JP2012115254A - Three-dimensional structural body of cell and method for producing the same - Google Patents
Three-dimensional structural body of cell and method for producing the same Download PDFInfo
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- JP2012115254A JP2012115254A JP2011071939A JP2011071939A JP2012115254A JP 2012115254 A JP2012115254 A JP 2012115254A JP 2011071939 A JP2011071939 A JP 2011071939A JP 2011071939 A JP2011071939 A JP 2011071939A JP 2012115254 A JP2012115254 A JP 2012115254A
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Abstract
Description
本発明は、細胞の三次元構造体、これを製造する方法、そのためのキット、並びに、この細胞の三次元構造体を用いた細胞の培養方法、及び、薬物の細胞応答を評価する方法に関する。 The present invention relates to a three-dimensional structure of a cell, a method for producing the same, a kit for the same, a method for culturing cells using the three-dimensional structure of the cell, and a method for evaluating a cellular response of a drug.
近年、生体外で細胞の三次元組織を構築する技術が知られている。
非特許文献１には、温度応答性の樹脂であるｐｏｌｙ（Ｎ−ｉｓｏｐｒｏｐｙｌａｃｒｙｌａｍｉｄｅ）（ＰＩＰＡＡｍ）を培養皿としてシート状の細胞外マトリックスを作製する技術が記載されている。
In recent years, a technique for constructing a three-dimensional tissue of a cell in vitro is known.
Non-Patent Document 1 describes a technique for producing a sheet-like extracellular matrix using poly (N-isopropyla amide) (PIPAAm), which is a temperature-responsive resin, as a culture dish.
非特許文献２には、細胞膜と静電相互作用をするナノ磁性微粒子を含むマグネタイトカチオニックリポソーム（ＭＣＬ）を細胞に取り込ませてこれを培養することで、細胞の三次元組織を構築する技術が記載されている。 Non-Patent Document 2 discloses a technique for constructing a three-dimensional tissue of a cell by incorporating a magnetite cationic liposome (MCL) containing nanomagnetic fine particles that electrostatically interact with a cell membrane into a cell and culturing it. Are listed.
特許文献１には、細胞層の形成と、前記細胞層を第１物質含有液と第２物質含有液とに交互に接触させる工程とを繰り返し行い、ナノメートルサイズの厚みのＥＣＭ（細胞外マトリックス）を介して連続的に細胞層を積層する技術が記載されている。これにより、単層の細胞シートの剥離や剥離した細胞シートの重ね合わせ等が不要であるため、優れた再現性・効率で、極めて簡便に三次元組織を製造できるとされている。 In Patent Document 1, the formation of a cell layer and the step of alternately contacting the cell layer with a first substance-containing liquid and a second substance-containing liquid are repeatedly performed, and an ECM (extracellular matrix) having a nanometer-sized thickness is disclosed. ), A technique for continuously laminating cell layers is described. This eliminates the need for exfoliation of a single-layer cell sheet or superposition of exfoliated cell sheets, and therefore it is said that a three-dimensional tissue can be produced very easily with excellent reproducibility and efficiency.
しかしながら、非特許文献１に記載の技術では、細胞シートの剥離を必須とするため、細胞の形状を維持させつつ剥離するのが困難だった。そのため、複雑な形状の構造体を作製する方法に適していなかった。また、同一平面に異種細胞を組織化させることが困難だった。 However, since the technique described in Non-Patent Document 1 requires the cell sheet to be peeled, it is difficult to peel the cell sheet while maintaining the shape of the cell. Therefore, the method is not suitable for a method of manufacturing a complex-shaped structure. It was also difficult to organize heterogeneous cells in the same plane.
また、非特許文献２に記載の技術は、細胞層の厚みや三次元配置の制御が困難だった。 In addition, with the technique described in Non-Patent Document 2, it is difficult to control the thickness of the cell layer and the three-dimensional arrangement.
特許文献１の技術では、薄い細胞層や複雑な三次元構造を有する組織を構築することができる。しかしながら、細胞層を一層ずつ培養して作製させる技術であるため、作製に長時間を要していた。また、同一平面内に多種細胞の配置をさせることが困難であるため、三次元配置のバリエーションに限界があった。 With the technique of Patent Document 1, it is possible to construct a tissue having a thin cell layer or a complicated three-dimensional structure. However, since it is a technique of culturing and producing cell layers one by one, it took a long time for production. In addition, since it is difficult to arrange multiple cells in the same plane, there is a limit to variations in three-dimensional arrangement.
本発明は上記事情に鑑みてなされたものであり、その目的とするところは、三次元配置のバリエーションが豊富な細胞組織を短時間で作製できる三次元構造体を提供することにある。 The present invention has been made in view of the above circumstances, and an object thereof is to provide a three-dimensional structure capable of producing a cell tissue rich in variations in three-dimensional arrangement in a short time.
本発明によれば、
培養細胞が互いに接着している細胞の三次元構造体であって、
前記培養細胞は、前記培養細胞の表面全体が接着膜で被覆された被覆細胞を含み、
前記接着膜を介して前記被覆細胞と前記培養細胞とが互いに接着している、細胞の三次元構造体が提供される。
According to the present invention,
A three-dimensional structure of cells in which cultured cells adhere to each other,
The cultured cell includes a coated cell in which the entire surface of the cultured cell is coated with an adhesive film,
There is provided a three-dimensional structure of cells in which the coated cells and the cultured cells are adhered to each other through the adhesive film.
また、本発明によれば、
培養細胞を互いに接着させて細胞の三次元構造体を製造する方法であって、
前記培養細胞の表面全体が接着膜で被覆された被覆細胞を培養する工程を含み、
前記被覆細胞を培養する前記工程において、前記接着膜を介して、前記被覆細胞と前記被覆細胞から増殖した前記培養細胞とを互いに接着させる、細胞の三次元構造体を製造する方法が提供される。
Moreover, according to the present invention,
A method for producing a three-dimensional structure of cells by adhering cultured cells to each other,
Culturing the coated cell in which the entire surface of the cultured cell is coated with an adhesive film,
In the step of culturing the coated cells, there is provided a method for producing a three-dimensional structure of cells, in which the coated cells and the cultured cells grown from the coated cells are adhered to each other via the adhesive film. .
また、本発明によれば、
培養細胞を互いに接着させる接着膜を前記培養細胞に形成させるための接着成分と、
前記接着成分で前記培養細胞の全体を被覆して被覆細胞を作製し、前記被覆細胞を培養するための使用説明書と、
を含む、三次元構造体を製造するためのキットが提供される。
Moreover, according to the present invention,
An adhesive component for forming the cultured cells with an adhesive film that adheres the cultured cells to each other;
An instruction manual for culturing the coated cell by coating the entire cultured cell with the adhesive component to produce a coated cell;
A kit for producing a three-dimensional structure is provided.
また、本発明によれば、上記の三次元構造体の上にさらに培養細胞を積層させる、細胞の培養方法が提供される。 Moreover, according to this invention, the culture | cultivation method of a cell which laminates | stacks a cultured cell further on said three-dimensional structure is provided.
また、本発明によれば、上記の三次元構造体を培養し、前記接着膜を介して接着した前記培養細胞と前記被覆細胞とを組織化させる、細胞の培養方法が提供される。 In addition, according to the present invention, there is provided a cell culturing method in which the above three-dimensional structure is cultured, and the cultured cells and the coated cells adhered through the adhesive film are organized.
さらに、本発明によれば、上記の三次元構造体を用いて薬物の細胞応答を評価する方法が提供される。 Furthermore, according to the present invention, there is provided a method for evaluating a cellular response of a drug using the above three-dimensional structure.
この発明によれば、培養細胞の表面全体が接着膜で被覆された被覆細胞を用いて細胞を培養する。これにより、個々の細胞に形成された接着膜同士が相互作用して、組織化することができる。したがって、一段階で同種又は異種の細胞の三次元構造体を作製することができるため、三次元配置のバリエーションが豊富な細胞組織を短時間で作製できる構造体が実現可能になる。 According to this invention, the cells are cultured using the coated cells in which the entire surface of the cultured cells is coated with the adhesive film. Thereby, the adhesive films formed on individual cells can interact and be organized. Therefore, since a three-dimensional structure of the same or different types of cells can be produced in a single step, it is possible to realize a structure that can produce a cell tissue rich in variations in three-dimensional arrangement in a short time.
本発明によれば、三次元配置のバリエーションが豊富な細胞組織を短時間で作製可能な三次元構造体を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the three-dimensional structure which can produce the cell tissue with abundant variations of three-dimensional arrangement in a short time can be provided.
以下、本発明の実施の形態について、図面を用いて説明する。尚、すべての図面において、同様な構成要素には同様の符号を付し、適宜説明を省略する。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings, the same reference numerals are given to the same components, and the description will be omitted as appropriate.
（第１の実施形態）
本実施の形態は、培養細胞が互いに接着している細胞の三次元構造体である。図１は、本実施の形態に係る三次元構造体の一例を示す模式的な断面図である。本実施の形態の三次元構造体を構成する培養細胞１０１には、培養細胞１０１の表面全体が接着膜１０２で被覆された被覆細胞１０３が含まれており、接着膜１０２を介して被覆細胞１０３と培養細胞１０１とが互いに接着している。
(First embodiment)
The present embodiment is a three-dimensional structure of cells in which cultured cells are adhered to each other. FIG. 1 is a schematic cross-sectional view showing an example of the three-dimensional structure according to the present embodiment. The cultured cells 101 constituting the three-dimensional structure of the present embodiment include coated cells 103 in which the entire surface of the cultured cells 101 is coated with the adhesive film 102, and the coated cells 103 are interposed via the adhesive film 102. And the cultured cell 101 are adhered to each other.
具体的に、接着膜１０２は、培養細胞１０１同士を接着できる物質であれば制限されないが、第１の物質を含む膜１０２ａと、第１の物質とは異なる第２の物質を含む膜１０２ｂとが積層された膜とすることが好ましく、第１の物質からなる膜１０２ａと、第２の物質からなる膜１０２ｂとが積層された膜とすることがより好ましい。 Specifically, the adhesive film 102 is not limited as long as it is a substance that can adhere the cultured cells 101 to each other, but the film 102a containing the first substance and the film 102b containing the second substance different from the first substance Are preferably stacked, and more preferably, the film is formed by stacking the film 102a made of the first substance and the film 102b made of the second substance.
また、接着膜１０２は、第１の物質を含む膜１０２ａと第２の物質を含む膜１０２ｂとがそれぞれ複数積層された多層膜であることがより好ましく、第１の物質からなる膜１０２ａと第２の物質からなる膜１０２ｂとがそれぞれ複数積層された多層膜とするとさらに好ましい。 The adhesive film 102 is more preferably a multilayer film in which a plurality of films 102a containing a first substance and a film 102b containing a second substance are stacked, and the film 102a made of the first substance and the second film 102b It is more preferable to form a multilayer film in which a plurality of films 102b made of two substances are stacked.
また、接着膜１０２の厚みは、１ｎｍ以上１×１０３ｎｍ以下であることが好ましく、２ｎｍ以上１×１０２ｎｍ以下とすることがより好ましい。また、第１の物質からなる膜１０２ａと第２の物質からなる膜１０２ｂとを積層させることで接着膜１０２の厚みを制御することができ、特に、３ｎｍ以上１×１０２ｎｍ以下の多層膜を形成させることがより好ましい。このような多層膜とすることで、より密に積層した構造体とすることができる。なお、本発明において、接着膜１０２の厚みは、位相差顕微鏡により１０箇所測定した平均値により求めることができる。また、接着膜１０２の厚みは、別途、基材上に接着膜の形成を行い、水晶発振子マイクロバランス（ＱＣＭ）測定法を用いて、行った処理（ステップ）数と形成される厚みとを測定し、その結果から細胞表面への接着膜形成時に行ったステップ数に応じて算出してもよい。 The thickness of the adhesive film 102 is preferably 1 nm or more and 1 × 10 3 nm or less, and more preferably 2 nm or more and 1 × 10 2 nm or less. Further, the thickness of the adhesive film 102 can be controlled by laminating the film 102a made of the first substance and the film 102b made of the second substance, and in particular, a multilayer film having a thickness of 3 nm to 1 × 10 2 nm. It is more preferable to form By setting it as such a multilayer film, it can be set as the structure laminated | stacked more densely. In the present invention, the thickness of the adhesive film 102 can be obtained from an average value measured at 10 points with a phase contrast microscope. The thickness of the adhesive film 102 is determined by separately forming the adhesive film on the substrate and using the quartz oscillator microbalance (QCM) measurement method and the number of treatments (steps) and the thickness to be formed. It may be calculated according to the number of steps measured at the time of forming an adhesive film on the cell surface.
第１、第２の物質は、それぞれ分子量１千以上１千万以下の高分子であることが好ましい。本明細書でいう高分子には、タンパク質などの天然高分子や化学的に得られる合成高分子も含むものである。第１の物質及び第２の物質の組合せとしては、インテグリンが結合するアルギニン−グリシン−アスパラギン酸（ＲＧＤ）配列を含む高分子とＲＧＤ配列を含む高分子と相互作用する高分子との組み合わせであることが好ましい。また、正の電荷を有する高分子と、負の電荷を有する高分子との組合せも用いることができる。本発明において、ＲＧＤ配列を含む高分子と「相互作用する」とは、例えば、静電的相互作用、疎水性相互作用、水素結合、電荷移動相互作用、共有結合形成、タンパク質間の特異的相互作用、ファンデルワールス力等により、化学的、物理的に、ＲＧＤ配列を含む高分子と結合、接着、吸着、または電子の授受が可能な程度に近接することを意味する。なお、第１、第２の物質は、生分解性であることが好ましい。 The first and second substances are each preferably a polymer having a molecular weight of 1,000 to 10 million. The polymer referred to in this specification includes natural polymers such as proteins and chemically obtained synthetic polymers. The combination of the first substance and the second substance is a combination of a polymer containing an arginine-glycine-aspartic acid (RGD) sequence to which integrin binds and a polymer interacting with a polymer containing an RGD sequence. It is preferable. A combination of a polymer having a positive charge and a polymer having a negative charge can also be used. In the present invention, “interact” with a polymer containing an RGD sequence means, for example, electrostatic interaction, hydrophobic interaction, hydrogen bond, charge transfer interaction, covalent bond formation, specific interaction between proteins. It means that it is close enough to bond, adhere, adsorb, or exchange electrons with a polymer containing an RGD sequence chemically or physically by action, van der Waals force, or the like. Note that the first and second substances are preferably biodegradable.
「ＲＧＤ配列を含む高分子」とは、元来、ＲＧＤ配列を有するタンパク質でもよいし、また、ＲＧＤ配列が化学的に結合されたタンパク質であってもよい。また、ＲＧＤ配列を有していればよく、タンパク質以外の天然由来高分子であってもよいし、合成高分子であってもよい。 The “polymer containing RGD sequence” may originally be a protein having an RGD sequence, or may be a protein in which an RGD sequence is chemically bound. Moreover, what is necessary is just to have a RGD arrangement | sequence, natural origin polymers other than protein may be sufficient, and synthetic polymers may be sufficient.
タンパク質からなるＲＧＤ配列を含む高分子としては、例えば、従来公知の接着性タンパク質が挙げられ、フィブロネクチン（分子量約５０万）、ビトロネクチン、ラミニン、カドヘリン、コラーゲン等が使用できる。 Examples of the polymer containing the RGD sequence composed of protein include conventionally known adhesive proteins, and fibronectin (molecular weight: about 500,000), vitronectin, laminin, cadherin, collagen, and the like can be used.
また、ＲＧＤ配列を有する水溶性タンパク質も使用でき、例えば、ＲＧＤを結合させたコラーゲン、ゼラチン、アルブミン、グロブリン、プロテオグリカン、酵素、抗体等があげられる。 Water-soluble proteins having an RGD sequence can also be used, and examples thereof include collagen, gelatin, albumin, globulin, proteoglycan, enzyme, antibody and the like to which RGD is bound.
