JP2009278960A - Method for producing spheroid and spheroid array - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a large amount of spheroid derived from an embryonic stem cell and having a uniform size at a time. <P>SOLUTION: The method for producing a spheroid comprises a step of seeding embryonic stem cells on a substrate containing a base material and a plurality of hydrophilic regions and hydrophobic regions formed by curing a photosensitive composition containing a branched polyalkylene glycol derivative placed on the base material and having ≥3 polyalkylene glycol groups having a polymerizable substituent at the terminal and a ≥3-valent linking group bonding with the polyalkylene glycol group, a step of culturing the seeded embryonic stem cells and a step of forming a spheroid derived from the cultured embryonic stem cell. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a spheroid and a spheroid array.

In recent years, it has been studied to artificially induce differentiation into various tissue cells using embryonic stem cells having the potential to differentiate into various tissue cells, and to apply them to regenerative medicine and the like. As a method for inducing differentiation of embryonic stem cells into various tissue cells in vitro, a method in which a cytokine or a growth factor is acted, a method in which a gene is introduced, and the like are known.
For example, a method of promoting differentiation into a specific tissue cell by introducing a specific gene into an embryoid body formed from embryonic stem cells using a viral vector is known (see, for example, Patent Document 1). ).

As methods for forming embryoid bodies from embryonic stem cells, a method of culturing embryonic stem cells in a hanging drop, a method of seeding in a petri dish, and the like are generally known. Furthermore, a method for efficiently forming a qualitatively stable embryoid body by culturing embryonic stem cells in a culture vessel having a specific shape is disclosed (for example, see Patent Document 2).
JP 2006-254872 A JP 2006-254622 A

  However, in the method described in Patent Document 1, the size of the embryoid bodies to be formed tends to be uneven, so that the efficiency of differentiation induction is not sufficient, and it is difficult to stably induce differentiation. It was. Further, in the method described in Patent Document 2, it is difficult to uniformly control the size of the formed lung-like bodies, and it is difficult to say that the number of lung-like bodies that can be formed at one time is sufficient. .

  An object of the present invention is to provide a method for producing a spheroid capable of forming a large amount of spheroids derived from embryonic stem cells having a uniform size at a time. Another object of the present invention is to provide a spheroid array in which embryonic stem cell-derived spheroids having a uniform size are arranged in an array.

The first aspect of the present invention is a base material, a trivalent or higher-valent linkage that binds to the polyalkylene glycol group and three or more polyalkylene glycol groups that are disposed on the base material and have a polymerizable substituent at the terminal. Seeding embryonic stem cells on a substrate comprising a plurality of hydrophilic regions and hydrophobic regions formed by curing a photosensitive composition containing a branched polyalkylene glycol derivative having a group, and seeding A method for producing spheroids, comprising culturing cultured embryonic stem cells and forming spheroids derived from cultured embryonic stem cells.
The branched polyalkylene glycol derivative preferably has 4 or more polyalkylene glycol groups having a polymerization degree of 5 to 1000.

  According to a second aspect of the present invention, the expression level of AFP that is an endoderm marker produced by the spheroid production method is at least one of FlK-1 that is a mesodermal marker and NCAM that is an ectoderm marker. It is a spheroid larger than the expression level.

  According to a third aspect of the present invention, there is provided a base material, three or more polyalkylene glycol groups disposed on the base material and having a polymerizable substituent at a terminal, and a trivalent or higher valent bond that binds to the polyalkylene glycol group. A substrate comprising a plurality of hydrophilic regions and hydrophobic regions formed in an array by curing a photosensitive composition containing a branched polyalkylene glycol derivative having a group, and disposed in the hydrophobic region A plurality of spheroids derived from embryonic stem cells.

  A fourth aspect of the present invention is a drug screening method using the spheroid array. It is preferable that the drug screening method includes contacting a drug encapsulated in polymer micelles with the spheroid.

  According to a fifth aspect of the present invention, there is provided a base material, three or more polyalkylene glycol groups that are disposed on the base material and have a polymerizable substituent at the terminal, and a trivalent or higher-valent bond that binds to the polyalkylene glycol group. Seeding embryonic stem cells on a substrate comprising a plurality of hydrophilic regions and hydrophobic regions formed by curing a photosensitive composition containing a branched polyalkylene glycol derivative having a group, and differentiation A method for inducing differentiation of embryonic stem cells, comprising culturing embryonic stem cells seeded in the presence of an inducer and obtaining cells induced to differentiate from the cultured embryonic stem cells. The differentiation-inducing factor is preferably added in a state of being encapsulated in polymer micelles.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the spheroid which can form the spheroid derived from the embryonic stem cell which has a uniform magnitude | size in large quantities at once can be provided. Furthermore, according to the present invention, it is possible to provide a spheroid array in which spheroids derived from embryonic stem cells having a uniform size are arranged in an array.

The method for producing a spheroid of the present invention comprises a base material, a trivalent or higher-valent linkage that binds to the polyalkylene glycol group and three or more polyalkylene glycol groups that are disposed on the base material and have a polymerizable substituent at the terminal. Seeding embryonic stem cells on a substrate comprising a plurality of hydrophilic regions and hydrophobic regions formed by curing a photosensitive composition containing a branched polyalkylene glycol derivative having a group, and seeding Culturing the cultured embryonic stem cells and forming spheroids derived from the cultured embryonic stem cells.
By using such a substrate, a large amount of embryonic stem cell-derived spheroids having a uniform size can be formed at a time.

  The substrate in the present invention includes a base material and a plurality of hydrophilic regions and hydrophobic regions formed on the base material using a photosensitive composition containing a branched polyalkylene glycol derivative having a specific structure described later. The hydrophilic region is a region where a crosslinked product obtained by curing the photosensitive composition is formed, and the hydrophobic region is a region other than the hydrophilic region where the substrate is exposed.

  The substrate in the present invention is preferably a substrate formed by partitioning a plurality of the hydrophilic regions and a plurality of hydrophobic regions. Further, the number, size, and shape of the hydrophilic region and the hydrophobic region are not particularly limited, and can be appropriately selected according to the purpose. In addition, the detail of the board | substrate in this invention and its manufacturing method is mentioned later.

In the present invention, it is preferable that the plurality of hydrophobic regions have a uniform size and shape from the viewpoint of forming a spheroid having a uniform size.
In addition, from the viewpoint of efficiently forming a large amount of homogeneous spheroids, the diameter is preferably 50 to 500 μm, preferably 100 to 300 μm as the size when the hydrophobic region is formed in a circular shape, for example. Is more preferable.

The method for producing spheroids of the present invention includes seeding embryonic stem cells on the substrate, culturing the seeded embryonic stem cells, and forming spheroids derived from the cultured embryonic stem cells. .
By seeding and culturing embryonic stem cells on the substrate, spheroids derived from embryonic stem cells can be formed in the hydrophobic region on the substrate. That is, embryonic stem cells are arranged only in the hydrophobic region on the substrate, and the embryonic stem cells do not adhere to the hydrophilic region, so that the embryonic stem cells according to the number, size, and shape of the hydrophobic region Origin spheroids can be formed.

The embryonic stem cell is a cell that can be differentiated into various cells having different functions. The origin of such embryonic stem cells is not particularly limited as long as it is a mammal, and includes primates (eg, humans, monkeys, etc.), ungulates (eg, pigs, cows, horses, etc.), small mammalian rodents. Depending on the purpose of use, it can be appropriately selected from a variety of animals (eg, mouse, rat, rabbit, etc.).
The method for preparing embryonic stem cells can be performed using methods well known in the art. For example, it can be obtained by culturing cells under culture conditions that allow only embryonic stem cells to be selectively grown.

For culture of embryonic stem cells, a commonly used medium can be used. The medium used for the culture may be a medium generally used for culturing animal cells, and examples thereof include Dulbecco's modified Eagle medium (DMEM).
To this medium, serum for promoting cell growth may be added, or as an alternative to serum, for example, cell growth factors such as FGF, EGF, PDGF, and known serum components such as transferrin may be added. Good. In addition, although the density | concentration in the case of adding serum can be suitably changed according to the culture state at that time, it can usually be 5 volume%-10 volume%. Moreover, what added antibiotics (AB), such as various vitamins and streptomycin, may be used suitably. Furthermore, the medium may contain a factor (for example, leukemia inhibitory factor (LIF) or the like) that suppresses differentiation into various tissue cells while maintaining the self-renewal ability of embryonic stem cells.

For culturing embryonic stem cells, normal culturing conditions such as culturing in an incubator with a temperature of 37 ° C. and a concentration of 5% CO ₂ are applied.
In the formation of spheroids derived from embryonic stem cells, spheroids can be formed by culturing for about 2 hours to 2 days under normal culture conditions.

  As a medium used for culturing embryonic stem cells in the present invention, it is preferable to use a medium that does not contain LIF, and more preferably a medium that does not contain LIF and contains retinoic acid, from the viewpoint of efficient spheroid formation.

There is no particular limitation on the method of seeding embryonic stem cells on the substrate, and a normal method can be used as appropriate. The density of embryonic stem cells to be seeded is not particularly limited as long as spheroids can be formed, and can be appropriately selected according to the size of the substrate, the number and size of the hydrophobic regions, and the like. For example, it can be set to 1 × 10 ⁴ to 1 × 10 ⁸ cells / mL, and is preferably 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL.

In the present invention, the formation of embryonic stem cell-derived spheroids can be determined based on morphological observation. That is, the seeded embryonic stem cells gather on the hydrophobic region as the culture period progresses to form cell aggregates (spheroids). As another indicator of spheroid formation, A concomitant decrease in alkaline phosphatase (ALP) activity may be used.

