JP2018134063A - Cell scaffold material, cell model, and production method for cell scaffold material - Google Patents
Cell scaffold material, cell model, and production method for cell scaffold material Download PDFInfo
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Abstract
Description
本発明は、細胞足場材料、細胞モデル、及び細胞足場材料の製造方法に関する。 The present invention relates to a cell scaffold material, a cell model, and a method for producing a cell scaffold material.
近年、生体を構成している細胞は、細胞外マトリクス（Extracellular Matrix、以下ＥＣＭ）の化学・力学特性を認識し、細胞機能を変化させていることが明らかになってきた。細胞は、細胞表面のタンパク質を介してECMと結合、接着し、ECMの硬さを認識していると考えられているが、そのメカニズムの詳細は不明である。特に、力学特性に関しては、生体組織の弾性率が不均一であることや加齢とともに弾性率が変化することが報告されているが、このような特性変化が細胞・組織に及ぼす影響は明らかになっていない（非特許文献１）。例えば、中枢神経系を構成する神経細胞やグリア細胞は、疾患や外傷による損傷を受けると再生が困難な組織であることが知られており、その原因の一端が損傷部位周辺のECM力学特性の変化である可能性が示唆されているが、未だ明らかになっていない（非特許文献２）。上記の、中枢神経系の再生も含め、再生医療の分野では、組織再生の際に細胞の足場となる基材が必要となるため、その基材の力学特性が細胞組織に与える影響を解明することは、基材の材料設計に重要となる。 In recent years, it has become clear that cells constituting a living body recognize the chemical and mechanical properties of an extracellular matrix (hereinafter referred to as ECM) and change cell functions. Cells are thought to bind and adhere to ECM via cell surface proteins and recognize the hardness of ECM, but the details of the mechanism are unknown. In particular, with regard to mechanical properties, it has been reported that the elastic modulus of living tissue is non-uniform and the elastic modulus changes with age, but the effect of such property changes on cells and tissues is clearly evident (Non-Patent Document 1). For example, nerve cells and glial cells that make up the central nervous system are known to be tissues that are difficult to regenerate when damaged by disease or trauma, and one of the causes is the ECM mechanical properties around the damaged site. Although the possibility of a change has been suggested, it has not yet been clarified (Non-Patent Document 2). In the field of regenerative medicine, including the regeneration of the central nervous system described above, a base material that serves as a scaffold for cells is required for tissue regeneration. Therefore, the influence of the mechanical properties of the base material on the cellular tissue is elucidated. This is important for the material design of the substrate.
このような観点から、ポリジメチルシロキサン（Polydimethylsiloxane、PDMS）を代表とするエラストマや、水溶性高分子から構成されるハイドロゲルといった、ソフトマテリアルからなるフィルムの力学特性を制御し、フィルムの硬さに対する細胞応答を調べる方法が提案されている。今後は、加齢や、組織損傷などによる生体内の動的な力学特性変化を再現することで、組織再生の際に用いられる足場材料の力学特性をスクリーニングするためのin vitroモデルとして利用することが期待される。 From this point of view, we control the mechanical properties of films made of soft materials such as elastomers typified by polydimethylsiloxane (PDMS) and hydrogels composed of water-soluble polymers. Methods for examining cellular responses have been proposed. In the future, it will be used as an in vitro model to screen the mechanical properties of scaffold materials used in tissue regeneration by reproducing dynamic changes in mechanical properties in vivo due to aging and tissue damage. There is expected.
脳組織のようにＥＣＭの深さが組織構造の重要な指標となる場合など、細胞が認識可能なＥＣＭ力学特性の深さを明らかにする必要がある。そのためには、厚さが制御された足場材料が求められる。
従来、様々な弾性率の足場材料上で細胞培養が行われてきたが、生体組織の弾性率は不均一であるため、生体内での細胞のＥＣＭ力学特性認識をin vitroで再現・検討するためには、同一基板上で弾性率が異なる領域を有する足場材料を用いることが望ましい。
また、生体内では外的刺激などで弾性率が変化するのに対し、これまでの足場材料は弾性率を変化させることが困難であった。ＥＣＭの力学特性に対する細胞・組織の応答をより詳細に調べるためには、細胞培養下においてin situで弾性率を変化させることができる機能的な足場材料が求められる。
It is necessary to clarify the depth of ECM mechanical characteristics that can be recognized by cells, such as when the ECM depth is an important index of tissue structure as in brain tissue. For this purpose, a scaffold material with a controlled thickness is required.
Conventionally, cell culture has been carried out on scaffold materials with various elastic moduli, but because the elastic modulus of living tissue is not uniform, the recognition of ECM mechanical properties of cells in vivo is reproduced and examined in vitro. For this purpose, it is desirable to use a scaffold material having regions having different elastic moduli on the same substrate.
In addition, while the elastic modulus changes in vivo due to an external stimulus, it has been difficult to change the elastic modulus of conventional scaffold materials. In order to investigate the response of cells and tissues to the mechanical properties of ECM in more detail, a functional scaffold material capable of changing the elastic modulus in situ under cell culture is required.
このような問題を解決するために、本発明は、フィルム状であり、光照射により力学特性を変化させることが可能な細胞足場材料を提供する。また本発明は、フィルム状であり、弾性率が異なる領域を有する細胞足場材料を提供する。また本発明は、前記細胞足場材料と前記細胞足場材料で培養される細胞とを備える細胞モデルを提供する。また本発明では前記細胞足場材料の製造方法を提供する。 In order to solve such problems, the present invention provides a cell scaffold material that is film-like and capable of changing mechanical properties by light irradiation. The present invention also provides a cell scaffold material having a film-like region having different elastic moduli. The present invention also provides a cell model comprising the cell scaffold material and cells cultured with the cell scaffold material. The present invention also provides a method for producing the cell scaffold material.
本発明の一態様は、高分子化合物を含む網目構造を形成するフィルム状のゲルを備え、前記網目構造は、光照射により前記網目構造を構成する化学結合が生成又は切断されるものであり、前記ゲルへ光照射によって領域Ａと領域Ｂとが分けられたとき、前記領域Ａよりも前記領域Ｂの弾性率が低くなるよう変化する、細胞足場材料である。 One embodiment of the present invention includes a film-like gel that forms a network structure containing a polymer compound, and the network structure is a structure in which a chemical bond that forms the network structure is generated or broken by light irradiation. It is a cell scaffold material that changes so that the elastic modulus of the region B is lower than that of the region A when the region A and the region B are separated by light irradiation on the gel.
本発明の一態様は、高分子化合物を含む網目構造を有するフィルム状のゲルを備え、前記ゲルは領域Ａと領域Ｂとを有し、前記領域Ａよりも前記領域Ｂの弾性率が低い、細胞足場材料である。 One embodiment of the present invention includes a film-like gel having a network structure including a polymer compound, the gel includes a region A and a region B, and the elastic modulus of the region B is lower than that of the region A. Cell scaffolding material.
本発明の一態様は、上記細胞足場材料であって、前記高分子化合物が、ポリアクリルアミド系樹脂を含む。 One embodiment of the present invention is the cell scaffold material described above, wherein the polymer compound includes a polyacrylamide resin.
本発明の一態様は、上記細胞足場材料であって、前記ゲルが、光異性化構造を有し光照射によって二本鎖が解離及び／又は形成する核酸を含む。 One embodiment of the present invention is the cell scaffold material described above, wherein the gel includes a nucleic acid that has a photoisomerization structure and a double strand is dissociated and / or formed by light irradiation.
本発明の一態様は、上記細胞足場材料と、上記細胞足場材料で培養される細胞と、を備える、細胞モデルである。 One embodiment of the present invention is a cell model including the cell scaffold material and cells cultured with the cell scaffold material.
本発明の一態様は、上記細胞モデルであって、前記細胞が神経細胞及び／又はグリア細胞を含む。 One embodiment of the present invention is the above cell model, wherein the cell includes a nerve cell and / or a glial cell.
本発明の一態様は、高分子化合物を含む網目構造を形成するフィルム状のゲル、又は前記ゲルの構成材料を含有する足場材料組成物の一部の領域に光照射して領域Ａと領域Ｂとに分け、前記領域Ａよりも前記領域Ｂの弾性率を低下させる光照射工程を有し、前記網目構造は、光照射により前記網目構造を構成する化学結合が生成又は切断されるものである、細胞足場材料の製造方法である。 In one embodiment of the present invention, a film-like gel that forms a network structure including a polymer compound, or a partial region of a scaffold material composition containing a constituent material of the gel is irradiated with light, and regions A and B And a light irradiation step for lowering the elastic modulus of the region B than the region A, and the network structure is one in which chemical bonds constituting the network structure are generated or broken by light irradiation. This is a method for producing a cell scaffold material.
