Method for manufacturing three-dimensional structure

Abstract

To provide novel means enabling formation to recovery of a three-dimensional structure to be easily performed.SOLUTION: A method for manufacturing a three-dimensional structure is provided, comprising the following steps (i) to (iii): (i) a step of preparing a container in which a top end is a first opening, and at least one second opening is provided for either one or both of a bottom portion and a sidewall; (ii) a step of filling a first composition into the container until at least the first composition comes in contact with from the inside of the container to the second opening, wherein the first composition has a ratio (X / Y) of 10 or more between viscosity X when measured under the conditions of a measuring temperature 23°C and a shear rate 0.1 sec-1 by using a rotational viscometer and viscosity Y when measured under the conditions of a measuring temperature 23°C and a shear rate 100 sec-1 by using the rotational viscometer; and (iii) a step of injecting the second composition into the first composition filled into the container in the step (ii).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for manufacturing a three-dimensional structure. More specifically, the present invention relates to a method for manufacturing a three-dimensional structure, a three-dimensional structure obtained by the manufacturing method, and a container for use in the method for manufacturing a three-dimensional structure.

Background Art

[0002] 3D printing refers to three-dimensionally fabricating a three-dimensional structure based on 3D model data. 3D bioprinting, which utilizes this technology to manufacture three-dimensional cell patterns and the like, has attracted attention in the field of regenerative medicine. In order to be used as a living tissue or organ, it is necessary for the three-dimensional cell pattern to be as complex and flexible as an actual living tissue or organ. In order to create such a pattern, 3D bioprinting is performed by applying shear to a support (a support exhibiting Bingham plastic behavior) that becomes liquid when stress is applied (during drawing) and solid when stress-free after drawing in a container filled with a liquid (Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] As a method for recovering the formed three-dimensional structure from the support, methods such as immersing it in an excessive amount of organic solvent and allowing it to stand still or increasing the electrolyte concentration of the support to destroy the network structure of the gel that is the support are known (Patent Documents 2 to 3), but there are problems such as requiring a long time to recover the three-dimensional structure and possibly damaging the three-dimensional structure. The problem to be solved by the present invention is to provide a new means that can easily perform the process from the formation to the recovery of the three-dimensional structure.

Means for Solving the Problem

[0005] On the other hand, the applicant of the present application has found that a composition containing a polymer having a structural unit derived from a specific N-vinylamide-based monomer and an aqueous medium is useful as a composition for a 3D printing support or a three-dimensional cell culture support, and previously filed a patent application (PCT / JP2023 / 024928). In the process of finding this technology, the viscosity X measured at a measurement temperature of 23°C and a shear rate of 0.1 sec using a rotational viscometer, and the viscosity Y measured at a measurement temperature of 23°C and a shear rate of 100 sec using a rotational viscometer -1 It was found that a composition for a support having a ratio (X / Y) of 10 or more between the viscosities measured under the conditions of has a viscosity characteristic similar to that of a plastic fluid and is likely to have a significantly reduced viscosity when shaken. -1 In addition, as the performance of the container used for filling the composition for the support, support performance such as robustness, durability, and resistance to the support and its stability are generally emphasized, and physical processing of the container for filling the composition for the support such as openings in the bottom and side walls has hardly been noticed.

[0006] ​Under such circumstances, as a result of intensive studies to solve the above problems, the inventors of the present invention have found that even when a support composition having a ratio (X / Y) of 10 or more is filled into a second opening of a container whose upper end is a first opening and at least one second opening is provided in either one or both of the bottom and side walls until it contacts the second opening, the composition is unexpectedly difficult to leak from the second opening due to the yield stress, and a three-dimensional structure can be manufactured using this as a support bath. Moreover, after the formation of the three-dimensional structure is completed, it has been found that the support leaks from the second opening when a simple operation typified by shaking is performed, and the three-dimensional structure can be recovered.

[0007] That is, the present invention provides the following <1> to <18>. <1> A method for manufacturing a three-dimensional structure (hereinafter, also referred to as the method for manufacturing a three-dimensional structure of the present invention or the manufacturing method of the present invention) comprising the following steps (i) to (iii). (i) A step of preparing a container having a first opening at the upper end and at least one second opening provided in either one or both of the bottom and side walls (ii) The viscosity X measured under the conditions of a measurement temperature of 23°C and a shear rate of 0.1 sec using a rotational viscometer, and the viscosity Y measured under the conditions of a measurement temperature of 23°C and a shear rate of 100 sec using a rotational viscometer, and a first composition having a ratio (X / Y) of 10 or more is filled into the container until at least the first composition contacts the second opening from the inside of the container. -1 -1 (iii) A step of injecting a second composition into the first composition filled in the container in step (ii)

[0008] <2> The manufacturing method according to <1>, further comprising the following step (iv). (iv) A step of shaking the container containing the three-dimensional structure and the first composition formed in step (iii) <3> The manufacturing method according to <2>, wherein step (iv) is a step of immersing the container in a solvent that dissolves the first composition until at least the solvent contacts the second opening from the outside of the container, and shaking the container immersed in the solvent. <4> The leakage rate of the first composition leaking from the second opening in step (ii) is 0% by mass or more and 10% by mass or less with respect to the first composition filled in step (ii), and the leakage rate of the first composition leaking from the second opening in step (iv) is 20% by mass or more with respect to the first composition after step (iii). The manufacturing method according to <2> or <3>. <5> The shaking speed in step (iv) is 10 rpm or more and 1000 rpm or less. The manufacturing method according to any one of <2> to <4>.

[0009] <6> The shaking speed in step (iv) is 80 rpm or more and 200 rpm or less. The manufacturing method according to any one of <2> to <4>. <7> Step (iii) is a step of injecting the second composition while applying shear to the first composition filled in the container in step (ii). The manufacturing method according to any one of <1> to <6>. <8> The number of the second openings of the container is 3 or more. The manufacturing method according to any one of <1> to <7>. <9> The number of the second openings of the container is 8 or more. The manufacturing method according to any one of <1> to <8>.

[0010] <10> The container has at least a second opening provided on the side wall, and the opening width in the circumferential direction of the container per second opening provided on the side wall is 0.5 mm or more and 10 mm or less. The manufacturing method according to any one of <1> to <9>. <11> The container has at least a second opening provided on the side wall, and the opening width in the circumferential direction of the container per second opening provided on the side wall is 3.5 mm or more and 4.5 mm or less. The manufacturing method according to any one of <1> to <10>. <12> The opening area per second opening is 15 mm 2 or more and 300 mm 2 or less. The manufacturing method according to any one of <1> to <11>. <13> In step (ii), the area where the second opening and the first composition are in contact per second opening is 15 mm 2 or more and 300 mm2 The production method according to any one of <1> to <12>, which is filled as follows.

[0011] <14> The production method according to any one of <1> to <13>, wherein the first composition contains the following components (A) and (B). (A) A polymer having at least one structural unit selected from a structural unit derived from an N-vinylamide-based monomer and a structural unit derived from an unsaturated carboxylic acid (B) An aqueous medium

[0012] <15> The production method according to <14>, wherein the component (A) is a polymer having a structural unit derived from an N-vinylamide-based monomer. <16> The production method according to any one of <1> to <15>, wherein the second composition contains cells.

[0013] <17> A container (hereinafter also referred to as the container of the present invention) having a first opening at the upper end and at least one second opening provided in either or both of the bottom and side walls, for use in the production method according to any one of <1> to <16>. <18> A three-dimensional structure (hereinafter also referred to as the three-dimensional structure of the present invention) obtained by the production method according to any one of <1> to <16>.

Advantages of the Invention

[0014] According to the production method of the present invention, it is possible to easily perform the steps from the formation to the recovery of the three-dimensional structure.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0016] 〔Method for Manufacturing a Three-Dimensional Structure〕 The method for manufacturing a three-dimensional structure of the present invention comprises the following steps (i) to (iii). (i) Step of preparing a container having a first opening at the upper end and at least one second opening provided in either or both of the bottom and side walls (ii) Viscosity X measured at a measurement temperature of 23°C and a shear rate of 0.1 sec -1 using a rotational viscometer, and the ratio (X / Y) of viscosity X to viscosity Y measured at a measurement temperature of 23°C and a shear rate of 100 sec -1 using a rotational viscometer is 10 or more. Filling the container with at least the first composition until at least the first composition contacts the second opening from the inside of the container (iii) Step of injecting a second composition into the first composition filled in the container in step (ii)

[0017] (Step (i)) The container prepared in step (i) has a first opening at the upper end and at least one second opening provided in either or both of the bottom and side walls. As long as the upper end has the first opening and at least one second opening is provided in either or both of the bottom and side walls, the shape of the container is not particularly limited. For example, there are those in which at least one second opening is provided in the bottom or side wall of a bottle, tube, canister, or cup. Examples of containers in which at least one second opening is provided in the bottom include bottomless cylindrical containers such as cylindrical containers, square cylindrical containers, elliptical cylindrical containers, and polygonal cylindrical containers. The region where the second opening is provided may be either the bottom or the side wall. However, in order to quickly leak the support from the second opening after the completion of the formation of the three-dimensional structure and simplify the recovery of the three-dimensional structure, at least the side wall among the bottom and the side wall is preferable. Examples of the opening shape of the second opening include a polygonal shape, a circular shape, and an elliptical shape. However, as the opening shape of the second opening provided on the side wall, a polygonal shape is preferable, a rectangular shape or a square shape is more preferable, and a rectangular shape is particularly preferable.

[0018] Further, when two or more second openings are provided on the side wall, in order to make the leakage of the support from the second opening after the completion of the formation of the three-dimensional structure uniform and efficient, it is preferable that the second openings are provided at substantially equal intervals in the circumferential direction of the container, and it is more preferable that the second openings are provided in one row (that is, one by one) at substantially equal intervals in the circumferential direction of the container.

[0019] The number of the second openings can be appropriately selected according to the size of the container. However, in order to quickly leak the support from the second opening after the completion of the formation of the three-dimensional structure and simplify the recovery of the three-dimensional structure (particularly for simplifying the recovery of the three-dimensional structure when the internal volume of the container is 50 mL or more and 100 mL or less), it is preferably 3 or more, more preferably 4 or more, still more preferably 8 or more, still more preferably 10 or more, and particularly preferably 12 or more. Also, in order to suppress the leakage of the support until the completion of the formation of the three-dimensional structure (particularly for suppressing the leakage of the support when the internal volume of the container is 50 mL or more and 100 mL or less), it is preferably 40 or less, more preferably 30 or less, still more preferably 25 or less, and particularly preferably 20 or less. As a specific range, 3 or more and 40 or less is preferable, 4 or more and 30 or less is more preferable, 8 or more and 25 or less is still more preferable, 10 or more and 25 or less is still more preferable, and 12 or more and 20 or less is particularly preferable. When the number of the second openings is 8, 10, or 12 or more, the recovery of the three-dimensional structure becomes particularly simple. Also, when the number of the second openings is 25 or less, it becomes easier to suppress the leakage of the support until the completion of the formation of the three-dimensional structure.