According to the method for producing spheroids of the present invention, a large amount of spheroids having a uniform size can be formed on a substrate. Since such spheroids have almost homogeneous cell properties, they can show the same tendency in various behaviors.
Therefore, by applying the spheroid formed by the spheroid production method of the present invention to, for example, drug screening, it becomes possible to perform drug screening with higher sensitivity and accuracy. Such spheroids can induce the same degree of differentiation in response to a predetermined stimulus.

In the spheroid derived from embryonic stem cells formed by the spheroid production method of the present invention, the expression level of AFP, which is an endoderm marker, is the expression level of Flk-1, which is a mesoderm marker, and the ectoderm marker. Although it is characterized by being larger than at least one expression level of NCAM, it is preferable that the expression level of Oct4, which is an undifferentiated marker, is further reduced.
Thereby, the spheroid of the present invention can be suitably applied to induce differentiation into endoderm tissue cells.

The expression level of AFP is preferably 50 times or more of at least one of the expression level of Flk-1 and the expression level of NCAM, and more preferably 200 times or more.
In the present invention, the expression level of a marker such as AFP means the expression level measured by a normal method using RT-PCR.

The spheroid array of the present invention comprises a base material, three or more polyalkylene glycol groups that are arranged on the base material and have a polymerizable substituent at a terminal, and a trivalent or higher linking group that binds to the polyalkylene glycol group. A substrate comprising a plurality of hydrophilic regions and hydrophobic regions formed in an array by curing a photosensitive composition containing a branched polyalkylene glycol derivative having a structure, and an embryo disposed in the hydrophobic region A plurality of spheroids derived from sex stem cells.
The substrate and embryonic stem cell in the present invention are the same as the substrate and embryonic stem cell in the spheroid production method.

  The arrangement and the number of spheroids in the spheroid array of the present invention are in accordance with the arrangement and the number of hydrophobic regions formed on the substrate. Here, the arrangement of the hydrophobic regions is not particularly limited as long as they are arranged in an array on the substrate, and can be appropriately changed according to the purpose of the spheroid array and the number of the hydrophobic regions.

  In the spheroid array of the present invention, each spheroid arranged in an array can be formed on a substrate in a large amount and efficiently so that the size is uniform. In such a spheroid array, properties such as drug sensitivity of each spheroid can be made uniform. For example, when this is used for drug screening, drug screening with high accuracy and good reproducibility can be realized. For example, when used for induction of differentiation into tissue cells, a large amount of tissue cell aggregates can be obtained with good reproducibility.

In the spheroid array of the present invention, a plurality of hydrophilic regions and hydrophobic regions formed on a substrate are partitioned and formed, and the hydrophobic regions surrounded by the hydrophilic regions are arranged in an array. . As a method for forming the hydrophobic region in such a shape, a method for producing a substrate in the present invention described later can be preferably exemplified.
In the present invention, the hydrophobic region preferably has a uniform size and shape from the viewpoint of the uniformity of the formed spheroids.
The spheroid array of the present invention can be manufactured using, for example, the spheroid manufacturing method.

The drug screening method of the present invention is characterized by using the spheroid array. Thereby, drug screening with high accuracy and good reproducibility can be realized.
In addition, the drug screening method of the present invention preferably includes bringing a drug encapsulated in a polymer micelle into contact with a spheroid constituting a spheroid array. Thereby, a spheroid and a drug can be contacted more efficiently, and drug screening can be performed with higher sensitivity.

The spheroid array in the drug screening of the present invention is configured by arranging embryonic stem cell-derived spheroids in an array. By screening using spheroids (cell aggregates), the cellular response to the drug can be determined more clearly and with high sensitivity. For example, when a cellular response to a drug is detected by color development, luminescence, fluorescence, or the like, it can be detected as a response from a plurality of aggregated cells, and thus can be determined clearly and with high sensitivity.
Further, since the spheroids are arranged in an array, the cellular response to the drug can be determined with high reproducibility and with high sensitivity, and the cellular responses to a plurality of drugs can be simultaneously determined.

The drug to be screened in the present invention is not particularly limited as long as it is a drug that causes some kind of response change in embryonic stem cell-derived spheroids. Specific examples include differentiation inducing factors and growth factors. In the present invention, it is preferably a differentiation inducing factor from the viewpoint of screening sensitivity.
The drug may be a low molecular compound or a high molecular compound. Examples of the polymer compound include proteins and polynucleotides.

In the drug screening method of the present invention, the method for bringing the drug into contact with the spheroid is not particularly limited, and a commonly used method can be applied.
For example, when the drug to be screened is a polynucleotide, a method using a viral vector (see, for example, JP-A-2006-254872) is generally used as a method for bringing the drug into contact with the spheroid. In the present invention, it is preferable to contact the spheroid in a state where the polynucleotide is encapsulated in the polymer micelle. Thereby, the expression of the cytotoxicity derived from a viral vector can be avoided, and a drug and a spheroid can be contacted more efficiently.

  The polymeric micelle that may be used in the present invention may be composed of a block copolymer containing a hydrophilic constituent unit and a hydrophobic constituent unit. The hydrophilic structural unit is preferably a nonionic hydrophilic structural unit, and more preferably a polyalkylene glycol. The hydrophobic structural unit can be appropriately selected depending on the drug to be encapsulated. Examples thereof include a structural unit that can interact with a metal complex such as polyaspartic acid, an anionic structural unit that can interact with a cationic protein, and a cationic structural unit that can interact with a polynucleotide.

  In the present invention, when the drug to be screened is, for example, a polynucleotide, the block copolymer constituting the polymer micelle preferably contains a cationic constituent unit as a hydrophobic constituent unit. As a specific example of such a block copolymer, for example, a polyethylene glycol-polycation block copolymer described in JP-A-2004-352972 can be preferably exemplified.

In the present invention, the embryonic stem cell preferably contains a response gene corresponding to the screening purpose. Thereby, drug screening can be performed with higher accuracy and higher sensitivity. The response gene is appropriately selected according to the purpose of screening. For example, when used for screening for a differentiation-inducing factor for a specific tissue cell, a promoter of a gene expressed specifically in the tissue cell can be suitably used as a response gene.
Specifically, for example, when applied to screening for differentiation-inducing factors into osteoblast-like cells, a type I or type II collagen promoter can be used as a response gene.

  In the present invention, the response gene preferably has a reporter gene whose expression is controlled thereby linked downstream thereof. As the reporter gene, those commonly used can be used without particular limitation, and for example, a gene encoding green fluorescent protein (GFP) can be preferably used.

  When the drug screening method of the present invention is applied to screening of differentiation-inducing factors into osteoblast-like cells, the embryonic stem cells that form the spheroids are type I or type II collagen promoters and reporter genes whose expression is controlled thereby Are preferably included.

The method for inducing differentiation of embryonic stem cells of the present invention comprises a base material, three or more polyalkylene glycol groups that are arranged on the base material and have a polymerizable substituent at the terminal, and a trivalent group that binds to the polyalkylene glycol group. Seeding embryonic stem cells on a substrate comprising a plurality of hydrophilic regions and hydrophobic regions formed by curing a photosensitive composition containing a branched polyalkylene glycol derivative having the above linking group. And culturing embryonic stem cells seeded in the presence of a differentiation-inducing factor, and obtaining differentiation-induced cells from the cultured embryonic stem cells.
The substrate and embryonic stem cell in the present invention are the same as the substrate and embryonic stem cell in the spheroid production method.

  In the present invention, differentiation induction of embryonic stem cells is performed on the substrate, so that cells induced to differentiate from embryonic stem cells form spheroids (cell aggregates) in the hydrophobic region of the substrate. Therefore, differentiation-induced cells can be efficiently obtained with high uniformity.

The differentiation induction in the present invention is not particularly limited as long as it induces differentiation from embryonic stem cells to cells having different properties from embryonic stem cells. For example, cells constituting somatoid bodies, somatic stem cells, tissues Examples include differentiation induction into cells and the like.
The differentiation-inducing factor in the present invention is not particularly limited as long as it is a compound that can induce differentiation of embryonic stem cells, and can be appropriately selected and used according to the purpose. The differentiation inducing factor may be a low molecular compound or a high molecular compound. Here, the polymer compound includes a protein, a polynucleotide and the like.

In the differentiation induction method of the present invention, after seeding embryonic stem cells on a substrate, embryonic stem cell-derived spheroids are formed in the hydrophobic region of the substrate, and then embryonic stem cell-derived in the presence of a differentiation-inducing factor. Spheroid culture is preferred. Differentiation can be induced more efficiently by forming spheroids derived from embryonic stem cells.
As a method for forming embryonic stem cell-derived spheroids in the hydrophobic region of the substrate, the method for producing spheroids can be suitably applied.

Examples of the method of culturing embryonic stem cells seeded in the presence of a differentiation-inducing factor in the present invention include a method of culturing embryonic stem cells in a medium to which a differentiation-inducing factor is added. The method for adding the differentiation-inducing factor is not particularly limited, and a commonly used method can be used.
In the present invention, it is preferable to add a differentiation-inducing factor encapsulated in polymer micelles from the viewpoint of differentiation induction efficiency. As the polymer micelle, the polymer micelle described in the drug screening method can be preferably applied in the present invention.

