JP2019162097A - Cell tissue producing method, producing apparatus, and producing program - Google Patents

Abstract

To provide a cell tissue producing method with a high survival rate of cells located thereon.SOLUTION: Provided is a cell tissue producing method including: a cell adhesive portion forming step of forming a cell adhesive portion formed of a cell adhesive material and having a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface; and a cell locating step of discharging a cell suspension containing at least cells and a cell drying inhibitor onto the cell adhesive portion in the form of a liquid droplet to locate the cells.SELECTED DRAWING: Figure 4F

Description

  The present invention relates to a method for manufacturing a cell tissue body, a manufacturing apparatus, and a manufacturing program.

In recent years, with the advancement of stem cell technology, development of technology for artificially forming tissue bodies composed of cells has been performed.
Since the technology to arrange cells arbitrarily is indispensable for artificial formation of tissues, various efforts have been made, and cell sheet method, spheroid lamination method, gel extrusion method, ink jet method, etc. are known. ing.
On the other hand, efforts are being made to devise the substrate itself in order to place cells.
For example, a layer of an aqueous cell support material precursor solution containing biocompatible particles and a cell support material precursor is formed on a substrate. Next, on the layer of the cell support material precursor aqueous solution, a cell support material precursor gelled aqueous solution that gels the cell support material precursor when contacted with the cell support material precursor aqueous solution is applied. Then, a method for producing a three-dimensional culture structure has been proposed in which a cell layer-forming material containing cells is provided on a layer of cell support material, thereby allowing cells to adhere to the cell support material (for example, patents). Reference 1). And in this proposal, it is described that a gel-like polysaccharide may be included as a cell support material.

  An object of the present invention is to provide a method for producing a cellular tissue body in which the survival rate of the arranged cells is high.

  The method for producing a cell tissue body of the present invention as a means for solving the above-described problem is that a cell adhesion portion made of a cell adhesive material is formed in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface. Forming a cell adhesion portion to be formed, and disposing a cell suspension containing at least cells and a cell desiccation inhibitor as droplets to the cell adhesion portion to arrange the cells. .

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of a cell tissue body with the high survival rate of the arrange | positioned cell can be provided.

FIG. 1 is a schematic diagram showing an example of an ink jet head used for discharging droplets. FIG. 2A is a schematic diagram illustrating an example of an input waveform to the inkjet head. FIG. 2B is a schematic diagram illustrating an example of an input waveform to the inkjet head. FIG. 3A is a schematic diagram illustrating an example of a manufacturing apparatus for manufacturing a cellular tissue body. FIG. 3B is a schematic diagram illustrating another example of a manufacturing apparatus for manufacturing a cellular tissue body. FIG. 3C is a schematic diagram illustrating another example of a manufacturing apparatus for manufacturing a cellular tissue body. FIG. 3D is a schematic diagram illustrating another example of a manufacturing apparatus for manufacturing a cellular tissue body. FIG. 4A is a schematic diagram illustrating an example for explaining a method for producing a cell tissue body. FIG. 4B is a schematic diagram illustrating an example for explaining a method of manufacturing a cell tissue body. FIG. 4C is a schematic diagram illustrating an example for explaining a method of manufacturing a cell tissue body. FIG. 4D is a schematic diagram illustrating an example for explaining a method of manufacturing a cell tissue body. FIG. 4E is a schematic diagram illustrating an example for explaining a method of manufacturing a cell tissue body. FIG. 4F is a schematic diagram illustrating an example for explaining a method of manufacturing a cell tissue body. FIG. 5A is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body. FIG. 5B is a schematic diagram illustrating another example for explaining a method of manufacturing a cell tissue body. FIG. 5C is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body. FIG. 5D is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body. FIG. 6A is a schematic view illustrating another example for explaining a method for producing a cell tissue body (contact printing method). FIG. 6B is a schematic view showing another example for explaining a method for producing a cell tissue body (contact printing method). FIG. 6C is a schematic view showing another example for explaining a method for producing a cell tissue body (contact printing method). FIG. 6D is a schematic view showing another example for explaining a method for producing a cell tissue body (contact printing method). FIG. 6E is a schematic view showing another example for explaining a method for producing a cell tissue body (contact printing method). FIG. 6F is a schematic view illustrating another example for explaining a method for producing a cell tissue body (contact printing method). FIG. 7A is a schematic view showing another example for explaining a method for producing a cell tissue body (negative printing method (mask)). FIG. 7B is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (negative printing method (mask)). FIG. 7C is a schematic view showing another example for explaining a method for producing a cell tissue body (negative printing method (mask)). FIG. 7D is a schematic view showing another example for explaining a method for producing a cell tissue body (negative printing method (mask)). FIG. 7E is a schematic view showing another example for explaining a method for producing a cell tissue body (negative printing method (mask)). FIG. 7F is a schematic view showing another example for explaining a method for producing a cell tissue body (negative printing method (mask)). FIG. 8A is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (negative printing method (IJ)). FIG. 8B is a schematic diagram showing another example for explaining a method for producing a cell tissue body (negative printing method (IJ)). FIG. 8C is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (negative printing method (IJ)). FIG. 8D is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (negative printing method (IJ)). FIG. 9A is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (negative printing method (contact)). FIG. 9B is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (negative printing method (contact)). FIG. 9C is a schematic view showing another example for explaining a method for producing a cell tissue body (negative printing method (contact)). FIG. 9D is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (negative printing method (contact)). FIG. 9E is a schematic view showing another example for explaining a method for producing a cell tissue body (negative printing method (contact)). FIG. 9F is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (negative printing method (contact)). FIG. 10A is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (latent image + negative printing method (IJ + IJ)). FIG. 10B is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (latent image + negative printing method (IJ + IJ)). FIG. 10C is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (latent image + negative printing method (IJ + IJ)). FIG. 10D is a schematic diagram illustrating another example for explaining a method for producing a cell tissue body (latent image + negative printing method (IJ + IJ)). FIG. 11A is a photograph showing an example in which a cell pattern in which 1-2 cells are arranged at a predetermined position on a substrate is formed. FIG. 11B is a photograph showing an example in which a cell pattern in which 3 to 4 cells are arranged at predetermined positions on the substrate is formed. FIG. 11C is a photograph showing an example in which a cell pattern in which about 10 cells are arranged at a predetermined position on the substrate is formed. FIG. 12A is a photograph showing an example in which a cell pattern in which two types of cells are arranged at predetermined positions on a substrate is formed. FIG. 12B is a photograph for explaining a cell pattern by two types of cells in FIG. 12A. FIG. 13 is a flowchart illustrating an example of a processing procedure of a cell tissue manufacturing program in the cell tissue manufacturing apparatus.

The technique of placing cells on a substrate by the inkjet method allows cells to be placed while suspended in a liquid, so the damage to the cells is small and the number of cells to be ejected is easy to control. Above, it is an effective technology.
However, there are many problems in stably discharging a cell suspension in which cells are suspended (hereinafter also referred to as “cell ink”) by an inkjet method. For example, an ink material that is usually used in image formation by an ink jet method often cannot be used with a cell ink because of its great damage to cells. Further, it is necessary to discharge the cell ink at a thinner dispersion concentration than the pigment in the ink conventionally used for image formation. In addition, after the cells have landed on the substrate, cell death is caused by drying of the cell ink droplets, so the ink blending method used in conventional image formation that is quickly dried can be used as it is. I can't do it. In addition, since the cell ink must be arranged within a range where the droplets do not coalesce, the arrangement density of the cells on the substrate cannot be increased.
Moreover, in the said patent document 1, the point of positioning (pattern arrangement | positioning) of biocompatible particle | grains is not described.
As a result of repeated studies, the following methods for producing a cellular tissue body that can produce a cellular tissue body having a cell pattern in which a desired number of cells are arranged at predetermined positions on a substrate are as follows. It has been found that the method for producing a cell tissue construct of the following constitution is effective.
According to the method for producing a cellular tissue body of the present invention, a cell pattern can be formed in a short time, the shape accuracy of the pattern shape can be increased, the accuracy of arranging a desired number of cells can be increased, and the survival of the arranged cells The rate can be increased.
Furthermore, according to the method for producing a cell tissue body of the present invention, a cell pattern can be formed using a plurality of cells, and a cell tissue body composed of a plurality of cells can be artificially formed.

