JP2019180394A - Cell arrangement plate production method, cell holding member and production method thereof and cell arrangement plate - Google Patents

Abstract

To provide a production method of a cell arrangement plate capable of reducing damage when cells discharged by an ink jet method land, and suppressing dryness of the cells without reduction of accuracy of positions of the landed cells.SOLUTION: A production method of a cell arrangement plate comprises: a step for laminating a structure layer in which a plurality of hole parts are formed on a layer formed of a cell non-adhesive material, to a plane of a substrate; a step for coating the plane on the substrate exposed from the hole parts in the hole parts with a coating fluid; and a step for discharging a cell suspension to the plane coated with the coating fluid.SELECTED DRAWING: Figure 9B

Description

  The present invention relates to a method for producing a cell arrangement plate, a method for producing a cell holding member, a cell holding member, and a cell arrangement plate.

  In recent years, with the progress of stem cell technology, development of a technique for artificially forming a tissue body composed of a plurality of cells has been performed. Since a technique for arbitrarily arranging cells is necessary to artificially form a tissue body, various efforts have already been made, and the cell sheet method, spheroid lamination method, gel extrusion method, ink jet method, etc. are known. ing.

Among these, the cell placement technique based on the inkjet method is considered to be one of the promising techniques capable of artificially forming a tissue body because it is excellent in controlling a plurality of cell types to an arbitrary place with an arbitrary number of cells. Various proposals have been made.
For example, in order to form a micropattern of cells on a substrate, a cell-immobilized substrate in which a cell adhesive material is arranged in a pattern on the substrate by an inkjet method has been proposed (see, for example, Patent Documents 1 and 2).
In order to prevent elution of the constituent material of the pressure-sensitive adhesive layer used for the cell culture substrate, in the cell culture substrate having a pressure-sensitive adhesive layer, an adhesive layer, a cell culture layer, and a protective layer on the substrate, the exposed surface of the pressure-sensitive adhesive layer A cell culture substrate or the like coated with a protective layer has been proposed (see, for example, Patent Document 3).
Furthermore, as a technique for precisely arranging cell aggregates on a cell-adhesive surface, a well is composed of a first substrate in which a through hole is formed and a second substrate that is not cell-adhesive. A cell assembly forming device is disclosed in which cells are seeded, one cell assembly is formed, and then transferred to a cell adhesive surface (see Patent Document 4).

  The present invention relates to a method for producing a cell arrangement plate that can reduce damage when cells ejected by the ink jet method land, reduce the accuracy of the position of the landed cells, and suppress cell drying. The purpose is to provide.

  The method for producing a cell arrangement plate of the present invention as means for solving the problem includes a step of laminating a structure layer having a plurality of holes formed in a layer made of a cell non-adhesive material on a plane of a substrate, A step of coating the flat surface of the substrate exposed from the hole portion with a coating solution in the hole portion, and a step of discharging a cell suspension onto the flat surface coated with the coating solution are included.

  According to the present invention, there is provided a cell arrangement plate that can reduce damage when cells ejected by the ink jet method land, reduce the accuracy of the position of the landed cells, and suppress cell drying. A manufacturing method can be provided.

FIG. 1A is an explanatory diagram illustrating an example of a state before a hole is formed in a structural layer by laser processing. FIG. 1B is an explanatory diagram showing an example of a state in which a structural layer in which holes are formed is arranged on a substrate. FIG. 1C is an explanatory diagram showing another example of a state in which the structural layer in which the hole is formed is arranged on the base. FIG. 2 is a schematic cross-sectional view showing an example of a droplet forming apparatus. FIG. 3 is an explanatory diagram illustrating an example of a discharge signal and a suppression signal. FIG. 4A is an explanatory diagram showing an example of the operation of the droplet forming apparatus. FIG. 4B is an explanatory diagram showing an example of the operation of the droplet forming apparatus. FIG. 4C is an explanatory diagram illustrating an example of the operation of the droplet forming apparatus. FIG. 5A is an explanatory diagram illustrating another example of the ejection signal and the suppression signal. FIG. 5B is an explanatory diagram illustrating another example of the ejection signal and the suppression signal. FIG. 6 is a schematic view showing an example of a cell holding member manufacturing apparatus equipped with a plurality of droplet forming apparatuses. FIG. 7 is an explanatory diagram illustrating an example of an operation of discharging the cell adhesive material ink. FIG. 8A is an explanatory diagram illustrating an example of an operation of discharging the coating liquid. FIG. 8B is an explanatory diagram illustrating an example of an operation of discharging the coating liquid. FIG. 9A is an explanatory diagram illustrating an example of an operation of discharging cell ink. FIG. 9B is an explanatory diagram illustrating an example of an operation of discharging cell ink. FIG. 10 is an explanatory diagram showing an example of an operation of discharging cell ink by a conventional ink jet method. FIG. 11A is a photograph showing a hole formed by laser processing of the structural layer in Example 1. FIG. 11B is an image showing a hole formed by laser processing of the structural layer in Example 1. FIG. 12A is a photograph showing a hole formed by laser processing of the structural layer in Example 2. 12B is an image showing a hole formed by laser processing of the structural layer in Example 2. FIG. FIG. 13 is an explanatory diagram showing an operation of discharging the cell adhesive material ink in Example 7. FIG. 14 is an explanatory diagram illustrating an operation of discharging the cell ink according to the eighth embodiment. FIG. 15 is a photograph when the cell ink is dyed and arranged in two types in Example 9.

(Cell retention member)
The cell holding member of the present invention has a structural layer formed of a non-cell-adhesive material and having a plurality of holes on at least a plane of a substrate having a plane, and is exposed from the holes in the holes. It has a coating liquid for covering the flat surface of the substrate, and further has other members as necessary. In other words, in the cell holding member of the present invention, a structural layer in which a plurality of holes are formed in a layer made of a cell non-adhesive material is laminated on a plane having at least a plane, and the holes are formed in the holes from the holes. A coating solution for coating the flat surface of the exposed substrate is added, and other members are provided as necessary.

The cell holding member of the present invention is based on the problems found by the present inventors from the prior art.
For example, in a cell-immobilized substrate in which a conventional cell-adhesive material is arranged in a pattern on a substrate by an ink-jet method, the cell-adhesive material is simply patterned on the substrate by an ink-jet method. Therefore, there is a possibility that the cells may be united with each other.
In addition, in the conventional cell culture substrate, since the cells are directly landed on the cell adhesion layer, there is a problem that the cells discharged by the ink jet method are easily damaged.
Furthermore, in the conventional cell assembly forming device, since it is assumed that cells are seeded, it is difficult to arrange a plurality of types of cells at desired positions, and thus it is difficult to manufacture a tissue body. There is a problem.

In the cell holding member of the present invention, by having a structural layer having a plurality of holes in the substrate, when the cell suspension is discharged into the plurality of holes, the cells can be divided and held for each hole, It is possible to prevent the landing droplets from coalescing to shift the cell position.
Further, in the cell holding member of the present invention, by holding the cell suspension that has landed in the hole, the surface area of the cell suspension in contact with the air can be reduced, so that the drying of the cells is suppressed. Can do.
Furthermore, in the cell holding member of the present invention, by having a coating liquid that covers the flat surface of the substrate exposed from the hole in the hole, damage when cells discharged by the ink jet method land, In addition, cell drying can be suppressed.
The cell holding member of the present invention is preferably used for holding a cell suspension containing cells in the pores.

<Cell suspension>
The cell suspension is not particularly limited as long as it contains cells, and can be appropriately selected according to the purpose. For example, the cell suspension includes cells to be patterned and a dispersion medium for dispersing the cells, and further if necessary. And other ingredients.
The “cell suspension” may be referred to as “cell ink” or simply “ink”.

<< cells >>
The cell is not particularly limited as to its type and can be appropriately selected according to the purpose, and taxonomically, for example, eukaryotic cells, prokaryotic cells, multicellular biological cells, unicellular biological cells, It can be used for all cells. These may be used individually by 1 type and may use 2 or more types together.