In the differentiation induction method of the present invention, cells induced to differentiate can be obtained according to the differentiation-inducing factor used.
For example, when osteoblast-like cells are obtained by the differentiation induction method of the present invention, the differentiation induction factor that can be used is TH (for example, Biochem. Biophys. Res. Commun., 357 (4), 854-860 (2007 ))), And genes (polynucleotides) such as caALK6, Runx2, caSmo, caLEF-1, and IRS-1. Among these, from the viewpoint of differentiation induction efficiency into osteoblast-like cells, at least one of caALK6 and Runx2 is preferable.
Moreover, for example, genes (polynucleotides) such as Sox5, Sox6, and Sox9 can be cited as factors for inducing differentiation into cartilage-like cells.
Details of the gene are described in, for example, FASEB J., 21, 1777-1787 (2007), Japanese Patent Application Laid-Open No. 2004-267052, and the like.

  Since the differentiation-induced cells obtained by the differentiation induction method of the present invention are cell aggregates having high uniformity, they can be suitably used for various applications such as regenerative medicine.

Hereinafter, the substrate in the present invention will be described in detail.
The branched polyalkylene glycol derivative in the present invention is characterized by having three or more polyalkylene glycol groups having a polymerizable substituent at the terminal and a trivalent or more linking group bonded to the polyalkylene glycol group.
The branched polyalkylene glycol derivative having such a configuration can form a hydrophilic crosslinked product. Such a hydrophilic cross-linked product has good cell non-adhesive stability over time. For example, a hydrophilic region and a hydrophobic region can be highly accurately formed on a substrate using the branched polyalkylene glycol derivative of the present invention. When the cells are cultured on the substrate, the cells adhere specifically only to the hydrophobic region, so that a cell assembly partitioned with high accuracy can be formed. Further, the hydrophilic region has good cell non-adhesive stability over time and can maintain a compartmented cell aggregate over a long period of time. Further, the cell aggregate can be, for example, a monolayer cell aggregate or a cell aggregate (spheroid) in which cells form a three-dimensional aggregated state.

In the branched polyalkylene glycol derivative in the present invention, the content of polyalkylene glycol group having a polymerizable substituent at the terminal is 3 or more. When the polyalkylene glycol group content is 2 or less, the non-adhesive temporal stability of the hydrophilic region formed thereby is insufficient, and the compartmentalized cell aggregate can be maintained for a long time. Can not.
In addition, the content of the polyalkylene glycol group is preferably 4 or more, more preferably 4 or more and 64 or less, and more preferably 4 or more and 16 or less from the viewpoint of stability over time and good spheroid formation. Is more preferable.

The polyalkylene glycol group in the present invention is not particularly limited as long as it is a polyalkylene glycol group having a polymerizable substituent at the terminal.
The polymerizable substituent is not particularly limited as long as it is a substituent having a polymerizable functional group and can be bonded to the terminal of the polyalkylene glycol. The bonding mode of the polymerizable substituent to the terminal of the polyalkylene glycol is a bond in which the terminal hydroxyl group of the polyalkylene glycol is substituted with another element even if it is a bonding mode through an oxygen atom derived from the polyalkylene glycol. An aspect may be sufficient.

The polymerizable substituent may be a polymerizable functional group itself or a substituent constituted by including a polymerizable functional group and a linking group.
As the polymerizable functional group in the present invention, a commonly used polymerizable functional group can be used without particular limitation, and examples thereof include a group having an ethylenically unsaturated bond and an azide group. In the present invention, from the viewpoint of pattern formation of the hydrophilic region, it is preferably at least one selected from a group having an ethylenically unsaturated bond and an azide group, and more preferably an azide group.

The linking group in the polymerizable substituent is not particularly limited as long as it is a group capable of linking a polymerizable functional group and a polyalkylene glycol group. For example, an alkylene group, an arylene group, a heteroarylene group, a carbonyl group, It can be configured to include at least one selected from an oxygen atom, a nitrogen atom, a sulfur atom, a disulfide and a hydrogen atom.
Specific examples include a carbonyl group, an arylene group, an alkylenecarbonyl group, a carbonylarylene group, a carbamoylarylene group, and the like.
Furthermore, the valence of the linking group may be at least divalent, and may be a linking group having a valence of 3 or more and linking a polyalkylene glycol and two or more polymerizable functional groups.

  The polyalkylene glycol group constituting the polyalkylene glycol group having a polymerizable substituent at the terminal is not particularly limited as long as it is a polyalkylene glycol group capable of imparting hydrophilicity to the branched polyalkylene glycol derivative in the present invention. For example, a polyalkylene glycol group containing an alkylene glycol structural unit having 2 to 4 carbon atoms (for example, ethyleneoxy, n-propyleneoxy, isopropyleneoxy, butyleneoxy, isobutyleneoxy, etc.) can be preferably used.

The alkylene glycol structural unit in the polyalkylene glycol group may be composed of one type of alkylene glycol structural unit or may be composed of a combination of two or more types of alkylene glycol structural units. When the polyalkylene glycol group is composed of a combination of two or more alkylene glycol structural units, it may be a block polymer or a random polymer.
The degree of polymerization of the polyalkylene glycol group may be 5 or more from the viewpoint of hydrophilicity, and a polyalkylene glycol group having a degree of polymerization of 5 to 1000 can be preferably used, and more preferably 10 to 500. is there.

In the present invention, a trivalent or higher valent linking group bonded to a polyalkylene glycol group having a polymerizable substituent at the terminal is bonded to the terminal of the at least three polyalkylene glycol groups to which the polymerizable substituent is not bonded. The polyalkylene glycol group is not particularly limited as long as it can be linked to each other. The bonding mode may be any of a covalent bond, a coordination bond, and an ionic bond.
Specifically, for example, a linking group derived from a saccharide, a linking group derived from a polyhydric alcohol, a linking group derived from a polyvalent carboxylic acid, or a group containing the polyalkylene glycol group can be bonded via a coordination bond. A metal atom etc. can be mentioned.

  Examples of the saccharide include glyceraldehyde, erythrose, ribose, glucose and the like. Examples of the polyhydric alcohol include glycerin, pentaerythritol, xylitol, sorbitol and the like. Furthermore, examples of the polyvalent carboxylic acid include propanetricarboxylic acid, citric acid, and benzenetricarboxylic acid. Examples of the metal atom include gold, silver, platinum, nickel, and copper.

  In the present invention, from the viewpoint of hydrophilicity and stability over time, it is preferably a linking group derived from a polyhydric alcohol, more preferably a linking group derived from glycerin or a linking group derived from pentaerythritol. A linking group derived from a compound selected from glycerin, pentaerythritol, and polypentaerythritol is particularly preferable.

  In the present invention, the polyalkylene glycol group having a polymerizable substituent at the terminal is preferably a substituted polyalkylene glycol group represented by the following general formula (1) from the viewpoint of temporal stability and good spheroid-forming properties. .

  In the general formula (1), m represents an integer of 2 to 4, preferably 2 or 3, and more preferably 2. Moreover, although n represents the integer of 5-1000, it is preferable that it is 10-500, and it is more preferable that it is 10-300.

In the general formula (1), X ¹ represents a polymerizable substituent. In the present invention, the polymerizable substituent is a polymerizable substituent represented by at least one of the following general formula (3) and general formula (4) from the viewpoint of cross-linking curability of the branched polyalkylene glycol derivative in the present invention. It is preferable that

In General Formula (3), L ³ represents a single bond or a divalent linking group. The divalent linking group is not particularly limited as long as it can link an ethylenically unsaturated group and a polyalkylene glycol group. For example, an alkylene group, an arylene group, a heteroarylene group, a carbonyl group, an oxygen atom, a nitrogen atom, an imino group, and a divalent linking group including at least one of a hydrogen atom can be exemplified, and a carbonyl group And a divalent linking group selected from an ester group, an amide group, a phenylene group, an alkylene group having 2 to 4 carbon atoms, or a combination thereof.
In the present invention, L ³ is more preferably a single bond or a divalent linking group selected from a carbonyl group, a carbonylphenylene group, and a carbamoylphenylene group.

R ³ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 3 carbon atoms include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. In the present invention, from the viewpoint of the crosslinking reactivity of the branched polyalkylene glycol derivative, R ³ is preferably a hydrogen atom or a methyl group, and more preferably a hydrogen atom.

L ⁴ in the general formula (4) represents a divalent linking group, and the definition and preferred range thereof are the same as those of the divalent linking group in L ³ .

  In the present invention, at least one of the polymerizable substituents is preferably a substituent represented by the following general formula (5). As a result, the reaction of the polymerizable substituent can be initiated by irradiation with light having a longer wavelength.

In general formula (5), L ⁵ represents a single bond or a divalent linking group. The divalent linking group represented by L ⁵ is the same as the divalent linking group in L ³ .
I represents 1 or 2.

In general formula (5), R ⁵ represents a substituent, and is not particularly limited as long as it is a substituent capable of changing the maximum absorption wavelength of the polymerizable substituent represented by general formula (5). Among these, a substituent capable of shifting the maximum absorption wavelength of the polymerizable substituent represented by the general formula (5) to the long wavelength side is preferable. Specific examples include a nitro group, a hydroxyl group, an alkyloxy group, a dialkylamino group, a cyano group, and a nitroso group.
When i is 2, two R ⁵ may be the same or different.

  The branched polyalkylene glycol derivative in the present invention is preferably a compound represented by the following general formula (2) from the viewpoint of hydrophilicity and crosslinking reactivity.

