provided in a server or in a personal computer.

The portion other than the measurement region of the container may be inside the container or outside the container as long as it is a portion other than the region to be measured.
The storage means is preferably provided detachably from the container. As a method for attaching and detaching the storage means, by providing perforations at the boundary between the container and the storage means, the storage means can be separated along the perforations as necessary. Thus, when inserting the container into the analyzer, the storage means can be separated from the container, and the separated storage means can be inserted into the reading device to associate them with each other.
Preferably, the storage means is attached to the container by a coupling member. This can prevent the storage means from being lost. Examples of the coupling member include a string and a magnet.

As a method of writing the information of the cell-containing container and the information about the cells contained in the concave portion to the storage means, there are a method of manually inputting, a method of directly writing data from a droplet discharge counting device for counting the number of cells, and a method of storing data in a server. Transfer of data stored in the cloud, and transfer of data stored in the cloud. Among these, the method of writing data directly from the droplet discharge counting device is preferable.
As the droplet discharge counting device, for example, Japanese Patent Application No. 2006-12260, Japanese Patent Application No. 2016-132021 and the like can be referred to. The droplet discharge counting device discharges a cell suspension in which cells are suspended in a liquid as droplets, and measures the number of cells contained in the droplets during the flight before the discharged droplets land on the recess. Cell counting means for counting the number of cells. Further, the droplet discharge counting device also has a cell number counting means for counting the number of cells dropped on the concave portion by a sensor.

  The operation method of the droplet discharge means in the droplet discharge counting device is not particularly limited and can be appropriately selected depending on the purpose. For example, a piezoelectric pressurization method using a piezoelectric element, a thermal method using a heater And an ink-jet head of an electrostatic type in which a liquid is pulled by electrostatic attraction.

  Reading of the information stored in the storage means can be performed using an external information reading device, and can be performed by a reading mechanism incorporated in the device when the container is mounted on the analyzer.

When the identification means is an identification display, the method of associating the identification means with the storage means is performed by causing the storage means to store the same identification display as the identification means. The storage of the identification display in the storage means includes a method of directly printing the identification display, a method of attaching a seal describing the identification display, and the like.
On the other hand, when the identification unit is the identification unit, the identification is performed by the storage unit storing the identification information of the identification unit. The storage of the information of the identification unit in the storage means includes manual input, writing by a writing device, and the like.

  In addition, the identification information of the identification unit as the identification means read when the container is mounted on the analyzer can be compared with the information of the container stored in the storage means. Thereby, it is possible to confirm whether or not the association between the identification unit and the storage unit has been correctly performed.

The cell-containing container of the present invention has at least two concave portions, the concave portions include cells, and the type of the cells is at least two types with respect to the container, and between the centers of two closest and adjacent concave portions. Since the shortest distance is 5.0 mm or less, since a plurality of types of cells are contained in a small area, an evaluation test using the cells can be efficiently performed in one container. Furthermore, the amount of the reagent used for the evaluation test using cells can be reduced.
Since the cell-containing container of the present invention has the above-described characteristics, it can be suitably used for a test for evaluating drug efficacy or toxicity using cells.

The reagent for evaluating the efficacy is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include oxazolone, benzoquinone, 2,4-dinitrocobenzine, 4-phenylenediamine, glutaraldehyde, benzoyl peroxide, 4-methylaminophenol sulfate, formaldehyde, cinnamaldehyde, ethylenediamine, 2-hydroxyethyl acrylate, isoeugenol, nickel (II) sulfate, benzylideneacetone, methyl 2-nonate, benzyl salicylate, diethylenetriamine, thioglycerol, -Mercaptobenzothiazole, phenylacetaldehyde, hexylcinnamaldehyde, dihydroeugenol, benzoisothiazolione, citral, resorcinol, phenyl benzoate, euge Lumpur, abietic acid, ethyl aminobenzoate, benzyl cinnamate, cinnamyl alcohol, hydroxy citronellal, imidazolidinyl urea, butyl glycidyl ether, ethylene glycol dimethacrylate, glyoxal, and 4-nitrobenzyl bromide and the like.
The reagent for evaluating toxicity is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include zinc chloride, 1-butanol, benzoic acid, ethyl vanillin, 4-hydroxybenzoic acid, sulfanilic acid, and tartaric acid. , Methyl salicylate, salicylic acid, sodium lauryl sulfate, lactic acid, benzyl alcohol, dextran, diethyl phthalate, glycerol, propylparaben, Tween 80, dimethyl isophthalate, phenol, chlorobenzene, sulfanilamide, octanoic acid and the like.

  The cell-containing container of the present invention can be widely used in the bio-related industry, life science industry, medical industry, and the like.

Here, the cell-containing container of the present invention will be described in detail with reference to the drawings. In each of the drawings, the same components are denoted by the same reference numerals, and redundant description may be omitted. In addition, the number, position, shape, and the like of the following constituent members are not limited to the present embodiment, but can be set to preferable numbers, positions, shapes, and the like in practicing the present invention.
FIG. 2 is a perspective view showing an example of the cell-containing container of the present invention. In the cell-containing container 1, a plurality of concave portions 3 are provided in a container 2, and a desired number of cells 4 are filled in the concave portions 3. In addition, 5 in FIGS. 3 and 4 is a sealing member.

Here, for example, as shown in FIGS. 3 and 4, information on uncertainty (certainty) relating to the cells 4 and the number of cells to be filled in each recess 3 or information associated with these information is stored. An IC chip or a bar code (identification means 6) is arranged between the sealing member 5 and the container 2 and at a position other than the opening of the concave portion. This is suitable for preventing unintended alteration of the identification means 6.
In addition, since the cell-containing container has the identification means, it can be distinguished from a general well plate having no identification means. For this reason, it is possible to prevent mixing.

(Method for producing cell-containing container)
The method for producing a cell-containing container of the present invention includes a step of dispensing a cell suspension containing cells into at least two recesses by an inkjet method.
By using the method for producing a cell-containing container of the present invention, a desired number of cells can be filled into a concave portion at a predetermined position, and the volume of a liquid necessary for filling the cells can be suppressed. The effect of the reagent contained in the test system on the user's experimental system can be reduced.

In the method for producing a cell-containing container of the present invention, for example, the dispensing of the cell suspension may be performed only by dispensing using an inkjet method, or dispensing using an inkjet method after dispensing using a dispenser. May be performed.
First, a case where the cell suspension is dispensed only by the inkjet method will be described below.

  Hereinafter, a flowchart showing an example of the method for producing the cell-containing container of the present invention is shown in FIGS. 5A and 5C, and each step (step) will be described below.

FIG. 5A is a flowchart showing an example of the method for producing a cell-containing container of the present invention.
The process of returning to step S101 in the case of “NO” in step S103 and the process of returning to step S101 in the case of “NO” in step S105 are performed by reducing the number of cells in the concave portion to be dispensed to a predetermined value. When the number of cells does not reach the predetermined value, the number of cells in the concave portion is regarded as a “correction process” for setting the number to the predetermined value.
Further, when there are a plurality of the concave portions, if “NO” in the step S105, the process returns to the step S101. The “correction process” is performed after the steps S101 to S104 are performed on each of the concave portions. You may go together.
Step S106 is a process to be performed on one of the recesses when dispensing a predetermined number of cells to at least one of the recesses when there are a plurality of the recesses.

  In step S101, the cell suspension is ejected as droplets.

  In step S102, the number of cells in the discharged droplet is counted.

In step S103, it is determined whether the number of cells in the at least one recess calculated based on the counted number of cells in the droplet and the number of droplets has reached a predetermined value.
Examples of a method for counting the number of cells in a droplet include a method for optically detecting the particles, which will be described later, and a method for detecting electric and magnetic droplets.
In step S103, the number of cells in the droplet is counted, and based on the number of droplets discharged into the at least one recess, a predetermined value (set number) of cells is dispensed into the at least one recess. Is determined. That is, the number of cells dispensed into the one concave portion is counted (estimated) based on the number of cells contained in the liquid droplet discharged into the one concave portion and the number of liquid droplets discharged into the one concave portion. . In step S103, if it is determined that a predetermined number of cells has been dispensed into at least one recess, the process proceeds to S104, and if it is determined that the predetermined number of cells has not been dispensed into at least one recess, processing proceeds. Is shifted to S101.

In step S104, the number of cells landed in at least one recess is counted.
Examples of a method for counting the number of cells that have landed in at least one concave portion include a method for detecting optically, a method for detecting electric and magnetic droplets described later, and the like.

In step S105, it is determined whether the number of cells that have landed in at least one recess has reached a predetermined value.
In step S105, if it is determined that the number of cells landed in at least one recess (actually existing in the recess) counted in step S104 has reached a predetermined value (set number), the process proceeds to step S106. Then, if it is determined that the number of cells landed in at least one recess (actually existing in the recess) counted in step S104 has not reached the predetermined value (the set number), the process proceeds to S101. When the process proceeds to S101, the cell suspension is ejected by the inkjet method, and an operation of correcting the number of cells in the concave portion is performed.

In step S106, it is determined whether or not the dispensing has been completed for the predetermined recess. By predetermined recess is meant an arbitrarily selected recess of a container having at least one recess.
In step S106, when the dispensing to the predetermined concave portion has not been completed, the process proceeds to S101, and the droplet is discharged to the remaining predetermined concave portion, and the dispensing to the predetermined concave portion is completed. If yes, the process ends.

When dispensing is performed only with a dispenser, a dead volume tends to be generated due to the necessity of an excess cell suspension in order to prevent air bubbles from being mixed into the recess during the suction operation. In addition, when dispensing is performed using only the dispenser, the amount of liquid to be dispensed tends to increase.
The dispensing of the cell suspension is performed only by the dispensing by the inkjet method, so that the amount of liquid to be dispensed can be reduced, and the dead volume can be reduced. There is no need to prepare.

  Further, as shown in FIG. 5B, the method for producing a cell-containing container of the present invention includes a B: droplet discharging step, a C: cell number counting step, and a D: droplet landing step, and further, if necessary, A: a cell suspension generation step; E: a step of calculating the likelihood of the estimated number of cells in the A to D steps; G: an output step; and H: a recording step. Further, if necessary, A: the number of nucleic acids contained in the cell suspension may be estimated in the cell suspension generation step, and C: the cell number before the ejection in the cell number counting step. And C3: a process of counting cells after impact.

<Cell suspension generation step>
The cell suspension generation step is a step of generating a cell suspension containing cells and liquid.
Liquid means a liquid used to disperse cells.
Suspension in a cell suspension means a state in which cells are dispersed and present in a liquid.
Generating means producing.

--- Cells--
The cells are the same as those that can be used in the cell-containing container of the present invention, and thus the description is omitted.

