JP7129668B2 - Container and its use - Google Patents

Description

The present invention relates to containers and uses thereof. More specifically, the present invention relates to a container, a kit, a method for producing a hydrogel having a concentration gradient of a target substance, a method for culturing cells, and a method for producing a pituitary gland.

Three-dimensional culture (3D culture) of cells is suitable for inducing differentiation of complex tissues and organs from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). It is believed that there are The key point is to reproduce fetal development in a test tube, and it is considered that 3D culture is easier to reproduce fetal development than planar culture.

The pituitary gland is one example of a complex organ. For example, in Patent Document 1, aggregates of human pluripotent stem cells are subjected to suspension culture (3D suspension culture) in a medium containing an activator of osteogenic factor signaling pathway and an active substance of Shh signaling pathway. A method for producing human cell aggregates containing glandular pituitary gland or its progenitor tissue is described.

WO2016/013669 WO2017/126551

The development of the pituitary gland is well studied and knowledge is accumulating. FIG. 1 is a schematic diagram showing mouse development after the formation of pituitary primordia, which can be called the latter half of pituitary differentiation. In FIG. 1, BMP, FGF, Shh, and Wnt represent differentiation-inducing signals. As shown in FIG. 1, it is believed that signals from the hypothalamus on the dorsal side and signals from the oral epidermis on the ventral side act on the pituitary primordium in a state in which a concentration gradient is formed. Neural crest-derived mesenchymal cells migrate to the periphery in the left-right direction, and signals are also secreted therefrom.

Patent Document 2 describes a further development of the manufacturing method of Patent Document 1. Specifically, by 3D culturing mouse or human ES cells or iPS cells, hypothalamus and oral ectoderm, which are essential for pituitary differentiation, can be simultaneously induced to differentiate into one 3D cell mass. can. The pituitary primordium is then spontaneously induced and ultimately differentiated into pituitary hormone-producing cells.

These pituitary hormone-producing cells have achieved a high level of differentiation, as in the living body, by secreting hormones in response to stimuli and being inhibited by the presence of sufficient hormones in the surroundings. Since pituitary hormone-producing cells with such responsiveness cannot be induced by planar culture, 3D culture is considered to have high potential.

On the other hand, conventional 3D culture methods have limitations. In the pituitary gland of a living body, a plurality of types of pituitary hormone-producing cells are unevenly distributed for each type on the rostral side, caudal side, ventral side, and dorsal side. In contrast, the conventional 3D culture method can induce pituitary hormone-producing cells one by one, but cannot simultaneously induce multiple types of pituitary hormone-producing cells by unevenly distributing them.

The inventors believe that the reason for this is that the concentration gradient of the inducer present in the fetus cannot be reproduced by the conventional 3D culture method, and the concentration of the inducer in the culture medium is constant. thought. Accordingly, an object of the present invention is to provide a technique for forming a concentration gradient of a target substance.

［式（Ａ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^１～Ｒ^４は、それぞれ独立に、水素原子、－Ｌ－Ｚ^１、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^１又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^１はアルキニル基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^２及びＲ^４はそれぞれ同一であってもよく異なっていてもよい。］

［式（Ｂ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^５～Ｒ^８は、それぞれ独立に、水素原子、－Ｌ－Ｚ^２、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^２又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^２はアジド基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^６及びＲ^８はそれぞれ同一であってもよく異なっていてもよい。］
［６］［１］～［３］のいずれかに記載の容器の前記孔にハイドロゲルを配置する工程と、前記複数の液体貯留部の少なくとも２つに、対象物質の濃度が互いに異なる液体をそれぞれ入れ、その結果、前記ハイドロゲルに前記対象物質の濃度勾配が形成される工程と、を含む、対象物質の濃度勾配を有する前記ハイドロゲルの製造方法。
［７］前記ハイドロゲルが、下記式（Ａ）で表される化合物、下記式（Ｂ）で表される化合物及び水系液体を混合することにより得られるものである、［６］に記載の製造方法。

［式（Ａ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^１～Ｒ^４は、それぞれ独立に、水素原子、－Ｌ－Ｚ^１、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^１又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^１はアルキニル基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^２及びＲ^４はそれぞれ同一であってもよく異なっていてもよい。］

［式（Ｂ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^５～Ｒ^８は、それぞれ独立に、水素原子、－Ｌ－Ｚ^２、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^２又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^２はアジド基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^６及びＲ^８はそれぞれ同一であってもよく異なっていてもよい。］
［８］［１］～［３］のいずれかに記載の容器の前記孔に細胞含有ハイドロゲルを配置する工程と、前記複数の液体貯留部の少なくとも２つに、対象物質の濃度が互いに異なる培地をそれぞれ入れ、その結果、前記細胞含有ハイドロゲルに前記対象物質の濃度勾配が形成される工程と、前記細胞含有ハイドロゲルをインキュベートする工程と、を含む、細胞培養方法。
［９］前記細胞含有ハイドロゲルが、下記式（Ａ）で表される化合物、下記式（Ｂ）で表される化合物、培地及び細胞を混合することにより得られるものである、［８］に記載の細胞培養方法。

［式（Ａ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^１～Ｒ^４は、それぞれ独立に、水素原子、－Ｌ－Ｚ^１、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^１又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^１はアルキニル基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^２及びＲ^４はそれぞれ同一であってもよく異なっていてもよい。］

［式（Ｂ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^５～Ｒ^８は、それぞれ独立に、水素原子、－Ｌ－Ｚ^２、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^２又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^２はアジド基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^６及びＲ^８はそれぞれ同一であってもよく異なっていてもよい。］
［１０］前記細胞が下垂体原基の性質を有する細胞塊であり、前記対象物質が、糖質コルチコイド、骨形成タンパク質（ＢＭＰ）、線維芽細胞増殖因子（ＦＧＦ）及びソニック・ヘッジホッグ（Ｓｈｈ）からなる群より選択される分化誘導因子である、［８］又は［９］に記載の細胞培養方法。
［１１］下垂体の製造方法であって、［１］～［３］のいずれかに記載の容器の前記孔に、下垂体原基の性質を有する細胞塊を含有する、細胞塊含有ハイドロゲルを配置する工程と、前記複数の液体貯留部の少なくとも２つに、糖質コルチコイド、ＢＭＰ、ＦＧＦ及びＳｈｈからなる群より選択される分化誘導因子の濃度が互いに異なる培地をそれぞれ入れ、その結果、前記細胞塊含有ハイドロゲルに前記分化誘導因子の濃度勾配が形成される工程と、前記細胞塊含有ハイドロゲルをインキュベートし、その結果、前記細胞塊から下垂体が形成される工程と、を含む、製造方法。
［１２］前記細胞塊含有ハイドロゲルが、下記式（Ａ）で表される化合物、下記式（Ｂ）で表される化合物、培地及び下垂体原基の性質を有する細胞塊を混合することにより得られるものである、［１１］に記載の製造方法。

［式（Ａ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^１～Ｒ^４は、それぞれ独立に、水素原子、－Ｌ－Ｚ^１、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^１又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^１はアルキニル基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^２及びＲ^４はそれぞれ同一であってもよく異なっていてもよい。］

［式（Ｂ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^５～Ｒ^８は、それぞれ独立に、水素原子、－Ｌ－Ｚ^２、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^２又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^２はアジド基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^６及びＲ^８はそれぞれ同一であってもよく異なっていてもよい。］ The present invention includes the following aspects.
[1] A container body in which an internal space capable of storing liquid is formed; a container formed with a hole that connects the two and that holds the hydrogel.
[2] The container according to [1], wherein the hole is formed at a position apart from the peripheral edge of the partition when viewed from the thickness direction of the partition.
[3] The container according to [1] or [2], wherein a compound for fixing the hydrogel is layered on at least part of the surfaces of the pores.
[4] A kit comprising the container according to any one of [1] to [3] and the material for the hydrogel.
[5] The kit according to [4], wherein the hydrogel material contains a compound represented by the following formula (A) and a compound represented by the following formula (B).

[In formula (A), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ¹ to R ⁴ each independently represent a hydrogen atom; , -LZ ¹ , -O(CH ₂ CH ₂ O) _n -LZ ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ¹ represents an alkynyl group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ² and R ⁴ may be the same or different. ]

[In formula (B), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ⁵ to R ⁸ each independently represent a hydrogen atom; , -LZ ² , -O(CH ₂ CH ₂ O) _n -LZ ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ² represents an azide group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ⁶ and R ⁸ may be the same or different. ]
[6] A step of disposing a hydrogel in the hole of the container according to any one of [1] to [3]; respectively, so that a concentration gradient of the target substance is formed in the hydrogel.
[7] The production according to [6], wherein the hydrogel is obtained by mixing a compound represented by the following formula (A), a compound represented by the following formula (B), and an aqueous liquid. Method.