本発明の一態様は、上記細胞足場材料の製造方法であって、前記光照射工程の前に、光照射により自らを構成する化学結合が生成又は切断される成分を含む再膨潤液に、前記ゲルを含浸させる再膨潤工程を有する。 One aspect of the present invention is a method for producing the cell scaffold material described above, wherein the re-swelling liquid containing a component that generates or cleaves a chemical bond constituting itself by light irradiation before the light irradiation step, Having a re-swelling step to impregnate the gel.
本発明によれば、光照射により力学特性を変化させることが可能な細胞足場材料を提供できる。
また本発明によれば、弾性率が異なる領域を有する細胞足場材料を提供できる。
また本発明によれば、前記細胞足場材料と前記細胞足場材料で培養される細胞とを備える細胞モデルを提供できる。
また本発明によれば、前記細胞足場材料の製造方法を提供できる。
ADVANTAGE OF THE INVENTION According to this invention, the cell scaffold material which can change a mechanical characteristic by light irradiation can be provided.
Moreover, according to this invention, the cell scaffold material which has the area | region from which an elasticity modulus differs can be provided.
Moreover, according to this invention, a cell model provided with the said cell scaffold material and the cell cultured with the said cell scaffold material can be provided.
Moreover, according to this invention, the manufacturing method of the said cell scaffold material can be provided.
以下、本発明の細胞足場材料、細胞モデル、及び細胞足場材料の製造方法の実施形態を説明する。 Hereinafter, embodiments of the cell scaffold material, the cell model, and the method for producing the cell scaffold material of the present invention will be described.
≪細胞足場材料≫
（１）細胞足場材料の第一の実施形態
本実施形態の細胞足場材料は、高分子化合物を含む網目構造を形成するフィルム状のゲルを備え、前記網目構造は、光照射により網目構造を構成する化学結合が生成又は切断されるものであり、前記ゲルへ光照射によって 領域Ａと領域Ｂとが分けられたとき、前記領域Ａよりも前記領域Ｂの弾性率が低くなるよう変化するものである。
≪Cell scaffold materials≫
(1) First Embodiment of Cell Scaffold Material The cell scaffold material of this embodiment includes a film-like gel that forms a network structure containing a polymer compound, and the network structure forms a network structure by light irradiation. The chemical bond is generated or broken, and when the region A and the region B are separated by light irradiation to the gel, the elastic modulus of the region B changes to be lower than that of the region A. is there.
前記網目構造は、ゲルを構成する分散質の少なくとも一部として、実施形態に係るゲルに含有される。網目構造は、ゲルの分散質の少なくとも一部又は全部を構成可能で、光照射により網目構造を構成する化学結合が生成又は切断されるものあれば特に制限されるものではない。 The network structure is contained in the gel according to the embodiment as at least part of the dispersoid constituting the gel. The network structure is not particularly limited as long as it can form at least a part or all of the dispersoid of the gel and can generate or break chemical bonds constituting the network structure by light irradiation.
光照射により網目構造の化学結合が生成又は切断されることにより、光照射された部分の網目構造が生成又は変化する。これにより、光照射によって領域Ａと領域Ｂとが分けられたとき、領域Ａと領域Ｂとで、弾性率の値に違いが生じる。
領域Ａへの光照射により化学結合が生成されるとすると、領域Ａの網目構造の結合が密になり、領域Ａよりも領域Ｂの弾性率が低くなる。
領域Ｂへの光照射により化学結合が切断されるとすると、領域Ｂの網目構造の結合が疎になり、領域Ａよりも領域Ｂの弾性率が低くなる。
When the chemical bonds of the network structure are generated or broken by light irradiation, the network structure of the light irradiated portion is generated or changed. As a result, when the region A and the region B are separated by light irradiation, a difference occurs in the value of the elastic modulus between the region A and the region B.
If a chemical bond is generated by light irradiation to the region A, the network structure bond in the region A becomes dense, and the elastic modulus of the region B is lower than that of the region A.
If the chemical bond is broken by light irradiation to the region B, the network structure bond in the region B becomes sparse, and the elastic modulus of the region B is lower than that of the region A.
ここでの化学結合の種類としては、網目構造の形成に寄与するものであれば特に制限されず、例えば、共有結合、配位結合、イオン結合、水素結合等が挙げられる。 The type of chemical bond here is not particularly limited as long as it contributes to the formation of a network structure, and examples thereof include a covalent bond, a coordinate bond, an ionic bond, and a hydrogen bond.
領域Ａと領域Ｂとは、光照射の有無、強度、照射時間、波長などの種々の光照射条件により分けることができる。これらの光照射条件を選択することで、フィルムの形状や力学特性の制御が容易に行える。 Region A and region B can be divided according to various light irradiation conditions such as the presence / absence of light irradiation, intensity, irradiation time, and wavelength. By selecting these light irradiation conditions, the shape and mechanical properties of the film can be easily controlled.
光の波長は、ゲルを構成する成分の種類等に応じて適宜定めればよく、２００〜５００ｎｍ程度とできる。 What is necessary is just to determine the wavelength of light suitably according to the kind etc. of component which comprises a gel, and can be about 200-500 nm.
光照射により領域Ａと領域Ｂを分けることは、該領域に対して局所的に光照射を行うことにより実施できる。該領域に対し局所的に光を照射する方法は、特に制限されるものでなく、フォトマスクを利用する方法の他、光ファイバーやレーザーを用いて局所的に光を照射する方法等を用いることができる。 The region A and the region B can be separated by light irradiation by locally irradiating the region with light. The method of locally irradiating light to the region is not particularly limited, and a method of locally irradiating light using an optical fiber or a laser can be used in addition to a method using a photomask. it can.
本発明および明細書におけるゲルの「弾性率」は原子間力顕微鏡を用いて、実施例に記載の方法により測定できる。 The “elastic modulus” of the gel in the present invention and specification can be measured by the method described in Examples using an atomic force microscope.
領域Ｂの弾性率は、例えば、１０Ｐａ以上８００Ｐａ未満であってよく、５０〜７００Ｐａであってよい。領域Ａの弾性率は、例えば、８００〜３００００Ｐａであってよく、９００〜２００００Ｐａであってよい。弾性率を上記値とすることで、ＥＣＭ力学特性を模倣することができる。
特に、実施形態の細胞足場材料を中枢神経系のモデルに適用する場合では、領域Ｂの弾性率が脳組織の弾性率１００〜５００Ｐａであり、領域Ａの弾性率が脳組織損傷時の弾性率１０００〜１００００Ｐａの範囲にあるフィルムを作製することで、ＥＣＭ力学特性を模倣することができる。
The elastic modulus of the region B may be, for example, 10 Pa or more and less than 800 Pa, or 50 to 700 Pa. The elastic modulus of the region A may be, for example, 800 to 30000 Pa or 900 to 20000 Pa. By setting the elastic modulus to the above value, ECM mechanical characteristics can be imitated.
In particular, when the cell scaffold material of the embodiment is applied to a central nervous system model, the elastic modulus of the region B is 100 to 500 Pa of the brain tissue, and the elastic modulus of the region A is the elastic modulus when the brain tissue is damaged. ECM mechanical properties can be mimicked by making films in the range of 1000-10000 Pa.
上記の観点から、領域Ａと領域Ｂとの弾性率の差は、１００〜９９００Ｐａであってよく、５００〜５０００Ｐａであってよい。 From the above viewpoint, the difference in elastic modulus between the region A and the region B may be 100 to 9900 Pa, and may be 500 to 5000 Pa.
網目構造は１つの成分から構成されていてもよく、２つ以上の成分から構成されていてもよい。 The network structure may be composed of one component, or may be composed of two or more components.
網目構造を構成する成分の一例として、高分子化合物が挙げられる。高分子化合物は、前記網目構造の少なくとも一部又は全部を構成する。
高分子化合物は、合成高分子、生体高分子等であってもよい。合成高分子としては、例えば、ポリエチレングリコール（ＰＥＧ）、ポリアクリルアミド、ポリジメチルシロキサン（ＰＤＭＳ）等のシリコーン、（３，４−エチレンジオキシチオフェン）／ポリ（４−スチレンスルホン酸）（ＰＥＤＯＴ−ＰＳＳ）、ポリピロール系高分子、ポリアニリン系高分子、ポリヒドロキシエチルアクリレートなどのアクリル系高分子などを挙げることができる。生体高分子としては、例えば、デキストラン、アルギン酸等の多糖類；ゼラチン、シルクフィブロインなどのタンパク質；キトサン、コラーゲンなどの細胞外マトリクスなどが挙げられる。
An example of a component constituting the network structure is a polymer compound. The polymer compound constitutes at least a part or all of the network structure.