その他のＲＧＤ配列を有する天然由来高分子としては、例えば、水溶性ポリペプチド、低分子ペプチド、α−ポリリジン、ε−ポリリジン等のポリアミノ酸、キチンやキトサン等の糖等においてＲＧＤ配列を有するものがあげられる。 Examples of other naturally-derived polymers having RGD sequences include those having RGD sequences in water-soluble polypeptides, low molecular peptides, polyamino acids such as α-polylysine and ε-polylysine, sugars such as chitin and chitosan, and the like. can give.
合成高分子としては、ＲＧＤ配列を有していればよく、特に制限されない。合成高分子は、例えば、ポリマーでも共重合体でもよく、重合の形態も特に制限されず、例えば、直鎖型、グラフト型、くし型、樹状型、星型等があげられる。具体例として、例えば、ポリウレタン、ポリカーボネート、ポリアミド、及び、これらの共重合体、並びに、ポリエステル、ポリアクリル酸、ポリメタクリル酸、ポリエチレングリコール−グラフト−ポリアクリル酸、ポリ(Ｎ−イソプロピルアクリルアミド−ｃｏ−ポリアクリル酸)、ポリアミドアミンデンドリマー、ポリエチレンオキサイド、ポリε−カプロラクタム、ポリアクリルアミド、ポリ(メタクリル酸メチル−γ−ポリメタクリル酸オキシエチレン)等があげられる。 The synthetic polymer is not particularly limited as long as it has an RGD sequence. The synthetic polymer may be, for example, a polymer or a copolymer, and the form of polymerization is not particularly limited, and examples thereof include a linear type, a graft type, a comb type, a dendritic type, and a star type. Specific examples include, for example, polyurethane, polycarbonate, polyamide, and copolymers thereof, and polyester, polyacrylic acid, polymethacrylic acid, polyethylene glycol-graft-polyacrylic acid, poly (N-isopropylacrylamide-co- Polyacrylic acid), polyamidoamine dendrimer, polyethylene oxide, polyε-caprolactam, polyacrylamide, poly (methyl methacrylate-γ-polyoxymethacrylate) and the like.
ＲＧＤ配列を含む高分子の具体例を下記表１に示すがこれらには限定されない。
また、「ＲＧＤ配列を含む高分子と相互作用する高分子」としては、「ＲＧＤ配列を含む高分子」との相互作用により、両物質が、例えば、結合、接着、吸着、電子の授受が可能な程度近接できる物質であれば特に制限されないが、「ＲＧＤ配列を含む高分子」と相互作用するタンパク質、天然由来高分子及び合成高分子のいずれかを使用できる。 In addition, as a “polymer interacting with a polymer containing an RGD sequence”, both substances can, for example, bind, bond, adsorb, and exchange electrons by interacting with a “polymer containing an RGD sequence”. Any substance can be used as long as it is close enough, but any of proteins interacting with “polymer containing RGD sequence”, naturally derived polymers and synthetic polymers can be used.
「ＲＧＤ配列を含む高分子」と相互作用するタンパク質としては、特に制限されないが、例えば、水溶性タンパク質があげられ、具体例として、コラーゲン、ゼラチン（一例として分子量１０万）、プロテオグリカン、インテグリン、酵素、抗体等が使用できる。 The protein that interacts with the “polymer containing RGD sequence” is not particularly limited, and examples thereof include water-soluble proteins. Specific examples include collagen, gelatin (molecular weight 100,000 as an example), proteoglycan, integrin, enzyme An antibody or the like can be used.
「ＲＧＤ配列を含む高分子」と相互作用する天然由来高分子としては、特に制限されないが、例えば、水溶性ポリペプチド、低分子ペプチド、α−ポリリジン，ε−ポリリジン（分子量５千）等のポリアミノ酸、ヘパリンやヘパラン硫酸、デキストラン硫酸（分子量５０万）、ヒアルロン酸（分子量１００万〜）等の糖等があげられる。 The naturally occurring polymer that interacts with the “polymer containing the RGD sequence” is not particularly limited, and examples thereof include water-soluble polypeptides, low-molecular peptides, α-polylysine, and ε-polylysine (molecular weight 5,000). Examples thereof include sugars such as amino acids, heparin, heparan sulfate, dextran sulfate (molecular weight 500,000), and hyaluronic acid (molecular weight 1 million).
合成高分子としては、特に制限されず、例えば、ポリマーでも共重合体でもよく、重合の形態も、例えば、直鎖型、グラフト型、くし型、樹状型、星型等があげられる。具体例として、例えば、ポリウレタン、ポリアミド、ポリカーボネート、及び、これらの共重合体、並びに、ポリエステル、ポリアクリル酸、ポリメタクリル酸、ポリエチレングリコール−グラフト−ポリアクリル酸、ポリ(Ｎ−イソプロピルアクリルアミド−ｃｏポリアクリル酸)、ポリアミドアミンデンドリマー、ポリエチレンオキサイド、ポリε−カプロラクタム、ポリアクリルアミド、ポリ(メタクリル酸メチル−γ−ポリメタクリル酸オキシエチレン)等があげられる。 The synthetic polymer is not particularly limited, and may be, for example, a polymer or a copolymer. Examples of the polymerization form include linear, graft, comb, dendritic, and star shapes. Specific examples include, for example, polyurethane, polyamide, polycarbonate, and copolymers thereof, and polyester, polyacrylic acid, polymethacrylic acid, polyethylene glycol-graft-polyacrylic acid, poly (N-isopropylacrylamide-copoly). Acrylic acid), polyamidoamine dendrimer, polyethylene oxide, polyε-caprolactam, polyacrylamide, poly (methyl methacrylate-γ-polyoxymethacrylate) and the like.
ＲＧＤ配列を含む高分子と相互作用する高分子の具体例を下記表２に示すがこれらには限定されない。 Specific examples of the polymer that interacts with the polymer containing the RGD sequence are shown in Table 2 below, but are not limited thereto.
このような第１の物質と第２の物質との組合せとしては、特に制限されず相互作用する異なる物質の組合せであればよく、いずれか一方がＲＧＤ配列を含む高分子であり、他方が、ＲＧＤ配列を含む高分子と相互作用する高分子であればよい。具体的には、フィブロネクチンとゼラチン、フィブロネクチンとε−ポリリジン、フィブロネクチンとヒアルロン酸、ラミニンとゼラチン、ビトロネクチンとゼラチン、フィブロネクチンとデキストラン硫酸、フィブロネクチンとへパリン、エラスチンとポリリジン、フィブロネクチンとコラーゲン、ラミニンとコラーゲン、ビトロネクチンとコラーゲン、ＲＧＤ結合コラーゲンまたはＲＧＤ結合ゼラチンとコラーゲンまたはゼラチン等の組合せがあげられ、中でもフィブロネクチンとゼラチン、フィブロネクチンとヘパリン、フィブロネクチンとε−ポリリジン、フィブロネクチンとヒアルロン酸、フィブロネクチンとデキストラン硫酸との組合せが好ましく、より好ましくはフィブロネクチンとゼラチンとの組合せである。なお、第１の物質と第２の物質は、それぞれ一種類ずつでもよいし、相互作用を示す範囲で二種類以上をそれぞれ併用してもよい。 Such a combination of the first substance and the second substance is not particularly limited as long as it is a combination of different substances that interact with each other, and one of them is a polymer containing an RGD sequence, and the other is Any polymer that interacts with a polymer containing an RGD sequence may be used. Specifically, fibronectin and gelatin, fibronectin and ε-polylysine, fibronectin and hyaluronic acid, laminin and gelatin, vitronectin and gelatin, fibronectin and dextran sulfate, fibronectin and heparin, elastin and polylysine, fibronectin and collagen, laminin and collagen, Combinations of vitronectin and collagen, RGD binding collagen or RGD binding gelatin and collagen or gelatin, etc. are mentioned. Among them, a combination of fibronectin and gelatin, fibronectin and heparin, fibronectin and ε-polylysine, fibronectin and hyaluronic acid, fibronectin and dextran sulfate More preferred is a combination of fibronectin and gelatin. Note that one kind of each of the first substance and the second substance may be used, or two or more kinds may be used in combination within a range showing the interaction.
正の電荷を有する高分子としては、正の電荷を有していれば、例えば、天然由来高分子でも合成高分子でもよい。正イオンに対応するカウンターの負イオンは、特に制限されないが、塩化物イオンが好ましい。天然由来高分子としては、例えば、水溶性タンパク質が好ましく、具体的には、塩基性コラーゲン、塩基性ゼラチン、リゾチーム、シトクロムｃ、ペルオキシダーゼ、ミオグロビン等があげられる。 The polymer having a positive charge may be, for example, a naturally derived polymer or a synthetic polymer as long as it has a positive charge. The negative ion of the counter corresponding to the positive ion is not particularly limited, but a chloride ion is preferable. As the naturally derived polymer, for example, a water-soluble protein is preferable, and specific examples include basic collagen, basic gelatin, lysozyme, cytochrome c, peroxidase, myoglobin, and the like.
また、その他の天然由来高分子としては、例えば、水溶性ポリペプチド、低分子ペプチド、α−ポリリジン塩、ε−ポリリジン塩（一例として、分子量５千）、ポリアルギニン塩、ポリヒスチジン塩等のポリアミノ酸、キチン、キトサン等の糖等があげられる。 Other naturally-occurring polymers include, for example, water-soluble polypeptides, low-molecular peptides, α-polylysine salts, ε-polylysine salts (for example, molecular weight 5,000), polyarginine salts, polyhistidine salts, and the like. Examples include sugars such as amino acids, chitin and chitosan.
また、合成高分子としては、特に制限されず、例えば、ポリマーでも共重合体でもよく、重合の形態も、例えば、直鎖型、グラフト型、くし型、樹状型、星型等があげられる。具体例として、例えば、ポリウレタン、ポリアミド、ポリカーボネート及びその共重合体等、並びに、ポリエステル、ポリジアリルジメチルアンモニウム塩（一例として、分子量７万）、ポリアリルアミン塩、ポリエチレンイミン、ポリビニルアミン、ポリアミドアミンデンドリマー等があげられる。 Further, the synthetic polymer is not particularly limited, and may be, for example, a polymer or a copolymer. Examples of polymerization forms include linear, graft, comb, dendritic, star, and the like. . Specific examples include, for example, polyurethane, polyamide, polycarbonate and copolymers thereof, polyester, polydiallyldimethylammonium salt (for example, molecular weight 70,000), polyallylamine salt, polyethyleneimine, polyvinylamine, polyamideamine dendrimer, and the like. Can be given.
また、負の電荷を有する高分子としては、負の電荷を有していれば、例えば、天然由来高分子でも合成高分子でもよい。負イオンに対応するカウンターの正イオンは、特に制限されないが、ナトリウムイオンが好ましい。天然由来高分子としては、特に制限されないが、例えば、負の電荷を有するタンパク質が好ましく、具体的には、酸性コラーゲン、酸性ゼラチン、アルブミン、グロブリン、カタラーゼ、β−ラクトグロブリン、チログロブリン、α−ラクトアルブミン、卵白アルブミン等があげられる。 The negatively charged polymer may be, for example, a naturally-derived polymer or a synthetic polymer as long as it has a negative charge. The positive ion of the counter corresponding to the negative ion is not particularly limited, but sodium ion is preferable. Although it does not restrict | limit especially as a natural origin polymer | macromolecule, For example, the protein which has a negative charge is preferable, Specifically, acidic collagen, acidic gelatin, albumin, globulin, catalase, beta-lactoglobulin, thyroglobulin, alpha- Examples include lactalbumin and ovalbumin.
また、その他の天然由来高分子としては、水溶性ポリペプチド、低分子ペプチド、α−ポリリジン、ε−ポリリジン等のポリアミノ酸、糖等があげられる。 Examples of other naturally-occurring polymers include water-soluble polypeptides, low-molecular peptides, polyamino acids such as α-polylysine and ε-polylysine, sugars, and the like.
合成高分子としては、特に制限されず、例えば、ポリマーでも共重合体でもよく、重合の形態も、例えば、直鎖型、グラフト型、くし型、樹状型、星型等があげられる。具体例として、例えば、ポリウレタン、ポリアミド、ポリカーボネートおよびその共重合体、並びに、ポリエステル、ポリアクリル酸（一例として、分子量２５万）、ポリメタクリル酸、ポリスチレンスルホン酸塩（一例として、分子量２０万）、ポリアクリルアミドメチルプロパンスルホン酸、末端カルボキシ化ポリエチレングリコール等があげられる。 The synthetic polymer is not particularly limited, and may be, for example, a polymer or a copolymer. Examples of the polymerization form include linear, graft, comb, dendritic, and star shapes. Specific examples include, for example, polyurethane, polyamide, polycarbonate and copolymers thereof, polyester, polyacrylic acid (for example, molecular weight 250,000), polymethacrylic acid, polystyrene sulfonate (for example, molecular weight 200,000), Polyacrylamide methyl propane sulfonic acid, terminal carboxylated polyethylene glycol and the like can be mentioned.
このような第１の物質と第２の物質との組合せとしては、特に制限されず相互作用する異なる物質の組合せであればよく、いずれか一方が正の電荷を有する物質であり、他方が、負の電荷を有する物質であればよい。具体的には、例えば、ε−ポリリジン塩とポリスチレンスルホン酸塩との組合せ、ポリジアリルジメチルアンモニウム塩とポリアクリル酸塩との組合せ、キトサンとデキストラン硫酸との組合せ、ポリアリルアミンハイドロクロライドとポリスチレンスルホン酸塩との組合せ、ε−ポリリジンとポリスチレンスルホン酸塩との組合せ、ポリジアリルジメチルアンモニウムクロライドとポリスチレンスルホン酸塩との組合せ等があげられ、好ましくはε−ポリリジン塩とポリスチレンスルホン酸塩との組合せ、ポリジアリルジメチルアンモニウム塩とポリアクリル酸塩との組合せである。なお、第１の物質と第２の物質とは、それぞれ一種類ずつでもよいし、相互作用を示す範囲で二種類以上をそれぞれ併用してもよい。 Such a combination of the first substance and the second substance is not particularly limited as long as it is a combination of different substances that interact with each other, one of which is a substance having a positive charge, and the other is Any substance having a negative charge may be used. Specifically, for example, a combination of ε-polylysine salt and polystyrene sulfonate, a combination of polydiallyldimethylammonium salt and polyacrylate, a combination of chitosan and dextran sulfate, polyallylamine hydrochloride and polystyrene sulfonic acid A combination of a salt, a combination of ε-polylysine and polystyrene sulfonate, a combination of polydiallyldimethylammonium chloride and polystyrene sulfonate, and the like, preferably a combination of ε-polylysine salt and polystyrene sulfonate, A combination of polydiallyldimethylammonium salt and polyacrylate. Note that one kind of each of the first substance and the second substance may be used, or two or more kinds may be used in combination within a range showing the interaction.
培養細胞１０１の種類は、組織を形成する多細胞生物の細胞であればどのような細胞でも用いることができる。動物細胞であってもよいし、植物細胞であってもよいが、動物細胞が好ましく、哺乳類由来の細胞がより好ましい。哺乳類由来の細胞として、肝細胞、血管内皮細胞、線維芽細胞、表皮細胞、上皮細胞、乳腺細胞、筋細胞、神経細胞、組織幹細胞、胚性幹細胞、骨細胞および免疫細胞等の接着性細胞があげられる。細胞は、一種類でもよいし、二種類以上を用いて共培養を行ってもよい。互いに接着している被覆細胞１０３と培養細胞１０１とが同一種類の細胞であってもよいし、異なる種類の細胞であってもよく、垂直方向に積層され接着している培養細胞１０１と被覆細胞１０３とが異なる細胞であってもよいし、平面視方向で接着している培養細胞１０１と被覆細胞１０３とが同一種類の細胞であってもよいし、異なる細胞であってもよい。 As the type of the cultured cell 101, any cell can be used as long as it is a cell of a multicellular organism that forms a tissue. Although it may be an animal cell or a plant cell, an animal cell is preferred, and a mammal-derived cell is more preferred. Mammal cells include hepatocytes, vascular endothelial cells, fibroblasts, epidermis cells, epithelial cells, mammary cells, muscle cells, nerve cells, tissue stem cells, embryonic stem cells, bone cells and immune cells. can give. One type of cells may be used, or two or more types of cells may be used for co-culture. The coated cell 103 and the cultured cell 101 that are adhered to each other may be the same type of cell or different types of cells, and the cultured cell 101 and the coated cell that are stacked and adhered in the vertical direction. 103 may be a different cell, or the cultured cell 101 and the coated cell 103 adhered in a plan view direction may be the same type of cell or different cells.