[In formula (A), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ¹ to R ⁴ each independently represent a hydrogen atom; , -LZ ¹ , -O(CH ₂ CH ₂ O) _n -LZ ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ¹ represents an alkynyl group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ² and R ⁴ may be the same or different. ]

[In formula (B), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ⁵ to R ⁸ each independently represent a hydrogen atom; , -LZ ² , -O(CH ₂ CH ₂ O) _n -LZ ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ² represents an azide group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ⁶ and R ⁸ may be the same or different. ]
[8] The step of disposing the cell-containing hydrogel in the holes of the container according to any one of [1] to [3]; A method for culturing cells, comprising the steps of adding a medium to each of the cells, thereby forming a concentration gradient of the target substance in the cell-containing hydrogel, and incubating the cell-containing hydrogel.
[9] The cell-containing hydrogel is obtained by mixing a compound represented by the following formula (A), a compound represented by the following formula (B), a medium and cells, [8] The cell culture method described.

[In formula (A), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ¹ to R ⁴ each independently represent a hydrogen atom; , -LZ ¹ , -O(CH ₂ CH ₂ O) _n -LZ ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ¹ represents an alkynyl group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ² and R ⁴ may be the same or different. ]

[In formula (B), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ⁵ to R ⁸ each independently represent a hydrogen atom; , -LZ ² , -O(CH ₂ CH ₂ O) _n -LZ ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ² represents an azide group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ⁶ and R ⁸ may be the same or different. ]
[10] The cells are cell masses having properties of pituitary primordium, and the target substances are glucocorticoid, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and sonic hedgehog (Shh ), the cell culture method of [8] or [9], wherein the differentiation-inducing factor is selected from the group consisting of
[11] A method for producing a pituitary gland, comprising a cell mass-containing hydrogel containing cell masses having properties of pituitary primordium in the hole of the container according to any one of [1] to [3]. and at least two of the plurality of liquid reservoirs are filled with media having different concentrations of a differentiation-inducing factor selected from the group consisting of glucocorticoid, BMP, FGF and Shh, and as a result, a step of forming a concentration gradient of the differentiation-inducing factor in the cell mass-containing hydrogel; and a step of incubating the cell mass-containing hydrogel, as a result of which a pituitary gland is formed from the cell mass, Production method.
[12] The cell mass-containing hydrogel is obtained by mixing a compound represented by the following formula (A), a compound represented by the following formula (B), a medium, and cell masses having properties of pituitary primordia. The production method according to [11], which is obtained.

[In formula (A), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ¹ to R ⁴ each independently represent a hydrogen atom; , -LZ ¹ , -O(CH ₂ CH ₂ O) _n -LZ ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ¹ represents an alkynyl group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ² and R ⁴ may be the same or different. ]

[In formula (B), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ⁵ to R ⁸ each independently represent a hydrogen atom; , -LZ ² , -O(CH ₂ CH ₂ O) _n -LZ ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ² represents an azide group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ⁶ and R ⁸ may be the same or different. ]

ADVANTAGE OF THE INVENTION According to this invention, the technique which forms the concentration gradient of a target substance can be provided.

FIG. 4 is a schematic diagram showing development after formation of pituitary primordia. 1 is a perspective view of a container according to one embodiment; FIG. Figure 3 is a plan view of the container of Figure 2; 4 is a YZ cross-sectional view taken along line II shown in FIG. 3; FIG. 4 is an XZ cross-sectional view along line II-II shown in FIG. 3; FIG. FIG. 3 is a YZ cross-sectional view of a culture apparatus using the container of FIG. 2; FIG. 3 is an XZ cross-sectional view of a culture apparatus using the container of FIG. 2; 1 is a top view showing an example of a container having three liquid reservoirs; FIG. It is a schematic diagram which shows the shaping|molding die of the container which concerns on 1 Embodiment. It is a perspective view which shows the shaping|molding die of the container which concerns on 1 Embodiment. It is a mimetic diagram explaining a manufacturing method of a container concerning one embodiment. It is a mimetic diagram explaining a manufacturing method of a container concerning one embodiment. It is a mimetic diagram explaining a manufacturing method of a container concerning one embodiment. 1 is a top view showing an example of a container with two liquid reservoirs; FIG. It is a top view of the container used in the experimental example. FIG. 16 is a YZ cross-sectional view along line III-III shown in FIG. 15; FIG. 16 is an XZ sectional view along line IV-IV shown in FIG. 15; 6(a) is a photograph showing a state in which a pre-gel solution was introduced into the holes of the container shown in FIGS. 3 to 5 in Experimental Example 3. FIG. 17(b) is a photograph showing how a pre-gel solution was introduced into the holes of the container shown in FIGS. 15 to 17 in Experimental Example 3. FIG. (a) and (b) are photographs showing a state in which liquids are respectively introduced into the first liquid reservoir and the second liquid reservoir of a container in which hydrogel is introduced into the pores. (a) and (b) are photographs of hydrogels when fluorescein was used as a target substance in Experimental Example 4. FIG. (c) is a graph showing the results when fluorescein was used as the target substance in Experimental Example 4. FIG. (a) and (b) are photographs of hydrogels when FITC dextran was used as a target substance in Experimental Example 4. FIG. (c) is a graph showing the results when FITC dextran was used as the target substance in Experimental Example 4. FIG. (a) and (b) are photographs of hydrogels when a YFP-CFP fusion protein was used as a target substance in Experimental Example 4. FIG. (c) is a graph showing the results when YFP-CFP fusion protein was used as the target substance in Experimental Example 4. FIG. (a) and (b) are fluorescence micrographs showing the results of immunostaining in Experimental Example 5. FIG. 11 is a fluorescence micrograph showing the results of immunostaining in Experimental Example 6. FIG.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as the case may be. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and overlapping descriptions are omitted. Note that the dimensional ratios in each drawing are exaggerated for the sake of explanation, and do not necessarily match the actual dimensional ratios. In some cases, the figures also show an XYZ coordinate system. Hereinafter, each direction will be explained based on each coordinate system as necessary.

[container]
In one embodiment, the present invention includes a container body having an internal space capable of storing a liquid, and a partition wall partitioning the internal space into a plurality of liquid reservoirs, wherein the partition wall stores the plurality of liquids. Provided is a container in which a hole communicating with at least two of the reservoirs and in which a hydrogel is retained is formed. As will be described later, by using the container of this embodiment, a hydrogel having a concentration gradient of the target substance can be produced.

FIG. 2 is a perspective view of a container 10, which is an example of the container of this embodiment. FIG. 3 is a plan view of the container 10. FIG. FIG. 4 is a YZ sectional view taken along line II shown in FIG. FIG. 5 is an XZ sectional view along line II-II shown in FIG.

2 to 5, the X direction is the longitudinal direction of the bottom plate 11 of the container body 1. As shown in FIG. The Y direction is a direction orthogonal to the X direction within the plane along the bottom plate 11 . The Z direction is a direction perpendicular to the X direction and the Y direction, and is the thickness direction of the bottom plate 11 . The Z direction is also referred to as a vertical direction or a height direction. The pair of side plates 12A, 12B and the pair of end plates 13A, 13B extend upward with respect to the bottom plate 11. As shown in FIG. Planar view means viewing from the Z direction.

As shown in FIG. 2, the container 10 has a container body 1 and a partition wall 2 . The container body 1 includes a bottom plate 11, a pair of side plates 12A and 12B, and a pair of end plates 13A and 13B. The side plates 12A, 12B, the end plates 13A, 13B, and the partition wall 2 constitute a main portion 14 of the container 10. As shown in FIG. The main portion 14 may be integrally formed.

The bottom plate 11 has a rectangular shape in plan view. The side plates 12A and 12B are erected on the first main surface 11a of the bottom plate 11. As shown in FIG. The side plates 12A, 12B are formed along the XZ plane. The side plates 12A and 12B are, for example, rectangular when viewed from the thickness direction (Y direction). The two side plates 12A and 12B are separated in the Y direction.

The end plates 13A and 13B are erected on the first major surface 11a of the bottom plate 11. As shown in FIG. The end plates 13A, 13B are formed along the YZ plane. The end plates 13A and 13B are rectangular when viewed from the thickness direction (X direction). The two end plates 13A and 13B are formed apart in the X direction.

A space surrounded by the bottom plate 11, the side plates 12A and 12B, and the end plates 13A and 13B is called an internal space 15. As shown in FIG. The lower edges of the side plates 12A and 12B, the lower edges of the end plates 13A and 13B, and the bottom plate 11 are liquid-tightly joined. Therefore, the container 10 can store liquid in the internal space 15 . The bottom plate 11 closes lower openings of liquid reservoirs 17A and 17B (described later).

