JP2020121531A - Method for manufacturing three-dimensional structure - Google Patents

Abstract

To provide a three-dimensional modeling method that has a high degree of freedom of an acceptive liquid, is more convenient, and has a low risk of failure.SOLUTION: A three-dimensional modeling method comprises steps of: adding a solution B that reacts with an acceptive liquid A to be a gel onto a liquid surface of the acceptive liquid A, to form a base layer; and laminating gels formed by dropping a solution C that reacts with the acceptive liquid A to be a gel on the base layer, to form a three-dimensional structure.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for manufacturing a three-dimensional structure.

As a method for manufacturing a three-dimensional structure, for example, there are conventional techniques using microbeads as in Patent Documents 1 and 2 and Non-Patent Document 1.

In Patent Document 1, by injecting the gel-forming liquid A into a water tank having a gel-forming liquid B (receptive liquid) that forms a gel when contacted with the liquid A, while changing the injection position, gel microsequentially occurs. A method of making a three-dimensional structure with adhered beads is disclosed.

In Patent Document 2, a composition containing cells and polymer particles is placed on a substrate placed on a heat source, and the composition is repeatedly changed from a sol state to a gel state by a temperature change to form a three-dimensional structure. A method of making a cell aggregate is disclosed.

Also, Non-Patent Document 1 discloses a method of stacking gel on an elevator stage that is vertically movable by using a 3D bioprinter.

JP, 2008-126459, A JP, 2016-111988, A

International Journal of Bioprinting(2016)-Volume 2, Issue 2, p.153-162

However, there is room for improvement in the above-mentioned conventional techniques. For example, in Patent Document 1, the gel forming the three-dimensional structure is not fixed in the receiving liquid. Therefore, the gel formed in the receiving liquid may easily move, and thus it may be difficult to form a three-dimensional structure having a desired shape. Further, in order to prevent the migration of gel in the receptive liquid, if a thickener is used to make the receptive liquid highly viscous to suppress the flow, when a three-dimensional structure using plant cells or animal cells is produced, It can be a factor that inhibits culture.

In Patent Document 2, if the laminated surface of the three-dimensional cell aggregate is too far from the heat source, the composition containing the polymer particles is unlikely to gel, and it is difficult to form a bulky laminated body. On the other hand, if the inkjet nozzle for dropping the composition containing the polymer particles is too close to the heat source, the composition may gel before dropping, so that the inkjet nozzle and the laminated surface are separated by a certain distance. .. However, in this case, the composition to be dropped is affected by wind or the like during flight, and the landing position is displaced. In addition, it cannot be denied that the cells contained in the composition may be damaged by heating.

Further, in Non-Patent Document 1, since the receiving liquid flows when the elevator stage is moved, the modeling accuracy may be reduced. Further, it takes a certain amount of time for the flow of the receiving liquid to settle down, resulting in poor work efficiency. In addition, due to the structure of the 3D bioprinter, there is a risk of leakage of the receiving liquid, which may lead to device failure.

An object of one embodiment of the present invention is to provide a method for manufacturing a three-dimensional structure that has a high degree of freedom of the receiving liquid, is simpler, and has a low risk of device failure.

In order to solve the above problems, as a result of earnest research, as a result of laminating a gel on an underlayer formed by using the receiving solution A and a solution which reacts with the receiving solution A to gel. The inventors have found that an arbitrary three-dimensional structure can be easily obtained, and completed the present invention. The present invention includes the following aspects.

<1> An underlayer forming step of forming a underlayer by adding a solution B which reacts with the receiving solution A to gel on the liquid surface of the receiving solution A, and reacting with the receiving solution A on the underlayer. And a gel forming step of forming a three-dimensional structure by stacking gels formed by dropping the solution C that gels.

<2> The method for producing a three-dimensional structure according to <1>, wherein in the underlying layer forming step, the underlying layer is formed by applying the solution B to the liquid surface of the receiving liquid A by inkjet. ..

<3> The method for producing a three-dimensional structure according to <1>, wherein in the underlayer forming step, the underlayer is formed by spray-coating the solution B on the liquid surface of the receiving liquid A.

<4> In any one of <1> to <3>, the area of the underlying layer viewed from above vertically is larger than the area where the three-dimensional structure having the desired shape and the underlying layer are in contact with each other. A method for producing the three-dimensional structure described.

<5> The method for producing a three-dimensional structure according to any one of <1> to <4>, in which the shape of the underlying layer viewed from above vertically is a regular polygon or a circle.

<6> In the underlayer forming step, the solution B is added so that the aspect ratio of the underlayer as viewed from above vertically is 1:2 or less. <1> to <5> Manufacturing method of the three-dimensional structure.

<7> Manufacture of the three-dimensional structure according to any one of <1> to <6>, in which pores having a diameter smaller than the diameter of the gel forming the three-dimensional structure are formed in the underlayer. Method.

<8> The method for producing a three-dimensional structure according to any one of <1> to <7>, in which the specific gravity of the underlayer is smaller than the specific gravity of the receiving liquid A.

