JP2005027598A - Cell culture chip and incubator and method for culturing cell by using those, cell-carrying module carrying spherical cell tissue body and spherical cell tissue body - Google Patents

Cell culture chip and incubator and method for culturing cell by using those, cell-carrying module carrying spherical cell tissue body and spherical cell tissue body Download PDF

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JP2005027598A
JP2005027598A JP2003272598A JP2003272598A JP2005027598A JP 2005027598 A JP2005027598 A JP 2005027598A JP 2003272598 A JP2003272598 A JP 2003272598A JP 2003272598 A JP2003272598 A JP 2003272598A JP 2005027598 A JP2005027598 A JP 2005027598A
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Koji Nakazawa
浩二 中澤
Junji Fukuda
淳二 福田
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Kitakyushu Foundation for Advancement of Industry Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell culture chip capable of embedding seed cells into regularly arranged cell culture cells, culturing these seed cells so as to become uniform and large spherical cell tissue bodies and carrying the spherical cell tissue bodies in a state each regularly arranged and keeping biological functions which the cells have originally in the long term in a high level without causing central necrosis, etc. <P>SOLUTION: The cell culture chip 10 is constituted so as to put the seed cells in dispersed culture mediums and embed the seed cells on a substrate surface 10a and culture the seed cells, and the chip 10 is equipped with cell culture cells 11 regularly arranged in an array shape or honeycomb shape on the substrate surface 10a. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

本発明は、細胞を培養して担持させるための細胞培養チップ及び培養器、それらを用いた球状細胞組織体の細胞培養方法、球状細胞組織体を担持させた細胞担持モジュール、球状細胞組織体に関する。   The present invention relates to a cell culture chip and an incubator for culturing and supporting cells, a cell culture method for spherical cell tissue bodies using them, a cell support module supporting spherical cell tissue bodies, and a spherical cell tissue body .

近年、細胞を組織培養させる技術は、再生医療や、ハイブリッド型人工臓器（バイオ人工臓器ともいう）、生体有用物質生産、生物組織や器官・臓器の機能の調査・探索、新薬のスクリーニング、内分泌かく乱物質等の影響を評価する動物実験代替法、センサ機能を有する細胞チップ等の各分野への産業利用が期待されている。
従来、動物細胞の培養方法としては、ポリスチレンやガラスなどの培養ディッシュや培養基板を用い、その表面を生体由来物質でコーティングしたり、あるいは化学的または物理化学的に処理したりして、その表面上で細胞を接着、伸展させる二次元培養法、いわゆる単層培養法によるものが一般的である。 In recent years, cell tissue culture techniques include regenerative medicine, hybrid artificial organs (also referred to as bioartificial organs), production of biologically useful substances, investigation / search for functions of biological tissues, organs / organs, screening for new drugs, endocrine disruption It is expected to be used in various fields such as an alternative to animal experiments for evaluating the effects of substances, etc., and a cell chip having a sensor function.
Conventionally, as a method for culturing animal cells, a culture dish such as polystyrene or glass or a culture substrate is used, and the surface thereof is coated with a biological substance, or chemically or physicochemically treated, and then the surface of the animal cell is cultured. A two-dimensional culture method in which cells are adhered and spread above, that is, a so-called monolayer culture method is generally used.

例えば、生体由来因子を有するコラーゲンをコートしたポリスチレンディッシュ上で細胞を培養すると、細胞はその表面に付着、伸展し、細胞質が扁平状に伸展した細胞形態をとる。
一方、生体組織や臓器などから単離した細胞、いわゆる初代細胞は、その由来する生体組織や臓器の特性や機能を保持している場合が多く、これらの細胞は産業上の利用価値が高い。しかし、ほとんどの場合、単層培養法では各種の細胞が持つ特性や機能が数日間から数週間で消失することが知られている。
特に、初代細胞の中でも高度に分化し、複雑多岐な機能を持つ初代肝細胞の場合、単層培養法ではその特性や機能が失われやすい。例えば、ラットの肝臓より単離した肝細胞を単層培養すると、肝臓の重要な機能であるタンパク質合成機能や解毒機能、薬物代謝機能などは培養開始から数日内に低下してしまう。これは単層培養法で培養された細胞では、細胞質が扁平した二次元状態となるため、本来生体内で細胞が有している細胞内構造や極性、隣接する細胞との結合による情報交換などの機能が低下、消失するためである。 For example, when a cell is cultured on a polystyrene dish coated with collagen having a biological factor, the cell adheres and spreads on the surface, and takes a cell form in which the cytoplasm is flattened.
On the other hand, cells isolated from biological tissues and organs, so-called primary cells, often retain the characteristics and functions of the biological tissues and organs from which they are derived, and these cells have high industrial utility value. However, in most cases, it is known that the characteristics and functions of various cells disappear in a few days to a few weeks in the monolayer culture method.
In particular, in the case of primary hepatocytes that are highly differentiated and have various functions among primary cells, the characteristics and functions are easily lost by the monolayer culture method. For example, when hepatocytes isolated from rat liver are cultured in a monolayer, protein synthesis functions, detoxification functions, drug metabolism functions, and the like, which are important functions of the liver, decrease within a few days from the start of culture. This is because cells cultured by the monolayer culture method have a two-dimensional state in which the cytoplasm is flattened, so the intracellular structure and polarity that cells originally have in the living body, information exchange by combining with adjacent cells, etc. This is because the function of is reduced and disappears.