  Examples of eukaryotic cells include animal cells, insect cells, plant cells, fungi and the like. These may be used alone or in combination of two or more. Among these, animal cells are preferable, and when the cells form a cell aggregate, the cells adhere to each other, and have adhesion properties that are not isolated unless physicochemical treatment is performed. Cells are more preferred.

Adhesive cells are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include differentiated cells and undifferentiated cells. These may be used individually by 1 type and may use 2 or more types together.
Examples of differentiated cells include hepatocytes that are liver parenchymal cells; stellate cells; Kupffer cells; vascular endothelial cells; endothelial cells such as epithelial endothelial cells and corneal endothelial cells; fibroblasts; osteoblasts; Periodontal ligament-derived cells; epidermal cells such as epidermal keratinocytes; tracheal epithelial cells; gastrointestinal epithelial cells; cervical epithelial cells; epithelial cells such as corneal epithelial cells; mammary cells; Examples include muscle cells such as cells; kidney cells; pancreatic islets of Langerhans; nerve cells such as peripheral nerve cells and optic nerve cells; chondrocytes; Adherent cells may be primary cells collected directly from tissues or organs, or may be passaged from them for several generations. These may be used individually by 1 type and may use 2 or more types together.
The undifferentiated cells are not particularly limited and can be appropriately selected according to the purpose. For example, pluripotent stem cells such as embryonic stem cells which are undifferentiated cells, mesenchymal stem cells having multipotency; Examples include unipotent stem cells such as vascular endothelial progenitor cells having unipotency; iPS cells and the like. These may be used individually by 1 type and may use 2 or more types together.
Examples of prokaryotic cells include eubacteria and archaea.

  As the dispersion medium, a medium for cell culture and a buffer solution are preferable as in the coating liquid described later. Examples of the dispersion medium include phosphate buffered saline.

  There is no restriction | limiting in particular as another component, According to the objective, it can select suitably, For example, a wetting agent, a cell distance adjustment material, a dispersing agent, a pH adjuster etc. are mentioned.

There is no restriction | limiting in particular as a wetting agent, According to the objective, it can select suitably, For example, a gel-like polysaccharide etc. are mentioned.
The gel-like polysaccharide is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include calcium alginate, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, and diyutan gum. And carboxymethyl cellulose. These may be used individually by 1 type and may use 2 or more types together. Among these, calcium alginate is preferable.
Calcium alginate is a salt in which calcium ions are bonded to the carboxyl group of alginate, and since calcium ions are divalent, the cell ink is bonded to two carboxyl groups (ionic cross-linking) and thickened to increase viscosity. Can be prevented from drying. At this time, calcium ions are contained in the dispersion medium and are considered to be combined with calcium ions that have become excessive due to concentration by drying, and a role of adjusting the osmotic pressure can also be expected.

  The intercellular distance adjusting material is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably biocompatible.

<Discharge>
As means for discharging droplets of cell ink onto the substrate, ink jet type liquid droplet discharging means is preferable.
As the droplet discharge means by the ink jet method, for example, a so-called piezo method (for example, Japanese Patent Publication No. 2), in which a droplet is discharged by changing the volume of the cell ink using a piezoelectric element as pressure generating means for pressurizing the cell ink. 51734), a so-called thermal method (for example, see Japanese Patent Publication No. 61-59911), in which cell ink is heated using a heating resistor to generate bubbles, and a diaphragm and an electrode are arranged to face each other, and vibration is generated. There is an electrostatic method (for example, see Japanese Patent Laid-Open No. 6-71882) in which the diaphragm is deformed by an electrostatic force generated between the plate and the electrode, thereby changing the volume of the cell ink and discharging a droplet. Can be mentioned.

There is no restriction | limiting in particular as a discharge speed, Although it can select suitably, 0.1 m / s or more and 10 m / s or less are preferable. When the discharge speed is 0.1 m / s or more and 10 m / s or less, damage to cells at the time of landing can be reduced if the minimum thickness of the coating liquid in the hole is at least 5 μm.
The method for obtaining the ejection speed is not particularly limited and can be appropriately selected according to the purpose.For example, when shooting the ink droplets in flight in order to count the number of cells of the ink droplets, For example, a calculation method based on the time interval between frames of a captured image and the movement distance of ink droplets may be used.

  There is no restriction | limiting in particular as the number of cells contained in the droplet formed by one discharge, Although it can select suitably, 1 or more and 2 or less are preferable. When the number of cells contained in a droplet formed by one ejection is 1 or more and 2 or less, non-ejection due to clogging of the nozzles of the inkjet head is less likely to occur. Moreover, since there is little variation in the number of cells, it is advantageous in that variation in cell concentration in each hole can be reduced.

<Substrate>
The shape of the substrate is not particularly limited as long as it has at least a flat surface, and can be appropriately selected according to the purpose, but is preferably flat. If the shape of the substrate is flat, that is, if the substrate is a substrate, it is advantageous in that it can be easily processed.
Here, the term “flat” means flat in a broad sense, and has a degree of flatness that does not leak even when liquid is injected into the hole when the structure layer in which the hole is formed is laminated. Microscopically, it may have irregularities. There is no restriction | limiting in particular as a range of a plane, Although it can select suitably according to the objective, It is preferable that the range applicable to the bottom face of a hole is a plane.

  The structure of the substrate is not particularly limited as long as it has a structure layer described later, and can be appropriately selected according to the purpose.

  There is no restriction | limiting in particular as a magnitude | size of a base | substrate, According to the objective, it can select suitably.

  The material of the substrate is not particularly limited as long as it is a cell non-adhesive material, and can be appropriately selected according to the purpose. Examples thereof include 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 cell adhesive materials are preferable. When the base material is easy to adsorb the cell adhesive material, when the cell adhesive material is discharged to the base corresponding to the bottom of the hole, the cell adhesive material can be stably attached.

-Cell non-adhesive material-
The cell non-adhesive means that the adhesiveness to the target cell is lower than at least the cell adhesive material to be used.
The measurement method for cell non-adhesion is not particularly limited and can be appropriately selected according to the purpose. For example, a needle-shaped AFM probe is inserted into a cell cultured on a material and pulled up. Examples of the method include measuring the load applied to the probe by AFM by peeling the cells from the substrate, and measuring / evaluating the adhesion of the cells to the material. Other methods include, for example, a method in which pure water or the like is flowed on cells cultured on the material, and evaluation is performed based on the adhesion peeling rate of the cells on the material before and after that.

There is no restriction | limiting in particular as a cell non-adhesive material, Although it can select suitably according to the objective, 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.
Moreover, there is no restriction | limiting in particular as a cell non-adhesive material, Although it can select suitably according to the objective, A silicon containing material is preferable.
There is no restriction | limiting in particular as a silicon-containing material, Although it can select suitably according to the objective, Polydimethylsiloxane (PDMS) is preferable from the point of biocompatibility.

There is no restriction | limiting in particular as an organic material, According to the objective, it can select suitably, For example, a polyethylene terephthalate (PET), a polystyrene (PS), a polycarbonate (PC), TAC (triacetylcellulose), a polyimide (PI) , Nylon (Ny), low density polyethylene (LDPE), medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, urethane acrylate and other acrylic materials, cellulose, Examples thereof include silicone materials such as polydimethylsiloxane (PDMS).
There is no restriction | limiting in particular as an inorganic material, According to the objective, it can select suitably, For example, glass, ceramics, etc. are mentioned.

Examples of the cell adhesive material include a protein selected from an extracellular matrix.
Examples of the protein 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 materials, Matrigel, PuraMatrix, and fibrin. Among these, collagen is preferable. There are various types of collagen, but it is known that the viscosity increases depending on the concentration and temperature. Inclusion in the cell ink increases the viscosity of the cell ink when the concentration of the cell ink increases. it can. Furthermore, examples of the protein selected from the extracellular matrix include basic polymers such as polylysine and basic compounds such as aminopropyltriethoxysilane.
When the cell adhesive material is discharged, it may be discharged as a solution containing the cell adhesive material. In this case, the solution may contain biocompatible particles.