[0020] The minimum opening width per second opening is preferably 0.5 mm or more, more preferably 1.5 mm or more, still more preferably 3 mm or more, and particularly preferably 3.5 mm or more in order to promptly leak the support from the second opening after completion of the formation of the three-dimensional structure and simplify the recovery of the three-dimensional structure. Also, in order to suppress the leakage of the support until completion of the formation of the three-dimensional structure, it is preferably 10 mm or less, more preferably 8 mm or less, still more preferably 5 mm or less, and particularly preferably 4.5 mm or less. As a specific range, 0.5 mm or more and 10 mm or less is preferable, 1.5 mm or more and 8 mm or less is more preferable, 3 mm or more and 5 mm or less is still more preferable, and 3.5 mm or more and 4.5 mm or less is particularly preferable. When the minimum opening width per second opening is 3.5 mm or more, the recovery of the three-dimensional structure becomes particularly simple. Also, when the minimum opening width per second opening is 4.5 mm or less, it becomes easier to suppress the leakage of the support until completion of the formation of the three-dimensional structure. Further, when the container has the second opening provided at least on the side wall, as the minimum opening width of the second opening provided on the side wall, the opening width in the circumferential direction of the container or the opening width in the height direction may be used, but the opening width in the circumferential direction of the container is preferable. In addition, when there is a difference between the opening width inside the container and the opening width outside the container, in this specification, the opening width shall mean the opening width inside the container.

[0021] The opening area per second opening can be appropriately selected according to the size of the container. However, in order to promptly leak the support from the second opening after completion of the formation of the three-dimensional structure and simplify the recovery of the three-dimensional structure (particularly for simplifying the recovery of the three-dimensional structure when the internal volume of the container is 50 mL or more and 100 mL or less), it is preferably 15 mm 2 or more, more preferably 45 mm 2 or more, still more preferably 90 mm 2 or more, and particularly preferably 105 mm 2The above, and in order to suppress the leakage of the support until the completion of the formation of the three-dimensional structure (particularly to suppress the leakage of the support when the internal volume of the container is 50 mL or more and 100 mL or less), it is preferably 300 mm 2 or less, more preferably 240 mm 2 or less, still more preferably 150 mm 2 or less, particularly preferably 130 mm 2 or less. As a specific range, it is preferably 15 mm 2 or more and 300 mm 2 or less, more preferably 45 mm 2 or more and 240 mm 2 or less, still more preferably 90 mm 2 or more and 150 mm 2 or less, even more preferably 105 mm 2 or more and 130 mm 2 or less is particularly preferred. When the opening area per one of the second openings is 90 mm 2 or more or 105 mm 2 or more, the recovery of the three-dimensional structure becomes particularly simple. Also, when the opening area per one of the second openings is 130 mm 2 or less, it becomes easier to suppress the leakage of the support until the completion of the formation of the three-dimensional structure. In addition, when there is a difference between the opening area inside the container and the opening area outside the container, in this specification, the above-mentioned opening area shall mean the opening area inside the container.

[0022] When the container has the second opening provided at least on the side wall, the opening ratio in the circumferential direction of the side wall is preferably 5% or more, more preferably 10% or more, still more preferably 15% or more, particularly preferably 25% or more in order to quickly leak the support from the second opening after the completion of the formation of the three-dimensional structure and simplify the recovery of the three-dimensional structure. Also, in order to suppress the leakage of the support until the completion of the formation of the three-dimensional structure, it is preferably 60% or less, more preferably 55% or less, still more preferably 50% or less, particularly preferably 45% or less. As a specific range, it is preferably 5% or more and 60% or less, more preferably 10% or more and 55% or less, still more preferably 15% or more and 50% or less, particularly preferably 25% or more and 45% or less. When the circumferential opening ratio of the side wall is 15% or more or 25% or more, the recovery of the three-dimensional structure becomes particularly simple. The circumferential opening ratio of the side wall can be calculated by the following formula. (Circumferential opening ratio of the side wall) = { (Circumferential opening width of the container per second opening (mm)) × (Number of second openings arranged in the circumferential direction of the container (When the second openings are provided one by one in the circumferential direction of the container, the number of second openings)) / (Inner circumference of the container (mm))} × 100

[0023] The thickness of the side wall is preferably 0.1 mm or more and 3 mm or less, more preferably 0.2 mm or more and 2 mm or less, and particularly preferably 0.5 mm or more and 1.5 mm or less in order to suppress the leakage of the support until the formation of the three-dimensional structure is completed.

[0024] In addition, the material of the container is not particularly limited, and examples include glass, plastic, metal, etc. However, since the situation of 3D printing can be confirmed and it can be directly used for cell culture, plastic is preferred. Also, a semi-transparent or transparent container is preferred.

[0025] Hereinafter, with reference to the accompanying drawings, the container prepared in step (i) (the container according to an embodiment of the present invention) will be described in more detail. In the description of the drawings, the same elements are denoted by the same reference numerals, and duplicate descriptions are omitted. As described above, the container prepared in step (i) only needs to have a first opening at the upper end and at least one second opening provided in either or both of the bottom and the side wall. However, as shown in FIGS. 1 and 2, a preferred embodiment of the container 1 has a bottom surface 2 and a side wall 3 erected on the outer periphery of the bottom surface 2, with a first opening 4 at the upper end, and at least one second opening 5 provided in at least the side wall 3 among the bottom surface 2 and the side wall 3. FIGS. 1 and 2 show a bottomed cylindrical container as an example, but as described above, the shape of the container is not particularly limited, and examples include a bottomed rectangular cylindrical shape, a bottomed elliptical cylindrical shape, a bottomed polygonal cylindrical shape, a deep dish shape, etc.

[0026] When the second opening 5 provided in the side wall 3 is rectangular, it is preferable that the opening width in the circumferential direction of the container is the minimum opening width of the second opening 5 provided in the side wall 3. The opening width in the height direction per one of the second openings 5 provided in the side wall 3 can be appropriately selected according to the size of the container. However, in order to suppress the leakage of the support until the completion of the formation of the three-dimensional structure and to quickly leak the support from the second opening after the completion of the formation of the three-dimensional structure to simplify the recovery of the three-dimensional structure, it is preferably 5 mm or more and 100 mm or less, more preferably 10 mm or more and 70 mm or less, and particularly preferably 20 mm or more and 40 mm or less.

[0027] The internal volume of the container 1 is preferably 10 mL or more and 1000 mL or less, more preferably 20 mL or more and 300 mL or less, and particularly preferably 50 mL or more and 100 mL or less.

[0028] As such a container, known ones or those manufactured according to known methods can be used. Here, when scaling up using a large container or when using a first composition having relatively high fluidity, etc., the container may have a shielding mechanism for shielding the second opening. The first composition can be filled by activating this mechanism, and the first composition can also be filled into the container by housing it in a shielding body that shields the second opening of the container. Further, it is preferable that the shielding mechanism is movable in parallel with the opening surface so as not to cause a pressure change when opening and closing, or is not in close contact with the second opening and there is a gap through which a certain amount of air can pass to relieve the pressure change. In the case of such a shielding mechanism, for example, when a situation such as taking out the container from the shielding mechanism occurs, the problem that the first composition is sucked out of the container due to reduced pressure or the like is less likely to occur. Even when the shielding mechanism or the shielding body is not used, in the method for manufacturing a three-dimensional structure of the present invention, the first composition is less likely to leak from the second opening until the completion of the formation of the three-dimensional structure due to the yield stress of the first composition.

[0029] (Step (ii)) Step (ii) is a step of filling the container with at least a first composition until the first composition contacts the second opening from the inner side of the container, wherein the ratio (X / Y) of the viscosity X measured at a measurement temperature of 23°C and a shear rate of 0.1 sec -1 using a rotational viscometer, to the viscosity Y measured at a measurement temperature of 23°C and a shear rate of 100 sec -1 using the rotational viscometer is 10 or more. The first composition acts as a support for manufacturing a three-dimensional structure. Also, by using the first composition having a ratio (X / Y) of 10 or more in this way, it becomes difficult for the first composition to leak from the second opening until the formation of the three-dimensional structure (for example, 3D printing, three-dimensional cell culture) is completed due to the yield stress. Further, after the completion of the formation of the three-dimensional structure, the three-dimensional structure can be recovered by performing a simple operation typified by shaking. It becomes less susceptible to the influence of contaminants during the formation of the three-dimensional structure, and is also suitable for manufacturing complex and flexible three-dimensional structures such as living tissues and organs.

[0030] Here, "3D printing" is printing that creates a desired structure in three-dimensional space. In 3D printing, the support is a material that is arranged to embed the material for the structure and supports the material for the structure in three-dimensional space. Also, "three-dimensional cell culture" is culturing desired cells in three-dimensional space. In three-dimensional cell culture, the support is a material that is arranged to embed the composition containing the desired cells and supports the composition containing the same cells in three-dimensional space.

[0031] Here, the first composition will be described in detail. In the first composition, the viscosity X measured at a measurement temperature of 23°C and a shear rate of 0.1 sec -1 using a rotational viscometer (for example, a rotational viscometer ViscoQC 300R, RheolabQC manufactured by Anton Paar), and the viscosity measured at a measurement temperature of 23°C and a shear rate of 100 sec -1The ratio (X / Y) with the viscosity Y measured under the conditions is 10 or more, preferably 50 or more, more preferably 75 or more, and particularly preferably 100 or more in order to suppress the leakage of the support until the completion of the three-dimensional structure formation and to quickly leak the support from the second opening after the completion of the three-dimensional structure formation to facilitate the recovery of the three-dimensional structure. Further, it is usually 10,000 or less, preferably 5,000 or less, and more preferably 3,000 or less. The larger this ratio is, the more it indicates having desirable viscosity characteristics like a plastic fluid. As a specific range, 10 or more and 10,000 or less is preferable, 50 or more and 10,000 or less is more preferable, 75 or more and 5,000 or less is still more preferable, and 100 or more and 3,000 or less is particularly preferable.