<9> The method for manufacturing a three-dimensional structure according to any one of <1> to <8>, which includes an underlayer removing step of removing the underlayer after the modeling step.

<10> The method for producing a three-dimensional structure according to <9>, wherein the solution B has a composition different from that of the solution C.

<11> The method for producing a three-dimensional structure according to any one of <1> to <10>, in which the three-dimensional structure contains cells.
<12> The method for producing a three-dimensional structure according to <11>, wherein the cells contained in the three-dimensional structure are plant cells.

According to one aspect of the present invention, it is possible to provide a three-dimensional modeling method that has a high degree of freedom in the receiving liquid, is simpler, and has a low risk of failure.

It is a schematic diagram showing an example of a process in a manufacturing method of a three-dimensional structure concerning the present invention. (A) is a diagram showing the moving direction of the inkjet head in Reference Example 1, and (b) is a diagram showing an image of the underlayer formed in Reference Example 1. (A) is a figure which showed the moving direction of the inkjet head in the reference example 2, (b) is a figure which showed the image which image|photographed the base layer formed in the reference example 2. (A) is a figure which showed the moving direction of the inkjet head in the reference example 3, (b) is a figure which showed the image which image|photographed the base layer formed in the reference example 3. FIG. (A)-(d) is the figure which showed the movement procedure of the inkjet head in the reference example 4. FIG. 3 is a diagram showing a moving direction of an inkjet head in Embodiment 1. FIG. 3 is a diagram showing an image obtained by photographing a three-dimensional structure formed in Example 1. FIG. 9 is a diagram showing a moving direction of an inkjet head in Embodiment 2. FIG. 6 is a diagram showing an image obtained by photographing a three-dimensional structure formed in Example 2. FIG. 11 is a diagram showing a moving direction of an inkjet head in Example 3. FIG. 8 is a diagram showing an image obtained by photographing a three-dimensional structure formed in Example 3; (A)-(f) is the figure which showed the movement procedure of the inkjet head in Example 4.

One embodiment of the present invention will be described below, but the present invention is not limited to this. The present invention is not limited to the respective configurations described below, and various modifications can be made within the scope shown in the claims, and the technical means disclosed in different embodiments and examples, respectively. Embodiments and examples obtained by appropriately combining the above are also included in the technical scope of the present invention. Further, all the academic documents and patent documents described in the present specification are incorporated herein by reference.

Unless otherwise specified in this specification, “A to B” indicating a numerical range means “A or more and B or less”. In the drawing, the X axis, Y axis, and Z axis define the directions in the three-dimensional space. The X axis and the Y axis represent the horizontal direction, and the X axis and the Y axis are orthogonal to each other. The Z axis represents the direction perpendicular to the XY plane.

[1. Method for manufacturing three-dimensional structure]
The three-dimensional modeling method according to one embodiment of the present invention includes a base layer forming step of forming a base layer by adding a solution B which reacts with the receiving liquid A and gels to the liquid surface of the receiving liquid A, and A modeling step of forming a three-dimensional structure by laminating a gel formed by dropping a solution C which reacts with the receiving solution A and gels on the underlayer.

<Receiving liquid A, solution B and solution C>
The solution B and the solution C according to one embodiment of the present invention are solutions that react with the receiving solution A and gelate. That is, when a droplet of the solution B or the solution C is dropped on the receiving liquid A, a gel film is formed on the surface of the droplet. This forms a gel. In the present specification, the solution B represents a solution used to form the underlayer, and the solution C represents a solution used to form a three-dimensional structure. The solution B may be the same solution as the solution C or a different solution.

As a combination of the receiving solution A and the solution B or the solution C, an aqueous solution of an alkaline earth metal salt and an aqueous solution of alginate; an aqueous solution containing two components of calcium salt, thrombin and fibrinogen and an aqueous solution containing the remaining one component; polyvinyl. Examples include, but are not limited to, an aqueous alcohol solution and an aqueous boric acid solution; an aqueous sodium chloride solution and an aqueous peptide hydrogel-forming peptide solution; warm water and an aqueous gelling-type thermoreversible hydrogel-forming hydrophilic polymer aqueous solution. is not. In the case of forming a three-dimensional structure containing cells, the combination of the receiving solution A and the solution B or the solution C is an aqueous solution of an alkaline earth metal salt and an alginate from the viewpoint of biocompatibility and the like. preferable.

Examples of the alkaline earth metal salt include calcium chloride, barium chloride, magnesium chloride and strontium chloride. Examples of the alginate include sodium alginate and potassium alginate. The gel obtained by the reaction between an aqueous solution of an alkaline earth metal salt and an aqueous solution of alginate contains an alkaline earth metal salt of alginic acid. Examples of the alkaline earth metal salt of alginic acid include calcium alginate, barium alginate, strontium alginate, magnesium alginate and the like.

In the above combination, it does not matter which is the receiving liquid A and which is the solution B or the solution C, but from the viewpoint of convenience when the solution B or the solution C is dropped, a solution having a low viscosity is selected. Solution B or solution C is preferred.