本実施の形態の三次元構造体は、基材上に作製されたものであってもよい。基材の種類は何ら制限されず、例えば、その形状、形態、材質等についても特に制限されず、従来公知のものが使用できる。基材の形状は平板なものが好ましく、これによりシート状の三次元構造体を作製することができる。基材の材質としては、特に制限されないが、例えば、ガラス、各種ポリマーからなるプラスチック、ろ紙、金属、ハイドロゲル等があげられる。その形態も、特に制限されず、例えば、非孔質基材、多孔質基材、ファイバー、織布や不織布等の布や、例えば、ハイドロゲル表面、医療用材料表面、人工臓器表面、細胞膜のようなリン脂質膜等の上にも形成できる。 The three-dimensional structure of the present embodiment may be produced on a substrate. The type of the substrate is not limited at all, and for example, its shape, form, material and the like are not particularly limited, and conventionally known materials can be used. The shape of the substrate is preferably a flat plate, whereby a sheet-like three-dimensional structure can be produced. The material of the substrate is not particularly limited, and examples thereof include glass, plastics made of various polymers, filter paper, metal, hydrogel, and the like. The form is not particularly limited, for example, a non-porous substrate, a porous substrate, a fiber, a cloth such as a woven fabric or a nonwoven fabric, for example, a hydrogel surface, a medical material surface, an artificial organ surface, a cell membrane It can also be formed on such phospholipid membranes.
つづいて、本実施の形態に係る細胞の三次元構造体を製造する方法の一例について図２を用いつつ説明する。
まず、培養細胞１０１を用意し（図２（ａ））、その表面全体を接着膜１０２で被覆して被覆細胞１０３を作製する（図２（ｂ））。
Next, an example of a method for producing a three-dimensional cell structure according to the present embodiment will be described with reference to FIG.
First, a cultured cell 101 is prepared (FIG. 2A), and the entire surface thereof is covered with an adhesive film 102 to produce a coated cell 103 (FIG. 2B).
被覆細胞１０３の作製工程を図３を用いてより具体的に説明する。図３で示すように、用意した培養細胞１０１を試験管に入れる（図３（ａ））。ここで用いる試験管としては、細胞の付着をできるだけ抑制しうるものであれば、どのようなものであってもよく、ガラス製であってもよいし、プラスチック製のものを用いてもよい。 The production process of the coated cell 103 will be described more specifically with reference to FIG. As shown in FIG. 3, the prepared cultured cell 101 is put into a test tube (FIG. 3 (a)). The test tube used here may be any tube as long as it can suppress the adhesion of cells as much as possible, and may be made of glass or plastic.
ついで、第１の物質を培養細胞１０１に接触させる（図３（ｂ））。培養細胞１０１と第１の物質との接触方法としては、特に制限されず、例えば、第１の物質を直接添加する方法、第１の物質の含有液に基材を浸漬する方法、培養細胞１０１に第１の物質の含有液を滴下または噴霧する方法等があげられるが、操作が容易であることから浸漬が好ましい。接触の条件は、特に制限されず、接触方法や使用する含有液の濃度等によって適宜決定できる。具体的には、接触時間は、特に制限されないが、例えば、１〜１４４０分であり、好ましくは５〜６０分、より好ましくは１０〜１５分であり、接触温度は、特に制限されないが、例えば、４〜６０℃であり、好ましくは２０〜４０℃、より好ましくは３０〜３７℃である。 Next, the first substance is brought into contact with the cultured cell 101 (FIG. 3B). The method for contacting the cultured cell 101 with the first substance is not particularly limited. For example, the method of directly adding the first substance, the method of immersing the substrate in the liquid containing the first substance, the cultured cell 101 Examples of the method include dripping or spraying the liquid containing the first substance, but immersion is preferable because the operation is easy. The contact conditions are not particularly limited, and can be appropriately determined depending on the contact method, the concentration of the contained liquid used, and the like. Specifically, the contact time is not particularly limited, and is, for example, 1 to 1440 minutes, preferably 5 to 60 minutes, more preferably 10 to 15 minutes, and the contact temperature is not particularly limited. It is 4-60 degreeC, Preferably it is 20-40 degreeC, More preferably, it is 30-37 degreeC.
第１の物質の含有液を調製する場合、溶媒としては、特に制限されないが、水や緩衝液等の水性溶媒があげられ、緩衝液としては、例えば、Ｔｒｉｓ−ＨＣｌ緩衝液等のＴｒｉｓ緩衝液、リン酸緩衝液、ＨＥＰＥＳ緩衝液、クエン酸−リン酸緩衝液、グリシルグリシン−水酸化ナトリウム緩衝液、Ｂｒｉｔｔｏｎ−Ｒｏｂｉｎｓｏｎ緩衝液、ＧＴＡ緩衝液等が使用できる。溶媒のｐＨも、特に制限されないが、例えば、３〜１１であり、好ましくは６〜８であり、より好ましくは７．２〜７．４である。第１の物質の濃度は、特に制限されないが、例えば、０．０００１〜１質量％であり、好ましくは０．０１〜０．５質量％であり、より好ましくは０．０２〜０．１質量％である。第１の物質は、このように溶媒に溶解もしくは分散させることによって、第１の物質の含有液を調製することができる。 When preparing the liquid containing the first substance, the solvent is not particularly limited, and examples thereof include water and an aqueous solvent such as a buffer. Examples of the buffer include Tris buffer such as Tris-HCl buffer. , Phosphate buffer, HEPES buffer, citrate-phosphate buffer, glycylglycine-sodium hydroxide buffer, Britton-Robinson buffer, GTA buffer and the like can be used. The pH of the solvent is not particularly limited, but is, for example, 3 to 11, preferably 6 to 8, and more preferably 7.2 to 7.4. The concentration of the first substance is not particularly limited, but is, for example, 0.0001 to 1% by mass, preferably 0.01 to 0.5% by mass, and more preferably 0.02 to 0.1% by mass. %. The first substance-containing solution can be prepared by dissolving or dispersing the first substance in the solvent as described above.
また、第１の物質の含有液は、さらに、塩化ナトリウム、塩化カルシウム、炭酸水素ナトリウム、酢酸ナトリウム、クエン酸ナトリウム、塩化カリウム、リン酸水素ナトリウム、硫酸マグネシウム、コハク酸ナトリウム等の塩を含有してもよい。いずれか一方の含有液のみでもよいし、両含有液が前記塩を含有してもよい。前記含有液における塩濃度は、特に制限されないが、例えば、１×１０-6〜２Ｍであり、好ましくは１×１０-4〜１Ｍであり、より好ましくは１×１０-4〜０．０５Ｍである。塩は、一種類でもよいし二種類以上を併用してもよい。 The liquid containing the first substance further contains a salt such as sodium chloride, calcium chloride, sodium hydrogen carbonate, sodium acetate, sodium citrate, potassium chloride, sodium hydrogen phosphate, magnesium sulfate, sodium succinate and the like. May be. Only one of the containing liquids may be sufficient, or both containing liquids may contain the salt. Although the salt concentration in the said containing liquid is not restrict | limited in particular, For example, it is 1 * 10 < -6 > -2M, Preferably it is 1 * 10 < -4 > -1M, More preferably, it is 1 * 10 < -4 > -0.05M. is there. One type of salt may be used, or two or more types may be used in combination.
第１の物質の含有液は、さらに、細胞成長因子、サイトカイン、ケモカイン、ホルモン、生理活性ペプチド等を含んでもよい。このように成長因子等を含有することによって、細胞層の形成の際に、例えば、細胞の増殖速度や増殖程度を調節することも可能となる。 The liquid containing the first substance may further contain a cell growth factor, cytokine, chemokine, hormone, bioactive peptide, and the like. By containing a growth factor or the like in this way, it is possible to adjust, for example, the cell growth rate and the degree of proliferation when the cell layer is formed.
また、第１の物質の含有液は、さらに、疾患の治療剤、予防剤、抑制剤、抗菌剤、抗炎症剤等の医薬組成物を含有してもよい。このような含有液を用いて製造した三次元組織は、前述のような医薬組成物を含むことから、例えば、ヒトやヒトを除く哺乳類、その他の動物の体内等に移植することによって、疾患の治療や予防を行うこともできる。 Further, the liquid containing the first substance may further contain a pharmaceutical composition such as a therapeutic agent, preventive agent, inhibitor, antibacterial agent, and anti-inflammatory agent for diseases. Since the three-dimensional tissue produced using such a containing liquid contains the pharmaceutical composition as described above, for example, it can be transplanted into the body of a human, mammals other than humans, other animals, etc. Treatment and prevention can also be performed.
このようにして培養細胞１０１を第１の物質からなる膜１０２ａにより被覆した後、遊離している第１の物質を除去する。第１の物質を除去する方法としては、特に制限されないが、上記の溶媒を用いて遠心分離を行い、培養細胞１０１と第１の物質の含有液とを分離して上澄みを取り除くことで、遊離している第１の物質を除去する方法が挙げられる（図３（ｃ））。こうすることで、第１の物質からなる膜１０２ａで被覆された被覆細胞を得ることができる。 Thus, after covering the cultured cell 101 with the film | membrane 102a which consists of a 1st substance, the free | released 1st substance is removed. The method for removing the first substance is not particularly limited, but it is released by centrifuging using the above solvent, separating the cultured cells 101 and the liquid containing the first substance, and removing the supernatant. A method of removing the first substance is shown (FIG. 3C). By doing so, coated cells coated with the film 102a made of the first substance can be obtained.
接着膜１０２として、第１の物質からなる膜１０２ａと第１の物質とは異なる第２の物質からなる膜１０２ｂとからなる積層膜を形成させる場合、第１の物質で被覆された培養細胞１０１を第２の物質と接触させることにより、第２の物質からなる膜１０２ｂで被覆する（図３（ｄ））。第２の物質との接触方法は、第１の物質と同様な方法で行うことができ、第２の物質の含有液を用いて接触させる場合は、第１の物質の含有液を調製する方法と同様にして第２の物質の含有液を調製することができる。 In the case where a stacked film including a film 102a made of the first substance and a film 102b made of the second substance different from the first substance is formed as the adhesive film 102, the cultured cell 101 coated with the first substance is formed. Is brought into contact with the second substance, thereby covering with a film 102b made of the second substance (FIG. 3D). The contact method with the second substance can be carried out in the same manner as the first substance. When the second substance-containing liquid is used for contact, the first substance-containing liquid is prepared. A liquid containing the second substance can be prepared in the same manner as described above.
こうすることで、第１の物質からなる膜１０２ａと、第２の物質からなる膜１０２ｂとからなる接着膜１０２で被覆された培養細胞１０１が被覆細胞１０３として得られる。 By doing so, the cultured cell 101 covered with the adhesive film 102 made of the film 102 a made of the first substance and the film 102 b made of the second substance is obtained as the coated cell 103.
なお、第１の物質及び第２の物質として用いる材料としては前述したものを用いることができるが、ＲＧＤ配列を含む高分子と、ＲＧＤ配列を含む高分子と相互作用する高分子とを組み合わせて用いる場合は、第１の物質としてＲＧＤ配列を含む高分子を用い、第２の物質としてＲＧＤ配列を含む高分子と相互作用する高分子を用いることが好ましい。具体的には、最初にＲＧＤ配列を含む高分子からなる膜（１０２ａ）を形成した後、ＲＧＤ配列を含む高分子と相互作用する高分子からなる膜（１０２ｂ）を形成させることが好ましい。また、正の電荷を有する高分子と、負の電荷を有する高分子とを組み合わせて用いる場合は、第１の物質として正の電荷を有する高分子を用い、第２の物質として負の電荷を有する高分子を用いることが好ましい。具体的には、最初に正の電荷を有する高分子からなる膜（１０２ａ）を形成した後、負の電荷を有する高分子からなる膜（１０２ｂ）を形成させることが好ましい。 Note that the above-described materials can be used as the first substance and the second substance, but a combination of a polymer containing an RGD sequence and a polymer that interacts with a polymer containing an RGD sequence is used. When used, it is preferable to use a polymer containing an RGD sequence as the first substance and a polymer that interacts with a polymer containing the RGD sequence as the second substance. Specifically, it is preferable to first form a film (102a) made of a polymer containing an RGD sequence and then form a film (102b) made of a polymer that interacts with the polymer containing an RGD sequence. When a polymer having a positive charge and a polymer having a negative charge are used in combination, a polymer having a positive charge is used as the first substance and a negative charge is given as the second substance. It is preferable to use a polymer having the same. Specifically, it is preferable to first form a film (102a) made of a polymer having a positive charge and then form a film (102b) made of a polymer having a negative charge.
例えば、第１の物質からなる膜１０２ａとしてフィブロネクチンからなる膜を形成した後、第２の物質からなる膜１０２ｂとしてゼラチン、ε−ポリリジン、ヒアルロン酸及びデキストラン硫酸の少なくとも一種からなる膜を形成する方法や、第１の物質からなる膜１０２ａとしてε−ポリリジン塩（特にε−ポリリジンの塩酸塩）からなる膜を形成した後、第２の物質からなる膜１０２ｂとしてポリスチレンスルホン酸塩（特にナトリウム塩）を形成する方法、第１の物質からなる膜１０２ａとしてポリジアリルジメチルアンモニウム塩（特にクロライド塩）を形成した後、第２の物質からなる膜１０２ｂとしてポリアクリル酸塩（特にナトリウム塩）を形成する方法が挙げられる。 For example, after forming a film made of fibronectin as the film 102a made of the first substance, a film made of at least one of gelatin, ε-polylysine, hyaluronic acid, and dextran sulfate is formed as the film 102b made of the second substance. Alternatively, after forming a film made of ε-polylysine salt (particularly ε-polylysine hydrochloride) as the film 102a made of the first substance, polystyrene sulfonate (particularly sodium salt) is made as the film 102b made of the second substance. After forming polydiallyldimethylammonium salt (especially chloride salt) as the film 102a made of the first substance, polyacrylic acid salt (especially sodium salt) is formed as the film 102b made of the second substance. A method is mentioned.
培養細胞１０１の表面の全面を接着膜１０２で覆うことができれば、本発明の効果を得ることはできる。そのため、第１の物質からなる膜１０２ａと第２の物質からなる膜１０２ｂとはそれぞれ１層ずつ形成させて、培養細胞１０１の表面の全面を接着膜１０２で覆うことができればよいが、第２の物質で被覆された培養細胞に第１の物質をさらに接触させて第１の物質からなる膜１０２ａで被覆し（図３（ｂ））、第１の物質からなる膜１０２ａと第２の物質からなる膜１０２ｂとを交互に積層させる（図３（ｂ）〜（ｄ））ことで、確実に培養細胞１０１の表面の全面を接着膜１０２で覆うことができる。 If the entire surface of the cultured cell 101 can be covered with the adhesive film 102, the effect of the present invention can be obtained. Therefore, the film 102a made of the first substance and the film 102b made of the second substance may be formed one by one so that the entire surface of the cultured cell 101 can be covered with the adhesive film 102. The first substance is further brought into contact with the cultured cells coated with the substance, and the film is coated with the film 102a made of the first substance (FIG. 3B), and the film 102a made of the first substance and the second substance are covered. By alternately laminating the film 102b made of (FIGS. 3B to 3D), the entire surface of the cultured cell 101 can be reliably covered with the adhesive film 102.