[0032] As the first composition, a plastic fluid composition is preferable. Here, "plastic fluid" means a kind of non-Newtonian fluid, which is a fluid that does not flow until a certain shear stress (yield stress) is applied. Also, as the first composition, a slurry composition is preferable. In this specification, a slurry refers to a mixture of a solid substance in a liquid. In the first composition, using a rotational viscometer (for example, the rotational viscometer ViscoQC 300R, RheolabQC manufactured by Anton Paar), at a measurement temperature of 23°C and a shear rate of 100 sec -1 The viscosity measured under the conditions is 1 mPa·s or more and 1×10 5 mPa·s or less is preferable, 5 mPa·s or more and 5×10 4 mPa·s or less is more preferable, 10 mPa·s or more and 1×10 4 mPa·s or less is still more preferable, 50 mPa·s or more and 5×10 3 mPa·s or less is particularly preferable. Also, the viscosity measured under the conditions of a measurement temperature of 23°C and a shear rate of 0.1 sec -1 is 1×103 Above 1×10 mPa·s 8 Preferably below 1×10 mPa·s, and more preferably 5×10 3 Above 5×10 mPa·s 7 More preferably below 5×10 mPa·s, and even more preferably 1×10 4 Above 1×10 mPa·s 7 Even more preferably below 1×10 mPa·s, and particularly preferably 5×10 4 Above 5×10 mPa·s 6 Particularly preferably below 5×10 mPa·s. The above viscosity can be determined in accordance with JIS Z 8803:2011. Specifically, it may be measured according to the method described in the examples below.

[0033] In the first composition, the viscosity change rate calculated according to the following formula (α) when NaCl is added is preferably -50% or more and 50% or less, more preferably -30% or more and 20% or less, even more preferably -20% or more and 10% or less, and particularly preferably -10% or more and 5% or less. Viscosity change rate (%) = {(Viscosity before adding NaCl) - (Viscosity after adding NaCl)} / (Viscosity before adding NaCl) × 100 ···(α) 〔In formula (α), the viscosity before adding NaCl means the viscosity (mPa·s) of the composition when measured at a measurement temperature of 23°C and a shear rate of 100 sec -1 using a rotational viscometer (for example, a rotational viscometer ViscoQC 300R or RheolabQC manufactured by Anton Paar). The viscosity after adding NaCl means the viscosity (mPa·s) measured using a rotational viscometer under the condition of a measurement temperature of 23°C for the composition after adding NaCl so that the NaCl concentration becomes 0.15 mol / L and 60 minutes have elapsed.〕 In addition, the viscosity of the first composition after adding NaCl when measured under the conditions of a measurement temperature of 23°C and a shear rate of 100 sec -1 is preferably 1 mPa·s or more and 1×10 5 mPa·s or less, more preferably 5 mPa·s or more and 5×10 4 mPa·s or less, even more preferably 10 mPa·s or more and 1×10 4 mPa·s or less, and particularly preferably 50 mPa·s or more and 5×10 3Less than mPa·s is particularly preferred.

[0034] As the first composition, in order to suppress the leakage of the support until the completion of the formation of the three-dimensional structure, and to quickly leak the support from the second opening after the completion of the formation of the three-dimensional structure to facilitate the recovery of the three-dimensional structure, to enhance the visibility during manufacturing (printing and cell culture), and to make it less susceptible to the influence of contaminants during the formation of the three-dimensional structure, those containing the following component (A) and component (B) are preferred. (A) A polymer having at least one structural unit selected from a structural unit derived from an N-vinylamide-based monomer and a structural unit derived from an unsaturated carboxylic acid (hereinafter also referred to as "structural unit (AS)") (B) An aqueous medium

[0035] (Component (A)) The polymer of component (A) has at least one structural unit selected from a structural unit derived from an N-vinylamide-based monomer and a structural unit derived from an unsaturated carboxylic acid, but those having a structural unit derived from an N-vinylamide-based monomer are preferred. When the polymer of component (A) has such a structural unit, excellent salt tolerance can be obtained while satisfying 3D printing performance and ease of three-dimensional cell culture. When the salt tolerance is excellent, the viscosity is less likely to decrease even when salts or ions are added. Therefore, when a polyvalent ion source (such as a calcium ion source) is added and used as a three-dimensional cell culture support, ions can easily reach the cells and the cells are easily grown. Also, for example, it can be used for 3D printing using an ink that cures in response to calcium ions. The above unsaturated carboxylic acid may be an unsaturated carboxylic acid or an unsaturated carboxylic anhydride, and examples thereof include (meth)acrylic acid, itaconic acid, maleic anhydride, crotonic acid, and salts thereof. These monomers can be used alone or in combination of two or more.

[0036] As the structural unit derived from the N-vinylamide-based monomer, in order to suppress the leakage of the support until the completion of the three-dimensional structure formation and to quickly leak the support from the second opening after the completion of the three-dimensional structure formation to facilitate the recovery of the three-dimensional structure, and also to obtain a desired viscosity when stress is applied while satisfying the salt resistance, the structural unit represented by the following formula (1) (hereinafter, also referred to as "structural unit (1)") is preferable.

[0037]

Chemical formula

[0038] 〔In formula (1), R 1 and R 2 each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, or R 1 and R 2 may be bonded to each other to form a ring structure having 3 to 10 carbon atoms.〕

[0039] In formula (1), the number of carbon atoms of the alkyl group represented by R 1 and R 2 is preferably 1 to 8, more preferably 1 to 4, and particularly preferably 1 or 2 in order to suppress the leakage of the support until the completion of the three-dimensional structure formation and to quickly leak the support from the second opening after the completion of the three-dimensional structure formation to facilitate the recovery of the three-dimensional structure, and also to obtain a desired viscosity when stress is applied while satisfying the salt resistance. The alkyl group may be linear or branched. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, and the like. Also, R 1For the purpose of suppressing the leakage of the support until the completion of the formation of the three-dimensional structure, and facilitating the recovery of the three-dimensional structure by promptly leaking the support from the second opening after the completion of the formation of the three-dimensional structure, and also for obtaining a desired viscosity when applying stress while satisfying salt resistance, a hydrogen atom or an alkyl group having 1 to 10 carbon atoms is preferable, a hydrogen atom or an alkyl group having 1 to 8 carbon atoms is more preferable, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms is still more preferable, and a hydrogen atom or an alkyl group having 1 or 2 carbon atoms is particularly preferable. R 2 For the purpose of suppressing the leakage of the support until the completion of the formation of the three-dimensional structure, and facilitating the recovery of the three-dimensional structure by promptly leaking the support from the second opening after the completion of the formation of the three-dimensional structure, and also for obtaining a desired viscosity when applying stress, a hydrogen atom is preferable. R 1 And R 2 The number of carbon atoms of the ring structure formed by R and R bonding to each other is preferably 4 to 8, and more preferably 4 to 6. R 1 And R 2 When R and R bond to each other to form a ring structure having 3 to 10 carbon atoms, as the structural unit (1), at least one selected from the structural unit represented by the following formula (1-1), the structural unit represented by the following formula (1-2), and the structural unit represented by the following formula (1-3) is preferable, and the structural unit represented by the following formula (1-1) is particularly preferable.

[0040]

Chemical formula

[0041] Examples of the monomer that induces the structural unit (1) include N-vinylformamide, N-vinylacetamide, N-vinylpropionamide, N-vinylbutylamide, N-vinylisobutylamide, N-vinyl-2-methylbutanamide, N-vinyl-3-methylbutanamide, N-vinyl-2,2-dimethylpropionamide, N-vinylvaleramide, N-methyl-N-vinylformamide, N-ethyl-N-vinylformamide, N-propyl-N-vinylformamide, N-isopropyl-N-vinylformamide, N-methyl-N-vinylacetamide, 1-vinyl-2-pyrrolidone, 1-vinyl-2-piperidone, N-vinylcaprolactam, etc. These monomers can be used alone or in combination of two or more. Among these, N-vinylacetamide, N-vinylformamide, 1-vinyl-2-pyrrolidone, and N-vinylpropionamide are preferred, and N-vinylacetamide is particularly preferred.

[0042] The content ratio of the structural unit (AS) is to suppress the leakage of the support until the completion of the three-dimensional structure formation, and to quickly leak the support from the second opening after the completion of the three-dimensional structure formation to simplify the recovery of the three-dimensional structure. To obtain a desired viscosity during stress application and improve the printing performance while satisfying the salt resistance, and to enhance the ease of culturing, it is preferably 50% by mass or more, more preferably 55% by mass or more, and particularly preferably 64% by mass or more with respect to all the structural units in the polymer of component (A). Also, to suppress the leakage of the support until the completion of the three-dimensional structure formation, and to quickly leak the support from the second opening after the completion of the three-dimensional structure formation to simplify the recovery of the three-dimensional structure, and to obtain the desired viscosity characteristics before and after stress application and satisfy the ease of manufacture, it is preferably 95% by mass or less, more preferably 90% by mass or less, and particularly preferably 85% by mass or less with respect to all the structural units in the polymer of component (A). As a specific range, it is preferably 50% by mass or more and 95% by mass or less, more preferably 55% by mass or more and 90% by mass or less, and particularly preferably 64% by mass or more and 85% by mass or less with respect to all the structural units in the polymer of component (A). When the content ratio of the structural unit (AS) is 64% by mass or more, the printing performance and ease of culturing are particularly good. In addition, the content ratio of the structural unit (AS) can be measured by pyrolysis gas chromatography-mass spectrometry (PyGC-MS), CHN elemental analysis, 1 1H-NMR, 13 13C-NMR, etc.

[0043] As the polymer of the component (A), in order to obtain desired viscosity characteristics before and after stress application and satisfy the ease of production by adjusting the interaction between polymers, in addition to the structural unit (AS), it preferably has a structural unit represented by the formula (2).

[0044]

Chemical formula

[0045] 〔In the formula (2), R 3 and R 4 each independently represent a hydrogen atom or a methyl group, R 5 represents an alkylene group having 2 to 4 carbon atoms, and n represents 1 to 1000 as an average value.〕

[0046] R 4 is preferably a methyl group in order to obtain desired viscosity characteristics before and after stress application and satisfy the ease of production. R 5 represents an alkylene group having 2 to 4 carbon atoms, and n R 5 may be the same or different. R 5 The number of carbon atoms of the alkylene group represented by is preferably 2 or 3, more preferably 2. Also, R 5The alkylene group represented by may be linear or branched. Examples of the alkylene group include an ethane-1,2-diyl group, a propane-1,2-diyl group, a propane-1,3-diyl group, a propane-2,2-diyl group, a butane-1,2-diyl group, a butane-1,3-diyl group, a butane-1,4-diyl group, etc. Among these, an ethane-1,2-diyl group is preferred.