(Cell tissue body manufacturing method, manufacturing apparatus, and manufacturing program)
The method for producing a cell tissue body according to the present invention includes a step of forming a cell adhesion portion that forms a cell adhesion portion made of a cell adhesive material in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface. .
The method for producing a cell tissue body of the present invention includes a cell placement step in which a cell suspension containing at least a cell and a cell desiccation inhibitor is discharged as a droplet to a cell adhesion portion to place the cell.
Moreover, the manufacturing method of the cell tissue body of this invention may also include another process as needed.

The apparatus for producing a cell tissue body of the present invention has a cell adhesion part forming means for forming a cell adhesion part made of a cell adhesive material in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface. .
The apparatus for producing a cell tissue body of the present invention has a cell placement means for placing cells by discharging a cell suspension containing at least cells and a cell desiccation inhibitor into droplets as a cell suspension.
Moreover, the manufacturing apparatus of the cell tissue body of this invention may have another means as needed.

The manufacturing program for a cell tissue body according to the present invention includes a process for forming a cell adhesion portion formed of a cell adhesive material in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface. Let the computer run.
The manufacturing program for a cell tissue body of the present invention causes a computer to execute a process of disposing a cell suspension containing at least cells and a cell desiccation inhibitor as droplets to a cell adhesion part and arranging the cells.

  The apparatus for producing a cellular tissue body of the present invention is synonymous with the implementation of the method for producing a cellular tissue body of the present invention, so the details of the production apparatus of the present invention will be described through the description of the production method of the present invention, or the present invention. The details of the manufacturing method of the present invention will be clarified through the description of the manufacturing apparatus. Moreover, since the manufacturing program of the cell tissue of the present invention realizes the method of manufacturing the cell tissue of the present invention by using a computer or the like as a hardware resource, the present invention is explained through the description of the manufacturing method of the present invention. The details of the manufacturing program are also clarified.

<Cell adhesion part forming step, forming means>
The cell adhesion portion forming step is a step of forming a cell adhesion portion made of a cell adhesive material in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface.
In the step of forming the cell adhesion portion, the cell adhesion portion is formed in a predetermined shape at a predetermined position on the substrate. In the present invention, the formation of a cell adhesive portion having a predetermined shape at a predetermined position in this way is also referred to as forming the cell adhesive portion in a pattern. Note that the position of the cell adhesion portion and the conditions of the shape can be arbitrarily set by the operator or the user. The cell adhesion part is formed on the substrate according to the size and interval of the cell adhesion part set by the operator or the user.
There is no restriction | limiting in particular as a method of forming a pattern-form cell adhesion part on the board | substrate which has a cell non-adhesive surface, According to the objective, it can select suitably, For example, the method as described below is mentioned.

As one method, the cell non-adhesive surface is covered with a mask formed in a pattern, and a region where the cell non-adhesive surface is exposed on the surface and a region hidden by the mask are formed. Next, a cell adhesive material is applied to the entire region to form a layer of the cell adhesive material. Then, the mask portion is removed. As a result, a layer made of the cell adhesion material existing in a region not covered with the mask remains, thereby forming a patterned cell adhesion portion (hereinafter, this method is also referred to as a mask pattern formation method).
As another method, a layer made of a cell adhesive material is formed by discharging droplets of the cell adhesive material onto a substrate having a cell non-adhesive surface using an inkjet head. At that time, a pattern-like cell adhesion portion is formed by adjusting the timing of ejecting the droplet and the ejection amount of the droplet (hereinafter, this method is also referred to as an inkjet forming method).
The inkjet head used when discharging the cell adhesive material droplets should be the same as the inkjet head used when discharging the cell suspension (cell ink) droplets described below. Can do. Therefore, the ink jet head will be described in detail in the following section <<< inkjet head for discharging droplets >>>.
Moreover, the specific embodiment about the formation process of a pattern-like cell adhesion part is demonstrated in detail below.

<< Substrate having cell non-adhesive surface >>
The substrate is not particularly limited as long as it has a non-cell-adhesive surface on the surface, and it is more preferable if the cell-adhesive material is easily adsorbed.
Cell non-adhesive means that the adhesiveness with respect to the target cell is lower than the cell adhesive material to be used at least.

Examples of the material of the substrate include organic materials and inorganic materials described below. These may be used alone or in combination of two or more.
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, Silicone materials such as polydimethylsiloxane (PDMS), polyvinyl alcohol (PVA), metal alginate such as calcium alginate, gel materials such as polyacrylamide, methylcellulose, and agarose Etc., and the like.
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.

Moreover, the board | substrate which has a cell non-adhesive surface can be obtained also by apply | coating the material which shows cell non-adhesiveness to arbitrary board | substrates.
For example, as a preferred embodiment of a substrate having a cell non-adhesive surface, a polydimethylsiloxane (PDMS) layer, a gel layer of a metal alginate (such as calcium alginate), a polyhydroxyethyl methacrylate (pHEMA) layer, polyethylene glycol ( And a substrate having a PEG) layer on the surface.

<< Cell adhesive material >>
Examples of the cell adhesive material include a protein selected from an extracellular matrix.
The protein selected from the extracellular matrix is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include collagen, laminin, fibronectin, elastin, fibrin, and matrigel. These may be used individually by 1 type and may use 2 or more types together.

Moreover, as the cell adhesive material, for example, particles having cell adhesiveness described below can be used.
The particle having cell adhesiveness is not particularly limited as long as it has an affinity with a living body such as a cell, and can be appropriately selected according to the purpose. Examples thereof include gelatin particles and collagen particles. These may be used individually by 1 type and may use 2 or more types together.
Gelatin particles that are particulate can improve the adhesion of cells to the substrate and are present in cellular tissues for a long time without being broken down into cells when compared to gelatin that is non-particulate. be able to. For this reason, particulate gelatin particles have the advantage of improving cell adhesion and being used as a nutrient source for cells over a long period of time.
There is no restriction | limiting in particular as particle | grains which have cell adhesiveness, A shape etc. can be selected suitably according to the objective.
Examples of the shape of the particle having cell adhesiveness include a spherical shape, a linear shape, a mesh shape, and an indefinite shape. These may be used individually by 1 type and may use 2 or more types together.
The cumulant diameter of the particles having cell adhesiveness is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.1 μm or more and 1.0 μm or less, more preferably 0.30 μm or more and 0.70 μm or less. preferable. The cumulant diameter was measured using the sample liquid obtained by the following sample liquid preparation example using a concentrated particle size analyzer (trade name: FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.). Can be measured.

  The substrate having a cell adhesive surface is not particularly limited as long as cells adhere to it, and may be a culture dish or a bottom of a microplate generally used in cell culture or the like. A coated substrate may also be used.

-Sample solution preparation-
In a pure water obtained using a pure water production apparatus (trade name: GSH-2000, manufactured by ADVANTEC), particles having cell adhesiveness are dispersed at a concentration of 0.5% by mass. The liquid volume for measurement is 5 mL, and the dispersion can be prepared by stirring a 20 mm rotor with a stirrer at 200 rpm for about 1 day.

-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

When the particles having cell adhesiveness are gelatin particles, the gelatin as a raw material for gelatin particles is not particularly limited and can be appropriately selected according to the purpose. For example, trade name: APH-250 (Nitta) Gelatin Corporation).
The particles having cell adhesiveness are preferably crosslinked with a crosslinking agent in the structure. By crosslinking with a cross-linking agent, the cumulant diameter of the particles having cell adhesiveness can be reduced, and the proliferation of cells can be promoted on the cell tissue containing the particles having cell adhesiveness.

  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 the particle | grains which have cell adhesiveness, and 2 mass% or more and 10 mass% or less are more preferable. When the content is 1% by mass or more and 20% by mass or less, the cumulant diameter of the cell-adhesive particles can be reduced, and cell proliferation can be achieved on the cell tissue including the particles having cell-adhesive properties. Can be promoted.