The concentration of the cells in the cell suspension is not particularly limited and can be appropriately selected depending on the purpose. However, the concentration is preferably 5 × 10 ⁴ cells / mL to 5 × 10 ⁸ cells / mL, and more preferably 5 × 10 ⁸ cells / mL. ^The number is more preferably 5 / ml or more and 5 × 10 ⁷ / ml or less. When the number of cells is 5 × 10 ⁵ cells / mL or more and 5 × 10 ⁸ cells / mL or less, the discharged droplets can surely contain cells. The number of cells can be measured using an automatic cell counter (trade name: Counts Automated Cell Counter, manufactured by invitrogen) in the same manner as the method for measuring the volume average particle diameter.

−−liquid−−
The liquid is not particularly limited as long as it can maintain an environment in which cells can survive, and can be appropriately selected depending on the purpose. , A polymer gel solution, a colloidal dispersion, an electrolyte aqueous solution, an inorganic salt aqueous solution, a metal aqueous solution, and a mixed liquid thereof. These may be used alone or in combination of two or more. Among them, a medium, a buffer, or a combination of the liquid and the polymer gel solution is preferable, and the medium, a phosphate buffered saline (PBS), a Tris-EDTA buffer (TE), and a polymer gel material used in combination are preferable. Is more preferably collagen, iMatrix 511, or the like.

--- Additives ---
The additive is not particularly limited as long as it can maintain an environment in which cells can survive, and can be appropriately selected depending on the purpose. Examples thereof include a surfactant. These may be used alone or in combination of two or more.

--- Surfactant ---
Surfactants can prevent aggregation of cells and improve continuous ejection stability.

  The surfactant is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include an ionic surfactant and a nonionic surfactant. These may be used alone or in combination of two or more. Among these, a nonionic surfactant is preferable, depending on the addition amount, from the viewpoint of not denaturing and inactivating the protein.

  Examples of the ionic surfactant include fatty acid sodium, fatty acid potassium, sodium alpha sulfo fatty acid ester, sodium linear alkyl benzene sulfonate, sodium alkyl sulfate, sodium alkyl ether sulfate, and sodium alpha olefin sulfonate. These may be used alone or in combination of two or more. Among these, sodium fatty acid is preferred, and sodium dodecyl sulfate (SDS) is more preferred.

  Examples of the nonionic surfactant include alkyl glycosides, alkyl polyoxyethylene ethers (such as Brij series), octylphenol ethoxylate (such as Triton X series, Igepal CA series, Nonidet P series, Nikkol OP series, etc.), polysorbates ( Tween 20, etc.), sorbitan fatty acid esters, polyoxyethylene fatty acid esters, alkyl maltosides, sucrose fatty acid esters, glycoside fatty acid esters, glycerin fatty acid esters, propylene glycol fatty acid esters, fatty acid monoglycerides, and the like. These may be used alone or in combination of two or more. Among these, polysorbates are preferred.

  The content of the surfactant is not particularly limited as long as it can maintain an environment in which cells can survive, and can be appropriately selected depending on the purpose. % By mass or less is preferred. When the content is 0.001% by mass or more, the effect of adding a surfactant can be obtained, and when the content is 30% by mass or less, cell aggregation can be suppressed.

−−Other materials−−
Other materials are not particularly limited as long as they can maintain an environment in which cells can survive, and can be appropriately selected depending on the purpose. Humectants, dispersants and the like.

[Method of dispersing cells]
The method for dispersing the cells is not particularly limited as long as the environment in which the cells can survive can be appropriately selected depending on the purpose. Examples of the method include a method using a pressure difference such as an ultrasonic method and a French press. These may be used alone or in combination of two or more. Among these, pipetting is more preferable because damage to cells is small. In the ultrasonic or media method, the crushing ability is strong, and there is a possibility that the cell membrane or the cell wall is destroyed, or the medium may be mixed as contamination.

[Cell screening method]
The cell screening method is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include wet classification, cell sorter, and screening using a filter. These may be used alone or in combination of two or more. Among these, screening with a cell sorter and a filter is preferable because damage to cells is small.

<Droplet ejection process>
The droplet discharging step is a step of discharging the cell suspension as droplets to a container having at least two concave portions by the droplet discharging means.
The term “droplet” refers to a liquid mass collected by surface tension.
Discharging means flying the cell suspension as droplets.

  As the droplet discharging means, a means for discharging the cell suspension as droplets (hereinafter, also referred to as an “ejection head” or an “ink-jet head”) or an automatic dispensing apparatus can be suitably used. Examples of the automatic dispensing device include Bravo Automated Liquid Handling Platform manufactured by Agilent.

The ejection head (inkjet head) includes a liquid holding unit that holds the cell suspension, a film-shaped member that applies vibration to the cell suspension to discharge droplets, and opens the atmosphere to the liquid holding unit. And at least a part.
Further, it is preferable that the droplet discharging means has at least two ink jet heads and is used simultaneously or alternately.

  Examples of a method of discharging the cell suspension as droplets include an on-demand method and a continuous method. Among these, in the case of the continuous method, for the reasons such as idle discharge until reaching a stable discharge state, adjustment of the amount of liquid droplets, continuous formation of liquid droplets even when moving between concave portions, etc. The dead volume of the cell suspension used tends to increase. In the present invention, it is preferable to reduce the influence of the dead volume from the viewpoint of adjusting the number of cells. Therefore, the on-demand method is more preferable in the above two methods.

  Examples of the on-demand method include a pressure application method in which a liquid is ejected by applying pressure to a liquid, a thermal method in which a liquid is ejected by film boiling due to heating, and a droplet being formed by pulling a droplet by electrostatic attraction. There are a plurality of known systems such as an electrostatic system. Among these, the pressure application method is preferable for the following reasons.

In the electrostatic method, it is necessary to dispose an electrode in opposition to a discharge unit that holds a cell suspension and forms droplets. In the method for producing a cell-containing container according to the present invention, the cell-containing container for receiving the liquid droplets is disposed to face each other, and it is preferable that no electrode is disposed in order to increase the degree of freedom in the configuration of the cell-containing container.
In the thermal method, since local heating occurs, there is a concern about the influence on cells, which are biological materials, and the scorching (kogation) on the heater. Since the influence of heat depends on the contents and the use of the cell-containing container, it is not necessary to exclude it unequivocally. However, the pressure application method is preferable in that there is no fear of scorching on the heater portion as compared with the thermal method.

Examples of the pressure application method include a method of applying pressure to a liquid using a film-like member such as a piezo element, and a method of applying pressure with a valve such as an electromagnetic valve. 6A to 6C show configuration examples of a droplet generation device that can be used for discharging droplets of a cell suspension.
FIG. 6A is a schematic diagram illustrating an example of a discharge head of the electromagnetic valve type. The discharge head of the electromagnetic valve type has a motor 13a, an electromagnetic valve 112, a liquid holding unit 11a, a cell suspension 300a, and a nozzle 111a.
As the discharge head of the electromagnetic valve type, for example, a dispenser manufactured by TechElan can be suitably used.
FIG. 6B is a schematic diagram illustrating an example of a piezo-type ejection head. The piezo-type ejection head includes a piezoelectric element 13b, a liquid holding unit 11b, a cell suspension 300b, and a nozzle 111b.
As a piezo type ejection head, a single cell printer manufactured by Cytena or the like can be preferably used.
Any of these discharge heads can be used, but the pressure application method using an electromagnetic valve cannot form droplets repeatedly at high speed.To increase the production throughput of cell-containing containers, a piezo method is used. Preferably, it is used. Further, in a piezo-type ejection head using a general piezoelectric element 13b, unevenness in cell density due to sedimentation and clogging of nozzles may occur as problems.
For this reason, a more preferable configuration includes the configuration shown in FIG. 6C. FIG. 6C is a schematic view of a modified example of a piezoelectric ejection head using the piezoelectric element in FIG. 6B. The ejection head of FIG. 6C includes a piezoelectric element 13c, a liquid holding unit 11c, a cell suspension 300c, and a nozzle 111c.
In the ejection head of FIG. 6C, by applying a voltage to the piezoelectric element 13c from a control device (not shown), a compressive stress is applied in the lateral direction of the paper, and the membrane can be deformed in the vertical direction of the paper. Accordingly, since the liquid droplets are formed while stirring the cell suspension 300c in the liquid holding unit 11c, nozzle clogging can be suppressed, and the liquid droplets can be repeatedly formed at high speed.

  As a method other than the on-demand method, for example, there is a continuous method in which droplets are continuously formed. In the continuous method, when a droplet is pressed and pushed out of a nozzle, a periodic fluctuation is given by a piezoelectric element or a heater, whereby a minute droplet can be continuously produced. Further, by controlling the discharge direction of the droplet during flight by applying a voltage, it is also possible to select whether to land on the concave portion or to collect the droplet in the collection unit. Such a method is used in a cell sorter or a flow cytometer, and for example, a device name: cell sorter SH800 manufactured by Sony Corporation can be used.

FIG. 7A is a schematic diagram illustrating an example of a voltage applied to the piezoelectric element. FIG. 7B is a schematic diagram showing another example of the voltage applied to the piezoelectric element. FIG. 7A shows a driving voltage for forming a droplet. Voltage _{(V A, V B, V} C) by intensity, it is possible to form a droplet. FIG. 7B shows a voltage for stirring the cell suspension without discharging the droplet.

  By inputting multiple pulses that are not strong enough to eject droplets during periods when droplets are not ejected, it is possible to stir the cell suspension in the liquid, and to achieve concentration distribution by cell sedimentation. Can be suppressed.

The droplet forming operation of the ejection head that can be used in the present invention will be described below.
The ejection head can eject droplets by applying a pulsed voltage to upper and lower electrodes formed on the piezoelectric element. 8A to 8C are schematic diagrams illustrating a state of formation of a droplet in a droplet discharging step. FIG. 8A shows that a high pressure is applied between the cell suspension held in the liquid holding portion 11c and the membrane 12c by applying a voltage to the piezoelectric element 13c, whereby the membrane 12c is rapidly deformed. This pressure causes the droplets to be pushed out of the nozzle portion by this pressure. Next, as shown in FIG. 8B, the liquid is continuously pushed out from the nozzle portion until the pressure is relaxed upward, and the droplet grows. Finally, as shown in FIG. 8C, when the membrane 12c returns to the original state, the liquid pressure near the interface between the cell suspension and the membrane 12c decreases, and a droplet 310 'is formed.

The container is not particularly limited as long as it is a member generally used in the field of biotechnology.For example, a hole, a concave portion, and a plate on which at least one section of the convex section is formed, a plate on which no section is formed, Containers such as tubes are exemplified. More specifically, as a plate on which a partition is formed, for example, a 24-hole, 96-hole, 384-hole, 1,536-hole plate or the like can be mentioned, and as a plate without a partition, for example, a slide glass And the like, and examples of the tube include an 8-unit PCR tube and a PCR tube used alone.
The concave portion is not particularly limited as long as it has a specific region capable of holding cells, and can be appropriately selected depending on the purpose. For example, the concave portion provided in the cell-containing container, the partition provided in the cell-containing container, An area other than the section is exemplified. More specifically, for example, wells in a 24-well, 96-well, 384-well, 1,536-well plate and the like can be mentioned.
The number of concave portions in the container is not particularly limited and can be appropriately selected depending on the purpose. The number of concave portions may be singular or plural. Here, the number of recesses means the number of partitions themselves in a plate in which partitions are formed, and the number of specific regions in which cells can be retained in a plate in which partitions are not formed. In the case of a tube, it means the number of tubes.
As the container having a plurality of concave portions, it is preferable to use a container having holes having a common number and size in the industry, such as 24, 96, 384, and 1,536 concave portions.
In addition, in this invention, a container may be called a plate. In the present invention, when the container is referred to as a plate, it means at least one of a container having irregularities and a container having no irregularities.