The polymer compound may be a synthetic polymer, a biopolymer, or the like. Examples of synthetic polymers include silicones such as polyethylene glycol (PEG), polyacrylamide, and polydimethylsiloxane (PDMS), (3,4-ethylenedioxythiophene) / poly (4-styrenesulfonic acid) (PEDOT-PSS). ), Acrylic polymers such as polypyrrole polymers, polyaniline polymers, and polyhydroxyethyl acrylate. Examples of the biopolymer include polysaccharides such as dextran and alginic acid; proteins such as gelatin and silk fibroin; and extracellular matrices such as chitosan and collagen.
これらのなかでも、高分子化合物はポリアクリルアミド系樹脂又はアクリル系樹脂を含むことが好ましく、透明性が良好でありモノマーと架橋剤との混合比で弾性率を容易に制御可能であるとの観点から、ポリアクリルアミド系樹脂を含むことがより好ましい。
ポリアクリルアミド系樹脂を構成するモノマーとしては、アミノ基を有する（メタ）アクリル酸エステルモノマーが挙げられ、アクリルアミド、メタクリルアミド等を例示できる。ポリアクリルアミド系樹脂は、上記モノマーの重合体であってよく、上記モノマーの重合体と該モノマーと共重合可能な他のモノマーとの共重合体であってよい。
アクリル系樹脂を構成するモノマーとしては、（メタ）アクリル酸エステルモノマーが挙げられ、（メタ）アクリル酸メチル、（メタ）アクリル酸エチル、（メタ）アクリル酸ヒドロキシメチル、（メタ）アクリル酸２−ヒドロキシエチル等の水酸基含有モノマー等を例示できる。ポリアクリルアミド系樹脂は、上記モノマーの重合体であってよく、上記モノマーの重合体と該モノマーと共重合可能な他のモノマーとの共重合体であってよい。
Among these, the polymer compound preferably contains a polyacrylamide resin or an acrylic resin, and has a good transparency, and the elastic modulus can be easily controlled by the mixing ratio of the monomer and the crosslinking agent. Therefore, it is more preferable to include a polyacrylamide resin.
Examples of the monomer constituting the polyacrylamide resin include (meth) acrylic acid ester monomers having an amino group, and examples include acrylamide and methacrylamide. The polyacrylamide-based resin may be a polymer of the monomer, or a copolymer of the polymer of the monomer and another monomer copolymerizable with the monomer.
Examples of the monomer constituting the acrylic resin include (meth) acrylic acid ester monomers, such as methyl (meth) acrylate, ethyl (meth) acrylate, hydroxymethyl (meth) acrylate, and (meth) acrylic acid 2- Examples thereof include hydroxyl group-containing monomers such as hydroxyethyl. The polyacrylamide-based resin may be a polymer of the monomer, or a copolymer of the polymer of the monomer and another monomer copolymerizable with the monomer.
実施形態に係るゲルが含有する高分子化合物は、１種のみでもよいし、２種以上でもよい。 The polymer compound contained in the gel according to the embodiment may be only one type or two or more types.
網目構造を構成する成分の一例として、架橋剤が挙げられる。架橋剤は前記高分子化合物同士を架橋し、網目構造を形成又は複雑化させる役割を果たし得る。
架橋剤は特に限定されるものではないが、メチレンビスアクリルアミドやエチレンビスアクリルアミド等のアクリルアミド系架橋剤が好ましい。アクリルアミド系架橋剤は、上記ポリアクリルアミド系樹脂と組み合わせて好適に用いることができる。
実施形態に係るゲルが含有してもよい架橋剤は、１種のみでもよいし、２種以上でもよい。
An example of a component constituting the network structure is a crosslinking agent. The crosslinking agent may serve to crosslink the polymer compounds to form or complicate a network structure.
The cross-linking agent is not particularly limited, but acrylamide-based cross-linking agents such as methylene bisacrylamide and ethylene bisacrylamide are preferable. The acrylamide crosslinking agent can be suitably used in combination with the polyacrylamide resin.
The crosslinking agent that may be contained in the gel according to the embodiment may be only one kind or two or more kinds.
光照射により網目構造において生成又は切断される化学結合は、網目構造の任意の位置にあってよい。
例えば、化学結合が生成される場合、生成される化学結合は、高分子化合物の一部として生成されてよい。このような反応として、該化学結合が生成されることにより高分子化合物の主鎖又は側鎖が伸長する反応が挙げられる。又は、生成される化学結合は、高分子化合物の一部として生成されなくともよい。このような反応として、架橋剤が高分子化合物同士を架橋する反応が挙げられる。
例えば、化学結合が切断される場合、切断される化学結合の位置は高分子化合物の一部であってよい。このような反応として、該化学結合が切断されることにより高分子化合物の鎖が分解する反応が挙げられる。又は、切断される化学結合の位置は高分子化合物の一部でなくともよい。このような反応として、架橋剤が分解し、高分子化合物同士の架橋が外れる反応が挙げられる。
The chemical bond generated or broken in the network structure by light irradiation may be at any position of the network structure.
For example, when a chemical bond is generated, the generated chemical bond may be generated as part of the polymer compound. An example of such a reaction is a reaction in which the main chain or side chain of the polymer compound is elongated by the generation of the chemical bond. Alternatively, the generated chemical bond may not be generated as part of the polymer compound. An example of such a reaction is a reaction in which a crosslinking agent crosslinks polymer compounds.
For example, when a chemical bond is cleaved, the position of the cleaved chemical bond may be a part of the polymer compound. An example of such a reaction is a reaction in which the chain of the polymer compound is decomposed by breaking the chemical bond. Alternatively, the position of the chemical bond to be cleaved may not be a part of the polymer compound. Examples of such a reaction include a reaction in which the crosslinking agent is decomposed and the crosslinking between the polymer compounds is released.
ゲルは、光照射により、自らを構成する化学結合が生成又は切断される成分、又は該成分に由来する構造を含んでよい。由来するとは、該成分が網目構造を形成する際に必要な変化を受けたことを言う。化学結合が生成されることで網目構造の少なくとも一部の構造が形成される。化学結合が切断されることで網目構造の少なくとも一部が切断される。該成分としては、前記高分子化合物であってもよく、高分子化合物の原材料であってもよく、架橋剤であってもよい。 The gel may include a component in which a chemical bond constituting itself is generated or broken by light irradiation, or a structure derived from the component. Deriving means that the component has undergone changes necessary to form a network structure. A chemical bond is generated to form at least a part of the network structure. By cutting the chemical bond, at least a part of the network structure is cut. The component may be the polymer compound, a raw material of the polymer compound, or a crosslinking agent.
光照射により自らを構成する化学結合が生成又は切断される成分として、光照射によって自らを構成する化学結合が生成または切断される官能基を有する成分が挙げられる。自らを構成する化学結合とは、自らと化学結合相手間との結合である。光照射によって化学結合が生成される官能基としては、下記式（１−Ａ）で表される構造を有するベンゾフェノン誘導体や、下記式（１−Ｂ）で表される構造を有するケイ皮酸誘導体などが挙げられる。光照射によって化学結合が切断される官能基として、例えば、下記式（１−Ｃ）で表されるニトロフェニル基を有する光解離性架橋剤や、下記式（１−Ｄ）で表される光解離性架橋剤などが挙げられる。 Examples of the component that generates or cleaves the chemical bond constituting itself by light irradiation include a component having a functional group that generates or cleaves the chemical bond constituting itself by light irradiation. A chemical bond that constitutes itself is a bond between itself and a chemical bond partner. Examples of the functional group that generates a chemical bond by light irradiation include a benzophenone derivative having a structure represented by the following formula (1-A) and a cinnamic acid derivative having a structure represented by the following formula (1-B). Etc. As a functional group whose chemical bond is cleaved by light irradiation, for example, a photolabile crosslinking agent having a nitrophenyl group represented by the following formula (1-C) or a light represented by the following formula (1-D) Examples include a dissociative crosslinking agent.
また、光照射により自らを構成する化学結合が生成される物質として、例えば光重合開始剤によって重合されるモノマー等の、高分子化合物の原材料が挙げられる。
光重合開始剤としては、下記式（２−Ａ）で表されるリチウムフェニル−２，４，６−トリメチルベンゾイルホスフィネート（ＬＡＰ）、下記式（２−Ｂ）で表される２−ヒドロキシ−４’−（２−ヒドロキシエトキシ）−２−メチルプロピオフェノン（Ｉｒｇａｃｕｒｅ２９５９）、下記式（２−Ｃ）で表されるＥｏｓｉｎＹ、下記式（２−Ｄ）で表されるＮ−ビニルピロリドン（ＮＭＰ）、下記式（２−Ｅ）で表されるトリエタノールアミン（ＴＥＯＡ）が例示できる。
In addition, examples of the substance that generates a chemical bond constituting itself by light irradiation include raw materials of polymer compounds such as a monomer that is polymerized by a photopolymerization initiator.