In one embodiment of the present invention, damage to cells can be eliminated by using a method of using a combination of gelling solutions as described above, that is, a method of easily performing gelation without temperature change. In addition, as a material which changes sol-gel by temperature change similarly to Patent Document 2, agar (90° C.), gelatin (50 to 60° C.), carrageenan (70° C.), pectin (80° C.), gellan gum (90° C.). ), xanthan gum+locust bean gum (80° C.), tamarind seed gum (85° C.), curdlan (50° C.) and the like. The numbers in the parentheses are melting temperatures.

The concentration of the alkaline earth metal ion is preferably 30 to 1,500 mM, from the viewpoint of suppressing the effect on cells during culture when forming a three-dimensional structure containing cells, and 45-1, More preferably, it is 000 mM. The concentration of the alginate is not particularly limited, but it is preferably 0.5% by weight to 1.5% by weight from the viewpoint of suppressing deterioration in handleability due to high viscosity.

The receiving liquid A, the solution B, and/or the solution C may contain an osmotic pressure adjusting agent, a pH adjusting agent and the like in addition to the above-mentioned components.

Examples of the osmotic pressure adjusting agent include mannitol, sorbitol and the like.

Examples of the pH adjusting agent include 2-morpholinoethanesulfonic acid (hereinafter abbreviated as MES), phosphate buffer, and the like. The pH range suitable for cell culture is generally about 5.5 to 6.

<Underlayer forming step>
The base layer forming step is a step of forming a base layer by adding a solution B which reacts with the receiving liquid A and gels to the liquid surface of the receiving liquid A. In one embodiment of the present invention, a base layer is formed on the liquid surface of the receiving liquid A before forming the target three-dimensional structure. Specifically, the water tank is filled with the receiving liquid A, and the solution B is brought into contact with the liquid surface of the receiving liquid A. Solution B reacts with receiving solution A, resulting in the formation of a gel. As a result, a base layer made of gel can be formed.

By forming the underlayer, the three-dimensional structure can be held without increasing the viscosity of the receiving liquid or installing an elevator stage in the water tank containing the receiving liquid. The accuracy can be increased. Further, since the receiving liquid does not flow when the elevator stage moves up and down, the process time does not become long. Furthermore, since there is no elevator stage, there is no risk of liquid leakage. Further, since there is no elevator stage, the cost of the device can be suppressed.

FIG. 1 is a schematic view showing an example of steps in the method for manufacturing a three-dimensional structure of the present invention. 1A and 1F show an example of a base layer forming step.

The method of bringing the solution B into contact with the liquid surface of the receiving liquid A is not particularly limited. An underlayer may be formed (hereinafter, also referred to as a “spray method”) by spray-coating the solution B on the liquid surface of the receiving liquid A. Alternatively, the underlayer may be formed by applying the solution B to the liquid surface of the receiving liquid A by inkjet (hereinafter, also referred to as “inkjet method”).

For example, FIG. 1A shows a step of forming a base layer in which a solution B3 which reacts with the receiving liquid A1 and gels is spray-coated on the liquid surface of the receiving liquid A1 filled in a water tank by using a spray nozzle 2. Indicates. At this time, in order to form a base layer of a desired size, it is preferable to spray-coat the solution B3 through the mask 4 that controls the range in which the solution B3 is applied. FIG. 1B shows the underlayer formed in the underlayer forming step. The receiving liquid B3 that has reacted with the receiving liquid A1 gels to form the underlayer 5. The underlayer 5 is formed near the liquid surface of the receiving liquid A1. The shape of the underlayer can be changed depending on the opening shape of the mask.

Alternatively, as shown in (f) of FIG. 1, by adding a solution B3 that gels by reacting with the receiving liquid A1 using an inkjet 6 to the liquid surface of the receiving liquid A1 filled in the water tank. 5 may be formed. At this time, it is preferable to drop the solution B3 while moving the inkjet in the X-axis direction and the Y-axis direction.

The spray method is suitable for easily forming the underlayer. Further, in the inkjet method, since the landing position and the spray amount can be controlled in detail, it is easy to control the structure and the film thickness of the underlayer. For example, according to the inkjet method, it is easy to form a base layer having holes and the like. Other methods of dropping the solution B include various contact/non-contact dispenser methods. An inkjet method or a non-contact jet dispenser method is preferable because the underlayer can be formed into an arbitrary shape.

The discharge amount and the flight speed of the solution B may be appropriately adjusted according to the composition of the solution and the like. The discharge amount of the solution B may be, for example, 200 to 700 pL. In this case, the diameter of the discharged droplet may be 70 to 120 μm. The flight speed of the liquid droplets may be 1.5 to 8 m/s.

