WO2019142614A1 - Container and use thereof - Google Patents

Abstract

This container comprises: a container body having formed therein an internal space in which a liquid can be stored; and partitions that divide the internal space into a plurality of liquid storage sections. Holes are formed in the partitions, the holes communicating at least two of the plurality of liquid storage sections and retaining a hydrogel therein.

Description

Container and its use

The present invention relates to a container and its use. More specifically, the present invention relates to a container, a kit, a method of producing a hydrogel having a concentration gradient of a target substance, a method of cell culture, and a method of producing a pituitary. Priority is claimed on Japanese Patent Application No. 2018-006810, filed January 18, 2018, the content of which is incorporated herein by reference.

When differentiating complex tissues and organs from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), it is suitable to culture the cells in three dimensions (3D culture) It is believed that The key point is to reproduce fetal development in a test tube, and it is thought that 3D culture is easier to reproduce fetal development than planar culture.

The pituitary is an example of a complex organ. For example, Patent Document 1 includes suspension culture (3D suspension culture) of aggregates of human pluripotent stem cells in a medium containing an osteogenic factor signal transduction pathway activator and an Shh signal pathway agonist. A method of producing human cell aggregates comprising the glandular pituitary or its precursor tissue is described.

International Publication No. 2016/013669 International Publication No. 2017/126551

Pituitary development is well studied and findings are accumulating. FIG. 1 is a schematic view showing the development of a mouse after formation of a pituitary primordia, which should also be referred to as the latter half of pituitary differentiation. In FIG. 1, BMP, FGF, Shh and Wnt represent differentiation induction signals. As shown in FIG. 1, it is believed that the signal from the dorsal hypothalamus and the signal from the ventral oral epidermis form a concentration gradient on the pituitary primordia. In addition, neural crest-derived mesenchymal cells migrate around the lateral direction, and the signal is also secreted from there.

Patent document 2 describes what developed the manufacturing method of patent document 1 further. Specifically, 3D culture of mouse or human ES cells or iPS cells can simultaneously induce differentiation of hypothalamus and oral ectoderm essential for pituitary differentiation into one 3D cell mass. it can. Subsequently, the pituitary primordia is induced spontaneously and eventually differentiates into pituitary hormone producing cells.

These pituitary hormone-producing cells secrete hormones in response to stimulation, as in the living body, and achieve high differentiation such that sufficient hormones in the surroundings cause suppression. It is thought that 3D culture has high potential also because planar culture can not induce such a responsive pituitary hormone-producing cell.

On the other hand, conventional 3D culture methods also have limitations. In the pituitary of a living body, a plurality of types of pituitary hormone-producing cells are present at unevenly distributed sites for each type in rostral and caudal, ventral and dorsal sides. On the other hand, in the conventional 3D culture method, pituitary hormone-producing cells can be induced for each type, but multiple types of pituitary hormone-producing cells can not be polarized and induced simultaneously.

As a cause of this, the present inventors have found that the concentration gradient of the inducer present in the fetus can not be reproduced by the conventional 3D culture method, and the concentration of the inducer in the culture solution has become constant. I thought. Then, an object of this invention is to provide the technique which forms the concentration gradient of an object 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］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、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、［１］～［５］のいずれかに記載の容器。

［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。］
［７］［１］～［３］のいずれかに記載の容器と、前記ハイドロゲルの材料とを含む、キット。
［８］前記ハイドロゲルの材料が、下記式（Ａ）で表される化合物及び下記式（Ｂ）で表される化合物を含む、［７］に記載のキット。

［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。］
［９］［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。
［１０］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、［９］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。］
［１１］［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。
［１２］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、［１１］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。］
［１３］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、［１１］又は［１２］に記載の細胞培養方法。
［１４］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。
［１５］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、［１４］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 main body in which an internal space capable of storing liquid is formed, and a partition dividing the internal space into a plurality of liquid reservoirs, and at least two of the plurality of liquid reservoirs in the partition A container communicating with one another, wherein a hole is formed in which a hydrogel is held.
[2] The container according to [1], wherein the hole is formed at a position away from the periphery of the partition wall as viewed in the thickness direction of the partition wall.
[3] The container according to [1] or [2], wherein a compound for fixing the hydrogel is laminated on at least a part of the surface of the hole.
[4] The container according to any one of claims 1 to 3, wherein the hydrogel is disposed in the hole.
[5] 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, [1] to [4] The container according to any of the above.

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ]
[6] The hydrogel is obtained by mixing a compound represented by the following formula (A), a compound represented by the following formula (B), a culture medium and cells, [1] to [5] The container according to any of the above.

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ]
[7] A kit comprising the container according to any one of [1] to [3] and the material of the hydrogel.
[8] The kit according to [7], wherein the material of the hydrogel contains a compound represented by the following formula (A) and a compound represented by the following formula (B).

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ]
[9] A step of disposing a hydrogel in the hole of the container according to any one of [1] to [3], and at least two of the plurality of liquid reservoirs, liquids having different concentrations of the target substance And D. a step of forming a concentration gradient of the target substance in the hydrogel as a result of each putting, thereby producing the hydrogel having a concentration gradient of the target substance.
[10] The production according to [9], 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 independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ]
[11] A step of disposing a cell-containing hydrogel in the pores of the container according to any one of [1] to [3], and at least two of the plurality of liquid reservoirs have different concentrations of the target substance from each other A cell culture method comprising the steps of: introducing a culture medium, and as a result, forming a concentration gradient of the target substance in the cell-containing hydrogel; and incubating the cell-containing hydrogel.
[12] The cell-containing hydrogel according to [11], which is obtained by mixing a compound represented by the following formula (A), a compound represented by the following formula (B), a culture medium and cells. Cell culture method described.