The biocompatible particles are not particularly limited as long as they have an affinity with a living body such as a cell, and can be appropriately selected. Examples thereof include gelatin particles and collagen particles. These may be used individually by 1 type and may use 2 or more types together.
When the biocompatible particles are gelatin particles, the gelatin as a raw material for the gelatin particles is not particularly limited and can be appropriately selected according to the purpose. For example, trade name: APH-250 (Nitta Gelatin Co., Ltd.) Company-made).
The gelatin particles that are particulate can improve the adhesion of the cells to the substrate, and may be present in the tissue without being broken down into cells for a long time compared to gelatin that is non-particulate. it can. Therefore, gelatin particles have the advantage of improving cell adhesion and being used as a nutrient source for cells over a long period of time.

  The biocompatible particles are preferably crosslinked with a crosslinking agent in the structure. By crosslinking with a crosslinking agent, the cumulant diameter of the biocompatible particles can be reduced, and cell growth can be promoted in a solution containing a cell adhesive material.

  The crosslinking agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include aldehydes such as glutaraldehyde and formaldehyde; ethylene propylene diglycidyl ether, glycerol polyglycidyl ether, diglycerol polyglycidyl ether, 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 individually by 1 type and may use 2 or more types together. Among these, aldehyde is preferable and glutaraldehyde is more preferable.

As content of a crosslinking agent, 1 mass% or more and 20 mass% or less are preferable with respect to the raw material whole quantity of bioaffinity particle | grains, and 2 mass% or more and 10 mass% or less are more preferable. When the content of the crosslinking agent is 1% by mass or more and 20% by mass or less, the cumulant diameter of the biocompatible particles can be reduced, and the proliferation of cells can be promoted in a solution containing a cell adhesive material. it can.
The content of the biocompatible particles is preferably 0.5% by mass or more and 10% by mass or less, and more preferably 1% by mass or more and 5% by mass or less with respect to the total amount of the solution containing the cell adhesive material. When the content of the biocompatible particles is 0.5% by mass or more and 10% by mass or less, the cells can be sufficiently adhered to the tissue body which is a three-dimensional cell aggregate, and the proliferation of the cells can be promoted.

  The range to which the cell adhesive material is attached is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include all or part of the bottom (substrate) of the hole, but the center of the bottom The vicinity is preferable. When the cell adhesion material is attached in the vicinity of the center of the bottom, it becomes difficult for cells to adhere to the wall surface (side surface) of the hole, so that the number of cells formed on the substrate can be controlled with high accuracy.

-Preparation 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 liquid volume for measurement is 5 mL, and the dispersion can be prepared by stirring a 20 mm rotator at 200 rpm for about one day using a stirrer.

-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 ND filter)
Measurement probe: Concentration probe Measurement routine: Measurement: 180 ° C. at 25 ° C. → Measurement: 600 ° C. at 25 ° C. (The liquid temperature gradually changes from 25 ° C. to 35 ° C. when the body side is changed to 35 ° C. Diameter change is monitored) → Measurement: 35 ° C for 180 seconds

<< Structural layer >>
The shape of the structural layer is not particularly limited as long as it has at least one of a plurality of holes, and can be appropriately selected according to the purpose.
There is no restriction | limiting in particular as average thickness of a structure layer, Although it can select suitably according to the objective, 10 micrometers or more are preferable. When the average thickness of the structural layer is 10 μm or more, it is advantageous in that a capacity for containing the cell ink can be secured and that the structural layer can be easily handled when laminated on the substrate.

  There is no restriction | limiting in particular as a structure of a structure layer, According to the objective, it can select suitably, For example, a single layer structure, a laminated structure, etc. are mentioned. As a laminated structure, what laminated | stacked the masking tape etc. are mentioned, for example. In this case, there is no restriction | limiting in particular as a masking tape, Although it can select suitably according to the objective, A thin adhesive layer and a weak adhesive force are preferable. If the adhesive layer of the masking tape is thin and has a weak adhesive force, it is advantageous in that the wall surface of the hole is difficult to be sticky with the adhesive layer even if the laminated masking tape is provided with holes by laser processing. Moreover, there is no restriction | limiting in particular as a material of a masking tape, Although it can select suitably according to the objective, Polyethylene is preferable.

  There is no restriction | limiting in particular as a magnitude | size of a structure layer, According to the objective, it can select suitably, For example, the magnitude | size similar to the magnitude | size of a base | substrate is mentioned.

  There is no restriction | limiting in particular as a material of a structural layer, According to the objective, it can select suitably, For example, what was illustrated by the material of the base | substrate is mentioned. These may be used alone or in combination of two or more. Among these, a cell non-adhesive material is preferable, and a material that is easily peeled off from the substrate is more preferable when a three-dimensional tissue body is produced. The material of the structural layer may be the same as or different from the material of the base.

-Hole-
There is no restriction | limiting in particular as long as it is a through-hole shape as a hole part, According to the objective, it can select suitably.

  There is no restriction | limiting in particular as a shape at the time of planarly viewing a hole, According to the objective, it can select suitably, For example, a circle, an ellipse, a triangle, a quadrangle etc. are mentioned.

  There is no restriction | limiting in particular as a structure of the wall surface (side surface) of a hole, According to the objective, it can select suitably, For example, cylindrical shape, a taper shape, uneven | corrugated shape etc. are mentioned.

  There is no restriction | limiting in particular as a formation method of a hole, According to the objective, it can select suitably, For example, laser processing, the process by a photolitho, the process by a drill, the mold shaping by a metal mold | die etc. are mentioned. Among these, laser processing is preferable. If the hole formation method is laser processing, the pattern shape that can form the desired tissue body can be easily and finely formed, and it is easy to obtain biocompatibility because it is not processed in direct contact with the structural layer. This is advantageous.

There is no restriction | limiting in particular as arrangement | positioning of a several hole part, According to the objective, it can select suitably, For example, you may make it respond | correspond to the pattern shape which can form a desired structure | tissue.
The distance between the centers of adjacent holes, that is, the pitch interval between the holes is preferably 300 μm or less. When the pitch interval of the holes is 300 μm or less, the landed cells are difficult to unite, and it is advantageous in that the accuracy of the discharge position of the cells can be maintained.

  The flat surface of the substrate exposed from the hole in the structural layer becomes the landing surface of the ink droplet ejected by the ink jet method.

  The substrate and the structural layer are preferably sterilized because they come into contact with the cell ink.

<Coating solution>
The coating solution is not particularly limited as long as it can cover the flat surface of the substrate exposed from the hole in the hole, and can be appropriately selected according to the purpose. preferable.
A medium is a solution that contains components necessary for the formation and maintenance of a tissue body, prevents dryness and adjusts the external environment such as osmotic pressure, and can be appropriately selected and used as long as it is known as a medium. . When it is not necessary to always immerse the cells in the medium solution, the medium can be appropriately removed from the cell ink.
The buffer is for adjusting the pH according to the cell and purpose, and a known one can be appropriately selected and used.

  There is no restriction | limiting in particular as a viscosity of a coating liquid, Although it can select suitably according to the objective, The higher one is preferable. A high viscosity of the coating solution is advantageous in that it is easy to reduce damage to the cells upon landing, and it is easy to suppress drying of the cells.

There is no restriction | limiting in particular as minimum thickness of a coating liquid, Although it can select suitably according to the objective, 5 micrometers or more are preferable. When the minimum thickness of the coating liquid is 5 μm or more, it is advantageous in that the rate at which cells are killed when the cell ink lands can be reduced.
As a method of obtaining the minimum thickness of the coating liquid, for example, using a spectral interference laser displacement meter (SI-F01, manufactured by Keyence Corporation), the minimum value of the result of measuring the thickness of the coating liquid at 10 points randomly is determined as the coating liquid. The method of making it the minimum thickness of these etc. is mentioned.

  Examples of the method of injecting the coating liquid into the hole include a method of seeding the coating liquid and a method of injecting by the ink jet method. Among these, the method of injecting by an inkjet method is preferable. The method of injecting by the ink jet method is advantageous in that variation in the amount of the coating liquid injected into the hole can be reduced.