In the underlayer forming step, it is preferable to add the solution B such that the aspect ratio of the underlayer as viewed from above vertically is 1:2 or less. In the underlayer forming step, it is more preferable to add the solution B while keeping the aspect ratio of the underlayer as viewed from above vertically at 1:2 or less. That is, it is preferable to form the underlayer while keeping the aspect ratio of the underlayer on the XY plane small. Further, it is preferable that the underlayer has a shape with a small aspect ratio. The underlayer is easily affected by the flow of the receiving liquid A. If the underlayer has a large aspect ratio, that is, if the underlayer has a rod-like shape, it may be largely deformed by the flow of the receiving liquid A. By reducing the aspect ratio, the deformation of the underlayer due to the influence of the flow of the receiving liquid A can be reduced. The aspect ratio of the underlayer on the XY plane is preferably 1:2 or less, for example. The aspect ratio of the underlayer on the XY plane of 1:2 means, for example, a case where two spherical gels are connected. In order to form the underlayer while keeping the aspect ratio of 1:2 or less, when the spherical gel is continuously drawn in the same direction, the length formed by the spherical gel continuously discharged in the drawing direction However, it is preferable that the drawing is performed so as not to exceed twice the length of the spherical gel already formed on the side in the direction orthogonal to the drawing direction. Further, as an example of a method for forming a base layer having a desired shape while keeping the aspect ratio small, there is a method of forming the base layer from the central portion toward the outer edge portion.

The shape of the underlayer viewed from above vertically, that is, the shape on the XY plane is preferably a regular polygon or a circle. By forming the underlayer into a shape having a small aspect ratio such as a regular polygon or a circle, it is possible to form the underlayer while keeping the aspect ratio small in the underlayer forming step.

The underlayer preferably has pores smaller than the diameter of the gel forming the three-dimensional structure. By providing the holes in the underlayer, the receiving liquid A oozes out from the holes, and the receiving liquid A is supplied to the surface of the underlayer. Therefore, the solution C and the receiving liquid A can be brought into contact with each other by dropping the solution C on the underlayer in the modeling step described later. Further, since the size of the pores is smaller than the diameter of the gel, it is possible to prevent the gel formed from the dropped solution C from passing through the pores.

The area of the underlayer viewed from above in the vertical direction, that is, the area in the XY plane (hereinafter referred to as “area of the underlayer”) is larger than the area where the three-dimensional structure having the desired shape and the underlayer are in contact with each other. It is preferably large. If the area of the underlayer is larger than the area where the target three-dimensional structure and the underlayer contact, the three-dimensional structure can be held sufficiently stably. However, if the area of the underlayer is too large, the edge of the underlayer may be turned over during the process of stacking the gel, and the gel may be caught in the three-dimensional structure. Therefore, it is preferable that 80% or more of the area of the underlayer on the XY plane is in contact with the target three-dimensional structure.

The specific gravity of the underlayer is preferably smaller than the specific gravity of the receiving liquid A. By setting the specific gravity of the underlayer to be smaller than the specific gravity of the receiving liquid A, the underlying layer can be arranged on the liquid surface of the receiving liquid A.

<Modeling process>
The modeling step is a step of forming a three-dimensional structure by laminating a gel formed by dropping a solution C which reacts with the receiving liquid A and gels on the underlayer. The underlayer exists near the liquid surface of the receiving liquid A. Therefore, by dropping the solution C toward the base layer, the solution C and the receiving solution A come into contact with each other. Solution C reacts with receiving solution A, resulting in the formation of a gel. Then, by repeating this, the gels can be laminated to form a three-dimensional structure.

The method of dropping the solution C is not particularly limited, but an inkjet method or a dispenser method is preferable. This makes it possible to control the landing position and the amount of spray, which makes it easy to control the shape of the three-dimensional structure.

1C to 1E show an example of a molding process for forming a three-dimensional structure.

In FIG. 1C, a solution C7 which reacts with the receiving liquid A1 and gels is dropped using an ink jet 6 onto the underlayer 5 formed near the liquid surface of the receiving liquid A1 to form a tertiary solution. The process of forming the first layer of the original structure 8 is shown. At this time, similarly to the case of FIG. 1F, it is preferable to drop the receiving liquid C7 while moving the inkjet 6 in the X-axis direction and the Y-axis direction. The underlayer 5 is formed near the liquid surface of the receiving liquid A1. By dropping the solution C7 on the underlayer 5, the underlayer 5 slightly sinks into the receiving solution A1, and the solution C7 reacts with the receiving solution A1 to react and gel. Then, the three-dimensional structure 8 can be formed by stacking the formed gels. Since the three-dimensional structure 8 is held on the underlayer 5, the gel can be formed at a desired position. Therefore, the modeling accuracy can be improved. FIG. 1D shows a state where the lamination of the first layer of the three-dimensional structure is completed. 1E shows a step of forming the second and subsequent layers of the three-dimensional structure 8 subsequent to FIG. 1C. Since the base layer can be gradually pushed into the receiving liquid each time the solution C is dropped in the second layer and thereafter, gels can be laminated one after another.

The nozzle diameter for ejecting the solution C in the inkjet method or the dispenser method is appropriately determined. For example, when the solution C contains plant cells, the size of the plant cells is about 20 to 50 μm, and therefore, if the nozzle diameter is 60 μm or more, the risk of nozzle clogging can be reduced. For example, the nozzle diameter may be 60 to 150 μm.