As a photopolymerization initiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) represented by the following formula (2-A), 2-hydroxy- represented by the following formula (2-B) 4 ′-(2-hydroxyethoxy) -2-methylpropiophenone (Irgacure 2959), EosinY represented by the following formula (2-C), N-vinylpyrrolidone (NMP represented by the following formula (2-D) ) And triethanolamine (TEOA) represented by the following formula (2-E).
また、光照射により自らを構成する化学結合が生成又は切断される成分としては、任意の波長の光照射によって分子構造が可逆的に変化する光異性化構造が挙げられる。当該構造を有する異性化分子としては、アゾベンゼン分子又はアゾベンゼン誘導体分子が挙げられる。
ゲルは、光異性化構造を有し光照射によって二本鎖が解離及び／又は形成する核酸を含んでもよい。当該核酸としては、アゾベンゼン又はアゾベンゼン誘導体から一つの水素原子を除いた基を有する核酸が挙げられ、アゾベンゼン又はアゾベンゼン誘導体から一つの水素原子を除いた基を有するＤＮＡ二重らせん（アゾＤＮＡ）が好ましい（図１）。アゾベンゼン又はアゾベンゼン誘導体は核酸の架橋剤として導入されたものとみなすこともできる。アゾＤＮＡは波長３６０ｎｍ前後の光を吸収すると解離し、波長４００ｎｍ以上の光を吸収すると再形成することが知られている。この性質を利用し、ゲルへの局所的な光照射によって可逆的な弾性率制御が可能となる。
Moreover, as a component by which the chemical bond which comprises self is produced | generated or cut | disconnected by light irradiation, the photoisomerization structure from which a molecular structure changes reversibly by light irradiation of arbitrary wavelengths is mentioned. Examples of the isomerized molecule having the structure include an azobenzene molecule or an azobenzene derivative molecule.
The gel may include a nucleic acid having a photoisomerization structure and having double strands dissociated and / or formed by light irradiation. Examples of the nucleic acid include a nucleic acid having a group obtained by removing one hydrogen atom from azobenzene or an azobenzene derivative, and a DNA double helix (azo DNA) having a group obtained by removing one hydrogen atom from azobenzene or an azobenzene derivative is preferable. (FIG. 1). Azobenzene or an azobenzene derivative can also be regarded as being introduced as a nucleic acid crosslinking agent. It is known that azo DNA dissociates when it absorbs light with a wavelength of around 360 nm and re-forms when it absorbs light with a wavelength of 400 nm or more. Utilizing this property, reversible elastic modulus control is possible by local light irradiation on the gel.
本実施形態の細胞足場材料は、ゲルを構成する分散媒の少なくとも一部として、液体成分を含有することができる。液体成分は、細胞培養に用いることのできるものであればよく、水、バッファー、液体培地等が挙げられる。液体成分は水を含有するものであることが好ましい。分散媒に水を含有するゲルはハイドロゲルと呼ばれる。 The cell scaffold material of the present embodiment can contain a liquid component as at least a part of the dispersion medium constituting the gel. The liquid component may be any liquid component that can be used for cell culture, and examples thereof include water, a buffer, and a liquid medium. The liquid component preferably contains water. A gel containing water in a dispersion medium is called a hydrogel.
本実施形態の細胞足場材料は、上記成分のいずれにも該当しないその他の添加剤を含有してもよい。添加剤としては、防腐剤、酸化防止剤、着色料、増粘剤、架橋促進剤、糖類、ビタミン類、抗生物質、細胞接着分子等が挙げられる。
細胞接着分子としては、ポリリジン、フィブロネクチン、コラーゲン、ビトロネクチン、エラスチン、ヒアルロン酸、ラミニン等が挙げられる。細胞接着分子は、ゲルの最表面に配置されてもよい。ゲル表面に修飾された細胞接着分子により、任意の細胞を効果的に接着可能とできる。
本実施形態の細胞足場材料は、神経細胞及び／又はグリア細胞の培養に好適に使用される。そのため、これらの培養に有用なラミニンやＮ−カドヘリンなどの細胞接着因子等の成分を含むことができる。
The cell scaffold material of this embodiment may contain other additives not corresponding to any of the above components. Examples of additives include preservatives, antioxidants, colorants, thickeners, crosslinking accelerators, sugars, vitamins, antibiotics, cell adhesion molecules, and the like.
Examples of cell adhesion molecules include polylysine, fibronectin, collagen, vitronectin, elastin, hyaluronic acid, laminin and the like. Cell adhesion molecules may be placed on the outermost surface of the gel. Arbitrary cells can be effectively adhered by the cell adhesion molecule modified on the gel surface.
The cell scaffold material of this embodiment is suitably used for culture of nerve cells and / or glial cells. Therefore, components such as cell adhesion factors such as laminin and N-cadherin useful for these cultures can be included.
実施形態に係るゲルは、フィルム状である。フィルム形状は特に限定されるものではなく、シート状、板状等の形状であることができる。
フィルム状のゲルの厚みは、１〜１０００μｍであってもよく、５〜５００μｍであってもよく、１０〜１００μｍであってもよい。
The gel which concerns on embodiment is a film form. The film shape is not particularly limited, and may be a sheet shape, a plate shape, or the like.
The thickness of the film-like gel may be 1 to 1000 μm, 5 to 500 μm, or 10 to 100 μm.
フィルム状のゲルは、単層であってもよく、複層であってもよい。複層の場合、各層は同一の種類及び／又は厚みのゲルが用いられていてもよく、互いに異なる種類及び／又は厚みのゲルが用いられていてもよい。 The film-like gel may be a single layer or a multilayer. In the case of multiple layers, gels of the same type and / or thickness may be used for each layer, or gels of different types and / or thicknesses may be used.
細胞足場材料は、フィルム状のゲルのみからなるものであってもよく、ゲルの他にその他の構造物を備えていてもよい。
その他の構造物としては、細胞培養に適用可能なものであれば特に制限はなく、ディッシュ、マルチディッシュ、フラスコ等の培養容器や、基板等を例示できる。一例として、細胞培養容器と、フィルム状のゲルを備え、培養容器内にゲルが配置された細胞足場材料が挙げられる。別の一例として、基板と、フィルム状のゲルを備え、基板上にゲルが積層された細胞足場材料が挙げられる。
The cell scaffold material may consist of only a film-like gel, and may include other structures in addition to the gel.
Other structures are not particularly limited as long as they are applicable to cell culture, and examples include culture containers such as dishes, multi-dish, and flasks, and substrates. An example is a cell scaffold material that includes a cell culture container and a film-like gel, and the gel is disposed in the culture container. Another example is a cell scaffold material that includes a substrate and a film-like gel, and the gel is laminated on the substrate.
培養容器及び基板の材質としては、金属、樹脂、セラミック、プラスチック、木材、布、紙、ガラス等が挙げられる。 Examples of the material for the culture vessel and the substrate include metals, resins, ceramics, plastics, wood, cloth, paper, and glass.
培養容器及び基板の表面は、骨格内にＳｉ原子を含むシラン化合物等によって、表面修飾されていてもよい。 The surfaces of the culture vessel and the substrate may be surface-modified with a silane compound containing Si atoms in the skeleton.
（２）細胞足場材料の第二の実施形態
本実施形態の細胞足場材料は、高分子化合物を含むフィルム状のゲルを備え、前記ゲルは領域Ａと領域Ｂとを有し、前記領域Ａよりも前記領域Ｂの弾性率が低いものである。
本実施形態の細胞足場材料に係るゲルは、上記第一実施形態の細胞足場材料の前記ゲルの一部の領域に光照射して領域Ａと領域Ｂとに分け、前記領域Ａよりも前記領域Ｂの弾性率を低下させることで、得ることができる。
(2) Second Embodiment of Cell Scaffold Material The cell scaffold material of the present embodiment includes a film-like gel containing a polymer compound, and the gel has a region A and a region B. From the region A, Also, the elastic modulus of the region B is low.
The gel according to the cell scaffold material of the present embodiment is divided into a region A and a region B by irradiating a partial region of the gel of the cell scaffold material of the first embodiment, and the region is more than the region A. It can be obtained by lowering the elastic modulus of B.
細胞足場材料を構成する成分や。細胞足場材料の構造としては、上記第一実施形態の細胞足場材料で説明したものが挙げられ、説明を省略する。 Components that make up cell scaffolding materials. Examples of the structure of the cell scaffold material include those described in the cell scaffold material of the first embodiment, and a description thereof is omitted.