  The material of the container is not particularly limited and may be appropriately selected depending on the purpose. However, it is preferable to use a material in which adhesion of cells or nucleic acids to the wall surface is suppressed for the subsequent treatment.

  As the container, it is preferable to use a container provided with a recognizable means that can be individually recognized. As a recognition unit, a bar code, a QR code (registered trademark), a Radio Frequency Identifier (hereinafter, also referred to as “RFID”), or the like can be used. When mass producing cell-containing containers, RFID that can be used wirelessly is preferable.

  It is preferable to use a 1-well microtube, 8-well tube, 96-well, 384-well, 1,536-well well plate, etc., but if there are a plurality of recesses, The same number of cells can be dispensed or different levels of numbers can be included. In addition, a concave portion that does not contain cells may be present.

<Cell counting step>
The cell counting step is a step of counting the number of cells contained in the droplet from two or more directions by a plurality of sensors while the droplet is flying to the concave portion.
Sensors are the mechanical, electromagnetic, thermal, acoustic, or chemical properties of natural phenomena or man-made objects, or the spatial or temporal information indicated by them, by applying some scientific principles to humans or humans. It means a device that replaces the signal with another medium that is easy to handle by the machine.
Counting means counting numbers.

  The cell number counting step is not particularly limited as long as the number of cells contained in the droplet is counted by a sensor during the flight of the droplet to the concave portion, and can be appropriately selected depending on the purpose. And the process of counting the cells after impact may be included.

  As a method of counting the number of cells contained in the droplet during the flight of the droplet into the concave portion, for example, the position at the position directly above the desired concave portion where the droplet is expected to reliably enter the concave portion of the container It is preferable to count the cells in the droplet at the timing. When the recess has an opening, it means a timing immediately above the opening of the desired recess.

  Methods for counting the number of cells in a droplet include, for example, a method of optically detecting a cell and a method of electrically and magnetically detecting the cell.

-Optical detection method-
The method for optical detection will be described below with reference to FIG. 10, FIG. 14, and FIG.
FIG. 10 is a schematic diagram illustrating an example of a droplet forming apparatus. FIGS. 14 and 15 are schematic diagrams illustrating another example of the droplet forming apparatus. As shown in FIG. 10, the droplet forming apparatus 1 includes a discharge head (droplet discharging unit) 10, a driving unit 20, a light source 30, a light receiving element 60, and a control unit 70.

In FIG. 10, a liquid obtained by fluorescently staining cells with a specific dye and then dispersing the cells in a predetermined solution is used as a cell suspension. Light having a specific wavelength emitted from a light source is applied to droplets formed from a discharge head. The counting is performed by detecting the fluorescence emitted from the cells by irradiation with a light receiving element. At this time, in addition to the method of staining cells with a fluorescent dye, autofluorescence generated by molecules originally contained in the cells may be used, or a fluorescent protein (for example, GFP (Green Fluorescent Protein)) may be produced in the cells. May be introduced in advance so that the cells emit fluorescence.
Irradiating light means irradiating light.

  The ejection head 10 has a liquid holding unit 11, a membrane 12, and a driving element 13, and can eject a cell suspension 300 in which fluorescently-stained cells 350 are suspended as droplets.

  The liquid holding unit 11 is a liquid holding unit that holds the cell suspension 300 in which the fluorescent stained cells 350 are suspended, and a nozzle 111 that is a through hole is formed on the lower surface side. The liquid holding unit 11 can be formed of, for example, metal, silicon, ceramic, or the like. Examples of the fluorescently stained cells 350 include inorganic fine particles and organic polymer particles stained with a fluorescent dye.

  The membrane 12 is a film-like member fixed to the upper end of the liquid holding unit 11. The planar shape of the membrane 12 may be, for example, a circle, but may be an ellipse, a square, or the like.

  The drive element 13 is provided on the upper surface side of the membrane 12. The shape of the drive element 13 can be designed according to the shape of the membrane 12. For example, when the planar shape of the membrane 12 is circular, it is preferable to provide the circular driving element 13.

  By supplying a drive signal from the drive unit 20 to the drive element 13, the membrane 12 can be vibrated. Due to the vibration of the membrane 12, the droplet 310 containing the fluorescent stained cell 350 can be ejected from the nozzle 111.

  When a piezoelectric element is used as the drive element 13, for example, a structure in which electrodes for applying a voltage to the upper and lower surfaces of the piezoelectric material can be provided. In this case, by applying a voltage between the upper and lower electrodes of the piezoelectric element from the driving means 20, a compressive stress is applied in the horizontal direction of the paper, and the membrane 12 can be vibrated in the vertical direction of the paper. As the piezoelectric material, for example, lead zirconate titanate (PZT) can be used. In addition, various piezoelectric materials such as bismuth iron oxide, metal niobate, barium titanate, or a material obtained by adding a metal or a different oxide to these materials can be used.

  The light source 30 irradiates the flying droplet 310 with light L. Note that “flying” means a state from when the droplet 310 is discharged from the droplet discharging means 10 to when the droplet lands on the landing target. The flying droplet 310 has a substantially spherical shape at the position where the light L is irradiated. The beam shape of the light L is substantially circular.

  Here, the beam diameter of the light L is preferably about 10 to 100 times the diameter of the droplet 310. This is to ensure that the light L from the light source 30 is applied to the droplet 310 even when the position of the droplet 310 varies.

  However, it is not preferable that the beam diameter of the light L greatly exceeds 100 times the diameter of the droplet 310. This is because the energy density of the light applied to the droplet 310 is reduced, so that the amount of the fluorescent light Lf that emits the light L as the excitation light is reduced, making it difficult for the light receiving element 60 to detect the light.

  The light L emitted from the light source 30 is preferably pulsed light, and for example, a solid laser, a semiconductor laser, a dye laser, or the like is suitably used. When the light L is pulse light, the pulse width is preferably 10 μs or less, more preferably 1 μs or less. The energy per unit pulse greatly depends on the optical system, such as the presence or absence of light collection, but is generally preferably 0.1 μJ or more, more preferably 1 μJ or more.

  The light receiving element 60 receives the fluorescent light Lf emitted when the fluorescent dyed cell 350 absorbs the light L as the excitation light when the flying liquid droplet 310 contains the fluorescent dyed cell 350. Since the fluorescent light Lf is emitted from the fluorescently stained cells 350 in all directions, the light receiving element 60 can be arranged at any position capable of receiving the fluorescent light Lf. At this time, in order to improve the contrast, it is preferable to arrange the light receiving element 60 at a position where the light L emitted from the light source 30 does not directly enter.

  The light receiving element 60 is not particularly limited as long as it can receive the fluorescence Lf emitted from the fluorescently stained cells 350, and can be appropriately selected according to the purpose. The light receiving element 60 irradiates the droplet with light having a specific wavelength. An optical sensor that receives the fluorescence from the cells in the droplet is preferred. As the light receiving element 60, for example, a one-dimensional element such as a photodiode or a photo sensor can be cited, but when high-sensitivity measurement is required, it is preferable to use a photomultiplier tube or an avalanche photodiode. As the light receiving element 60, for example, a two-dimensional element such as a charge coupled device (CCD), a complementary metal oxide semiconductor (CMOS), or a gate CCD may be used.

  Since the fluorescence Lf emitted by the fluorescently stained cells 350 is weaker than the light L emitted by the light source 30, a filter for attenuating the wavelength range of the light L may be provided at a stage preceding the light receiving element 60 (on the light receiving surface side). . Thereby, in the light receiving element 60, an image of the fluorescently stained cells 350 having a very high contrast can be obtained. As the filter, for example, a notch filter that attenuates a specific wavelength range including the wavelength of the light L can be used.

  Further, as described above, the light L emitted from the light source 30 is preferably a pulsed light, but the light L emitted from the light source 30 may be a continuous oscillation light. In this case, it is preferable to control the light receiving element 60 to be able to capture the light at the timing when the continuous oscillation light is applied to the flying droplet 310, and to make the light receiving element 60 receive the fluorescence Lf.

  The control means 70 has a function of controlling the driving means 20 and the light source 30. In addition, the control unit 70 has a function of obtaining information based on the amount of light received by the light receiving element 60 and counting the number (including zero) of the fluorescent stained cells 350 contained in the droplet 310. I have. Hereinafter, the operation of the droplet forming apparatus 1 including the operation of the control unit 70 will be described with reference to FIGS.

  FIG. 11 is a diagram illustrating a hardware block of the control unit in FIG. FIG. 12 is a diagram exemplifying functional blocks of the control means of FIG. FIG. 13 is a flowchart illustrating an example of the operation of the droplet forming apparatus.

  As shown in FIG. 11, the control means 70 has a CPU 71, a ROM 72, a RAM 73, an I / F 74, and a bus line 75. The CPU 71, the ROM 72, the RAM 73, and the I / F 74 are mutually connected via a bus line 75.

  The CPU 71 controls each function of the control means 70. The ROM 72 serving as a storage unit stores a program executed by the CPU 71 to control each function of the control unit 70 and various information. The RAM 73 serving as a storage unit is used as a work area of the CPU 71 and the like. Further, the RAM 73 can temporarily store predetermined information. The I / F 74 is an interface for connecting the droplet forming apparatus 1 to another device or the like. The droplet forming device 1 may be connected to an external network or the like via the I / F 74.

  As shown in FIG. 12, the control unit 70 has, as functional blocks, an ejection control unit 701, a light source control unit 702, and a cell number counting unit (cell number detecting unit) 703.

  The counting of the number of particles in the droplet forming apparatus 1 will be described with reference to FIGS. First, in step S11, the discharge control unit 701 of the control unit 70 issues a discharge command to the driving unit 20. The drive unit 20 that has received a discharge command from the discharge control unit 701 supplies a drive signal to the drive element 13 to vibrate the membrane 12. The droplets 310 containing the fluorescent stained cells 350 are ejected from the nozzles 111 by the vibration of the membrane 12.

  Next, in step S12, the light source control unit 702 of the control unit 70 controls the light source 30 in synchronization with the ejection of the droplet 310 (in synchronization with the drive signal supplied from the driving unit 20 to the droplet ejection unit 10). Issue a lighting command. Thereby, the light source 30 is turned on, and the light L is applied to the flying droplet 310.