このような細胞が本来持つ特性や機能の低下や消失を回避するために、細胞同士を集合化させ、生体組織に類似した三次元組織構造を備える球状細胞組織体（以下スフェロイドともいう）を構築させる三次元培養法（三次元組織化培養法）が注目されている。この球状細胞組織体を培養する技術に関連して、例えば以下のような技術が開示されている。   In order to avoid such deterioration and disappearance of the characteristics and functions inherent in cells, cells are assembled to form a spherical cell tissue body (hereinafter also referred to as spheroid) having a three-dimensional tissue structure similar to living tissue. The three-dimensional culture method (three-dimensional organized culture method) is attracting attention. For example, the following techniques are disclosed in relation to the technique for culturing the spherical cell tissue.

特許文献１には、肝細胞増殖因子及び上皮細胞増殖因子を有効成分として含有するスフェロイド形成促進剤を用いて、ラット初代肝細胞をポリウレタンフォームを用いた培養担体内で肝細胞スフェロイドに形成させる技術が記載されている。   Patent Document 1 discloses a technique for forming rat primary hepatocytes into hepatocyte spheroids in a culture carrier using polyurethane foam using a spheroid formation promoter containing hepatocyte growth factor and epidermal growth factor as active ingredients. Is described.

非特許文献１には三次元培養法として、５００μｍ程度の平均孔径を持つ多孔質材のポリウレタン発泡体孔内において、約２００個程度の肝細胞が集合して形成される球状細胞組織体が記載され、このスフェロイドは培養条件下で数週間以上の機能発現ができることが示されている。   Non-Patent Document 1 describes a spherical cell tissue formed by assembling about 200 hepatocytes in a polyurethane foam pore of a porous material having an average pore size of about 500 μm as a three-dimensional culture method. This spheroid has been shown to be capable of functioning over several weeks under culture conditions.

特許文献２には、アミノ基とカルボキシル基とを有する高分子により表面処理されたポリスチレン基板上で肝細胞を培養すると、スフェロイドが形成され、数週間の機能発現ができることが記載されている。また、フェニルボロン酸基を有するモノマー、アミノ基を有するモノマー、および２−ヒドロキシエチルメタクリレートの共重合体からなる高分子材料によって基板（シャーレ）表面をコーティングした培養床においても、肝細胞スフェロイドが形成され、アルブミン分泌能が維持されることが記載されている。   Patent Document 2 describes that when hepatocytes are cultured on a polystyrene substrate surface-treated with a polymer having an amino group and a carboxyl group, spheroids are formed and functional expression can be achieved for several weeks. Hepatocyte spheroids are also formed on a culture bed where the surface of a substrate (petriet) is coated with a polymer material comprising a monomer having a phenylboronic acid group, a monomer having an amino group, and a copolymer of 2-hydroxyethyl methacrylate. And albumin secretion ability is maintained.

非特許文献２には、スピナーフラスコ内で浮遊培養した肝細胞がスフェロイドを形成し、機能発現を長期的に維持できることが記載されている。
特開平１０−２９９５１号公報 特許第３０２０９３０号公報 Ｈ．Ｉｊｉｍａ等"Ｔｉｓｓｕｅ Ｅｎｇｉｎｅｅｒｉｎｇ"第４巻、第２号、２１３−２２６頁（１９９８）） （Ｙ．Ｓａｋａｉ等"Ｉｎｔｅｒｎａｔｉｏｎａ１ Ｊｏｕｒｎａ１ ｏｆ Ａｒｔｉｆｉｃｉａｌ Ｏｒｇａｎｓ"第１９巻、２９４−３０１頁（１９９６）） Non-Patent Document 2 describes that hepatocytes cultured in suspension in a spinner flask can form spheroids and maintain functional expression for a long time.
Japanese Patent Laid-Open No. 10-29951 Japanese Patent No. 3020930 H. Ijima et al., “Tissue Engineering” Vol. 4, No. 2, pages 213-226 (1998)) (Y. Sakai et al., “International 1 Journal 1 of Artificial Organs”, Volume 19, pages 294-301 (1996)).

前記従来の球状細胞組織体の培養法では以下のような課題を有していた。
（ａ）特許文献１や非特許文献１に記載のスフェロイド形成促進剤を用いてポリウレタンフォームの培養担体内で肝細胞スフェロイドを培養する方法や、特許文献２に記載の表面処理されたポリスチレン基板上に培養させる方法では、培養担体内や基板上に形成されるスフェロイドの粒径や担持位置のバラツキが大きく、スフェロイドの形態制御が困難であるため、球形に揃った球状細胞組織体が得られないという課題があった。
（ｂ）このため培養された球状細胞組織体を生体機能のチェックや新薬のスクリーニング等にバイオセンサとして用いる場合などに必要な一定の生体反応機能が得られず、センサなどとしての応答性や信頼性に欠けると共に、その適用範囲が限定され汎用性に欠けるという課題があった。
（ｃ）非特許文献２に記載の方法では、形成されるスフェロイドは培養液中に浮遊した状態となって規定の位置に確実に固定化できないため、培養されたスフェロイドを検知体として光ビームなどを所定パターンでスキャンして光学的な測定を行う新薬スクリーニング等のための自動測定機器に適用できないという問題があった。
（ｄ）さらに、前記特許文献１、２や非特許文献１、２に記載の方法ではスフェロイドの粒径制御が困難であるために、粒径が過度に大きくなるとその中心部の細胞には酸素や栄養分が充分に供給されずに中心部が壊死してしまい全体の細胞を有効に利用できないという問題があった。 The conventional method for culturing a spherical cell tissue has the following problems.
(A) A method of culturing hepatocyte spheroids in a polyurethane foam culture carrier using the spheroid formation promoter described in Patent Document 1 or Non-Patent Document 1, or on a surface-treated polystyrene substrate described in Patent Document 2 In the method of culturing the spheroids, since the spheroids formed on the culture support or on the substrate have a large variation in the particle size and position of the spheroids, it is difficult to control the spheroid morphology, so that spherical cell structures that are spherically shaped cannot be obtained. There was a problem.
(B) For this reason, a certain biological reaction function required when the cultured spherical cell tissue body is used as a biosensor for biological function check, new drug screening, etc. cannot be obtained. In addition to lacking in properties, there is a problem in that its application range is limited and versatility is lacking.
(C) In the method described in Non-Patent Document 2, since the formed spheroids are in a floating state in the culture solution and cannot be reliably fixed at a specified position, the cultured spheroids are used as a detector and a light beam or the like There is a problem that it cannot be applied to an automatic measuring instrument for new drug screening or the like that performs optical measurement by scanning in a predetermined pattern.
(D) Furthermore, since it is difficult to control the particle size of the spheroids by the methods described in Patent Documents 1 and 2 and Non-Patent Documents 1 and 2, oxygen is not contained in the central cell when the particle size becomes excessively large. In addition, there is a problem in that the whole cell cannot be effectively used because the central part is necrotized without sufficient supply of nutrients.