In General Formula (2), L ² represents a single bond or a methylene group, and p represents 1 or 2. When p is 1, L ² is preferably a single bond, and when p is 2, L ² is preferably a methylene group.
q represents an integer of 1 to 70. In the present invention, from the viewpoints of hydrophilicity and crosslinking reactivity, when p is 1, q is preferably 1 to 64, and more preferably 2 to 10. When p is 2, q is preferably 1 to 32, and more preferably 1 to 5.

In general formula (2), R ² represents a polyalkylene glycol group having a polymerizable substituent at the terminal or a polyalkylene glycol group having a hydroxyl group at the terminal. Among these, from the viewpoint of crosslinking reactivity, R ² is preferably a polyalkylene glycol group having a polymerizable substituent at the terminal, and more preferably a substituted polyalkylene glycol group represented by the general formula (1). .

  The branched polyalkylene glycol derivative in the present invention has from 4 to 16 polyalkylene glycol groups having a polymerizable substituent at the terminal from the viewpoint of stability over time and spheroid formation, and the polyalkylene glycol group is represented by the general formula It is preferable that the polymerizable substituent is represented by at least one of the general formula (3), the general formula (4), and the general formula (5). .

  Specific examples of the branched polyalkylene glycol derivative in the present invention are illustrated below, but the present invention is not limited thereto. In the following specific examples, the degree of polymerization (n) of the polyalkylene glycol means an average degree of polymerization calculated from the weight average molecular weight of the branched polyalkylene glycol derivative. The weight average molecular weight of the branched polyalkylene glycol derivative can be measured by, for example, gel permeation chromatography (GPC).

  The branched polyalkylene glycol derivative in the present invention is, for example, a compound having three or more polyethylene glycol groups (hereinafter, sometimes referred to as “multi-arm PEG”. For example, Sunbrigh (registered trademark) PTE series manufactured by NOF Corporation). , HGEO series, etc.) can be synthesized by bonding a polymerizable substituent with an ester bond, an ether bond or the like using a commonly used method. For example, the ester bond can be formed by an acid chloride method, an active ester method, or the like.

  The branched polyalkylene glycol derivative in the present invention can be, for example, a component of the photosensitive composition described later. Moreover, a hydrophilic crosslinked body can be formed by a crosslinking reaction.

The photosensitive composition in the present invention contains at least one of the branched polyalkylene glycol derivatives. By using such a photosensitive composition, for example, a hydrophilic region and a hydrophobic region can be formed on the substrate with high accuracy.
In the said photosensitive composition, the said branched polyalkylene glycol derivative can also be contained individually by 1 type, and can also contain 2 or more types.
In addition to the branched polyalkylene glycol derivative, the photosensitive composition contains various additives such as a photopolymerization initiator, a solvent, a cell culture solution, a surfactant, a buffer solution, an antifoaming agent, and an antiseptic. Can be configured.

  The photopolymerization initiator is not particularly limited as long as it can initiate a polymerization reaction by light irradiation, but is preferably one that has low damage to living cells. Specific examples include IRGACURE 2959, IRGACURE 184 (both manufactured by Ciba Specialty Chemicals), and IRGACURE 2959 is preferred from the viewpoint of cytotoxicity and water solubility.

The solvent is not particularly limited as long as it can dissolve the branched polyalkylene glycol derivative. The term “dissolvable” as used herein means that the branched polyalkylene glycol derivative can be dissolved by 0.1% or more on a mass basis.
Specifically, organic solvents such as benzene, toluene, THF, DMF, and chloroform, and water can be preferably used as the solvent. Moreover, you may use a solvent individually by 1 type or in mixture of 2 or more types.

  As content rate of the said branched polyalkylene glycol derivative in the said photosensitive composition, it can be 0.1-50 mass%, for example, and it is preferable that it is 0.1-20 mass%.

  The crosslinked body in the present invention is formed by crosslinking and curing the photosensitive composition. The cross-linking curing is not particularly limited as long as it is caused by a polymerization reaction by light irradiation, and the cross-linking curing conditions can be appropriately selected according to the photosensitive composition.

  The crosslinked body in the present invention is, for example, the photosensitive composition in a dispersed state in a medium even if the tourism composition layer made of the photosensitive composition formed on a substrate is cured. May be cured. By curing the photosensitive composition layer formed on the substrate, a hydrophilic region made of a crosslinked product can be formed on the substrate. Moreover, the particulate crosslinked body which has a hydrophilic surface can be formed by hardening the said photosensitive composition made into the dispersion state in the medium.

  The board | substrate in this invention contains the base material and the said bridge | crosslinking body arrange | positioned on the said base material, It is characterized by the above-mentioned. As a base material in this invention, the base material used normally can be especially used without a restriction | limiting. Examples of the material of the base material include glass, thermoplastic resin, thermosetting resin, silicone, diamond, metal, and ceramics. In this invention, it is preferable that it is glass or a thermoplastic resin from a viewpoint of the adhesiveness of a base material and a crosslinked body, and it is more preferable that it is glass.

Moreover, it is preferable that the base material in this invention is a base material surface-treated by at least 1 sort (s) chosen from the silane coupling agent which has an amino group, the silane coupling agent which has an ethylenically unsaturated group, and polylysine. Thereby, the bond stability of a base material and the crosslinked body formed on it can be improved.
In addition, the substrate is preferably a substrate surface-treated with at least one kind of cell adhesion protein, a silane coupling agent having an amino group, a silane coupling agent having an ethylenically unsaturated group, More preferably, the base material is surface-treated with at least one selected from polylysine and further surface-treated with at least one cell adhesion protein.
By constructing the substrate using the surface-treated base material, for example, when cells are cultured on the substrate, cell aggregates can be formed more efficiently.
Here, examples of the cell adhesion protein include collagen, gelatin, fibronectin, vitronectin, laminin, tainesin, and elastin. Among these, collagen, gelatin, fibronectin, Vitronectin is preferable, and collagen and gelatin are more preferable.

The method for producing a substrate in the present invention includes, for example, a step of applying a photosensitive composition containing the branched polyalkylene glycol derivative on a base material to form a photosensitive composition layer, and a step of forming the photosensitive composition layer. And a curing step of performing an exposure process. Thereby, the board | substrate with which the said crosslinked body was formed on a base material is producible.
The method for producing the substrate may further include a heating step, a washing step, a drying step, a sterilization step, and the like after the curing step, if necessary.

In the present invention, a normal thin film forming method can be applied to the step of forming the photosensitive composition layer on the substrate without any particular limitation. For example, a coating method, a dip coating method, a spin coating method and the like are preferable. Can be applied to.
There is no restriction | limiting in particular as layer thickness of the photosensitive composition layer formed on the base material, According to the intended purpose of a board | substrate, it can select suitably, For example, it can be set as 5 nm-1000 micrometers. In particular, when the substrate of the present invention is used as a cell culture substrate described later, the thickness is preferably 10 nm to 1000 nm, and more preferably 10 nm to 500 nm.

  The step of forming the photosensitive composition layer on the substrate can include a step of removing the solvent in the photosensitive composition, if necessary. The process for removing the solvent can be appropriately selected according to the solvent, and may be room temperature drying or heat drying. For example, it may be 1 minute to 10 hours at 30 to 150 ° C., and preferably 3 minutes to 1 hour at 35 to 120 ° C.

  The exposure process in the curing step may be a step of exposing the entire surface of the photosensitive composition layer, or a step of partially exposing the photosensitive composition layer in a desired pattern. In this invention, it is preferable that it is the process of partial exposure like a desired pattern, and it is more preferable to include the image development process after the said partial exposure process. Thereby, the board | substrate with which the hydrophilic region which consists of the said crosslinked body, and the hydrophobic area | region in which the crosslinked body is not formed was formed on the base material like a pattern can be produced.

  The step of partially exposing in the desired pattern is preferably a step of partially exposing through a mask (photomask) having light transmittance in the desired pattern. Moreover, by exposing the mask to the photosensitive composition layer and performing partial exposure, pattern exposure can be performed with higher accuracy.

The light source used for exposure is not particularly limited as long as it is a light source capable of curing the photosensitive composition layer. Examples of the light source include X-rays, electron beams, excimer lasers, xenon lamps, metal halide lamps, low-pressure mercury lamps, and high-pressure mercury lamps. Among them, a low-pressure or high-pressure mercury lamp can be suitably used, and a high-pressure mercury lamp of 10 to 2000 W is preferable.
Moreover, there is no restriction | limiting in particular also about an exposure wavelength and an exposure amount, According to the said photosensitive composition, it can select suitably. As an exposure wavelength, it can be 200-400 nm, for example, and it is preferable that it is 280-400 nm. The exposure amount, for example, be a 0.1~1000mJ / cm ^2, it is preferably from 1 to 200 mJ / cm ^2, more preferably 10~20mJ / cm ^2.

  The development step is not particularly limited as long as it can remove an unexposed area in the photosensitive composition layer from the substrate, and examples thereof include washing with a solvent and immersion in a solvent. In the present invention, washing with water as a solvent and immersion in water are preferred.

  The substrate in the present invention is produced, for example, as a substrate formed on a base material obtained by partitioning (patterning) a hydrophilic region composed of a crosslinked body and a hydrophobic region where the base material is exposed. It can be suitably used as a substrate. That is, a cell culture substrate on which cells can be arranged in a desired pattern can be obtained by arranging cells only in the hydrophobic region and not adhering cells to the hydrophilic region.

  As a method of using the substrate in the present invention as a substrate for culturing embryonic stem cells, a normal method for culturing embryonic stem cells can be applied without limitation. For example, by placing a cell culture medium on the substrate of the present invention, seeding desired embryonic stem cells in the cell culture medium, and then applying the culture conditions selected according to the desired embryonic stem cells, Desired embryonic stem cells can be selectively placed in the upper hydrophobic region.