  Here, to synchronize is not to emit light simultaneously with the ejection of the droplet 310 by the droplet discharge means 10 (at the same time as the drive means 20 supplies a drive signal to the droplet discharge means 10), but to emit light at the same time. This means that the light source 30 emits light at the timing when the light L is applied to the droplet 310 when the droplet 310 flies and reaches a predetermined position. That is, the light source control unit 702 emits light with a delay of a predetermined time with respect to the ejection of the droplet 310 by the droplet ejection unit 10 (a driving signal supplied from the driving unit 20 to the droplet ejection unit 10). The light source 30 is controlled.

  For example, the speed v of the droplet 310 ejected when a drive signal is supplied to the droplet ejection means 10 is measured in advance. Then, based on the measured velocity v, a time t from when the droplet 310 is discharged to reach a predetermined position is calculated, and the light source 30 emits light with respect to the timing of supplying a driving signal to the droplet discharging means 10. Is delayed by t. Thereby, good light emission control can be performed, and the light from the light source 30 can be reliably applied to the droplet 310.

  Next, in step S13, the cell number counting means 703 of the control means 70 determines the number of fluorescent stained cells 350 contained in the droplet 310 (including the case where the number is zero) based on the information from the light receiving element 60. Count. Here, the information from the light receiving element 60 is a luminance value (light amount) or an area value of the fluorescent stained cell 350.

  The cell number counting means 703 can count the number of fluorescently stained cells 350 by comparing, for example, the amount of light received by the light receiving element 60 with a preset threshold value. In this case, a one-dimensional element or a two-dimensional element may be used as the light receiving element 60.

  When a two-dimensional element is used as the light receiving element 60, the cell number counting unit 703 performs image processing for calculating a luminance value or an area of the fluorescent stained cell 350 based on the two-dimensional image obtained from the light receiving element 60. You may use the technique of performing. In this case, the cell number counting unit 703 calculates the luminance value or the area value of the fluorescently stained cells 350 by image processing, and compares the calculated luminance value or the area value with a preset threshold to thereby obtain the fluorescence value. The number of stained cells 350 can be counted.

  Note that the fluorescently stained cells 350 may be cells or stained cells. The stained cells mean cells stained with a fluorescent dye or cells capable of expressing a fluorescent protein.

In the stained cells, the fluorescent dye is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include fluoresceins, rhodamines, coumarins, pyrenes, cyanines, and azos. These may be used alone or in combination of two or more. Among them, eosin, Evans blue, trypan blue, rhodamine 6G, rhodamine B, and rhodamine 123 are more preferable.
Examples of fluorescent proteins include, for example, Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, TurboGFP, AcGFP, TagGFP, Azami-Green, EGFP, EGFP, EGFP, EFP2 , YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, KusabiraOrange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRedRomTrmRobRomRomRomRomArmRomRonArmRomRomArmRomRomArmRomBrm d, mCherry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, and the like KikumeGR. These may be used alone or in combination of two or more.

  As described above, in the droplet forming apparatus 1, the driving signal is supplied from the driving unit 20 to the droplet discharging unit 10 that holds the cell suspension 300 in which the fluorescent staining cells 350 are suspended, and the fluorescent staining cells 350 are The droplet 310 is ejected, and light L is irradiated from the light source 30 to the flying droplet 310. Then, the fluorescent stained cells 350 contained in the flying droplet 310 emit fluorescence Lf using the light L as excitation light, and the light receiving element 60 receives the fluorescence Lf. Further, based on the information from the light receiving element 60, the cell number counting means 703 counts the number of the fluorescently stained cells 350 contained in the flying droplet 310.

  That is, in the droplet forming apparatus 1, since the number of the fluorescently stained cells 350 contained in the flying droplet 310 is actually observed on the spot, the counting accuracy of the number of the fluorescently stained cells 350 is improved as compared with the conventional technique. Becomes possible. Further, since the fluorescent stained cells 350 contained in the flying droplet 310 are irradiated with the light L to emit the fluorescent light Lf and the fluorescent light Lf is received by the light receiving element 60, an image of the fluorescent stained cells 350 is obtained with high contrast. This makes it possible to reduce the frequency of erroneous counting of the number of fluorescently stained cells 350.

  FIG. 14 is a schematic diagram showing a modification of the droplet forming apparatus of FIG. As shown in FIG. 14, the droplet forming apparatus 1A is different from the droplet forming apparatus 1 (see FIG. 10) in that a mirror 40 is arranged in front of the light receiving element 60. The description of the same components as those of the embodiment described above may be omitted.

  As described above, in the droplet forming apparatus 1A, the freedom of the layout of the light receiving element 60 can be improved by disposing the mirror 40 before the light receiving element 60.

  For example, when the nozzle 111 and the landing target are brought close to each other, the landing target (not shown in FIG. 10, but corresponds to, for example, the cell-containing container 700 in FIG. 9) and the droplet forming apparatus 1 in the layout of FIG. May interfere with the optical system (particularly, the light receiving element 60), but the layout shown in FIG. 11 can avoid the occurrence of the interference.

  That is, as shown in FIG. 14, the light receiving element 60 is disposed in a region opposite to the droplet discharge direction from the discharge surface of the droplet discharge means, so that the landing object on which the droplet 310 lands and the nozzle 111 Can be reduced, and variations in the landing position can be suppressed. As a result, the accuracy of dispensing can be improved.

FIG. 15 is a schematic diagram showing another modified example of the droplet forming apparatus of FIG. As shown in FIG. 15, the droplet forming apparatus 1B, in addition to the light receiving element 60 for receiving the fluorescence Lf ₁ emitted from the fluorescent stained cells 350, a light receiving element 61 for receiving the fluorescence Lf ₂ emitted from the fluorescent stained cells 350 This point is different from the droplet forming apparatus 1 (see FIG. 10). The description of the same components as those of the embodiment described above may be omitted.

Here, the fluorescences Lf ₁ and Lf ₂ indicate a part of the fluorescence emitted from the fluorescently stained cells 350 in all directions. The light receiving elements 60 and 61 can be arranged at any positions where the fluorescence emitted from the fluorescent stained cells 350 in different directions can be received. Note that three or more light receiving elements may be arranged at positions where the fluorescence emitted from the fluorescent stained cells 350 in different directions can be received. Further, each light receiving element may have the same specification or different specifications.

  When the number of the light-receiving elements is one, when the flying droplet 310 includes a plurality of fluorescently-stained cells 350, the cell number counting means 703 causes the cell number counting means 703 to overlap the fluorescently-stained cells 350. Erroneously counts the number of the fluorescently stained cells 350 contained in (1) (count error may occur).

FIG. 16 is a diagram exemplifying a case in which a flying droplet contains two fluorescently stained cells. For example, as shown in FIG. 16A, and if the overlap and staining cells 350 ₁ and 350 ₂ is generated, as shown in FIG. 16B, may overlap the fluorescent stained cells 350 ₁ and 350 ₂ will not occur obtain. By providing two or more light receiving elements, it is possible to reduce the influence of overlapping fluorescently stained cells.

  As described above, the cell number counting unit 703 calculates the luminance value or the area value of the fluorescent particles by image processing, compares the calculated luminance value or the area value with a preset threshold value, and The number of particles can be counted.

  When two or more light receiving elements are provided, it is possible to suppress the occurrence of a count error by adopting data indicating the maximum value among the luminance values or the area values obtained from the respective light receiving elements. This will be described in more detail with reference to FIG.

  FIG. 17 is a diagram illustrating a relationship between the luminance value Li when particles do not overlap with each other and the actually measured luminance value Le. As shown in FIG. 17, when there is no overlap between the particles in the droplet, Le = Li. For example, assuming that the luminance value of one cell is Lu, Le = Lu when the number of cells / drops = 1, and Le = nLu when the number of particles / drops = n (n: natural number).

  However, in practice, when n is 2 or more, particles may overlap with each other, so that the actually measured luminance value is Lu ≦ Le ≦ nLu (the shaded portion in FIG. 14). Therefore, when the number of cells / drop is n, for example, the threshold value can be set as (nLu-Lu / 2) ≦ threshold value <(nLu + Lu / 2). In the case where a plurality of light receiving elements are installed, it is possible to suppress the occurrence of a count error by adopting the data indicating the maximum value from the data obtained from each of the light receiving elements. Note that an area value may be used instead of the luminance value.

When a plurality of light receiving elements are provided, the number of particles may be determined by an algorithm for estimating the number of cells based on a plurality of obtained shape data.
As described above, in the droplet forming apparatus 1B, since the fluorescently stained cells 350 have the plurality of light receiving elements that receive the fluorescence emitted in different directions, the frequency of erroneous counting of the number of the fluorescently stained cells 350 is further reduced. Can be reduced.

  FIG. 18 is a schematic diagram showing another modified example of the droplet forming apparatus of FIG. As shown in FIG. 18, the droplet forming apparatus 1C is different from the droplet forming apparatus 1 (see FIG. 10) in that the droplet discharging unit 10 is replaced with a droplet discharging unit 10C. The description of the same components as those of the embodiment described above may be omitted.

  The droplet discharge unit 10C has a liquid holding unit 11C, a membrane 12C, and a driving element 13C. The liquid holding part 11C has an air opening part 115 for opening the inside of the liquid holding part 11C to the atmosphere, and is configured so that air bubbles mixed in the cell suspension 300 can be discharged from the air opening part 115. I have.

  The membrane 12C is a film-like member fixed to the lower end of the liquid holding unit 11C. A nozzle 121, which is a through hole, is formed substantially at the center of the membrane 12C, and the cell suspension 300 held in the liquid holding unit 11C is ejected as a droplet 310 from the nozzle 121 by the vibration of the membrane 12C. Since the droplets 310 are formed by the inertia of the vibration of the membrane 12C, the cell suspension 300 having a high surface tension (high viscosity) can be discharged. The planar shape of the membrane 12C may be, for example, a circle, but may be an ellipse, a square, or the like.

  The material of the membrane 12C is not particularly limited, but if it is too soft, the membrane 12C easily vibrates, and it is difficult to suppress the vibration immediately when no ejection is performed. Therefore, it is preferable to use a material having a certain degree of hardness. . As a material of the membrane 12C, for example, a metal material, a ceramic material, a polymer material having a certain degree of hardness, or the like can be used.

  In particular, when a cell is used as the fluorescently stained cell 350, a material having low adhesion to cells and proteins is preferable. It is generally said that the cell adhesion depends on the contact angle of the material with water. When the material has high hydrophilicity or high hydrophobicity, the cell adhesion is low. Various materials and ceramics (metal oxides) can be used as the material having high hydrophilicity, and a fluorine resin or the like can be used as the material having high hydrophobicity.

  Other examples of such a material include stainless steel, nickel, aluminum, etc., silicon dioxide, alumina, zirconia, and the like. In addition to this, it is conceivable that the cell adhesion is reduced by coating the material surface. For example, it is possible to coat the material surface with the above-mentioned metal or metal oxide material, or to coat with a synthetic phospholipid polymer (for example, Lipidure, manufactured by NOF Corporation) that simulates a cell membrane.