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ]
[13] The cell is a cell mass having pituitary primordium-like properties, and the target substance is glucocorticoid, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and sonic hedgehog (Shh) [11] or [12], which is a differentiation inducer selected from the group consisting of
[14] A method for producing a pituitary, comprising: a cell mass-containing hydrogel containing a cell mass having a property of pituitary primordium in the hole of the container according to any one of [1] to [3]. Placing a medium having different concentrations of differentiation inducers selected from the group consisting of glucocorticoid, BMP, FGF and Shh in at least two of the plurality of liquid reservoirs, Including the steps of: forming a concentration gradient of the differentiation inducer in the cell mass-containing hydrogel; and incubating the cell mass-containing hydrogel so that a pituitary is formed from the cell mass. Production method.
[15] The cell mass-containing hydrogel may be prepared by mixing a compound represented by the following formula (A), a compound represented by the following formula (B), a medium, and a cell mass having a property of pituitary primordia The production method according to [14], which is obtained.

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ]

According to the present invention, it is possible to provide a technique for forming a concentration gradient of a target substance.

It is a schematic diagram which shows the generation after forming a pituitary primordia. It is a perspective view of a container concerning one embodiment. Figure 3 is a plan view of the container of Figure 2; FIG. 4 is a YZ sectional view taken along the line II shown in FIG. 3; FIG. 4 is a cross-sectional view taken along the line II-II shown in FIG. It is YZ sectional drawing of the culture apparatus using the container of FIG. It is XZ sectional drawing of the culture apparatus which used the container of FIG. It is a top view which shows an example of a container which has three liquid storage parts. It is a schematic diagram which shows the shaping | molding die of the container which concerns on 1 embodiment. It is a perspective view showing a forming die of a container concerning one embodiment. It is a schematic diagram explaining the manufacturing method of the container which concerns on 1 embodiment. It is a schematic diagram explaining the manufacturing method of the container which concerns on 1 embodiment. It is a schematic diagram explaining the manufacturing method of the container which concerns on 1 embodiment. It is a top view which shows an example of a container which has two liquid storage parts. It is a top view of the container used by the experiment example. FIG. 16 is a YZ sectional view taken along the line III-III shown in FIG. FIG. 16 is a cross-sectional view taken along line IV-IV shown in FIG. (A) is a photograph which shows a mode that the pregel solution was introduce | transduced into the hole of the container shown to FIGS. 3-5 in Experimental example 3. FIG. (B) is a photograph which shows a mode that the pregel solution was introduce | transduced into the hole of the container shown to FIGS. 15-17 in Example 3 of an experiment. (A) And (b) is a photograph which shows the state which introduced the liquid into the 1st liquid storage part and the 2nd liquid storage part of the container which introduced the hydrogel to the hole, respectively. (A) And (b) is a photograph of hydrogel at the time of using fluorescein as an object substance in Experimental example 4. (C) is a graph which shows the result at the time of using a fluorescein as an object substance in Experimental example 4. (A) And (b) is a photograph of hydrogel at the time of using FITC dextran as an object substance in Experimental example 4. (C) is a graph which shows the result at the time of using FITC dextran as an object substance in example 4 of an experiment. (A) And (b) is a photograph of hydrogel at the time of using YFP-CFP fusion protein as an object substance in Experimental example 4. (C) is a graph which shows the result at the time of using YFP-CFP fusion protein as an object substance in Experimental example 4. (A) And (b) is a fluorescence-microscope photograph which shows the result of the immunostaining in Experimental example 5. FIG. It is a fluorescence-microscope photograph which shows the result of the immunostaining in Experimental example 6.

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 will be denoted by the same or corresponding reference symbols, without redundant description. In addition, the dimensional ratio in each figure has the part which exaggerates for description, and does not necessarily correspond with an actual dimensional ratio. Also, in the figures, an XYZ coordinate system is shown as the case may be. Hereinafter, each direction will be described based on each coordinate system as needed.

[container]
In one embodiment, the present invention includes a container main body in which an internal space capable of storing liquid is formed, and a partition dividing the internal space into a plurality of liquid reservoirs, and the partition is provided with the plurality of liquids Provided is a container in which at least two of the reservoirs communicate with each other and in which a hole for holding a hydrogel is formed. As described later, by using the container of this embodiment, a hydrogel having a concentration gradient of the target substance can be produced.

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

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

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

The bottom plate 11 is rectangular in plan view. The side plates 12A and 12B are provided upright on the first major surface 11a of the bottom plate 11. The side plates 12A and 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 formed apart in the Y direction.

The end plates 13A and 13B are provided upright on the first major surface 11a of the bottom plate 11. The end plates 13A and 13B are formed along the YZ plane. The end plates 13A and 13B are rectangular when viewed in 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 referred to as an internal space 15. The lower edges of the side plates 12A and 12B and the lower edges of the end plates 13A and 13B and the bottom plate 11 are joined in a liquid tight manner. Therefore, the container 10 can store liquid in the internal space 15. The bottom plate 11 closes the lower openings of the liquid reservoirs 17A and 17B (described later).

The partition 2 is provided upright on the first major surface 11 a of the bottom plate 11. The partition 2 is formed along the XZ plane. The partition 2 is rectangular when viewed from the thickness direction (Y direction). The partition 2 is parallel to the side plates 12A and 12B. The partition wall 2 is formed between the side plates 12A and 12B at a distance from the side plates 12A and 12B. The partition wall 2 has a constant thickness.

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

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

As shown in FIG. 4 and FIG. 5, a hole 21 is formed in the partition 2. The hole 21 is a through hole which penetrates the partition 2 in the thickness direction (Y direction). The holes 21 communicate the first liquid reservoir 17A with the second liquid reservoir 17B.