領域Ａ及び領域Ｂの面積は、それぞれ独立に０．０００１〜１０００ｍｍ２であってよく、０．００２５〜５００ｍｍ２であってよく、０．０１〜１０ｍｍ２であってよい。 The area of the region A and the region B are each independently be a 0.0001～1000Mm 2, may be 0.0025～500Mm 2, may be 0.01 to 10 mm 2.
領域Ａと領域Ｂとは、同一平面上に配置されていることが好ましい。領域Ａと領域Ｂとは、縦方向や横方向等に互い違いに並んでいることが好ましい。領域Ａと領域Ｂとの並びのパターンは、領域Ａと領域Ｂとが互い違いに並んだ、縞状、市松模様状、同心円状とすることができる。領域Ａと領域Ｂとは互いの境界を共有するよう直接に接して配置されていることが好ましい。領域Ａ及び領域Ｂの短手方向の長さは０．０５〜５０ｍｍとしてもよく、０．１〜５ｍｍとしてもよい。このような構成とすることで、領域Ａと領域Ｂとの間で培養される細胞特性の差が検出されやすい。また、神経細胞及び／又はグリア細胞を含む細胞の培養に使用される場合には、グリア細胞は高弾性領域（領域Ａ）で接着しやすく、神経細胞は低弾性領域（領域Ｂ）で接着しやすいため、両者を共培養する際に、接着領域の住み分けが可能になる。 It is preferable that the region A and the region B are arranged on the same plane. The region A and the region B are preferably arranged alternately in the vertical direction, the horizontal direction, and the like. The arrangement pattern of the region A and the region B can be a striped pattern, a checkered pattern, or a concentric circle in which the regions A and B are alternately arranged. It is preferable that the region A and the region B are arranged in direct contact with each other so as to share a boundary with each other. The length of the region A and the region B in the short direction may be 0.05 to 50 mm, or 0.1 to 5 mm. By setting it as such a structure, the difference of the cell characteristic cultured between the area | region A and the area | region B is easy to be detected. In addition, when used for culturing cells containing nerve cells and / or glial cells, the glial cells are likely to adhere in the high elasticity region (region A), and the nerve cells adhere in the low elasticity region (region B). Since it is easy, when cocultivating both, the adhesion | attachment area | region can be segregated.
なお、本実施形態の細胞足場材料は、上記方法で得られたものに限定されるものではない。 In addition, the cell scaffold material of this embodiment is not limited to what was obtained by the said method.
以上に説明した第一〜第二の実施形態の細胞足場材料によれば、フィルム状であるため厚さが制御され、厚さの安定性に優れる。また、実施形態の細胞足場材料は、領域Ａよりも領域Ｂの弾性率が低い、又は低くなるよう変化するので、高弾性領域Ａと低弾性領域Ｂとの両方で細胞を培養でき、これまで明らかとなっていなかった、細胞・組織がＥＣＭ力学特性を認識・応答するメカニズムの解明が期待できる。 According to the cell scaffold material of 1st-2nd embodiment demonstrated above, since it is a film form, thickness is controlled and it is excellent in stability of thickness. In addition, since the cell scaffold material of the embodiment changes so that the elastic modulus of the region B is lower or lower than that of the region A, cells can be cultured in both the high elastic region A and the low elastic region B. The elucidation of the mechanism by which cells / tissues recognize and respond to ECM mechanical properties, which has not been clarified, can be expected.
≪細胞足場材料の製造方法≫
（１）細胞足場材料の製造方法の第一の実施形態
本実施形態の製造方法として、上記第一の実施形態の細胞足場材料の製造方法について説明する。
≪Method for producing cell scaffolding material≫
(1) First Embodiment of Manufacturing Method of Cell Scaffold Material As a manufacturing method of the present embodiment, the manufacturing method of the cell scaffold material of the first embodiment will be described.
第一の実施形態の細胞足場材料は、前記ゲルの構成材料である各成分を混合して、これら各成分を含有する足場材料組成物をフィルム状にゲル化させることで製造できる。
各成分としては、上記に例示した高分子化合物及び／又はその原材料、並びに液体成分を含み、必要により、上記に例示した架橋剤、添加剤等の各種成分を含んでもよい。高分子化合物、高分子化合物の原材料及び架橋剤は、上記に例示した自らを構成する化学結合が生成又は切断される成分であってよい。
高分子化合物の原材料が、光重合開始剤によって重合されるモノマー等の原材料を含む場合、足場材料組成物は、該当する光重合開始剤を含むことが好ましい。
The cell scaffold material of 1st embodiment can be manufactured by mixing each component which is the constituent material of the said gel, and gelatinizing the scaffold material composition containing these each component in a film form.
Each component includes the polymer compound exemplified above and / or its raw materials and a liquid component, and may contain various components such as the crosslinking agent and additives exemplified above as necessary. The polymer compound, the raw material of the polymer compound, and the cross-linking agent may be components that generate or cleave the chemical bond constituting the self exemplified above.
When the raw material of the polymer compound includes a raw material such as a monomer that is polymerized by a photopolymerization initiator, the scaffold material composition preferably includes the corresponding photopolymerization initiator.
足場材料組成物をフィルム状にゲル化させる方法の一例としては、培養容器や基板等の上に足場材料組成物を注ぎ、その上で硬化させることを例示できる。
ゲルを硬化させるため、必要に応じ足場材料組成物に対し光照射を行ってもよい。当該光照射は、ゲル全体を硬化させる目的でなされるものであり、光照射して領域Ａと領域Ｂとに分けることには該当しない操作である。
As an example of the method for gelling the scaffold material composition into a film, it is possible to exemplify pouring the scaffold material composition on a culture vessel, a substrate, or the like, and then curing it.
In order to cure the gel, if necessary, the scaffold material composition may be irradiated with light. The light irradiation is performed for the purpose of curing the entire gel, and is an operation that does not correspond to dividing the region A and the region B by light irradiation.
図２は、その上で足場材料組成物を硬化させるガラス基板の模式図である。図２（Ａ）は、表面にスペーサーを有するガラス基板の上面図であり、図２（Ｂ）は図２（Ａ）の側面図である。ガラス基板の両端にスペーサーを配置することで、フィルム状のゲルの厚みを均一にすることができる（図２（Ａ），（Ｂ））。また、配置する複数のスペーサーの厚みを互いに異なるものとすることで、フィルム状のゲルの厚みに勾配をつけることができる（図２（Ｃ））。
スペーサーの厚みの範囲は、ガラス基板への影響がなく、かつ、ゲルの膨潤による皺構造形成の影響が少ない、１０〜５０μｍが望ましい。
スペーサーの素材として、厚さが均一で耐水性を有していればよいが、例として、サランラップ（登録商標、旭化成、１１μｍ）、スコッチテープ（登録商標、３Ｍ、５０μｍ）、ＰＥＴ基材片面接着テープ（日東電工，１０，２０，３０μｍ）が挙げられる。また、ＳＵ−８等のネガティブフォトレジストを用いて任意のスペーサー厚としてもよい。
FIG. 2 is a schematic diagram of a glass substrate on which the scaffold material composition is cured. 2A is a top view of a glass substrate having a spacer on the surface, and FIG. 2B is a side view of FIG. By disposing spacers on both ends of the glass substrate, the thickness of the film-like gel can be made uniform (FIGS. 2A and 2B). In addition, by making the thicknesses of the plurality of spacers different from each other, a gradient can be given to the thickness of the film-like gel (FIG. 2C).
The range of the thickness of the spacer is preferably 10 to 50 μm, which has no influence on the glass substrate and has little influence on the formation of the wrinkle structure due to the swelling of the gel.
As a material for the spacer, it is only necessary to have a uniform thickness and water resistance. Tape (Nitto Denko, 10, 20, 30 μm) can be mentioned. Moreover, it is good also as arbitrary spacer thickness using negative photoresists, such as SU-8.
（２）細胞足場材料の製造方法の第二の実施形態
本実施形態の製造方法として、上記第二の実施形態の細胞足場材料の製造方法について説明する。
(2) Second Embodiment of Manufacturing Method of Cell Scaffold Material As a manufacturing method of the present embodiment, a manufacturing method of the cell scaffold material of the second embodiment will be described.
（光照射工程）
本実施形態の製造方法は、光照射工程を有する。
光照射工程は、高分子化合物を含む網目構造を形成するフィルム状のゲル、又は前記ゲルの構成材料を含有する足場材料組成物の一部の領域に光照射して領域Ａと領域Ｂとに分け、前記領域Ａよりも前記領域Ｂの弾性率を低下させる工程である。
第二実施形態のゲルは、第一実施形態のゲルに光照射工程を施すことにより製造できる。当該ゲル、足場材料組成物、及び光の照射については、上記の≪細胞足場材料≫および細胞足場材料の製造方法の第一の実施形態において例示したものが挙げられ、説明を省略する。
光照射して領域Ａと領域Ｂとに分けることについては以下の例が挙げられる。
１）領域Ａに光Ｌを照射し、光Ｌが照射されていない領域を領域Ｂとし、光Ｌによって領域Ａの弾性率を高める。
２）領域Ｂに光Ｌを照射し、光Ｌが照射されていない領域を領域Ａとし、光Ｌによって領域Ｂの弾性率を低下させる。
(Light irradiation process)
The manufacturing method of this embodiment has a light irradiation process.