  In the above description, the structure layer has a hole portion having a through-hole shape. However, the structure layer is not limited thereto, and the structure layer may have a hole portion having a bottom shape with an opening at one end. In other words, in the cell holding member of the present invention, a plurality of holes are formed in the layer made of the cell non-adhesive material, and a coating solution for covering the bottom surface of the hole is added to the holes. Good. In this case, the hole has a coating liquid for covering the bottom surface of the hole. In addition, the bottom surface of the hole in the structural layer is a landing surface of ink droplets ejected by the ink jet method. In addition, you may form a hole in a base | substrate, without having a structure layer.

(Method for producing cell holding member)
The method for producing a cell holding member includes a step of laminating a structure layer in which a plurality of pores are formed in a layer made of a cell non-adhesive material on a plane of a substrate, and a step of attaching a cell adhesive material in the pores And a step of coating the surface of the substrate exposed from the hole with a coating liquid in the hole.

(Manufacturing method of cell arrangement plate)
The method for producing a cell arrangement plate includes a step of laminating a structure layer in which a plurality of holes are formed in a layer made of a cell non-adhesive material on a plane of a substrate, and a plane in the substrate exposed from the holes in the hole. A step of coating with a coating solution, and a step of discharging a cell suspension onto a plane coated with the coating solution. Moreover, the manufacturing method of a cell arrangement | positioning plate may further be made to include the process of attaching a cell-adhesive material in a hole.

<Method for producing cell holding member>
[Hole formation of structural layer]
FIG. 1A is an explanatory diagram illustrating an example of a state before a hole is formed in a structural layer by laser processing.
As shown in FIG. 1A, by attaching a mask film M or the like to the laser incident side surface of the structural layer 110, it is possible to suppress the processing powder generated at the time of laser processing. Can be improved. Moreover, since the workability can be improved by using the mask film M having higher laser absorption than the structural layer 110, finer processing is possible and the cell resolution can be improved.
In addition, when the structural layer 110 is formed of an organic material, it is applied to a jig J having flatness such as glass in order to reduce variation in the height of each hole 120, and then bulked by thermosetting or the like. Is made. When the jig J is made of glass and PDMS having excellent biocompatibility is used as the structural layer 110, an interference material I such as a polyimide-based material is interposed between the jig J and the structural layer 110 so as not to be bonded by heat. It is preferable to provide it.

[Arrangement of structural layer on substrate]
FIG. 1B is an explanatory diagram showing an example of a state in which a structural layer in which holes are formed is arranged on a substrate.
As shown in FIG. 1B, when alignment of the structural layer 110 with respect to the base 130 is necessary, alignment is performed by forming alignment marks L on the base 130 and the structural layer 110. At this time, if a camera or the like is used, highly accurate alignment is possible. Further, even in the case of a three-dimensional tissue body, the position of each hole 120 (cell) can be aligned by aligning each structural layer 110 with the base body 130.
In addition, when the hole part of the through-hole shape formed in the structure layer is made into the hole part which is an end-opening bottomed shape, it will become as shown to FIG. 1C.

(Cell holding member)
The cell holding member of the present invention is obtained by holding cells on the above-described cell holding member.
The cell holding member of the present invention includes the above-described cell holding member and cells arranged in the hole portion or the hole portion of the cell holding member, and further includes other members as necessary. In other words, cells are ejected into the hole or hole in the cell holding member by the ink jet method, and further includes other steps as necessary.

There is no restriction | limiting in particular as a cell to hold | maintain, Although it can select suitably according to the objective, It is preferable that the kind of cell distribute | arranged in the hole part or a hole part is 2 or more types.
Further, the cell adhesive material may be discharged in advance into the hole.

The cell holding member is preferably a cell arrangement plate.
Here, the cell arrangement plate means a plate in which what kind of cells are contained in which hole is known. When the cell holding member is a cell arrangement plate, it is advantageous in that, when screening with a predetermined drug, it is easy to obtain a result in which the experimental conditions are not easily affected regardless of the skill of the experimenter. In addition, since the cell arrangement plate can be reduced in size as compared with general wells, the amount of a predetermined drug used can be reduced when screening.

  There is no restriction | limiting in particular as culture time of a cell, Although it can select suitably according to the objective, 24 hours or more are preferable from a viewpoint of making it closely_contact | adhere with a cell adhesive material.

<Other members>
There is no restriction | limiting in particular as another member, Although it can select suitably according to the objective, The protective cover which covers the cell holding member and the cell holding member is preferable. A protective cover is advantageous in that it can suppress contamination and can be stacked.
Examples of the protective cover include a sheet-like cover and a lid that seals the hole.

[Method for producing cell holding member]
Hereinafter, a method for producing a cell holding member will be described with reference to the drawings. In the drawings, the same components are denoted by the same reference numerals, and redundant description may be omitted.

<Droplet forming device>
FIG. 2 is a schematic cross-sectional view showing an example of a droplet forming apparatus. Hereinafter, the “droplet forming apparatus” may be referred to as an “inkjet head”.
As shown in FIG. 2, the droplet forming apparatus 1 outputs a drive signal to the liquid chamber 2 that stores the liquid, the membrane 3 in which the discharge holes (nozzles) 3 a are formed, the piezoelectric element 4, and the piezoelectric element 4. And a drive unit 5 for
In the present embodiment, for the sake of convenience, the side where the liquid level of the liquid chamber 2 exists is the upper side, and the side where the piezoelectric element 4 exists is the lower side. In each part, the surface on which the liquid chamber 2 is present is the upper surface, and the surface on which the piezoelectric element 4 is present is the lower surface.

The liquid chamber 2 has a membrane 3 at the bottom and can accommodate the cell ink A.
There is no restriction | limiting in particular as the cell ink A, According to the objective, it can select suitably.
Examples of the material of the liquid chamber 2 include metal, silicon, and ceramic.
There is no restriction | limiting in particular as a magnitude | size of the liquid chamber 2, According to the objective, it can select suitably.
There is no restriction | limiting in particular as the quantity of the cell ink A which can be accommodated in the liquid chamber 2, It can select suitably according to the objective, For example, it is good also as 1 microliter-1 mL, and the cell in which the cell ink A disperse | distributed the cell. In the case of a suspension or the like, it may be 1 μL to 50 μL.

The membrane 3 is disposed as the bottom of the liquid chamber 2 and is fixed to the end of the lower surface of the liquid chamber 2. A discharge hole 3 a that is a through hole is formed at substantially the center of the membrane 3, and the cell ink A accommodated in the liquid chamber 2 is discharged as a droplet D from the discharge hole 3 a by deformation of the membrane 3.
The membrane 3 is deformed by the piezoelectric element 4.

The shape of the membrane 3 when viewed in plan is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include a circle, an ellipse, and a rectangle, and the shape corresponds to the shape of the bottom of the liquid chamber 2. Those are preferred.
There is no restriction | limiting in particular as a material of the membrane 3, According to the objective, it can select suitably, For example, a metal material, a ceramic material, a polymeric material etc. are mentioned, It should be a material with a certain amount of hardness. preferable. When the material of the membrane 3 has a certain degree of hardness, the membrane 3 does not easily vibrate and it is easy to suppress vibration.
Examples of the metal material include stainless steel, nickel, and aluminum.
Examples of the ceramic material include silicon dioxide, alumina, zirconia, and the like.

In the present embodiment, the discharge hole 3 a is formed in a substantially circular shape substantially at the center of the membrane 3.
There is no restriction | limiting in particular as a shape of the discharge hole 3a, According to the objective, it can select suitably, For example, a perfect circle shape etc. are mentioned.
When the shape of the discharge hole 3a is a perfect circle, the diameter of the discharge hole 3a is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 20 μm or more and 200 μm or less. When the diameter of the discharge hole 3a is within a preferable range, it is advantageous in terms of stabilizing the shape of the discharged droplet.

The piezoelectric element 4 is disposed on the lower surface side of the membrane 3.
The shape of the piezoelectric element 4 is preferably a shape that matches the shape of the membrane 3. For example, when the shape of the membrane 3 when viewed from above is circular, it is preferable to dispose the annular (ring-shaped) piezoelectric element 4 having a planar shape around the discharge hole 3a.