As shown in FIG. 5, the shape of the hole 21 viewed from the thickness direction (Y direction) of the partition 2 is rectangular. The lower edge 21 a and the upper edge 21 c of the hole 21 extend in the X direction. The side edges 21ba and 21bb of the holes 21 extend in 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 23 ba and 23 bb of the hole 21 are along the YZ plane. The side surfaces 23ba and 23bb are parallel to one another. The bottom surface 23 a coincides with the lower edge 21 a when viewed from the thickness direction (Y direction) of the partition 2. The side surfaces 23ba and 23bb respectively coincide with the side edges 21ba and 21bb when viewed from the Y direction. The top surface 23c coincides with the upper edge 21c when viewed from the Y direction.

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

The holes 21 hold the hydrogel. As will be described later, the pores 21 are filled with the pregel solution which is a hydrogel material to fill the pores 21 and subsequently, the hydrogel is retained in the pores 21 by gelling the pregel solution to form a hydrogel. It can be done. The hydrogel will be described later.

Here, when a part of the hole 21 of the container 10 is in contact with the peripheral edge of the partition 2 as described later in the embodiment, when the pregel solution is injected into the hole 21, the pregel solution leaks from the hole 21. The gelation of the pregel solution having such a shape results in the formation of a gel having a spherical surface, in which case the concentration gradient of the target substance from the surrounding medium is disturbed.

On the other hand, when the pregel solution is injected into the hole 21 by forming the hole 21 of the container 10 at a position away from the peripheral edge of the partition 2, the inventors keep the pregel solution inside the hole 21. I found that I could do it. When the pregel solution is gelled in this state, a gel having a flat surface in contact with the medium can be obtained. As a result, when the medium is filled, a concentration gradient of the target substance from the surrounding medium can be formed linearly, for example, as shown in the examples. As described above, it is important that the surface of the gel in contact with the medium is flat, so it is important to form the holes 21 of the container 10 at positions away from the periphery of the partition wall 2.

Therefore, the hole 21 of the container 10 is formed at a position away from the peripheral edge of the partition 2, thereby filling the hole 21 with the hole 21 (shape for closing the hole 21, substantially the same shape as the hole 21) Can form a hydrogel. When the hydrogel is formed in a state where the holes 21 of the container 10 are filled, the liquid contained in the liquid storage portion 17A does not flow out to the liquid storage portion 17B as it is.

The hydrogel may contain cells as described later. The cells are not particularly limited, and may be cells of human origin, cells of non-human animals, cells of plant origin, or cells of insect origin. Good. The cells may be single cells dissociated into pieces one by one or may be cell masses. The number of cells constituting the cell mass is not particularly limited, and can be, for example, 2 to 10 ⁸ .

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

In the container 10, a compound for fixing a hydrogel is preferably laminated on at least a part of the surface of the hole 21.

In the present specification, laminating the compound on the surface of the hole 21 means that the compound is attached to the surface of the hole 21 by physical adsorption, covalent bonding or the like.

As described below, as the hydrogel, for example, a hydrogel formed by reacting a compound having an azide group with a compound having an alkynyl group can be suitably used. Therefore, when such a hydrogel is used, a compound having an azide group or an alkynyl group can be used as a compound for fixing the hydrogel. More specifically, examples thereof include azide poly-L-lysine and the like described later in the Examples. Moreover, as an alkynyl group, the dibenzo cyclooctyl (DBCO) group etc. are mentioned, for example.

The compound for fixing the hydrogel does not necessarily have to form a covalent bond with the constituents of the hydrogel. From such a viewpoint, as the compound for fixing the hydrogel, for example, poly-L-lysine, agarose, gelatin, polyhydroxyethyl methacrylate and the like can be used in addition to those described above.

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

The container 10 in which the pores 21 are filled with the hydrogel can be said to be a culture apparatus. 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 apparatus 30 includes the container body 1, the partition 2, and the hydrogel 3. That is, the culture apparatus 30 has a configuration in which the hydrogel 3 is provided in the hole 21 of the container 10.

The hydrogel 3 is filled in the entire pores 21 with the cells 4 contained therein. As shown in FIG. 6, the hydrogel 3 is formed in the hole 21 over the entire thickness direction of the partition 2. The first main surface 3a of the hydrogel 3 faces the first liquid reservoir 17A. The first main surface 3 a of the hydrogel 3 is a surface along the XZ plane, and is flush with the first main surface 2 a of the partition 2. The second major surface 3b of the hydrogel 3 faces the second liquid reservoir 17B. The second major surface 3 b of the hydrogel 3 is a plane along the XZ plane, and is flush with the second major surface 2 b of the partition 2.

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

(Modification)
The container of the present embodiment is not limited to the container 10. For example, the bottom plate 11 of the container 10 is rectangular in plan view, but the shape of the bottom plate 11 is not limited thereto. The shape of the bottom plate 11 may be, for example, a circle, an ellipse, or a polygon such as a triangle, a pentagon, or a hexagon.

Moreover, although the outer surface of the container 10 is a substantially cube shape, it is not restricted to this. The outer surface of the container may be, for example, a rectangular parallelepiped shape, or may be, for example, a spherical shape, an ellipsoidal shape, or the like.

Moreover, although the container 10 has one dividing wall 2, the number of dividing walls 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 a container has multiple partitions, it is preferable that the hole 21 is formed in the position where several partitions contact | connect. This makes it possible to form concentration gradients of a plurality of target substances in the hydrogel formed inside the holes 21.

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

Further, in the container 10 shown in FIGS. 2 to 5, the thickness of the partition 2 is constant, but the thickness of the partition 2 may not be constant.

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

Further, the shape of the holes 21 viewed from the Y direction is not limited to a rectangle, and may be a circle, an ellipse, a triangle, a pentagon, a polygon such as a hexagon, or the like.

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

Moreover, the preferable relationship of the thickness of the partition 2 and the magnitude | size of the cross-sectional area in XZ plane of the hole 21 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 2, the same area as the cross-sectional area of the hole 21 viewed from the thickness direction (Y direction) of the partition 2 Assuming a circle 22 (area equivalent circle), let the diameter (area equivalent diameter) of the circle 22 be “d”. In addition, 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 in the XZ plane) is t.