  The nozzle 121 is preferably formed as a substantially perfect circular through hole substantially at the center of the membrane 12C. In this case, the diameter of the nozzle 121 is not particularly limited, but is preferably at least twice the size of the fluorescent stained cell 350 in order to prevent the fluorescent stained cell 350 from clogging the nozzle 121. When the fluorescent stained cell 350 is, for example, an animal cell, particularly a human cell, the size of the human cell is generally about 5 μm to 50 μm. Therefore, the diameter of the nozzle 121 should be 10 μm or more in accordance with the cell to be used. Preferably, it is 100 μm or more.

  On the other hand, if the size of the droplet is too large, it is difficult to achieve the purpose of forming a minute droplet. Therefore, the diameter of the nozzle 121 is preferably 200 μm or less. That is, in the droplet discharging means 10C, the diameter of the nozzle 121 is typically in the range of 10 μm to 200 μm.

  The drive element 13C is formed on the lower surface side of the membrane 12C. The shape of the driving element 13C can be designed according to the shape of the membrane 12C. For example, when the planar shape of the membrane 12C is circular, it is preferable to form a driving element 13C having a circular shape (ring shape) around the nozzle 121. The driving method of the driving element 13C can be the same as that of the driving element 13.

  The driving means 20 selectively (for example, alternately) the ejection waveform for forming the droplet 310 by vibrating the membrane 12C and the stirring waveform for vibrating the membrane 12C in a range where the droplet 310 is not formed. ) Can be granted.

  For example, both the ejection waveform and the stirring waveform are rectangular waves, and the driving voltage of the stirring waveform is lower than the driving voltage of the ejection waveform, whereby the droplet 310 can be prevented from being formed by the application of the stirring waveform. That is, the vibration state (degree of vibration) of the membrane 12C can be controlled by the level of the drive voltage.

  In the droplet discharging means 10C, since the driving element 13C is formed on the lower surface side of the membrane 12C, when the membrane 12 is vibrated by the driving element 13C, a flow from the lower direction to the upper direction of the liquid holding unit 11C is generated. Is possible.

  At this time, the movement of the fluorescently stained cells 350 moves from bottom to top, convection occurs in the liquid holding unit 11C, and the cell suspension 300 containing the fluorescently stained cells 350 is stirred. Due to the flow from the lower part to the upper part of the liquid holding part 11C, the sedimented and aggregated fluorescent stained cells 350 are uniformly dispersed inside the liquid holding part 11C.

  That is, the driving unit 20 applies the ejection waveform to the driving element 13C and controls the vibration state of the membrane 12C, thereby ejecting the cell suspension 300 held in the liquid holding unit 11C as the droplet 310 from the nozzle 121. be able to. In addition, the driving unit 20 can stir the cell suspension 300 held in the liquid holding unit 11C by adding a stirring waveform to the driving element 13C and controlling the vibration state of the membrane 12C. During the stirring, the droplets 310 are not discharged from the nozzles 121.

  As described above, by stirring the cell suspension 300 while the droplet 310 is not being formed, the fluorescence stained cells 350 are prevented from settling and aggregating on the membrane 12C, and the fluorescence stained cells 350 are suspended. It can be evenly dispersed in the suspension 300. Accordingly, it is possible to suppress clogging of the nozzle 121 and variation in the number of the fluorescently stained cells 350 in the discharged droplet 310. As a result, the cell suspension 300 containing the fluorescently stained cells 350 can be continuously and stably discharged as the droplet 310 for a long time.

  Further, in the droplet forming device 1C, bubbles may be mixed in the cell suspension 300 in the liquid holding unit 11C. Even in this case, in the droplet forming apparatus 1C, since the air release unit 115 is provided above the liquid holding unit 11C, air bubbles mixed in the cell suspension 300 can be discharged to the outside air through the air release unit 115. . This makes it possible to continuously and stably form the droplet 310 without discarding a large amount of liquid for discharging bubbles.

  That is, when bubbles are mixed in the vicinity of the nozzle 121 or when a large number of bubbles are mixed on the membrane 12C, the ejection state is affected. Therefore, in order to stably form droplets for a long time, It is necessary to discharge the mixed air bubbles. Normally, air bubbles mixed into the membrane 12C move upward naturally or by the vibration of the membrane 12C. However, since the liquid holding unit 11C is provided with the air opening unit 115, the air bubbles mixed into the air opening unit 115 are removed. Can be discharged from Therefore, it is possible to prevent the occurrence of non-discharge even if bubbles are mixed in the liquid holding unit 11C, and it is possible to form the droplet 310 continuously and stably.

  Note that at the timing when the droplet is not formed, the membrane 12C may be vibrated within the range where the droplet is not formed, and the bubble may be positively moved above the liquid holding unit 11C.

-Electrical or magnetic detection method-
As an electrical or magnetic detection method, as shown in FIG. 19, the cell count is placed immediately below a discharge head that discharges a cell suspension from a liquid holding unit 11 ′ as a droplet 310 ′ to a cell-containing container 700 ′. A coil 200 for counting is provided as a sensor. The cells are covered with magnetic beads that are modified by a specific protein and can adhere to the cells.The induced current generated when the cells with the magnetic beads pass through the coil causes the cells in the flying droplet It is possible to detect the presence or absence. Generally, a cell has a cell-specific protein on its surface, and it is possible to attach the magnetic bead to the cell by modifying the antibody capable of adhering to this protein to the magnetic bead. . Off-the-shelf products can be used as such magnetic beads. For example, Dynabeads (registered trademark) manufactured by Veritas Corporation can be used.

The position of the sensor is not particularly limited and can be appropriately selected depending on the intended purpose.For example, the position of the sensor may be other than between the droplet discharging unit and the cell-containing container or between the cell-containing container and the droplet discharging unit. Examples of the region include a region in the direction opposite to the droplet discharge direction from the discharge surface of the droplet discharge unit.
The region in the direction opposite to the droplet discharge direction from the discharge surface of the droplet discharge means means the space on the droplet discharge means side of the surface from which the droplet is discharged.

<< Process for observing cells before ejection >>
As a process of observing cells before ejection, a method of counting the cells 350 ′ that have passed through the microchannel 250 shown in FIG. 20, a method of acquiring an image near the nozzle portion of the ejection head shown in FIG. 21, and the like Is mentioned. FIG. 20 shows a method used in a cell sorter apparatus. For example, a cell sorter SH800 manufactured by Sony Corporation can be used. In FIG. 20, the presence / absence of a cell and the type of a cell are identified by irradiating a laser beam from a light source 260 into a micro channel 250 and detecting scattered light and fluorescence by a detector 255 using a condenser lens 265. It is possible to form droplets while performing. By using this method, it is possible to estimate the number of cells that have landed in a predetermined recess from the number of cells that have passed through the microchannel 250. As the ejection head 10 'shown in FIG. 21, a single cell printer manufactured by Cytena can be used. In FIG. 21, before ejection, it is estimated that cells 350 ″ near the nozzle portion have been ejected from the result of image acquisition by the image acquisition unit 255 ′ via the lens 265 ′ near the nozzle portion, By estimating the number of cells that are considered to have been ejected by the difference from the above, it is possible to estimate the number of cells that have landed in the predetermined concave portion.The cells that have passed through the microchannel shown in FIG. In the method, the droplets are generated continuously, while FIG. 21 is more preferable because the droplets can be formed on demand.

<< Process for counting cells after impact >>
Examples of the process of counting the cells after landing include a step of counting the number of cells in at least one concave portion into which the cell suspension has been dispensed. Specifically, it is possible to adopt a method of detecting fluorescently stained cells by observing the concave portion in the cell-containing container with a fluorescence microscope or the like. This method is described, for example, in Sangjun et al. , PLoS One, Volume 6 (3), e17455.

  The method of observing the cells before and after the ejection of the droplet has the following problems, but it is most preferable to observe the cells in the droplet during ejection depending on the type of the cell-containing container to be manufactured. In the method of observing cells before ejection, the number of cells that seem to have landed is counted from the number of cells that have passed through the flow channel and the image observation before (and after) ejection. Has not been verified, and unexpected errors may occur. For example, a case occurs in which the droplets are not ejected correctly due to the dirty nozzle portion, adhere to the nozzle plate, and the cells in the droplets do not land accordingly. In addition, there may be a problem that cells remain in a narrow region of the nozzle portion, and cells move more than expected due to a discharging operation and come out of an observation range. There is also a problem in a method for detecting cells on a cell-containing container after landing. First, it is necessary to prepare a container that allows microscopic observation. As the cell-containing container that can be observed, a container having a transparent and flat bottom surface, particularly a container having a bottom surface made of glass is generally used. For example, there is a problem that a well cannot be used. In addition, when the number of cells is large, such as several tens, there is a problem that accurate counting cannot be performed because cells are overlapped. Therefore, in addition to counting the number of cells contained in the droplet by the sensor and the particle number (cell number) counting means after the droplet is ejected and before the droplet lands on the concave portion, the cell is counted before the ejection. It is preferable to perform a process of observing and a process of counting cells after landing.

  As a step of counting the number of cells in at least one concave portion into which the cell suspension has been dispensed, for example, an image is taken from the bottom side of each concave portion, and image processing such as binarization is performed to determine the number of cells. Measure and output / save as data file.

Further, in addition to the step of counting the number of cells in at least one concave portion into which the cell suspension has been dispensed, a step of calculating a difference between the measured number of cells and a predetermined number of cells, It is preferable that the method further includes a step of dispensing the number into the one concave portion by an inkjet method.
The step of calculating a difference between the measured number of cells and a predetermined number of cells includes calculating a difference between the measured data and a target number of cells. It is preferable that the calculated difference data is linked with the position information of the concave portion.
The step of dispensing the calculated number of cells of the difference into the one recess by the ink jet method includes dispensing the calculated number of cells to each recess based on the calculated value of the difference. Thus, even when the number of cells actually dispensed into the concave portion and the target (desired) cell number are different, the cells are ejected to the concave portion by the ink jet method so that the target cell number is accurately obtained. can do.
Calculating the difference between the measured number of cells and a predetermined number of cells, and dispensing the calculated number of cells of the difference into the one recess by an inkjet method, the number of cells in at least one recess. This is performed for the purpose of correcting.

  Further, it is preferable that the method further includes a step of adjusting the cell concentration of the cell suspension held in the liquid holding unit using the counted result of the cells after landing. By including the step of adjusting the cell concentration of the cell suspension, it is possible to suppress the ejection operation of the liquid droplets that do not contain cells. Therefore, waste of the solvent constituting the cell suspension can be reduced, and the operation time can be shortened.