The partition wall 2 is erected on the first main surface 11 a of the bottom plate 11 . The partition wall 2 is formed along the XZ plane. The partition wall 2 has a rectangular shape when viewed from the thickness direction (Y direction). The partition wall 2 is parallel to the side plates 12A and 12B. The partition wall 2 is formed between the side plates 12A and 12B with a space therebetween. The partition 2 has a constant thickness.

A lower edge 2d of the partition wall 2 is joined to the first main surface 11a of the bottom plate 11 in a liquid-tight manner. Side edges 2fa and 2fb of partition 2 reach inner surfaces 13Aa and 13Ba of end plates 13A and 13B, respectively. The upper edge 2e of the partition wall 2 is positioned at the same height as the upper edges 12Ab and 12Bb of the side plates 12A and 12B and the upper edges 13Ab and 13Bb of the end plates 13A and 13B.

The partition wall 2 divides the internal space 15 into two liquid reservoirs 17A and 17B in the container body 1 . The partition wall 2 separates the two liquid reservoirs 17A and 17B. The first liquid reservoir 17A is a space formed between one surface 2a (first main surface 2a) of the partition wall 2 and the first side plate 12A. The second liquid reservoir 17B is a space formed between the other surface 2b (second main surface 2b) of the partition wall 2 and the second side plate 12B.

As shown in FIGS. 4 and 5, the partition wall 2 is formed with holes 21 . The hole 21 is a through hole penetrating through the partition wall 2 in the thickness direction (Y direction). Hole 21 communicates between first liquid reservoir 17A and second liquid reservoir 17B.

As shown in FIG. 5, the shape of the hole 21 seen from the thickness direction (Y direction) of the partition wall 2 is rectangular. A lower edge 21a and an upper edge 21c of the hole 21 extend along the X direction. Side edges 21ba and 21bb of the hole 21 extend along the Z direction.

As shown in FIG. 5, the bottom surface 23a and the top surface 23c of the hole 21 are along the XY plane. The bottom surface 23a and the top surface 23c are parallel to each other. As shown in FIG. 5, the pair of side surfaces 23ba and 23bb of the hole 21 are along the YZ plane. Sides 23ba and 23bb are parallel to each other. The bottom surface 23 a coincides with the lower edge 21 a when viewed from the thickness direction (Y direction) of the partition wall 2 . The side surfaces 23ba and 23bb match the side edges 21ba and 21bb, respectively, when viewed from the Y direction. The top surface 23c matches the upper edge 21c when viewed from the Y direction.

Hole 21 is formed at a position away from the peripheral edges of partition wall 2 (lower edge 2d, upper edge 2e, and side edges 2fa and 2fb). That is, the lower edge 21a of the hole 21 is located higher than the lower edge 2d of the partition wall 2. As shown in FIG. Side edges 21ba and 21bb of the hole 21 are positioned closer to the inner side than the side edges 2fa and 2fb of the partition wall 2 (the direction in which the side edges 2fa and 2fb approach each other). The upper edge 21c of the hole 21 is positioned lower than the upper edge 2e of the partition wall 2. As shown in FIG.

The pores 21 are intended to hold the hydrogel. As will be described later, the holes 21 are filled with a pregel solution, which is the material of the hydrogel, and then the pregel solution is gelled to form a hydrogel, thereby holding the hydrogel in the holes 21. can be made Hydrogel will be described later.

Here, as will be described later in Examples, when a part of the hole 21 of the container 10 is in contact with the peripheral edge of the partition wall 2 , when the pre-gel solution is injected into the hole 21 , the pre-gel solution leaks out from the hole 21 . When the pre-gel solution having such a shape is gelled, a gel with a spherical surface is formed, in which case the concentration gradient of the target substance from the surrounding medium is disturbed.

In contrast, the inventors have found that by forming the hole 21 of the container 10 at a position away from the peripheral edge of the partition wall 2, the pre-gel solution is retained inside the hole 21 when the pre-gel solution is injected into the hole 21. I found that it can be done. By gelling the pre-gel solution in this state, a gel having a flat surface in contact with the culture medium can be obtained. As a result, when the medium is filled, the concentration gradient of the target substance from the surrounding medium can be linearly formed, for example, as shown in the examples. Since it is important that the surface of the gel in contact with the culture medium is flat, it is important to form the hole 21 of the container 10 at a position away from the peripheral edge of the partition wall 2 .

Therefore, since the hole 21 of the container 10 is formed at a position away from the peripheral edge of the partition wall 2, the hole 21 has a shape that fills the hole 21 (a shape that closes the hole 21, a shape that is substantially the same as the hole 21). of hydrogels can be formed. When the hydrogel is formed in a state in which the holes 21 of the container 10 are filled, the liquid contained in the liquid reservoir 17A does not directly flow out to the liquid reservoir 17B.

As described below, the hydrogel may contain cells. The cells are not particularly limited, and may be human-derived cells, non-human animal-derived cells, plant-derived cells, or insect-derived cells. good. The cells may be single cells that are individually dissociated, or may be cell clusters. The number of cells forming a cell cluster is not particularly limited, and can be exemplified by 2 to 10 ⁸ cells, for example.

The container of the present embodiment can be suitably used for culturing cells, for example. That is, it can be said that the container of this embodiment is a culture container.

In the container 10, it is preferable that at least part of the surface of the hole 21 is laminated with a compound for fixing the hydrogel.

In the present specification, laminating a compound on the surface of the pores 21 means attaching the compound to the surfaces of the pores 21 by physical adsorption, covalent bonding, or the like.

As will be described later, as the hydrogel, for example, a hydrogel formed by reacting a compound having an azide group and a compound having an alkynyl group can be preferably used. Therefore, when using such a hydrogel, a compound having an azide group or an alkynyl group can be used as a compound for fixing the hydrogel. More specific examples include poly-L-lysine azide, which will be described later in Examples. Moreover, as an alkynyl group, a dibenzocyclooctyl (DBCO) group etc. are mentioned, for example.

The compound for immobilizing the hydrogel does not necessarily have to form covalent bonds with the constituents of the hydrogel. From this point of view, as compounds for immobilizing hydrogels, in addition to the compounds described above, for example, poly-L-lysine, agarose, gelatin, polyhydroxyethyl methacrylate, and the like can be used.

By laminating a compound for fixing the hydrogel on the surface of the hole 21 , the hydrogel can be fixed in the hole 21 and the hydrogel can be prevented from coming off the hole 21 . Hereinafter, the operation of laminating the compound for fixing the hydrogel on the surfaces of the holes 21 may be referred to as "surface coating treatment".

It can be said that the container 10 in which the pores 21 are filled with hydrogel is a culture device. FIG. 6 is a YZ sectional view showing the culture device 30 in which the holes 21 of the container 10 are filled with hydrogel. FIG. 7 is an XZ sectional view of the culture device 30. As shown in FIG. As shown in FIGS. 6 and 7, the culture device 30 includes a container body 1, partition walls 2, and hydrogels 3. As shown in FIGS. That is, the culture device 30 has a structure in which the hydrogel 3 is provided in the hole 21 of the container 10 .

The hydrogel 3 fills the entire pores 21 while including the cells 4 . As shown in FIG. 6 , the hydrogel 3 is formed in the pores 21 over the entire thickness of the partition walls 2 . The first main surface 3a of the hydrogel 3 faces the first liquid reservoir 17A. The first main surface 3a of the hydrogel 3 is a surface along the XZ plane and is flush with the first main surface 2a of the partition wall 2 . The second main surface 3b of the hydrogel 3 faces the second liquid reservoir 17B. The second main surface 3b of the hydrogel 3 is a surface along the XZ plane and is flush with the second main surface 2b of the partition wall 2 .

The gel 3 is in contact with the bottom surface 23a, the side surfaces 21ba and 21bb (see FIG. 6), and the top surface 23c of the hole 21 over its entire surface. As will be described later in Examples, the culture device 30 can be used to culture cells under the influence of the density gradient of the target substance.

(Modification)
The container of this embodiment is not limited to the container 10 . For example, the bottom plate 11 of the container 10 has a rectangular shape in plan view, but the shape of the bottom plate 11 is not limited to this. The shape of the bottom plate 11 may be, for example, circular, elliptical, or polygonal such as triangular, pentagonal, or hexagonal.

Moreover, although the outer surface of the container 10 has a substantially cubic shape, it is not limited thereto. The outer surface of the container may be, for example, rectangular parallelepiped, spherical, ellipsoidal, or the like.

Moreover, although the container 10 has one partition 2, the number of partitions may be two or more. In other words, the container 10 has two liquid reservoirs 17A and 17B, but the number of liquid reservoirs may be three or more. FIG. 8 is a top view showing an example of a container having three liquid reservoirs.

When the container has a plurality of partition walls, the thickness of each partition wall may be the same, or may be different from each other as shown in FIG.

Moreover, when the container has a plurality of partition walls, it is preferable that the hole 21 is formed at a position where the plurality of partition walls are in contact with each other. This makes it possible to form concentration gradients of a plurality of target substances in the hydrogel formed inside the pores 21 .