  As content of the particle | grains which have cell adhesiveness, 0.5 mass% or more and 10 mass% or less are preferable with respect to the solution whole quantity of the material containing the particle | grains which have cell adhesiveness, 1 mass% or more and 5 mass% or less. Is more preferable. When the content is 0.5% by mass or more and 10% by mass or less, it is possible to sufficiently adhere cells to a cell tissue body that is a three-dimensional cell aggregate and promote cell proliferation.

-Method for producing particles having cell adhesion-
Examples of the particles having cell adhesiveness can be prepared as follows, using gelatin as an example.
As a particle having cell adhesion, gelatin is mixed with water so as to be 2% by mass and dissolved in a 60 ° C. hot water bath to obtain a 2% by mass gelatin aqueous solution. Next, 40 mL of a 2% by weight gelatin aqueous solution heated to 40 ° C. is placed in a 200 mL beaker and stirred. Thereafter, 60 g of acetone is added all at once to obtain a cloudy liquid (coacervation).
For example, 160 μL of glutaraldehyde aqueous solution of 24% by mass or more and 26% by mass or less as a cross-linking agent is added to the white turbid solution, and the content of glutaraldehyde is 5% by mass with respect to the total amount of gelatin. Hold for 30 minutes while stirring at about. Gradually the cloudy liquid turns creamy and can form gelatin particles.
Next, the temperature is returned to room temperature (25 ° C.), 100 g of acetone is added, and gelatin particles are aggregated and precipitated to obtain a precipitate.
The removal of the supernatant and the acetone washing are repeated several times, moisture and unreacted crosslinking agent are removed from the resulting precipitate, and filtration is performed as necessary. Thereafter, the precipitate is dried on a 60 ° C. hot plate and dried under reduced pressure on a 50 ° C. hot plate for 1 hour to obtain a powder of gelatin particles (particles having cell adhesiveness).

<Cell placement process, placement means>
The cell arranging step is a step of arranging cells by discharging a cell suspension containing at least cells and a cell drying inhibitor as droplets to a cell adhesion part.
In the cell placement step, by discharging a droplet of the cell suspension to the patterned cell adhesion part, the cells can be seeded on the pattern cell adhesion part, and a cell pattern can be formed. .
Hereinafter, the cell placement step will be described by dividing it into a droplet discharge step for discharging the cell suspension as a droplet and a step for discharging the droplet to the cell adhesion portion and arranging the cell on the cell adhesion portion.

<< Droplet ejection process, droplet ejection means >>
The droplet discharge step refers to a step of discharging a cell suspension (cell ink) containing at least cells and a cell drying inhibitor as droplets.
As means for causing droplets of a cell suspension (cell ink) containing cells and a cell desiccation inhibitor to fly onto the substrate, ink jet type droplet discharge means is preferable.
As the droplet discharge means by the ink jet method, for example, a so-called piezo that discharges droplets by changing the volume of the cell suspension using a piezoelectric element as pressure generating means for pressurizing the cell suspension (cell ink). Method (for example, refer to Japanese Patent Publication No. 2-51734), so-called thermal method (for example, refer to Japanese Patent Publication No. 61-59911), vibration in which a cell suspension is heated using a heating resistor to generate bubbles. An electrostatic method (for example, discharging a droplet by changing the volume of a cell suspension by deforming the diaphragm by an electrostatic force generated between the diaphragm and the electrode by disposing the plate and the electrode oppositely) And JP-A-6-71882).
A specific embodiment of an inkjet head used for discharging a cell suspension (cell ink) containing cells and a cell drying inhibitor as droplets will be described below.

<<< Inkjet Head for Droplet Discharge >>>
FIG. 1 is a schematic diagram showing an example of an ink jet head used for discharging droplets.
In FIG. 1, a piezoelectric element is used as the pressure generating means.
2A and 2B are schematic diagrams illustrating an example of an input waveform to the inkjet head.
The droplet discharge head 10 vibrates the liquid chamber 12 that holds the cell suspension 11, the nozzle 15, the membrane 13 that is a membrane member, the vibration unit 16 that vibrates the membrane 13, and the vibration unit 16. In order to achieve this, a drive unit 14 is provided that applies a voltage to the excitation unit 16 as a specific drive signal.
The liquid chamber 12 is provided with an atmosphere opening portion 17 for opening the liquid chamber to the atmosphere.
The droplet ejection head 10 ejects droplets of the cell suspension from the nozzle by applying vibration to the cell suspension.
The drive unit 14 can apply the ejection waveform Pj as a drive signal to the excitation unit 16, and the cell suspension 11 held in the liquid chamber 12 can be controlled by controlling the vibration state of the membrane 13. It can be ejected in the form of droplets. The discharge waveform Pj may be set to a drive signal including the natural vibration period To of the membrane 13 in order to resonate the membrane 13 and discharge the cell suspension 11 with a smaller voltage. As the ejection waveform Pj, not only a triangular wave and a sine wave but also a triangular wave with a gentle edge by applying a low-pass filter can be used. Further, the drive unit 14 can apply a vibration isolation waveform Ps that suppresses the membrane residual vibration after droplet discharge as a drive signal to the vibration unit 16. As a result, the membrane residual vibration after droplet formation is quickly suppressed, thereby enabling higher frequency continuous ejection. Further, by reducing satellites and mist, it becomes possible to control a smaller amount of droplets. As the vibration isolation waveform Ps, not only a triangular wave and a sine wave but also a triangular wave with a gentle edge by applying a low-pass filter can be used.

The amount of the cell suspension 11 held in the liquid chamber 12 is not particularly limited. For example, a liquid of about 1 μL to 1 mL can be held. In particular, when an expensive liquid such as a cell suspension in which cells are dispersed is used, it is preferable that droplets can be formed with a small amount of liquid, and the liquid volume is about 1 μL to 50 μL. It is good that the structure can be held.
The shape of the membrane 13 may be circular, oval or quadrangular.
The material of the membrane 13 is not particularly limited, and if it is too soft, the membrane will vibrate easily, making it difficult to suppress vibration immediately when no droplets are ejected. There should be. As a material of the membrane 13, for example, a metal material, a ceramic material, a polymer material having a certain degree of hardness, or the like can be used.
The nozzle 15 is preferably formed as a substantially circular through hole in the center of the membrane 13.

As the vibration part 16, a piezoelectric element is mentioned, for example. By applying a voltage, the membrane 13 can be deformed by applying a compressive stress in the lateral direction of the drawing. As a material of the piezoelectric element, for example, commonly used lead zirconate titanate 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 means for applying vibration to the membrane 13 for deforming the membrane 13 is not limited to the piezoelectric element. For example, a material having a different linear expansion coefficient from that of the membrane is stuck on the membrane 13 and is heated. It is also possible to deform the membrane 13 using the difference in linear expansion coefficient. At this time, for example, heaters are formed on materials having different linear expansion coefficients, and the heaters are heated by energization so that the membrane 13 can be deformed.

Next, the cell suspension (cell ink) will be described.
The cell suspension (cell ink) contains at least cells and a cell desiccation inhibitor. Furthermore, the cell suspension (cell ink) contains a dispersion medium that disperses the cells, and may contain other additive materials such as a dispersant and a pH adjuster as necessary.

<<< 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, 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.

<<< Cell drying inhibitor >>>
The cell drying inhibitor has a function of covering the cell surface and suppressing cell drying, and examples thereof include proteins selected from polyhydric alcohols, gelled polysaccharides, and extracellular substrates. .

-Polyhydric alcohol-
The polyhydric alcohol is not particularly limited as long as it does not damage the cells. For example, glycerin, diglycerin, diethylene glycol, 1,3-butanediol, 1,2,3-butanetriol, 1,2,4-butane. Examples include triol, triethylene glycol, tetraethylene glycol, propylene glycol, and polyethylene glycol. These may be used individually by 1 type and may use 2 or more types together. Among these, glycerin is preferable. The toxicity to cells is low, and the effect of inhibiting drying can be expected even with a low addition amount.

-Gelled polysaccharide-
A gel-like polysaccharide refers to a polysaccharide in a gel state.
The gel polysaccharide is not particularly limited and may be appropriately selected depending on the intended purpose. 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.