The piezoelectric element 4 has, for example, a structure in which electrodes for applying a voltage are provided on the upper surface and the lower surface of a piezoelectric material. By applying a voltage to the upper and lower electrodes of the piezoelectric element 4, a compressive stress is applied in the lateral direction of the paper surface, and the membrane. 3 can be deformed or vibrated.
Examples of the piezoelectric material include lead zirconate titanate, bismuth iron oxide, metal niobate, barium titanate, and materials obtained by adding metals or different oxides to these.

  In the present embodiment, the piezoelectric element 4 deforms the membrane 3. However, the present invention is not limited to this, and other modes may be employed. As another aspect, for example, a material having a linear expansion coefficient different from that of the membrane 3 may be attached to the membrane 3 and heated to deform the membrane 3 using the difference in linear expansion coefficient. In the case of this aspect, it is preferable that a heater is disposed in the vicinity of materials having different linear expansion coefficients, and the membrane 3 is deformed or vibrated by ON / OFF of the heater.

  The drive unit 5 can output an ejection signal Pj as a drive signal to the piezoelectric element 4. The drive unit 5 outputs the ejection signal Pj to the piezoelectric element 4, thereby deforming the membrane 3 and ejecting the cell ink A accommodated in the liquid chamber 2 as the droplet D. In addition, by performing deformation of the membrane 3 with the discharge signal Pj set at a predetermined cycle, the membrane 3 can be discharged by resonance vibration.

  Further, the drive unit 5 can output a suppression signal Ps as a drive signal to the piezoelectric element 4. The drive unit 5 can suppress the residual vibration of the membrane 3 by outputting the suppression signal Ps to the piezoelectric element 4 after discharging the droplet D. As a result, the droplet forming apparatus 1 can quickly suppress the residual vibration of the membrane 3 without waiting for the residual vibration to naturally decay, so that the number of ejections per unit time can be increased. Furthermore, since the droplet forming apparatus 1 can reduce the occurrence of defects due to residual vibrations such as satellites from which droplets are separated and mist that is finely scattered, it is possible to control a smaller droplet amount.

[Droplet formation process (operation) by droplet forming device]
A process in which the droplet forming apparatus forms droplets will be described.
FIG. 3 is an explanatory diagram illustrating an example of a discharge signal and a suppression signal. 4A to 4C are explanatory diagrams illustrating an example of the operation of the droplet forming apparatus.
When the ejection signal Pj and the suppression signal Ps shown in FIG. 3 are output to the piezoelectric element 4, the droplet D is formed as shown in FIGS. 4A to 4C, and the residual vibration of the membrane 3 is suppressed. it can.

  First, when the ejection signal Pj shown in FIG. 3 is output, the membrane 3 is rapidly deformed as shown in FIG. 4A, so that the cell ink A accommodated in the liquid chamber 2 moves downward from the ejection holes 3a. Extruded.

There is no restriction | limiting in particular as the discharge signal Pj, According to the objective, it can select suitably. The ejection signal Pj is preferably a signal based on the natural vibration period T ₀ of the membrane 3 in that the cellular ink A is ejected at a lower voltage by the membrane 3. In the present embodiment, the output time of the ejection signal Pj, by words the time for raising the applied voltage and T _0/2, can be discharged at a lower voltage cells ink A in the membrane 3.
The natural vibration period To of the membrane 3 can be measured using, for example, a laser Doppler vibrometer (LV-1800, manufactured by Ono Sokki Co., Ltd.).

  Next, in the time during which the constant voltage shown in FIG. 3 is applied to the piezoelectric element 4, that is, the interval time Ti from the end of the output of the ejection signal Pj to the start of the output of the suppression signal Ps, FIG. As shown in FIG. 4, the droplet D from the discharge hole 3a grows. In this interval time Ti, the membrane 3 remains vibrated due to deformation at the time of ejection.

  When the suppression signal Ps shown in FIG. 3 is output, when the membrane 3 returns to its original state as shown in FIG. 4A, droplets D are formed and residual vibration of the membrane 3 is suppressed. The

The suppression signal Ps is not particularly limited and may be appropriately selected according to the purpose. However, a signal based on the natural vibration period To of the membrane 3 is preferable. When the suppression signal Ps is a signal based on the natural vibration period To of the membrane 3, the residual vibration of the membrane 3 can be suppressed with low energy and in a short time.
The voltage of the suppression signal Ps is set below the maximum voltage of the ejection signal Pj. When the voltage of the suppression signal Ps exceeds the maximum voltage of the ejection signal Pj, unnecessary vibration is generated by the suppression signal Ps, and the residual vibration is likely to be prolonged.

  In the above description, among the pulsed drive signals shown in FIG. 3, the voltage signal at the rising edge is set as the ejection signal and the voltage signal at the falling edge is set as the suppression signal. The suppression signal may be as shown in FIGS. 5A and 5B.

As shown in FIG. 5A, the ejection signal Pj may be, for example, a triangular wave, a sine wave, a rectangular wave, or a triangular wave with a gentle edge through a low-pass filter. In this case, it is preferable to match the period of a triangular wave or the like with the natural vibration period To of the membrane 3.
The suppression signal Ps is not particularly limited as long as it is a signal based on the natural vibration period To of the membrane 3, and can be appropriately selected according to the purpose. For example, via a triangular wave, sine wave, rectangular wave, or low-pass filter A triangular wave with a gentle edge may be used. In this case, the period of a triangular wave or the like is adjusted to the natural vibration period To of the membrane 3.

  Further, as shown in FIG. 5B, the droplet forming apparatus 1 may output a plurality of suppression signals Ps when the residual vibration of the membrane 3 cannot be suppressed only by outputting the suppression signal Ps once. Also in this case, the period of the triangular wave or the like is adjusted to the natural vibration period To of the membrane 3.

[Cell holding member manufacturing apparatus equipped with a droplet forming apparatus]
FIG. 6 is a schematic view showing an example of a cell holding member manufacturing apparatus equipped with a plurality of droplet forming apparatuses.
As shown in FIG. 6, the cell holding member manufacturing apparatus 200 includes at least a stage unit 40 that fixes the substrate, the droplet forming apparatus 1 and the droplet forming apparatus 11 that discharge cell ink, and the coating liquid discharge head 21. The cell adhesive material discharge head 31 is mounted.
The coating liquid discharge head 21 includes a drive unit 22 and a liquid chamber 23 that holds the coating liquid. Further, the cell adhesive material discharge head 31 includes a drive unit 32 and a liquid chamber 33 that holds the cell adhesive material ink.
In addition, you may change the quantity of each apparatus and each head as needed.
As the cell adhesive material discharge head 31, the droplet forming apparatus 1 may be used, or another inkjet head may be used.
Examples of other inkjet heads include an industrial inkjet head (MH2420). A holding member that holds the inkjet head and a mechanism that controls the relative position between the stage and the inkjet head may also be mounted.

(Description of manufacturing method of tissue)
-Preparation of cell adhesive material ink-
The cell adhesive material is dissolved in pure water and the cell adhesive material ink B is prepared. At this time, the content of the cell adhesive material is preferably 0.001% to 5%, and more preferably 0.005% to 1%. When the content of the cell adhesive material is in a preferred range, it is advantageous in that the cell adhesion function is easily developed and the ink is easily ejected by an ink jet method.
Moreover, you may contain other additive materials, such as a wetting agent, a dispersing agent, and a pH adjuster as needed, and it is still more preferable if the boiling point is about room temperature to 90 degreeC.

-Discharge of cell adhesive material ink into the hole-
In order to improve the adhesion between the cells and the substrate 130, a cell adhesive material is disposed on the surface of the substrate 130. Examples of the method of arranging the cell adhesive material include a method of seeding on the entire surface, a method of discharging into the hole 120 using the manufacturing apparatus 200 of FIG. Among these, the method using the manufacturing apparatus 200 of FIG. 6 is preferable from the viewpoint of accurately discharging to a desired position.