For example, in the container shown in FIG. 8, by introducing different target substances into the liquid reservoirs 17A1 and 17A2, concentration gradients of a plurality of target substances can be formed in one hydrogel.

Moreover, although the thickness of the partition wall 2 is constant in the container 10 shown in FIGS. 2 to 5, the thickness of the partition wall 2 may not be constant.

Further, although the number of holes 21 in the container 10 shown in FIGS. 2 to 5 is one, the number of holes 21 may be two or more as long as at least two liquid reservoirs are communicated. . Moreover, even when a plurality of holes 21 are present, it is preferable that all of the plurality of holes 21 be formed at positions away from the peripheral edge of the partition wall 2 .

Moreover, the shape of the hole 21 when viewed in the Y direction is not limited to a rectangle, and may be a polygon such as a circle, an ellipse, a triangle, a pentagon, and a hexagon.

Also, the size of the opening of the hole 21 to the first liquid reservoir 17A and the size of the opening to the second liquid reservoir 17B may be the same or different. By changing the size of the openings of the pores 21, the concentration gradient of the target substance formed in the hydrogel can be changed.

A preferable relationship between the thickness of the partition wall 2 and the size of the cross-sectional area of the hole 21 in the XZ plane is as follows.

First, when the cross-sectional area of the hole 21 in the XZ plane is constant in the thickness direction (Y direction) of the partition wall 2, the cross-sectional area of the hole 21 when viewed from the thickness direction (Y direction) of the partition wall 2 A circle 22 (equivalent area circle) is assumed, and the diameter of the circle 22 (equivalent area diameter) is defined as "d". Let t be the thickness of the partition wall 2 at the location where the hole 21 is formed (the center of gravity of the cross section of the hole 21 on the XZ plane).

At this time, t:d is preferably 1:0.5 to 1:5, more preferably 1:0.5 to 1:2, and 1:0.5 to 1:1.5. is more preferred, and about 1:1 is particularly preferred.

Further, when the cross-sectional area of the hole 21 on the XZ plane differs in the thickness direction (Y direction) of the partition wall 2, a circle 22 having the same area as the minimum value of the cross-sectional area of the hole 21 on the XZ plane (area equivalent A circle) is assumed, and the diameter (equivalent area diameter) of the circle 22 is assumed to be "d". Also, let t be the thickness of the partition wall 2 at the center of gravity of the cross section of the hole 21 assuming an area-equivalent circle.

At this time, t:d is preferably 1:0.5 to 1:5, more preferably 1:0.5 to 1:2, and 1:0.5 to 1:1.5. is more preferred, and about 1:1 is particularly preferred.

When t:d is in the above range, for example, when the cell mass is placed in the hydrogel, it becomes easy to increase the density gradient of the target substance in contact with the cell mass. In other words, it becomes easy to greatly change the concentration of the target substance in contact with the cell mass for each position of the cell mass.

Also, the volume per hole 21 is, for example, preferably 0.5 μL to 1 mL, more preferably 1 μL to 500 μL, even more preferably 1 μL to 100 μL. When the volume per hole 21 is within the above range, it is easy to inject the hydrogel into the hole 21 .

(Container manufacturing method)
A method of manufacturing the container 10 will now be described with reference to FIGS. The container 10 can be molded using, for example, a mold 100 shown in FIG. The forming die 100 includes a first die 101, a second die 102, a hole-forming piece 103, lower pieces 104 and 105, and a bottom plate .

As shown in FIGS. 9 and 10, the first mold 101 includes a first forming portion 111 shaped to fit the first liquid reservoir 17A and a second forming portion 112 shaped to fit the second liquid reservoir 17B. and The first formation portion 111 and the second formation portion 112 are separated from each other.

The hole-forming piece 103 spans between the first forming portion 111 and the second forming portion 112 . The forming piece 103 can be attached to and removed from the first forming portion 111 and the second forming portion 112 . The lower pieces 104 and 105 are provided at the distal end portions of the first forming portion 111 and the second forming portion 112, respectively. The lower pieces 104 and 105 can be attached to and removed from the first forming portion 111 and the second forming portion 112, respectively.

To mold the culture vessel 10, the gap between the first mold 101 and the second mold 102 is filled with a resin P and hardened. Thereby, the main portion 14 (see FIGS. 2 to 5) is formed. The resin P filled between the first formation portion 111 and the second formation portion 112 becomes the partition walls 2 (see FIGS. 2 to 5). A hole 21 is formed in the partition wall 2 by a hole forming piece 103 .

Next, as shown in FIG. 11, the bottom plate 106 is removed from the second die 102, and the lower pieces 104 and 105 are removed from the first forming portion 111 and the second forming portion 112. Next, as shown in FIG. As a result, the hole forming piece 103 can be easily removed from the forming portions 111 and 112 .

Next, as shown in FIG. 12, the first die 101 is pulled out from the second die 102 . Next, as shown in FIG. 13, the molded main portion 14 is removed from the second die 102, and the hole forming piece 103 is removed from the main portion 14. Next, as shown in FIG. Next, the separately prepared bottom plate 11 (see FIGS. 2 to 5) is joined to the lower portion of the main portion 14 .

Bonding between the main portion 14 and the bottom plate 11 may be performed using, for example, a silicone-based adhesive or the like, or may be performed by ultrasonic fusion or the like. By the method described above, the container 10 shown in FIGS. 2 to 5 can be manufactured.

Examples of the resin P forming the container 10 include silicone resins such as polydimethylsiloxane (PDMS), thermoplastic resins such as polystyrene, polypropylene, polyethylene, and cycloolefin resins.

[Method for producing hydrogel having concentration gradient of target substance]
In one embodiment, the present invention comprises the steps of placing a hydrogel in the pores of the container described above, and filling at least two of the plurality of liquid reservoirs with liquids having different concentrations of the substance of interest, resulting in and forming a concentration gradient of the target substance in the hydrogel.

FIG. 14 is a top view showing a container 10 with two liquid reservoirs. In FIG. 14, hydrogel 3 is placed in hole 21 of container 10 . Liquids having different target substance concentrations are introduced into the first liquid storage section 17A and the second liquid storage section 17B, respectively. In the example of FIG. 14, the target substance is dissolved only in the liquid introduced into the first liquid reservoir 17A, and the liquid introduced into the second liquid reservoir 17B does not contain the target substance.

As a result, as shown in FIG. 14 , the target substance diffuses from the first liquid reservoir 17A side of the hydrogel 3 , and a concentration gradient of the target substance is formed in the hydrogel 3 .

(hydrogel)
Hydrogel means a gel in which water is incorporated inside the polymer network structure. In the production method of the present embodiment, the hydrogel is not particularly limited, and examples include agarose gel, low melting point agarose gel, alginate gel, carrageenan gel, chitosan gel, collagen gel, fibrin gel, methylcellulose gel, hydroxypropylcellulose gel, Carboxymethyl cellulose gel, copolymer of polylactic acid and polyethylene glycol, acrylic acid-based synthetic gel, click-crosslinked gel, and the like can be mentioned.

The click-crosslinked gel includes, for example, a hydrogel obtained by mixing a compound represented by the following formula (A), a compound represented by the following formula (B), and an aqueous liquid.

［式（Ａ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^１～Ｒ^４は、それぞれ独立に、水素原子、－Ｌ－Ｚ^１、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^１又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^１はアルキニル基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^２及びＲ^４はそれぞれ同一であってもよく異なっていてもよい。］

[In formula (A), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ¹ to R ⁴ each independently represent a hydrogen atom; , -LZ ¹ , -O(CH ₂ CH ₂ O) _n -LZ ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ¹ represents an alkynyl group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ² and R ⁴ may be the same or different. ]

［式（Ｂ）中、Ａ^１は、それぞれ独立に、単結合又は炭素数１～２０の直鎖状若しくは分岐鎖状のアルキレン基を表し、Ｒ^５～Ｒ^８は、それぞれ独立に、水素原子、－Ｌ－Ｚ^２、－Ｏ（ＣＨ_２ＣＨ_２Ｏ）_ｎ－Ｌ－Ｚ^２又は炭素原子数１～２０の直鎖状若しくは分岐鎖状のアルキル基を表す。ここで、Ｌは、それぞれ独立して、単結合又は炭素数１～２０の２価の基を表し、Ｚ^２はアジド基を表し、ｎは２０～５００の整数を表す。ｐは０以上の整数を表す。ｐが２以上の整数である場合、複数存在するＲ^６及びＲ^８はそれぞれ同一であってもよく異なっていてもよい。］

[In formula (B), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ⁵ to R ⁸ each independently represent a hydrogen atom; , -LZ ² , -O(CH ₂ CH ₂ O) _n -LZ ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ² represents an azide group, and n represents an integer of 20 to 500. p represents an integer of 0 or more. When p is an integer of 2 or more, multiple R ⁶ and R ⁸ may be the same or different. ]

<<Compound Represented by Formula (A)>>
The compound represented by the above formula (A) has a water-soluble basic skeleton and a group that undergoes a click cross-linking reaction with an azide group.