The inkjet method is preferable from the viewpoint that the solution C in picoliter to nanoliter units can be discharged. A piezo method is preferably used as the inkjet method. In the piezo method, a voltage is applied to deform the piezoelectric element. The solution is discharged by the pressure generated by this. The piezo method is preferable from the viewpoint that the droplet size of the ejected ink can be uniformly controlled by an electric pulse signal. In addition, the piezo method has less effect on cells than the thermal method of heating a solution.

As the dispenser method, for example, a jet dispenser method may be used. The jet dispenser method is also non-contact like the ink jet method, and is therefore suitable for modeling using delicate cells. The jet dispenser method is also preferable from the viewpoint that a relatively small amount of liquid droplets can be stably ejected, as in the inkjet method.

The discharge amount, the flight speed, and the like of the solution C may be appropriately adjusted according to the composition of the solution and the like as in the case of the solution B.

In one embodiment of the present invention, the water tank containing the receiving liquid A does not have a vertically movable mechanism inside the water tank. Non-Patent Document 1 discloses that gel is fixed to an elevator stage in order to prevent migration of the formed gel. In one embodiment of the present invention, the three-dimensional structure is held by the base layer, and the base layer sinks into the liquid in the process of forming the three-dimensional structure, so that the base layer can move in the vertical direction. No mechanism is needed. Further, since it is not necessary to make the receiving solution A highly viscous, it is possible to design the receiving solution optimal for cell culture.

Further, the specific gravity of the solution B is preferably smaller than that of the solution C. In this case, the base layer is easily pushed into the receiving liquid when the solution C is dropped. Therefore, the gel can be easily laminated.

In one embodiment of the present invention, the three-dimensional structure may contain cells. That is, the solution C used for forming the three-dimensional structure may contain cells. The cells contained in the three-dimensional structure may be plant cells or animal cells.

The cells may be cells having a differentiation ability, that is, undifferentiated cells, cells differentiated into a specific tissue, or both.

Examples of plant cells having differentiating ability include dedifferentiated cells. Examples of dedifferentiated cells include cells obtained from callus obtained from cells collected from plant tissue. Examples of the plant tissue include roots, stems, leaves, petals, seeds, embryos, ovules, ovaries, anthers, pollen and growth points (stem apical meristems and root apical meristems). Further, cells of these plant tissues can also be used as cells differentiated into specific tissues.

Examples of the animal cell having a differentiating ability include stem cells such as artificial multifunctional stem cells and embryonic stem cells. Animal cells differentiated into specific tissues include vascular endothelial cells, fibroblasts, smooth muscle cells, erythrocytes, leukocytes, platelets, cancer cells, and bacteria (single cells) such as Escherichia coli and lactic acid bacteria.

<Underlayer removal step>
The method for manufacturing a three-dimensional structure according to an embodiment of the present invention may include an underlayer removal step of removing the underlayer after the modeling step. In another embodiment, the underlying layer may not be removed and may be part of the three-dimensional structure.

The method for removing the underlayer is not particularly limited. For example, when an alginate is used as the solution B for forming the underlayer and an aqueous calcium chloride solution is used as the receiving solution A, a chelating agent that supplements the alkaline earth metal can be used to facilitate the removal. The underlayer can be removed.

In order to remove the underlayer more easily, it is preferable that the material of the underlayer and the material of the three-dimensional structure are different, that is, the solution B has a composition different from that of the solution C. At this time, only the underlayer can be removed by using a chemically removable material for the underlayer.

[2. Three-dimensional structure]
In the present specification, the “three-dimensional structure” is a structure formed by laminating a plurality of gels. The shape of the three-dimensional structure may be a sheet, a tube, a sphere, a polyhedron, a cone, a complex thereof, or the like, and is not particularly limited.

The three-dimensional structure may or may not include plant cells or animal cells.

In one embodiment of the present invention, when the three-dimensional structure contains a plant cell, the three-dimensional structure is a structure formed by stacking a plurality of plant cells, and the outline of the target plant is It is preferable that the structure has a shape equal to the shape. Here, the target plant may be the whole plant including roots, stems and leaves, or may be part of the organs.

By the method for manufacturing a three-dimensional structure according to the embodiment of the present invention, it is possible to form a three-dimensional structure having a shape different from the shape when produced by a normal production method or when grown under natural conditions. Becomes For example, a three-dimensional structure having large leaves, a large number of leaves, or different leaf arrangements makes it possible to obtain a plant that efficiently performs photosynthesis. Further, a three-dimensional structure having thick roots, long roots, a large number of roots, or thick stems makes it possible to obtain a plant body having a high absorbency of water or nutrients.

In one embodiment of the present invention, when the three-dimensional structure includes animal cells, the three-dimensional structure is a structure formed by stacking a plurality of animal cells, and the target organ and organ It is preferable that the structure is formed in a shape equal to the general shape such as.