本発明は上記課題を解決するためになされたもので、球状細胞組織体を均一な所定の大きさに培養してこれを規則配列させた状態で担持させることのできる細胞培養チップや培養器を提供すると共に、培養される球状細胞組織体の中心部を壊死させることなく維持して生産性に優れた球状細胞組織体の細胞培養方法を提供し、生体反応機能を有する球状細胞組織体を担持させてなる細胞培養チップを複数配列して新薬等をスクリーニング処理する自動検査システム等を効率的に構成できる細胞担持モジュールを提供し、かつ生体反応の応答性と信頼性に優れた球状細胞組織体を提供することを目的する。   The present invention has been made in order to solve the above-described problems. A cell culture chip and an incubator capable of culturing spherical cell tissue bodies in a uniform predetermined size and carrying them in a regularly arranged state are provided. In addition to providing a cell culture method for a spherical cell tissue body with excellent productivity by maintaining the central part of the cultured spherical cell tissue body without necrosis, and carrying a spherical cell tissue body having a biological reaction function A cell-bearing module capable of efficiently configuring an automatic inspection system or the like for screening a new drug by arranging a plurality of cell culture chips, and a spherical cell tissue body excellent in responsiveness and reliability of biological reaction The purpose is to provide.

本発明の請求項１に記載の細胞培養チップは、基板表面上に種細胞を着床させる細胞培養チップであって、前記基板表面上にアレイ状やハニカム状等に規則配列され前記種細胞を凝集させて保持する細胞培養セルを備えた構成を有している。
この構成によって、以下の作用を有する。
（ａ）細胞培養セルがチップ基板表面上に規則配列されているので、培養液中の種細胞を規則配列された細胞培養セル内に着床させ、この種細胞を均一な大きさの球状細胞組織体に培養してこれらをそれぞれ規則配列させた状態で担持させることができる。
（ｂ）このような球状細胞組織体を規則配列させた細胞培養チップを用いて信頼性に優れた自動分析システムなどを構築でき、新薬スクリーニングなどの検査処理を効率的に行うことができる。
（ｃ）細胞培養チップの細胞培養セル内で、バラツキのない所定大きさの球状細胞組織体を培養できるので、中心壊死などを生じさせることがなく、しかも細胞が本来有する生体機能を高いレベルで長期的に維持させることができる。
（ｄ）このような球状細胞組織体は、アルブミンなどの特定タンパク質の分泌特性に優れているので、再生医療や、ハイブリッド型人工臓器、有用物質生産、生物組織や器官、臓器の機能の調査、探索、新薬のスクリーニングや内分泌かく乱物質などの影響を評価するための動物実験代替法、センサ電極上に細胞を担持させて構築される細胞チップ関連技術などの各分野を開発するために好適に利用できる。 The cell culture chip according to claim 1 of the present invention is a cell culture chip in which seed cells are implanted on the surface of the substrate, and the seed cells are regularly arranged in an array shape or a honeycomb shape on the substrate surface. It has the structure provided with the cell culture cell which aggregates and hold | maintains.
This configuration has the following effects.
(A) Since the cell culture cells are regularly arranged on the surface of the chip substrate, the seed cells in the culture solution are implanted in the regularly arranged cell culture cells, and the seed cells are uniformly spherical cells. These can be cultured in a tissue and supported in a state in which they are regularly arranged.
(B) A highly reliable automatic analysis system or the like can be constructed by using such a cell culture chip in which spherical cell tissues are regularly arranged, and examination processing such as new drug screening can be performed efficiently.
(C) Since a spherical cell tissue body of a predetermined size without variation can be cultured in the cell culture cell of the cell culture chip, it does not cause central necrosis and the biological function inherent in the cell is high. It can be maintained over the long term.
(D) Since such a spherical cell tissue body is excellent in the secretion characteristics of specific proteins such as albumin, regenerative medicine, hybrid artificial organs, production of useful substances, biological tissues and organs, investigation of organ functions, Suitable for use in various fields such as exploration, screening for new drugs, alternative animal experiments to evaluate the effects of endocrine disrupting substances, and cell chip-related technologies constructed by supporting cells on sensor electrodes it can.