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. More preferably, it is about 1: 1.

When the cross-sectional area of hole 21 in the XZ plane is different in the thickness direction (Y direction) of partition wall 2, circle 22 (area equivalent to the minimum value of the cross-sectional area of hole 21 in the XZ plane Assuming a circle), let the diameter (area equivalent diameter) of the circle 22 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. More preferably, it is about 1: 1.

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

In addition, the volume per hole 21 is, for example, preferably 0.5 μL to 1 mL, more preferably 1 μL to 500 μL, and still more preferably 1 μL to 100 μL. When the volume per hole 21 is in the above range, the hydrogel can be easily injected into the hole 21.

(Method of manufacturing container)
The method of manufacturing the container 10 will 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 106.

As shown in FIGS. 9 and 10, the first mold 101 has a first forming portion 111 shaped according to the first liquid reservoir 17A and a second forming portion 112 shaped according to 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 is bridged between the first forming portion 111 and the second forming portion 112. The formation piece 103 is attachable to and detachable from the first formation portion 111 and the second formation portion 112. The lower pieces 104 and 105 are provided at the tip of the first forming portion 111 and the second forming portion 112, respectively. The lower pieces 104 and 105 are attachable to and removable from the first forming portion 111 and the second forming portion 112, respectively.

In order to form the culture container 10, the space between the first mold 101 and the second mold 102 is filled with the resin P and cured. 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 wall 2 (see FIGS. 2 to 5). A hole 21 is formed in the partition 2 by the hole forming piece 103.

Next, as shown in FIG. 11, the bottom plate 106 is removed from the second mold 102, and the lower pieces 104 and 105 are removed from the first forming portion 111 and the second forming portion 112. 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 mold 101 is removed from the second mold 102. Next, as shown in FIG. 13, the formed main portion 14 is removed from the second mold 102, and the hole forming piece 103 is removed from the main portion 14. Next, the bottom plate 11 (see FIGS. 2 to 5) prepared separately is joined to the lower part of the main part 14.

The 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 welding or the like. The container 10 shown in FIGS. 2 to 5 can be manufactured by the above method.

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

[Method of producing hydrogel having concentration gradient of target substance]
In one embodiment, the present invention places a liquid having different concentrations of the target substance in at least two of the step of disposing the hydrogel in the hole of the container described above, and at least two of the plurality of liquid reservoirs. A process for producing the hydrogel having a concentration gradient of a target substance, comprising: a step of forming a concentration gradient of the target substance on the hydrogel.

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

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

(Hydrogel)
By hydrogel is meant a gel in which water is incorporated inside the polymer network. In the manufacturing method of the present embodiment, the hydrogel is not particularly limited. For example, agarose gel, low melting point agarose gel, alginic acid gel, carrageenan gel, chitosan gel, collagen gel, fibrin gel, fibrin gel, methyl cellulose gel, hydroxypropyl cellulose gel, Examples thereof include carboxymethyl cellulose gel, copolymer of polylactic acid and polyethylene glycol, synthetic gel of acrylic acid type, click cross-linked gel and the like.

Examples of the click crosslinkable gel include hydrogels 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 independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

［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 independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ]

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

Here, that the basic skeleton is water-soluble means that the compound represented by the above formula (A) containing the compound to be the basic skeleton or the basic skeleton is water or substantially neutral buffer at a temperature from normal temperature to 0 ° C. It means dissolving 10% by mass or more in the solution. A specific water solubility is a compound having the basic skeleton or the compound represented by the above formula (A) containing the basic skeleton in a buffer solution such as HEPES buffer at a concentration of about 1 to 100 mg / mL (pH 7.0 to It can be assessed by visual observation whether it is dispersed in 7.6) and dissolved.

The azide group and the group which click-crosslinks with each other are groups which easily and specifically cause a crosslinking reaction with the azide group. Such groups include alkynyl groups. More specifically, groups having a cyclooctin ring or an azacyclooctic ring can be mentioned.

In the 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 two or more of —CH ₂ — in the alkylene group are And each may be independently substituted by —CHCHCH—, —C≡C—, —O—, —CO—, —COO—, —OCO—, or cyclohexylene group. Also, p may be 0 to 50.

In addition, at least two, preferably three or more of R ¹ to R ⁴ are -L-Z ¹ and two or more are -O (CH ₂ CH ₂ O) _n -L-Z ¹ Is preferred. By including the -L-Z ¹ 3 or more, it is possible to enhance the strength of cross points is sufficiently large, the formed hydrogel is formed between the compound of formula (B).

In addition, 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 number of repetitions of ethylene glycol. n is 20 to 500, preferably 30 to 250, and more preferably 40 to 125. By setting the average number of repetitions of ethylene glycol in the above range, the solubility of the compound represented by the formula (A) in an aqueous liquid can be increased, and handling becomes easy at the time of production and use.

The average repetition 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 a group having a cyclooctin ring or an azacyclooctin ring. The alkynyl group in the cyclooctin ring and the azacyclooctin ring is highly reactive to an azide group, and can be click-reacted with an azide group without using a catalyst such as a copper catalyst. The group having a cyclooctin ring or an azacyclooctin ring is preferably a group represented by any of the following formulas (1) to (4).

［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 a bonding position to L in the above formula (A). ]

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

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

Also, at least two, preferably three or more of R ⁵ to R ⁸ are -L-Z ² and two or more are -O (CH ₂ CH ₂ O) _n -L-Z ² Is preferred. By including the -L-Z ² 3 or more, it is possible to enhance the strength of cross points is sufficiently large, the formed hydrogel is formed between the compound of formula (A).