Here, the fact that the basic skeleton is water-soluble means that the compound serving as the basic skeleton or the compound represented by the above formula (A) containing the basic skeleton is water or a substantially neutral buffer at a temperature from room temperature to 0 degrees. It means that 10% by mass or more is dissolved in a liquid. Specific water-solubility is obtained by adding a compound represented by the above formula (A) containing a basic skeleton or a compound represented by the above formula (A) at a concentration of about 1 to 100 mg/mL in a buffer solution such as HEPES buffer (pH 7.0 to 7.6) It can be evaluated by visually observing whether or not it is dissolved.

A group that undergoes a click cross-linking reaction with an azide group is a group that readily and specifically undergoes a cross-linking reaction with an azide group. Such groups include alkynyl groups. More specific examples include groups having a cyclooctyne ring or an azacyclooctyne ring.

In formula (A), when a plurality of A ¹ is a linear or branched alkylene group having 1 to 20 carbon atoms, one or two or more non-adjacent —CH ₂ — in the alkylene group each independently may be substituted with -CH=CH-, -C≡C-, -O-, -CO-, -COO-, -OCO- or a cyclohexylene group. Also, p may be 0-50.

At least two, preferably three or more, of R ¹ to R ⁴ are —LZ ¹ , and two or more are —O(CH ₂ CH ₂ O) _n —LZ ¹ . is preferred. By containing three or more -LZ ¹ , the number of cross-linking points formed with the compound represented by formula (B) is sufficiently increased, and the strength of the formed hydrogel can be increased.

Also, L may be an ester bond, an ether bond, an amide bond, a thioester bond, a carbamate bond, a carbonyl group, an alkylene group, a combination thereof, or the like.

In formula (A), n is the average repeating number of ethylene glycol. n is 20-500, preferably 30-250, more preferably 40-125. By setting the average repeating number of ethylene glycol within the above range, the solubility of the compound represented by formula (A) in an aqueous liquid can be increased, and handling during production and use is facilitated.

The average repeating number n of ethylene glycol can be estimated by measuring the molecular weight by gel filtration chromatography or mass spectrometry, and estimating the number of R ¹ to R ⁴ groups by NMR.

In formula (A), Z ¹ represents an alkynyl group, and specific examples thereof include groups having a cyclooctyne ring or an azacyclooctyne ring. The alkynyl group in the cyclooctyne ring and the azacyclooctyne ring has high reactivity with the azide group and can undergo a click reaction with the azide group without using a catalyst such as a copper catalyst. As the group having a cyclooctyne ring or an azacyclooctyne ring, a group represented by any one of the following formulas (1) to (4) is preferable.

［式（１）～（４）中、＊は上記式（Ａ）におけるＬとの結合位置を表す。］

[In the formulas (1) to (4), * represents the bonding position with L in the above formula (A). ]

More specifically, compounds represented by the following formulas (5) to (7) are preferably used as the compound represented by the above formula (A). In formulas (5) to (7) below, L, Z ¹ , n, and p are the same as described above for formula (A). In the following formulas (5) to (7), p is preferably 1-3.

<<Compound Represented by Formula (B)>>
The compound represented by the above formula (B) has a water-soluble basic skeleton and an azide group (--N ³ ). The water-soluble basic skeleton is the same as described above for the compound represented by formula (A). In formula (B), A ¹ , L, n, and p are the same as described above for formula (A).

At least two, preferably three or more, of R ⁵ to R ⁸ are —LZ ² , and two or more are —O(CH ₂ CH ₂ O) _n —LZ ² . is preferred. By containing three or more ^-LZ2 , the number of cross-linking points formed with the compound represented by formula (A) is sufficiently increased, and the strength of the formed hydrogel can be increased.

More specifically, compounds represented by the following formulas (8) to (10) are preferably used as the compound represented by the formula (B). In formulas (8) to (10) below, L, n, and p are the same as described above for formula (A). In formulas (8) to (10) below, p is preferably 1 to 3.

《Water-based liquid》
A hydrogel can be obtained by mixing the compound represented by the above formula (A), the compound represented by the above formula (B), and an aqueous liquid. Here, the water-based liquid refers to a liquid containing water as a main component. In addition, "mainly containing water" means that the proportion of water contained in the aqueous liquid is 50% by mass or more, preferably 60% by mass or more, and more preferably 70% by mass or more.

Examples of water-based liquids include water, cell culture media, pH buffer solutions, and the like. The cell culture medium may contain serum such as fetal bovine serum, antibiotics, differentiation-inducing factors, and the like.

《Hydrogel with concentration gradient of target substance》
A hydrogel having a concentration gradient of a target substance can be produced by the production method of the present embodiment. That the hydrogel has a target substance concentration gradient means that the amount of the target substance contained in the hydrogel changes depending on the position on the hydrogel.

[Cell culture method]
In one embodiment, the present invention comprises the steps of placing the cell-containing hydrogel in the holes of the container described above, and placing media having different target substance concentrations in at least two of the plurality of liquid reservoirs, respectively, As a result, there is provided a cell culture method comprising the steps of: forming a concentration gradient of the target substance in the cell-containing hydrogel; and incubating the cell-containing hydrogel. According to the cell culture method of this embodiment, cells can be cultured in a concentration gradient of a target substance. Cells may be similar to those described above.

The cell culture method of the present embodiment can be suitably used, for example, for induction of differentiation of complex tissues and organs that require culture in a concentration gradient of a differentiation-inducing factor. Examples of such tissues and organs include pituitary gland, brain, eye, liver, kidney, lung, digestive tract, pancreas, and the like.

The cell culture method of the present embodiment can be used, for example, in regenerative medicine in which HLA-compatible pluripotent stem cells are induced in a patient to differentiate into complex tissues or organs and transplanted into the patient. . Alternatively, differentiation-induced tissues and organs can be used for screening drug candidate compounds, compound safety tests, and the like.

In the cell culture method of this embodiment, the same hydrogel as described above can be used as the hydrogel. Among them, a hydrogel obtained by mixing the compound represented by the above formula (A), the compound represented by the above formula (B), and an aqueous liquid can be preferably used.

A cell-containing hydrogel can be prepared by incorporating cells into the hydrogel. For example, if the hydrogel is low-melting agarose, mix the low-melting-point agarose with an aqueous liquid such as a medium, heat it to dissolve it, and then heat it to a temperature that does not cause the cells to die and the low-melting-point agarose not to gel. Cool to The cells are then mixed and introduced into the pores 21 of the container 10 where they are allowed to gel. Thereby, the cell-containing hydrogel can be arranged in the holes 21 of the container 10 .

Alternatively, when the hydrogel is a click-crosslinked gel, for example, the compound represented by the above formula (A), the compound represented by the above formula (B) and a medium are mixed to prepare a pregel solution, and the pregel solution is A cell-containing pre-gel solution can be prepared by mixing cells in .

Subsequently, the cell-containing pre-gel solution is introduced into the holes 21 of the container 10 and allowed to gel there. Gelation can be performed simply by standing at room temperature. Thereby, the cell-containing hydrogel can be arranged in the holes 21 of the container 10 . Since the click-crosslinking gel can be gelled at room temperature and has low toxicity to cells, the cell-containing hydrogel can be easily arranged in the pores 21 .

In the cell culture method of the present embodiment, for example, the cells are cell masses having properties of pituitary primordia, and the target substance is a differentiation-inducing factor selected from the group consisting of glucocorticoids, BMP, FGF and Shh. There may be. As a result, the concentration gradient of the differentiation-inducing factor can be applied to the pituitary primordium to induce differentiation of the pituitary gland, which has been impossible in the past.

Examples of cell masses having properties of pituitary gland primordia include LIM Homeobox 3 ( LHX3) positive cell clusters.

[Method for producing pituitary gland]
In one embodiment, the present invention is a method for producing a pituitary gland, comprising the step of placing a cell mass-containing hydrogel containing cell masses having properties of pituitary primordium into the hole of the container described above. , at least two of the plurality of liquid reservoirs are filled with media having different concentrations of a differentiation-inducing factor selected from the group consisting of glucocorticoid, BMP, FGF and Shh, and as a result, the cell mass-containing hydro Provided is a production method comprising the steps of: forming a concentration gradient of the differentiation-inducing factor in the gel; and incubating the cell mass-containing hydrogel, and as a result, forming a pituitary gland from the cell mass. .

As will be described later in Examples, according to the production method of the present embodiment, a plurality of types of pituitary hormone-producing cells can be unevenly distributed and simultaneously induced to produce a functional pituitary gland.