Also, a three-dimensional structure containing no cells can be used as a scaffold for cell culture. By culturing cells on such a three-dimensional structure, a plant, organ, organ or the like having a desired shape may be formed.

The present invention is not limited to the above-described embodiments, but various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining the technical means disclosed in the different embodiments Is also included in the technical scope of the present invention.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the following examples.

[Reference Example 1: Formation of Square (2 cm x 2 cm) Underlayer by Inkjet Method (1)]
<Material>
The following solutions were used as the receiving solution A and the solution B. The pH of each of the receiving liquid A and the solution B was adjusted to 5.8 with MES-KOH.
-Receptor A: calcium chloride 20 mM aqueous solution (0.4 M mannitol, 15 mM magnesium chloride, 5 mM MES-KOH is included)
Solution B: 0.6% by weight aqueous solution of sodium alginate (containing 0.4 M mannitol, 15 mM magnesium chloride, 5 mM MES-KOH)
<Device>
For dropping the solution B, an inkjet is provided on a stage (hereinafter, also referred to as “XY automatic stage”) that has a receiving liquid container holding stage for holding a container filled with the receiving liquid A and is movable in the XY directions. An inkjet device equipped with a head was used. As the ink jet head, a piezo type single nozzle ink jet head was used. A nozzle having an opening for discharge having a diameter of 60 μm was used.

<Formation of base layer>
A petri dish was used as a container for filling the receiving liquid A. The petri dish filled with the receiving liquid A prepared as described above is placed on the receiving liquid container holding stage. The solution B prepared as described above was discharged onto the liquid surface of the receiving liquid A from the inkjet head while moving the XY automatic stage of the inkjet device along the path shown in FIG. The discharge amount of the solution B from the inkjet head was set to about 350 pL per one drop. At this time, the diameter of the droplet of the solution B was about 87 μm, and the flight speed was about 4 m/s. The interval of dropping the solution B was 43 μm in both the X-axis direction and the Y-axis direction. The “dropping interval” is the distance from the center of the droplet dropped immediately before to the center of the droplet dropped next. 43 μm is approximately the same distance as the radius of the droplets of the solution B, and the droplets of the solution B were connected so that adjacent droplets overlap each other by about half, and the underlayer was formed. The thickness of the formed underlayer was about 100 μm.

FIG. 2B shows the formed underlayer. Although slightly deformed, a base layer close to a square could be formed. When the underlayer is formed as shown in FIG. 2A, the underlayer has a large aspect ratio in the XY plane for a while after the formation of the underlayer is started. Therefore, it is easily affected by the flow of the receiving liquid. Therefore, it is considered that the shape of the underlayer was slightly deformed.

[Reference Example 2: Formation of square (2 cm x 2 cm) underlayer by inkjet method (2)]
<Material>
Receptor liquid A and solution B were prepared in the same manner as in Reference Example 1.

<Device>
The same inkjet device as in Reference Example 1 was used.

<Formation of base layer>
The same procedure as in Example 1 was performed except that the solution B was discharged onto the liquid surface of the receiving liquid A while the XY automatic stage of the inkjet device was moved along the path shown in FIG. FIG. 3B shows the formed underlayer. The square base layer could be formed with high precision. Since the underlayer was formed while keeping the aspect ratio small, it is considered that the underlayer could be formed more accurately than in Manufacturing Example 1.

[Reference Example 3: Formation of circular (diameter 3 cm) underlayer by inkjet method]
<Material>
The following solutions were used as the receiving solution A and the receiving solution B. The pH of each of the receiving liquid A and the solution B was adjusted to 5.8 with MES-KOH.
-Receptor A: 40 mM calcium chloride aqueous solution (including 0.4 M mannitol, 15 mM magnesium chloride, 5 mM MES-KOH)
-Solution B: 1.2% by weight aqueous solution of sodium alginate (containing 0.4 M mannitol, 15 mM magnesium chloride, 5 mM MES-KOH)
<Device>
The same inkjet device as in Reference Example 1 was used.

<Formation of base layer>
The petri dish filled with the receiving liquid A prepared as described above is placed on the receiving liquid container holding stage. The solution B prepared as described above was discharged from the inkjet head onto the liquid surface of the receiving liquid A while moving the XY automatic stage of the inkjet device along the path shown in FIG. The discharge amount of the solution B from the inkjet head was set to about 350 pL per one drop. At this time, the diameter of the droplet of the solution B was about 87 μm, and the flight speed was about 4 m/s. The interval at which the solution B was dropped was 64 μm in both the X-axis direction and the Y-axis direction. 64 μm is a distance of about 1.5 times the radius of the droplet of the solution B, and the droplets of the solution B are connected so that adjacent droplets overlap each other by about ¼, and the underlayer is formed. Was formed.

FIG. 3B shows the formed underlayer. The circular underlayer could be formed accurately. Since the underlayer was formed while keeping the aspect ratio small as in Reference Example 2, it is considered that the underlayer could be formed with high accuracy.