また、接着膜１０２は一層でも本発明の効果を得ることができるが、薄膜を積層させることでより密な細胞構造体を得ることができる。そこで、第２の物質で被覆された培養細胞にさらに第１の物質を接触させ、図３（ｂ）で示す第１の物質との接触工程と、図３（ｄ）で示す第２の物質との接触工程とを繰り返す。こうすることで、第１の物質からなる膜１０２ａと第２の物質からなる膜１０２ｂとを交互に積層させ、所望の厚みからなる接着膜１０２を形成させることができる。図３（ｂ）で示す第１の物質との接触工程と、図３（ｄ）で示す第２の物質との接触工程は、５段階以上繰り返すと好ましく、９段階以上繰り返すとより好ましい。こうすることで、３ｎｍ以上１×１０２ｎｍ以下の厚みの接着膜１０２を形成させることができる。 The adhesive film 102 can obtain the effects of the present invention even with a single layer, but a denser cell structure can be obtained by laminating thin films. Therefore, the first substance is further brought into contact with the cultured cells coated with the second substance, and the contact process with the first substance shown in FIG. 3B and the second substance shown in FIG. Repeat the contact process. Thus, the adhesive film 102 having a desired thickness can be formed by alternately laminating the film 102a made of the first substance and the film 102b made of the second substance. The contact process with the first substance shown in FIG. 3 (b) and the contact process with the second substance shown in FIG. 3 (d) are preferably repeated in 5 stages or more, more preferably in 9 stages or more. By doing so, the adhesive film 102 having a thickness of 3 nm or more and 1 × 10 2 nm or less can be formed.
図２に戻り、得られた被覆細胞１０３を基材に播種する（図２（ｃ））。基材の種類は何ら制限されず、例えば、その形状、形態、材質等についても特に制限されず、従来公知のものが使用できる。基材の材質としては、特に制限されないが、例えば、ガラス、各種ポリマー、ろ紙、金属、ハイドロゲル等があげられる。その形態も、特に制限されず、例えば、非孔質基材、多孔質基材、ファイバー、織布や不織布等の布等があげられる。また、例えば、ハイドロゲル表面、医療用材料表面、人工臓器表面、細胞膜のようなリン脂質膜等の上にも形成できる。 Returning to FIG. 2, the obtained coated cells 103 are seeded on a substrate (FIG. 2 (c)). The type of the substrate is not limited at all, and for example, its shape, form, material and the like are not particularly limited, and conventionally known materials can be used. The material for the substrate is not particularly limited, and examples thereof include glass, various polymers, filter paper, metal, and hydrogel. The form is not particularly limited, and examples thereof include non-porous substrates, porous substrates, fibers, fabrics such as woven fabrics and nonwoven fabrics, and the like. For example, it can be formed on a hydrogel surface, a medical material surface, an artificial organ surface, a phospholipid membrane such as a cell membrane, and the like.
基材には、細胞の培養に必要な培地を形成し、培地上に被覆細胞１０３を播種させてもよい。培地は特に制限されず、細胞に応じて適宜使用でき、例えば、Ｅａｇｌｅ'ｓ ＭＥＭ培地、Ｄｕｌｂｅｃｃｏ'ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ培地 (ＤＭＥＭ)、Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ培地(ＭＥＭ)、Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ培地、ＲＤＭＩ、ＧｌｕｔａＭａｘ培地、無血清培地等が使用できる。播種する細胞数は、特に制限されないが、例えば、０．０１×１０4〜１００×１０4ｃｅｌｌｓ／ｃｍ2であり、好ましくは１×１０4〜１０×１０4ｃｅｌｌｓ／ｃｍ2である。 A medium necessary for cell culture may be formed on the substrate, and the coated cells 103 may be seeded on the medium. The medium is not particularly limited, and can be appropriately used depending on the cell.For example, Eagle's MEM medium, Dulbecco's Modified Eagle medium (DMEM), Modified Eagle medium (MEM), Minimum Essential medium, RDMI, GlutaMax medium, A serum medium or the like can be used. The number of cells to be seeded is not particularly limited, but is, for example, 0.01 × 10 4 to 100 × 10 4 cells / cm 2 , and preferably 1 × 10 4 to 10 × 10 4 cells / cm 2 .
その後、培養細胞１０１表面全体が接着層１０２で被覆された被覆細胞１０３を培養する（図２（ｄ））。細胞の培養条件は、特に制限されず、培養する細胞に応じて適宜決定できる。一般的な条件としては、培養温度が、例えば、４〜６０℃であり、好ましくは２０〜４０℃、より好ましくは３０〜３７℃であり、培養時間は、例えば、１〜１６８時間であり、好ましくは３〜２４時間、より好ましくは３〜１２時間である。これにより、接着膜１０２を介して、被覆細胞１０３同士を接着させたり、被覆細胞１０３から増殖した培養細胞１０１と被覆細胞１０３とを互いに接着させたりして、細胞の三次元構造体を製造することができる。 Thereafter, the coated cell 103 in which the entire surface of the cultured cell 101 is coated with the adhesive layer 102 is cultured (FIG. 2D). Cell culture conditions are not particularly limited, and can be determined appropriately according to the cells to be cultured. As general conditions, culture | cultivation temperature is 4-60 degreeC, for example, Preferably it is 20-40 degreeC, More preferably, it is 30-37 degreeC, Culture | cultivation time is 1-168 hours, for example, Preferably it is 3-24 hours, More preferably, it is 3-12 hours. Thereby, the coated cells 103 are adhered to each other through the adhesive film 102, or the cultured cells 101 and the coated cells 103 grown from the coated cells 103 are adhered to each other to produce a three-dimensional structure of cells. be able to.
三次元構造体の厚みは、培養時間により制御させることができるが、例えば、２４時間培養させることで、シート状の三次元構造体を７層以上形成させることができ、厚みは、１０μｍ以上１００μｍ以下にすることができる。 Although the thickness of the three-dimensional structure can be controlled by the culture time, for example, by culturing for 24 hours, seven or more layers of the sheet-like three-dimensional structure can be formed, and the thickness is 10 μm or more and 100 μm. It can be:
接着膜１０２を形成させる成分（例えば、第１の物質及び第２の物質）と、上記説明した三次元構造体の製造方法を記載した使用説明書とを備えることで、本発明の三次元構造体を製造するためのキットとすることができる。こうしたキットには、さらに基材を備えてもよく、板状の基材を備えることで、シート状の細胞の三次元構造体を製造するためのキットとすることができる。 The three-dimensional structure of the present invention is provided with a component (for example, a first substance and a second substance) for forming the adhesive film 102 and an instruction manual describing the manufacturing method of the three-dimensional structure described above. It can be set as the kit for manufacturing a body. Such a kit may further include a base material, and by providing a plate-shaped base material, a kit for producing a sheet-like three-dimensional structure of cells can be obtained.
また、こうして得られた細胞の三次元構造体の上にさらに培養細胞からなる細胞層を積層させてもよい。このとき用いられる培養細胞としては、被覆細胞１０３を構成する培養細胞１０１と同種であっても異種であってもよいし、接着膜１０２で被覆された被覆細胞１０３であってもよいし、接着膜１０２で被覆されていない培養細胞１０１であってもよい。得られる三次元構造体を用いて薬物の細胞応答を評価することができる。こうした薬物の細胞応答評価は特に限定されず、例えば、皮膚細胞を培養させて化粧品のアレルギー反応の評価に用いることもできる。 In addition, a cell layer made of cultured cells may be further laminated on the three-dimensional structure of cells thus obtained. The cultured cells used at this time may be the same or different from the cultured cells 101 constituting the coated cells 103, may be the coated cells 103 coated with the adhesive film 102, or may be adhered. The cultured cells 101 not covered with the membrane 102 may be used. The resulting three-dimensional structure can be used to evaluate the cellular response of the drug. The cell response evaluation of such drugs is not particularly limited. For example, skin cells can be cultured and used for evaluation of allergic reactions in cosmetics.
また、本発明の細胞の三次元構造体の上にヒトやヒト以外のほ乳類から分離した肝細胞、血管内皮細胞、線維芽細胞、表皮細胞、上皮細胞、乳腺細胞、筋細胞、神経細胞、組織幹細胞、胚性幹細胞、骨細胞、歯髄細胞および免疫細胞等の接着性細胞を培養させてもよい。こうすることで、異常な組織を除去して健常な組織のみを再生し、ヒトやヒト以外のほ乳類の体内に戻すことで治療に役立てることができる。また、血液、皮膚、歯、骨などの再生にも役立てることができる。 In addition, hepatocytes, vascular endothelial cells, fibroblasts, epidermal cells, epithelial cells, mammary cells, muscle cells, nerve cells, tissues isolated from humans or non-human mammals on the three-dimensional structure of the cells of the present invention Adherent cells such as stem cells, embryonic stem cells, bone cells, dental pulp cells and immune cells may be cultured. In this way, abnormal tissues can be removed, only healthy tissues can be regenerated, and returned to the body of humans or non-human mammals, which can be used for treatment. It can also be used to regenerate blood, skin, teeth, bones, and the like.
なお、これら細胞の培養条件は、前述と同様である。 The culture conditions for these cells are the same as described above.
（第２の実施形態）
図１０は、本実施の形態に係る三次元構造体の一例を示す模式的な断面図である。本実施の形態では、第１の実施形態と異なる点を説明し、第１の実施形態と同様な説明は適宜省略する。本実施の形態での三次元構造体には、第１の実施形態において説明した培養細胞１０１として、第一培養細胞１０１ａ及び第一培養細胞１０１ａとは異なる第二培養細胞１０１ｂが用いられる。また、第１の実施形態において被覆細胞１０３として、第一培養細胞１０１ａが接着膜１０２で被覆された第一被覆細胞１０３ａ、及び、第二培養細胞１０１ｂが接着膜１０２で被覆された第二被覆細胞１０３ｂが用いられる。そして、第一培養細胞１０１ａと第一被覆細胞１０３ａとが接着膜１０２を介して互いに接着して第一の積層構造１０を形成し、第二培養細胞１０１ｂと第二被覆細胞１０３ｂとが接着膜１０２を介して互いに接着している第二の積層構造２０を形成し、第一の積層構造１０上に第二の積層構造２０が積層されることで三次元構造体が構築されている。
(Second Embodiment)
FIG. 10 is a schematic cross-sectional view showing an example of the three-dimensional structure according to the present embodiment. In the present embodiment, differences from the first embodiment will be described, and descriptions similar to those of the first embodiment will be omitted as appropriate. In the three-dimensional structure in the present embodiment, the first cultured cell 101a and the second cultured cell 101b different from the first cultured cell 101a are used as the cultured cell 101 described in the first embodiment. In the first embodiment, as the coated cell 103, the first coated cell 103a in which the first cultured cell 101a is coated with the adhesive film 102 and the second coated cell in which the second cultured cell 101b is coated with the adhesive film 102 Cells 103b are used. Then, the first cultured cell 101a and the first coated cell 103a adhere to each other through the adhesive film 102 to form the first laminated structure 10, and the second cultured cell 101b and the second coated cell 103b are bonded to each other. A second stacked structure 20 bonded to each other through 102 is formed, and the second stacked structure 20 is stacked on the first stacked structure 10 to construct a three-dimensional structure.
第一培養細胞１０１ａ及び第二培養細胞１０１ｂは、互いに異なる細胞であればよく、第１の実施の形態で培養細胞１０１として例示された任意の細胞を組み合わせて用いることができる。 The first cultured cell 101a and the second cultured cell 101b may be different cells, and any cell exemplified as the cultured cell 101 in the first embodiment can be used in combination.
接着膜１０２は、第１の実施形態で説明した接着膜１０２と同様にすることができる。具体的には、本実施の形態の接着膜１０２もまた、第１の実施の形態と同様に、第１の物質を含む膜１０２ａと第２の物質を含む膜１０２ｂとが積層された構成を採用することができる。第一被覆細胞１０３ａと第二の被覆細胞１０３ｂとは、接着膜１０２の構成が同じであってもよいし、異なっていてもよいが、同じであることが好ましい。 The adhesive film 102 can be the same as the adhesive film 102 described in the first embodiment. Specifically, the adhesive film 102 of this embodiment also has a structure in which a film 102a containing a first substance and a film 102b containing a second substance are stacked, as in the first embodiment. Can be adopted. The first coated cell 103a and the second coated cell 103b may have the same or different structure of the adhesive film 102, but are preferably the same.
第一の積層構造１０と第二の積層構造２０との間には、ナノ細胞外マトリックス（ナノＥＣＭ）３０が形成されており、第一の積層構造１０と第二の積層構造２０とを接着している。ナノＥＣＭ３０は、接着膜１０２と同成分から構築されており、好ましくは、第１の実施形態で説明した、第１の物質と第２の物質とが交互に積層されることで形成されている。第１の物質及び第２の物質の種類は、第１培養細胞１０１ａや第２培養細胞１０１ｂを覆う接着膜１０２の構成成分と同じであってもよいし異なってもよいが、同じにするのが好ましい。ナノＥＣＭ３０の厚みは、特に制限されないが、１ｎｍ以上が好ましく、１〜１０００ｎｍがより好ましく、１〜１００ｎｍがさらに好ましい。 A nano-extracellular matrix (nano-ECM) 30 is formed between the first laminated structure 10 and the second laminated structure 20, and the first laminated structure 10 and the second laminated structure 20 are bonded to each other. is doing. The nano-ECM 30 is constructed from the same components as the adhesive film 102, and is preferably formed by alternately laminating the first substance and the second substance described in the first embodiment. . The types of the first substance and the second substance may be the same as or different from the constituent components of the adhesive film 102 that covers the first cultured cell 101a and the second cultured cell 101b. Is preferred. The thickness of the nano ECM 30 is not particularly limited, but is preferably 1 nm or more, more preferably 1 to 1000 nm, and further preferably 1 to 100 nm.
つづいて、図１１を用いて本実施の形態の三次元構造体の製造方法について説明する。まず、第１の実施形態で説明した方法と同様に、第一培養細胞１０１ａに、第１の物質からなる膜１０２ａと第２の物質からなる膜１０２ｂとを積層させて、第一培養細胞１０１ａが接着膜１０２で被覆された第一被覆細胞１０３ａを形成する（図１１（ａ））。また、同様に、第二培養細胞１０１ｂに、第１の物質からなる膜１０２ａと第２の物質からなる膜１０２ｂとを積層させて、第二培養細胞１０１ｂが接着膜１０２で被覆された第二被覆細胞１０３ｂを形成する。 Next, a manufacturing method of the three-dimensional structure according to the present embodiment will be described with reference to FIG. First, similarly to the method described in the first embodiment, the first cultured cell 101a is formed by laminating the film 102a made of the first substance and the film 102b made of the second substance on the first cultured cell 101a. Form a first coated cell 103a coated with an adhesive film 102 (FIG. 11A). Similarly, the second cultured cell 101b is laminated with the film 102a made of the first substance and the film 102b made of the second substance, and the second cultured cell 101b is covered with the adhesive film 102. Coated cells 103b are formed.
ついで、第一被覆細胞１０３ａを基材に播種する。基材及び播種する細胞の数は、第１の実施形態と同様にすればよい。 Next, the first coated cells 103a are seeded on the substrate. The number of substrates and the number of cells to be seeded may be the same as in the first embodiment.
その後、第１の実施形態と同様に第一被覆細胞１０３ａを培養し、第一の積層構造１０を形成する（第一の培養工程、図１１（ｂ））。 Thereafter, the first coated cells 103a are cultured in the same manner as in the first embodiment to form the first laminated structure 10 (first culture step, FIG. 11B).
ここで、第一の積層構造１０の厚みは、１０μｍ以上１００μｍ以下にすることが好ましい。こうすることで、第一被覆細胞１０３ａを良好に培養させることができる。これにより、数時間の培養で、１〜１０層のシート状の第一の積層構造１０を、一度に作成することができる。 Here, the thickness of the first laminated structure 10 is preferably 10 μm or more and 100 μm or less. By doing so, the first coated cells 103a can be cultured well. Thereby, 1 to 10 layers of sheet-like first laminated structure 10 can be formed at a time by culturing for several hours.