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

Water-based liquid
A hydrogel can be obtained by mixing the compound represented by the said Formula (A), the compound represented by the said Formula (B), and an aqueous liquid. Here, the aqueous liquid refers to a liquid containing water as a main component. Further, having water as the main component 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 the aqueous liquid include water, a medium for cell culture, a pH buffer solution and the like. The cell culture medium may contain serum such as fetal calf serum, an antibiotic, a differentiation inducer, and the like.

<< Hydrogel with concentration gradient of target substance >>
According to the production method of the present embodiment, a hydrogel having a concentration gradient of the target substance can be produced. The hydrogel having a concentration gradient of the target substance 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 places media having different concentrations of a target substance in at least two of the step of disposing the cell-containing hydrogel in the pores of the container described above and the plurality of liquid reservoirs, 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. By the cell culture method of the present embodiment, cells can be cultured in a concentration gradient of a target substance. The cells may be similar to those described above.

The cell culture method of the present embodiment can be suitably used, for example, for inducing differentiation of complex tissues and organs which need to be cultured in a concentration gradient of differentiation inducer. Examples of such tissues and organs include the pituitary, brain, eye, liver, kidney, lung, digestive tract, pancreas and the like.

According to the cell culture method of the present embodiment, for example, pluripotent stem cells having HLA compatibility in a patient can be induced to differentiate, differentiation of a complex tissue or organ can be induced, and it can be used for regenerative medicine etc. . Alternatively, it can be used for screening of drug candidate compounds, safety testing of compounds, etc. using differentiated tissues and organs.

In the cell culture method of the present embodiment, as the hydrogel, one similar to that described above can be used. Among them, a hydrogel obtained by mixing a compound represented by the above formula (A), a compound represented by the above formula (B) and an aqueous liquid can be suitably used.

Cell-containing hydrogels can be prepared by including cells in the hydrogel. For example, when the hydrogel is a low melting point agarose, the low melting point agarose is mixed with an aqueous liquid such as a medium, heated and dissolved, and then the low melting point agarose does not gel and the cells are not killed. Cool down. Subsequently, the cells are mixed and introduced into the pores 21 of the container 10 and gelated there. Thereby, the cell-containing hydrogel can be disposed in the hole 21 of the container 10.

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

Subsequently, the cell-containing pregel solution is introduced into the pores 21 of the container 10 and gelated there. Gelation can be carried out simply by leaving at room temperature. Thereby, the cell-containing hydrogel can be disposed in the hole 21 of the container 10. The click cross-linked gel can be gelled at room temperature and has low toxicity to cells, so the cell-containing hydrogel can be easily placed in the pores 21.

In the cell culture method of the present embodiment, for example, the cell is a cell mass having the property of pituitary primordia, and the target substance is a differentiation inducer selected from the group consisting of glucocorticoid, BMP, FGF and Shh. It may be. In this way, it is possible to cause the concentration gradient of the differentiation inducer to act on the pituitary primordia, and to perform differentiation induction of the pituitary which has hitherto been impossible.

As a cell mass having a property of pituitary primordium, for example, LIM Homeobox 3 (3D suspension culture) obtained by suspension culture (3D suspension culture) of a human clump of human pluripotent stem cells in a medium containing BMP and Shh LHX3) positive cell clusters are included.

[Method of producing pituitary]
In one embodiment, the present invention provides a method for producing a pituitary, comprising the step of disposing a cell mass-containing hydrogel containing a cell mass having a property of pituitary primordium in the hole of the container described above; The medium containing different concentrations of differentiation-inducing factors selected from the group consisting of glucocorticoid, BMP, FGF and Shh are respectively put in at least two of the plurality of liquid reservoirs, and as a result, the cell mass-containing hydro There is provided a manufacturing method comprising the steps of: forming a concentration gradient of the differentiation-inducing factor in a gel; and incubating the cell mass-containing hydrogel so that a pituitary body is formed from the cell mass. .

As described later in the Examples, according to the production method of the present embodiment, it is possible to produce a functional pituitary gland by ubiquitously inducing and simultaneously inducing a plurality of types of pituitary hormone-producing cells.

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

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

In the kit of the present embodiment, the material of the hydrogel may include the compound represented by the above formula (A) and the compound represented by the above formula (B). Here, the compound represented by the above-mentioned formula (A) and the compound represented by the above-mentioned formula (B) may be separately stored in the container, or may be mixed and stored in the same container. It is also good. When the compound represented by the formula (A), the compound represented by the formula (B), and the aqueous liquid are mixed, the click crosslinking reaction can proceed to form a hydrogel.

EXAMPLES The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

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

<< Synthesis of DBCO-4 arm PEG >>
First, 1 mL of pentaerythritol tetra (aminopropyl) polyoxyethylene (type “PTE-400PA”, manufactured by NOF Corporation, sometimes referred to as “4armPEG” in the present specification), and 20 mL of dimethyl sulfoxide (DMSO) in 1 g. In addition, it was dissolved by stirring at 40 ° C.

In addition, 52 mg of dibenzocyclooctin (DBCO) -sulfo-NHS (type “A124”, Click chemistry tools) was dissolved in 10 mL of DMSO.

Subsequently, the above DBCO-sulfo-NHS solution was added to the above 4 arm PEG solution, and reacted while stirring at 40 ° C. for 3 hours. As a result, DBCO-4armPEG was obtained.

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

Subsequently, an excitation wavelength of 360 nm was irradiated, and fluorescence at a wavelength of 465 nm was measured using a microplate reader (model "GENios", manufactured by Tecan) to measure the introduction rate of DBCO group to 4arm PEG.

Subsequently, DBCO-4 arm PEG was dialyzed. The dialysis was performed using a dialysis membrane with a molecular weight cut off of 6,000 to 8,000 and water (Milli Q water) as an external solution. The external fluid was replaced after 1 hour, 17 hours, 21 hours, 25 hours and 3 days from the start of dialysis.