FIG. 7 is an explanatory diagram illustrating an example of an operation of discharging the cell adhesive material ink.
As shown in FIG. 7, by using the cell adhesive material discharge head 31, the cell adhesive material ink B is discharged into the hole 120, thereby controlling the cell adhesive material in a quantitative manner along the pattern shape. Is possible. At this time, the drive signal is not particularly limited as long as the cell adhesive material ink B can be ejected from the nozzle. The cell adhesive material may be disposed before the structural layer is laminated on the substrate.

-Injection of coating liquid into the hole-
FIG. 8A is an explanatory diagram illustrating an example of an operation of discharging the coating liquid.
As shown in FIG. 8A, by using the coating liquid discharge head 21, the coating liquid C is controlled in a fixed amount along the pattern shape by discharging the coating liquid C into the hole 120 having the base 130 as the bottom surface. Is possible. The driving signal is not particularly limited as long as the coating liquid C can be discharged from the nozzle. As shown in FIG. 8B, it is preferable to discharge the cell adhesive material ink B before discharging the coating liquid C.
As described above, before discharging the cell ink A from the cell discharge head 1, the coating liquid C is injected into the hole 120 in advance in order to suppress the drying of the cells and the impact to the cells when the substrate is landed. .

-Cell ink injection into the hole-
9A and 9B are explanatory diagrams illustrating an example of an operation of ejecting cell ink.
As shown in FIGS. 9A and 9B, by using the cell discharge head 1, the cell ink A is quantitatively controlled along the pattern shape by discharging the cell ink A into the hole 120 having the base 130 as the bottom surface. Is possible.
The relationship between the depth of the coating liquid C and the cell viability when the droplet ejection speed of the cell ink A from the cell ejection head 1 is 0.1 m / s, 1.0 m / s, and 10 m / s is shown below. It was evaluated according to the criteria. The results are shown in Table 1.
○: Average cell survival rate per pore is 75% or more ×: Average cell survival rate per pore is less than 75%

From the results of Table 1, when the discharge speed is in the range of 0.1 m / s to 10 m / s, the coating liquid C is injected into the hole 120 even if the liquid thickness is about 5 μm regardless of the discharge speed. Thus, it was confirmed that the cell viability was significantly improved as compared with the case where the coating liquid C was not present.
Considering the suppression of cell drying and the impact on the cells upon landing, it is more effective to increase the amount of the coating liquid C. However, the cell ink A that can be held in the pores 120 is relatively reduced, and the cell density is reduced. Since it will fall, you may adjust the liquid quantity of the coating liquid C according to the shape of a desired structure | tissue.

The cell ink A is discharged from the cell discharge head 1 into the hole 120. The coalescence of adjacent droplets is suppressed due to the partition of the structural layer 110. Moreover, since the number of droplets to the same location can be increased, the cell density can also be increased. In addition, since the coating liquid C is injected into the hole 120, the influence on the cells when landing on the base 130 is suppressed. Further, the cell viability can be improved by using the coating liquid C as a solution containing components necessary for the formation and maintenance of the tissue body and preventing the drying and adjusting the external environment such as osmotic pressure. Can cope with expansion and complexity.
In addition, it is possible to construct a three-dimensional complex tissue by repeating the above steps. Whether the structural layer 110 is peeled or left for each layer configuration is not limited because it depends on the use of the tissue structure and the biocompatibility of the structural layer.

Here, FIG. 10 shows an explanatory diagram showing an example of the operation of discharging the cell ink by the conventional ink jet method.
As shown in FIG. 10, since the cells are dispersed in the cell ink, when the cell resolution and cell density are increased, when the droplets are landed on the substrate 130, if the distance between adjacent droplets is close, the liquid Droplets are united, and the cell position and the number of cells are not targeted. For this reason, when several cell types unite, the quality of a tissue body falls. Further, since the droplets of the cell ink discharged from the cell discharge head 1 directly collide with the base body 130, there is a high possibility that the cells are damaged. In addition, since the cell ink A is exposed to the atmosphere after being discharged and starts to dry, it is conceivable that drying of the previously discharged cells proceeds due to the enlargement and complication of the tissue, and the cell survival rate deteriorates.

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

Example 1
<Formation of hole in structure layer-formation of cell adhesive material>
After four layers of masking tape (N-380, manufactured by Nitto Denko Corporation) with a thickness of 70 μm are laminated on the slide glass (thickness: 280 μm), a laser processing machine (Spectra-Physics H10-106QW J80-8ss42 Hippo ), A structural layer was formed with holes of φ100 μm as shown in FIGS. 11A and 11B at a pitch of 150 μm. In addition, an alignment mark of φ100 μm was also formed so that the alignment with the evaluation substrate as a base was possible.
Next, after peeling off the four-layer masking tape from the slide glass, alignment was performed using the alignment mark of the evaluation substrate and the alignment mark of the masking tape to form a desired evaluation substrate.
Then, Matrigel (Corning Matrigel basement membrane matrix) as a cell adhesive material was prepared with a cell adhesive material solution so as to have a weight ratio of 0.1%. The cell adhesive solution was filled in the liquid chamber of the cell adhesive material discharge head, and droplets were discharged from the nozzle into each hole at 900 pL (300 pL × 3 droplets) and dried to form a cell adhesive material.

<Preparation of cell ink>
A green fluorescent dye (trade name: Cell Tracker Green, manufactured by Life Technologies) is dissolved in dimethyl sulfoxide at a concentration of 10 mmol / L (mM), mixed with serum-free Dulbecco's modified Eagle's medium (produced by Life Technologies), and concentrated. A serum-free medium containing 10 μmol / L (μM) of a fluorescent dye was prepared.

Next, 5 mL of a fluorescent dye-containing serum-free medium was added to a dish of cultured NIH / 3T3 cells (Clone 5611, JCRB Cell Bank), and an incubator (KM-CC17RU2, Panasonic Corporation, 37 ° C., 5 vol% CO _2). For 30 minutes. Thereafter, the supernatant was removed using an aspirator. 5 mL of phosphate buffered saline (manufactured by Life Technologies, hereinafter also referred to as “PBS (−)”) was added to the dish, and PBS (−) was removed by suction with an aspirator, and the surface was washed. After washing with PBS (−) twice, 2 mL of 0.05 mass% trypsin-0.05 mass% EDTA solution (manufactured by life technologies) was added to each dish and added for 5 minutes in the incubator. After warming and detaching the cells from the dish, it contains 10% by weight fetal bovine serum (hereinafter also referred to as “FBS”) and 1% by weight antibiotics (Antibiotic-Antilytic Mixed Solution (100x), manufactured by Nacalai Tesque, Inc.) 4 mL of D-MEM was added, the cell ink in which the trypsin was deactivated was transferred to one 50 ml centrifuge tube, and centrifuged (trade name: H-19FM, manufactured by KOKUSAN, 1200 rpm, 5 minutes, 5 ° C.). And the supernatant was removed using an aspirator. After removal, 2 mL of D-MEM containing 10% by mass FBS and 1% by mass antibiotics was added to the centrifuge tube, and pipetting was performed gently to disperse the cells and obtain a cell ink.

10 μL from the lifetime cell ink was taken out into an Eppendorf tube, 70 μL of the medium was added, 10 μL was taken out into another Eppendorf tube, and 10 μL of 0.4 mass% trypan blue staining solution was added and pipetting was performed. 10 μL of the stained cell ink is taken out and placed on a plastic slide made of PMMA, and the number of cells is determined by measuring the number of cells using a trade name: Countess Automated Cell Counter (manufactured by Invitrogen). Got. PBS (−) was used as the dispersion medium. Glycerin (molecular biology grade, Wako Pure Chemical Industries, Ltd.) as a wetting agent is dissolved in the PBS (−) so as to have a mass ratio of 0.5%, and the NIH / 3T3 cell ink becomes 6 × 10 ⁶ cells / mL. A cell ink was obtained by dispersing in the dispersion medium.