ついで、接着膜１０２の構成材料を第１の積層構造１０の表面に接触させて、ナノ細胞外マトリックス（ナノＥＣＭ）３０を形成する（図１１（ｃ））。ナノＥＣＭ３０を形成する方法としては、例えば、第１の実施形態で説明した第１の物質と第２の物質とを交互に積層させる方法がある。具体的には、第１の物質と第２の物質をそれぞれ含む溶液に交互に浸積する方法が挙げられる。ナノＥＣＭ３０は、例えば、特許文献１記載の技術を用いて形成してもよい。 Next, the constituent material of the adhesive film 102 is brought into contact with the surface of the first laminated structure 10 to form a nano extracellular matrix (nano ECM) 30 (FIG. 11C). As a method of forming the nano ECM 30, for example, there is a method of alternately laminating the first substance and the second substance described in the first embodiment. Specifically, a method of alternately immersing in a solution containing each of the first substance and the second substance can be given. Nano ECM30 may be formed using the technique of patent document 1, for example.
その後、あらかじめ、第１の実施形態で説明した方法と同様に作製された第二被覆細胞１０３ｂをナノＥＣＭ３０上に播種する。このとき播種する細胞の密度も第１の実施の形態と同様にすることができる。ついで、第一被覆細胞１０３ａと同様に第二被覆細胞１０３ｂを培養し、第一の積層構造１０上に第二の積層構造２０を形成する（第二の培養工程、図１１（ｄ））。 Thereafter, the second coated cells 103b prepared in the same manner as in the method described in the first embodiment are seeded on the nano ECM 30 in advance. At this time, the density of the cells to be seeded can be the same as that in the first embodiment. Next, the second coated cell 103b is cultured in the same manner as the first coated cell 103a, and the second laminated structure 20 is formed on the first laminated structure 10 (second culturing step, FIG. 11 (d)).
第二の積層構造２０の厚みもまた、１０μｍ以上１００μｍ以下にすることが好ましい。これにより、数時間の培養で、１〜１０層のシート状の第二の積層構造２０を、一度に作成することができる。 The thickness of the second laminated structure 20 is also preferably 10 μm or more and 100 μm or less. Thereby, 1 to 10 layers of sheet-like second laminated structure 20 can be formed at a time in culture for several hours.
この後、さらに第二の積層構造２０にナノＥＣＭ３０を形成した後に第一の被覆細胞１０を播種し、第一の培養工程を繰り返してもよい。こうすることで、第二の積層構造２０上に、第一の積層構造１０を形成させることができる（図１２）。 Thereafter, after the nano-ECM 30 is further formed on the second laminated structure 20, the first coated cells 10 may be seeded and the first culture step may be repeated. By doing so, the first laminated structure 10 can be formed on the second laminated structure 20 (FIG. 12).
図１２の三次元構造体においては、例えば、第一培養細胞１０１ａとして繊維芽細胞を用い、第二培養細胞１０１ｂとして血管内皮細胞を用いることができる。このとき、図１２で示す三次元構造体を、好ましくは３５〜３９℃、より好ましくは３７℃の温度条件下で、０．５日〜７日間培養してもよい。これにより、第二の積層構造２０には、血管内皮細胞から血管枝が成長し、これを分岐して組織化させることができる。したがって、第二の積層構造２０に血管網を形成させることも可能である。 In the three-dimensional structure of FIG. 12, for example, fibroblasts can be used as the first cultured cells 101a, and vascular endothelial cells can be used as the second cultured cells 101b. At this time, the three-dimensional structure shown in FIG. 12 may be cultured at a temperature of preferably 35 to 39 ° C., more preferably 37 ° C., for 0.5 to 7 days. Thereby, a blood vessel branch grows from the vascular endothelial cell in the second laminated structure 20 and can be branched and organized. Therefore, it is possible to form a vascular network in the second laminated structure 20.
このように本実施の形態においても、三次元配置のバリエーションが豊富な細胞組織を短時間で作製することができる。 As described above, also in this embodiment, a cell tissue rich in three-dimensional arrangement variations can be produced in a short time.
（第３の実施形態）
図１３は、本実施の形態に係る三次元構造体の一例を示す模式的な断面図である。本実施の形態では、第１の実施形態と異なる点を説明し、第１の実施形態と同様な説明は適宜省略する。本実施の形態の三次元構造体には、第１の実施形態において説明した培養細胞１０１として、第一培養細胞１０１ａ及び第一培養細胞１０１ａとは異なる第二培養細胞１０１ｂが用いられる。また、第１の実施形態において被覆細胞１０３として、第一培養細胞１０１ａが接着膜１０２で被覆された第一被覆細胞１０３ａ、及び、第二培養細胞１０１ｂが接着膜１０２で被覆された第二被覆細胞１０３ｂが用いられる。そして、第一被覆細胞１０３ａと第二培養細胞１０１ｂとが接着膜１０２を介して互いに接着し、第二被覆細胞１０３ｂと第一培養細胞１０１ａとが接着膜１０２を介して互いに接着することで、三次元構造体が構築されている。
(Third embodiment)
FIG. 13 is a schematic cross-sectional view showing an example of the three-dimensional structure according to the present embodiment. In the present embodiment, differences from the first embodiment will be described, and descriptions similar to those of the first embodiment will be omitted as appropriate. In the three-dimensional structure of the present embodiment, the first cultured cell 101a and the second cultured cell 101b different from the first cultured cell 101a are used as the cultured cell 101 described in the first embodiment. In the first embodiment, as the coated cell 103, the first coated cell 103a in which the first cultured cell 101a is coated with the adhesive film 102 and the second coated cell in which the second cultured cell 101b is coated with the adhesive film 102 Cells 103b are used. Then, the first coated cell 103a and the second cultured cell 101b adhere to each other through the adhesive film 102, and the second coated cell 103b and the first cultured cell 101a adhere to each other through the adhesive film 102, A three-dimensional structure is constructed.
第一培養細胞１０１ａ及び第二培養細胞１０１ｂは、互いに異なる細胞であればよく、第１の実施の形態で培養細胞１０１として例示された任意の細胞を組み合わせて用いることができる。 The first cultured cell 101a and the second cultured cell 101b may be different cells, and any cell exemplified as the cultured cell 101 in the first embodiment can be used in combination.
接着膜１０２は、第１の実施形態で説明した接着膜１０２と同様にすることができる。具体的には、本実施の形態の接着膜１０２もまた、第１の実施の形態と同様に、第１の物質を含む膜１０２ａと第２の物質を含む膜１０２ｂとが積層された構成を採用する。第一被覆細胞１０３ａと第二の被覆細胞１０３ｂとは、接着膜１０２の構成が同じであってもよいし、異なっていてもよいが、同じであることがより好ましい。 The adhesive film 102 can be the same as the adhesive film 102 described in the first embodiment. Specifically, the adhesive film 102 of this embodiment also has a structure in which a film 102a containing a first substance and a film 102b containing a second substance are stacked, as in the first embodiment. adopt. The first coated cell 103a and the second coated cell 103b may have the same or different structure of the adhesive film 102, but are more preferably the same.
つづいて、本実施の形態の三次元構造体の製造方法について説明する。まず、第１の実施形態で説明した方法と同様に、第一培養細胞１０１に、第１の物質からなる膜１０２ａと第２の物質からなる膜１０２ｂとを積層させて、第一培養細胞１０１ａが接着膜１０２で被覆された第一被覆細胞１０３ａを形成する。また、第１の物質からなる膜１０２ａと第２の物質からなる膜１０２ｂとを積層させて、第二培養細胞１０１ｂが接着膜１０２で被覆された第二被覆細胞１０３ｂを形成する。 Next, a method for manufacturing the three-dimensional structure according to the present embodiment will be described. First, similarly to the method described in the first embodiment, the first cultured cell 101a is formed by laminating the film 102a made of the first substance and the film 102b made of the second substance on the first cultured cell 101. Forms a first coated cell 103a coated with an adhesive film 102. In addition, the film 102 a made of the first substance and the film 102 b made of the second substance are laminated to form the second coated cell 103 b in which the second cultured cell 101 b is covered with the adhesive film 102.
ついで、第一被覆細胞１０３ａ及び第二被覆細胞１０３ｂを混合して基材に播種する。基材及び播種する細胞の数も第１の実施の形態と同様にすればよい。ついで、第１の実施形態と同様に第一被覆細胞１０３ａ及び第二被覆細胞１０３ｂを培養し、図１３で示す三次元構造体を形成する。 Next, the first coated cell 103a and the second coated cell 103b are mixed and seeded on a substrate. The number of base materials and cells to be seeded may be the same as in the first embodiment. Next, as in the first embodiment, the first coated cell 103a and the second coated cell 103b are cultured to form the three-dimensional structure shown in FIG.
ここで、図１３で示す三次元構造体の厚みは、１０μｍ以上１００μｍ以下にすることが好ましい。こうすることで、第一被覆細胞１０３ａ及び第二被覆細胞１０３ｂを良好に培養させることができる。これにより、数時間の培養で、１〜１０層のシート状の細胞構造体を、一度に作成することができる。 Here, the thickness of the three-dimensional structure shown in FIG. 13 is preferably 10 μm or more and 100 μm or less. By carrying out like this, the 1st covering cell 103a and the 2nd covering cell 103b can be cultured favorably. Thereby, a 1-10 layer sheet-like cell structure can be produced at a time by culture | cultivation for several hours.
図１３の三次元構造体において、例えば、第一培養細胞１０１ａとして繊維芽細胞を用い、第二培養細胞１０１ｂとして血管内皮細胞を用いることができる。このとき、図１３で示す三次元構造体を、好ましくは３５〜３９℃、より好ましくは３７℃の温度条件下で、０．５日〜７日間培養してもよい。これにより、第２の実施形態に比して効率は劣る一方、接着膜１０２を介して繊維芽細胞が接着した血管内皮細胞から血管枝が成長し、これが分岐して組織化し、血管網を形成させることが可能である。 In the three-dimensional structure of FIG. 13, for example, fibroblasts can be used as the first cultured cells 101a, and vascular endothelial cells can be used as the second cultured cells 101b. At this time, the three-dimensional structure shown in FIG. 13 may be cultured at a temperature of preferably 35 to 39 ° C., more preferably 37 ° C., for 0.5 to 7 days. As a result, the efficiency is inferior to that of the second embodiment, but a vascular branch grows from a vascular endothelial cell to which fibroblasts adhere via the adhesive film 102, and this branches and organizes to form a vascular network. It is possible to make it.
このように本実施の形態においても、三次元配置のバリエーションが豊富な細胞組織を短時間で作製することができる。 As described above, also in this embodiment, a cell tissue rich in three-dimensional arrangement variations can be produced in a short time.
以上、図面を参照して本発明の実施形態について述べたが、これらは本発明の例示であり、上記以外の様々な構成を採用することもできる。 As mentioned above, although embodiment of this invention was described with reference to drawings, these are the illustrations of this invention, Various structures other than the above are also employable.
（実施例１）
第１の物質としてフィブロネクチンを用い、第２の物質としてゼラチンを用い、フィブロネクチンとゼラチンとからなる接着膜１０２で細胞表面全面を被覆したヒト繊維芽細胞（Ｃａｍｂｒｅｘ社製、正常ヒト皮膚繊維芽細胞 ＮＨＤＦ）を用いて図１に示す細胞の三次元構造体を作製した。作製方法は、図２、３に示す方法に従った。具体的には、１ウェルあたりヒト繊維芽細胞４×１０６個をそれぞれ用意し、０．２ｍｇ／ｍＬのフィブロネクチン（ウシ由来）（シグマアルドリッチ社製、Ｆ−４７５９）の５０ｍＭトリス緩衝液（ｐＨ７．４）１ｍＬを加えて３７℃で１分間インキュベーションした。その後、２０００ｒｐｍで２分間遠心分離を行い、上澄みを除去した。続いて、０．２ｍｇ／ｍＬのゼラチン（和光純薬工業社製、０７７−０３１５５）の５０ｍＭトリス緩衝液（ｐＨ７．４）１ｍＬを加えて３７℃で１分間インキュベートした。その後、２０００ｒｐｍで２分間遠心分離を行い、上澄みを除去した。これを１ステップとして、９ステップ実行し、フィブロネクチンからなる膜とゼラチンからなる膜とを交互に積層して、膜厚６ｎｍの接着膜をヒト繊維芽細胞に形成した。なお、膜厚は、位相差顕微鏡により１０箇所測定し、得られた測定値を平均化することで求めた。その後、接着膜で被覆されたヒト繊維芽細胞２×１０６個をセルカルチャーインサート（ベクトンディッキンソン社製、３５３０９０）のメンブレンフィルターに播種し、さらに、１０重量％ウシ胎仔血清（ＦＡＢ）を含むＥａｇｌｅ'ｓＭＥＭ培地を添加してから３７℃で４８時間インキュベートして培養した。これにより厚み４０μｍのシート状のヒト繊維芽細胞の三次元構造体が得られた。得られた実施例１の三次元組織をＨＥ染色した後、位相差顕微鏡より評価した。図４に結果を示す。図４で図示するように、細胞の重なりが観察された。なお、２４時間インキュベート後には、７層のヒト繊維芽細胞の三次元構造体が観察された。
Example 1
Human fibroblasts using fibronectin as the first substance, gelatin as the second substance, and the entire cell surface covered with an adhesive film 102 composed of fibronectin and gelatin (manufactured by Cambrex, normal human skin fibroblasts NHDF The three-dimensional structure of the cell shown in FIG. The manufacturing method followed the method shown in FIGS. Specifically, 4 × 10 6 human fibroblasts were prepared per well, and 50 mM Tris buffer (pH 7) of 0.2 mg / mL fibronectin (derived from bovine) (manufactured by Sigma-Aldrich, F-4759). .4) 1 mL was added and incubated at 37 ° C. for 1 minute. Thereafter, centrifugation was performed at 2000 rpm for 2 minutes, and the supernatant was removed. Subsequently, 1 mL of 50 mg Tris buffer (pH 7.4) of 0.2 mg / mL gelatin (manufactured by Wako Pure Chemical Industries, Ltd., 077-03155) was added and incubated at 37 ° C. for 1 minute. Thereafter, centrifugation was performed at 2000 rpm for 2 minutes, and the supernatant was removed. This was performed as one step, and 9 steps were executed, and a film made of fibronectin and a film made of gelatin were alternately laminated to form a 6 nm-thick adhesive film on human fibroblasts. The film thickness was determined by measuring 10 points with a phase contrast microscope and averaging the obtained measurement values. Thereafter, 2 × 10 6 human fibroblasts coated with an adhesive film were seeded on a membrane filter of a cell culture insert (Becton Dickinson, 353090), and further, Eagle containing 10 wt% fetal bovine serum (FAB). 'sMEM medium was added and incubated at 37 ° C for 48 hours. As a result, a sheet-like three-dimensional structure of human fibroblasts having a thickness of 40 μm was obtained. The obtained three-dimensional structure of Example 1 was stained with HE and then evaluated with a phase contrast microscope. The results are shown in FIG. As illustrated in FIG. 4, cell overlap was observed. In addition, after incubation for 24 hours, a three-dimensional structure of seven layers of human fibroblasts was observed.
（比較例１）
実施例１において、接着膜を形成していないヒト繊維芽細胞にセルカルチャーインサートに播種し、同様に培養した。得られた実施例１の三次元組織をＨＥ染色した後、位相差顕微鏡より評価した。図５に結果を示す。空隙が観察されており、細胞が接着できず組織化できないことが示された。
(Comparative Example 1)
In Example 1, human fibroblasts without an adhesive film were seeded on a cell culture insert and cultured in the same manner. The obtained three-dimensional structure of Example 1 was stained with HE and then evaluated with a phase contrast microscope. The results are shown in FIG. Voids were observed, indicating that the cells could not adhere and could not be organized.