  There is no restriction | limiting in particular in the shape of the hydrophilic region and hydrophobic region in this invention, According to the objective, it can select suitably. For example, the hydrophobic region can be formed in a circular shape, a polygonal shape including a triangle, an oval shape, a stripe shape, or the like. Moreover, there is no restriction | limiting in particular also about the magnitude | size of a hydrophilic region and a hydrophobic region, According to the objective, it can select suitably.

When the substrate in the present invention is used as a culture substrate for embryonic stem cells, the size when the hydrophobic region is formed in a circle is preferably 5 to 1000 μm in diameter, and preferably 50 to 500 μm. More preferred. Further, the width of the hydrophilic region separating adjacent hydrophobic regions is preferably 50 to 500 μm, and more preferably 100 to 200 μm. Furthermore, the thickness of the layer in the hydrophilic region is preferably 10 nm to 1000 nm, and more preferably 10 nm to 500 nm.
The shape and size of the hydrophilic region and the hydrophobic region in the present invention can be controlled easily and with high accuracy by making the exposure process in the above-described curing step an exposure process through a mask.

The substrate in the present invention can be suitably used as a cell culture substrate for forming spheroids derived from embryonic stem cells. Since the substrate in the present invention has a hydrophilic region composed of the branched polyalkylene glycol derivative, a hydrophobic region patterned with high precision can be formed. Further, the hydrophilic region has extremely good cell non-adhesive stability over time, and can maintain a spheroid exhibiting good functionality over a long period of time. That is, a function specific to each parenchymal cell forming a spheroid can be maintained. For example, hepatocytes have a high level of hepatocyte function (for example, high level of albumin production ability and drug metabolic activity), pancreatic β cells have an insulin secretion function, and cardiomyocytes have a pulsatile motor function. Etc. are maintained over a long period of time.
The substrate on which such spheroids are formed can be used, for example, for screening for environments or substances that can affect various cells. The influence on various cells can be evaluated by monitoring changes in the spheroid morphology and production ability of products (eg, albumin in hepatocytes, metabolites of model drugs, etc.).

  In the substrate in the present invention, the hydrophilic region is formed as a branched polyalkylene glycol derivative using a compound having 4 or more polyalkylene glycol groups having a polymerization degree of 5 to 1000 and having a polymerizable substituent at the terminal. Thus, the temporal stability of the formed spheroids is remarkably improved.

When using the board | substrate in this invention as a board | substrate for spheroid formation, as a magnitude | size at the time of forming a hydrophobic region in circular shape, it can be set as 5-1000 micrometers in diameter. Preferably it is 50-500 micrometers, More preferably, it is 100-300 micrometers. Further, the width of the hydrophilic region separating adjacent hydrophobic regions is preferably 50 to 500 μm, and more preferably 100 to 200 μm. The thickness of the layer in the hydrophilic region is preferably 10 nm to 1000 nm, and more preferably 10 nm to 500 nm.
By configuring the hydrophilic region and the hydrophobic region with the above-mentioned sizes, highly functional spheroids can be created more efficiently and can be maintained for a longer period of time.

When using the board | substrate in this invention as a board | substrate for spheroid formation, a feeder cell can be previously arrange | positioned to the said hydrophobic area | region as needed. That is, in the present invention, a feeder cell layer can be formed in the hydrophobic region on the substrate of the present invention, and embryonic stem cells can be cultured on the formed feeder cell layer.
By forming the feeder cell layer in advance, the formation of embryonic stem cell-derived spheroids proceeds more efficiently, and the stability of the spheroids is improved. Examples of the feeder cells include COS-1 cells, vascular endothelial cells (for example, “Human Umbilical Vein Endothelial Cells” manufactured by Dainippon Pharmaceutical), fibroblasts, and the like.

  Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto. Unless otherwise specified, “%” is based on mass, and the average molecular weight is a weight average molecular weight.

First, the branched polyalkylene glycol derivative used in the present invention and a substrate produced using the photosensitive composition containing the polyalkylene glycol will be specifically described.
(Reference Example 1)
-Synthesis of branched polyalkylene glycol derivative (4PA20K)-
4-Azido-benzoic acid 12 g (93.6 mmol) was dissolved in 40 mL of thionyl chloride and heated to reflux for 1.5 hours. The reaction mixture was concentrated under reduced pressure, a small amount of hexane was added, and the mixture was again concentrated under reduced pressure, and then dried under vacuum, and 9.3 g (51.2 mmol, yield 70) of the desired 4-azido-benzoic acid chloride as a white solid. %).
¹ H-NMR (CDCl ₃ ) δ: 8.11-8.15 (2H, m), 7.11-7.16 (2H, m).

Next, 81 mg (0.8 mmol) of triethylamine and 147 mg (1.2 mol) of 4-dimethylaminopyridine were added to 3 mL of dichloromethane (dehydrated), followed by stirring while cooling in an ice bath. To this solution was added dropwise a dichloromethane (dehydrated) solution (5 mL) of 363 mg (2.0 mmol, 5 molar equivalents to the terminal OH group of multi-arm PEG) of 4-azido-benzoic acid chloride obtained above. After stirring for 5 minutes, the reaction vessel was shielded from light, and multi-arm PEG (SUNBRIGHT (registered trademark) PTE-20000 manufactured by NOF Corp., pentaerythritol derivative having four polyethylene glycol groups) 2 g (0.1 mmol) Of dichloromethane (dehydrated) (20 mL) was slowly added dropwise. The reaction solution was removed from the ice bath and stirred as such for 18 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and suspended by adding benzene, the salt was removed by filtration, and then concentrated again under reduced pressure. The process of dissolving the crude product in a small amount of benzene and dripping it into isopropyl ether cooled to 0 ° C. and collecting the resulting precipitate by filtration was repeated three times, and the resulting white solid was dried under reduced pressure. As a result, 1.74 g (yield 85%) of a branched polyalkylene glycol derivative (4PA20K) was obtained.
The substitution ratio of the terminal hydroxyl group to the polymerizable substituent calculated from the integral ratio of ¹ H-NMR was 85%.
¹ H-NMR (CDCl ₃ ) δ: 8.05 (8H, d, J = 8.6 Hz), 7.07 (8H, d, J = 8.6 Hz), 4.46 (8H, t, J = 4.9 Hz), 3.89-3.39 ( 2145H, m).

  A branched polyalkylene glycol derivative was synthesized in the same manner as Reference Example 1, except that the multi-arm PEG shown in Table 1 below was used instead of PTE-20000 as the multi-arm PEG. The yield, properties, etc. are shown in Table 1.

(Reference Example 2)
5-Amino-salicylic acid (15.3 g, 0.1 mol) was suspended in a mixed solution of distilled water (80 mL) and concentrated hydrochloric acid (20 mL), and stirred at room temperature for 30 minutes. After cooling the mixed solution in an ice bath, 10 mL of an aqueous solution of 6.9 g (0.1 mol) of sodium nitrite was added dropwise at such a rate that the temperature of the reaction solution did not exceed 5 ° C., and the mixture was stirred for 1 hour. did. Subsequently, 30 mL of an aqueous solution of 7.15 g (0.11 mol) of sodium azide was added dropwise at a rate such that the temperature of the reaction solution did not exceed 10 ° C. While removing the ice bath and returning to room temperature, the mixture was vigorously stirred until no bubbles were generated. The produced precipitate was collected by filtration, and the precipitate was further washed with distilled water. The obtained solid was air-dried in a dark place and then completely dried under reduced pressure to obtain 12.0 g (67.0 mmol, yield = 67%) of 5-azido-salicylic acid as a white solid.
¹ H-NMR (DMSO-d ₆ ) δ: 11.14 (1H, bs), 7.41 (1H, d, J = 3.0 Hz), 6.88 (1H, dd, J = 8.5, 3.0 Hz), 6.68 (1H, d , J = 8.4 Hz).

5 g (27.9 mmol) of 5-azido-salicylic acid obtained was suspended in 50 mL of thionyl chloride and stirred at 70 ° C. for 1 hour. The reaction mixture was allowed to cool to room temperature, excess thionyl chloride was removed under reduced pressure, and a red solid of 5-azido-salicylic acid chloride was obtained quantitatively.
¹ H-NMR (DMSO-d ₆ ) δ: 7.39 (1H, d, J = 2.9 Hz), 7.26 (1H, dd, J = 8.8, 2.9 Hz), 7.00 (1H, d, J = 8.8 Hz).

Next, 81 mg (0.8 mmol) of triethylamine and 147 mg (1.2 mol) of dimethylaminopyridine were added to 3 mL of dichloromethane (dehydrated), followed by stirring while cooling in an ice bath. To this solution was added dropwise a solution (5 mL) of 5-azido-salicylic acid chloride obtained above in 358 mg (2 mmol, 5 molar equivalents relative to the terminal OH group of multi-arm PEG) in dichloromethane (dehydrated), and the mixture was stirred for 5 minutes. Then, the reaction vessel was shielded from light, and 2 g (0.1 mmol) of dichloromethane (dehydration) of multi-arm PEG (SUNBRIGHT (registered trademark) PTE-20000, a compound having four polyethylene glycol groups, manufactured by NOF Corporation) was used. The solution (20 mL) was slowly added dropwise. The reaction solution was removed from the ice bath and stirred as such for 18 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and suspended by adding benzene, the salt was removed by filtration, and then concentrated again under reduced pressure. The process of dissolving the crude product in a small amount of benzene and dripping it into isopropyl ether cooled to 0 ° C. and collecting the resulting precipitate by filtration was repeated three times, and the resulting white solid was dried under reduced pressure. As a result, 1.77 g (yield: 85%) of a branched polyalkylene glycol derivative (4PB20K) was obtained.
The substitution ratio of the terminal hydroxyl group to the polymerizable substituent calculated from the integral ratio of ¹ H-NMR was 85%.
¹ H-NMR (CDCl ₃ ) δ: 8.05 (8H, d, J = 8.6 Hz), 7.07 (8H, d, J = 8.6 Hz), 4.46 (8H, t, J = 4.9 Hz), 3.89-3.39 ( 2145H, m).