<Injection of coating liquid into pores-formation of cell pattern>
Next, a medium (D-MEM containing 10% by mass FBS and 1% by mass antibiotic) is filled in the liquid chamber of the coating liquid discharge head as a coating liquid in the hole of the evaluation substrate on which the cell adhesive material is formed. Then, 300 pL (300 pL × 1 drop) of liquid droplets was discharged from the nozzle into each hole (liquid depth 40 μm).
Subsequently, the prepared cell ink is filled in the liquid chamber of the cell discharge head, and droplets are discharged from the nozzle diameter into each hole at a droplet speed of 1.0 m / s, aiming at 10 cells and 1,500 pL (300 pL × 5 drops) were discharged.
After the last cell ink landed, it was allowed to stand for 10 minutes, and then cultured for 24 hours in an incubator (KM-CC17RU2, Panasonic Corporation, 37 ° C., 5 vol% CO ₂ environment).
The following evaluation was performed on the obtained cell pattern.

[Cell pattern shape accuracy (evaluation of pattern resolution)]
The accuracy of the shape of the cell pattern was evaluated according to the following criteria using a fluorescence microscope (CKX41, manufactured by Olympus).
○: Pitch of cell adhesion pattern is 300 μm or less ×: Pitch of cell adhesion pattern is larger than 300 μm

[Probability that a predetermined number of cells can be placed in a predetermined cell adhesion part (evaluation of cell number control)]
The number of cells held in each hole of φ100 μm was calculated based on the fluorescence image and evaluated according to the following criteria. The evaluation object was all the holes discharged aiming at 10 cells.
○: 80% or more of the holes where the number of cells is ± 20% or less with respect to the target number ×: Less than 80% of the holes where the number of cells is ± 20% or less with respect to the target number In the example, the target number is used, but the average number of cells may be used.

[Probability that the number of arranged cells is the desired number (evaluation of cell density)]
The number of cells held in each hole of φ100 μm was calculated based on the fluorescence image and evaluated according to the following criteria. The evaluation target was 20 holes ejected aiming at 10 cells.
○: 80% or more of pores having 8 or more cells ×: Less than 80% of pores having 8 or more cells

[Viability of landed cells after elapse of a predetermined time (evaluation of cell viability)]
A stock solution in which propidium iodide (Cellstein-PI, Dojindo Laboratories) is dissolved in distilled water at a concentration of 1 mg / mL as a nuclear stain for dead cells is 10 μL in 5 mL against a cell pattern that is allowed to stand for 10 minutes. Were added. Then, after culturing for 10 minutes in an incubator, the ratio of dead cells present in each hole was calculated using a fluorescence microscope and evaluated according to the following criteria. The evaluation target was 20 holes ejected aiming at 10 cells.
○: Average cell survival rate per pore is 75% or more ×: Average cell survival rate per pore is less than 75%

(Example 2)
In Example 1, it evaluated similarly to Example 1 except having changed the structural layer from the masking tape to PDMS. The results are shown in Table 2.
The PDMS bulk formation method was performed by applying PDMS (SYLGARD 184, manufactured by Toray Dow Corning Co., Ltd.) obtained by mixing and defoaming a main agent and a curing agent at a ratio of 10: 1 to a slide glass (MICRO SLIDE GLASS S1112 manufactured by Matsunami Glass Industrial Co., Ltd.) Heat curing was performed at 70 ° C. for 2 hours. Moreover, the hole as shown in FIG. 12A and FIG. 12B was formed by laser processing of PDMS of the structural layer.

(Example 3)
In Example 2, it evaluated similarly to Example 2 except having changed the cell-adhesive material into the mass ratio 0.05% collagen (Cellmatrix Type1-A, Nitta gelatin). The results are shown in Table 2.

Example 4
In Example 2, evaluation was performed in the same manner as in Example 2 except that the cell adhesive material was changed to fibrin. Fibrinogen (fibrinogen from bovine plasma F8630, SIGMA-ALDRICH) mass ratio 0.2% and thrombin (Thrombin from bovine plasma T4648, SIGMA-ALDRICH) mass ratio 0.2% Obtained by standing. The results are shown in Table 2.

(Example 5)
In Example 2, it evaluated similarly to Example 2 except having changed the cell adhesive material into RGD peptide (for biochemistry, Wako Pure Chemical Industries). The results are shown in Table 2.

(Example 6)
Evaluation was performed in the same manner as in Example 2 except that the cell adhesive material was changed to gelatin fine particles (APH-250, manufactured by Nitta Gelatin) having a mass ratio of 2% in Example 2. The results are shown in Table 2.

(Example 7)
In Example 2, evaluation was performed in the same manner as in Example 2 except that the number of droplets in each hole of the cell adhesive material was changed to 1. The results are shown in Table 2.
FIG. 13 is an explanatory diagram showing an example of the operation of discharging the cell adhesive material ink in Example 7.
As shown in FIG. 13, by adjusting the amount of the cell adhesive material that lands on the substrate, the formation of the cell adhesive material on the side wall of the partition wall can be suppressed. Thereby, since all the discharged cells can be formed on the substrate, the number of cells formed on the substrate can be controlled with higher accuracy.
In addition, when the cells formed on the substrate are observed with a microscope, it is possible to suppress measurement errors on the partition wall that have been dispersed due to the meniscus of the liquid.

(Example 8)
In Example 2, the shape of the hole was evaluated in the same manner as in Example 2 except that the opening area on the surface on the substrate side was smaller than the opening area on the other side. Results are shown.
FIG. 14 is an explanatory diagram showing the operation of discharging the cell ink in the eighth embodiment.
As shown in FIG. 14, it is possible to increase the hole area on the droplet discharge side while maintaining the cell resolution and cell density, and to suppress variations in the number of cells due to variations in landing positions in the cell discharge head. It becomes possible.

Example 9
In Example 2, a cell ink 1 in which NIH / 3T3 is dispersed and a cell ink 2 in which normal human derma fibroblast (CC-2509, Lonza, hereinafter referred to as “NHDF”) is dispersed are prepared. Evaluation was performed in the same manner as in Example 2 except that the evaluation of a plurality of cells alternately arranged in the part was performed.
Cell ink 2 was prepared in the same manner as cell ink 1 except that the cells were changed to NHDF and the dye was changed to a red fluorescent dye (Cell Tracker RED, Life Technologies). The results are shown in Table 2. Moreover, the photograph at the time of arrange | positioning dyeing | staining cell ink into two types is shown in FIG.

Further, in Example 9, since a plurality of types of cells are handled, the pattern accuracy in a state where the cells are adhered is evaluated using a fluorescence microscope (CKX41, manufactured by Olympus) according to the following criteria. went.
○: Pitch where alternately arranged cell patterns are 300 μm or less ×: Pitch where alternately arranged cell patterns are larger than 300 μm

(Comparative Example 1)
In Example 9, evaluation was performed in the same manner as in Example 9 except that the structural layer was not disposed on the substrate. The results are shown in Table 2.
In Comparative Example 1, since there is no hole formed in the structural layer, adjacent droplets are united. Since cells could not adhere to the target attachment position, pattern formation could not be achieved with the target cell resolution and cell density. In addition, since the coating solution could not be retained, cell viability could not be achieved due to collision with the substrate.

(Comparative Example 2)
In Example 9, it evaluated similarly to Example 9 except not having formed the coating liquid. The results are shown in Table 2.
In Comparative Example 2, since there was a hole formed in the structural layer, in addition to cell resolution, cell number control, and cell density, patterning of multiple cells could be achieved. The standard of cell viability could not be achieved because there was no coating solution.

(Comparative Example 3)
In Example 9, instead of arranging the cells by the inkjet method, cell inks were prepared for NIH / 3T3 and NHDF, respectively, and manually, 5 × 10 ⁴ cells / mL each, 1 × 10 ⁵ cells / mL in total. Evaluation was performed in the same manner as in Example 9 except that seeding was performed. The results are shown in Table 2.
In Comparative Example 3, if the cells are not arranged by the ink jet method, the position accuracy of seeding deteriorates, so the cells cannot be arranged accurately in the targeted hole, and the hole on the adjacent hole or on the structural layer. Since the cells are arranged outside and interfere with the droplets in the adjacent holes, they are unable to achieve the cell number control standard. In addition, NIH / 3T3 and NHDF were combined with each other, and the standard could not be achieved.
In addition, the comparative example 3 corresponds to an example as a “cell holding member” which is a state before seeding cells.