（実施例２）
実施例１において、フィブロネクチンとゼラチンとの接触を１ステップのみ行い、膜厚２ｎｍの接着膜を形成した。その他は、実施例１と同様にし、厚み５２μｍのシート状のヒト繊維芽細胞の三次元構造体を得た。
(Example 2)
In Example 1, fibronectin and gelatin were contacted for only one step to form an adhesive film having a thickness of 2 nm. Others were the same as in Example 1, and a sheet-shaped three-dimensional structure of human fibroblasts having a thickness of 52 μm was obtained.
（実施例３）
実施例１において、フィブロネクチンとゼラチンとの接触を５ステップ行い、膜厚４ｎｍの接着膜を形成した。その他は、実施例１と同様にし、厚み４５μｍのシート状のヒト繊維芽細胞の三次元構造体を得た。
(Example 3)
In Example 1, fibronectin and gelatin were contacted for 5 steps to form a 4 nm thick adhesive film. Others were the same as in Example 1, and a three-dimensional structure of a sheet-like human fibroblast having a thickness of 45 μm was obtained.
（比較例２）
特開２００７−２２８９２１号公報の実施例４において、ヒト臍帯動脈平滑筋細胞（ＵＡＳＭＣ）にかえてヒト繊維芽細胞を用い、ゼラチンとフィブロネクチンとからなるナノＥＣＭ（細胞外マトリックス）と細胞層とを順に積層させる構造を作製した。１層の細胞層を形成させるために、３７℃で６時間インキュベーションさせる必要があるため、７層の細胞層を作製するのに４２時間要し、実施例１の２倍の時間がかかった。
(Comparative Example 2)
In Example 4 of Japanese Patent Application Laid-Open No. 2007-228921, human fibroblasts are used instead of human umbilical artery smooth muscle cells (UASMC), nano-ECM (extracellular matrix) composed of gelatin and fibronectin, and cell layer. A structure in which layers were stacked in order was prepared. Since it was necessary to incubate at 37 ° C. for 6 hours in order to form one cell layer, it took 42 hours to produce 7 cell layers, which was twice as long as in Example 1.
（実験例１）
培養細胞に接着膜が形成されていることの確認実験を図３に従った方法で行った。
具体的には、まず、フィブロネクチン（シグマアルドリッチ社製、Ｆ−４７５９）をファロイジン−ローダミン（Ｒｈ）を用いて標識した。また、ゼラチン（和光純薬工業社製、０７７−０３１５５）は、ｆｌｕｏｒｅｓｃｅｉｎ ｉｓｏｔｈｉｏｃｙａｎａｔｅ（ＦＩＴＣ）を用いて標識した。そして、５×１０６個のＬ９２９マウス繊維芽細胞（ＤＳファーマバイオメディカル、ＥＣ８５０１１４２５）に０．２ｍｇ／ｍＬのＲｈラベル化フィブロネクチンの５０ｍＭトリス緩衝液（ｐＨ７．４）１ｍＬを加えて３７℃で１分間インキュベーションした。その後、２０００ｒｐｍで２分間遠心分離を行い、上澄みを除去した。続いて、０．２ｍｇ／ｍＬのＦＩＴＣラベル化ゼラチンの５０ｍＭトリス緩衝液（ｐＨ７．４）１ｍＬを加えて３７℃で１分間インキュベートした。その後、２０００ｒｐｍで２分間遠心分離を行い、上澄みを除去した。これを１ステップとして、９ステップ実行し、フィブロネクチンからなる膜とゼラチンからなる膜とを交互に積層して、膜厚６ｎｍの接着膜をマウス繊維芽細胞に形成した。膜厚は、水晶発振子マイクロバランス（ＱＣＭ）により求めたが、位相差顕微鏡の観察による測定結果と同様の結果が得られた。
ＱＣＭ測定法を用いた測定は、具体的には以下のように行った。ＱＣＭ水晶センサをピランハ溶液（過酸化水素水：濃硫酸（１：３、ｖ／ｖ）混合液）で１分間洗浄してから、０．２ｍｇ／ｍＬフィブロネクチンの５０ｍＭトリス緩衝液（ｐＨ７．４）に３７℃で１分間浸漬し、５０ｍＭトリス緩衝液（ｐＨ７．４）で洗浄して風乾した後、振動数シフトを測定した（ステップ１）。ついで０．２ｍｇ／ｍＬゼラチンの５０ｍＭトリス緩衝液（ｐＨ７．４）に３７℃で１分間浸漬し、５０ｍＭトリス緩衝液（ｐＨ７．４）で洗浄して風乾した後、振動数シフトを測定した（ステップ２）。このステップ１及び２を交互に繰り返すことによりＱＣＭ水晶センサに接着膜を形成すると共に、振動数シフトの測定を行った。得られた振動数シフトに基づき、ステップの数とそれにより形成される接着膜の厚みとを得た。特許文献１の図２に示すように、ステップを繰り返すことによって、振動数シフトが減少し、ＱＣＭ基材上にナノレベルの層が連続的に形成されることがわかる。また、振動数の減少が、ステップ数と相関関係を示していることから、ステップ数の増加によって形成するナノＥＣＭの膜厚を増加させ、ステップ数の減少によって形成するナノＥＣＭの膜厚を低減できる。
(Experimental example 1)
An experiment for confirming that an adhesive film was formed on the cultured cells was performed by the method according to FIG.
Specifically, fibronectin (Sigma Aldrich, F-4759) was first labeled with phalloidin-rhodamine (Rh). Gelatin (077-03155, manufactured by Wako Pure Chemical Industries, Ltd.) was labeled with fluorescein isothiocyanate (FITC). Then, 1 mL of 0.2 mg / mL Rh-labeled fibronectin 50 mM Tris buffer (pH 7.4) was added to 5 × 10 6 L929 mouse fibroblasts (DS Pharma Biomedical, EC85011425), and 1 at 37 ° C. Incubated for minutes. Thereafter, centrifugation was performed at 2000 rpm for 2 minutes, and the supernatant was removed. Subsequently, 1 mL of 50 mM Tris buffer (pH 7.4) of 0.2 mg / mL FITC-labeled gelatin was added and incubated at 37 ° C. for 1 minute. Thereafter, centrifugation was performed at 2000 rpm for 2 minutes, and the supernatant was removed. This was performed as one step, and 9 steps were performed, and a film made of fibronectin and a film made of gelatin were alternately laminated to form an adhesive film having a thickness of 6 nm on mouse fibroblasts. The film thickness was determined by quartz crystal microbalance (QCM), and the same result as the measurement result by observation with a phase contrast microscope was obtained.
Specifically, the measurement using the QCM measurement method was performed as follows. The QCM quartz sensor is washed with a Piranha solution (hydrogen peroxide water: concentrated sulfuric acid (1: 3, v / v) mixed solution) for 1 minute, and then 0.2 mg / mL fibronectin 50 mM Tris buffer (pH 7.4). The sample was soaked at 37 ° C. for 1 minute, washed with 50 mM Tris buffer (pH 7.4) and air-dried, and then the frequency shift was measured (step 1). Subsequently, it was immersed in 0.2 mg / mL gelatin 50 mM Tris buffer (pH 7.4) at 37 ° C. for 1 minute, washed with 50 mM Tris buffer (pH 7.4) and air-dried, and then the frequency shift was measured ( Step 2). By repeating Steps 1 and 2 alternately, an adhesive film was formed on the QCM quartz sensor, and the frequency shift was measured. Based on the obtained frequency shift, the number of steps and the thickness of the adhesive film formed thereby were obtained. As shown in FIG. 2 of Patent Document 1, it can be seen that by repeating the steps, the frequency shift is reduced and nano-level layers are continuously formed on the QCM substrate. In addition, since the decrease in the frequency shows a correlation with the number of steps, the film thickness of the nano-ECM formed by increasing the number of steps is increased, and the film thickness of the nano-ECM formed by decreasing the number of steps is reduced. it can.
実験例１のＬ９２９マウス繊維芽細胞の位相差顕微鏡の画像を図６に示す。図６（ａ）が接着膜を形成する前の画像であり、図６（ｂ）が１ステップ目のフィブロネクチンからなる膜を形成した直後の画像であり、図６（ｃ）が２ステップ目のゼラチンからなる膜を形成した直後の画像であり、図６（ｄ）が５ステップ目のフィブロネクチンからなる膜を形成した直後の画像であり、図６（ｅ）が９ステップ目のフィブロネクチンからなる膜を形成した直後の画像であり、図６（ｆ）が図６（ｄ）で示す画像と５ステップ目のゼラチンからなる膜を形成した直後の画像との重ね合わせ画像である。各ステップにおいて、個々の細胞表面の全面がフィブロネクチンからなる膜、及び、ゼラチンからなら膜で覆われていることがわかる。 The phase-contrast microscope image of the L929 mouse fibroblasts of Experimental Example 1 is shown in FIG. FIG. 6A is an image before forming an adhesive film, FIG. 6B is an image immediately after forming a film made of fibronectin at the first step, and FIG. 6C is a second step. FIG. 6D is an image immediately after forming a film made of gelatin, FIG. 6D is an image immediately after forming a film made of fibronectin at the fifth step, and FIG. 6E is a film made of fibronectin at the ninth step. 6 (f) is a superimposed image of the image shown in FIG. 6 (d) and the image immediately after the formation of the fifth step gelatin film. In each step, it can be seen that the entire surface of each cell surface is covered with a film made of fibronectin and a film made of gelatin.
（実験例２〜７）
接着膜の形成と細胞毒性との関係を評価した。接着膜の形成は、５×１０６個のＬ９２９マウス繊維芽細胞（理化学研究所、ＣｅｌｌＢａｎｋ）を用いて、図３に従った方法で行った。第１の物質として、フィブロネクチン（シグマアルドリッチ社製、Ｆ−４７５９）、ε−ポリリシン塩酸塩（チッソ社製、分子量４７００）、ポリアリルアミン塩酸塩（日東紡社製、ＰＡＡ−ＨＣＬ）に５０ｍＭトリス緩衝液（ｐＨ７．４）を加え、０．２ｍｇ／ｍＬの第１の物質の含有液をそれぞれ調製した。また、第２の物質として、ゼラチン（和光純薬工業社製、０７７−０３１５５）、ヘパリン（和光純薬 ０８１−００１３６ １００，０００ユニット）、デキストラン硫酸（和光純薬工業社製、１９９−０９９８３、分子量５００，０００）、ポリスチレンスルホン酸ナトリウム塩、ポリアクリル酸ナトリウム塩に５０ｍＭトリス緩衝液（ｐＨ７．４）を加え、０．２ｍｇ／ｍＬの第２の物質の含有液をそれぞれ調製した。Ｌ９２９マウス繊維芽細胞を第１の物質の含有液１ｍＬに１分間浸積した後、２０００ｒｐｍで２分間遠心分離を行った。上澄みを取り除いて第１の物質を除去した。その後、第２の物質の含有液１ｍＬを添加して１分間浸積し、同様に遠心分離を行って、上澄みを取り除くことで第２の物質を除去した。これを１ステップとして、１０ステップ繰り返した。
(Experimental Examples 2-7)
The relationship between adhesion film formation and cytotoxicity was evaluated. Formation of the adhesive film was performed by the method according to FIG. 3 using 5 × 10 6 L929 mouse fibroblasts (RIKEN, CellBank). As a first substance, fibronectin (manufactured by Sigma-Aldrich, F-4759), ε-polylysine hydrochloride (manufactured by Chisso, molecular weight 4700), polyallylamine hydrochloride (manufactured by Nittobo, PAA-HCL) with 50 mM Tris buffer The liquid (pH 7.4) was added, and the liquid containing 0.2 mg / mL of the first substance was prepared. Further, as the second substance, gelatin (manufactured by Wako Pure Chemical Industries, 077-03155), heparin (Wako Pure Chemical 081-00136 100,000 units), dextran sulfate (manufactured by Wako Pure Chemical Industries, 199-09983, Molecular weight 500,000), polystyrenesulfonic acid sodium salt, polyacrylic acid sodium salt were added with 50 mM Tris buffer (pH 7.4) to prepare 0.2 mg / mL second substance-containing liquids. L929 mouse fibroblasts were immersed in 1 mL of the first substance-containing solution for 1 minute, and then centrifuged at 2000 rpm for 2 minutes. The supernatant was removed to remove the first material. Thereafter, 1 mL of the second substance-containing solution was added and immersed for 1 minute, similarly centrifuged, and the second substance was removed by removing the supernatant. This was taken as one step and repeated 10 steps.
遠心分離直後、上澄みを除去前に、ＣｅｌｌＴｒａｃｋｅｒ Ｇｒｅｅｎ（ＣＭＦＤＡ、インビトロジェン社製）及びＨＥ染色して細胞の挙動を確認したところ、第１の物質としてフィブロネクチンを用い第２の物質としてゼラチン又はε−ポリリシン塩酸塩を用いた場合（実験例２、５）は、細胞の凝集が見られなかったが、第１の物質としてフィブロネクチンを用い第２の物質としてヘパリンを用いた場合（実験例４）は、細胞の凝集が認められた。ただし、実験例４においても、５０ｍＭトリス緩衝液（ｐＨ７．４）を１ｍＬ加えることで細胞は分散した。１０ステップ処理後の被覆細胞の膜厚を位相差顕微鏡により観察したところ、表３で示す結果が得られた。また、各ステップの処理後の細胞の構造体の生存数を確認し、接着膜を形成する前の５×１０６個を１００として、生存率を求めた。ステップ数と生存率との関係を図７に示す。 Immediately after centrifugation, before removing the supernatant, CellTracker Green (CMFDA, manufactured by Invitrogen) and HE staining were used to confirm the behavior of the cells. Fibronectin was used as the first substance and gelatin or ε-polylysine was used as the second substance. When hydrochloride was used (Experimental Examples 2 and 5), cell aggregation was not observed, but when fibronectin was used as the first substance and heparin was used as the second substance (Experimental Example 4), Cell aggregation was observed. However, also in Experimental Example 4, cells were dispersed by adding 1 mL of 50 mM Tris buffer (pH 7.4). When the thickness of the coated cells after the 10-step treatment was observed with a phase contrast microscope, the results shown in Table 3 were obtained. In addition, the number of surviving cell structures after the treatment in each step was confirmed, and the survival rate was determined with 5 × 10 6 before forming the adhesive film as 100. The relationship between the number of steps and the survival rate is shown in FIG.
（実験例８）
実験例２で作製したＬ９２９マウス繊維芽細胞の被覆細胞と、接着膜で被覆されていないＬ９２９マウス繊維芽細胞を、１０重量％ウシ胎仔血清（ＦＡＢ）を含むＥａｇｌｅ'ｓＭＥＭ培地が添加されたシャーレに１．８×１０３個／ｃｍ２播種し、３７℃でインキュベートして培養した。２４時間後及び９６時間後の位相差顕微鏡の画像を図８に示す。図８（ａ）（ｂ）が、接着膜が形成されていないＬ９２９マウス繊維芽細胞を培養させた結果であり、図８（ｃ）（ｄ）が実施例２のＬ９２９マウス繊維芽細胞を培養させた結果である。図８（ａ）（ｃ）が培養２４時間後の結果であり、図８（ｂ）（ｄ）が培養９６時間後の結果である。また、培養後２４、４８、７２、９６時間における細胞数を位相差顕微鏡で観察することで計測した。図９が培養時間と細胞数との関係を示す図である。倍加時間はＬ９２９マウス繊維芽細胞で２３時間であり、実験例２の被覆細胞で２５時間だった。
(Experimental example 8)
A petri dish to which Eagle's MEM medium containing 10 wt% fetal bovine serum (FAB) was added to the coated cells of L929 mouse fibroblasts prepared in Experimental Example 2 and L929 mouse fibroblasts not coated with an adhesive film. Were seeded at 1.8 × 10 3 cells / cm 2 and incubated at 37 ° C. for cultivation. The images of the phase contrast microscope after 24 hours and after 96 hours are shown in FIG. 8 (a) and 8 (b) show the results of culturing L929 mouse fibroblasts without an adhesive film, and FIGS. 8 (c) and 8 (d) show the culture of L929 mouse fibroblasts of Example 2. This is the result. 8A and 8C show the results after 24 hours of culture, and FIGS. 8B and 8D show the results after 96 hours of culture. In addition, the number of cells at 24, 48, 72, and 96 hours after culturing was measured by observing with a phase contrast microscope. FIG. 9 is a diagram showing the relationship between the culture time and the number of cells. The doubling time was 23 hours for L929 mouse fibroblasts and 25 hours for the coated cells of Experimental Example 2.