(Reference Example 3)
In Reference Example 2, in the same manner as in Example 4 except that HGEO-20000 (manufactured by NOF Corporation, hexaglycerin derivative having 8 polyethylene glycol groups) was used instead of PTE-20000 as multi-arm PEG, 1.72 g (yield 85%) of the branched polyalkylene glycol derivative (8PB20K) as the target product was obtained.
The substitution ratio of the terminal hydroxyl group to the polymerizable substituent calculated from the integral ratio of ¹ H-NMR was 85%.
¹ H-NMR (CDCl ₃ ) δ: 8.05 (8H, d, J = 8.6 Hz), 7.07 (8H, d, J = 8.6 Hz), 4.46 (8H, t, J = 4.9 Hz), 3.89-3.39 ( 2145H, m).

(Reference Example 4)
18.1 g (0.1 mol) of 5-amino-2-nitrobenzoic acid was suspended in a mixed solution of 80 mL of distilled water and 20 mL of concentrated hydrochloric acid, and stirred at room temperature for 30 minutes. After cooling the mixed solution in an ice bath, 10 mL of an aqueous solution of 6.9 g (0.1 mol) of sodium nitrite was added dropwise at such a rate that the temperature of the reaction solution did not exceed 5 ° C., and the mixture was stirred for 1 hour. did. Subsequently, 30 mL of an aqueous solution of 7.15 g (0.11 mol) of sodium azide was added dropwise at a rate such that the temperature of the reaction solution did not exceed 10 ° C. While removing the ice bath and returning to room temperature, the mixture was vigorously stirred until no bubbles were generated. The produced precipitate was collected by filtration, and the precipitate was further washed with distilled water. The obtained solid was air-dried in the dark and then completely dried under reduced pressure to obtain 19.2 g (92.4 mmol, yield = 92%) of 5-azido-2-nitrobenzoic acid as a white solid. It was.
¹ H-NMR (DMSO-d ₆ ) δ: 13.99 (1H, bs), 8.09-8.06 (1H, m), 7.45-7.42 (2H, m).

The obtained 5-azido-2-nitrobenzoic acid 1.0 g (4.8 mmol) was suspended in 10 mL of thionyl chloride and stirred at 70 ° C. for 1 hour. The reaction mixture was allowed to cool to room temperature, excess thionyl chloride was removed under reduced pressure, and a red solid of 5-azido-salicylic acid chloride was obtained quantitatively.
¹ H-NMR (DMSO-d ₆ ) δ: 8.10-8.07 (1H, m), 7.46-7.42 (2H, m).

Next, 81 mg (0.8 mmol) of triethylamine and 147 mg (1.2 mol) of dimethylaminopyridine were added to 3 mL of dichloromethane (dehydrated), followed by stirring while cooling in an ice bath. To this solution was added dropwise a dichloromethane (dehydrated) solution (5 mL) of 417 mg (2 mmol, 5 molar equivalents relative to the terminal OH group of multi-arm PEG) of 5-azido-2-nitrobenzoic acid chloride obtained above, After stirring for 5 minutes as it was, the reaction vessel was shielded from light, and 2 g (0.1 mmol) of multi-arm PEG (SUNBRIGHT (registered trademark) PTE-20000, a compound having four polyethylene glycol groups, manufactured by NOF Corporation) was added. Dichloromethane (dehydrated) solution (20 mL) was slowly added dropwise. The reaction solution was removed from the ice bath and stirred as such for 18 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and suspended by adding benzene, the salt was removed by filtration, and then concentrated again under reduced pressure. The process of dissolving the crude product in a small amount of benzene and dripping it into isopropyl ether cooled to 0 ° C. and collecting the resulting precipitate by filtration was repeated three times, and the resulting white solid was dried under reduced pressure. A branched polyalkylene glycol derivative (4PC20K) 1.81 (yield 85%) was obtained.
The substitution ratio of the terminal hydroxyl group to the polymerizable substituent calculated from the integral ratio of ¹ H-NMR was 85%.
¹ H-NMR (CDCl ₃ ) δ: 8.05 (8H, d, J = 8.6 Hz), 7.07 (8H, d, J = 8.6 Hz), 4.46 (8H, t, J = 4.9 Hz), 3.89-3.39 ( 2145H, m).

(Reference Example 5)
In Reference Example 4, in the same manner as Reference Example 4 except that HGEO-20000 (manufactured by NOF Corporation, hexaglycerin derivative having 8 polyethylene glycol groups) was used as the multi-arm PEG instead of PTE-20000, 1.71 g (yield 85%) of the branched polyalkylene glycol derivative (8PC20K) as the target product was obtained.
The substitution ratio of the terminal hydroxyl group to the polymerizable substituent calculated from the integral ratio of ¹ H-NMR was 85%.
¹ H-NMR (CDCl ₃ ) δ: 8.05 (8H, d, J = 8.6 Hz), 7.07 (8H, d, J = 8.6 Hz), 4.46 (8H, t, J = 4.9 Hz), 3.89-3.39 ( 2145H, m).

(Reference Example 6)
A benzene (dehydrated) solution (5 mL) of 181 mg of acryloyl chloride (2.0 mmol, 5 molar equivalents relative to the terminal OH group of the multi-arm PEG) was dropped, and the stirring was continued for 5 minutes. A benzene (dehydrated) solution (20 mL) of 2 g (0.1 mmol) of multi-arm PEG (SUNBRIGHT (registered trademark) PTE-20000, 4 pentaerythritol derivatives having four polyethylene glycol groups) manufactured by NOF Corporation was slowly added dropwise. The reaction solution was removed from the ice bath and stirred as such for 18 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and suspended by adding benzene, the salt was removed by filtration, and then concentrated again under reduced pressure. The process of dissolving the crude product in a small amount of benzene and dripping it into isopropyl ether cooled to 0 ° C. and collecting the resulting precipitate by filtration was repeated three times, and the resulting white solid was dried under reduced pressure. As a result, 1.74 g (yield 85%) of a branched polyalkylene glycol derivative (4PD20K) was obtained.
The substitution ratio of the terminal hydroxyl group to the polymerizable substituent calculated from the integral ratio of ¹ H-NMR was 85%.
¹ H-NMR (CDCl ₃ ) δ: 8.05 (8H, d, J = 8.6 Hz), 7.07 (8H, d, J = 8.6 Hz), 4.46 (8H, t, J = 4.9 Hz), 3.89-3.39 ( 2145H, m).

(Reference Example 7)
In Reference Example 6, in the same manner as Reference Example 6 except that HGEO-20000 (manufactured by NOF Corporation, hexaglycerin derivative having 8 polyethylene glycol groups) was used as the multi-arm PEG instead of PTE-20000. As a result, 1.74 g (yield: 85%) of a branched polyalkylene glycol derivative (8PD20K) as a target product was obtained.
The substitution ratio of the terminal hydroxyl group to the polymerizable substituent calculated from the integral ratio of ¹ H-NMR was 85%.
¹ H-NMR (CDCl ₃ ) δ: 8.05 (8H, d, J = 8.6 Hz), 7.07 (8H, d, J = 8.6 Hz), 4.46 (8H, t, J = 4.9 Hz), 3.89-3.39 ( 2145H, m).

(Reference Example 8)
-Fabrication of substrate-
The following operations were all performed in the yellow room.
The branched polyalkylene glycol derivative (multi-arm PEG-azide: 4PA20K) prepared in Reference Example 1 was dissolved in toluene to prepare a 4PA20K toluene solution (1%) as photosensitive composition A. Poly-L-lysine-coated slide glass (manufactured by Matsunami Glass Industry Co., Ltd., white cut-off NO.1 slide glass round shape 21 mmΦ, hereinafter abbreviated as “PLL-coated glass”) is used as a base material. Then, after 110 μL of the photosensitive composition A was dropped, a film was formed by a spin coating method (500 rpm × 5 seconds + 3000 rpm × 20 seconds + 6000 rpm × 1 second) and left to dry at room temperature. A quartz glass photomask (with a large number of circular patterns with a diameter of 200 μm) is adhered to this, and after exposure for 40 seconds using a high-pressure mercury lamp (200 W), it is washed with deionized water (development process: Running water for 15 seconds + immersion for 20 minutes). The substrate was dried at room temperature to obtain a substrate having a hydrophilic crosslinked body finely processed on the surface. When the surface of the said board | substrate was observed with the phase-contrast optical microscope (magnification x100), it has confirmed that the favorable micro pattern was formed with high precision.

(Reference Example 9)
In Reference Example 8, instead of PLL-coated glass as a substrate, aminopropylsilane-coated glass (manufactured by Matsunami Glass Industry Co., Ltd. APS Coat No. 1 Cover Glass Round 21 mmΦ, hereinafter abbreviated as “APS Coated Glass”). A substrate was produced in the same manner as in Reference Example 8 except that it was used, and a substrate having a hydrophilic cross-linked body finely processed on its surface was obtained. When the surface of the substrate was observed with a phase-contrast optical microscope, it was confirmed that a good micropattern was formed with high accuracy.