[0047] n represents an average value of 1 to 1000. However, in order to obtain desired viscosity characteristics before and after stress application to improve printing performance, to enhance ease of cultivation, and to satisfy manufacturability, an average value of 2 or more is preferred, an average value of 4 or more is more preferred, an average value of 8 or more is further preferred, an average value of 10 or more is particularly preferred. Also, in order to obtain desired viscosity characteristics before and after stress application to improve printing performance, to enhance ease of cultivation, and to satisfy manufacturability, an average value of 500 or less is preferred, an average value of 250 or less is more preferred, an average value of 100 or less is further preferred, an average value of 50 or less is further preferred, an average value of 35 or less is particularly preferred. Specifically, in order to obtain desired viscosity characteristics before and after stress application to improve printing performance, to enhance ease of cultivation, and to satisfy manufacturability, preferably the average value is 2 to 500, more preferably the average value is 4 to 250, further preferably the average value is 8 to 100, further preferably the average value is 10 to 50, and particularly preferably the average value is 10 to 35. When n in formula (2) is 35 or less on average, the printing performance and ease of cultivation are particularly good. In addition, each "average value" in this specification can be measured by NMR. For example, when R 4 in the above formula (2) is a methyl group, for the structure of the above formula (2), 1 1H-NMR is measured, and by comparing the integral values of the respective proton peaks of the alkylene group having 2 to 4 carbon atoms represented by R 5 and the methyl group represented by R 4 , the average value of n can be calculated.

[0048] Further, when the content ratio of the aforementioned structural unit (AS) is 64% by mass or more and n in the formula (2) is 35 or less on average, particularly when the content ratio of the structural unit (AS) is 64% by mass or more and 85% by mass or less and n in the formula (2) is 10 to 35 on average, the printing performance and ease of culturing are particularly good.

[0049] Examples of the monomer that induces the structural unit (2) include ethylene glycol mono(meth)acrylate, diethylene glycol mono(meth)acrylate, triethylene glycol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, 2-methoxyethyl (meth)acrylate, methoxydiethylene glycol (meth)acrylate, methoxytriethylene glycol (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, polypropylene glycol mono(meth)acrylate, methoxypolypropylene glycol (meth)acrylate, and the like. These monomers can be used alone or in combination of two or more.

[0050] The content ratio of the structural unit (2) is preferably 5% by mass or more, more preferably 7% by mass or more, particularly preferably 10% by mass or more, based on all the structural units in the polymer of the component (A) in order to obtain desired viscosity characteristics before and after stress application and to satisfy the ease of production. Also, in order to improve the printing performance, enhance the ease of culturing, and obtain sufficient viscosity to satisfy the shape maintainability of the three-dimensional structure, it is preferably 50% by mass or less, more preferably 40% by mass or less, particularly preferably 30% by mass or less, based on all the structural units in the polymer of the component (A). As a specific range, it is preferably 5% by mass or more and 50% by mass or less, more preferably 7% by mass or more and 40% by mass or less, particularly preferably 10% by mass or more and 30% by mass or less, based on all the structural units in the polymer of the component (A). When the content ratio of the structural unit (2) is 30% by mass or less, the printing performance and ease of culturing are particularly good. The content ratio of the structural unit (2) may be measured in the same manner as the content ratio of the structural unit (AS).

[0051] Also, when the content ratio of the structural unit (2) is 30% by mass or less and n in the above formula (2) is 35 or less on average, particularly when the content ratio of the structural unit (2) is 10% by mass or more and 30% by mass or less and n in the above formula (2) is 10 to 35 on average, the printing performance and ease of culturing are particularly good.

[0052] As the content ratio [(AS):(2)] of the structural unit (AS) and the structural unit (2) contained in the polymer of the component (A), in order to obtain desired viscosity characteristics before and after stress application and improve the printing performance, to enhance the ease of culturing, and to satisfy the ease of manufacturing, the mass ratio is preferably 50:50 to 95:5, more preferably 58:42 to 92:8, and particularly preferably 68:32 to 89:11. When the content ratio [(AS):(2)] is 68:32 or more, the printing performance and ease of culturing are particularly good.

[0053] Also, when the content ratio [(AS):(2)] is 68:32 or more and n in the above formula (2) is 35 or less on average, when the content ratio [(AS):(2)] is 68:32 to 89:11 and n in the above formula (2) is 10 to 35 on average, the printing performance and ease of culturing are particularly good.

[0054] As the polymer of the component (A), in order to reduce the drawability and obtain a desired viscosity before and after stress application, it is preferable to have a structural unit derived from a crosslinkable monomer in addition to the structural unit (AS). Note that as the polymer of the component (A), it is preferable to have both the above structural unit (2) and the structural unit derived from the crosslinkable monomer in addition to the structural unit (AS).

[0055] Examples of the structural unit derived from the crosslinkable monomer include one or more selected from the structural unit derived from a vinyl-based crosslinkable monomer, the structural unit derived from an allyl-based crosslinkable monomer, the structural unit derived from a (meth)acrylate-based crosslinkable monomer, and the structural unit derived from a (meth)acrylamide-based crosslinkable monomer. Further, as the crosslinkable monomer, a crosslinkable monomer having 2 to 5 functional groups is preferable, and a crosslinkable monomer having 2 to 4 functional groups is more preferable. Among the crosslinkable monomers, an allyl-based crosslinkable monomer and a (meth)acrylate-based crosslinkable monomer are preferable, and an allyl-based crosslinkable monomer is more preferable in order to obtain high visibility. Further, as the crosslinkable monomer, a nonionic crosslinkable monomer is preferable in order to improve the salt resistance.

[0056] In addition, as the crosslinkable monomer, a crosslinkable monomer having a degradable partial structure may be preferable. Examples of the degradable partial structure include a disulfide bond, an ester bond, a thioester bond, an acetal bond, a benzyl ester structure, and a nitrobenzyl structure. As an example, when a crosslinkable monomer having a disulfide bond is used and a reducing agent such as dithiothreitol or glutathione is added, a three-dimensional structure can be easily obtained from the container. As the crosslinkable monomer having a degradable partial structure, an allyl-based crosslinkable monomer having a degradable partial structure is preferable because of its high availability.

[0057] Examples of vinyl crosslinkable monomers include aromatic vinyl crosslinkable monomers such as divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene; N,N'-methylenebis(N-vinylacetamide), N,N'-ethylenebis(N-vinylacetamide), N,N'-propylenebis(N-vinylacetamide), N,N'-butylenebis(N-vinylacetamide), N,N'-hexylenebis(N-vinylacetamide) and other N,N'-alkylenebis(N-vinylacetamide); N,N'-alkylenebis(N-vinylformamide) such as N,N'-butylenebis(N-vinylformamide), and in addition, divinyl ether, N,N'-(diacetyl)-N,N'-(divinyl)-1,3-bis(aminomethyl)cyclohexane and the like. These can be used alone or in combination of two or more kinds.

[0058] Examples of allyl crosslinkable monomers include pentaerythritol diallyl ether, pentaerythritol triallyl ether, pentaerythritol tetraallyl ether, tetraallyloxyethane, triallyl phosphate, trimethylolpropane diallyl ether, trimethylolpropane triallyl ether, allylsucrose, diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconate, triallyl trimellitate, diallyl disulfide, bis(1,3-bis(allyloxy)propan-2-yl)disulfide and the like. These can be used alone or in combination of two or more kinds.

[0059] Examples of the (meth)acrylate crosslinkable monomer include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, tetrapropylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, 1,4-butanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, glycerin di(meth)acrylate, trimethylolethane di(meth)acrylate, trimethylolpropane di(meth)acrylate, trimethylolpropane tri(meth)acrylate, butanetriol di(meth)acrylate, pentaerythritol di(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, glucose di(meth)acrylate, glucose tri(meth)acrylate, glucose tetra(meth)acrylate, dipentaerythritol di(meth)acrylate, dipentaerythritol tri(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, inositol di(meth)acrylate, inositol tri(meth)acrylate, inositol tetra(meth)acrylate, mannitol di(meth)acrylate, mannitol tri(meth)acrylate, mannitol tetra(meth)acrylate, mannitol penta(meth)acrylate, bis(2-(meth)acryloyl)oxyethyl disulfide, and the like. These can be used alone or in combination of two or more kinds.

[0060] Examples of the (meth)acrylamide-based crosslinkable monomer include N,N'-methylenebis(meth)acrylamide, N,N'-ethylenebis(meth)acrylamide, N,N'-propylenebis(meth)acrylamide, N,N'-butylenebis(meth)acrylamide, N,N'-hexylenebis(meth)acrylamide, N,N'-bis((meth)acryloyl)cystamine, etc. These can be used alone or in combination of two or more.

[0061] The content ratio of the structural unit derived from the crosslinkable monomer (hereinafter also referred to as "structural unit (3)") is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, still more preferably 1% by mass or more, still more preferably 2% by mass or more, particularly preferably 2.5% by mass or more, based on all the structural units in the polymer of component (A) in order to reduce the drawability and obtain a desired viscosity before and after stress application. Also, in order to maintain a high swelling degree and obtain a high viscosity, it is preferably 10% by mass or less, more preferably 7% by mass or less, particularly preferably 5% by mass or less, based on all the structural units in the polymer of component (A). As a specific range, it is preferably 0.1% by mass or more and 10% by mass or less, more preferably 0.5% by mass or more and 7% by mass or less, still more preferably 1% by mass or more and 7% by mass or less, still more preferably 2% by mass or more and 7% by mass or less, particularly preferably 2.5% by mass or more and 5% by mass or less, based on all the structural units in the polymer of component (A). The content ratio of the structural unit (3) may be measured in the same manner as the content ratio of the structural unit (AS).

[0062] The content ratio [(AS):(3)] of the structural unit (AS) and the structural unit (3) contained in the polymer of component (A) is preferably 83:17 to 99.99:0.01, more preferably 89:11 to 99.9:0.1, still more preferably 93:7 to 99:1, particularly preferably 94:6 to 99:1, by mass ratio, in order to enhance the transparency (visibility during printing or cell culture) and adjust the swelling degree to obtain a high viscosity. When the content ratio [(AS):(3)] is 94:6 or more, the transparency (visibility during printing or cell culture) becomes particularly good.

[0063] The polymer of component (A) may have structural units other than the structural unit (AS), the structural unit (2), and the structural unit derived from the crosslinkable monomer. For example, structural units derived from non-crosslinkable monomers other than the structural unit (AS) and the structural unit (2) can be mentioned, and preferably, they are structural units derived from nonionic non-crosslinkable monomers. Specific examples of such non-crosslinkable monomers include methyl (meth)acrylate, ethyl (meth)acrylate, hydroxymethyl (meth)acrylate, 2,3-dihydroxypropyl (meth)acrylate, (meth)acrylamide, N-methyl (meth)acrylamide, N,N-dimethyl (meth)acrylamide, N-hydroxymethyl (meth)acrylamide, N-hydroxyethyl (meth)acrylamide, N-methoxymethyl (meth)acrylamide, N-ethoxymethyl (meth)acrylamide, polyethylene glycol mono(meth)acrylamide, methoxypolyethylene glycol (meth)acrylamide, polypropylene glycol mono(meth)acrylamide, methoxypolypropylene glycol (meth)acrylamide, allyl alcohol, allyl methyl ether, allyl ethyl ether, ethylene glycol monoallyl ether, pentaerythritol monoallyl ether, polyethylene glycol monoallyl ether, and the like.