[Reference Example 4: Formation of reticulated (3 cm x 3 cm) underlayer by inkjet method]
<Material>
Receptor solution A and solution B were prepared in the same manner as in Reference Example 3.

<Device>
The same inkjet device as in Reference Example 1 was used.

<Formation of base layer>
In the same manner as in Reference Example 3 except that the solution B was discharged from the inkjet head onto the liquid surface of the receiving liquid A while the XY automatic stage of the inkjet device was moved in the procedure shown in FIGS. 5A to 5D. went. Specifically, as shown in FIG. 5A, first, the solution B is dropped while moving the XY automatic stage in the X-axis direction, and the gel is linearly formed so as to be parallel to the X-axis direction. I placed it. Then, the XY automatic stage was moved in the Y-axis direction by 120 μm, and then the solution B was dropped while moving in the X-axis direction in the same manner as in (a), and the gel was arranged in a line. This procedure was repeated until the desired size was reached. That is, a gel was formed as shown in FIG. Next, in order to form a net-like underlayer, the solution is moved in the Y-axis direction as shown in (c) of FIG. 5 in the same manner as the procedure performed in (a) and (b) of FIG. B was dropped, and the gel was arranged in a line so as to be parallel to the Y-axis direction. As shown in FIG. 5D, the formed mesh-like underlayer had pores of about 30 μm×about 30 μm. It should be noted that when the gel was arranged in a line, the aspect ratio was large, and therefore some deformation was observed.

[Reference Example 5: Formation of Square (2 cm x 2 cm) Underlayer by Spray Method]
<Material>
Receptor liquid A and solution B were prepared in the same manner as in Reference Example 1.

<Device>
A commercially available spray nozzle of the type that can be sprayed in a mist state was used. Further, a polyethylene terephthalate (PET) film provided with a square opening of 2 cm×2 cm was used as a mask.

<Formation of base layer>
A base layer was formed in the same manner as in FIG. Specifically, a mask was placed above the petri dish filled with the receiving liquid A as close as possible so as not to contact the surface of the receiving liquid. The opening of the mask is smaller than the petri dish so that the solution B does not overflow the petri dish. From several cm above the mask, the solution B was sprayed onto a larger area than the mask using a spray nozzle, and applied onto the liquid surface of the receiving liquid A.

As in Reference Example 2, a square base layer could be formed.

[Example 1: Modeling of a three-dimensional structure on an underlayer produced by an inkjet method (1)]
<Material>
(Receptor A)
The same solution as in Reference Example 2 was used.

(Solution C)
A solution having the same composition as the solution B in Reference Example 2 was used.

(Underlayer)
The 2 cm×2 cm square underlayer produced in Reference Example 2 was used.

<Device>
The same inkjet device as in Reference Example 1 was used.

<Modeling of three-dimensional structure>
While moving the XY automatic stage of the inkjet device along the path shown in FIG. 6, the solution C was discharged onto the underlayer, and the gel was arranged so that the letter “K” was obtained. This was repeated 3 times. Other than that, it performed on the conditions similar to Example 1.

An image of the obtained three-dimensional structure is shown in FIG. In this way, the desired shaping was possible. The thickness of the three-dimensional structure was about 300 μm. When the three-dimensional structure was formed by the method of Example 1 in the absence of the underlayer, the three-dimensional structure in which the letter “K” could be discriminated was not obtained.

[Example 2: Modeling of a three-dimensional structure on an underlayer produced by an inkjet method (2)]
<Material>
(Receptor A)
The same solution as in Reference Example 2 was used.

(Solution C)
A solution in which cells (protoplasts) were dispersed in a solution having the same composition as the solution B in Reference Example 2 so that the cell density was 1.5×10 ⁶ cells/mL was used.

(Underlayer)
A 7 mm×7 mm square underlayer manufactured by the same method as in Reference Example 2 was used.

<Modeling of three-dimensional structure>
While moving the XY automatic stage of the inkjet device along the path shown in FIG. 8, the solution C prepared as described above is discharged from the inkjet head onto the underlayer, and the gel is formed into a square of 5 mm×5 mm. I placed it. This was repeated 25 times. The discharge amount of the solution C from the inkjet head was set to about 300 pL per one drop. At this time, the droplet diameter of the solution C was about 83 μm, and the flight speed was about 3.5 m/s. The interval at which the solution C was dropped was 60 μm in both the X-axis direction and the Y-axis direction.

An image of the obtained three-dimensional structure is shown in FIG. In this way, the desired shaping was possible. The thickness of the three-dimensional structure was about 2.5 mm. When the three-dimensional structure was formed by the method of Example 1 in the absence of the underlayer, only a large number of gel-like gels were seen, and the intended three-dimensional structure was not obtained. It was

[Example 3: Modeling of a three-dimensional structure on an underlayer produced by an inkjet method (3)]
<Material>
(Receptor A)
The same solution as in Reference Example 3 was used.

(Solution C)
A solution having the same composition as the solution B in Reference Example 3 was used.

(Underlayer)
The 3 cm×3 cm reticulated underlayer produced in Reference Example 4 was used.