ここで、種細胞としては、１種類の細胞を単独で、又は２種以上のものを混合した状態で用いることができる。例えばヒト、ブタ、イヌ、ラット、マウス等の動物由来の肝臓、膵臓、腎臓、神経、皮膚などから得られる初代細胞、または樹立された株化細胞を単独で用いたり、もしくは複数種のものを混合したりして用いることができる。特に、単層培養法では短期間で機能が低下するような細胞を本発明の対象となる種細胞として球状細胞組織体に培養することにより長期間に渡ってその生体機能を良好に保持させることができる。
なお、球状細胞組織体（スフェロイド）とは、分散状態から出発した細胞同士が高度に接着した状態、または高頻度に接着した状態の細胞群であり、細胞は単層でなく規則的又は不規則的に重なり合った複数細胞層を形成している球状またはそれに近い形状のものをいう。
細胞培養チップの基板用材料としては、ガラス等の無機材料、ステンレス鋼等の金属材料、もしくは、ポリスチレン、ポリエチレン、ポリプロピレン、ポリカーボネート、ポリアミド、ポリアセタールなどや、不飽和ポリエステル、ポリウレタン、シリコン樹脂、ＥＰＤＭ等の可撓性合成樹脂などの合成樹脂、ゴムが適用できる。これら可撓性のものを用いることによって、全体を湾曲させて円筒状などに形成して培養器中に配置できる。また、これらを平板状に用いてもよいし、又はこれらを円筒形や楕円筒形に加工して用いてもよい。
基板には、その基板表面を必要に応じてコラーゲンやホスファチジルコリン、ホスファチジルセリン、レシチンなどやこれらの不飽和脂肪酸を水添したもの等で被覆して、着床される種細胞に対する親和性や付着性などの表面性状を調整したものや、これら異なる素材のものを複数組み合わせて積層させた複合構造のもの等も用いることができる。
細胞培養チップの基板はその全体が略矩形板状や円盤状でその表面が平滑状に形成される。この基板表面上に規則配列される細胞培養セルは、マシニングセンタ、マイクロマシンなどを用いた穿孔加工、エッチング加工、フォトグラフィック加工などの手段で形成したり、又は射出成形型やプレス成形型などを用いて基板成形時に形成したりするようにしてもよい。 Here, as a seed cell, one type of cell can be used alone or in a state where two or more types are mixed. For example, primary cells obtained from livers, pancreas, kidneys, nerves, skin, etc. derived from animals such as humans, pigs, dogs, rats, mice, etc., or established cell lines are used alone, or multiple types of cells are used. It can be used by mixing. In particular, by culturing a cell whose function is reduced in a short period of time in a monolayer culture method as a seed cell of the present invention in a spherical cell tissue, the biological function of the cell can be maintained well over a long period of time. Can do.
A spherical cell tissue (spheroid) is a cell group in which cells starting from a dispersed state are in a highly adhered state or in a highly adhered state, and the cells are not monolayers but regular or irregular. A spherical shape or a shape close to it that forms a plurality of overlapping cell layers.
Cell culture chip substrate materials include inorganic materials such as glass, metal materials such as stainless steel, polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, unsaturated polyester, polyurethane, silicone resin, EPDM, etc. A synthetic resin such as a flexible synthetic resin or rubber can be applied. By using these flexible materials, the whole can be curved and formed into a cylindrical shape or the like and placed in an incubator. These may be used in a flat plate shape, or may be processed into a cylindrical shape or an elliptical cylindrical shape.
The substrate surface is coated with collagen, phosphatidylcholine, phosphatidylserine, lecithin, etc., as well as those hydrogenated with these unsaturated fatty acids as necessary, and the affinity and adhesion to the seed cells to be implanted For example, those having the surface properties adjusted, and those having a composite structure in which a plurality of these different materials are combined and laminated can be used.
The substrate of the cell culture chip is generally formed in a substantially rectangular plate shape or disk shape, and its surface is formed in a smooth shape. The cell culture cells regularly arranged on the surface of the substrate are formed by means such as drilling using a machining center or a micromachine, etching, or photolithography, or using an injection mold or a press mold. You may make it form at the time of board | substrate shaping | molding.

細胞培養セルは、基板表面上に窪み状などに形成された有底孔や、基板に穿設された無底孔の開口部を透水膜や非透水膜で覆設して形成したセル状部などであって、その開口部が円形状や多角形などに形成される。これら複数個の細胞培養セルを細胞培養チップの基板表面上に規則的に配列させてアレイ構造やハニカム構造などを有するセグメントとすることができる。また、一基板内に多数のセグメントを配置することもできる。
細胞培養セルの大きさは、その内部に所定数の種細胞が保持できる状態であればよい。また、その壁面及び／又は底面は、培養液や栄養液の不透過状態としたり、培養液や栄養液が透過できるように通液孔を設けたり、通液性の材質で構成したりすることができる。このようにセル内に培養液が流れるようにした場合には、セル内に栄養分等を供給できると共に、細胞培養セル内で培養される細胞の周囲に下降流や旋回流が形成されるので、球状細胞組織体をセル中心に位置付けしかも球状細胞組織体の生成を同時に助長させることができる。 A cell culture cell is a cell-shaped part formed by covering a bottomed hole formed in the shape of a depression on the surface of a substrate or a bottomless hole formed in a substrate with a permeable film or a non-permeable film. And the opening is formed in a circular shape or a polygonal shape. The plurality of cell culture cells can be regularly arranged on the substrate surface of the cell culture chip to form a segment having an array structure or a honeycomb structure. In addition, a large number of segments can be arranged in one substrate.
The size of the cell culture cell may be in a state where a predetermined number of seed cells can be held therein. In addition, the wall surface and / or the bottom surface thereof should be impermeable to the culture solution and nutrient solution, provided with a fluid passage hole so that the culture solution and nutrient solution can permeate, or configured with a liquid-permeable material. Can do. In this way, when the culture fluid is allowed to flow in the cell, nutrients and the like can be supplied into the cell, and a downward flow and swirl flow are formed around the cells cultured in the cell culture cell. The spherical cell tissue body can be positioned at the center of the cell and the generation of the spherical cell tissue body can be promoted simultaneously.