Subsequently, the dialyzed DBCO-4 arm PEG was lyophilized and stored at -20 ° C. until use. The introduction rate of DBCO group to 4 arm PEG was 93.4%, and the yield of DBCO-4 arm PEG was 776.3 mg. In addition, the molecular weight of the synthesized DBCO-4 armPEG was 43,168.

<< Synthesis of Azide-4 arm PEG >>
The above procedure was repeated except that Azide-PEG4-NHS solution in which 38 mg of Azide-PEG4-NHS (type “AZ103”, Click chemistry tools) was dissolved in 10 mL of DMSO was used instead of the above DBCO-sulfo-NHS solution. Azide-4arm PEG was synthesized, dialyzed, lyophilized, and stored at -20 ° C. until use.

The rate of introduction of the Azide group to 4 arm PEG was 99.4%, and the yield of Azide-4 arm PEG was 1.0743 g. In addition, the molecular weight of the synthesized Azide-4 arm PEG was 42, 006.

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

First, 1 mL of the above-mentioned solvent was added to 41.3 mg of DBCO-4 arm PEG, and was dissolved by stirring with a vortex mixer. As a result, a 1 mM solution of DBCO-4armPEG was obtained.

Subsequently, a 1 mM solution of DBCO-4 arm PEG is diluted with the above solvent, and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0. .9 mM solutions were prepared respectively.

In addition, 1 mL of the above solvent was added to 41.2 mg of Azide-4arm PEG, and was dissolved by stirring with a vortex mixer. As a result, a 1 mM solution of Azide-4 arm PEG was obtained. Subsequently, a 1 mM solution of Azide-4 arm PEG is diluted with the above solvent, and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0. .9 mM solutions were prepared respectively.

Subsequently, 10 μL each of DBCO-4 arm PEG solution and Azide-4 arm PEG solution respectively prepared to the same molar concentration were mixed to prepare a pregel solution. Subsequently, each pregel solution was allowed to stand at room temperature for 30 minutes to proceed with the gelation reaction, and the state of gelation was visually observed. When the pregel solution after reaction did not dissolve even after addition and removal of phosphate buffer solution (PBS), it was evaluated as solification or gelation. Among them, those which maintained their shape even when lightly touched with a pipette tip were evaluated as gelation. The state of gelation was evaluated by the following evaluation criteria. The evaluation results are shown in Table 1 below.

(Evaluation criteria)
X ... did not sol gelation.
... ... solified.
○ ... gelled.

As a result, when the concentration of the pregel solution was 0.15 mM or more, a tendency of forming a hydrogel was observed. In the following experiments, a DBCO-4 arm PEG solution and an Azide-4 arm PEG solution were each prepared at 0.5 mM, and 0.25 mM pregel solution was prepared by mixing equal volumes to form a hydrogel.

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

The container 10 ′ is mainly different from the container 10 shown in FIGS. 2 to 5 in that a part of the hole 21 is in contact with the peripheral edge of the partition 2. As shown in FIGS. 16 and 17, the lower edge 21 a of the hole 21 of the container 10 ′ coincides with the lower edge 2 d of the partition 2. The hole 21 of the container 10 'had a cubic shape of length x width x height 3 mm x 3 mm x 3 mm.

<< Formation of Hydrogel >>
The DBCO-4 arm PEG solution and Azide-4 arm PEG solution were each prepared at 0.5 mM. As a solvent, gfCDM medium containing 5% KSR (Invitrogen) was used. Subsequently, a 0.25 mM pre-gel solution was prepared by mixing equal volumes of DBCO-4 arm PEG solution and Azide 4 arm PEG solution. Subsequently, 30 μL of the pregel solution was injected into the hole 21 of the container 10 ′.

FIG. 18A is a photograph showing the pregel solution introduced into the holes 21 of the container 10. As shown in FIG. 18 (a), when the container 10 was used, it became clear that the pregel solution was held without leaking from the holes 21. FIG. 18 (b) is a photograph showing the pregel solution introduced into the hole 21 of the container 10 '. As shown in FIG. 18 (b), it has become clear that the pregel solution leaks from the holes 21 when the container 10 ′ is used.

[Experimental Example 4]
(Formation of concentration gradient of target substance)
The same container 10 as shown in FIGS. 2 to 5 was used to prepare a hydrogel having a concentration gradient of the target substance. The hole 21 of the container 10 had a cubic shape of length × width × height 3 mm × 3 mm × 3 mm.

Target substance
As target substances, a fusion protein of fluorescein (molecular weight 332.31), FITC dextran (molecular weight 10,000), 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 introduces 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) and expressed in E. coli Let it be prepared. 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 >>
The DBCO-4 arm PEG solution and Azide-4 arm PEG solution were each prepared at 0.5 mM. Water was used as the solvent. Subsequently, a 0.25 mM pre-gel solution was prepared by mixing equal volumes of DBCO-4 arm PEG solution and Azide 4 arm PEG solution. Subsequently, 30 μL of the pregel solution was injected into the hole 21 of the container 10. The pregel solution could be retained inside the holes 21 without leaking out of the holes 21. Subsequently, it was allowed to stand at room temperature for 30 minutes to form a hydrogel. As a result, the holes 21 were blocked by the hydrogel.

<< Formation of concentration gradients >>
A plurality of containers 10 in which hydrogels are formed in the holes 21 are prepared, and fluorescein (final concentration 10 μM), FITC dextran (final concentration 10 μM) or YFP-CFP (final concentration 600 μg / final) in the first liquid reservoir 17A of each container 10 1.5 mL of each aqueous solution of mL) was introduced. In addition, 1.5 mL of water was introduced into the second liquid reservoir 17B of each container 10.

FIGS. 19 (a) and 19 (b) are photographs showing the liquid 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 holes 21, respectively. FIG. 19B is a photograph taken from the opening side of the liquid storage portion. The dotted line shows the position of hydrogel in FIG.19 (b).