(Reference Example 10)
In Reference Example 8, a substrate was prepared in the same manner as in Reference Example 8 except that MAS-coated glass (manufactured by Matsunami Glass Industry Co., Ltd.) was used instead of PLL-coated glass as the base material, and the substrate was finely processed on the surface. A substrate having a hydrophilic cross-linked product was obtained. When the surface of the substrate was observed with a phase-contrast optical microscope, it was confirmed that a good micropattern was formed with high accuracy.

(Reference Example 11)
In Reference Example 8, a substrate was prepared in the same manner as in Reference Example 8 except that “collagen-coated glass” in which collagen was further coated on the PLL coat was used instead of PLL-coated glass as a base material, and the surface was finely formed. A substrate having a processed hydrophilic crosslinked body was obtained. When the surface of the said board | substrate was observed with the phase-contrast optical microscope (magnification x100), it has confirmed that the favorable micro pattern was formed with high precision.
The collagen-coated glass was dropped on a PLL-coated glass with 400 μL of 0.1% aqueous solution of porcine type I collagen (manufactured by Nippon Ham Co., Ltd.) and spin-coated (350 rpm × 5 seconds + 500 rpm × 5 seconds + 1000 rpm × 10). Second + 1500 rpm × 10 seconds + 6000 rpm × 1 second), and the process of drying at room temperature was repeated twice.

(Reference Example 12)
In Reference Example 8, a substrate was prepared in the same manner as in Reference Example 8 except that “gelatin-coated glass” in which gelatin was further coated on the PLL coat was used instead of PLL-coated glass as the base material, and the surface was made fine. A substrate having a processed hydrophilic crosslinked body was obtained. When the surface of the substrate was observed with a phase-contrast optical microscope, it was confirmed that a good micropattern was formed with high accuracy.
As the gelatin-coated glass, PLL coated glass is used, and 400 μL of a 0.1% solution of gelatin (manufactured by Nitta Gelatin Co., Ltd.) is dropped, and spin coating (350 rpm × 5 seconds + 500 rpm × 5 seconds + 1000 rpm × 10 seconds + 1500 rpm ×) The film was formed at 10 seconds + 6000 rpm × 1 second) and then dried at room temperature.

(Reference Example 13)
In Reference Example 11, the method for producing the collagen-coated glass was as follows: the PLL-coated glass was immersed in a 0.02% aqueous solution of porcine type I collagen (manufactured by Nippon Ham Co., Ltd.) for 3 hours, washed with running deionized water, and dried. A substrate was produced in the same manner as in Reference Example 11 except that the method was changed. When the surface of the substrate was observed with a phase-contrast optical microscope, it was confirmed that a good micropattern was formed with high accuracy.

(Reference Example 14)
In Reference Example 8, instead of Photosensitive Composition A, the same method as Reference Example 8 was used, except that Photosensitive Composition B in which the concentration of multi-arm PEG-azide (4PA20K) was 0.5% was used. A substrate was produced. When the surface of the substrate was observed with a phase-contrast optical microscope, it was confirmed that a good micropattern was formed with high accuracy.

(Reference Example 15)
In Reference Example 8, instead of 4PA20K as the branched polyalkylene glycol derivative, a substrate was prepared in the same manner as in Reference Example 8 except that the various branched polyalkylene glycol derivatives synthesized in Reference Examples 2 to 5 were used. When the surface of the substrate was observed with a phase-contrast optical microscope, it was confirmed that a good micropattern was formed with high accuracy.

(Example 16)
In Reference Example 8, instead of the branched polyalkylene glycol derivative (4PA20K), the branched polyalkylene glycol derivative (4PC20K) synthesized in Reference Example 4 was used, and the exposure condition was 3 seconds with a high-pressure mercury lamp (200 W). A substrate was produced in the same manner as in Reference Example 8. When the surface of the said board | substrate was observed with the phase-contrast optical microscope (magnification x100), it has confirmed that the favorable micro pattern was formed with high precision.

(Reference Example 17)
In Reference Example 8, instead of the branched polyalkylene glycol derivative (4PA20K), the branched polyalkylene glycol derivative (4PC20K) synthesized in Reference Example 4 was used, and the exposure conditions were filtered on a photomask (manufactured by Sigma Koki Co., Ltd.). , UTVAF36U) and a substrate was produced in the same manner as in Example 10 except that the exposure was performed with a high-pressure mercury lamp (200 W) for 10 seconds. When the surface of the said board | substrate was observed with the phase-contrast optical microscope (magnification x100), it has confirmed that the favorable micro pattern was formed with high precision.

(Reference Example 18)
In Reference Examples 9 to 13, instead of the branched polyalkylene glycol derivative (4PA20K), the branched polyalkylene glycol derivative (4PB20K) synthesized in Reference Example 2 or the branched polyalkylene glycol derivative (4PC20K) synthesized in Reference Example 4 was used. The same exposure method as in Reference Examples 9 to 13 except that a filter (Sigma TV Co., UTVAF36U) was placed on the photomask and the exposure was performed for 10 seconds with a high-pressure mercury lamp (200 W). A substrate was prepared. When the surface of the substrate was observed with a phase-contrast optical microscope, it was confirmed that a good micropattern was formed with high accuracy.

(Reference Example 19)
The branched polyalkylene glycol derivatives (4PD20K, 8PD20K) prepared in Reference Example 6 and Reference Example 7 were dissolved in toluene so that the concentration of IRGACURE2959 as a photopolymerization initiator was 0.05%. A photosensitive composition D was prepared. Poly-L-lysine-coated slide glass (manufactured by Matsunami Glass Industry Co., Ltd., white cut-off NO.1 slide glass round shape 21 mmΦ, hereinafter abbreviated as “PLL-coated glass”) is used as a base material. After 110 μL of the photosensitive composition D was dropped, a film was formed by a spin coating method (500 rpm × 5 seconds + 3000 rpm × 20 seconds + 6000 rpm × 1 second), and left to dry at room temperature. A quartz glass photomask (having a large number of circular patterns with a diameter of 100 μm arranged thereon) is adhered to this, and after exposure for 40 seconds using a high-pressure mercury lamp (200 W), it is washed with deionized water (development process: Running water for 15 seconds + immersion for 20 minutes). The substrate was dried at room temperature to obtain a substrate having a hydrophilic crosslinked body finely processed on the surface. When the surface of the substrate was observed with a phase-contrast optical microscope (magnification x100), it was confirmed that a good micropattern was formed with high accuracy when any branched polyalkylene glycol derivative was used. It was.

Example 1
-Preparation of embryonic stem cells-
An embryonic stem cell (COL1-GFP) derived from a transgenic mouse recombined with a GFP gene whose expression is controlled by a type I collagen α promoter was expressed by FASEB J. et al. , 21, 1777-1787 (2007), and subcultured using the following LIF (+) medium.
LIF (+) medium: DMEM (manufactured by Sigma-Aldrich) 500 mL, FBS 75 mL, AB 5 mL, GlutaMAX 5 mL, non-essential amino acid solution 5 mL, α-LIF 50 μL, 2-mercaptoethanol 3.8 μL.
Similarly, embryonic stem cells (COL2-GFP) derived from WT mice recombined with a GFP gene whose expression was controlled by a type II collagen promoter were prepared, and subcultured in the same manner using LIF (+) medium. It was.

~ Spheroid formation ~
Embryonic stem cells (COL1-GFP) that have been subcultured in LIF (+) medium were treated with trypsin (1X), and after centrifugation, the cells were dispersed in LIF (+) medium.
After the preparation in Reference Example 8, the sterilized substrate is set on the bottom of a 12-well plate manufactured by FALCON, LIF (+) medium is added as a medium, and embryonic stem cells are dispersed in the LIF (+) medium. (COL1-GFP) was seeded at a cell concentration of 1.5 × 10 ⁵ cells / mL. Next, the medium was replaced with the following LIF (−) ATRA (+) medium, and the cells were cultured for 6 days at 37 ° C. under culture conditions 5% CO ₂ .
LIF (−) ATRA (+) medium: In the above LIF (+) medium, 50 μL of retinoic acid (ATRA) was added instead of 50 μL of α-LIF.

  A large number of embryonic stem cell-derived spheroids (cell aggregates) having a uniform size arranged in an array corresponding to the hydrophobic regions formed on the substrate within one day were obtained. A photomicrograph (magnification x400) one day after the start of culture is shown in FIG. 1, and a photomicrograph after six days (magnification x400) is shown in FIG.

(Example 2)
In Example 1, instead of using a circular pattern with a diameter of 100 μm and a circular pattern with a diameter of 500 μm instead of a circular pattern with a diameter of 200 μm as the substrate, embryonic stem cells ( COL1-GFP) was cultured.
When a substrate on which a circular pattern having a diameter of 100 μm was formed, spheroids derived from embryonic stem cells having a uniform size as in Example 1 and arranged in an array were obtained. When a 500 μm circular pattern was used, embryonic stem cell-derived spheroids were formed in the hydrophobic region at the center of the substrate.

(Example 3)
In Example 1, embryonic stem cells (COL1-GFP) were cultured in the same manner as in Example 1 except that the substrates prepared in Reference Examples 9 to 19 were used instead of the substrate prepared in Reference Example 8.
Whichever substrate was used, as in Example 1, a large number of embryonic stem cell-derived spheroids having a uniform size and arranged in an array were obtained.