In the light irradiation step, a film-form gel that forms a network structure containing a polymer compound, or a partial region of a scaffold material composition containing the constituent material of the gel is irradiated with light to form regions A and B. This is a step of lowering the elastic modulus of the region B than the region A.
The gel of the second embodiment can be produced by subjecting the gel of the first embodiment to a light irradiation process. Examples of the gel, the scaffold material composition, and the light irradiation include those exemplified in the first embodiment of the above-described << cell scaffold material >> and the method for producing the cell scaffold material, and the description thereof is omitted.
The following examples can be given for dividing light into region A and region B by light irradiation.
1) The region L is irradiated with the light L, the region not irradiated with the light L is defined as a region B, and the elastic modulus of the region A is increased by the light L.
2) The region B is irradiated with the light L, the region where the light L is not irradiated is defined as the region A, and the elastic modulus of the region B is decreased by the light L.
ゲルに対し光照射工程を行って細胞足場材料の備えるゲルを得てもよく、足場材料組成物に対し光照射工程を行って細胞足場材料の備えるゲルを得てもよい。足場材料組成物に対する前記光照射工程のみで、足場材料組成物がゲル化しない場合には、領域Ａと領域Ｂとの弾性率の違いが打ち消されないよう、足場材料組成物全体を硬化する操作を行ってもよい。 A gel with which the cell scaffold material is provided may be obtained by performing a light irradiation step on the gel, or a gel with which the cell scaffold material is provided by performing a light irradiation step on the scaffold material composition. When the scaffold material composition is not gelated only by the light irradiation step for the scaffold material composition, the entire scaffold material composition is cured so that the difference in elastic modulus between the region A and the region B is not negated. May be performed.
（再膨潤工程）
本実施形態の製造方法は、さらに再膨潤工程を有していてもよい。
再膨潤工程は、前記照射工程の前に、光照射により自らを構成する化学結合が生成又は切断される成分を含む再膨潤液にゲルを含浸させる工程である。
(Re-swell process)
The manufacturing method of this embodiment may further have a re-swelling process.
The re-swelling step is a step of impregnating the gel with a re-swelling liquid containing a component that generates or breaks a chemical bond constituting itself by light irradiation before the irradiation step.
光照射により自らを構成する化学結合が生成又は切断される成分については、上記≪細胞足場材料≫で例示したものが挙げられる。当該成分は高分子化合物の原材料を含んでもよい。高分子化合物の原材料が、光重合開始剤によって重合されるモノマー等の原材料を含む場合、再膨潤液は、該当する光重合開始剤を含むことが好ましい。
再膨潤させたゲルの任意の領域に光照射を行うことで、ゲルの弾性率を局所的に変化させることが可能となる。再膨潤液の組成は、ゲルが膨潤する溶媒に溶解する分子でることが好ましい。再膨潤液中の分子組成によって、ゲルに様々な機能を付与することが可能となる。
Examples of the component in which a chemical bond constituting itself is generated or cleaved by light irradiation include those exemplified in the above << cell scaffold material >>. The component may include a raw material of a polymer compound. When the raw material of the polymer compound includes a raw material such as a monomer that is polymerized by a photopolymerization initiator, the re-swell liquid preferably includes the corresponding photopolymerization initiator.
By irradiating light to an arbitrary region of the re-swelled gel, the elastic modulus of the gel can be locally changed. The composition of the re-swell liquid is preferably a molecule that dissolves in the solvent in which the gel swells. Various functions can be imparted to the gel depending on the molecular composition in the re-swelling liquid.
実施形態の細胞足場材料の製造方法によれば、実施形態の細胞足場材料を容易に製造できる。 According to the method for producing a cell scaffold material of the embodiment, the cell scaffold material of the embodiment can be easily produced.
≪細胞モデル≫
本実施形態の細胞モデルは、本発明の細胞足場材料と、前記細胞足場材料で培養される細胞と、を備えるものである。
培養材料としては、上記の≪細胞足場材料≫において例示したものが挙げられ、説明を省略する。
細胞は、細胞足場材料のゲルの表面に位置していてもよく、ゲルの内部に位置していてもよい。
細胞としては、特に制限されるものではなく、動物細胞、植物細胞等の細胞や、微生物が挙げられる。前記細胞は、組織、器官等の細胞集合体を構成していてもよい。
実施形態の細胞モデルは、中枢神経系モデルとして好適に用いることができる。細胞は、神経細胞及び／又はグリア細胞を含むことが好ましい。
≪Cell model≫
The cell model of the present embodiment includes the cell scaffold material of the present invention and cells cultured with the cell scaffold material.
Examples of the culture material include those exemplified in the above << cell scaffold material >>, and description thereof is omitted.
The cells may be located on the surface of the gel of cell scaffold material or may be located inside the gel.
The cells are not particularly limited, and include cells such as animal cells and plant cells, and microorganisms. The cells may constitute cell aggregates such as tissues and organs.
The cell model of the embodiment can be suitably used as a central nervous system model. The cells preferably include nerve cells and / or glial cells.
一態様として、細胞モデルの製造方法は、前記細胞モデルの第一の実施形態の細胞培養基材のゲルの一部の領域に光照射して領域Ａと領域Ｂとに分け、前記領域Ａよりも前記領域Ｂの弾性率を低下させる光照射工程を含む、方法である。これにより、細胞足場材料と、前記細胞足場材料で培養される細胞と、を備え、前記ゲルは領域Ａと領域Ｂとを有し、前記領域Ａよりも前記領域Ｂの弾性率が低い細胞モデルを製造することができる。光が照射される領域には、予め細胞が含まれていてもよく、含まれていないくともよい。 As one aspect, the method for producing a cell model comprises irradiating a part of the gel of the cell culture substrate of the first embodiment of the cell model with light to divide the region A and the region B; Is a light irradiation step of reducing the elastic modulus of the region B. Accordingly, a cell model comprising a cell scaffold material and cells cultured with the cell scaffold material, wherein the gel has a region A and a region B, and the elastic modulus of the region B is lower than that of the region A. Can be manufactured. The region irradiated with light may or may not contain cells in advance.
実施形態の細胞モデルによれば、領域Ａよりも前記領域Ｂの弾性率が低い、又は低くなるよう変化するので、生体内のＥＣＭの力学特性を模倣し、細胞・組織が外部環境の力学特性を認識するメカニズムを解明するためのin vitroモデルとすることができる。特に、疾患や外的損傷の再生が困難な中枢神経系に注目し、中枢神経系のＥＣＭ力学特性を模倣した細胞モデルを提供でる。神経細胞またはグリア細胞の培養下、in situでゲルの力学特性変化を実現することで疾患・外傷のモデルとし、組織再生に最適な足場材料の力学特性をスクリーニングするための細胞モデルとして利用可能である。
再生医療の観点からは、中枢神経系の疾患・損傷モデルとすることで、組織再生に最適な足場材料の力学特性をスクリーニングできる。
According to the cell model of the embodiment, since the elastic modulus of the region B is lower or lower than that of the region A, it mimics the mechanical properties of the ECM in the living body, and the cells / tissues have mechanical properties of the external environment. It can be used as an in vitro model for elucidating the mechanism that recognizes. In particular, focusing on the central nervous system where it is difficult to regenerate diseases and external damage, it is possible to provide a cell model that mimics the ECM mechanical properties of the central nervous system. It can be used as a model of disease and trauma by realizing the change of mechanical properties of gel in situ in culture of nerve cells or glial cells, and as a cell model for screening the mechanical properties of scaffold materials optimal for tissue regeneration. is there.
From the viewpoint of regenerative medicine, it is possible to screen the mechanical properties of the scaffold material optimal for tissue regeneration by using a disease / damage model of the central nervous system.
以上、この発明の実施形態について図面を参照して詳述してきたが、具体的な構成はこの実施形態に限られるものではなく、この発明の要旨を逸脱しない範囲の設計等も含まれる。 The embodiment of the present invention has been described in detail with reference to the drawings. However, the specific configuration is not limited to this embodiment, and includes designs and the like that do not depart from the gist of the present invention.