Thereafter, the hydrogel of each container was observed with a fluorescence microscope for 9 days. Here, a group in which the liquid in the first liquid storage section 17A and the second liquid storage section 17B is replaced with a new one and a group in which the liquid is not replaced for nine days are prepared for every two to three days. I also went. The fluorescence intensities of fluorescein, FITC and YFP-CFP in fluorescence microscopy images were analyzed and graphed using Image J software.

FIGS. 20 (a) to 20 (c) use fluorescein (molecular weight 332.31) as the target substance, and refresh the liquid in the first liquid reservoir 17A and the second liquid reservoir 17B every two to three days. It is the result in the case of exchange. 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 showing numerical values of fluorescence micrographs of hydrogels 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 first liquid reservoir side, 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 showing numerical values of fluorescence micrographs of hydrogels 1, 2, 4, 7, 9 days after the start of the experiment. In FIG. 21 (c), the horizontal axis shows the distance (mm) from the end of the hydrogel on the first liquid reservoir side, and the vertical axis shows the fluorescence intensity (relative value) of FITC.

FIGS. 22 (a) to 22 (c) show the results when YFP-CFP (molecular weight 56,000 Da) is used as a 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 showing numerical values of fluorescence micrographs of hydrogels 1, 2, 3, 6, 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 first liquid storage portion side, and the vertical axis indicates the fluorescence intensity (relative value) of YFP-CFP.

As a result, it became clear that a concentration gradient could be formed in the hydrogel for any of the target substances. Moreover, the concentration gradient formed could be stably maintained for 9 days or more. In addition, for low molecular weight compounds (fluorescein), it was revealed that the concentration gradient was flattened in several days if liquid exchange was not performed. In addition, it was revealed that the concentration gradient can be maintained for at least 8 days for molecules having a molecular weight of about 10,000 or more (FITC dextran, YFP-CFP) regardless of whether or not liquid exchange is performed.

[Experimental Example 5]
(Examination of cell toxicity)
Using a container 10 similar to that shown in FIGS. 2 to 5, the cell mass (LIM Homeobox 3 (LHX3) -positive cell mass) having pituitary primordium properties is cultured, and the toxicity to the cell mass is examined did. It is thought that the toxicity of the hydrogel, the oxygen permeability of the hydrogel, the permeability of nutritional components by the hydrogel, etc. affect the cells. The hole 21 of the container 10 had a cubic shape of length × width × height 3 mm × 3 mm × 3 mm.

<< Preparation of LHX3-positive cell mass >>
Human ES cells (KhES-1) were treated with enzymes and dispersed into single cells. Subsequently, it was 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).

The day of seeding was designated as differentiation culture day 0, and a final concentration of 20 μM of Y-27632 (ROCK inhibitor) was added from day 0 to day 3. On the 3rd and 6th day of culture, half volume medium exchange was performed with medium without Y-27632.

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

By immunostaining on the 60th day of culture to confirm that LHX3 positive cells appeared, it was judged that the cell mass having the property of pituitary primordia could be differentiated. Cell clusters with pituitary primordium properties were used in the following experiments.

<< Formation of Hydrogel >>
The DBCO-4 arm PEG solution and Azide-4 arm PEG solution were each prepared at 0.5 mM. A gfCDM medium containing 20% KSR (Invitrogen) was used as a solvent. Subsequently, a 0.25 mM pre-gel solution was prepared by mixing equal volumes of DBCO-4 arm PEG solution and Azide 4 arm PEG solution.

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

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

The hydrogel was collected one week, two weeks and three weeks after the start of culture in the container 10, and the cell mass was fixed with paraformaldehyde to prepare thin film sections. Subsequently, thin film sections were immunostained with anti-adrenocorticotropin (ACTH) antibody, anti-PitX1 antibody and anti-E-cadherin antibody, and observed with a fluorescence microscope. The expression of ACTH indicates the presence of adrenocorticotropic hormone-producing cells, PitX1 is a marker for pituitary progenitor cells, and E-cadherin is a marker for oral ectoderm including pituitary progenitor cells.

FIG. 23 (a) and (b) are fluorescence micrographs showing the results of immunostaining. Fig. 23 (a) is a photograph one week after the start of culture in the container 10, and Fig. 23 (b) is a photograph two weeks after the start of culture in the container 10. As a result, it was revealed that there was no adverse effect on cultured cells for at least 2 weeks from the start of culture.

Further, it was revealed that the hydrogel was detached from the hole 21 of the container 10 three weeks after the start of the culture when the hole 21 was not surface-coated.

[Experimental Example 6]
(Culture of cell mass)
Cell clumps were cultured in a concentration gradient of differentiation inducer using a container 10 similar to that shown in FIGS. The hole 21 of the container 10 had a cubic shape of length × width × height 3 mm × 3 mm × 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 properties of pituitary primordium was prepared.

Surface coating
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 irradiation with ultraviolet light for 30 minutes. Subsequently, 30 μL of poly-L-lysine solution (hereinafter referred to as “PLL solution”) was injected into the hole 21 of the container 10 in a clean bench. The PLL solution was prepared by dissolving poly-L-lysine (type "P2636", Sigma) at a concentration of 50 μL / mL in water and filter-sterilizing.

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

<< Formation of Hydrogel >>
The DBCO-4 arm PEG solution and Azide-4 arm PEG solution were each prepared at 0.5 mM. A gfCDM medium containing 5% KSR (Invitrogen) was used as a solvent. Subsequently, a 0.25 mM pre-gel solution was prepared by mixing equal volumes of DBCO-4 arm PEG solution and Azide 4 arm PEG solution.