-Proteins selected from extracellular matrix-
There is no restriction | limiting in particular as protein selected from an extracellular matrix, According to the objective, it can select suitably, For example, collagen, laminin, fibronectin, elastin, fibrin etc. are mentioned. These may be used individually by 1 type and may use 2 or more types together. 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. By including it in the cell ink, a trigger to increase the viscosity when the concentration is increased can be provided.

<<< Distributed culture >>>
As the dispersion culture, a culture medium or buffer for cell culture is preferable.
The medium is a solution that contains components necessary for the formation and maintenance of the cell tissue, prevents the drying 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. it can. When it is not necessary to always immerse the cells in the medium solution, the medium can be appropriately removed from the cell suspension.
The buffer is for adjusting the pH according to the cell and purpose, and a known one can be appropriately selected and used.

<< Step of arranging cells, means for arranging >>
The step of arranging the cells is a step of discharging the droplets to the cell adhesion part and arranging the cells on the cell adhesion part.
As a more specific aspect, a cell pattern in which cells are adhered on the patterned cell adhesion part is formed by discharging droplets to the pattern cell adhesion part and seeding the cells on the pattern cell adhesion part. be able to.
In the present invention, arranging a desired number of cells at a predetermined position on the substrate is also referred to as forming a cell pattern.
In the step of arranging the cells, a droplet of cell ink is ejected onto or near the patterned cell adhesion portion. Then, the cells in the droplet landed in the vicinity of the cell adhesion part migrate to the cell adhesion part and are adhered on the cell adhesion part.
The operator or the user can arbitrarily set the conditions of the cell position and the number of cells arranged in the cell pattern.
For example, the position of the cell adhered to the cell adhesion part can be adjusted by adjusting the timing of discharging the droplets according to the position of the pattern cell adhesion part. Further, the number of cells adhered to the cell adhesion portion can be adjusted by adjusting the discharge amount (number of droplets or droplet amount) of the cell ink and the cell concentration at that time. In this way, by arranging the cells on the patterned cell adhesion part, a cell pattern in which a predetermined number of cells are regularly arranged at predetermined positions on the substrate can be formed on the substrate.

As a preferred embodiment of the method for producing a cell tissue body of the present invention, a patterned cell adhesion part is formed, and a cell ink droplet containing a cell drying inhibitor is discharged to the pattern cell adhesion part. Forming a cell pattern. Together, these steps show the effects described below.
The droplets of the cell ink land on the substrate, and the cells adhere to the cell adhesion portion due to the flow in the droplet generated at the time of landing and the migration of the cells. Since the droplets of cell ink are very small compared to the procedure, it takes a very short time until the cells adhere to the cell adhesion part. Therefore, the method for producing a cellular tissue body of the present invention can form a cell pattern in a short time. Moreover, the method for producing a cellular tissue body of the present invention has high cell pattern shape accuracy in which cells are arranged at predetermined positions. In addition, the method for producing a cellular tissue body according to the present invention has a high probability that a predetermined number of cells can be arranged in a predetermined cell adhesion portion, or the probability that the number of arranged cells is a desired number. The accuracy of placing is high. In addition, the method for producing a cellular tissue body of the present invention has a high survival rate after a predetermined time elapses of the arranged cells.
Furthermore, since the method for producing a cellular tissue body of the present invention has high cell pattern shape accuracy, a cell pattern composed of a plurality of cells can be accurately formed using a plurality of cells.

<Embodiment of manufacturing method and manufacturing apparatus of cell tissue>
Hereinafter, specific embodiments of the method for producing a cellular tissue body of the present invention and a production apparatus for carrying out the production method will be described. However, the present invention is not limited to these embodiments.
An apparatus for manufacturing a cell tissue body includes a stage unit and a droplet discharge head (inkjet head) that is a droplet discharge unit that discharges a droplet of a cell suspension.
The stage unit holds the substrate.
The configuration of the droplet discharge head is as described above in <<< inkjet head for droplet discharge >>>.

FIG. 3A to FIG. 3D show an example of a manufacturing apparatus for manufacturing a cell tissue equipped with the above-described inkjet head for discharging droplets.
3A includes a stage unit 31 and a droplet discharge head (inkjet head) 21 that is a droplet discharge unit that discharges a droplet of a cell suspension.
As described above, the droplet discharge head (inkjet head) 21 includes the liquid chamber 25, the vibration unit 27, the drive unit 26, and the membrane 28. The droplet discharge head (inkjet head) 21 is formed with an atmosphere opening portion 24.
The apparatus for manufacturing a cell tissue body can also form a patterned cell adhesion part by discharging a droplet of a solution containing a cell adhesive material. In that case, as shown in FIG. 3B, in addition to the droplet discharge head (inkjet head) 21 that discharges the cell suspension droplet, the droplet discharge that discharges the droplet of the solution containing the cell adhesive material. A head (inkjet head) 23 is provided. The basic configuration of the droplet discharge head (inkjet head) 23 is the same as that of the droplet discharge head (inkjet head) 21. In FIG. 3B, reference numeral 29 denotes a drive unit, and reference numeral 30 denotes a liquid chamber that holds a solution of an adhesive material. Like the droplet discharge head (inkjet head) 21, the droplet discharge head (inkjet head) 23 is not shown in the drawing, but is provided with a vibrating portion and a membrane.
In the apparatus for producing a cellular tissue body of the present invention, a cell pattern composed of two or more types of cells can also be formed. In that case, as shown in FIG. 3C, a plurality of droplet discharge heads (inkjet heads) 21 that discharge droplets of the cell suspension can be provided. FIG. 3C shows an example in which two droplet discharge heads (inkjet heads) 21 and two inkjet heads 22 having the same configuration as the inkjet head 21 are provided.
FIG. 3D shows an example of an apparatus for manufacturing a cellular tissue body in which two droplet discharge heads (inkjet heads) 21 and 22 are provided and a droplet discharge head (inkjet head) 23 is also provided.
The cell tissue body manufacturing apparatus of the present invention may be provided with a holding unit for holding the ink jet head, a mechanism unit for controlling the relative position of the stage and the ink jet head, and the like as means other than the above means.

Next, a method for manufacturing a cell tissue body using a cell tissue body manufacturing apparatus as shown in FIGS. 3A to 3D will be specifically described.
A method for producing a cell tissue body will be described with reference to FIGS. 4A to 4F or FIGS. 5A to 5D.

[Formation Step 1 of Patterned Cell Adhesion Part (Formation Method Using Mask Pattern)]
A mask film 43 is formed on a substrate 41 having a cell non-adhesive surface 42 by drawing a pattern on a film, a weak adhesive tape, or the like with a laser processing machine or the like. The mask film is attached to the non-cell-adhesive surface (FIG. 4A).
Next, the cell adhesive material 44 is applied (FIG. 4B). If necessary, the layer of cell adhesive material may be dried (FIG. 4C).
Thereafter, by peeling off the mask film, a layer pattern 45 made of a cell adhesive material can be formed on a substrate having a cell non-adhesive surface (FIG. 4D).
At this time, the cell adhesive material is preferably applied after being dissolved in pure water or the like. The content of the cell adhesive material with respect to the cell adhesive material solution is not particularly limited, but is preferably 0.001% by mass to 10% by mass, and more preferably 0.01% by mass to 5% by mass. . When the content is within this range, unevenness during coating can be effectively prevented.
It is preferable to form the patterned cell adhesion part with a mask pattern because the cell adhesion part forming process is simple and the formed cell adhesion part is a solid flat film and has excellent adhesion to cells.

[Formation Step 2 of Patterned Cell Adhesion Part (Formation Method Using Inkjet)]
-Preparation of cell adhesive material ink-
Cell adhesive material is dissolved in pure water or the like, and cell adhesive material ink is prepared. At this time, the content of the cell adhesive material with respect to the cell adhesive material solution is not particularly limited, but is preferably 0.001% by mass to 5% by mass, and preferably 0.005% by mass to 1% by mass. Is more preferable. When the content is within this range, the cell adhesion function can be sufficiently expressed, and droplets can be discharged well in the ink jet method.
If necessary, the cell adhesive material ink may contain other additive materials such as a wetting agent, a dispersing agent, and a pH adjusting agent.