[0064] Examples of the polymer of component (A) include particulate polymers, monolithic polymers, plate-like polymers, film-like polymers, fibrous polymers, and chip-like polymers. However, in order to reduce the drawability and obtain a desired viscosity before and after stress application, particulate polymers are preferred, and gel particulate polymers are more preferred. As the polymer of component (A), a nonionic polymer is preferred in order to improve the salt resistance.

[0065] When the polymer of component (A) is a particulate polymer, the volume average particle diameter is preferably 0.05 to 100 μm, more preferably 0.1 to 50 μm. Also, the coefficient of variation of the volume average particle diameter is preferably 30% or less, more preferably 25% or less. Incidentally, the volume average particle diameter and the coefficient of variation can be measured by means of observation with an atomic force microscope in liquid, observation with a phase contrast microscope, measurement of the particle size distribution by laser diffraction / scattering, etc. Further, after fluorescently staining the particles, measurement can also be carried out by means of observation with a confocal laser microscope, etc.

[0066] In the polymer of component (A), while dispersing in pure water, using a rotational viscometer (for example, the rotational viscometer ViscoQC 300R, RheolabQC manufactured by Anton Paar), at a measurement temperature of 23°C and a shear rate of 100 sec -1 When measuring the viscosity of the dispersion under the conditions of, the content of pure water at which the viscosity of the dispersion reaches a value less than 1000 mPa·s is preferably 70% by mass or more, more preferably 80% by mass or more, still more preferably 90% by mass or more, and particularly preferably 95% by mass or more. Further, it is usually 99.9% by mass or less.

[0067] The polymer of component (A) can be produced by appropriately combining known methods described in JP-A-10-226715, JP-A-2002-239380, etc.

[0068] The content ratio of the polymer of component (A) suppresses the leakage of the support until the completion of the formation of the three-dimensional structure, and after the completion of the formation of the three-dimensional structure, causes the support to quickly leak from the second opening to facilitate the recovery of the three-dimensional structure. In order to enhance the shape maintainability of the three-dimensional structure, in the first composition, it is preferably 0.1% by mass or more, more preferably 0.2% by mass or more, and particularly preferably 0.5% by mass or more. Also, in order to suppress the leakage of the support until the completion of the formation of the three-dimensional structure, cause the support to quickly leak from the second opening after the completion of the formation of the three-dimensional structure to facilitate the recovery of the three-dimensional structure, enhance the ease of three-dimensional culture, or improve the visibility, in the first composition, it is preferably 30% by mass or less, more preferably 20% by mass or less, and particularly preferably 10% by mass or less. As a specific range, in the first composition, 0.1% by mass or more and 30% by mass or less is preferable, 0.2% by mass or more and 20% by mass or less is more preferable, and 0.5% by mass or more and 10% by mass or less is particularly preferable.

[0069] (Component (B)) Examples of the aqueous medium of Component (B) include one or more selected from water, alcohol, and a culture medium, with water, a culture medium, a mixture of water and alcohol, and a mixture of water and a culture medium being preferred. When performing three-dimensional cell culture for the production of the three-dimensional structure, as Component (B), a culture medium or a mixture of water and a culture medium is particularly preferred.

[0070] As the alcohol, a lower alcohol is preferred, and a linear or branched monohydric alcohol having 1 to 6 carbon atoms is more preferred. For example, ethanol, isopropanol, n-propanol, etc. may be mentioned. One of these can be used alone or in combination of two or more. In the above mixture of water and alcohol, when performing three-dimensional cell culture for the production of the three-dimensional structure, the content ratio of water is preferably 80% by mass or more and less than 100% by mass, more preferably 90% by mass or more and less than 100% by mass.

[0071] The above culture medium is not particularly limited as long as cells can survive or grow. For example, Eagle's medium, Ham's medium, Fisher's medium, Dulbecco's modified MEM (DMEM) medium, MEM medium, F12 medium, RPMI1640 medium, MCDB104 medium, 199 medium, MCDB153 medium, L15 medium, SkBM medium, Basal medium, and media containing these mixed media may be mentioned. Also, the culture medium may be either a serum medium or a serum-free medium. Examples of the serum include fetal bovine serum. Further, nutrients, energy sources, minerals, etc. may be added to the culture medium. Specifically, minerals, carbon sources (such as glucose, carbon dioxide, etc.), nitrogen sources (such as glutamine, etc.), antibiotics, vitamin sources, mineral sources, proteins, peptides, etc. may be mentioned. As the minerals, a polyvalent ion source is preferred. Examples of the polyvalent ion source include calcium ion sources such as calcium carbonate, calcium hydrogen phosphate, and calcium chloride. According to the present invention, even when such a polyvalent ion source is used as the minerals, three-dimensional cell culture can be efficiently performed. The culture medium used in the present invention may be a medium containing the above-mentioned plurality of media and additives.

[0072] In order to enhance the ease of three-dimensional culture and improve visibility, the content ratio of the aqueous medium of component (B) in the first composition is preferably 70% by mass or more, more preferably 80% by mass or more, and particularly preferably 90% by mass or more. Also, in order to enhance the shape retention of the three-dimensional structure, in the first composition, it is preferably 99.9% by mass or less, more preferably 99.8% by mass or less, and particularly preferably 99.5% by mass or less. As a specific range, in the first composition, 70% by mass or more and 99.9% by mass or less is preferable, 80% by mass or more and 99.8% by mass or less is more preferable, and 90% by mass or more and 99.5% by mass or less is particularly preferable.

[0073] As the content ratio [(A):(B)] of the polymer of component (A) and the aqueous medium of component (B) contained in the first composition, in order to balance the shape retention of the three-dimensional structure and the ease of three-dimensional culture, the mass ratio is preferably 0.1:99.9 to 30:70, more preferably 0.2:99.8 to 20:80, and particularly preferably 0.5:99.5 to 10:90.

[0074] The first composition can contain components other than the above (hereinafter also referred to as other components) as needed. Examples of other components include surfactants, isotonic agents (such as sodium chloride), chelating agents, pH adjusters, buffers, thickeners, stabilizers, etc. One of these can be used alone or in combination of two or more.

[0075] The filling of the first composition into the container may be performed until at least the first composition contacts the second opening from the inside of the container. In the case where two or more second openings are provided and they are provided at different heights, if the first composition contacts a part of the second openings among the two or more second openings, it is not necessary for the first composition to contact the other second openings. Here, while referring to FIG. 3, step (ii) of the manufacturing method according to an embodiment of the present invention will be described in detail. FIG. 3 is a schematic cross-sectional view showing an example of a state in which a container 1 (a bottomed cylindrical container of Production Example 1 in FIG. 3) is filled with a first composition 6 such that the first composition 6 contacts the second opening 5 from the inside of the container 1. In FIG. 3, a part of the opening surface of the second opening 5 is in contact with the first composition 6, but the entire opening surface of the second opening 5 may be in contact with the first composition 6. Although step (ii) fills the container with the first composition so that at least the first composition contacts the second opening from the inside of the container (fills the container so that a region where the first composition contacts the second opening is formed), in the method for manufacturing a three-dimensional structure of the present invention, the first composition hardly leaks from the second opening until the formation of the three-dimensional structure is completed.

[0076] The leakage rate of the first composition leaking from the second opening in step (ii) is preferably 0% by mass or more and 10% by mass or less, more preferably 0% by mass or more and 6% by mass or less, still more preferably 0% by mass or more and 4% by mass or less, still more preferably 0% by mass or more and 2% by mass or less, and particularly preferably 0% by mass, based on the first composition filled in step (ii). The leakage rate of the first composition in the above step (ii) can be calculated from the initial filling weight of the first composition filled in the container and the remaining weight of the first composition remaining in the container after the state has stabilized after the filling is completed.

[0077] The filling in step (ii) suppresses the leakage of the support until the formation of the three-dimensional structure is completed, and allows the support to quickly leak from the second opening after the formation of the three-dimensional structure is completed to facilitate the recovery of the three-dimensional structure. Therefore, the area of the contact surface between the second opening and the first composition per second opening is 15 mm 2 or more and 300 mm 2 or less. This contact area is more preferably 20 mm 2 or more and 240 mm 2 or less, still more preferably 30 mm 2 or more and 160 mm 2 or less, still more preferably 40 mm 2 or more and 100 mm 2 or less, and particularly preferably 60 mm 280 mm or more 2 or less. When the area of contact between the second opening and the first composition per second opening is 60 mm 2 80 mm or more 2 or less, leakage of the support until completion of the formation of the three-dimensional structure is easily suppressed, and recovery of the three-dimensional structure becomes particularly simple. The contact area can be measured, for example, according to the method described in the examples described later.

[0078] (Step (iii)) Step (iii) is a step of injecting a second composition into the first composition filled in the container in step (ii). This step can be carried out with reference to known 3D printing methods such as the inkjet method, the material extrusion method, and the stereolithography method, and 3D bioprinting methods. Specifically, it may be carried out with reference to the descriptions in WO2015 / 129881 pamphlet, WO2016 / 090286 pamphlet, WO2018 / 165584 pamphlet, WO2018 / 187595 pamphlet, WO2018 / 187780 pamphlet, etc. The second composition may be any composition that acts as an ink or a bioink in 3D printing or three-dimensional cell culture. Examples thereof include a curable composition in addition to a composition containing cells and, if necessary, an aqueous medium. When a composition containing cells and, if necessary, an aqueous medium is used as the second composition, three-dimensional cells can be produced as the three-dimensional structure. Examples of the three-dimensional cells include organoids, spheroids, embryoid bodies, tumors, cysts, and microtissues. Organoids and spheroids are preferable, and organoids are particularly preferable. The method for producing a three-dimensional structure of the present invention is suitable for producing a complex and flexible three-dimensional structure such as a biological tissue or an organ, and is particularly suitable for producing an organoid. Examples of the cells include anchorage-dependent cells and floating cells (e.g., blood cells such as leukocytes, erythrocytes, and platelets). Examples of the anchorage-dependent cells include cancer cells such as HeLa cells and F9 cells; fibroblasts such as 3T3 cells; stem cells such as ES cells, iPS cells, and mesenchymal stem cells; kidney cells such as HEK293 cells; nerve cells such as NT2 cells; endothelial cells such as UV♀2 cells, HMEC-1 cells, and HUVEC; cardiomyocytes such as H9c2 cells; and epithelial cells such as Caco-2 cells. Examples of the aqueous medium include one or more selected from water, alcohol, and a medium. Further, it may further contain a surfactant, an isotonic agent (e.g., sodium chloride), a chelating agent, a pH adjuster, a buffer, a thickening agent, a stabilizer, and the like.

[0079] When using, as the second composition, one containing cells and, if necessary, an aqueous medium, the second composition preferably contains at least one selected from an extracellular matrix and a hydrogel, and more preferably contains an extracellular matrix.