Subsequently, one LHX3 positive cell mass was suspended in 30 μL of a pregel solution to prepare a cell mass-containing pregel solution. Subsequently, 30 μL of a cell mass-containing pregel solution was injected into the holes 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), a final concentration of 1 μM dexamethasone, and a final concentration of 2 μM SAG was introduced into the first liquid reservoir 17A of the container 10 in which the cell mass-containing hydrogel was formed. In addition, 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 10 days of culture, the hydrogel was recovered and the cell mass was fixed with paraformaldehyde to prepare thin film sections. Subsequently, the thin film sections were immunostained with an anti-adrenocorticotropin (ACTH) antibody, an anti-growth hormone (GH) antibody, and an anti-growth hormone releasing hormone receptor (GHRH-R) antibody, and observed with a fluorescence microscope. The expression of ACTH indicates the presence of adrenocorticotropin-producing cells, the expression of GH indicates the presence of growth hormone-producing cells, and the expression of GHRH-R indicates the maturation of growth hormone-producing cells.

FIG. 24 is a fluorescence micrograph showing the result of immunostaining. In FIG. 24, “dexamethasone (+)” indicates that the result is a region on the side of the first liquid reservoir 17A to which dexamethasone is added among cell aggregates, and “dexamethasone (−)” is a portion of cell aggregates. It shows that it is a result of the field by the side of the 2nd fluid storage section 17B which did not add dexamethasone.

As a result, it was revealed that growth hormone-producing cells were evident on the first liquid reservoir 17A side and that they were functionally mature because they expressed growth hormone releasing hormone receptor on the surface .

On the other hand, it was considered that the second liquid reservoir 17B side was slightly affected by dexamethasone, and a phenomenon in which adrenocorticotropin-producing cells slightly expressed growth hormone was observed.

In the conventional 3D culture method, although it is possible to induce pituitary hormone-producing cells one by one, it has not been possible to induce two or more kinds of pituitary hormone-producing cells at one time and at the same time. On the other hand, in this experiment example, it became clear that a plurality of types of pituitary hormone-producing cells could be localized and differentiated according to the concentration gradient of dexamethasone.

According to the present invention, it is possible to provide a technique for forming a concentration gradient of a target substance.

2 ... Partition wall 2a, 2b, 3a, 3b, 11a, 13Ba ... surface, 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 part, 17A, 17A1, 17A2, 17B ... liquid reservoir, DESCRIPTION OF SYMBOLS 21 ... hole 23a, 23ba, 23bb, 23c ... surface 30, 30 culture apparatus 100 100 forming type 101 first type 102 second type 103 hole forming piece 104 105 lower piece 106: Bottom plate, 111: First formation portion, 112: Second formation portion, P: Resin.

Claims (15)

A container body in which an internal space capable of storing liquid is formed;
And a partition dividing the internal space into a plurality of liquid reservoirs,
The container is a hole which makes at least two of the plurality of liquid reservoirs communicate with each other in the partition wall, and a hole in which a hydrogel is held is formed. The container according to claim 1, wherein the hole is formed at a position away from the periphery of the partition wall as viewed in the thickness direction of the partition wall. The container according to claim 1 or 2, wherein a compound for fixing the hydrogel is laminated on at least a part of the surface of the hole. The container according to any one of the preceding claims, wherein the hydrogel is arranged in the hole. The hydrogel according to any one of claims 1 to 4, 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. The container described in.

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ] The hydrogel according to any one of claims 1 to 5, wherein the hydrogel is obtained by mixing a compound represented by the following formula (A), a compound represented by the following formula (B), a culture medium and cells. The container described in the section.

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ] A kit comprising the container according to any one of claims 1 to 3 and a material of the hydrogel. The kit according to claim 7, wherein the material of the hydrogel contains a compound represented by the following formula (A) and a compound represented by the following formula (B).

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ] Placing a hydrogel in the pores of the container according to any one of claims 1 to 3;
Placing at least two of the plurality of liquid reservoirs with liquids having different concentrations of the target substance, thereby forming a concentration gradient of the target substance in the hydrogel;
A method for producing the hydrogel having a concentration gradient of a target substance, comprising: The method according to claim 9, 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.

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ] Placing a cell-containing hydrogel in the pores of the container according to any one of claims 1 to 3;
A step of forming a concentration gradient of the target substance in the cell-containing hydrogel as a result of respectively putting media having different concentrations of the target substance into at least two of the plurality of liquid reservoirs;
Incubating the cell-containing hydrogel;
Cell culture methods, including: The cell according to claim 11, wherein 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. Culture method.

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ] The cell is a cell mass having pituitary primordium properties, and the target substance comprises glucocorticoid, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and sonic hedgehog (Shh) The cell culture method according to claim 11 or 12, which is a differentiation inducer selected from the group. A method of producing a pituitary,
Placing a cell mass-containing hydrogel containing cell masses having the property of pituitary primordia into the pores of the container according to any one of claims 1 to 3;
Media containing different concentrations of differentiation-inducing factors selected from the group consisting of glucocorticoid, BMP, FGF and Shh are respectively added to at least two of the plurality of liquid reservoirs, and as a result, the cell mass-containing hydrogel Forming a concentration gradient of said differentiation inducer in
Incubating the cell mass containing hydrogel so that a pituitary body is formed from the cell mass;
Including the manufacturing method. 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 a cell mass having a property of pituitary primordia The manufacturing method according to claim 14 which is.

[In Formula (A), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ¹ to R ⁴ each independently represent a hydrogen atom -L-Z ¹ , -O (CH ₂ CH ₂ O) _n -L-Z ¹ or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ² and R ⁴ may be identical to or different from each other. ]

[In Formula (B), A ¹ independently represents a single bond or a linear or branched alkylene group having 1 to 20 carbon atoms, and R ⁵ to R ⁸ each independently represent a hydrogen atom And -L-Z ² , -O (CH ₂ CH ₂ O) _n -L-Z ² or a linear or branched alkyl group having 1 to 20 carbon atoms. Here, L each 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, a plurality of R ⁶ and R ⁸ may be identical to or different from each other. ]

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