Further, as the light receiving element, a light receiving element having one or a small number of light receiving portions, for example, a photodiode, an avalanche photodiode, a photomultiplier tube, and a light receiving element provided in a two-dimensional array can be used. It is also possible to use a two-dimensional sensor such as a CCD (Charge Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor), and a gate CCD.
When using a light-receiving element having one or a small number of light-receiving parts, it is conceivable to determine how many cells are contained from the fluorescence intensity using a calibration curve prepared in advance. Binary detection of cell presence is performed. When the cell concentration of the cell suspension is sufficiently low and the ejection is performed in a state where only one or zero cells are almost contained in the droplet, counting is performed with sufficient accuracy by the binary detection described above. It is possible. Assuming that cells are randomly arranged in the cell suspension, the number of cells in the flying droplet is considered to follow the Poisson distribution, and the probability P ( > 2) is represented by the following equation (1). FIG. 22 is a graph showing the relationship between the probability P (> 2) and the average cell number. Here, λ is the average number of cells in the droplet, which is obtained by multiplying the cell concentration in the cell suspension by the volume of the discharged droplet.
P (> 2) = 1− (1 + λ) × e− ^λ Equation (1)

  When the cell count is performed by binary detection, it is preferable that the probability P (> 2) is a sufficiently small value in order to ensure accuracy, and the probability P (> 2) is 1% or less. <0.15 is preferred. The light source is not particularly limited as long as it can excite the fluorescence of the cells, and can be appropriately selected depending on the purpose.The light source can irradiate a general lamp such as a mercury lamp or a halogen lamp with a specific wavelength. It is possible to use a filter, an LED (Light Emitting Diode), a laser, or the like. However, it is necessary to irradiate a narrow area with high light intensity particularly when forming a fine droplet of 1 nL or less, and therefore, it is preferable to use a laser. As the laser light source, various types of commonly known lasers such as a solid-state laser, a gas laser, and a semiconductor laser can be used. The excitation light source may be a light source that continuously irradiates a region through which a droplet passes, or at a timing with a predetermined time delay with respect to the droplet discharge operation in synchronization with the discharge of the droplet. Irradiation may be performed in a pulsed manner.

<Droplet landing process>
The droplet landing step is a step of landing droplets such that a predetermined number of cells are arranged in at least one recess.
The predetermined number means an arbitrarily set number. Here, it means that the number of cells to be arranged in each recess is arbitrarily set.
As the predetermined number, for example, the number of cells to be arranged in all the recesses of the cell-containing container may be the same, and a plurality of groups (sometimes referred to as levels) having the same number of cells may be provided in each of the recesses. .
The arrangement means that a predetermined object is arranged at a predetermined position.
Landing means that the droplets reach the recesses.

The method of landing the droplet is not particularly limited and can be appropriately selected depending on the purpose.For example, after arranging the droplet until a predetermined number set in one concave portion is reached, the droplet is placed in another concave portion. There are a method of repeating landing of liquid droplets until the number reaches a predetermined number, a method of sequentially arranging liquid droplets until the number reaches a predetermined number set in each recess, and the like.
Sequential means to follow one after another.

  In FIG. 9, in the method for producing a cell-containing container according to the present invention, the cell-containing container having the recess (recess) formed thereon is fixed on a movable stage, and the driving of the stage and the formation of droplets from the ejection head are combined. In this way, the liquid droplets sequentially land on the concave portions (recesses). Here, the method of moving the cell-containing container as the movement of the stage has been described, but the ejection head may be moved as a matter of course.

FIG. 9 is a schematic view showing an example of an apparatus for sequentially landing droplets in the concave portion of the cell-containing container.
As shown in FIG. 9, a device (dispensing device) 2 for landing a droplet includes a droplet forming unit 1, a stage 800, and a control device 900.

  The cell-containing container 700 is arranged on a stage 800 configured to be movable. The cell-containing container 700 has a plurality of recesses 710 (recesses) where the droplets 310 discharged from the discharge head of the droplet forming means 1 land. The control device 900 moves the stage 800 to control the relative positional relationship between the ejection head of the droplet forming unit 1 and each of the recesses 710. Thus, the droplets 310 containing the fluorescent stained cells 350 can be sequentially discharged from the discharge head of the droplet forming means 1 into the respective concave portions 710.

  The control device 900 can be configured to include, for example, a CPU, a ROM, a RAM, a main memory, and the like. In this case, various functions of the control device 900 can be realized by reading a program stored in a ROM or the like into the main memory and executing the program by the CPU. However, part or all of the control device 900 may be realized only by hardware. Further, the control device 900 may be physically configured by a plurality of devices and the like.

As the droplet to be ejected, it is preferable that when the cell suspension lands in the recess, the droplet lands in the recess so as to obtain a plurality of levels.
A plurality of levels means a plurality of standards that are standard.
The plurality of levels means that the cell holding member has a predetermined concentration gradient by, for example, arranging a plurality of cells each having a nucleic acid having a specific base sequence in a different number for each recess. By having a concentration gradient, it can be suitably used as a calibration curve reagent. The plurality of levels can be controlled using a value counted by the sensor.

<Step of calculating the certainty of the number of cells to be estimated in the cell suspension generation step and the droplet landing step>
The step of calculating the likelihood of the number of cells to be estimated in the cell suspension generation step and the droplet landing step is a step of calculating the likelihood in each of the cell suspension generation step and the droplet landing step. is there.
The calculation of the certainty of the estimated number of cells can be performed in the same manner as the certainty in the cell suspension generation step.
The calculation timing of the certainty may be calculated in the next step of the cell number counting step as shown in FIG. 1, or may be calculated at the end of each step of the cell suspension generation step and the droplet landing step. The uncertainty may be calculated and calculated in the next step of the cell number counting step. In other words, the certainty in each of the above steps may be appropriately calculated before the synthesis calculation.

<Output process>
The output step is a step of outputting a value obtained by counting the number of cells contained in the cell suspension landed in the concave portion by the particle number counting means based on the detection result measured by the sensor.
The counted value means the total number of cells contained in the concave portion calculated by the particle counting means from the detection result measured by the sensor.
The particle counting means counts the number of cells measured by the sensor and calculates a total value.
Output means that a device such as a prime mover, a communication device, or a computer receives an input and transmits the counted value to a server as an external counting result storage device as electronic information, or prints the counted value as a printed matter. Means that.

In the output step, at the time of manufacturing the cell-containing container, the number of cells in each concave portion in the cell-containing container is observed or estimated, and the observed value or the estimated value is output to an external storage unit.
The output may be performed simultaneously with the cell counting step, or may be performed after the cell counting step.

<Recording process>
The recording step is a step of recording the output observed value or estimated value in the output step.
The recording step can be suitably performed in the recording unit.
The recording may be performed simultaneously with the output step, or may be performed after the output step.
Recording means not only adding information to a recording medium but also storing information in a recording unit.

Next, FIG. 5C is a flow chart showing an example of the method for producing the cell-containing container of the present invention in the case where the dispensing of the cell suspension is performed by the dispenser and then the dispensing is performed by the inkjet method. Each step will be described below.
FIG. 5C is a flowchart showing an example of the method for producing a cell-containing container of the present invention. FIG. 5C is a diagram illustrating a case where dispensing by the inkjet method is performed after dispensing by the dispenser, except that a step of dispensing the cell suspension by the dispenser is performed before S101 shown in FIG. 5A. Is the same as the case of dispensing when dispensing is performed only by the inkjet method.

In step S201, the cell suspension is dispensed into at least one recess with a dispenser.
The dispensing by the dispenser in step S101 is performed by the operation shown in FIGS. 26A to 26D.
As shown in FIG. 26A, in dispensing by a dispenser, a dispenser head 1001 equipped with a pipette tip 1002 and a reservoir 1004 for storing a cell suspension (dispensed liquid) 1003 adjusted to a predetermined concentration in advance are used.
First, as shown in FIG. 26B, the dispenser head 1001 is lowered to suck the cell suspension 1003 stored in the reservoir 1004 into each pipette tip 1002. At this time, as shown in FIG. 26C, by placing excess cell suspension 1003 in the reservoir 1004 with respect to the required dispensed amount, variation in the amount of suction to pipette tip 1002 and variation in pipette volume Air bubbles can be prevented from being trapped. If air bubbles are involved, there is a possibility that observing, photographing, and counting the number of cells in the concave portion may be hindered when the cell suspension 1003 is dispensed into the concave portion. In the suction operation, it is preferable that the cell suspension 1003 is suctioned into the pipette tip 1002 in excess of the amount of the cell suspension required for dispensing into the concave portion. By sucking an excessive amount of the cell suspension into the pipette tip 1002, it is possible to prevent air bubbles from being mixed into the concave portion when the entire cell suspension 1003 in the pipette tip 1002 is discharged. .
Next, as shown in FIG. 26D, the dispenser head 1001 after the suction operation is moved onto the target concave portions, and a desired amount of the cell suspension 1003 sucked into each concave portion is dispensed.

  In step S202, the number of cells dispensed into at least one recess is counted. As a method for counting the number of cells in the concave portion, a method similar to the method described above can be used.

In step S203, it is determined whether the number of cells in at least one recess has reached a predetermined value. That is, in step S203, when it is determined that the number of cells dispensed into the at least one concave portion (actually existing in the concave portion) counted in step S202 has not reached the predetermined value (the set number), the process proceeds to step S101. Then, if it is determined that the number of cells dispensed into at least one of the recesses has reached the predetermined value, the process is terminated.
The process after step S101 is the same as the case where the cell suspension is dispensed only by the inkjet method, and therefore, the description is omitted.

  By dispensing the cell suspension by a dispenser and then by the inkjet method, the dead volume can be reduced while improving the productivity.

  The cell-containing container produced by the method for producing a cell-containing container of the present invention is widely used in the bio-related industry, the life science industry, the medical industry, and the like, and is preferably used for applications such as evaluation tests using cells. Can be used.

(Cell chip)
The cell chip of the present invention is a cell chip having at least two recesses accommodating cells, wherein the recesses are a first recess containing a first cell type and a second recess containing a second cell type. And the minimum center-to-center distance between the recesses is 5.0 mm or less.

The cells in the cell chip of the present invention are not particularly limited as long as they are at least two types of cells, a first cell type and a second cell type, and may be the same as the cells used in the cell-containing container of the present invention. The description is omitted because it can be used.
The concave portion in the cell chip of the present invention is not particularly limited as long as it has at least a first concave portion containing a first cell type and a second concave portion containing a second cell type. Since the same recess as that of the cell-containing container of the invention can be used, the description is omitted.
Further, the minimum center-to-center distance between the concave portions in the cell chip of the present invention has the same meaning as the shortest distance between the centers of two nearest and adjacent concave portions in the cell-containing container of the present invention. Is omitted from the description.

  Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.

(Production Example 1)
<Preparation of cell-containing container 1>
-Preparation Example of Cell Dispersion A-
After seeding iPS cells A prepared from a human in a 10 cm dish and culturing them at 37 ° C. for 7 days in Stemfit AK02N (manufactured by Ajinomoto Co., Inc.), the iPS cells A were detached, and when the seeds were again seeded on a 24-well plate, the medium was cultured. Change to Medium N9 in the Quick-Neuron Mixed SeV component kit from Elixirgen Scientific, and add the Quick-Neuron Mixon solution from Elixirgen Scientific's Quick-Neuron Mixon with the Mix-Neuron Mixon from Nixon so that the final concentration is 0.1%. By culturing the cells at 33 ° C. and 5% CO _{2 for one} day, the differentiation induction of the nerve cells was started. On the third day of the culture, the cells were detached from the dish and dispersed in a PBS (phosphate buffered saline) solution to prepare a cell dispersion A. The iPS cells A have been genetically modified so as to express free GFP protein when differentiated into neurons A.