次に実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
＜弾性率測定方法＞
実施例で得られた各ゲルの弾性率は、下記方法により求めた。
原子間力顕微鏡を用い、温度２５℃の測定条件で測定を行った。なお、当該測定は、ヒトの体液とほぼ等張に調整したPBS水溶液中で行われるものとする。
<Elastic modulus measurement method>
The elastic modulus of each gel obtained in the examples was determined by the following method.
Using an atomic force microscope, the measurement was performed at a temperature of 25 ° C. It is assumed that the measurement is performed in a PBS aqueous solution adjusted to be approximately isotonic with human body fluid.
［参考例１］弾性率が一定のハイドロゲルフィルム（定弾性フィルム）の製造
図３は、参考例１の定弾性フィルムの製造手順を模式的に示す図である。
３−（トリメチルシリル）プロピルメタクリレートで表面修飾されたガラス基板（以下、「シラン化ガラス基板」という。）の両端に、スペーサーとしてサランラップ（登録商標）を配置した（図２（Ａ）参照）。アクリルアミドを５重量％、Ｎ，Ｎ’−メチレンビスアクリルアミド（以下、「ビスアクリルアミド」という。）を０．０５重量％又は０．５重量％、及び光重合開始剤ＬＡＰを１ｍＭ含む溶液（以下、「足場材料組成物」という。）を、シラン化ガラス基板中央に滴下した。足場材料組成物のビスアクリルアミド濃度を０．０５重量％とした場合、低弾性ゲル（１００〜３００Ｐａ）が生成され、０．５重量％とした場合、高弾性ゲル（１〜３ｋＰａ）が生成された。この基板に、カバーガラス（以下、「シール基板」という。）を上面からのせ、足場材料組成物をシラン化ガラス基板とシール基板とで挟み込んだ。波長３６０ｎｍのバンドパスフィルターを用い、１０ｍＷ／ｃｍ２で１０分間、足場材料組成物へ光照射し、ハイドロゲルフィルムとした。光照射終了後、ＰＢＳ溶液に浸漬し、未反応の成分を除去した。シール基板を取り除き、ハイドロゲルフィルム表面にポリ−Ｄ−リジンを提示するためのクロスリンカーとして２ｍＭのｓｕｌｆｏｓｕｃｃｉｎｉｍｉｄｙｌ ６-（４‘-ａｚｉｄｏ-２’-ｎｉｔｒｏｐｈｅｎｙｌａｍｉｎｏ）ｈｅｘａｎｏａｔｅ（以下、「ｓｕｌｆｏ−ＳＡＮＰＡＨ」という。）溶液を１００ｕｌハイドロゲルフィルム表面に滴下した。波長３３０ｎｍバンドパスフィルターを介してハイドロゲルフィルムへ１０分間光照射した。１ｍｇ／ｍｌのポリ−Ｄ−リジン（以下、「ＰＤＬ」という。）溶液を１００ｕｌ滴下し、ハイドロゲルフィルム表面にＰＤＬ分子を修飾し、ポリアクリルアミドを含むゲルからなる定弾性フィルムを得た。
Reference Example 1 Production of Hydrogel Film (Constant Elastic Film) with Constant Elastic Modulus FIG. 3 is a diagram schematically showing the production procedure of the constant elastic film of Reference Example 1.
Saran Wrap (registered trademark) was disposed as a spacer on both ends of a glass substrate (hereinafter referred to as “silanized glass substrate”) surface-modified with 3- (trimethylsilyl) propyl methacrylate (see FIG. 2A). A solution containing 5% by weight of acrylamide, 0.05% by weight or 0.5% by weight of N, N′-methylenebisacrylamide (hereinafter referred to as “bisacrylamide”), and 1 mM of a photopolymerization initiator LAP (hereinafter, (Referred to as “scaffold material composition”) was dropped onto the center of the silanized glass substrate. When the bisacrylamide concentration of the scaffold material composition is 0.05% by weight, a low elastic gel (100 to 300 Pa) is generated, and when it is 0.5% by weight, a high elastic gel (1 to 3 kPa) is generated. It was. A cover glass (hereinafter referred to as “seal substrate”) was placed on this substrate from the top surface, and the scaffold material composition was sandwiched between the silanized glass substrate and the seal substrate. Using a bandpass filter with a wavelength of 360 nm, the scaffold material composition was irradiated with light at 10 mW / cm 2 for 10 minutes to obtain a hydrogel film. After completion of light irradiation, it was immersed in a PBS solution to remove unreacted components. The sealing substrate is removed and 2 mM sulfosuccinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate (hereinafter referred to as “sulfo-SANPAH”) is used as a crosslinker for presenting poly-D-lysine on the surface of the hydrogel film. ) The solution was dropped on the surface of 100ul hydrogel film. The hydrogel film was irradiated with light for 10 minutes through a bandpass filter having a wavelength of 330 nm. 100 μl of a 1 mg / ml poly-D-lysine (hereinafter referred to as “PDL”) solution was added dropwise to modify PDL molecules on the surface of the hydrogel film, thereby obtaining a constant elastic film made of a gel containing polyacrylamide.
［実施例１］同一基板上で異なる弾性率の領域を有するハイドロゲルフィルム（パターン化フィルム）の製造
図４は、実施例１のパターン化フィルムの製造手順を模式的に示す図である。
金またはアルミのフォトマスクを基板の片面に有するシール基板（以下、「フォトマスク付きシール基板」という。）を用い、足場材料組成物のビスアクリルアミド濃度を０．５重量％とした以外は、参考例１のハイドロゲルフィルムと同様にして、パターン化フィルムを得た。得られたパターン化フィルムでは、光照射の際のマスク下の部分の弾性率が３００〜５００Ｐａの低弾性領域、マスクのない部分の弾性率が２ｋ〜５ｋＰａの高弾性領域であることを確認した（図５）。
[Example 1] Manufacture of hydrogel film (patterned film) having regions of different elastic modulus on the same substrate FIG. 4 is a diagram schematically showing the procedure for manufacturing the patterned film of Example 1.
Except for using a sealing substrate having a gold or aluminum photomask on one side of the substrate (hereinafter referred to as “sealing substrate with a photomask”) and setting the bisacrylamide concentration of the scaffold material composition to 0.5% by weight. In the same manner as the hydrogel film of Example 1, a patterned film was obtained. In the obtained patterned film, it was confirmed that the elastic modulus of the portion under the mask during light irradiation was a low elastic region of 300 to 500 Pa, and the elastic modulus of the portion without the mask was a high elastic region of 2 k to 5 kPa. (FIG. 5).
［実施例２］光解離性官能基を架橋剤としたパターン化フィルム（光反応性パターン化フィルム）の製造
図６は、実施例２の光反応性パターン化フィルムの製造手順を模式的に示す図である。
アクリルアミドを５重量％、ビスアクリルアミドを０．０５重量％、上記式（１−D）で表される化合物（光解離性架橋剤）を０．４５重量％、並びに重合開始剤としてＥｏｓｉｎＹを０．０１ｍＭ、ＴＥＯＡを１０重量％、及びＮＶＰを３７．５ｕＭの割合で含む溶液（以下、「光解離性官能基含有足場材料組成物」という。）を、シラン化ガラス基板中央に滴下した。この基板に、シール基板を上面からのせ、光解離性官能基含有足場材料組成物をシラン化ガラス基板とシール基板とで挟み込んだ。波長４００ｎｍのハイパスフィルタを介して１０分間、光解離性官能基含有足場材料組成物へ光照射し、ハイドロゲルフィルムとした。ＰＢＳ溶液で未反応の成分を取り除き、ハイドロゲルフィルムと基板を反転させ、基板の上面（ハイドロゲルフィルムと接していない面）にフォトマスクを設置した。波長３６０ｎｍの光を１０分間、ハイドロゲルフィルムへ照射し、ＰＢＳ溶液に再度浸漬した。シール基板を取り除き、２ｍＭのｓｕｌｆｏ−ＳＡＮＰＡＨ溶液を１００ｕｌハイドロゲルフィルム表面に滴下した。波長４００ｎｍのハイパスフィルタを使用して、ハイドロゲルフィルムへ３０分間光照射した。その後は、前述の参考例１と同様にＰＤＬをハイドロゲル表面に修飾し、光反応性パターン化フィルムとした。
Example 2 Production of Patterned Film (Photoreactive Patterned Film) Using Photolabile Functional Group as Crosslinking Agent FIG. 6 schematically shows the production procedure of the photoreactive patterned film of Example 2. FIG.