(Comparative Example 1)
In Example 1, embryonic stem cells (COL1-GFP) were cultured in the same manner as in Example 1 except that a petri dish was used instead of the substrate prepared in Reference Example 8.
A photomicrograph (magnification × 400) of the formed lung-like body (EB) after 6 days of culture is shown in FIG.

Example 4
Embryonic stem cells (COL1-GFP) subcultured in LIF (+) medium were seeded on the substrate prepared in Reference Example 1. The medium was replaced with LIF (−) ATRA (+) when seeded on the substrate. Cells immediately before seeding on the substrate were set to 0 day, and cultured cells were collected 1 day and 6 days after seeding, and alkaline phosphatase (ALP) activity was measured by a conventional method.
All cultured cells were collected by trypsin treatment, washed with PBS (-) (counting the number of cells at this time), added with 1% SDS, and allowed to stand for 5 minutes. This was used as a sample. In a 96-well plate, 100 μL of the substrate solution was previously added, and 20 μL of the sample solution was added thereto. From this point of time, the mixture was incubated at 37 ° C. for 15 minutes, and the reaction was stopped by adding 80 μL of a reaction stop solution. Thereafter, the absorbance at 405 nm was measured with a plate reader, the p-nitrophenol concentration was calculated from a calibration curve prepared in advance, and ALP activity was determined. The results are shown in FIG.

  FIG. 4 shows that ALP activity decreases with time by culturing embryonic stem cells on the substrate according to the present invention.

(Example 5)
-Evaluation of marker gene expression by RT-PCR-
Embryonic stem cells (COL1-GFP) subcultured in LIF (+) medium were seeded on the substrate prepared in Reference Example 1. The medium was replaced with LIF (−) ATRA (+) medium when seeded on the substrate. The cells immediately before seeding on the substrate were set to 0 day, and the cultured cells were collected 1 day, 5 days and 7 days after seeding. The expression levels of the undifferentiated marker Oct4, the ectoderm marker NCAM, the mesoderm marker Flk-1, and the endoderm marker AFP were evaluated according to the following protocol by a conventional method using RT-PCR. The results are shown in FIGS.

RNA extraction: The medium on the substrate was removed, and after washing twice with PBS (−), 500 μL of TRIzol was added and allowed to stand at room temperature for 5 minutes. After pipetting well, it was transferred to an Eppendorf tube and stored at -80 ° C.
RNA purification: 100 μL of chloroform was added to 500 μL of TRIzol, and the mixture was thoroughly stirred by hand, followed by centrifugation at 12,000 rpm for 5 minutes at 4 ° C. Subsequently, 200 μL of the supernatant after centrifugation was added to another Eppendorf tube containing 400 μL of IPA, mixed by inversion, and centrifuged at 15000 rpm for 10 minutes at 4 ° C. The supernatant was removed by decantation and further laid down on a Kimwipe to completely remove the supernatant. After adding 500 μL of 75% EtOH and stirring with VORTEX, centrifugation was performed at 15000 rpm for 5 minutes at 4 ° C. The supernatant was again removed by decantation, and the supernatant was further completely removed with an eppen, and 20 μL of RNA Free Water was added and dissolved.
RT: Using Takara ExScript RT reagent Kit, the operation was performed according to the attached protocol.
PCR: Setting conditions are as follows. 94 ° C., 5 min; 94 ° C., 30 sec; 58 ° C., 30 sec; 72 ° C., 30 sec; 72 ° C., 7 min; 4 ° C., ∞;

  From FIG. 5 to FIG. 8, it can be seen that the expression level of Oct4 is decreased and the expression level of the three germ layer markers is increased by culturing embryonic stem cells over time. Furthermore, it can be seen that the expression level of the endoderm marker is prominently increased.

(Example 6)
-Differentiation induction using polymer micelles-
As a non-viral gene vector, a diethylenetriamine adduct (hereinafter referred to as “PEG-DET”) of a polyethylene glycol-poly (β-benzyl-L-aspartate) -Ac copolymer described in JP-A-2004-352972. Was used to prepare polymer micelles containing Runx2 and caALK6 genes.
Note that the Runx2 and caALK6 genes are FASEB J. et al. , 21, 1777-1787 (2007). In addition, these genes are known to have the ability to induce cartilage differentiation on human dermal fibroblasts.
The preparation conditions of polymer micelles and the addition conditions to cultured cells are described in Mol. Ther. 15 (9), 1655-1662 (2007).

A substrate (spheroid array) in which embryonic stem cell-derived spheroids are arranged in an array is prepared in the same manner as in Example 1, and polymer micelles containing Runx2 and caALK6 genes are added thereto to add 5% CO _2. When cultured at 37 ° C. for 5 days, GFP-derived fluorescence could be observed.

(Example 7)
-Differentiation induction using polymer micelles-
As a non-viral gene vector, a diethylenetriamine adduct (hereinafter referred to as “PEG-DET”) of a polyethylene glycol-poly (β-benzyl-L-aspartate) -Ac copolymer described in JP-A-2004-352972. Was used to prepare polymer micelles containing the Sox5, Sox6, and Sox9 genes.
The Sox5, Sox6, and Sox9 genes were prepared according to the method described in Japanese Patent Application Laid-Open No. 2004-267052. In addition, these genes are known to have the ability to induce cartilage differentiation on human dermal fibroblasts.
The preparation conditions of polymer micelles and the addition conditions to cultured cells are described in Mol. Ther. 15 (9), 1655-1662 (2007).

In Example 1, embryonic stem cell-derived spheroids were arranged in an array in the same manner as in Example 1 except that embryonic stem cells (COL2-GFP) were used instead of embryonic stem cells (COL1-GFP). A substrate (spheroid array) was produced.
When polymer micelles containing Sox5, Sox6, and Sox9 genes were added thereto and cultured at 5% CO ₂ and 37 ° C. for 5 days, GFP-derived fluorescence could be observed.

(Example 8)
~ Induction of differentiation by low molecular weight compounds ~
In Example 1, embryonic stem cell-derived spheroids were arranged in an array in the same manner as in Example 1 except that embryonic stem cells (COL2-GFP) were used instead of embryonic stem cells (COL1-GFP). A substrate (spheroid array) was produced.
To this, a low molecular compound TH (see Biochem. Biophys. Res. Com., 357 (4), 854-860 (2007)) was added and cultured at 5% CO ₂ at 37 ° C. for 5 days. The origin fluorescence could be observed.

From the above results, it was possible to produce a large amount of embryonic stem cell-derived spheroids having a uniform size by culturing embryonic stem cells on the substrate in the present invention.
Moreover, the spheroid array by which the embryonic stem cell origin spheroid was formed in the hydrophobic area | region was obtained by forming the hydrophobic area | region arrange | positioned at the array form on the board | substrate in this invention.
Furthermore, by using the spheroid array of the present invention, drug screening and induction of embryonic stem cell differentiation were possible.

It is an enlarged photograph of embryonic stem cells after 1 day of culture. It is an enlarged photograph of embryonic stem cells after 6 days in culture. It is an enlarged photograph of an embryoid body after 6 days of culture. It is a figure which shows the change of ALP activity. It is a figure which shows the expression level change of Oct4 gene. It is a figure which shows the expression level change of a NCAM gene. It is a figure which shows the expression level change of Flk-1 gene. It is a figure which shows the expression level change of an AFP gene.

Claims (8)

A branched polyalkylene glycol derivative having a base material, three or more polyalkylene glycol groups having a polymerizable substituent at the terminal and a trivalent or higher linking group bonded to the polyalkylene glycol group, disposed on the base material On a substrate including a plurality of hydrophilic regions and hydrophobic regions formed by curing a photosensitive composition containing
Seeding embryonic stem cells;
Culturing the seeded embryonic stem cells;
Forming spheroids derived from cultured embryonic stem cells;
A method for producing spheroids.   The method for producing a spheroid according to claim 1, wherein the branched polyalkylene glycol derivative has 4 or more polyalkylene glycol groups having a polymerization degree of 5 to 1000.   The spheroid production method according to claim 1 or 2, wherein the expression level of AFP as an endoderm marker is at least one of Flk-1 as a mesodermal marker and NCAM as an ectoderm marker Spheroids larger than the expression level. A branched polyalkylene glycol derivative having a base material, three or more polyalkylene glycol groups having a polymerizable substituent at the terminal and a trivalent or higher linking group bonded to the polyalkylene glycol group, disposed on the base material A substrate comprising a plurality of hydrophilic regions and hydrophobic regions formed in an array by curing a photosensitive composition containing
A plurality of embryonic stem cell-derived spheroids arranged in the hydrophobic region;
A spheroid array.   A drug screening method using the spheroid array according to claim 4.   The drug screening method according to claim 5, comprising bringing a drug encapsulated in a polymer micelle into contact with the spheroid. A branched polyalkylene glycol derivative having a base material, three or more polyalkylene glycol groups having a polymerizable substituent at the terminal and a trivalent or higher linking group bonded to the polyalkylene glycol group, disposed on the base material On a substrate including a plurality of hydrophilic regions and hydrophobic regions formed by curing a photosensitive composition containing
Seeding embryonic stem cells;
Culturing embryonic stem cells in the presence of a differentiation-inducing factor;
Obtaining differentiated cells from cultured embryonic stem cells;
A method for inducing differentiation of embryonic stem cells, comprising:   The method for inducing differentiation of embryonic stem cells according to claim 7, further comprising adding the differentiation-inducing factor in a state of being encapsulated in polymer micelles.