-Preparation Example of Cell Dispersion B-
A cell dispersion B was prepared in the same manner as in the preparation example of the cell dispersion A, except that iPS cells B prepared from different humans were used. The iPS cells B have been genetically modified so as to express free GFP protein upon differentiation into nerve cells B.

-Arrangement and filling of cells into recesses-
A 384-well plate (manufactured by Thermo Scientific, shortest pitch: 4.5 mm) in which 200 μL of Medium N9 in a Quick-Neuron Mixed SeV component kit manufactured by Elixirgen Scientific was dispensed into each recess was used for automatic dispensing by an automatic illuminator (Agilent). It is installed on a stage of Automated Liquid Handling Platform (dispenser method). Before installing the well plate in the apparatus, a UV lamp is irradiated for about 15 minutes to sterilize the inside of the apparatus.
The cell dispersing liquid A (cell concentration 5 × 10 ⁵ cells / mL) and the cell dispersing liquid B (cell concentration 5 × 10 ⁵ cells / mL) (mL) (S201 in FIG. 5C), and the cells were filled at 16 locations so that the number of cells became 500, 1,000, and 1,500, respectively. At this time, the number of cells in the recesses that were continuously filled was counted (S202 in FIG. 5C), and it was determined whether the number reached a predetermined number (S203 in FIG. 5C).
When the number of cells dispensed into the recess by the automatic dispensing device has not reached the predetermined number, the dispersing process of the cell dispersion A and the cell dispersion B by the ink jet device was performed (FIG. 5C). S101).
Discharge heads each filled with the prepared cell dispersion liquid A and cell dispersion liquid B were installed in an ink jet apparatus (developing apparatus name: IJ-MINI, Ricoh Co., Ltd.), and the droplet deposition position on the center of the concave portion was adjusted to ensure stable discharge. And adjustment of the ink jet device.
After the adjustment of the ink jet device, the cell dispersion A and the cell dispersion B were respectively filled into the 96-well concave portion at the center of the 384-well plate, and the 96-well concave portion filled above was respectively filled. The difference from a predetermined value was ejected by an ink jet method while referring to the result of counting the number of cells in the concave portion, thereby filling the cells. In discharging the cell dispersion liquid A and the cell dispersion liquid B, the number of cells in the droplet after the discharge and in flight is counted with a laser beam (S102 in FIG. 5C). The number of cells is counted with a microscope (S104 in FIG. 5C). When the “number of cells in the droplet after ejection” and the “number of cells in the concave portion after impact” have not reached the predetermined number of cells, Filling was performed while performing correction processing as shown in steps S103 and S105 in FIG. 5C (S106 in FIG. 5C). After the filling was completed, the well plate was cultured in a 5% CO ₂ incubator (Panasonic) for 30 minutes to promote adhesion to the well plate. After culturing for 30 minutes, 200 μL of the medium N9 medium in the Quick-Neuron Mixed SeV component kit manufactured by Elixirgen Scientific was dispensed into each of the wells filled with the cells in the well plate and the wells surrounding the wells filled with the cells. The cells were cultured for 5 days in a 5% CO ₂ incubator to produce a cell-containing container 1.

-Cell count-
The well plate after 4 days of culture was observed with a fluorescence microscope (Axio Observer D1 manufactured by Carl Zeiss Co., Ltd.) by irradiating each recess with 488 nm excitation light. The image obtained by the fluorescence microscope observation was used to create a binarized image using Image J, which is image processing software, and the number of cells was counted. From the obtained results, an average value x, a standard deviation s, and a filling accuracy CV value were calculated. Table 2 shows the results.

(Production Example 2)
<Preparation of cell-containing container 2>
-Preparation Example of Cell Dispersion C-
After seeding iPS cells C prepared from a human in a 10 cm dish and culturing them at 37 ° C. for 7 days in Stemfit AK02N (Ajinomoto Co., Inc.), the iPS cells C are detached, and when the seeds are again seeded on a 24-well plate, the medium is used for Elixirgen Scientific. Change to Medium N9 in the Quick-Neuro Mixed SeV component kit manufactured by the company, and add the Quick-Neuron Mixed SeV component kit manufactured by Elixirgen Scientific to the final concentration of 0.1% and add the D1 solution in the D1 solution for 2 days. By incubating the cells at 33 ° C. and 5% CO ₂ concentration, the induction of differentiation into nerve cells C was started. On the third day of the culture, the cells were detached from the dish and dispersed in a PBS (phosphate buffered saline) solution to prepare a cell dispersion C.

-Preparation Example of Cell Dispersion D-
Cell dispersion liquid D was prepared in the same manner as in cell dispersion liquid C except that iPS cells D prepared from different humans were used.

-Preparation of container-
A dimethylpolysiloxane (PDMS) sheet (trade name: Sylgard (registered trademark) 184, manufactured by Dow Corning Toray Co., Ltd., 25 mm ×) having 192 holes with a diameter of 1.5 mm so that the shortest pitch is 2.25 mm. Molding and heat curing at 100 ° C. for 40 minutes to a size of 75 mm × h 0.75 mm) and a plastic slide made of polystyrene (manufactured by Pernomax) are immersed in 100% ethanol for 5 minutes for sterilization. I do. The sterilized PDMS sheet and the plastic slide are attached in a wet state and dried at room temperature. Hereinafter, the laminated product is referred to as a container.

A cell adhesive material was prepared by diluting iMatrix 511 (manufactured by Nippi Co., Ltd.) to a final concentration of 1.5 μL / 100 μL in PBS, mixed well and stirred, and then dropped 1.0 μL / recessed cell adhesive material. Then, the mixture is allowed to stand at 4 ° C. for 8 hours to 12 hours (or at 37 ° C. for 2 hours in an incubator).
After allowing the container to stand for a predetermined period of time, the liquid is removed while paying attention to drying in the concave portion, and a washing operation is performed twice with PBS, and then N2 medium is dropped in 200 μL portions.

-Filling cells into recesses-
The manufactured container is set on the stage of an ink jet device (developing device name: IJ-MINI, Ricoh Co., Ltd.), and a UV lamp is irradiated for about 15 minutes to sterilize the inside of the device.
An ejection head filled with the prepared cell dispersion liquid C and the prepared cell dispersion liquid D was installed in the ink jet apparatus, and the position of the liquid droplet landing on the center of the concave portion was adjusted, and the adjustment for the discharge stability was performed. .
After the adjustment of the ink jet apparatus, 500, 1,000, and 1,500 drops of the cell dispersion liquid C and the cell dispersion liquid D are respectively discharged to the 96-well portion at the center of the container at 16 locations. , Filling was performed. After completion of the filling, the container was cultured in a 5% CO ₂ incubator (manufactured by Panasonic) for 30 minutes to promote adhesion to the container. After culturing for 30 minutes, N9 medium (manufactured by ELIXIRGEN Scientific) was dispensed into the concave portion filled with cells in the container and the concave portion around the concave portion filled with cells in 200 μL portions, and the mixture was placed in a 5% CO ₂ incubator for 4 days. After culturing, a cell-containing container 2 was produced.

-Cell count-
After culturing for 4 days, the medium in each concave portion of the cell-containing container is removed, and after washing with PBS, the PDMS sheet of the cell-containing container is peeled off. A frame was prepared with a pup pen (trade name: Super Pap Pen Liquid Blocker, manufactured by Cosmo Bio Co., Ltd.) so as to surround the entire 96 concave portions where the cell culture was performed, and 500 μL of 4% paraformaldehyde was dropped into the frame. After fixing at 4 ° C. for 10 minutes, 4% paraformaldehyde is extracted, and PBS is washed with 200 μL.

Next, Normal Goat Serum (manufactured by Thermo Fisher Scientific) and Triton X-100 (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) are adjusted to a final concentration of 10% and PBS, respectively, so that the final concentration is 1%. The added blocking solution was dropped on a 500 μL slide, and left standing for 1 hour while blocking light and keeping moisture to perform blocking. Next, an anti-beta-III Tubulin antibody (product name: Purified anti-Tubulin Beta 3 (TUBB3) Antibody, BioLegend) as a primary antibody was diluted 5,000-fold with PBS to prepare a primary antibody dilution. did. After washing the slide with PBS, 200 μL of the primary antibody diluent is dropped and left at 4 ° C. overnight.
Next, Alexa 555 anti-mouse secondary-antibody was used as a secondary antibody in a solution in which Normal Goat Serum was added to a final concentration of 3% and Triton X-100 was added to a final concentration of 0.2%. (Abcam) was diluted 500 times to prepare a secondary antibody diluent. After washing the slide with PBS, 200 μL of a secondary antibody diluent is dropped, and the slide is allowed to stand at 4 ° C. for 1 hour in a light-shielded state.
Next, after washing with PBS, a coverslip sterilized with 100% ethanol is coated with an encapsulating solution containing DAPI (manufactured by Thermo Fisher Scientific), placed on a slide, and allowed to stand at 4 ° C. for 1 hour.

The prepared slide was irradiated with excitation light at 364 nm and 555 nm using a fluorescence microscope to confirm that nuclei and tubulin were stained, and a fluorescence microscope image of the slide was obtained.
The number of cells in each recess was counted using Image J, which is image processing software, for the obtained image. From the obtained results, an average value x, a standard deviation s, and a filling accuracy CV value were calculated. Table 3 shows the results.

(Production Example 3)
In Production Example 2, cells were filled in the recesses in the same manner as in Production Example 2 except that a predetermined number of cells was filled into each recess using a technique (multichannel pipette, trade name: P8X10L, manufactured by Gilson). The cell-containing container 3 was manufactured. Table 4 shows the results.

(Example 1)
<Efficacy screening>
In Production Example 2, Staurosporine (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was dissolved in DMSO to prepare a 2,000 μM drug solution on the third day of culture after the cells were filled into the recesses, and the final concentration was 0%. 0.1 μM, 0.3 μM, 1.0 μM, 3 μM, or 10 μM was added to each recess using a pipette (trade name: Pipetteman, manufactured by Eppendorf) using a 10 μL tip. The drug solution used at this time was 50 pL to 5000 pL.

On the day after the addition of the drug solution, the medium in each recess of the cell-containing container is removed, and each recess is washed with 200 μL of Hank's balanced salt solution (HBSS, manufactured by Thermo Fisher Scientific) at room temperature. After washing, a staining liquid mixture was prepared by diluting Hoechst 33342 (manufactured by Thermo Fisher Scientific), PI (propidium iodide, manufactured by Cosmo Bio Inc.), and Calsein AM (manufactured by Takara Bio Inc.) 1,000 times with HBSS. Then, 200 μL of the prepared staining mixture is added. Let stand in a 5% CO ₂ incubator for 20 minutes.