<Modeling of three-dimensional structure>
While moving the XY automatic stage of the inkjet device along the path shown in FIG. 10, the solution C prepared as described above was discharged from the inkjet head onto the underlayer, and the gel was formed into a circle with a diameter of 0.7 cm. Was placed. This was repeated 3 times. The other conditions were the same as in Reference Example 3.

An image of the obtained three-dimensional structure is shown in FIG. In this way, the desired shaping was possible. When the three-dimensional structure was formed by the method of Example 1 in the absence of the underlayer, only a large number of gel-like gels were seen, and the intended three-dimensional structure was not obtained. It was

[Example 4: Modeling of a three-dimensional structure on a base layer prepared by a spray method]
<Material>
(Receptor A)
The same solution as in Reference Example 5 was used.

(Solution C)
A solution having the same composition as the solution B in Reference Example 5 was used.

(Underlayer)
The 2 cm×2 cm square underlayer produced in Reference Example 5 was used.

<Modeling of three-dimensional structure>
While moving the XY automatic stage of the inkjet device along the path shown in FIG. 12, the solution C prepared as described above is discharged from the inkjet head onto the underlayer, and the gel is formed into a circle with a diameter of 1.0 cm. Was placed. Specifically, first, as shown in FIG. 12A, the solution B was continuously discharged in the X-axis direction, and the liquid droplets were arranged in a line so that adjacent droplets did not overlap each other. Then, after arranging the gel in one line, as shown in (b) of FIG. 12, the gel arranged immediately before the line was displaced by 80 μm from the starting point of the gel arranged immediately before in the Y direction. Repeatedly placing the gel in the next row in the same direction as. FIG. 12C is a diagram of the arranged gel viewed from the Y-axis direction. Next, as shown in (d) of FIG. 12, the gel observed in (b) of FIG. 12 is filled in a linear manner in the X-axis direction in the same manner as in (b) of FIG. I placed it. FIG. 12E is a view of the gel arranged in FIG. 12D as seen from the Y-axis direction. This (b) and (d) of FIG. 12 was repeated 3 times. 12B and 12D, for convenience, the gel is arranged in a rectangular shape, but a three-dimensional structure is formed so that the final shape becomes a circle shown in FIG. 12F. Formed. The discharge amount of the solution C from the inkjet head was set to about 350 pL per droplet. At this time, the diameter of the droplet of the solution C was about 87 μm, and the flight speed was about 4 m/s. The interval at which the solution C was dropped was 160 μm in the X-axis direction and 80 μm in the Y-axis direction.

As described above, even when the solution C was not dropped so that the gel was connected, the desired shaping was possible as in the case of Example 3.

According to one aspect of the present invention, a three-dimensional structure can be easily manufactured.

1 Receptor liquid A
2 Spray nozzle 3 Solution B
4 Mask 5 Underlayer 6 Inkjet 7 Solution C
8 three-dimensional structure

Claims (12)

An underlayer forming step of forming an underlayer by adding a solution B which reacts with the receiving solution A to gel on the liquid surface of the receiving solution A,
A three-dimensional structure including a modeling step of forming a three-dimensional structure by laminating a gel formed by dropping a solution C which reacts with a receiving liquid A and gels on the underlayer. Manufacturing method. The method for producing a three-dimensional structure according to claim 1, wherein, in the underlayer forming step, the underlayer is formed by applying the solution B onto the liquid surface of the receiving liquid A by inkjet. The method for producing a three-dimensional structure according to claim 1, wherein, in the underlayer forming step, the underlayer is formed by spray-coating the solution B on the liquid surface of the receiving liquid A. The three-dimensional structure according to any one of claims 1 to 3, wherein an area of the underlayer viewed from above in the vertical direction is larger than an area where a three-dimensional structure having a desired shape and the underlayer contact. Body manufacturing method. The method for manufacturing a three-dimensional structure according to any one of claims 1 to 4, wherein the shape of the underlayer viewed from above in the vertical direction is a regular polygon or a circle. The three-dimensional structure according to any one of claims 1 to 5, wherein, in the step of forming the underlayer, the solution B is added so that the aspect ratio of the underlayer viewed from above vertically is 1:2 or less. Manufacturing method. The method for manufacturing a three-dimensional structure according to any one of claims 1 to 6, wherein pores having a diameter smaller than a diameter of a gel forming the three-dimensional structure are formed in the underlayer. The method for producing a three-dimensional structure according to any one of claims 1 to 7, wherein the specific gravity of the underlayer is smaller than the specific gravity of the receiving liquid A. The method for manufacturing a three-dimensional structure according to any one of claims 1 to 8, further comprising a base layer removing step of removing the base layer after the modeling step. The method for manufacturing a three-dimensional structure according to claim 9, wherein the solution B has a composition different from that of the solution C. The method for producing a three-dimensional structure according to any one of claims 1 to 10, wherein the three-dimensional structure contains cells. The method for producing a three-dimensional structure according to claim 11, wherein the cells contained in the three-dimensional structure are plant cells.