-Pattern formation process of cell adhesion part by inkjet-
As a manufacturing apparatus, an apparatus having the cell adhesive material discharge head 23 shown in FIG. 3B can be used.
As shown in FIG. 5A to FIG. 5B, a cell adhesion part pattern 55 made of a cell adhesive material is formed on a substrate 51 having a cell non-adhesive surface 52. The cell adhesive material ink is filled in the liquid chamber of the cell adhesive material discharge head 61, and the droplets 53 of the cell adhesive material ink are discharged onto the substrate 51 having the cell non-adhesive surface 52 (FIG. 5A). When the droplet 53 is discharged, the cell adhesion portion having a desired pattern shape is formed by adjusting the discharge timing, the amount of the discharged droplet, and the like. Thereby, the pattern-shaped cell adhesion part 55 can be formed on the board | substrate which has a cell non-adhesion surface (FIG. 5B).

Further, as shown in FIGS. 6A to 6F, it is possible to form a pattern of the cell adhesion portion by plate transfer.
As shown in FIG. 6B, the cell adhesive material 63 is applied to the plate 61, and the cell adhesive material 63 is transferred to the substrate 65 having the cell non-adhesive surface 64 to form the cell adhesive material pattern 63a. Except for this, it is the same as the pattern forming process of the cell adhesion part by the ink jet.
6A to 6F, 66a represents a flying droplet, 66b represents a landing droplet, 67 represents a cell, 68 represents a cell suspension, and 69 represents a cell ejection head.
The material used for the plate is not particularly limited as long as the cell adhesive material can be held, but generally softer than the base material is preferable because the followability during transfer is improved. Further, the pressure at the time of transfer is not particularly limited as long as it does not break the plate pattern.

[Cell pattern formation process]
-Preparation of cell suspension (cell ink)-
Dissolve the cell drying inhibitor in the dispersion medium and prepare the dispersion medium for cell ink. At this time, the content of the cell drying inhibitor is not particularly limited, but is preferably 0.001% by mass or more and 20% by mass or less, and 0.01% by mass or more by mass ratio of the cell drying inhibitor in the cell suspension. 10 mass% or less is more preferable. Within this range of content, although depending on the type of cells, the effect of inhibiting drying can be exhibited and the damage to the cells is small. Next, the cell ink is obtained by dispersing the cells in a cell culture dispersion medium and gently stirring by pipetting. At this time, the cell content in the cell suspension is preferably 5 × 10 ⁵ cells / mL to 1 × 10 ⁸ cells / mL, more preferably 1 × 10 ⁶ cells / mL to 5 × 10 ⁷ cells / mL. . If it is in the range of this content, the cell density will not be low, and it will not be difficult to eject by the ink jet method.
The ambient temperature in the cell ink preparation step is preferably 4 ° C to 40 ° C, more preferably 15 ° C to 37 ° C. Even if the temperature exceeds 37 ° C., the cells do not die immediately. However, if the temperature exceeds 37 ° C. or if the cells are exposed for a long time, the cells may be damaged. When the environmental temperature is lower than 4 ° C., the effect on the life and death of the cells is small, but the activity of the cells decreases, and there is a tendency that it takes time for cell adhesion in this process.

-Formation of cell pattern-
As shown in FIG. 4E, the cell ink is ejected and arranged on the substrate on which the patterned cell adhesion portion is formed. The cell ink 46 is filled into the liquid chamber of the cell ejection head 47 and ejected along the patterned cell adhesion portion formed on the substrate. At this time, if the cell ink is ejected from the nozzle, the drive signal is not particularly limited. Since the cells are dispersed in the ink droplet, it is difficult to control the position after landing. However, since the cell adhesion part has a pattern shape, the cells that have landed in the vicinity of the cell adhesion part migrate to the cell adhesion part and can adhere to the cell adhesion part. In FIG. 4E, reference numeral 48 indicates a flying droplet, reference numeral 49 indicates a landed droplet, and reference numeral 50 indicates a cell. Thereby, a cell pattern in which cells are regularly arranged at predetermined positions on the substrate can be formed (FIG. 4F).
Similarly, FIG. 5C can form a cell pattern.
As shown in FIG. 5C, the cell ink is discharged and arranged on the substrate on which the patterned cell adhesion portion is formed. The cell ink 56 is filled into the liquid chamber of the cell ejection head 57 and is ejected along the patterned cell adhesion portion formed on the substrate. The cells that land near the cell adhesion part migrate to the cell adhesion part and adhere to the cell adhesion part. In FIG. 5C, reference numeral 58 denotes a flying droplet, reference numeral 59 denotes a landed droplet, and reference numeral 60 denotes a cell. Thereby, the cell pattern in which the cells are regularly arranged at predetermined positions on the substrate can be formed (FIG. 5D).
In FIG. 4E and FIG. 5C, the number of cells arranged per unit area in the cell pattern can be controlled by changing the number of ejected droplets or changing the cell dispersion concentration in the cell ink. By controlling the number of cells per unit area, interactions such as adhesion between cells and exchange of proteins can be arbitrarily designed. For this reason, the possibility of not only mere cell evaluation but functional evaluation as a cell tissue body can be presented. In addition, since ink droplets are landed using ink jets, the time after the droplets land on the substrate, migrate to the cell adhesion area, and adhere to the cell adhesion area is extremely short. Can be done in time. In addition, according to the present invention, it is possible to stably discharge droplets of cellular ink. However, even if the discharge becomes unstable, stirring in the liquid chamber including the vicinity of the nozzles of the inkjet head is performed. If implemented, it becomes possible to discharge stably again.

<Specific Example of Cell Tissue with Cell Pattern>
A specific example of a cell tissue formed by the method for producing a cell tissue of the present invention is shown.
A dot of a cell adhesion part having a diameter (φ) of 150 μm was formed on the substrate. A cell ink droplet was ejected toward each dot so that one cell was arranged in one dot.
The cells used NIH / 3T3. A photograph of the result of placing one cell at each dot is shown in FIG. 11A.
In the same manner, FIG. 11B shows a photograph of the result of placing three cells aiming at each dot.
Similarly, FIG. 11C shows a photograph of the result of arranging 10 cells to each dot.
FIG. 12A shows a photograph of the results (showing a cell pattern composed of two types of cells) when two types of cells are used and the cells are regularly arranged.
The cells used NIH / 3T3 and NHDF.
Note that the photograph in FIG. 12A was originally a color photograph and was color-coded on a black background in green and blue. Therefore, it was possible to easily visually recognize the state in which each cell is arranged. However, in FIG. 12A below, since the display is black and white, FIG. 12B is attached as a supplementary explanation. In FIG. 12B, the symbols a and b indicate two different types of cells.

<Manufacturing program of cell tissue>
The cell tissue manufacturing program causes a cell tissue manufacturing method to be executed in the cell tissue manufacturing apparatus of the present invention by using a computer or the like as a hardware resource.
The processing by the cell tissue body manufacturing program can be executed using a computer having a control unit that constitutes the above-described cell tissue body manufacturing apparatus (manufacturing apparatuses such as FIGS. 3A to 3D).

  An example of the processing procedure of the manufacturing program of the cell tissue body of this invention is shown. FIG. 13 is a flowchart illustrating an example of a processing procedure of a cell tissue manufacturing program in the cell tissue manufacturing apparatus.