In the production method of the present embodiment, the cell mass-containing hydrogel comprises a compound represented by the above formula (A), a compound represented by the above formula (B), a medium, and cell masses having properties of pituitary primordia. It may be obtained by mixing.

[kit]
In one embodiment, the invention provides a kit comprising a container as described above and a hydrogel material as described above. The kit of the present embodiment can be said to be, for example, a hydrogel production kit having a target substance concentration gradient, a cell culture kit, a pituitary gland production kit, or the like.

In the kit of the present embodiment, the hydrogel material may contain the compound represented by the above formula (A) and the compound represented by the above formula (B). Here, the compound represented by the above formula (A) and the compound represented by the above formula (B) may be stored separately in containers, or may be mixed and stored in the same container. good too. When the compound represented by the above formula (A), the compound represented by the above formula (B) and an aqueous liquid are mixed, a click cross-linking reaction proceeds to form a hydrogel.

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental example 1]
(Synthesis of reagent for hydrogel preparation)
DBCO-4armPEG and Azide-4armPEG were synthesized as reagents for hydrogel preparation.

<<Synthesis of DBCO-4armPEG>>
First, pentaerythritol tetra (aminopropyl) polyoxyethylene (model "PTE-400PA" manufactured by NOF Corporation, sometimes referred to as "4arm PEG" in this specification.) 1 g of 20 mL of dimethyl sulfoxide (DMSO) It was added and dissolved by stirring at 40°C.

In addition, 52 mg of dibenzocyclooctyne (DBCO)-sulfo-NHS (model "A124", Click chemistry tools) was dissolved in 10 mL of DMSO.

Subsequently, the above DBCO-sulfo-NHS solution was added to the above 4armPEG solution, and the mixture was allowed to react with stirring at 40° C. for 3 hours. This resulted in DBCO-4armPEG.

Subsequently, the above reaction solution was reacted with fluorescamine, and the rate of introduction of DBCO groups into 4armPEG was measured. Specifically, first, fluorescamine was dissolved in DMSO to prepare a 3 mg/mL solution. Subsequently, 90 μL of the 100-fold diluted solution of the above DBCO-4armPEG solution and 30 μL of the fluorescamine solution were mixed and allowed to react at room temperature for 30 minutes in the dark.

Subsequently, the plate was irradiated with an excitation wavelength of 360 nm, and fluorescence at a wavelength of 465 nm was measured using a microplate reader (model "GENios", Tecan) to measure the rate of introduction of DBCO groups into 4armPEG.

The DBCO-4armPEG was subsequently dialyzed. Dialysis was performed using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000 and water (milli-Q water) as an external liquid. After 1 hour, 17 hours, 21 hours, 25 hours and 3 days from the start of dialysis, the external solution was exchanged.

The dialyzed DBCO-4armPEG was then lyophilized and stored at −20° C. until use. The introduction rate of DBCO groups into 4armPEG was 93.4%, and the yield of DBCO-4armPEG was 776.3 mg. Also, the molecular weight of the synthesized DBCO-4armPEG was 43,168.

<<Synthesis of Azide-4armPEG>>
Instead of the above DBCO-sulfo-NHS solution, 38 mg of Azide-PEG4-NHS (model "AZ103", Click chemistry tools) was dissolved in 10 mL of DMSO, except that the above was used. Azide-4armPEG was synthesized, dialyzed, lyophilized and stored at −20° C. until use.

The introduction rate of Azide groups into 4armPEG was 99.4%, and the yield of Azide-4armPEG was 1.0743 g. Also, the molecular weight of the synthesized Azide-4armPEG was 42,006.

[Experimental example 2]
(Examination of hydrogel formation conditions)
A hydrogel was prepared using DBCO-4armPEG (flocculant) and Azide-4armPEG (flocculant) synthesized in Experimental Example 1. MEM medium supplemented with 10 v/v % fetal bovine serum (FBS) and 1 v/v % penicillin/streptomycin was used as a solvent for DBCO-4armPEG and Azide-4armPEG.

First, 1 mL of the above solvent was added to 41.3 mg of DBCO-4armPEG and dissolved by stirring with a vortex mixer. This resulted in a 1 mM solution of DBCO-4armPEG.

Subsequently, a 1 mM solution of DBCO-4armPEG was diluted with the above solvents to give 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0. A .9 mM solution was prepared for each.

Also, 1 mL of the above solvent was added to 41.2 mg of Azide-4armPEG, and dissolved by stirring with a vortex mixer. This resulted in a 1 mM solution of Azide-4armPEG. Subsequently, a 1 mM solution of Azide-4armPEG was diluted with the above solvents to give 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0. A .9 mM solution was prepared for each.

Subsequently, 10 μL each of the DBCO-4armPEG solution and the Azide-4armPEG solution prepared to have the same molar concentration were mixed to prepare a pregel solution. Subsequently, each pre-gel solution was allowed to stand at room temperature for 30 minutes to promote the gelation reaction, and then the state of gelation was visually observed. When the pre-gel solution after the reaction did not dissolve even when phosphate buffer (PBS) was added and removed, it was evaluated as solified or gelled. Among them, those that maintained their shape even when lightly touched with the tip of a pipette were evaluated as gelled. The state of gelation was evaluated according to the following evaluation criteria. The evaluation results are shown in Table 1 below.

(Evaluation criteria)
×: No sol-gelation occurred.
(triangle|delta)...Solized.
◯: gelled.

As a result, it was found that a hydrogel tends to be formed when the concentration of the pregel solution is 0.15 mM or more. In subsequent experiments, a 0.5 mM DBCO-4armPEG solution and an Azide-4armPEG solution were each prepared and mixed in equal volume to prepare a 0.25 mM pregel solution to form a hydrogel.

[Experimental example 3]
(Consideration of container)
A hydrogel was formed using a container 10' having the shape shown in Figures 15-17. FIG. 15 is a plan view of container 10'. FIG. 16 is a YZ sectional view taken along line III-III shown in FIG. FIG. 17 is an XZ sectional view along line IV-IV shown in FIG.

The container 10 ′ mainly differs from the container 10 shown in FIGS. As shown in FIGS. 16 and 17, the lower edge 21a of the hole 21 of the container 10' matches the lower edge 2d of the partition wall 2. As shown in FIGS. The hole 21 of the container 10' had a cubic shape of length x width x height of 3 mm x 3 mm x 3 mm.

《Formation of hydrogel》
A DBCO-4armPEG solution and an Azide-4armPEG solution were prepared at 0.5 mM each. A gfCDM medium containing 5% KSR (Invitrogen) was used as a solvent. Subsequently, a 0.25 mM pregel solution was prepared by mixing equal volumes of the DBCO-4armPEG solution and the Azide-4armPEG solution. Subsequently, 30 μL of the pregel solution was injected into the hole 21 of the container 10'.

FIG. 18( a ) is a photograph showing how the pre-gel solution is introduced into the hole 21 of the container 10 . As shown in FIG. 18( a ), it was found that the pre-gel solution was retained without leaking out from the holes 21 when the container 10 was used. Also, FIG. 18(b) is a photograph showing a state in which the pre-gel solution is introduced into the hole 21 of the container 10'. As shown in FIG. 18(b), it was found that the pre-gel solution leaked out from the holes 21 when the container 10' was used.

[Experimental example 4]
(Formation of concentration gradient of target substance)
Using a container 10 similar to that shown in FIGS. 2 to 5, a hydrogel having a concentration gradient of the substance of interest was produced. The hole 21 of the container 10 had a cubic shape of length x width x height of 3 mm x 3 mm x 3 mm.

《Target substance》
As target substances, fluorescein (molecular weight 332.31), FITC dextran (molecular weight 10,000), fusion protein of yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) (molecular weight 56,000 Da, hereinafter referred to as "YFP-CFP" .)It was used. YFP-CFP is expressed in E. coli by introducing a gene encoding a fusion protein in which ECFP is fused to the C-terminal side of EYFP via a linker peptide (GGNSSVDGG: SEQ ID NO: 1) into the expression vector pET21(b). It was prepared by The expressed YFP-CFP was purified using a nickel column using a histidine tag attached to the C-terminal side of YFP-CFP.

《Formation of hydrogel》
A DBCO-4armPEG solution and an Azide-4armPEG solution were prepared at 0.5 mM each. Water was used as the solvent. Subsequently, a 0.25 mM pregel solution was prepared by mixing equal volumes of the DBCO-4armPEG solution and the Azide-4armPEG solution. Subsequently, 30 μL of the pregel solution was injected into the hole 21 of the container 10 . The pre-gel solution could be retained inside the holes 21 without leaking out from the holes 21 . Subsequently, it was allowed to stand at room temperature for 30 minutes to form a hydrogel. As a result, the pores 21 were blocked with hydrogel.