[0080] Examples of the extracellular matrix components include components contained in the basement membrane and glycoproteins present in the intercellular space. Examples of the components contained in the basement membrane include type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. Examples of the glycoproteins present in the intercellular space include collagen, laminin, entactin, fibronectin, fibrinogen, and heparin sulfate. One of these or a combination of two or more thereof can be used. Examples of the hydrogel include a combination of a polysaccharide and a coagulant corresponding to the polysaccharide as needed. Examples of the polysaccharide include hyaluronic acid, hyaluronate, alginic acid, alginate, carrageenan, glucomannan, agarose, cellulose, pectin, gellan gum, chitin, chitosan, chondroitin sulfate, etc. Those obtained by subjecting natural substances to hydrolysis treatment with an acid or a base or chemical modification treatment such as acetylation can also be used. One of these can be used alone or two or more thereof can be used in combination. Examples of the coagulant include divalent metal salts. Examples of the divalent metal salts include barium salts, calcium salts, magnesium salts, etc. According to the present invention, even when such a divalent metal salt is used as a coagulant, three-dimensional cell culture can be efficiently performed.

[0081] Further, when the above curable composition is used as the second composition, a 3D printing molded article can be produced as a three-dimensional structure. Examples of such molded articles include models for design images, industrial parts, medical devices, etc. Examples of the curable composition include, in addition to the extracellular matrix component and the hydrogel, thermoplastic resins such as ABS resin, polyethylene, polypropylene, vinyl chloride resin, polyethylene terephthalate, polycarbonate, polyacetal, polyimide, etc.; thermosetting resins such as phenol resin, epoxy resin, melamine resin, silicone resin, etc.; photocurable resins such as acrylic resin; compositions containing inorganic substances such as silica and hydroxyapatite. One of these can be used alone or two or more thereof can be used in combination. Further, it may further contain a monomer, an oligomer, a reaction initiator, a solvent, etc. One of these can be used alone or two or more thereof can be used in combination.

[0082] As step (iii), it is preferable to inject the second composition into the first composition filled in the container while applying shear. When the force applied by shear is equal to or less than the yield value of the first composition, the first composition filled in the container is solid or semi-solid, but when the force applied by shear exceeds the yield value of the first composition, the first composition filled in the container becomes a liquid. At this time, the second composition is injected into the first composition, thereby drawing (applying stress). Also, when the applied stress is removed, the first composition becomes solid or semi-solid again.

[0083] The above shear may be applied by any energy such as mechanical, electrical, radiation or light. Also, the second composition is injected through, for example, an injector, a dispenser, a microchannel, etc. When producing three-dimensional cells, injection may be performed with a syringe, a pipette, an auto cell injector. For example, when using a computer-controlled cell injector, while applying shear to the first composition filled in the container, at a plurality of positions along a path forming a desired 3D pattern, the second composition containing cells and, if necessary, further an aqueous medium is injected.

[0084] In this way, by injecting the second composition (preferably injecting the second composition while applying shear) into the first composition filled in the container in step (ii), a three-dimensional structure of a desired shape can be produced. In particular, when using, as the second composition, one containing cells and, if necessary, further an aqueous medium, the cells can be arranged at desired positions. Also, when using, as the second composition, one containing cells and, if necessary, further an aqueous medium, after injecting the second composition, it is preferable to culture the injected cells. Thereby, the cells adhere, spread and proliferate, and three-dimensional cells are formed. Also, when using, as the second composition, one containing cells and, if necessary, an aqueous medium, the production method of the present invention may further include, after step (iii), adding to the second composition injected into the first composition one or more components selected from a nutrient source, an energy source, and a mineral. Specifically, examples include minerals, carbon sources (such as glucose and carbon dioxide), nitrogen sources (such as glutamine), antibiotics, vitamin sources, mineral sources, proteins, peptides, etc. As the minerals, a polyvalent ion source is preferred. Examples of the polyvalent ion source include calcium ion sources such as calcium carbonate, calcium hydrogen phosphate, and calcium chloride.

[0085] (Step (iv)) As the method for producing a three-dimensional structure of the present invention, in addition to steps (i) to (iii), it is preferably further provided with the following step (iv). When the method for producing a three-dimensional structure of the present invention includes step (iv), the first composition (support) after the production of the three-dimensional structure is significantly reduced in viscosity by the shaking in step (iv), and this leaks out from the second opening, and the three-dimensional structure from which the support has been removed can be easily recovered. When using, as the second composition, one containing cells and, if necessary, an aqueous medium, the three-dimensional cells can be recovered, for example, by suction with a syringe or a pipette. (iv) A step of shaking the container containing the three-dimensional structure formed in step (iii) and the first composition

[0086] The leakage rate of the first composition leaking from the second opening in step (iv) is preferably 20% by mass or more, more preferably 30% by mass or more, still more preferably 50% by mass or more, and particularly preferably 55% by mass or more, with respect to the first composition after step (iii). Also, it is preferably 100% by mass or less, more preferably 96% by mass or less, still more preferably 93% by mass or less, and particularly preferably 90% by mass or less, with respect to the first composition after step (iii). As a specific range, it is preferably 20% by mass or more and 100% by mass or less, more preferably 30% by mass or more and 96% by mass or less, still more preferably 50% by mass or more and 93% by mass or less, and particularly preferably 55% by mass or more and 90% by mass or less, with respect to the first composition after step (iii). The leakage rate of the first composition in step (iv) can be calculated from the weight of the first composition in the container after step (iii) and the remaining weight of the first composition remaining in the container after step (iv).

[0087] The shaking speed in step (iv) is preferably 10 rpm or more, more preferably 35 rpm or more, still more preferably 60 rpm or more, and particularly preferably 80 rpm or more, in order to quickly leak the support from the second opening after the completion of the three-dimensional structure formation and simplify the recovery of the three-dimensional structure. Also, in order to reduce damage to the three-dimensional structure, it is preferably 2000 rpm or less, more preferably 1000 rpm or less, still more preferably 350 rpm or less, and particularly preferably 200 rpm or less. As a specific range, it is preferably 10 rpm or more and 1000 rpm or less, more preferably 35 rpm or more and 500 rpm or less, still more preferably 60 rpm or more and 350 rpm or less, and particularly preferably 80 rpm or more and 200 rpm or less. When the shaking speed in step (iv) is 80 rpm or more, the recovery of the three-dimensional structure becomes particularly simple. The shaking time in step (iv) is preferably 0.5 to 600 minutes, more preferably 1 to 300 minutes, still more preferably 3 to 150 minutes, and particularly preferably 5 to 60 minutes. The shaking in step (iv) can be carried out using a shaking device of a known method such as a rotary shaking method, a reciprocating shaking method, an 8-shaped shaking method, etc.

[0088] As step (iv), in order to quickly leak the support from the second opening after the completion of the formation of the three-dimensional structure and simplify the recovery of the three-dimensional structure, it is preferable to immerse the container in a liquid agent that dissolves the first composition until at least the liquid agent contacts the second opening from the outside of the container, and then shake the container immersed in the liquid agent. When the manufacturing method of the present invention includes this step, not only does the viscosity of the first composition (support) after the production of the three-dimensional structure significantly decrease due to shaking, but also when the support and the liquid agent come into contact with each other through the second opening due to the above immersion, the low-viscosity support and the liquid agent are quickly mixed by the above immersion and shaking, and the support flows out of the container, enabling the support to be removed more efficiently.

[0089] The liquid agent that dissolves the first composition may be any agent that can dissolve the first composition when shaken. For example, water; various buffer solutions such as phosphate buffer, glycine buffer, Good buffer, Tris buffer, ammonia buffer, borate buffer, HEPES buffer; culture medium; aqueous solutions of various salts such as sodium chloride, potassium chloride, calcium chloride; various organic solvents such as methanol, ethanol, acetone, acetonitrile; aqueous solutions of various chelating agents such as ethylenediaminetetraacetic acid, nitrilotriacetic acid, citric acid; acidic solutions such as hydrochloric acid, sulfuric acid; basic solutions such as aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, etc. These liquid agents can be used alone or in combination of two or more. Among these, in order to reduce damage to the three-dimensional structure, water; various buffer solutions such as phosphate buffer, glycine buffer, Good buffer, Tris buffer, ammonia buffer, borate buffer, HEPES buffer; culture medium are preferable. As the above culture medium, the same medium as the culture medium used as the aqueous medium of component (B) contained in the first composition can be used.

[0090] The amount of the solvent used to dissolve the first composition is preferably 50 parts by volume or more, more preferably 75 parts by volume or more, particularly preferably 100 parts by volume or more, with respect to 100 parts by volume of the first composition filled in step (ii). Also, it is preferably 2000 parts by volume or less, more preferably 1000 parts by volume or less, particularly preferably 500 parts by volume or less, with respect to 100 parts by volume of the first composition filled in step (ii). As a specific range, it is preferably 50 parts by volume or more and 2000 parts by volume or less, more preferably 75 parts by volume or more and 1000 parts by volume or less, particularly preferably 100 parts by volume or more and 500 parts by volume or less, with respect to 100 parts by volume of the first composition filled in step (ii).

[0091] The immersion in the solvent for dissolving the first composition is preferably performed such that the area where the second opening per one second opening and the solvent for dissolving the first composition are in contact is 10 mm 2 or more and 400 mm 2 or less. This contact area is more preferably 20 mm 2 or more and 280 mm 2 or less.

[0092] And according to the method for manufacturing a three-dimensional structure of the present invention, it is possible to simply perform from the formation to the recovery of the three-dimensional structure. Further, the method for manufacturing a three-dimensional structure of the present invention can efficiently perform 3D printing (drawing) and three-dimensional cell culture, and is also suitable for manufacturing complex and flexible three-dimensional structures such as living tissues and organs. In addition, the obtained three-dimensional cells can be used, for example, for toxicity evaluation of substances, efficacy evaluation of drugs, elucidation of biochemical functions of cells, search for biomarkers, etc.

[0093] 〔Container, three-dimensional structure〕 The container of the present invention is a container for use in the method for manufacturing a three-dimensional structure of the present invention, having a first opening at the upper end and at least one second opening provided in either one or both of the bottom and the side wall. The container of the present invention is suitable for use in the method for manufacturing a three-dimensional structure of the present invention. The three-dimensional structure of the present invention is obtained by the method for manufacturing a three-dimensional structure of the present invention. In the container of the present invention and the three-dimensional structure of the present invention, the meanings of various statements, the contents of each component, etc. are the same as those described for the method for manufacturing the three-dimensional structure of the present invention.

Examples

[0094] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

[0095] The viscosity measurement conditions in the examples are as shown below unless otherwise specified. That is, in accordance with JIS Z 8803:2011, using a rotational rheometer RheolabQC and an adapter jig CC27 (manufactured by Anton Paar), at 23 °C and a shear rate of 100 s -1 the viscosity was measured under the conditions.