培養液中に分散される種細胞の濃度は、１個の細胞培養セル内に着床する種細胞の数が２〜１．５×１０5個となるように設定することが好ましく、より好ましくは５０〜３．０×１０4個の範囲である。これは適用する種細胞や培養液の種類、温度や時間などの培養条件にもよるが以下の理由による。すなわち、細胞培養セル中に着床して保持される種細胞数が５０個より少なくなるにつれ、バイオセンサなどに必要なタンパク質分泌機能などを効果的に発揮させることが困難となる傾向が現れ、逆に種細胞数が３．０×１０4個を超えるにつれ、セル内で培養される球状細胞組織体が大きくなりすぎてその中心部まで酸素や栄養分を行き渡らせることができず球状細胞組織体の中心部に壊死を生じさせるような傾向が生じ、これらの傾向は２個より少なくなるか又は１．５×１０5個を超えるとさらに顕著になるからである。
なお、細胞培養チップに入れられる培養液中の種細胞の濃度は、細胞培養チップの実効着床面積、前記細胞培養チップが配置される培養器の底面積、培養液量などの条件に応じて、前記細胞培養セル内の細胞数が前記適正細胞数となるように実験的に又は推定計算によりに設定することができる。 The concentration of the seed cells dispersed in the culture solution is preferably set so that the number of seed cells implanted in one cell culture cell is 2 to 1.5 × 10 5 , more preferably. Is in the range of 50 to 3.0 × 10 4 . This is due to the following reasons, although it depends on the type of seed cells and culture medium to be applied, and culture conditions such as temperature and time. That is, as the number of seed cells that are implanted and retained in the cell culture cell is less than 50, it tends to become difficult to effectively exert the protein secretion function necessary for biosensors, etc. On the other hand, as the number of seed cells exceeds 3.0 × 10 4 , the spherical cell tissue cultured in the cell becomes too large, and oxygen and nutrients cannot be distributed to the center of the spherical cell tissue. This is because there is a tendency to cause necrosis in the center of the film, and these tendencies become more prominent when the number is less than 2 or exceeds 1.5 × 10 5 .
Note that the concentration of seed cells in the culture solution put into the cell culture chip depends on conditions such as the effective implantation area of the cell culture chip, the bottom area of the incubator in which the cell culture chip is placed, and the amount of the culture solution. The number of cells in the cell culture cell can be set experimentally or by estimation calculation so that the number of cells is the appropriate number.

本発明の請求項２に記載の発明は、請求項１に記載の細胞培養チップであって、前記細胞培養セルが、前記基板に窪み状に形成された有底孔を備えた構成を有している。
この構成によって、請求項１に記載の作用に加えて以下の作用を有する。
（ａ）細胞培養セルが有底孔で形成されるので、基板が合成樹脂などからなる場合にエンボス加工やドリルを用いた穿孔加工など適用して、規則配列された細胞培養セルを精度よく基板上に形成させることができる。
（ｂ）細胞培養セルを有底孔としているので、その深さや孔径、内壁面の形状などを適正に設定することで内部に着床される種細胞の保持性を高め、凝集される球状細胞組織体の球形度を高めることができる。 Invention of Claim 2 of this invention is a cell culture chip of Claim 1, Comprising: The said cell culture cell has the structure provided with the bottomed hole formed in the said substrate at the hollow shape ing.
With this configuration, in addition to the operation of the first aspect, the following operation is provided.
(A) Since the cell culture cell is formed with a bottomed hole, when the substrate is made of a synthetic resin or the like, the substrate can be accurately arranged by applying embossing or drilling using a drill. Can be formed on top.
(B) Since the cell culture cell has a bottomed hole, by appropriately setting the depth, hole diameter, shape of the inner wall surface, etc., the retention of the seed cells implanted inside is improved, and the spherical cells that are aggregated The sphericity of the tissue can be increased.