《Formation of Concentration Gradient》
A plurality of containers 10 with hydrogel formed in the pores 21 are prepared, and the first liquid storage portion 17A of each container 10 is filled with fluorescein (final concentration 10 μM), FITC dextran (final concentration 10 μM) or YFP-CFP (final concentration 600 μg / mL) were introduced respectively. Also, 1.5 mL of water was introduced into the second liquid reservoir 17B of each container 10 .

FIGS. 19A and 19B are photographs showing a state in which the liquid is introduced into the first liquid reservoir 17A and the second liquid reservoir 17B of the container 10 in which the hydrogel is introduced into the hole 21, respectively. FIG. 19(b) is a photograph taken from the opening side of the liquid reservoir. In FIG. 19(b), the dotted line indicates the position of the hydrogel.

After that, the hydrogel in each container was observed under a fluorescence microscope for 9 days. Here, a group in which the liquids in the first liquid reservoir 17A and the second liquid reservoir 17B were replaced with new ones every 2 to 3 days and a group in which the liquids were not replaced for 9 days were prepared, and the two were compared. also went The fluorescence intensities of fluorescein, FITC and YFP-CFP in the fluorescence microscope image were analyzed and graphed using Image J software.

20(a) to (c), fluorescein (molecular weight: 332.31) is used as the target substance, and the liquids in the first liquid reservoir 17A and the second liquid reservoir 17B are renewed every 2 to 3 days. This is the result of the replacement. FIG. 20(a) is a fluorescence micrograph of the hydrogel one day after the start of the experiment. FIG. 20(b) is a fluorescence micrograph of the hydrogel 9 days after the start of the experiment. FIG. 20(c) is a graph digitizing the fluorescence micrographs of the hydrogel 1, 4, 7, and 9 days after the start of the experiment. In FIG. 20(c), the horizontal axis indicates the distance (mm) from the end of the hydrogel on the side of the first liquid reservoir, and the vertical axis indicates the fluorescence intensity (relative value) of fluorescein.

FIGS. 21(a) to (c) show the results when FITC dextran (molecular weight 10,000) was used as the target substance. The liquids in the first liquid reservoir 17A and the second liquid reservoir 17B were not replaced for nine days. FIG. 21(a) is a fluorescence micrograph of the hydrogel one day after the start of the experiment. FIG. 21(b) is a fluorescence micrograph of the hydrogel 9 days after the start of the experiment. FIG. 21(c) is a graph digitizing the fluorescence micrographs of the hydrogel 1, 2, 4, 7 and 9 days after the start of the experiment. In FIG. 21(c), the horizontal axis indicates the distance (mm) from the end of the hydrogel on the side of the first liquid reservoir, and the vertical axis indicates the fluorescence intensity (relative value) of FITC.

22(a) to (c) show the results when YFP-CFP (molecular weight 56,000 Da) was used as the target substance. The liquids in the first liquid reservoir 17A and the second liquid reservoir 17B were not replaced for nine days. FIG. 22(a) is a fluorescence micrograph of the hydrogel one day after the start of the experiment. FIG. 22(b) is a fluorescence micrograph of the hydrogel 9 days after the start of the experiment. FIG. 22(c) is a graph digitizing the fluorescence micrographs of the hydrogel 1, 2, 3, 6, and 8 days after the start of the experiment. In FIG. 22(c), the horizontal axis indicates the distance (mm) from the end of the hydrogel on the side of the first liquid reservoir, and the vertical axis indicates the fluorescence intensity (relative value) of YFP-CFP.

As a result, it was clarified that concentration gradients could be formed in hydrogels for all target substances. In addition, the formed concentration gradient could be stably maintained for 9 days or more. It was also found that the concentration gradient of the low-molecular-weight compound (fluorescein) flattened out in a few days if the liquid was not exchanged. It was also revealed that molecules with a molecular weight of about 10,000 or more (FITC dextran, YFP-CFP) can maintain a concentration gradient for at least 8 days regardless of whether liquid exchange is performed or not.

[Experimental example 5]
(Examination of toxicity to cells)
Using a container 10 similar to those shown in FIGS. 2 to 5, cell clusters having the properties of pituitary primordia (LIM Homeobox 3 (LHX3)-positive cell clusters) were cultured, and toxicity to the cell clusters was examined. did. The toxicity of hydrogel, the oxygen permeability of hydrogel, the permeability of nutrient components by hydrogel, etc. were thought to affect cells. The hole 21 of the container 10 had a cubic shape of length x width x height of 3 mm x 3 mm x 3 mm.

<<Preparation of LHX3-positive cell mass>>
Human ES cells (KhES-1) were enzymatically treated to disperse into single cells. Subsequently, the cells were reaggregated using a low cell adhesion V-bottom 96-well plate (Sumitomo Bakelite Co., Ltd.). 5,000 cells were seeded per well and cultured in gfCDM medium containing 5% KSR (Invitrogen).

Y-27632 (ROCK inhibitor) was added at a final concentration of 20 μM from day 0 to day 3, with the day of seeding set to day 0 of the differentiation culture. On days 3 and 6 of culture, a half medium exchange was performed with medium without Y-27632.

From day 6 to day 18 of culture, Bone Morphogenetic Protein 4 (BMP4) at a final concentration of 5 nM was added to the medium. After 6 days of culture, 3-Chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]benzo[b]thiophene-2 at a final concentration of 2 μM - continued to add carboxamide (SAG) to the medium. After the 18th day of culture, the oxygen partial pressure during culture was set to 40%. After 30 days of culture, KSR (Invitrogen) contained in the gfCDM medium was changed from 5% to 10%. After 50 days of culture, the KSR (Invitrogen) contained in the gfCDM medium was changed from 10% to 20%.

By confirming the emergence of LHX3-positive cells by immunostaining on the 60th day of culture, it was determined that cell clusters having the properties of pituitary primordia could be differentiated. Cell clusters with pituitary primordium properties were used for the following experiments.

《Formation of hydrogel》
A DBCO-4armPEG solution and an Azide-4armPEG solution were prepared at 0.5 mM each. A gfCDM medium containing 20% KSR (Invitrogen) was used as a solvent. Subsequently, a 0.25 mM pregel solution was prepared by mixing equal volumes of the DBCO-4armPEG solution and the Azide-4armPEG solution.

Subsequently, one LHX3-positive cell mass was suspended in 30 μL of pregel solution to prepare a cell mass-containing pregel solution. Subsequently, 30 μL of the cell mass-containing pre-gel solution was injected into the hole 21 of the container 10 and allowed to stand at room temperature for 30 minutes to form a cell mass-containing hydrogel.

《Study of Toxicity》
1.5 mL each of gfCDM medium containing 20% KSR (Invitrogen) and SAG at a final concentration of 2 μM was introduced into the first liquid reservoir 17A and the second liquid reservoir 17B of the container 10 in which the cell mass-containing hydrogel was formed. did. Subsequently, the container 10 was placed in an incubator to culture the cell mass.

After 1 week, 2 weeks and 3 weeks from the start of culture in the container 10, the hydrogel was recovered, the cell mass was fixed with paraformaldehyde, and thin slices were prepared. Subsequently, thin sections were immunostained with anti-adrenocorticotropic hormone (ACTH) antibody, anti-PitX1 antibody and anti-E-cadherin antibody and observed under a fluorescence microscope. Expression of ACTH indicates the presence of adrenocorticotropic hormone-producing cells, PitX1 is a pituitary progenitor cell marker, and E-cadherin is a marker of oral ectoderm containing pituitary progenitor cells.

Figures 23(a) and (b) are fluorescence micrographs showing the results of immunostaining. FIG. 23(a) is a photograph taken one week after the start of culture in container 10, and FIG. 23(b) is a photograph taken two weeks after the start of culture in container 10. FIG. As a result, it was clarified that there was no adverse effect on cultured cells for at least two weeks after the start of culture.

Moreover, it was found that the hydrogel was removed from the holes 21 of the container 10 three weeks after the start of the culture when the holes 21 were not surface-coated.

[Experimental example 6]
(Culture of cell mass)
Using a vessel 10 similar to those shown in FIGS. 2 to 5, cell clusters were cultured in a concentration gradient of a differentiation-inducing factor. The hole 21 of the container 10 had a cubic shape of length x width x height of 3 mm x 3 mm x 3 mm.

<<Preparation of LHX3-positive cell mass>>
In the same manner as in Experimental Example 4, a cell mass (LHX3-positive cell mass) having pituitary primordium properties was prepared.

《Surface coating treatment》
The surface of the hole 21 of the container 10 was coated with poly-L-lysine, and an azide group was further introduced. First, the container 10 was sterilized by irradiating it with ultraviolet rays for 30 minutes. Subsequently, 30 μL of a poly-L-lysine solution (hereinafter referred to as “PLL solution”) was injected into the hole 21 of the container 10 within the clean bench. The PLL solution was prepared by dissolving poly-L-lysine (model “P2636”, Sigma) in water at a concentration of 50 μL/mL, followed by filter sterilization.