なお、実験例２〜７で得られた被覆細胞を実施例１と同様に培養することで、Ｌ９２９マウス繊維芽細胞の三次元構造体が得られる。 The three-dimensional structure of L929 mouse fibroblasts can be obtained by culturing the coated cells obtained in Experimental Examples 2 to 7 in the same manner as in Example 1.
（実施例４）
図１０で示す三次元構造体を作製した。図１１は、異種細胞の積層組織を構築する方法を説明する模式的な図である。異種細胞のモデルとして、ヒト繊維芽細胞（Ｃａｍｂｒｅｘ社製、正常ヒト皮膚繊維芽細胞 ＮＨＤＦ）を赤色色素（タカラバイオ、Ｃｅｌｌ Ｔｒａｃｋｅｒ Ｏｒａｎｇｅ Ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｂｅ、ＰＡ−３０１２）と緑色色素（タカラバイオ、Ｃｅｌｌ Ｔｒａｃｋｅｒ Ｇｒｅｅｎ Ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｂｅ、ＰＡ−３０１１）に着色したものをそれぞれ用意し、第１の物質としてフィブロネクチン、第２の物質としてゼラチンを用いて、実施例１と同様な手技により、フィブロネクチンとゼラチンとからなる接着膜１０２（膜厚６ｎｍ）を成膜した。これにより、緑色色素に着色したヒト繊維芽細胞が、フィブロネクチンとゼラチンとからなる接着膜１０２で被覆された第一被覆細胞１０３ａと、赤色色素に着色したヒト繊維芽細胞がフィブロネクチンとゼラチンとからなる接着膜１０２で被覆された第二被覆細胞１０３ｂとが得られた。
その後、第一被覆細胞１０３ａを１．１×１０６個／ｃｍ２セルカルチャーインサート（ベクトンディッキンソン社製、３５３０９０）のメンブレンフィルターに播種し、さらに、１０重量％ウシ胎仔血清（ＦＡＢ）を含むＥａｇｌｅ'ｓＭＥＭ培地を添加した後、３７℃で２４時間インキュベートして培養し、４層の第一の積層構造１０を構築した。
ついで、実施例１で用いたフィブロネクチン含有緩衝液とゼラチン含有緩衝液への浸積を交互に合計９ステップ行い、フィブロネクチンとゼラチンとが交互に積層されたナノＥＣＭ３０を作成した。
その後、第二被覆細胞１０３ｂを１．１×１０６個／ｃｍ２播種し、さらに、１０重量％ウシ胎仔血清（ＦＡＢ）を含むＥａｇｌｅ'ｓＭＥＭ培地を添加した後、３７℃で２４時間インキュベートして培養し、４層の第二の積層構造２０を構築した。
得られた三次元構造体を共焦点レーザー顕微鏡（Ｃｏｎｆｏｃａｌ Ｌａｓｅｒ Ｓｃａｎｎｉｎｇ Ｍｉｃｒｏｓｃｏｐｅ：ＣＬＳＭ）で観察したところ、図１４で示す結果が得られ、４層からなる第一被覆細胞１０３ａの積層組織の上に４層からなる第二被覆細胞１０３ｂの積層組織が構築できたことを確認した。
Example 4
A three-dimensional structure shown in FIG. 10 was produced. FIG. 11 is a schematic diagram for explaining a method for constructing a laminated tissue of heterogeneous cells. As a heterogeneous cell model, human fibroblasts (manufactured by Cambrex, normal human skin fibroblasts NHDF), red pigment (Takara Bio, Cell Tracker Orange Fluorescent Probe, PA-3012) and green pigment (Takara Bio, Cell Tracker Green) Fluorescent Probe, PA-3011), each prepared, and using fibronectin as the first substance and gelatin as the second substance, and an adhesive film comprising fibronectin and gelatin by the same procedure as in Example 1. 102 (film thickness 6 nm) was formed. Thereby, the human fibroblasts colored with the green pigment are coated with the adhesive film 102 made of fibronectin and gelatin, and the first fibroblasts colored with the red pigment are made of fibronectin and gelatin. A second coated cell 103b coated with an adhesive film 102 was obtained.
Thereafter, the first coated cells 103a were seeded on a membrane filter of 1.1 × 10 6 cells / cm 2 cell culture insert (Becton Dickinson, 353090), and further, Eagle containing 10 wt% fetal bovine serum (FAB). After adding 'sMEM medium, incubation was performed at 37 ° C. for 24 hours to culture, and a four-layer first laminated structure 10 was constructed.
Subsequently, immersion in the fibronectin-containing buffer solution and gelatin-containing buffer solution used in Example 1 was alternately carried out for a total of 9 steps to prepare nano-ECM30 in which fibronectin and gelatin were alternately laminated.
Thereafter, the second coated cells 103b were seeded at 1.1 × 10 6 cells / cm 2 , and further added with Eagle's MEM medium containing 10 wt% fetal bovine serum (FAB), followed by incubation at 37 ° C. for 24 hours. 4 layers of the second laminated structure 20 was constructed.
The obtained three-dimensional structure was observed with a confocal laser scanning microscope (CLSM). As a result, the result shown in FIG. 14 was obtained, and four layers were formed on the laminated structure of the first coated cells 103a composed of four layers. It was confirmed that a layered tissue of the second coated cells 103b composed of layers could be constructed.
（実施例５）
図１３で示す三次元構造体を作製した。実施例４で得られた第一被覆細胞１０３ａを１．１×１０６個／ｃｍ２、及び、第二被覆細胞１０３ｂを１．１×１０６個／ｃｍ２セルカルチャーインサート（ベクトンディッキンソン社製、３５３０９０）のメンブレンフィルターに播種し、さらに、１０重量％ウシ胎仔血清（ＦＡＢ）を含むＥａｇｌｅ'ｓＭＥＭ培地を添加した後、３７℃で２４時間インキュベートして培養し、８層の積層組織を構築した。得られた積層組織を共焦点レーザー顕微鏡（Ｃｏｎｆｏｃａｌ Ｌａｓｅｓｒ Ｓｃａｎｎｉｎｇ Ｍｉｃｒｏｓｃｏｐｅ：ＣＬＳＭ）で観察したところ、図１５で示す結果が得られ、第一被覆細胞１０３ａと第二被覆細胞１０３ｂとがランダムに積層した８層の積層組織が構築できたことを確認した。
(Example 5)
A three-dimensional structure shown in FIG. 13 was produced. EXAMPLE first coated cells 103a obtained in 4 1.1 × 10 6 cells / cm 2, and the second coated cells 103b to 1.1 × 10 6 cells / cm 2 cell culture insert (manufactured by Becton Dickinson , 353090), and after addition of Eagle's MEM medium containing 10% by weight fetal bovine serum (FAB), incubation at 37 ° C. for 24 hours is carried out to construct an 8 layered tissue did. When the obtained laminated structure was observed with a confocal laser scanning microscope (CLSM), the results shown in FIG. 15 were obtained, and the first coated cells 103a and the second coated cells 103b were randomly laminated. It was confirmed that a layered structure of layers could be constructed.
（実施例６）
図１２で示す三次元構造体を図１１に示す方法に従って作製した。第一培養細胞１０１ａとして、ヒト繊維芽細胞（Ｃａｍｂｒｅｘ社製、正常ヒト皮膚繊維芽細胞 ＮＨＤＦ）を用意し、緑色色素（タカラバイオ、Ｃｅｌｌ Ｔｒａｃｋｅｒ Ｇｒｅｅｎ Ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｂｅ、ＰＡ−３０１１）に着色した。また、第二培養細胞１０１ｂとして、ヒト血管内皮細胞（Ｃａｍｂｒｅｘ社製、正常ヒト臍帯静脈血管内皮細胞）を用意した。第一培養細胞１０１ａ及び第二培養細胞１０１ｂのそれぞれの表面に、第１の物質としてフィブロネクチン、第２の物質としてゼラチンを用いて、実施例１と同様な手技により、フィブロネクチンとゼラチンとからなる接着膜１０２（膜厚６ｎｍ）を成膜した。これにより、ヒト繊維芽細胞が接着膜１０２で被覆された第一被覆細胞１０３ａ、及び、血管内皮細胞が接着膜１０２で被覆された第二被覆細胞１０３ｂを得た。
その後、第一被覆細胞１０３ａ１．１×１０６個／ｃｍ２セルカルチャーインサート（ベクトンディッキンソン社製、３５３０９０）のメンブレンフィルターに播種し、１０重量％ウシ胎仔血清（ＦＡＢ）を含むＥａｇｌｅ'ｓＭＥＭ培地を添加した後、３７℃で２４時間インキュベートして培養し、４層の第一の積層構造１０を構築した。
ついで、実施例１で用いたフィブロネクチン含有緩衝液とゼラチン含有緩衝液への浸積を交互に合計９ステップ行い、フィブロネクチンとゼラチンとが交互に積層されたナノＥＣＭ３０を第一の積層構造１０上に作成した。
その後、第二の被覆細胞１０３ｂを０．２×１０６個／ｃｍ２播種し、１０重量％ウシ胎仔血清（ＦＡＢ）を含むＥａｇｌｅ'ｓＭＥＭ培地を添加した後、３７℃で２４時間インキュベートして培養し、ナノＥＣＭ３０上に１層の第二の積層構造２０を構築した。
ついで、実施例１で用いたフィブロネクチン含有緩衝液とゼラチン含有緩衝液への浸積を交互に合計９ステップ行い、第二の積層構造２０上に、フィブロネクチンとゼラチンとが交互に積層されたナノＥＣＭ３０を作成した。
さらに、第一被覆細胞１０３ａを１．１×１０６個／ｃｍ２を播種し、１０重量％ウシ胎仔血清（ＦＡＢ）を含むＥａｇｌｅ'ｓＭＥＭ培地を添加した後、３７℃で２４時間インキュベートして培養し、ナノＥＣＭ３０上に４層の第一の積層構造１０を構築した。
(Example 6)
The three-dimensional structure shown in FIG. 12 was produced according to the method shown in FIG. Human fibroblasts (manufactured by Cambrex, normal human skin fibroblasts NHDF) were prepared as the first cultured cells 101a, and were colored green pigment (Takara Bio, Cell Tracker Green Fluorescent Probe, PA-3011). In addition, human vascular endothelial cells (manufactured by Cambrex, normal human umbilical vein endothelial cells) were prepared as the second cultured cells 101b. Adhesion comprising fibronectin and gelatin by the same procedure as in Example 1 using fibronectin as the first substance and gelatin as the second substance on the surface of each of the first cultured cell 101a and the second cultured cell 101b A film 102 (film thickness 6 nm) was formed. As a result, a first coated cell 103a in which human fibroblasts were coated with the adhesive film 102 and a second coated cell 103b in which vascular endothelial cells were coated with the adhesive film 102 were obtained.
Thereafter, the cells were seeded on a membrane filter of a first coated cell 103a1.1 × 10 6 cells / cm 2 cell culture insert (Becton Dickinson, 353090), and Eagle's MEM medium containing 10 wt% fetal bovine serum (FAB) was added. After the addition, incubation was carried out at 37 ° C. for 24 hours to culture, and a four-layer first laminated structure 10 was constructed.
Subsequently, the immersion into the fibronectin-containing buffer solution and gelatin-containing buffer solution used in Example 1 was alternately performed for a total of 9 steps, and nano-ECM30 in which fibronectin and gelatin were alternately laminated was formed on the first laminated structure 10. Created.
Thereafter, the second coated cells 103b were seeded at 0.2 × 10 6 cells / cm 2 , Eagle's MEM medium containing 10% by weight fetal bovine serum (FAB) was added, and incubated at 37 ° C. for 24 hours. After culturing, one layer of the second laminated structure 20 was constructed on the nano-ECM30.
Subsequently, the nano-ECM30 in which fibronectin and gelatin were alternately laminated on the second laminated structure 20 was performed by alternately submerging the fibronectin-containing buffer solution and gelatin-containing buffer solution used in Example 1 for nine steps. It was created.
Furthermore, 1.1 × 10 6 cells / cm 2 of the first coated cells 103a were seeded, and Eagle's MEM medium containing 10% by weight fetal bovine serum (FAB) was added, followed by incubation at 37 ° C. for 24 hours. After culturing, a four-layer first laminated structure 10 was constructed on the nano-ECM30.
得られた三次元構造体を、３７℃で７日間培養した。ｍｏｎｏｃｌｏｃａｌ ｍｏｕｓｅ ａｎｔｉ−ｈｕｍａｎ ＣＤ３１抗体（製品コード：ＪＣ７０Ａ、Ｄａｋｏ社製）及びＧｏａｔ ａｎｔｉ−ｍｏｕｓｅ Ａｌｅｘａ Ｆｌｕｏｒ５４６−ｃｏｎｊｕｇａｔｅｄ ＩｇＧ抗体（製品コード：Ａ１１００１、Ｉｎｖｉｔｒｏｇｅｎ社製）にて第二培養細胞１０１ｂであるヒト血管内皮細胞を選択的に着色し、培養１日目、及び、７日目の積層組織を、それぞれ、共焦点レーザー顕微鏡（Ｃｏｎｆｏｃａｌ Ｌａｓｅｒ Ｓｃａｎｎｉｎｇ Ｍｉｃｒｏｓｃｏｐｅ：ＣＬＳＭ）で観察した。結果を図１６（ｅ）、（ｆ）、（ｇ）、（ｈ）に示す。図１６（ｅ）、（ｆ）は、培養１日目を示し、図１６（ｇ）、（ｈ）が培養７日目を示す。図１６（ｆ）は、図１６（ｅ）の拡大図であり、図１６（ｈ）は、図１６（ｇ）の拡大図である。図１６では、血管内皮細胞が白く描出されるように示している。図１６（ｅ）、（ｆ）で示すように、培養１日目では、血管内皮細胞から血枝が成長しているのが示される。また、図１６（ｇ）、（ｈ）で示すように、培養７日目には、緻密な血管網が形成されている。 The obtained three-dimensional structure was cultured at 37 ° C. for 7 days. Monoclonal mouse anti-human CD31 antibody (product code: JC70A, manufactured by Dako) and Goat anti-mouse Alexa Fluor 546-conjugated IgG antibody (product code: A11001, manufactured by Invitrogen) are human cultured endothelial cells 101b. The cells were selectively colored, and the laminated tissues on day 1 and day 7 of the culture were observed with a confocal laser scanning microscope (CLSM), respectively. The results are shown in FIGS. 16 (e), (f), (g) and (h). 16 (e) and (f) show the first day of culture, and FIGS. 16 (g) and (h) show the seventh day of culture. FIG. 16 (f) is an enlarged view of FIG. 16 (e), and FIG. 16 (h) is an enlarged view of FIG. 16 (g). In FIG. 16, the vascular endothelial cells are depicted in white. As shown in FIGS. 16 (e) and 16 (f), it is shown that blood branches grow from vascular endothelial cells on the first day of culture. Further, as shown in FIGS. 16 (g) and (h), a dense vascular network is formed on the seventh day of culture.
図１７に第二の積層構造２０の平面視で上記共焦点レーザー顕微鏡により観察した結果を示す。第二の積層構造２０の断面中、血管網占有面積は、４５−６０％であり、血管網の間隔は、５０−１００μｍであった。 FIG. 17 shows the result of observation by the confocal laser microscope in a plan view of the second laminated structure 20. In the cross section of the second laminated structure 20, the area occupied by the vascular network was 45-60%, and the interval between the vascular networks was 50-100 μm.