[0096] (Synthesis Example 1) (1) 270 g of ethyl acetate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 21.6 g of N-vinylacetamide (manufactured by Showa Denko K.K.), 7.5 g of M-230G (manufactured by Shin-Nakamura Chemical Co., Ltd.) as methoxypolyethylene glycol (23) monomethacrylate, and 0.9 g of pentaerythritol allyl ether (manufactured by Sigma-Aldrich (a mixture mainly composed of pentaerythritol diallyl ether, pentaerythritol triallyl ether, and pentaerythritol tetraallyl ether)) were all charged into a separable flask and stirred to dissolve. This was heated to 65 °C, and 0.3 g of 2,2'-azoisobutyronitrile (manufactured by Wako Pure Chemical Industries, Ltd.) was added while stirring under a nitrogen atmosphere to initiate polymerization. Stirring was continued for 6 hours while maintaining the temperature. The obtained polymer was filtered and washed with ethyl acetate, and then dried under reduced pressure to obtain Polymer A1. (2) Thereafter, 10 g of Polymer A1 was added to 190 g of pure water in a polypropylene container and dispersed to obtain Composition B1 for a support. (3) Composition B1 for a support was diluted with water so that the viscosity at a shear rate of 100 (1 / s) was 1000 mPa·s to obtain Composition B2 for a support. (4) Weigh out 100 g of the support composition B1, sterilize it in an autoclave, add 1.34 g of Dulbecco's modified Eagle medium powder (manufactured by Sigma-Aldrich) and 11 mL of fetal bovine serum (manufactured by Sigma-Aldrich), and mix them uniformly. Dilute this with Dulbecco's modified Eagle medium containing 10% fetal bovine serum so that the viscosity at a shear rate of 100 (1 / s) becomes 1000 mPa·s to obtain the support composition B3 containing the medium.

[0097] (Synthesis Example 2) (1) Charge 270 g of ethyl acetate, 29.7 g of acrylic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.3 g of pentaerythritol allyl ether into a separable flask, and stir to dissolve. Heat this to 65°C, and while stirring under a nitrogen atmosphere, add 0.3 g of 2,2'-azobisisobutyronitrile (manufactured by Wako Pure Chemical Industries, Ltd.) to initiate polymerization. Continue stirring for 6 hours while maintaining the temperature. Dry the obtained polymer under reduced pressure to obtain Polymer A2. (2) Then, add 2 g of Polymer A2 to 198 g of pure water in a polypropylene container, disperse it, and then neutralize it to pH 7 with a 5 mol / L sodium hydroxide solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain the support composition B4. (3) Dilute the support composition B4 with water so that the viscosity at a shear rate of 100 (1 / s) becomes 1000 mPa·s to obtain the support composition B5. (4) Weigh out 100 g of the support composition B4, sterilize it in an autoclave, add 1.34 g of Dulbecco's modified Eagle medium powder (manufactured by Sigma-Aldrich) and 11 mL of fetal bovine serum (manufactured by Sigma-Aldrich), and mix them uniformly. Dilute this with Dulbecco's modified Eagle medium containing 10% fetal bovine serum so that the viscosity at a shear rate of 100 (1 / s) becomes 1000 mPa·s to obtain the support composition B6 containing the medium.

[0098] (Synthesis Example 3) The amount of N-vinylacetamide was changed to 17.8 g and the amount of M-230G was changed to 11.3 g, and the support composition B7 was obtained in the same manner as the support composition B2 of Synthesis Example 1 except for these changes.

[0099] (Synthesis Example 4) The support composition B8 was obtained in the same manner as the support composition B2 of Synthesis Example 1 except that M-230G was changed to M-450G (manufactured by Shin-Nakamura Chemical Co., Ltd.) as methoxypolyethylene glycol (45) monomethacrylate.

[0100] (Synthesis Example 5) The amount of N-vinylacetamide was changed to 21.0 g and the amount of pentaerythritol allyl ether was changed to 1.5 g, and the support composition B9 was obtained in the same manner as the support composition B2 of Synthesis Example 1 except for these changes.

[0101] Test Example 1 (Evaluation of Shear Rate Dependence of Viscosity) The shear rate dependence of the viscosity was evaluated for the support compositions shown in Table 1. That is, in accordance with JIS Z 8803:2011, using a rotational rheometer RheolabQC and an adapter jig CC18 (manufactured by Anton Paar), the viscosity of the support compositions shown in Table 1 was measured at 23°C and a shear rate of 0.1 (1 / s), and the obtained viscosity value was designated as X. Also, the viscosity (1000 mPa·s) at a shear rate of 100 (1 / s) measured in advance was designated as viscosity value Y. The ratio (X / Y) of viscosity value X to viscosity value Y was determined, and the printing performance was evaluated according to the following criteria. The results are shown in Table 1. (Printing Performance Evaluation Criteria) AAA (better than AA): 100 or more AA (better than A): 75 or more and less than 100 A (good): 50 or more and less than 75

[0102]

Table 1

[0103] (Production Example 1) The bottomed cylindrical container shown in Fig. 1 was fabricated by 3D printing. The printer used was the L-DEVO (registered trademark) TP series medical model (manufactured by Fusion Technology Co., Ltd.), and the filament used was PolyLite PLA (manufactured by Polymaker). As shown in Fig. 1, the bottomed cylindrical container obtained in Production Example 1 has a bottom surface and a side wall erected on the outer periphery of the bottom surface. The upper end forms a first opening, and 13 second openings are provided in the side wall at equal intervals in the circumferential direction of the bottomed cylindrical container. The detailed specifications of the bottomed cylindrical container are as follows. Inner diameter (diameter): 52 mm, inner height: 33 mm, outer diameter (diameter): 55 mm, outer height: 35 mm, second opening: rectangular shape of 27 mm (length in the inner height side height direction) × 4 mm (width in the inner diameter side circumferential direction), length in the inner height side height direction from the inner bottom to the second opening: 2 mm, opening ratio in the inner diameter side circumferential direction of the side wall calculated by the following formula: 32% (Opening ratio in the inner diameter side circumferential direction of the side wall) = { (width in the inner diameter side circumferential direction (mm) of each second opening) × (number of second openings) / (inner circumference (mm) of the bottomed cylindrical container)} × 100 Also, for use in Test Examples 2 to 5, a shielding body that houses the above bottomed cylindrical container and shields the second opening of the bottomed cylindrical container was fabricated by 3D printing using the same printer and filament as above. The shielding body has a bottom surface and a side wall erected on the outer periphery of the bottom surface, and the upper end forms an opening (not shown). The detailed specifications of the shielding body are as follows, and the side wall has no opening. Inner diameter (diameter): 57 mm, inner height: 35 mm, outer diameter (diameter): 60 mm, outer height: 37 mm

[0104] (Production Examples 2 to 6) A bottomed cylindrical container was fabricated in the same manner as in Production Example 1, except that the number of second openings, the width in the inner diameter side circumferential direction of the second opening, and the opening ratio in the inner diameter side circumferential direction of the side wall were made as shown in Table 2.

[0105]

Table 2

[0106] Test Example 2 (Evaluation of Low Leakage of Support during Manufacturing) The difficulty of leakage of the support from the container when manufacturing the three-dimensional structure was evaluated. · Examples 1, 6 to 9 That is, the bottomed cylindrical container obtained in Production Example 1 was housed in a shielding body, the composition B2 for the support was filled up to a height of 20 mm from the inner bottom of the bottomed cylindrical container, and the initial filling weight X1 was recorded. After removing the shielding body, it was left for 60 seconds, and the remaining weight Y1 of the composition B2 for the support in the container after leaving was recorded. The value calculated by ((X1 - Y1) / X1)×100 was defined as "leakage rate 1", and this value was calculated. The obtained leakage rate 1 was recorded as the leakage rate 1 of Example 1 and the leakage rates 1 of Examples 6 to 9. In addition, when the remaining weight Y1 was recorded, the area where the second opening and the composition for the support were in contact per one second opening was calculated by the following formula, and it was 72 mm 2 It was. (Area (mm 2 ) where the second opening and the composition for the support are in contact per one second opening) = (Filling height (mm) when the remaining weight Y1 was recorded - Length in the inner height side height direction from the inner bottom to the second opening (mm)) × Width in the inner diameter side circumferential direction of the second opening

[0107] · Examples 2 to 5, 10 In addition, except that the bottomed cylindrical container obtained in Production Example 1 was changed to the bottomed cylindrical containers obtained in Production Examples 2 to 5 respectively, the leakage rate 1 of Examples 2 to 5 was calculated in the same manner as the leakage rate 1 of Example 1 above. In addition, except that the composition B2 for the support was changed to the composition B5 for the support, the leakage rate 1 of Example 10 was calculated in the same manner as the leakage rate 1 of Example 1 above. Also, the area where the second opening and the composition for the support were in contact per one second opening when the remaining weight Y1 was recorded was as follows: Example 2: 72 mm 2 Example 3: 72 mm 2 Example 4: 54 mm 2 Example 5: 84 mm 2 Example 10: 70 mm 2 It was. The leakage rate 1 of Examples 1 to 10 is shown in Table 3. The smaller the value of the leakage rate 1, the higher the performance of retaining the composition for the support in a stationary state even when the container is filled so that the composition for the support contacts the second opening, and it can be said that the support is less likely to leak from the second opening during the production of the three-dimensional structure.

[0108] Test Example 3 (Evaluation of the removal efficiency of the support after production completion) The removal efficiency of the support from the container after producing the three-dimensional structure was evaluated. · Example 1 That is, the bottomed cylindrical container obtained in Production Example 1 was housed in a shielding body, the composition B2 for the support was filled up to a height of 20 mm from the inner bottom of the bottomed cylindrical container, and the initial filling weight X2 was recorded. After removing the shielding body, the bottomed cylindrical container was left stationary in an acrylic cube container with a side length of 100 mm, and water was filled as the dissolving liquid up to a height of 25 mm from the inner bottom of the cube container. In this way, water was added to the cube container so as to contact the composition B2 for the support in the bottomed cylindrical container through the second opening of the bottomed cylindrical container. This was placed on a bioshaker BR-22FH (manufactured by Taitec Corporation) and shaken at a shaking speed of 90 rpm for 15 minutes. During the shaking, the composition B2 for the support in the bottomed cylindrical container moved into the aqueous phase in the cube container through the second opening. After completion of the shaking, the remaining weight Y2 of the composition B2 for the support in the container was recorded. The value calculated by ((X2 - Y2) / X2)×100 was defined as the "leakage rate 2", and this value was calculated. The obtained leakage rate 2 was recorded as the leakage rate 2 of Example 1.