5% by weight of acrylamide, 0.05% by weight of bisacrylamide, 0.45% by weight of the compound represented by the above formula (1-D) (photodissociable cross-linking agent), and EosinY as a polymerization initiator in an amount of 0. A solution containing 01 mM, 10% by weight of TEOA, and 37.5 uM of NVP (hereinafter referred to as “photodissociative functional group-containing scaffold material composition”) was dropped onto the center of the silanized glass substrate. A seal substrate was placed on the substrate from above, and the photodissociative functional group-containing scaffold material composition was sandwiched between the silanized glass substrate and the seal substrate. The photodissociative functional group-containing scaffold material composition was irradiated with light through a high-pass filter having a wavelength of 400 nm for 10 minutes to obtain a hydrogel film. Unreacted components were removed with a PBS solution, the hydrogel film and the substrate were inverted, and a photomask was placed on the upper surface of the substrate (the surface not in contact with the hydrogel film). The hydrogel film was irradiated with light having a wavelength of 360 nm for 10 minutes and immersed again in a PBS solution. The sealing substrate was removed, and 2 mM sulfo-SANPAH solution was dropped onto the surface of 100 ul hydrogel film. The hydrogel film was irradiated with light for 30 minutes using a high-pass filter having a wavelength of 400 nm. Thereafter, PDL was modified on the surface of the hydrogel in the same manner as in Reference Example 1 to obtain a photoreactive patterned film.
［実施例３］フィルムの再膨潤によるパターン化フィルム（再膨潤パターン化フィルム）の製造
図７は、実施例３の再膨潤パターン化フィルムの製造手順を模式的に示す図である。
上述と参考例１と同様の方法で、ポリアクリルアミドを含むゲルからなる定弾性フィルムを作製した。この定弾性フィルムを再膨潤溶液（アクリルアミドを５重量％、ビスアクリルアミドを０．０５重量％、及び光重合開始剤としてＬＡＰを１ｍＭ含む溶液）に１晩以上浸漬し、ハイドロゲルフィルムを膨潤させ、再膨潤フィルムとした。この再膨潤フィルム上にフォトマスクを設置して、再膨潤フィルムへ波長３６０ｎｍの光を照射した。ＰＢＳ溶液に浸漬し、未反応の再膨潤溶液を取り除くことで、再膨潤パターン化フィルムを得た。
[Example 3] Manufacture of patterned film (re-swelled patterned film) by re-swelling of film Fig. 7 is a diagram schematically showing a manufacturing procedure of the re-swelled patterned film of Example 3.
A constant elastic film made of a gel containing polyacrylamide was produced in the same manner as described above and in Reference Example 1. This constant elastic film is immersed in a re-swelling solution (a solution containing 5% by weight of acrylamide, 0.05% by weight of bisacrylamide, and 1 mM of LAP as a photopolymerization initiator) for more than one night to swell the hydrogel film, A re-swelled film was obtained. A photomask was placed on the re-swelled film, and the re-swelled film was irradiated with light having a wavelength of 360 nm. A re-swelled patterned film was obtained by immersing in a PBS solution and removing the unreacted re-swelled solution.
［実施例４］光異性化分子を用いた光応答性パターン化フィルム（光応答性フィルム）の製造
図８は、実施例４の光応答性フィルムの製造手順を模式的に示す図である。
アクリルアミドを５重量％、ビスアクリルアミドを０．０５重量％、アゾＤＮＡを２．９ｍＭ、並びに重合開始剤としてＥｏｓｉｎＹを０．０１ｍＭ、ＴＥＯＡを１０重量％、及びＮＶＰを３７．５ｕＭの割合で含む溶液（以下、「光異性化分子含有足場材料組成物」という）を、シラン化ガラス基板中央に滴下した。この基板に、シール基板を上面からのせ、光異性化分子含有足場材料組成物をシラン化ガラス基板とシール基板とで挟み込んだ。波長４００ｎｍのハイパスフィルタを介して１０分間、光異性化分子含有足場材料組成物へ光照射し、ハイドロゲルフィルムとした。光照射終了後、ＰＢＳ溶液に浸漬し、未反応の成分を取り除いた。２ｍＭのｓｕｌｆｏ−ＳＡＮＰＡＨ溶液１００ｕｌをハイドロゲルフィルム表面に滴下し、波長４００ｎｍのハイパスフィルタを使用して、３０分間光照射した。その後は、参考例１のハイドロゲルフィルムと同様にＰＤＬをハイドロゲル表面に修飾し、光応答性フィルムを得た。次いで光応答性フィルム基板の底面（光応答性フィルムと接していない面）にフォトマスクを設置した。３６０ｎｍの光を光応答性フィルムへ１０分間照射することで、光照射部分が低弾性領域、未照射の領域が高弾性領域となった。
Example 4 Manufacture of Photoresponsive Patterned Film (Photoresponsive Film) Using Photoisomerized Molecules FIG. 8 is a diagram schematically showing the manufacturing procedure of the photoresponsive film of Example 4.
A solution containing 5% by weight of acrylamide, 0.05% by weight of bisacrylamide, 2.9 mM of azo DNA, 0.01 mM of EosinY as a polymerization initiator, 10% by weight of TEOA, and 37.5 uM of NVP (Hereinafter referred to as “photoisomerized molecule-containing scaffold material composition”) was dropped onto the center of the silanized glass substrate. A seal substrate was placed on this substrate from above, and the photoisomerizable molecule-containing scaffold material composition was sandwiched between the silanized glass substrate and the seal substrate. The photo-isomerized molecule-containing scaffold material composition was irradiated with light through a high-pass filter having a wavelength of 400 nm for 10 minutes to obtain a hydrogel film. After completion of light irradiation, it was immersed in a PBS solution to remove unreacted components. 100 ul of 2 mM sulfo-SANPAH solution was dropped on the surface of the hydrogel film, and irradiated with light for 30 minutes using a high-pass filter having a wavelength of 400 nm. Thereafter, PDL was modified on the surface of the hydrogel in the same manner as the hydrogel film of Reference Example 1 to obtain a photoresponsive film. Next, a photomask was placed on the bottom surface of the photoresponsive film substrate (the surface not in contact with the photoresponsive film). By irradiating the light-responsive film with 360 nm light for 10 minutes, the light-irradiated portion became a low elastic region and the unirradiated region became a high elastic region.
Claims (8)
前記網目構造は、光照射により前記網目構造を構成する化学結合が生成又は切断されるものであり、
前記ゲルへ光照射によって領域Ａと領域Ｂとが分けられたとき、前記領域Ａよりも前記領域Ｂの弾性率が低くなるよう変化する、細胞足場材料。 Provided with a film-like gel that forms a network structure containing a polymer compound,
The network structure is one in which chemical bonds constituting the network structure are generated or broken by light irradiation,
A cell scaffold material that changes so that the elastic modulus of the region B is lower than that of the region A when the region A and the region B are separated by light irradiation on the gel.
前記ゲルは領域Ａと領域Ｂとを有し、前記領域Ａよりも前記領域Ｂの弾性率が低い、細胞足場材料。 A film-like gel having a network structure containing a polymer compound is provided,
The cell scaffold material, wherein the gel has a region A and a region B, and the elastic modulus of the region B is lower than that of the region A.
前記網目構造は、光照射により前記網目構造を構成する化学結合が生成又は切断されるものである、細胞足場材料の製造方法。 A film-like gel forming a network structure containing a polymer compound, or a partial region of a culture material composition containing the constituent material of the gel is irradiated with light to divide the region A and region B, and the region A A light irradiation step of lowering the elastic modulus of the region B than
The said network structure is a manufacturing method of the cell scaffold material by which the chemical bond which comprises the said network structure is produced | generated or cut | disconnected by light irradiation.
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| JP2011514145A (en) * | 2008-01-30 | 2011-05-06 | ジェロン・コーポレーション | Cell culture products and screening |
| JP2010046012A (en) * | 2008-08-21 | 2010-03-04 | Konica Minolta Holdings Inc | Substrate and method for cell culture |
| JP2012080844A (en) * | 2010-10-14 | 2012-04-26 | National Institute Of Advanced Industrial Science & Technology | Photodegradable crosslinking agent, photodegradable gel, cell culture tool, cell sequence/separation device, method for sequencing cell, and method for separating cell |
| JP2015501640A (en) * | 2011-12-07 | 2015-01-19 | ツィンファ ユニバーシティ | Cell colony in vitro culture apparatus and use thereof |
| WO2014188911A1 (en) * | 2013-05-22 | 2014-11-27 | 独立行政法人産業技術総合研究所 | Photodegradable crosslinking agent, photodegradable gel, cell culture device, cell arrangement/separation device, cell arrangement method, cell separation method, method of forming tissue material, and tissue material |
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| JP2019038999A (en) * | 2017-08-22 | 2019-03-14 | 住友化学株式会社 | Compound, resin, resist composition, and method for producing resist pattern |
| JP7167539B2 (en) | 2017-08-22 | 2022-11-09 | 住友化学株式会社 | Compound, resin, resist composition, and method for producing resist pattern |
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