本発明の請求項３に記載の発明は、請求項１に記載の細胞培養チップであって、前記細胞培養セルが、前記基板に形成された無底孔と、前記無底孔の底部を形成して前記基板の一面に積層されたメンブレンフィルム等の透水膜と、を備えた構成を有している。
この構成によって、請求項１に記載の作用に加えて以下の作用を有する。
（ａ）透水膜を介して細胞培養セル内の種細胞凝集体の周囲に培養液などによる旋回流や下降流を形成することができるので、球状細胞組織体の形成を促進させることができる。
（ｂ）培養中における細胞培養セル内への培養液の循環流通を良好に維持させることができ、培養効率性に優れた細胞培養チップを提供できる。
（ｃ）培養液による上下方向への循環流を細胞培養チップが配置される培養器や細胞担持モジュール内に形成できるので、細胞培養チップを一定スペース内に多数積層させて配置したり、それぞれ異なる種類の細胞培養チップを高密度で平面配置させたりすることができる。
（ｄ）これによって種々の医療システムのデバイスとして適用できると共に、有用細胞の培養生産性やセンサ検知性能の向上をさらに図ることができる。 The invention according to claim 3 of the present invention is the cell culture chip according to claim 1, wherein the cell culture cell forms a bottomless hole formed in the substrate and a bottom portion of the bottomless hole. And a water permeable film such as a membrane film laminated on one surface of the substrate.
With this configuration, in addition to the operation of the first aspect, the following operation is provided.
(A) Since a swirling flow or a downward flow due to a culture solution or the like can be formed around the seed cell aggregate in the cell culture cell via the water permeable membrane, formation of a spherical cell tissue body can be promoted.
(B) It is possible to satisfactorily maintain the circulation of the culture solution into the cell culture cell during the culture, and to provide a cell culture chip excellent in culture efficiency.
(C) Since a circulating flow in the vertical direction by the culture solution can be formed in the incubator or the cell carrying module in which the cell culture chip is arranged, a large number of cell culture chips are stacked in a certain space, or different from each other. Various types of cell culture chips can be arranged in a plane at high density.
(D) As a result, it can be applied as a device for various medical systems, and the culture productivity of useful cells and sensor detection performance can be further improved.

ここで、透水膜としては、細胞培養セルの内部に細胞を保持しておくために適用する種細胞より小さい孔径、例えば０．００１〜５μｍの範囲の孔径を有する細孔が形成されたものが好適に用いられる。また、細孔密度としては１０5〜１０11個／ｃｍ2、開孔率としては１４〜５０％のものが好適に用いられる。これにより、種細胞がセル外に流出するのを防止するとともに、セル内に形成される培養液や栄養液の流れを維持して種細胞を凝集させて球形度に優れた所定サイズの球状細胞組織体に成長させることができる。 Here, the water permeable membrane has a pore diameter smaller than that of the seed cell applied to hold the cells inside the cell culture cell, for example, pores having a pore diameter in the range of 0.001 to 5 μm. Preferably used. A pore density of 10 5 to 10 11 pores / cm 2 and a porosity of 14 to 50% are preferably used. This prevents the seed cells from flowing out of the cell and maintains the flow of the culture solution and nutrient solution formed in the cell to agglomerate the seed cells to give a spherical cell of a predetermined size with excellent sphericity. Can grow into an organization.

本発明の請求項４に記載の発明は、請求項１乃至３の内いずれか１項に記載の細胞培養チップであって、前記細胞培養セルが円形又は多角形状の開口部を有し、その開口径が培養される前記種細胞の大きさに対して２〜５０倍の範囲である構成を有している。
この構成によって、請求項１乃至３の内いずれか１項に記載の作用に加えて、以下の作用を有する。
（ａ）セル形状を特定範囲に設定しているので、各セル内で培養される種細胞同士が凝集して球状化するのをさらに促進させることができる。
（ｂ）各セル内にそれぞれ均一サイズの球状細胞組織体を効率的に培養することができ、常時一定の反応特性を有するバイオセンサなどとして機能させることができる。
ここで細胞培養セルは、開口径が種細胞の大きさに対して２〜５０倍、好ましくは４〜３０倍に形成される。その開口径が培養液を介して供給される種細胞の大きさに対してその４倍より小さくなるにつれ、所望の生体機能を発揮させるのに必要な所定数以上の種細胞をセル内に着床させるのが困難になるような傾向が生じ、逆に３０倍を超えるにつれ、球状細胞組織体が肥大して栄養分の供給不足により中心組織の壊死が生じたり、あるいは１つのセル内に複数個の球状細胞組織体が生じたりする傾向が生じ、これらの傾向は開口径が２倍より小さくなるかまたは５０倍を超えるとさらに顕著になるので好ましくない。
また、細胞培養セルが有底孔である場合には、細胞培養セルの深さは種細胞の大きさに対して１〜５０倍、好ましくは２〜３０倍に形成される。細胞培養セルの深さが２倍より少なくなるにつれ、セル内に保持できる種細胞の絶対数が不足したり、着床率が極端に低下したりするような弊害が生じ、逆に３０倍を超えるにつれ、培養液のセル内への流れが少なくなって酸素や養分の供給が阻害されるなどの弊害を生じ、特に、１倍未満又は５０倍を超えると更にこの傾向が種細胞の種類によって顕著になるので好ましくない。
なお、細胞培養セルが基板に形成された無底孔と無底孔の底部を形成する透水膜とを備えている場合は、細胞培養セルの深さは限定する必要がない。透水膜を介して、酸素や養分が豊富な新鮮な培養液をセル内に供給して老廃物が含まれる培養液と交換させることができ種細胞の培養を促進できるからである。また、透水膜を介して培養液に旋回流や下降流を形成することができるので、培養される球状細胞組織体の形成を促進させることができるからである。 Invention of Claim 4 of this invention is a cell culture chip of any one of Claims 1 thru | or 3, Comprising: The said cell culture cell has a circular or polygonal opening part, The opening diameter is in the range of 2 to 50 times the size of the seed cell to be cultured.
With this configuration, in addition to the operation described in any one of claims 1 to 3, the following operation is provided.
(A) Since the cell shape is set to a specific range, it is possible to further promote the aggregation and spheroidization of seed cells cultured in each cell.
(B) A spherical cell tissue of uniform size can be efficiently cultured in each cell, and can function as a biosensor having constant reaction characteristics at all times.
Here, the cell culture cell has an opening diameter of 2 to 50 times, preferably 4 to 30 times the size of the seed cell. As the opening diameter becomes smaller than four times the size of the seed cells supplied through the culture solution, a predetermined number or more of seed cells necessary for exerting a desired biological function are placed in the cells. The tendency to become difficult to lay occurs, and conversely, as it exceeds 30 times, the globular cell tissue is enlarged and necrosis of the central tissue occurs due to insufficient nutrient supply, or a plurality of cells are contained in one cell. Or the like, and these tendencies become undesirable when the opening diameter becomes smaller than 2 times or exceeds 50 times.
When the cell culture cell is a bottomed hole, the depth of the cell culture cell is 1 to 50 times, preferably 2 to 30 times the seed cell size. As the depth of the cell culture cell becomes less than 2 times, there is a detrimental effect that the absolute number of seed cells that can be held in the cell is insufficient, or the implantation rate is extremely reduced. As it exceeds the limit, the flow of the culture broth into the cell is reduced and the supply of oxygen and nutrients is hindered. Especially when the ratio is less than 1 or more than 50 times, this tendency further depends on the type of seed cell. Since it becomes remarkable, it is not preferable.
In addition, when the cell culture cell is provided with a bottomless hole formed in the substrate and a water permeable film forming the bottom of the bottomless hole, the depth of the cell culture cell does not need to be limited. This is because a fresh culture solution rich in oxygen and nutrients can be supplied into the cell through the water permeable membrane and exchanged with a culture solution containing waste products, thereby promoting the culture of seed cells. Moreover, since a swirl flow or a downward flow can be formed in the culture solution via the water permeable membrane, the formation of the spherical cell tissue to be cultured can be promoted.