（実施例７）
図１０で示す三次元構造体を、図１１に示す方法に従って作製した。第一培養細胞１０１ａとして、ヒト繊維芽細胞を用意し、第二培養細胞１０１ｂとして、ヒト血管内皮細胞を用意した。第二の積層構造２０上に、ナノＥＣＭ３０及び第一の積層構造１０を形成しない以外は、実施例６と同様にした。
(Example 7)
The three-dimensional structure shown in FIG. 10 was produced according to the method shown in FIG. Human fibroblasts were prepared as the first cultured cells 101a, and human vascular endothelial cells were prepared as the second cultured cells 101b. The same procedure as in Example 6 was performed except that the nano-ECM 30 and the first stacked structure 10 were not formed on the second stacked structure 20.
得られた三次元構造体を、３７℃で７日間培養した。ｍｏｎｏｃｌｏｃａｌ ｍｏｕｓｅ ａｎｔｉ−ｈｕｍａｎ ＣＤ３１抗体（製品コード：ＪＣ７０Ａ、Ｄａｋｏ社製）及びＧｏａｔ ａｎｔｉ−ｍｏｕｓｅ Ａｌｅｘａ Ｆｌｕｏｒ５４６−ｃｏｎｊｕｇａｔｅｄ ＩｇＧ抗体（製品コード：Ａ１１００１、Ｉｎｖｉｔｒｏｇｅｎ社製）にて第二培養細胞１０１ｂであるヒト血管内皮細胞を選択的に着色し、培養１日目、及び、７日目の積層組織を、それぞれ、ＣＬＳＭで観察した。結果を図１６（ａ）、（ｂ）、（ｃ）、（ｄ）に示す。図１６（ａ）、（ｂ）は、培養１日目を示し、図１６（ｃ）、（ｄ）が培養７日目を示す。図１６（ｂ）は、図１６（ａ）の拡大図であり、図１６（ｄ）は、図１６（ｃ）の拡大図である。図１６では、血管内皮細胞が白く描出されるように示している。図１６（ａ）〜（ｄ）で示すように、実施例６とは異なり、培養１日目、７日目のいずれも、血枝の成長が観察されず、血管網は観察できなかった。 The obtained three-dimensional structure was cultured at 37 ° C. for 7 days. Monoclonal mouse anti-human CD31 antibody (product code: JC70A, manufactured by Dako) and Goat anti-mouse Alexa Fluor 546-conjugated IgG antibody (product code: A11001, manufactured by Invitrogen) are human cultured endothelial cells 101b. The cells were selectively colored, and the laminated tissues on day 1 and day 7 were observed with CLSM. The results are shown in FIGS. 16 (a), (b), (c) and (d). 16 (a) and 16 (b) show the first day of culture, and FIGS. 16 (c) and 16 (d) show the seventh day of culture. 16 (b) is an enlarged view of FIG. 16 (a), and FIG. 16 (d) is an enlarged view of FIG. 16 (c). In FIG. 16, the vascular endothelial cells are depicted in white. As shown in FIGS. 16 (a) to (d), unlike Example 6, no growth of blood branches was observed on day 1 and day 7 of culture, and a vascular network could not be observed.
（実施例８）
図１３で示す三次元構造体を図１１に示す方法に従って作製した。第一培養細胞１０１ａとして、ヒト繊維芽細胞（Ｃａｍｂｒｅｘ社製、正常ヒト皮膚繊維芽細胞 ＮＨＤＦ）を用意し、緑色色素（タカラバイオ、Ｃｅｌｌ Ｔｒａｃｋｅｒ Ｇｒｅｅｎ Ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｂｅ、ＰＡ−３０１１）に着色した。また、第二培養細胞１０１ｂとして、ヒト血管内皮細胞（Ｃａｍｂｒｅｘ社製、正常ヒト臍帯静脈血管内皮細胞）を用意した。第一培養細胞１０１ａ及び第二培養細胞１０１ｂのそれぞれの表面に、第１の物質としてフィブロネクチン、第２の物質としてゼラチンを用いて、実施例１と同様な手技により、フィブロネクチンとゼラチンとからなる接着膜１０２（膜厚６ｎｍ）を成膜した。これにより、ヒト繊維芽細胞が接着膜１０２で被覆された第一被覆細胞１０３ａ、及び、血管内皮細胞が接着膜１０２で被覆された第二被覆細胞１０３ｂを得た。
得られた第一被覆細胞１０３ａを１．１×１０６個／ｃｍ２、及び、第二被覆細胞１０３ｂを１．１×１０６個／ｃｍ２、セルカルチャーインサート（ベクトンディッキンソン社製、３５３０９０）のメンブレンフィルターに播種した。さらに、１０重量％ウシ胎仔血清（ＦＡＢ）を含むＥａｇｌｅ'ｓＭＥＭ培地を添加した後、３７℃で２４時間インキュベートして培養し、５層の積層組織を構築した。
(Example 8)
The three-dimensional structure shown in FIG. 13 was produced according to the method shown in FIG. Human fibroblasts (manufactured by Cambrex, normal human skin fibroblasts NHDF) were prepared as the first cultured cells 101a, and were colored green pigment (Takara Bio, Cell Tracker Green Fluorescent Probe, PA-3011). In addition, human vascular endothelial cells (manufactured by Cambrex, normal human umbilical vein endothelial cells) were prepared as the second cultured cells 101b. Adhesion comprising fibronectin and gelatin by the same procedure as in Example 1 using fibronectin as the first substance and gelatin as the second substance on the surface of each of the first cultured cell 101a and the second cultured cell 101b A film 102 (film thickness 6 nm) was formed. As a result, a first coated cell 103a in which human fibroblasts were coated with the adhesive film 102 and a second coated cell 103b in which vascular endothelial cells were coated with the adhesive film 102 were obtained.
The obtained first coated cells 103a were 1.1 × 10 6 cells / cm 2 , and the second coated cells 103b were 1.1 × 10 6 cells / cm 2 , cell culture insert (Becton Dickinson, 353090). Seeded on a membrane filter. Furthermore, after adding Eagle's MEM medium containing 10% by weight fetal bovine serum (FAB), incubation was carried out at 37 ° C. for 24 hours to construct a 5-layer laminated tissue.
得られた三次元構造体を、３７℃で７日間培養した。ｍｏｎｏｃｌｏｃａｌ ｍｏｕｓｅ ａｎｔｉ−ｈｕｍａｎ ＣＤ３１抗体（製品コード：ＪＣ７０Ａ、Ｄａｋｏ社製）及びＧｏａｔ ａｎｔｉ−ｍｏｕｓｅ Ａｌｅｘａ Ｆｌｕｏｒ５４６−ｃｏｎｊｕｇａｔｅｄ ＩｇＧ抗体（製品コード：Ａ１１００１、Ｉｎｖｉｔｒｏｇｅｎ社製）にて第二培養細胞１０１ｂであるヒト血管内皮細胞を選択的に着色し、培養１日目、及び、７日目の積層組織を、それぞれ、ＣＬＳＭで観察した。結果を図１６（ｉ）、（ｊ）、（ｋ）、（ｌ）に示す。図１６（ｉ）、（ｊ）は、培養１日目を示し、図１６（ｋ）、（ｌ）が培養７日目を示す。図１６（ｊ）は、図１６（ｉ）の拡大図であり、図１６（ｌ）は、図１６（ｋ）の拡大図である。図１６では、血管内皮細胞が白く描出されるように示している。図１６（ｈ）、（ｉ）で示すように、培養１日目は、血枝の成長が観察されなかったが、培養７日目で微量の血枝の成長が観察され、実施例６ほど血管網占有面積は高くないものの、血管網も観察することができた。 The obtained three-dimensional structure was cultured at 37 ° C. for 7 days. Monoclonal mouse anti-human CD31 antibody (product code: JC70A, manufactured by Dako) and Goat anti-mouse Alexa Fluor 546-conjugated IgG antibody (product code: A11001, manufactured by Invitrogen) are human cultured endothelial cells 101b. The cells were selectively colored, and the laminated tissues on day 1 and day 7 were observed with CLSM. The results are shown in FIGS. 16 (i), (j), (k), and (l). FIGS. 16 (i) and (j) show the first day of culture, and FIGS. 16 (k) and (l) show the seventh day of culture. 16 (j) is an enlarged view of FIG. 16 (i), and FIG. 16 (l) is an enlarged view of FIG. 16 (k). In FIG. 16, the vascular endothelial cells are depicted in white. As shown in FIGS. 16 (h) and (i), no growth of blood branch was observed on the first day of culture, but a small amount of blood branch was observed on the seventh day of culture. Although the area occupied by the vascular network was not high, the vascular network could also be observed.
１０ 第一の積層構造
２０ 第二の積層構造
３０ ナノ細胞外マトリックス
１０１ 培養細胞
１０１ａ 第一培養細胞
１０１ｂ 第二培養細胞
１０２ 接着膜
１０２ａ 第１の物質からなる膜
１０２ｂ 第２の物質からなる膜
１０３ 被覆細胞
１０３ａ 第一被覆細胞
１０３ｂ 第二被覆細胞
DESCRIPTION OF SYMBOLS 10 1st laminated structure 20 2nd laminated structure 30 Nano extracellular matrix 101 Cultured cell 101a 1st cultured cell 101b 2nd cultured cell 102 Adhesion film | membrane 102a Film | membrane 102b which consists of 1st substance Film | membrane 103 which consists of 2nd substance Coated cell 103a First coated cell 103b Second coated cell
Claims (29)
前記培養細胞は、前記培養細胞の表面全体が接着膜で被覆された被覆細胞を含み、
前記接着膜を介して前記被覆細胞と前記培養細胞とが互いに接着している、細胞の三次元構造体。 A three-dimensional structure of cells in which cultured cells adhere to each other,
The cultured cell includes a coated cell in which the entire surface of the cultured cell is coated with an adhesive film,
A three-dimensional structure of cells in which the coated cells and the cultured cells are adhered to each other through the adhesive film.
前記被覆細胞が、前記第一培養細胞が前記接着膜で被覆された第一被覆細胞と、前記第二培養細胞が前記接着膜で被覆された第二被覆細胞とを含み、
前記第一培養細胞と前記第一被覆細胞とが前記接着膜を介して互いに接着している第一の積層構造をなし、
前記第二培養細胞と前記第二被覆細胞とが前記接着膜を介して互いに接着している第二の積層構造をなし、
前記第一の積層構造上に前記第二の積層構造が積層されている、請求項１乃至１１いずれか１項に記載の三次元構造体。 The cultured cell includes a first cultured cell and a second cultured cell different from the first cultured cell,
The coated cell includes a first coated cell in which the first cultured cell is coated with the adhesive film, and a second coated cell in which the second cultured cell is coated with the adhesive film,
The first cultured cells and the first coated cells form a first laminated structure in which the first cultured cells are bonded to each other through the adhesive film,
The second cultured cells and the second coated cells form a second laminated structure in which they are adhered to each other via the adhesive film,
The three-dimensional structure according to any one of claims 1 to 11, wherein the second laminated structure is laminated on the first laminated structure.
前記被覆細胞が、前記第一培養細胞が前記接着膜で被覆された第一被覆細胞と、前記第二培養細胞が前記接着膜で被覆された第二被覆細胞とを含み、
前記第一被覆細胞と前記第二培養細胞とが前記接着膜を介して互いに接着し、前記第二被覆細胞と前記第一培養細胞とが前記接着膜を介して互いに接着している、請求項１乃至１１いずれか１項に記載の三次元構造体。 The cultured cell includes a first cultured cell and a second cultured cell different from the first cultured cell,
The coated cell includes a first coated cell in which the first cultured cell is coated with the adhesive film, and a second coated cell in which the second cultured cell is coated with the adhesive film,
The first coated cell and the second cultured cell are adhered to each other through the adhesive film, and the second coated cell and the first cultured cell are adhered to each other through the adhesive film. The three-dimensional structure according to any one of 1 to 11.
前記培養細胞の表面全体が接着膜で被覆された被覆細胞を培養する工程を含み、
前記被覆細胞を培養する前記工程において、前記接着膜を介して、前記被覆細胞と前記培養細胞とを互いに接着させる、細胞の三次元構造体を製造する方法。 A method for producing a three-dimensional structure of cells by adhering cultured cells to each other,
Culturing the coated cell in which the entire surface of the cultured cell is coated with an adhesive film,
A method for producing a three-dimensional structure of cells, wherein in the step of culturing the coated cells, the coated cells and the cultured cells are adhered to each other via the adhesive film.
前記被覆細胞を作製する工程は、
第１の物質を含む膜で前記培養細胞を被覆する工程と、
前記第１の物質を含む膜で被覆された前記培養細胞を、前記第１の物質とは異なる第２の物質を含む膜で被覆する工程と、
を含む、請求項１７に記載の三次元構造体を製造する方法。 Further comprising the step of coating the entire surface of the cultured cell with the adhesive film to produce the coated cell,
The step of producing the coated cell comprises:
Coating the cultured cells with a membrane containing a first substance;
Coating the cultured cells coated with a film containing the first substance with a film containing a second substance different from the first substance;
The method of manufacturing the three-dimensional structure of Claim 17 containing these.
前記被覆細胞が、前記第一培養細胞が前記接着膜で被覆された第一被覆細胞と、前記第二培養細胞が前記接着膜で被覆された第二被覆細胞とを含み、
前記被覆細胞を培養する前記工程において、
前記接着膜を介して、前記第一培養細胞と前記第一被覆細胞とを接着させて、第一の積層構造を形成する第一の培養工程と、
前記接着膜を介して、前記第二培養細胞と前記第二被覆細胞とを接着させて、前記第一の積層構造上に第二の積層構造を形成する第二の培養工程と、
を含む、請求項１７乃至２０いずれか１項に記載の三次元構造体を製造する方法。 The cultured cell includes a first cultured cell and a second cultured cell different from the first cultured cell,
The coated cell includes a first coated cell in which the first cultured cell is coated with the adhesive film, and a second coated cell in which the second cultured cell is coated with the adhesive film,
In the step of culturing the coated cells,
A first culturing step for forming a first laminated structure by bonding the first cultured cells and the first coated cells via the adhesive film;
A second culturing step for forming the second laminated structure on the first laminated structure by adhering the second cultured cells and the second coated cells via the adhesive film;
The method of manufacturing the three-dimensional structure according to any one of claims 17 to 20, comprising:
前記被覆細胞が、前記第一培養細胞が前記接着膜で被覆された第一被覆細胞と、前記第二培養細胞が前記接着膜で被覆された第二被覆細胞とを含み、
前記被覆細胞を培養する前記工程において、前記接着膜を介して、前記第一被覆細胞と前記第二培養細胞とを互いに接着させるとともに、前記第二被覆細胞と前記第一培養細胞とを互いに接着させる、請求項１７乃至２０いずれか１項に記載の三次元構造体を製造する方法。 The cultured cell includes a first cultured cell and a second cultured cell different from the first cultured cell,
The coated cell includes a first coated cell in which the first cultured cell is coated with the adhesive film, and a second coated cell in which the second cultured cell is coated with the adhesive film,
In the step of culturing the coated cell, the first coated cell and the second cultured cell are adhered to each other through the adhesive film, and the second coated cell and the first cultured cell are adhered to each other. A method for manufacturing a three-dimensional structure according to any one of claims 17 to 20.
前記接着成分で前記培養細胞の全体を被覆して被覆細胞を作製し、前記被覆細胞を培養するための使用説明書と、
を含む、三次元構造体を製造するためのキット。 An adhesive component for forming the cultured cells with an adhesive film that adheres the cultured cells to each other;
An instruction manual for culturing the coated cell by coating the entire cultured cell with the adhesive component to produce a coated cell;
A kit for manufacturing a three-dimensional structure, comprising:
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