The cells can be arbitrarily arranged by patterning the non-cell-adhesive material in the same manner as the above-mentioned cell-adhesive material is patterned.
For example, as shown in FIG. 7A to FIG. 7F, FIG. 8A to FIG. 8D, and FIG. 9A to FIG. 9F, a cell adhesive material pattern is formed on a substrate (71) having a cell adhesive surface (72, 81a, 94). 81, 95), it is possible to place cells in locations other than the cell non-adhesive material pattern portions (74, 83c, 93a).
Further, as shown in FIGS. 10A to 10D, by arranging the cell adhesive material pattern 103c and the cell non-adhesive material pattern 102 by an inkjet (IJ) apparatus or the like, the cells can be obtained regardless of the characteristics of the substrate 101. Can also be arranged.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(Example 1)
<Formation of patterned cell adhesion part>
In order to form a non-cell-adhesive surface on a slide glass, the following were prepared. A sodium alginate solution in which sodium alginate (SKAT ONE, manufactured by Kimika Co., Ltd.) was dissolved in distilled water to a mass ratio of 2% was prepared. In addition, a calcium chloride solution in which calcium chloride dihydrate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water so as to be 100 mmol / L was prepared. The sodium alginate solution and the calcium chloride solution were mixed and applied on the slide glass so as to have a mass ratio of 1: 1, thereby forming a calcium alginate gel layer as a non-cell-adhesive surface layer.
Next, using a laser processing machine (H10-106QW J80-8ss42Hippo, manufactured by Spectra-Physics), a polyimide film (3-1966-03, manufactured by ASONE Corporation) having a diameter (φ) of 100 μm is used. Holes were drilled at a pitch of 300 μm to form a mask film. The substrate having the cell non-adhesive surface was covered with the formed mask film. A cell adhesive material solution was prepared in distilled water so that the mass ratio of fibronectin (F-2006, Fibronectin from human plasma, manufactured by SIGMA-ALDRICH) as a cell adhesive material was 0.10% by mass. A cell adhesive material solution was applied onto a substrate having a cell non-adhesive surface to which a mask film was bonded, and dried. Then, the pattern layer (patterned cell adhesion part) of cell adhesive material was formed in the board | substrate which has a cell non-adhesive surface by peeling a mask film.

<Preparation of cell ink>
Cells were stained to evaluate the cell pattern formed. A green fluorescent dye (trade name: Cell Tracker Green, manufactured by Life Technologies) was dissolved in dimethyl sulfoxide (hereinafter referred to as “DMSO”) at a concentration of 10 mmol / L (mM), and serum-free Dulbecco's modified Eagle medium (Life) And a serum-free medium containing a green fluorescent dye having a concentration of 10 μmol / L (μM). Next, 5 mL of a serum-free medium containing a green fluorescent dye was added to a dish of cultured NIH / 3T3 cells (Clone 5611, JCRB Cell Bank), and an incubator (KM-CC17RU2, manufactured by Panasonic Corporation, 37 ° C., 5% by volume CO 2). Incubated for 30 minutes in ₂ environments)). 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 per dish.
Next, after heating in an incubator for 5 minutes and detaching the cells from the dish, 10% by weight fetal bovine serum (hereinafter also referred to as “FBS”) and 1% by weight antibiotic (antibiotic-antimixic mixed stock solution) 100 x), 4 mL of D-MEM containing Nacalai Tesque Corporation) was added. Next, the cell suspension in which trypsin is inactivated is transferred to one 50 mL centrifuge tube, and centrifuged (trade name: H-19FM, manufactured by KOKUSAN, 1,200 rpm, 5 minutes, 5 ° C.). Was used to remove the supernatant. 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 suspension. 10 μL was taken out from the cell suspension into an Eppendorf tube, 70 μL of 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 was taken out from the stained cell suspension and placed on a PMMA plastic slide. Product name: A cell suspension was obtained by measuring the number of cells using a Countess Automated Cell Counter (manufactured by Invitrogen) to determine the number of cells. PBS (-) was used as a dispersion medium. Glycerin (molecular biology grade, manufactured by Wako Pure Chemical Industries, Ltd.) as a cell drying inhibitor is dissolved in PBS (−) so that the mass ratio becomes 0.5 mass%, and the NIH / 3T3 cell suspension is dissolved. Cell ink was obtained by dispersing in a dispersion medium to 6 × 10 ⁶ cells / mL.

<Formation of cell pattern>
Cell ink was filled into the liquid chamber of the cell discharge head of the apparatus for manufacturing the cell tissue body of FIG. 3A. Next, for each dot (diameter (φ) 100 μm) of the pattern-shaped cell adhesion portion prepared above, 3 droplets of cell ink (targeting 6 cells) were dropped onto 20 dots. In addition, 5 droplets of cell ink (targeting 10 cells) were dropped onto 20 dots.
After leaving the last droplet of cell ink to land for 10 minutes, D-MEM containing 10% by mass FBS and 1% by mass antibiotic was gently added.
In this manner, a cell tissue body in which a cell pattern was formed was produced.

<Evaluation of cell pattern>
The following evaluation was performed on the cell pattern of the obtained cell tissue. The evaluation results are shown in Table 1.

[Initial adhesion time of cells]
Whether or not the cells adhered to the patterned cell adhesion portion after standing for 10 minutes was evaluated using a fluorescence microscope (CKX41, manufactured by Olympus Corporation) according to the following criteria.
○: It was confirmed that the cells were able to adhere onto the pattern-like cell adhesion part. ×: It was not possible to confirm that the cells were able to adhere onto the pattern-like cell adhesion part.

[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 Corporation).
○: The cell pattern was formed with a pitch of 300 μm ×: The cell pattern was a pitch 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 arranged in each cell adhesion portion dot having a diameter (φ) of 100 μm was calculated based on the fluorescence image, and evaluated according to the following criteria.
The dots used for the evaluation were targeted for all 40 dots in total, each of which was dropped 6 aiming at 6 or 10 pieces.
○: 80% or more of dots where the number of cells per dot is ± 20% or less of the target number ×: Less than 80% of dots where the number of cells per dot is ± 20% or less of the target number

[Probability that the number of arranged cells is the desired number (evaluation of cell density)]
The number of cells arranged in each cell adhesion portion dot having a diameter (φ) of 100 μm was calculated based on the fluorescence image, and evaluated according to the following criteria.
The dots used for the evaluation were 20 dots dropped to aim at 10 cells.
○: 8 or more cells per dot is 80% or more ×: 8 or more cells per dot is less than 80%

[Viability of arranged cells after a predetermined time (evaluation of cell viability)]
A stock solution in which propidium iodide (Cellstein-PI, manufactured by Dojindo Laboratories) was dissolved in distilled water at 1 mg / mL as a nuclear staining agent for dead cells against a cell pattern that was allowed to stand for 10 minutes was 10 μL in 5 mL. It added so that it might become. Then, after culturing for 10 minutes in an incubator, the ratio of dead cells present in each dot was calculated using a fluorescence microscope and evaluated according to the following criteria.
The dots used for the evaluation were 20 dots dropped to aim at 10 cells.
○: The average cell survival rate per dot is 75% or more ×: The average cell survival rate per dot is less than 75%

(Example 2)
A cell tissue in which a cell pattern was formed was produced in the same manner as in Example 1 except that the cell drying inhibitor was changed to 0.1% by mass of sodium alginate, and the same evaluation as in Example 1 was performed. The evaluation results of Example 2 are shown in Table 1.

(Example 3)
A cell tissue body in which a cell pattern was formed was produced in the same manner as in Example 1, except that the cell drying inhibitor and the substrate having a non-adhesive surface were changed to those shown below. Was evaluated. The evaluation results of Example 3 are shown in Table 1.
The cell drying inhibitor is 0.05% by mass collagen (Cellmatrix Type1-A, Nitta Gelatin Co., Ltd.), and the substrate having a non-adhesive surface is dimethylpolysiloxane (SYLGARD 184, manufactured by Toray Dow Corning, (Hereinafter referred to as PDMS).
PDMS is obtained by mixing a base material and a curing agent at a mass ratio of 10: 1, defoaming, and heating at 70 ° C. for 2 hours.

Example 4
A cell tissue body in which a cell pattern was formed was produced in the same manner as in Example 3 except that the cell adhesive material was changed to collagen. Evaluation similar to Example 1 was performed. The evaluation results of Example 4 are shown in Table 1.

(Example 5)
A cell tissue in which a cell pattern was formed was produced in the same manner as in Example 3 except that the cell adhesive material was changed to fibrin. Evaluation similar to Example 1 was performed. The evaluation results of Example 5 are shown in Table 1.
Fibrin used was fibrinogen (Fibrinogen from bovine plasma F8630, manufactured by SIGMA-ALDRICH) mass ratio 0.2 mass% and thrombin (Thrombin from bovine plasma T4648, manufactured by SIGMA-ALCH mass ratio 0.2% by mass). Were mixed and then allowed to stand at room temperature overnight.