本発明の請求項５に記載の発明は、請求項１乃至４の内いずれか１項に記載の細胞培養チップであって、前記細胞培養セルの底面や壁面の一部又は全部が下方中心に向けて傾斜状に形成されたテーパ部や湾曲部を備えた構成を有している。
この構成によって、請求項１乃至４の内のいずれか１項に記載の作用に加えて、以下の作用を有する。
（ａ）細胞培養セルがその内壁部や底部にテーパ部やＵ字状などの湾曲部を備えるので、細胞培養セル内に種細胞を着床させて細胞培養チップを静置したり、又は同細胞培養チップに所定の水平回転や揺動を付与したりすることによって、そのセル内の種細胞をセル中心部に効率的に凝集させて担持させることができる。
（ｂ）球状細胞組織体に培養される担持位置をさらに正確に規定できるので、この球状細胞組織体が担持された細胞培養チップをバイオセンサなどに適用して、光ビームを所定のパターンで細胞培養チップ上のそれぞれの球状細胞組織体にあてるようにスキャンさせて、その反射光や透過光を検出してセル内の球状細胞組織体の薬剤に対する反応特性を取得する際の測定信頼性などを高めることができる。 The invention according to claim 5 of the present invention is the cell culture chip according to any one of claims 1 to 4, wherein a part or all of the bottom surface and wall surface of the cell culture cell is centered downward. It has the structure provided with the taper part and curved part which were formed in the inclined form.
With this configuration, in addition to the operation described in any one of claims 1 to 4, the following operation is provided.
(A) Since the cell culture cell has a curved portion such as a taper portion or a U-shape on the inner wall portion or bottom portion thereof, seed cells are placed in the cell culture cell and the cell culture chip is left still, or the same By applying predetermined horizontal rotation and swinging to the cell culture chip, seed cells in the cell can be efficiently aggregated and supported on the center of the cell.
(B) Since the carrying position to be cultured in the spherical cell tissue can be more accurately defined, the cell culture chip carrying the spherical cell tissue is applied to a biosensor or the like, and the light beam is emitted in a predetermined pattern. The measurement reliability when acquiring the response characteristics to the drug of the spherical cell tissue in the cell by scanning each spherical cell tissue on the culture chip and detecting the reflected or transmitted light. Can be increased.

ここで、細胞培養セルの壁面に形成されるテーパ部は、基板の開口表面側や底面側の一部もしくは壁面及び底面の全面に渡って形成することができる。   Here, the tapered portion formed on the wall surface of the cell culture cell can be formed over a part of the opening surface side or the bottom surface side of the substrate or the entire wall surface and bottom surface.

本発明の請求項６に記載の発明は、請求項１乃至５の内いずれか１項に記載の細胞培養チップであって、前記基板表面や前記細胞培養セルの内表面が親水性を備えた構成を有している。
この構成によって、請求項１乃至５の内いずれか１項に記載の作用に加えて以下の作用を有する。
（ａ）基板表面に着床した種細胞を付着させることなく、その近傍に穿設された細胞培養セルに落とし込むことができ、少ない種細胞濃度の培養液であっても所定の細胞数をセル内に確保することができる。
（ｂ）セルの内表面が親水性高分子などで形成されるので、種細胞の細胞培養セル壁への付着が抑制され、球状細胞組織体の形成を促進できる。
ここで、親水性を備えた細胞培養チップは、基板が疎水性の場合は、親水性高分子膜でコートしたりプラズマ処理等の表面処理等によって形成することができる。また、基板をポリアセタール、ポリビニール、ポリエステル、ポリアミド等の親水性高分子で形成してもよい。 The invention according to claim 6 of the present invention is the cell culture chip according to any one of claims 1 to 5, wherein the substrate surface and the inner surface of the cell culture cell have hydrophilicity. It has a configuration.
With this configuration, in addition to the operation described in any one of claims 1 to 5, the following operation is provided.
(A) It is possible to drop seed cells that have been implanted on the surface of the substrate into a cell culture cell that has been drilled in the vicinity of the seed cells. Can be secured within.
(B) Since the inner surface of the cell is formed of a hydrophilic polymer or the like, the adhesion of seed cells to the cell culture cell wall is suppressed, and the formation of a spherical cell tissue body can be promoted.
Here, when the substrate is hydrophobic, the cell culture chip having hydrophilicity can be formed by coating with a hydrophilic polymer film or surface treatment such as plasma treatment. Moreover, you may form a board | substrate with hydrophilic polymers, such as a polyacetal, polyvinyl, polyester, and polyamide.