  A fluorescence microscope image of the cell-containing container after completion of the staining was obtained using a fluorescence microscope. The obtained fluorescence microscope image was processed using Image J which is image processing software, and the number of cells and the number of dead cells were counted. The results are shown in Table 5 below.

  When drug efficacy screening is performed using the cell-containing container of the present invention, the drug efficacy rate can be quantified as the ratio of the number of cells and dead cells. In Example 1, a cell-containing container having a CV value of about 20% or less can be quantitatively evaluated. In addition, since the cell-containing container of the present invention has a small volume per recess, the amount of reagents (cells, drugs, and the like) used in one test can be reduced. That is, experimental data necessary for quantitative analysis can be efficiently obtained in one container without using a large amount of sample.

(Example 2)
<Cytotoxicity evaluation>
-Cell viability-
100 μL of an N9 medium (manufactured by ELIXIRGEN Scientific) to which zinc chloride at each of four concentration conditions of 0 μM, 100 μM, 160 μM, and 220 μM was added as a test substance was added to the evaluation container of Production Example 2 with a micropipette overnight. (20 hours), it was left still in a CO ₂ incubator at 37 ° C.
Next, in order to evaluate the effect of zinc chloride on cell viability, 10 μL of a WST-1 reagent (trade name: Premix WST-1 Cell Proliferation Assay System, manufactured by Takara Bio Inc.) was added per well (recess). The reaction was allowed to stand for 1 hour in a 37 ° C. CO ₂ incubator. Thereafter, the absorbance at 450 nm and a reference value of 570 nm were measured using a plate reader (device name: Cytation 5, manufactured by BioTek) to evaluate the "cell viability". The results are shown in FIG.

(Cell membrane damage rate)
In the same manner as in the evaluation of the cell viability, the above-mentioned N9 medium supplemented with zinc chloride was added, and the mixture was allowed to stand overnight (20 hours) in a CO ₂ incubator at 37 ° C.
To evaluate the effect of zinc chloride on cell membrane damage, only the medium was collected using a micropipette, placed in a 96-well cell-containing container, and furthermore, an LDH measurement reagent (trade name: Cytotoxicity Detection Kit, manufactured by Roche) Was added, and reacted at room temperature (23 ° C.) for 5 minutes. Thereafter, the absorbance at 492 nm and a reference value of 620 nm were measured using a plate reader to evaluate the “cell membrane damage rate”. The results are shown in FIG.

(Inflammatory substance secretion)
In the same manner as in the evaluation of the cell viability, an N9 medium (manufactured by ELIXIRGEN Scientific) supplemented with zinc chloride was added, and the mixture was allowed to stand overnight (20 hours) in a CO ₂ incubator at 37 ° C.
To evaluate the effect of zinc chloride on secretion of inflammatory substances, only the medium was collected using a micropipette. Using a protein assay kit (trade name: Human IL-8 ELISA Ready-SET-Go! (Registered trademark), manufactured by Affymetrix), the amount of IL-8 secreted by ELISA was measured at 450 nm using a plate reader. The absorbance at 570 nm was measured as a reference value. The results are shown in FIG.

  As can be seen from the results of FIG. 23 to FIG. 25, the cell viability, the cell membrane damage rate, and the secretion amount of the inflammatory substance with respect to zinc chloride are different between the nerve cells C and the nerve cells D in the cell-containing container 2 in Production Example 2. It showed almost the same tendency of toxic reaction. These results show that the cell-containing container of the present invention is effective for efficiently evaluating a toxic reaction.

Aspects of the present invention are, for example, as follows.
<1> having at least two recesses, wherein the recesses include cells,
The type of cells is at least two types for the container,
A cell-containing container, wherein the shortest distance between the centers of two nearest neighboring recesses is 9.0 mm or less.
<2> The cell-containing container according to <1>, wherein the recess contains a liquid, and a total amount of the liquid in the container is 10.0 μL or less.
<3> The cell-containing container according to any one of <1> to <2>, wherein the filling accuracy of the number of cells contained in the recess is 30% or less.
<4> The cell-containing container according to any one of <1> to <2>, wherein the filling accuracy of the number of cells contained in the recess is 15% or less.
<5> The above-mentioned <5>, wherein the cell is at least one of an induced pluripotent stem (iPS) cell, a differentiated cell derived from an iPS cell, an Embronic Stem (ES) cell, a differentiated cell derived from an ES cell, and a stem cell obtained from a human body. The cell-containing container according to any one of 1> to <4>.
<6> The cell-containing container according to any one of <1> to <5>, wherein the shortest distance between the centers of the two concave portions is 4.5 mm or less.
<7> The cell-containing container according to any one of <1> to <6>, wherein the shortest distance between the centers of the two concave portions is 2.25 mm or less.
<8> The cell-containing container according to any one of <1> to <7>, wherein the recess further includes a cell culture solution.
<9> identification means for identifying the cell-containing container,
The cell-containing container according to any one of <1> to <8>, further including a storage unit configured to store at least one of information on the cell-containing container and information on the cell included in the recess. is there.
<10> The cell-containing container according to <9>, wherein the information on the cell is at least one of a cell type, a cell differentiation history, the number of cells in the recess, and the survival rate of the cell in the recess. It is.
<11> The cell-containing container according to any one of <9> to <10>, wherein the storage unit is separated from the container.
<12> The cell-containing container according to any one of <9> to <11>, wherein the identification unit is provided on the container.
<13> Any one of <9> to <12>, wherein the identification means is at least one of a barcode, a QR code (registered trademark), a Radio Frequency Identifier (RFID), a character, a symbol, a graphic, and a color. 4. The cell-containing container according to item 1.
<14> The cell-containing container according to any one of <1> to <13>, wherein each of the concave portions has one type of the cell.
<15> The method for producing a cell-containing container according to any one of <1> to <14>, wherein the dispensing of a cell suspension containing cells into at least two recesses is performed by an inkjet method. A method for producing a cell-containing container, comprising the steps of:
<16> The inkjet head in the inkjet method,
A liquid holding unit that holds the cell suspension, a film-shaped member that applies vibration to the cell suspension to discharge droplets, and has at least an atmosphere opening unit that opens the liquid holding unit to the atmosphere. A method for producing the cell-containing container according to <15>.
<16> The method for producing a cell-containing container according to <16>, wherein at least two of the inkjet heads are used and used simultaneously or alternately.
<17> The method for producing a cell-containing container according to any one of <15> to <16>, further including a step of counting the number of cells in at least one concave portion into which the cell suspension is dispensed.
<18> a step of calculating a difference between the measured number of cells and a predetermined number of cells;
The method according to any one of <15> to <17>, further including a step of dispensing the calculated difference in the number of cells into the one recess by an inkjet method.
<20> The method for producing a cell-containing container according to any one of <15> to <17>, further including a step of adjusting a concentration of cells in the cell suspension.
<21> The method according to <15>, wherein the dispensing of the cell suspension containing the cells to the at least two recesses includes dispensing by a dispenser and then dispensing by an inkjet method. 20> The method for producing a cell-containing container according to any one of the above items.
<22> A cell chip having at least two recesses accommodating cells,
The recess has at least a first recess containing a first cell type and a second recess containing a second cell type,
A cell chip, wherein a minimum center-to-center distance between the concave portions is 5.0 mm or less.

  According to the cell-containing container according to any one of <1> to <14>, the method for producing the cell-containing container according to <15> to <21>, and the cell chip according to <22>, The above-mentioned problems can be solved and the object of the present invention can be achieved.

1 Cell-containing container 2 Container (substrate)
3 recess 4 cell 6 identification means

JP 2015-47077 A JP 2010-112839 A

Claims (21)

Having at least two recesses, wherein the recesses include cells;
The type of cells is at least two types for the container,
A cell-containing container, wherein a shortest distance between the centers of two nearest and adjacent recesses is 9.0 mm or less.   The cell-containing container according to claim 1, wherein the concave portion contains a liquid, and a total amount of the liquid with respect to the concave portion is 10.0 µL or less.   3. The cell-containing container according to claim 1, wherein the filling accuracy of the number of cells contained in the recess is 30% or less. 4.   3. The cell-containing container according to claim 1, wherein the filling accuracy of the number of cells contained in the recess is 15% or less. 4.   5. The cells according to claim 1, wherein the cells are at least one of an induced pluripotent stem (iPS) cell, a differentiated cell derived from an iPS cell, an Embronic Stem (ES) cell, a differentiated cell derived from an ES cell, and a stem cell obtained from a human body. The cell-containing container according to any one of the above.   The cell-containing container according to any one of claims 1 to 5, wherein a shortest distance between centers of the two concave portions is 4.5 mm or less.   The cell-containing container according to any one of claims 1 to 6, wherein the shortest distance between the centers of the two concave portions is 2.25 mm or less.   The cell-containing container according to claim 1, wherein the recess further contains a cell culture solution. Identification means for identifying the cell-containing container,
9. The cell-containing container according to claim 1, further comprising: a storage unit configured to store at least one of information on the cell-containing container and information on the cell included in the concave portion. 10.   The cell-containing container according to claim 9, wherein the information on the cell is at least one of a cell type, a cell differentiation history, the number of cells in the recess, and a survival rate of the cell in the recess.   The cell-containing container according to any one of claims 9 to 10, wherein the storage means is separated from the container.   The cell-containing container according to any one of claims 9 to 11, wherein the identification means is provided on the container.   The cell-containing container according to any one of claims 9 to 12, wherein the identification means is at least one of a barcode, a QR code (registered trademark), a Radio Frequency Identifier (RFID), a character, a symbol, a graphic, and a color. .   14. The method for producing a cell-containing container according to any one of claims 1 to 13, wherein the step of dispensing a cell suspension containing cells into at least two recesses includes an ink jet method. A method for producing a cell-containing container. The inkjet head in the inkjet method,
A liquid holding unit that holds the cell suspension, a film-shaped member that applies vibration to the cell suspension to discharge droplets, and has at least an atmosphere opening unit that opens the liquid holding unit to the atmosphere. A method for producing the cell-containing container according to claim 14.   The method for producing a cell-containing container according to claim 14, comprising at least two of the inkjet heads, wherein the inkjet heads are used simultaneously or alternately.   The method for producing a cell-containing container according to any one of claims 14 to 17, further comprising a step of counting the number of cells in at least one concave portion into which the cell suspension has been dispensed. A step of calculating a difference between the measured number of cells and a predetermined number of cells,
18. The method for producing a cell-containing container according to claim 17, further comprising: dispensing the calculated number of cells into the one concave portion by an inkjet method.   The method for producing a cell-containing container according to any one of claims 14 to 18, further comprising a step of adjusting the concentration of cells in the cell suspension.   20. The method according to claim 14, wherein dispensing the cell suspension containing the cells to the at least two recesses includes dispensing by an inkjet method after dispensing by a dispenser. A method for producing the cell-containing container according to the above. A cell chip having at least two recesses in which cells are accommodated,
The recess has at least a first recess containing a first cell type and a second recess containing a second cell type,
A cell chip, wherein a minimum center-to-center distance between the recesses is 5.0 mm or less.