(Example 6)
A cell tissue body in which a cell pattern is formed is produced in the same manner as in Example 3 except that the cell adhesive material is changed to 0.1% by mass of RGD peptide (for biochemistry, manufactured by Wako Pure Chemical Industries, Ltd.). did. Evaluation similar to Example 1 was performed. The evaluation results of Example 6 are shown in Table 1.

(Example 7)
A cell tissue body in which a cell pattern was formed was produced in the same manner as in Example 3 except that the cell adhesive material was changed to gelatin fine particles having a mass ratio of 2% by mass. Evaluation similar to Example 1 was performed. The evaluation results of Example 7 are shown in Table 1.
The gelatin microparticles were prepared by using gelatin (APH-250, manufactured by Nitta Gelatin Co., Ltd.) according to the method described in the above-mentioned section “Method for producing particles having cell adhesion”.

(Example 8)
Cell ink 1 in which NIH / 3T3 was dispersed and cell ink 2 in which normal human derma fibroblast (CC-2509, manufactured by Lonza, hereinafter referred to as NHDF) was prepared. These cell inks were alternately arranged on the cell-adhesion portions having a pattern shape.
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).
In addition to the same evaluation as in Example 1, the following evaluation of multiple cells was also performed. The evaluation results of Example 8 are shown in Table 2.

[Arrangement of multiple cells]
The accuracy of the shape of the cell pattern was evaluated according to the following criteria using a fluorescence microscope (CKX41, manufactured by Olympus Corporation).
○: A cell pattern in which NIH / 3T3 and NHDF were alternately arranged was formed at a pitch of 300 μm or less. ×: A cell pattern in which NIH / 3T3 and NHDF were alternately arranged was a pitch larger than 300 μm.

Example 9
A cell tissue body in which a cell pattern was formed was produced in the same manner as in Example 8, except that the inkjet method was used as the method for forming the patterned cell adhesion part. Evaluation similar to Example 8 was performed. The evaluation results of Example 9 are shown in Table 2.

<Formation of patterned cell adhesion part>
-Preparation of cell adhesive material ink-
As a cell adhesive material, fibronectin was dissolved in distilled water so as to have a mass ratio of 0.05% by mass to prepare a cell adhesive material ink.
-Formation process of patterned cell adhesion by inkjet-
The cell adhesive material ink was filled into the liquid chamber of the cell adhesive material discharge head of the manufacturing apparatus of FIG. 3D. After discharging onto a PDMS substrate at a pitch of 300 μm so as to form dots with a diameter (φ) of 100 μm, drying was performed to form a patterned cell adhesion portion on a substrate having a cell non-adhesive surface.

(Example 10)
−Contact printing method−
As a method for forming a patterned cell adhesion part, Example 1 and Example 1 were used except that the contact printing method shown in FIGS. 6A to 6F was used and Matrigel (Matrigel GFR REF 354230, manufactured by CORNING) was used as the cell adhesion material. Similarly, a cell tissue body in which a cell pattern was formed was produced and evaluated in the same manner as in Example 1. The evaluation results of Example 10 are shown in Table 3.

<Formation of patterned cell adhesion part>
-Preparation of cell adhesive material ink-
As a cell adhesive material, Matrigel was dissolved in distilled water to a mass ratio of 0.05% by mass to prepare a cell adhesive material ink.
-Process for forming patterned cell adhesion by contact printing-
A cell adhesive ink was applied on a PDMS plate having a column having a diameter (φ) of 100 μm and a height of 50 μm, and transferred onto a substrate (PDMS) having a cell non-adhesive surface to form a cell adhesion portion. .

(Example 11)
-Cell non-adhesion pattern formation with Poly-HEMA (negative printing, mask process)-
As a method for forming a cell non-adhesive portion on the pattern, the method shown in FIGS. 7A to 7F is used. As a cell non-adhesive material, poly-HEMA (Poly (2-hydroxyethyl methacrylate), manufactured by SIGMA-ALDRICH) and cells are used. A cell non-adhesive pattern was formed in the same manner as in Example 1 except that 60 mm dish (IWAKI Tissue Culture Disc code 3010-060, manufactured by AGC Techno Glass Co., Ltd.) was used as the adhesive substrate. A cell tissue having a cell pattern formed thereon was produced and evaluated in the same manner as in Example 1. The evaluation results of Example 11 are shown in Table 3.

<Formation of patterned cell adhesion part>
-Preparation of non-cell adhesive material ink-
As a cell non-adhesive material, 50 μL of poly-HEMA was dissolved in 1 mL of ethanol (Ethanol (99.5), manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a cell non-adhesive material ink.

Example 12
-Cell non-adhesion pattern formation with alginic acid (negative printing, IJ process)-
As a method for forming a cell non-adhesive portion on the pattern, the method shown in FIGS. 8A to 8D was used, and a calcium alginate gel similar to the above was used as the cell non-adhesive material in the same manner as in Example 11, A cell non-adhesive pattern was formed, and a cell tissue body in which the cell pattern was formed on the cell adhesive substrate was produced. The same evaluation as in Example 1 was performed. The evaluation results of Example 12 are shown in Table 3.

<Formation of patterned cell adhesion part>
-Preparation of non-cell adhesive material ink-
Sodium alginate solution in which sodium alginate (SKAT ONE, manufactured by Kimika Co., Ltd.) as a cell non-adhesive material is dissolved in distilled water to a mass ratio of 2% by mass, and calcium chloride dihydrate (Wako Pure Chemical Industries, Ltd.) A calcium chloride solution prepared by dissolving in a distilled water so as to be 100 mmol / L was prepared.

-Formation process of non-adhered cell pattern by inkjet-
The sodium alginate solution and the calcium chloride solution were each ejected onto a cell adhesive substrate by inkjet, and mixed and gelled on the substrate to form a cell non-adhesive pattern on the cell adhesive substrate.

(Example 13)
-Cell non-adhesion pattern formation with Poly-HEMA (negative printing, contact printing)-
A cell non-adhesive pattern was formed in the same manner as in Example 11 except that the contact print method shown in FIGS. 9A to 9F was used as a method for forming the patterned cell non-adhesive part. A cell tissue having a cell pattern formed thereon was produced and evaluated in the same manner as in Example 1. The evaluation results of Example 13 are shown in Table 3.

(Example 14)
-Cell non-adhesion pattern formation with collagen + PDMS (negative printing, IJ process)-
As a method for forming a cell adhesive pattern and a cell non-adhesive pattern on a substrate, the method shown in FIGS. 10A to 10D is used, a slide glass (S1111 manufactured by Matsunami Glass Industrial Co., Ltd.), and Matrigel is used as a cell adhesive material. In the same manner as in Example 1 and Example 11 except that PDMS was used as the non-cell-adhesive material, a cell-adhesive pattern was formed, and a cell tissue body in which the cell pattern was formed on the cell-adhesive substrate was obtained. The same evaluation as in Example 1 was performed. The evaluation results of Example 14 are shown in Table 3.

<Formation of patterned cell adhesion part>
After coating on the mask film used in Example 1, the cell film was peeled off to form a cell non-adhesive pattern.
In the same manner as in Example 9, collagen was ejected to a portion having no non-adhesive pattern by ink jetting to form a cell adhesive pattern.

(Comparative Example 1)
A cell tissue body was produced in the same manner as in Example 8 except that fibronectin was used as the cell adhesive material, and the cell adhesive material was applied without a mask pattern (not a cell adhesive portion having a pattern). . Evaluation similar to Example 8 was performed. The evaluation results of Comparative Example 1 are shown in Table 2.

(Comparative Example 2)
A cell tissue body was produced in the same manner as in Example 8, except that the cell ink did not contain a cell drying inhibitor. Evaluation similar to Example 8 was performed. The evaluation results of Comparative Example 2 are shown in Table 2.

(Comparative Example 3)
Instead of arranging the cells by the inkjet method, NIH / 3T3 and NHDF were prepared as cell suspensions containing no cell desiccation inhibitor. Cell tissue bodies were produced in the same manner as in Example 9, except that each cell suspension was seeded by hand so that 5 × 10 ⁴ cells / mL totaled 1 × 10 ⁵ cells / mL. did. Evaluation similar to Example 8 was performed. The evaluation results of Comparative Example 3 are shown in Table 2.