[0109] · Examples 2 to 10 In addition, the leakage rate 2 of Examples 2 to 5 was calculated in the same manner as that of Example 1 except that the bottomed cylindrical container obtained in Production Example 1 was changed to the bottomed cylindrical containers obtained in Production Examples 2 to 5, respectively. Also, the leakage rate 2 of Examples 6 and 7 was calculated in the same manner as that of Example 1 except that the shaking speed was changed to 110 rpm and 70 rpm, respectively. Further, the leakage rate 2 of Examples 8 and 9 was calculated in the same manner as that of Example 1 except that the dissolution solution was changed to PBS and Dulbecco's modified Eagle medium containing 10% fetal bovine serum, respectively. Also, the leakage rate 2 of Example 10 was calculated in the same manner as that of Example 1 except that the composition B2 for the support was changed to the composition B5 for the support. The leakage rate 2 of Examples 1 to 10 is shown in Table 3. The larger the value of the leakage rate 2, the more efficiently the support can be removed by shaking after the completion of the formation of the three-dimensional structure.

[0110] Test Example 4 (Evaluation of Ease of Removal of Three-Dimensional Structure after Production Completion) Using an acrylamide gel sheet (10 mm square, 1 mm thick) as a simple model of the three-dimensional structure, the ease of removal of the three-dimensional structure after producing the three-dimensional structure was evaluated. · Example 1 That is, the bottomed cylindrical container obtained in Production Example 1 was placed in a shielding body, and the composition B2 for the support was filled from the inner bottom of the bottomed cylindrical container up to a height of 10 mm. Next, an acrylamide gel sheet (10 mm square, 1 mm thick) was placed statically on the composition B2 for the support at the center of the container, and the composition B2 for the support was additionally filled thereon up to a height of 20 mm from the inner bottom. After removing the shielding body, the bottomed cylindrical container was placed statically in an acrylic cube container with a side length of 100 mm, and water was filled as the dissolving solution up to a height of 25 mm from the inner bottom of the cube container. In this way, water was added to the cube container so as to contact the composition B2 for the support in the bottomed cylindrical container through the second opening of the bottomed cylindrical container. This was placed on a bioshaker BR-22FH (manufactured by Taitec Corporation) and shaken at a shaking speed of 90 rpm for 15 minutes. During the shaking, the composition B2 for the support in the bottomed cylindrical container moved into the aqueous phase in the cube container through the second opening. The time from the start of shaking until the support around the acrylamide gel sheet disappeared and the acrylamide gel sheet began to swim due to shaking was recorded. This time was taken as the extraction time (minutes) of Example 1.

[0111] ·Examples 2 to 10 Also, except that the bottomed cylindrical container obtained in Production Example 1 was changed to the bottomed cylindrical containers obtained in Production Examples 2 to 5 respectively, the extraction times of Examples 2 to 5 were measured in the same manner as the extraction time of Example 1 above. Also, except that the shaking speed was changed to 110 rpm and 70 rpm respectively, the extraction times of Examples 6 and 7 were measured in the same manner as the extraction time of Example 1 above. Also, except that the dissolving solution was changed to PBS and Dulbecco's modified Eagle medium containing 10% fetal bovine serum respectively, the extraction times of Examples 8 and 9 were measured in the same manner as the extraction time of Example 1 above. Also, except that the composition B2 for the support was changed to the composition B5 for the support, the extraction time of Example 10 was measured in the same manner as the extraction time of Example 1 above.

[0112] ·Comparative Example Further, the bottomed cylindrical container obtained in Production Example 6 was filled with the support composition B2 up to a height of 10 mm from the inner bottom. Next, an acrylamide gel sheet (10 mm square, 1 mm thick) was placed statically on the support composition B2 at the center of the container, and the support composition B2 was additionally filled thereon up to a height of 20 mm from the inner bottom. Then, the bottomed cylindrical container was placed statically in an acrylic cube container with a side length of 100 mm, and water was filled up to a height of 50 mm from the inner bottom of the cube container. By this operation, the entire bottomed cylindrical container was completely submerged and water flowed in from the first opening, and the inside of the bottomed cylindrical container was also filled with water. This was placed on a bioshaker BR-22FH (manufactured by Taitec Corporation) and shaken at a shaking speed of 90 rpm for 15 minutes. The time from the start of shaking until the support around the acrylamide gel sheet disappeared and the acrylamide gel sheet began to swim due to shaking was recorded. This time was taken as the extraction time (minutes) of the comparative example. Table 3 shows the extraction times of Examples 1 to 10 and the comparative example. It can be said that the shorter the extraction time, the easier it is to simply extract the three-dimensional structure after the completion of the formation of the three-dimensional structure.

[0113] Test Example 5 (Cell culture evaluation) · Examples 1, 6 to 9 HEK293 cells were mixed with the support composition B3 at a concentration of 1.0×10 5 / mL to prepare a support composition containing HEK293 cells. The bottomed cylindrical container obtained in Production Example 1 was housed in a shield, and the support composition containing HEK293 cells was filled up to a height of 20 mm from the inner bottom of the bottomed cylindrical container. After removing the shield, the bottomed cylindrical container was placed statically in a glass petri dish with a diameter of 75 mm, and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum was filled up to a height of 25 mm from the inner bottom of the glass petri dish, and cultured in an incubator with a carbon dioxide concentration of 5% for 96 hours. At that time, the medium filled in the petri dish was exchanged once at the time point when 48 hours had elapsed. After culturing, the total amount of cells in the support in the bottomed cylindrical container was recovered using 100 mL of PBS, and the number of living cells was counted by the trypan blue staining method after trypsin treatment. The growth rate of the living cells was evaluated according to the following criteria. The obtained cell culture evaluation was recorded as the cell culture evaluation of Example 1 and the cell culture evaluations of Examples 6 to 9.

[0114] (Growth rate of viable cells) AA: The number of viable cells is 10 times or more that at the time of seeding A: The number of viable cells is 2 times or more and less than 10 times that at the time of seeding B: The number of viable cells is less than 2 times that at the time of seeding

[0115] · Examples 2 to 5, 10 In addition, except that the bottomed cylindrical container obtained in Production Example 1 was changed to the bottomed cylindrical containers obtained in Production Examples 2 to 5, respectively, the cell culture evaluations of Examples 2 to 5 were calculated in the same manner as the cell culture evaluation of Example 1 above. In addition, except that the composition B3 for the support was changed to the composition B6 for the support, the cell culture evaluation of Example 10 was calculated in the same manner as the cell culture evaluation of Example 1 above. The cell culture evaluations of Examples 1 to 10 are shown in Table 3.

[0116] [Table 3]

[0117] Reference Example To 100 g of the composition B2 for the support or the composition B5 for the support, sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added so that the final concentration became 0.15 mol / L, and calcium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added so that the final concentration became 10 mmol / L, and the mixture was stirred and dissolved to prepare a calcium ion-containing 3D printing support. Using a 3D bioprinter INKREDIBLE (manufactured by Cellink), CELLINK XPLORE (manufactured by Cellink) that cures in response to calcium ions was used as the ink, and a test pattern was formed in the calcium ion-containing 3D printing support. The test pattern when using the composition B2 for the support is shown in FIG. 4, and the test pattern when using the composition B5 for the support is shown in FIG. 5. As shown in FIG. 4, when using the composition B2 for the support, the transparency of the bath was high, but turbidity was confirmed when using the composition B5 for the support. The composition B5 for the support is considered to cause turbidity when a salt such as calcium chloride is added.

Description of Symbols

[0118] 1: Container (bottomed cylindrical container) 2: Bottom (bottom surface) 3: Side wall 4: First opening 5: Second opening 6: First composition

Claims

1. A method for manufacturing a three-dimensional structure, comprising the following steps (i) to (iii). (i) A step of preparing a container having a first opening at the upper end and at least one second opening provided in either or both of the bottom and the side wall (ii) The viscosity X measured using a rotational viscometer under the conditions of a measurement temperature of 23°C and a shear rate of 0.1 sec －１ and the ratio (X / Y) of the viscosity X measured using a rotational viscometer under the conditions of a measurement temperature of 23°C and a shear rate of 100 sec －１ is 10 or more. A step of filling the container with at least the first composition until at least the first composition contacts the second opening from the inside of the container (iii) A step of injecting a second composition into the first composition filled in the container in step (ii)

2. The manufacturing method according to Claim 1, further comprising the following step (iv). (iv) A step of shaking the container containing the three-dimensional structure formed in step (iii) and the first composition

3. The manufacturing method according to Claim 2, wherein step (iv) is a step of immersing the container in a solvent that dissolves the first composition until at least the solvent contacts the second opening from the outside of the container, and shaking the container immersed in the solvent.

4. The manufacturing method according to Claim 2, wherein the leakage rate of the first composition leaking from the second opening in step (ii) is 0 mass% or more and 10 mass% or less with respect to the first composition filled in step (ii), and the leakage rate of the first composition leaking from the second opening in step (iv) is 20 mass% or more with respect to the first composition after step (iii).

5. The manufacturing method according to Claim 2, wherein the shaking speed in step (iv) is 10 rpm or more and 1000 rpm or less.

6. The manufacturing method according to Claim 2, wherein the shaking speed in step (iv) is 80 rpm or more and 200 rpm or less.

7. The manufacturing method according to Claim 1, wherein step (iii) is a step of injecting the second composition into the first composition filled in the container while applying shear.

8. The manufacturing method according to Claim 1, wherein the number of the second openings of the container is 3 or more.

9. The manufacturing method according to Claim 1, wherein the number of the second openings of the container is 8 or more.

10. The manufacturing method according to Claim 1, wherein the container has a second opening provided at least in the side wall, and the opening width in the circumferential direction of the container per second opening provided in the side wall is 0.5 mm or more and 10 mm or less.

11. The manufacturing method according to Claim 1, wherein the container has a second opening provided at least in the side wall, and the opening width in the circumferential direction of the container per second opening provided in the side wall is 3.5 mm or more and 4.5 mm or less.

12. The opening area per second opening is 15 mm ２ or more and 300 mm ２ or less, the manufacturing method according to claim 1.

13. Step (ii) fills such that the area where the second opening and the first composition are in contact per second opening is 15 mm ２ or more and 300 mm ２ or less. The manufacturing method according to claim 1

14. The production method according to claim 1, wherein the first composition contains the following components (A) and (B). (A) A polymer having at least one structural unit selected from a structural unit derived from an N-vinylamide-based monomer and a structural unit derived from an unsaturated carboxylic acid (B) An aqueous medium

15. The production method according to claim 14, wherein component (A) is a polymer having a structural unit derived from an N-vinylamide-based monomer.

16. The production method according to claim 1, wherein the second composition contains cells.

17. A container having a first opening at the upper end and at least one second opening provided in either or both of the bottom and the side wall, for use in the production method according to any one of claims 1 to 16.

18. A three-dimensional structure obtained by the production method according to any one of claims 1 to 16.

Images