Subsequently, the container 10 was dried in a dryer set at 80°C. Subsequently, 30 μL of the Azide-PEG4-NHS solution was injected into the hole 21 of the container 10 within the clean bench and left for 30 minutes. Azide-PEG4-NHS solution was prepared by dissolving Azide-PEG4-NHS (model "AZ103", Click chemistry tools) in DMSO (model "276855", Sigma) at a concentration of 1 w/v%. Subsequently, in a clean bench, the Azide-PEG4-NHS solution was sucked from the hole 21, washed with sterilized water four times, and dried.

《Formation of hydrogel》
A DBCO-4armPEG solution and an Azide-4armPEG solution were prepared at 0.5 mM each. A gfCDM medium containing 5% KSR (Invitrogen) was used as a solvent. Subsequently, a 0.25 mM pregel solution was prepared by mixing equal volumes of the DBCO-4armPEG solution and the Azide-4armPEG solution.

Subsequently, one LHX3-positive cell mass was suspended in 30 μL of pregel solution to prepare a cell mass-containing pregel solution. Subsequently, 30 μL of the cell mass-containing pre-gel solution was injected into the hole 21 of the container 10 and allowed to stand at room temperature for 30 minutes to form a cell mass-containing hydrogel.

《Culture of cell mass》
1.5 mL of gfCDM medium containing 20% KSR (Invitrogen), dexamethasone at a final concentration of 1 μM, and SAG at a final concentration of 2 μM was introduced into the first liquid reservoir 17A of the container 10 in which the cell mass-containing hydrogel was formed. Also, 1.5 mL of gfCDM medium containing 20% KSR (Invitrogen) and SAG at a final concentration of 2 μM was introduced into the second liquid reservoir 17B.

Subsequently, the container 10 was placed in an incubator to culture the cell mass. After culturing for 10 days, the hydrogel was recovered, the cell mass was fixed with paraformaldehyde, and thin slices were prepared. Subsequently, thin sections were immunostained with anti-adrenocorticotropic hormone (ACTH), anti-growth hormone (GH), and anti-growth hormone-releasing hormone receptor (GHRH-R) antibodies and observed under a fluorescence microscope. Expression of ACTH indicates the presence of adrenocorticotropic hormone-producing cells, expression of GH indicates the presence of growth hormone-producing cells, and expression of GHRH-R indicates maturation of growth hormone-producing cells.

FIG. 24 is a fluorescence micrograph showing the results of immunostaining. In FIG. 24, "Dexamethasone (+)" indicates the result of the area of the cell mass on the side of the first liquid reservoir 17A to which dexamethasone was added, and "Dexamethasone (-)" indicates the result of the cell mass. , indicates the result of the area on the side of the second liquid reservoir 17B to which dexamethasone was not added.

As a result, on the first liquid reservoir 17A side, growth hormone-producing cells were revealed, and growth hormone-releasing hormone receptors were expressed on the surface, indicating that the cells were functionally mature. .

On the other hand, the second liquid reservoir 17B side was thought to be slightly affected by dexamethasone, and a phenomenon was observed in which the adrenocorticotrophic hormone-producing cells slightly expressed growth hormone.

In conventional 3D culture methods, pituitary hormone-producing cells can be induced individually, but multiple types of pituitary hormone-producing cells cannot be ubiquitously distributed and induced simultaneously. On the other hand, in this experimental example, it was revealed that multiple types of pituitary hormone-producing cells could be unevenly distributed and differentiated according to the concentration gradient of dexamethasone.

ADVANTAGE OF THE INVENTION According to this invention, the technique which forms the concentration gradient of a target substance can be provided.

2 Partition walls 2a, 2b, 3a, 3b, 11a, 13Aa, 13Ba Surfaces 2e, 12Ab, 12Bb, 13Ab, 13Bb, 21c Upper edge 2d, 21a Lower edge 2fa, 2fb, 21ba, 21bb... Side edge 3 Hydrogel 4 Cell 10 Container 11 Bottom plate 12A, 12B Side plate 13A, 13B End plate 14 Main portion 17A, 17A1, 17A2, 17B Liquid reservoir, 21... Hole, 23a, 23ba, 23bb, 23c... Surface, 30... Culture device, 100... Mold, 101... First mold, 102... Second mold, 103... Hole forming piece, 104, 105... Lower piece, 106... Bottom plate, 111... First forming part, 112... Second forming part, P... Resin.

Claims (10)

a container body having an internal space capable of storing a liquid;
a partition partitioning the internal space into a plurality of liquid reservoirs,
a hole is formed in the partition wall for communicating a first liquid reservoir and a second liquid reservoir among the plurality of liquid reservoirs, and hydrogel is held in the hole;
The opening of the hole is formed at a position away from all peripheral edges of the partition wall when viewed from the thickness direction of the partition wall,
When a circle having the same area as the cross-sectional area of the hole seen from the thickness direction of the partition wall is assumed on one surface and the other surface of the partition wall in the thickness direction, the diameter of the circle (area equivalent diameter) is d and where t is the thickness of the partition wall at the location where the hole is formed, t:d is 1:0.5 to 1:5,
The volume per hole is 0.5 μL to 1 mL,
The hydrogel is filled in the entire pores while containing cells, and the hydrogel has a first main surface that is flush with one surface of the partition wall in the thickness direction, and the partition wall. A container having a second main surface that is flush with the other surface in the thickness direction of the container. 2. The container according to claim 1, wherein at least part of the surface of said pores is laminated with a compound for fixing said hydrogel. The hydrogel according to claim 1 or 2, which is obtained by mixing a compound represented by the following formula (A), a compound represented by the following formula (B), a medium and the cells. container.

[In formula (A), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ¹ to R ⁴ each independently represent a hydrogen atom; , -LZ ¹ , -O(CH ₂ CH ₂ O) _n -LZ ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms , and R ¹ to R ⁴ Two or more of them are —O(CH ₂ CH ₂ O) _n —LZ ¹ . Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ¹ represents an alkynyl group, and n represents an integer of 20 to 500. p represents an integer of 1 to 3 ; When p is an integer of 2 or more, multiple R ² and R ⁴ may be the same or different. ]

[In formula (B), A ¹ each independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms; R ⁵ to R ⁸ each independently represent a hydrogen atom; , -LZ ² , -O(CH ₂ CH ₂ O) _n -LZ ₂ or a linear or branched alkyl group having 1 to 20 carbon atoms , and R ⁵ to R ⁸ Two or more of them are —O(CH ₂ CH ₂ O) _n —LZ ² . Here, each L independently represents a single bond or a divalent group having 1 to 20 carbon atoms, Z ² represents an azide group, and n represents an integer of 20 to 500. p represents an integer of 1 to 3 ; When p is an integer of 2 or more, multiple R ⁶ and R ⁸ may be the same or different. ] 4. The container according to claim 3, wherein said alkynyl group is a group having a cyclooctyne ring or an azacyclooctyne ring. 4. The container according to claim 3, wherein the compound represented by formula (A) is DBCO-4armPEG and the compound represented by formula (B) is Azide-4armPEG. A method for manufacturing a container according to any one of claims 1 to 5 ,
A method for manufacturing a container, comprising the step of disposing the hydrogel containing the cells in the hole. The step of disposing the hydrogel containing the cells in the pores,
a step of filling the pores by injecting a pregel solution prepared by mixing the cells into the hydrogel material into the pores;
gelling the pre-gel solution to form the hydrogel;
A method of manufacturing a container according to claim 6 , comprising: A step of manufacturing the container by the container manufacturing method according to claim 6 or 7 ;
a step of filling media having different concentrations of a target substance into at least two of the plurality of liquid reservoirs, respectively, thereby forming a concentration gradient of the target substance in the hydrogel containing the cells;
incubating the hydrogel containing the cells;
A cell culture method, comprising: The cells are cell masses having properties of pituitary primordium, and the target substance consists of glucocorticoid, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and sonic hedgehog (Shh). The cell culture method according to claim 8 , which is a differentiation-inducing factor selected from the group. A method for producing a pituitary gland comprising:
a step of manufacturing the container by the container manufacturing method according to claim 6 or 7 , wherein the cells are cell masses having properties of pituitary primordium;
Media containing different concentrations of differentiation-inducing factors selected from the group consisting of glucocorticoids, BMP, FGF and Shh were placed in at least two of the plurality of liquid reservoirs, thereby containing the cell clusters. A step of forming a concentration gradient of the differentiation-inducing factor in the hydrogel;
incubating the hydrogel containing the cell mass, as a result of which a pituitary gland is formed from the cell mass;
A manufacturing method, including:

Images