As a result of Comparative Example 1, since the pattern-like cell adhesion portion was not formed, it was impossible to control the location where the cells adhered when landed. Therefore, not only the cell pattern could not be formed at the target pitch, but also the cell density in the set diameter (φ) 100 μm region was low.
As a result of Comparative Example 2, since the cell drying inhibitor was not contained in the cell ink, the cell survival rate was low. The dead cells were not attached to the substrate.
As a result of Comparative Example 3, since the cells were not arranged by ejecting droplets by the ink jet method, the cells adhered to the entire substrate, and the pattern shape accuracy of the cell pattern was poor. By washing with gentle flow of serum-free D-MEM, cells in areas with weak cell adhesion were peeled off, and cells adhered along the cell adhesion part of the pattern shape, but NIH / 3T3 and NHDF were random The two types of cells could not be arbitrarily arranged. In addition, the pattern-shaped cell adhesion part was also peeled off by washing.

  According to the present invention, a cell tissue body showing good results for cell viability, cell pattern resolution, cell density, and control of the number of cells, which are important elements in forming a cell pattern. The manufacturing method of can be provided. Furthermore, according to the present invention, it is possible to form a cell pattern in which two or more types of cells are arbitrarily arranged.

Aspects of the present invention are as follows, for example.
<1> a step of forming a cell adhesive portion made of a cell adhesive material in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface;
A cell disposing step of disposing at least the cells and a cell suspension containing the cell drying inhibitor as droplets, discharging the cells to the cell adhesion part, and disposing the cells;
It is a manufacturing method of the cell structure | tissue characterized by including.
<2> The step of forming the cell adhesion part is performed by discharging the solution containing the cell adhesion material as droplets and placing the droplets on the substrate having the cell non-adhesive surface. 1>. The method for producing a cellular tissue body according to 1>.
<3> The cell tissue according to any one of <1> to <2>, wherein the cell drying inhibitor includes at least one of a protein selected from polyhydric alcohols, gelled polysaccharides, and extracellular matrix. It is a manufacturing method of a body.
<4> The method for producing a cellular tissue body according to any one of <1> to <3>, wherein the cell adhesive material contains a protein composed of an extracellular matrix.
<5> a cell adhesive portion forming means for forming a cell adhesive portion made of a cell adhesive material in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface;
A cell disposing means for disposing at least the cells and a cell suspension containing the cell drying inhibitor as droplets, and discharging the cells to the cell adhesion part to dispose the cells;
It is a manufacturing apparatus of the cell tissue body characterized by having.
<6> A cell adhesion portion made of a cell adhesive material is formed in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface, and a cell adhesion portion is formed.
Disposing the cell suspension containing at least cells and a cell desiccation inhibitor as droplets, discharging the cells to the cell adhesion part, and arranging the cells,
A program for manufacturing a cellular tissue body, which causes a computer to execute processing.
<7> a cell adhesion part forming step of forming a cell non-adhesion part made of a cell non-adhesive material on a substrate having a cell adhesion surface in a predetermined shape;
A cell disposing step of disposing at least the cells and a cell suspension containing the cell drying inhibitor as droplets, discharging the cells to the cell adhesion part, and disposing the cells;
It is a manufacturing method of the cell structure | tissue characterized by including.
<8> The step of forming the cell adhesion portion is performed by discharging a solution containing the cell non-adhesive material as droplets, and disposing the droplets on the substrate having the cell adhesion surface. <7> The method for producing a cellular tissue body according to <7>.
<9> With respect to the substrate on which the cell non-adhesive portion made of the cell non-adhesive material is formed, the cell adhesive portion made of the cell adhesive material is applied to the substrate without the cell non-adhesive portion by the method according to claim 2. The method for producing a cell tissue body according to any one of <7> to <8>, wherein the step of forming the cell adhesion portion is performed by forming.
<10> a cell adhesion part forming means for forming a cell nonadhesion part made of a cell nonadhesive material on a substrate having a cell adhesion surface in a predetermined shape;
A cell disposing means for disposing at least the cells and a cell suspension containing the cell drying inhibitor as droplets, and discharging the cells to the cell adhesion part to dispose the cells;
It is a manufacturing apparatus of the cell tissue body characterized by having.
<11> A cell non-adhesive portion made of a cell non-adhesive material is formed in a predetermined shape on a substrate having a cell adhesive surface, and a cell adhesive portion is formed.
Disposing the cell suspension containing at least cells and a cell desiccation inhibitor as droplets, discharging the cells to the cell adhesion part, and arranging the cells,
A program for manufacturing a cellular tissue body, which causes a computer to execute processing.

  <1> to <4> and <7> to <9> the method for producing a cell tissue body according to any one of the above, <5> and <10> the cell tissue body production apparatus according to any one of the above, In addition, according to the program for producing a cell tissue body according to any one of <6> and <11>, the conventional problems can be solved and the object of the present invention can be achieved.

JP 2017-169560 A

DESCRIPTION OF SYMBOLS 10 Droplet discharge head 11 Cell suspension 12 Liquid chamber 13 Membrane 14 Drive part 15 Nozzle 16 Excitation part

Claims (11)

Forming a cell adhesion portion made of a cell adhesion material in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface; and
A cell disposing step of disposing at least the cells and a cell suspension containing the cell drying inhibitor as droplets, discharging the cells to the cell adhesion part, and disposing the cells;
A method for producing a cellular tissue body, comprising:   The said cell adhesion part formation process discharges the solution containing the said cell adhesive material as a droplet, and arrange | positions the said droplet on the said board | substrate which has the said cell non-adhesive surface. A method for producing a cell tissue body.   The method for producing a cellular tissue body according to any one of claims 1 to 2, wherein the cell drying inhibitor contains at least one of a protein selected from polyhydric alcohols, gelled polysaccharides, and extracellular matrix.   The method for producing a cellular tissue body according to any one of claims 1 to 3, wherein the cell adhesive material contains a protein comprising an extracellular matrix. A cell adhesion part forming means for forming a cell adhesion part made of a cell adhesive material in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface;
A cell disposing means for disposing at least the cells and a cell suspension containing the cell drying inhibitor as droplets, and discharging the cells to the cell adhesion part to dispose the cells;
An apparatus for producing a cellular tissue body, comprising: Forming a cell adhesive portion made of a cell adhesive material in a predetermined shape at a predetermined position on a substrate having a cell non-adhesive surface, and forming a cell adhesive portion;
Disposing the cell suspension containing at least cells and a cell desiccation inhibitor as droplets, discharging the cells to the cell adhesion part, and arranging the cells,
A program for manufacturing a cellular tissue, which causes a computer to execute processing. A step of forming a cell adhesion part that forms a cell non-adhesion part made of a cell non-adhesive material in a predetermined shape on a substrate having a cell adhesion surface;
A cell disposing step of disposing at least the cells and a cell suspension containing the cell drying inhibitor as droplets, discharging the cells to the cell adhesion part, and disposing the cells;
A method for producing a cellular tissue body, comprising:   The forming step of the cell adhesion part is performed by discharging the solution containing the cell non-adhesive material as droplets and placing the droplets on the substrate having the cell adhesion surface. A method for producing the cell tissue described above.   3. Forming a cell adhesion portion made of a cell adhesive material on a substrate without the cell non-adhesion portion by the method according to claim 2 for a substrate on which a cell non-adhesion portion made of a cell non-adhesion material is formed. The manufacturing method of the cell tissue body in any one of Claim 7 to 8 which performs the formation process of the said cell adhesion part. A cell adhesion part forming means for forming a cell nonadhesion part made of a cell nonadhesive material on a substrate having a cell adhesion surface in a predetermined shape;
A cell disposing means for disposing at least the cells and a cell suspension containing the cell drying inhibitor as droplets, and discharging the cells to the cell adhesion part to dispose the cells;
An apparatus for producing a cellular tissue body, comprising: A cell non-adhesive portion made of a cell non-adhesive material is formed in a predetermined shape on a substrate having a cell adhesive surface, and a cell adhesive portion is formed,
Disposing the cell suspension containing at least cells and a cell desiccation inhibitor as droplets, discharging the cells to the cell adhesion part, and arranging the cells,
A program for manufacturing a cellular tissue, which causes a computer to execute processing.