As described above, the cell holding member of the present invention has a structural layer having a plurality of pores in the substrate, so that when a cell suspension is discharged into the plurality of pores, cells are collected for each of the pores. Since they can be held separately, it is possible to prevent the landing droplets from being united and the position of the cells to shift.
Further, in the cell holding member of the present invention, by holding the cell suspension that has landed in the hole, the surface area of the cell suspension in contact with the air can be reduced, so that the drying of the cells is suppressed. Can do.
Furthermore, in the cell holding member of the present invention, by having a coating liquid that covers the flat surface of the substrate exposed from the hole in the hole, damage when cells discharged by the ink jet method land, In addition, cell drying can be suppressed.

As an aspect of this invention, it is as follows, for example.
<1> a step of laminating a structure layer in which a plurality of pores are formed in a layer made of a cell non-adhesive material on a plane of a substrate;
Coating the flat surface of the substrate exposed from the hole with a coating liquid in the hole; and
Discharging the cell suspension onto the plane coated with the coating solution;
A method for producing a cell arrangement plate, comprising:
<2> The method for producing a cell arrangement plate according to <1>, further including a step of attaching a cell adhesive material in the hole.
<3> The method for producing a cell arrangement plate according to <2>, wherein the cell adhesive material is not in contact with a side surface of the hole.
<4> Any one of <1> to <3>, wherein the flat surface of the substrate exposed from the hole in the structural layer is a landing surface of a droplet of a cell suspension discharged by an inkjet method. A method for producing the cell arrangement plate according to the above.
<5> The method for producing a cell arrangement plate according to any one of <1> to <4>, wherein the non-cell-adhesive material is a water-repellent material or a silicon-containing material.
<6> The method for producing a cell arrangement plate according to any one of <1> to <5>, wherein the minimum thickness of the coating solution is 5 μm or more.
<7> The method for producing a cell arrangement plate according to any one of <1> to <6>, wherein the coating solution is a cell culture medium.
<8> The method for producing a cell arrangement plate according to any one of <1> to <7>, wherein a pitch interval between the holes adjacent to each other is 300 μm or less.
<9> The cell arrangement according to any one of <1> to <8>, wherein the number of cells arranged in the hole is ± 20% of an average value, and the hole is 80% or more It is a manufacturing method of a plate.
<10> a step of laminating a structure layer in which a plurality of pores are formed in a layer made of a non-cell-adhesive material on a plane of a substrate;
A step of attaching a cell adhesive material in the hole;
Coating the flat surface of the substrate exposed from the hole with a coating liquid in the hole; and
It is a manufacturing method of the member for cell holding characterized by including.
<11> On the plane of the substrate having at least a plane,
A structural layer formed of a non-cell-adhesive material and having a plurality of pores;
A cell holding member, wherein a cell adhesive material is adhered in the hole.
<12> The cell holding member according to <11>, wherein the hole includes a coating solution that covers the flat surface of the substrate exposed from the hole.
<13> The cell holding member according to <12>, wherein the cell adhesive material is not in contact with a side surface of the hole.
<14> The cell holding according to any one of <11> to 13>, wherein the plane of the substrate exposed from the hole in the structural layer is a landing surface of an ink droplet ejected by an inkjet method. It is a member for use.
<15> The cell holding member according to any one of <11> to <14>, wherein a minimum thickness of the coating liquid is 5 μm or more.
<16> The cell holding member according to any one of <11> to <15>, wherein a pitch interval between the holes adjacent to each other is 300 μm or less.
<17> The cell holding member according to any one of <11 to <16>,
Cells arranged in the plurality of holes in the cell holding member;
It is a cell arrangement | positioning plate characterized by having.
<18> The cell arrangement plate according to <17>, wherein the number of cells arranged in the hole is ± 20% of an average value, and the hole is 80% or more.
<19> The cell arrangement plate according to any one of <17> to <18>, wherein cells arranged in the hole are discharged by an ink jet method.
<20> The cell arrangement plate according to any one of <17> to <19>, wherein the number of types of cells arranged in the hole is two or more.

  The method for producing a cell arrangement plate according to any one of <1> to <9>, the method for producing a cell holding member according to <10>, or any one of <11> to <16>. According to the cell holding member and the cell arrangement plate according to any one of <17> to <20>, the conventional problems can be solved and the object of the present invention can be achieved.

JP 2012-187072 A JP 2009-131240 A JP 2013-099271 A JP 2010-233456 A

1,11 Droplet forming device (cell ejection head)
DESCRIPTION OF SYMBOLS 21 Coating liquid discharge head 31 Cell-adhesive material discharge head 100 Cell holding member 110 Structural layer 120 Hole part 121 Hole part 130 Substrate A Cell ink (ink)
B Cell adhesive material ink C Coating liquid L Alignment mark

Claims (20)

Laminating a structure layer in which a plurality of pores are formed in a layer made of a cell non-adhesive material on a plane of a substrate;
Coating the flat surface of the substrate exposed from the hole with a coating liquid in the hole; and
Discharging the cell suspension onto the plane coated with the coating solution;
A method for producing a cell arrangement plate, comprising:   The manufacturing method of the cell arrangement | positioning plate of Claim 1 which further includes the process of making a cell-adhesive material adhere in the said hole.   The manufacturing method of the cell arrangement | positioning plate of Claim 2 with which the said cell adhesive material is not contacting the side surface of the said hole.   The cell arrangement plate according to any one of claims 1 to 3, wherein the flat surface of the substrate exposed from the hole in the structural layer is a landing surface of a droplet of a cell suspension discharged by an inkjet method. Manufacturing method.   The manufacturing method of the cell arrangement | positioning plate in any one of Claim 1 to 4 whose said cell non-adhesive material is a water repellent material or a silicon-containing material.   The manufacturing method of the cell arrangement | positioning plate in any one of Claim 1 to 5 whose minimum thickness of the said coating liquid is 5 micrometers or more.   The manufacturing method of the cell arrangement | positioning plate in any one of Claim 1 to 6 whose said coating liquid is a cell culture medium.   The manufacturing method of the cell arrangement | positioning plate in any one of Claim 1 to 7 whose pitch space | interval of the said adjacent hole part is 300 micrometers or less.   The method for producing a cell arrangement plate according to any one of claims 1 to 8, wherein the number of cells arranged in the hole is ± 20% relative to an average value, and the hole is 80% or more. Laminating a structure layer in which a plurality of pores are formed in a layer made of a cell non-adhesive material on a plane of a substrate;
A step of attaching a cell adhesive material in the hole;
Coating the flat surface of the substrate exposed from the hole with a coating liquid in the hole; and
The manufacturing method of the member for cell holding characterized by including. On the plane of the substrate having at least a plane;
A structural layer formed of a non-cell-adhesive material and having a plurality of pores;
A cell holding member, wherein a cell adhesive material is adhered in the hole.   The cell holding member according to claim 11, wherein the hole has a coating solution that covers the flat surface of the substrate exposed from the hole.   The cell holding member according to claim 12, wherein the cell adhesive material is not in contact with a side surface of the hole.   The cell holding member according to any one of claims 11 to 13, wherein the flat surface of the substrate exposed from the hole in the structural layer is a landing surface of an ink droplet ejected by an ink jet method.   The cell holding member according to claim 11, wherein a minimum thickness of the coating solution is 5 μm or more.   The cell holding member according to any one of claims 11 to 15, wherein a pitch interval between the holes adjacent to each other is 300 µm or less. The cell holding member according to any one of claims 11 to 16,
Cells arranged in the plurality of holes in the cell holding member;
The cell arrangement | positioning plate characterized by having.   18. The cell arrangement plate according to claim 17, wherein the number of cells arranged in the hole is ± 20% relative to the average value, and the hole is 80% or more.   The cell arrangement | positioning plate in any one of Claims 17-18 formed by discharging the cell distribute | arranged to the said hole part by the inkjet method.   The cell arrangement plate according to any one of claims 17 to 19, wherein the number of types of cells arranged in the hole is two or more.