本発明の請求項７に記載の培養器は、請求項１乃至６の内いずれか１項に記載の細胞培養チップの前記基板の外周部の一方向に立設された側壁を備えた構成を有している。
この構成によって、請求項１乃至６の内いずれか１項に記載の作用に加えて、以下の作用を有する。
（ａ）培養器本体と細胞培養チップとが一体に形成されるので、耐久性や取り扱い性に優れている。
（ｂ）培養器内に培養液を供給して、細胞培養セル内に種細胞を着床させたり、培養器を揺動させたりする際に、培養器の底部が平面状に形成されているので、培養液の流れが妨げられることがなく、その培養条件を常時適正に維持させることができる。 The incubator according to claim 7 of the present invention has a configuration including a side wall erected in one direction of the outer peripheral portion of the substrate of the cell culture chip according to any one of claims 1 to 6. Have.
With this configuration, in addition to the operation described in any one of claims 1 to 6, the following operation is provided.
(A) Since the incubator body and the cell culture chip are integrally formed, they are excellent in durability and handleability.
(B) When the culture solution is supplied into the incubator and the seed cells are implanted in the cell culture cell or the incubator is rocked, the bottom of the incubator is formed flat. Therefore, the flow of the culture solution is not hindered, and the culture conditions can be always properly maintained.

本発明の請求項８に記載の細胞培養方法は、請求項１乃至６の内いずれか１項に記載の細胞培養チップを培養器の底部に固定して、又は、請求項７に記載の培養器を用いて、前記培養器に所定濃度の種細胞が分散された培養液を充填して静置し、前記種細胞を前記細胞培養セル内に着床させる細胞着床工程と、前記培養器を連続的又は間欠的に所定加速度で水平回転又は往復揺動させながら前記細胞培養セル内の前記種細胞同士を凝集培養させて球状細胞組織体に成長させる細胞培養工程と、を備えた構成を有している。
この構成によって、以下の作用を有する。
（ａ）所定種細胞濃度の培養液中に細胞培養チップを保持させた培養器を静置する静置工程を有するので、種細胞が沈降して細胞培養セル内に所定数の種細胞を着床させることができる。
（ｂ）培養器を水平回転又は揺動させながら培養する細胞培養工程を有するので、細胞培養セル内に保持された種細胞を効率的にセル中心部分に凝集、担持させ、所定大きさで球形に整った形状の球状細胞組織体に培養できる。
（ｃ）培養される球状細胞組織体を細胞培養セルの中心に位置付けて培養できるので、球形度に優れた球状細胞組織体を生産することができる。 In the cell culture method according to claim 8 of the present invention, the cell culture chip according to any one of claims 1 to 6 is fixed to the bottom of the incubator, or the culture according to claim 7. A cell implantation step of filling the culture vessel with a culture solution in which seed cells of a predetermined concentration are dispersed, and allowing the seed cells to be implanted in the cell culture cell, and the incubator. A cell culture step in which the seed cells in the cell culture cell are coagulated and cultured to grow into a spherical cell tissue body while rotating horizontally or reciprocally swinging at a predetermined acceleration continuously or intermittently. Have.
This configuration has the following effects.
(A) Since there is a stationary step in which a culture vessel holding a cell culture chip in a culture solution having a predetermined seed cell concentration is left, the seed cells settle and a predetermined number of seed cells are deposited in the cell culture cell. Can be floored.
(B) Since it has a cell culturing step of culturing while incubating the incubator horizontally or swinging, the seed cells held in the cell culture cell are efficiently aggregated and supported on the central part of the cell, and spherical with a predetermined size. Can be cultured in a spherical cell tissue with a well-defined shape.
(C) Since the cultured spherical cell tissue can be positioned and cultured at the center of the cell culture cell, a spherical cell tissue excellent in sphericity can be produced.

ここで、培養器を水平方向に回転または往復動させるなどの揺動操作は、培養器を一方向に回転させる場合や、交互に逆方向に回転させる場合などが含まれる。また、この揺動操作には、往復運動の方向を所定パターンで変化させる場合や、超音波振動などを付加する場合などが含まれる。これによって、種細胞が細胞培養セルのセル壁に沿って旋回しながら凝集成長して球状細胞組織体に培養され、三次元細胞組織化が促進される。この細胞培養セル内の種細胞やその凝集体に付加される揺動や回転の加速度及び付加時間などの揺動培養条件は、種細胞の種類、目的とする球状細胞組織体の大きさや生体機能、テーパ部などを有する細胞培養セルの形状や大きさなどに応じて、実験的にその適正範囲を定めることができる。   Here, the swinging operation such as rotating or reciprocating the incubator in the horizontal direction includes a case where the incubator is rotated in one direction, a case where the incubator is alternately rotated in the opposite direction, and the like. This swinging operation includes a case where the direction of reciprocating motion is changed in a predetermined pattern, a case where ultrasonic vibration is added, and the like. As a result, the seed cells coagulate and grow along the cell wall of the cell culture cell and are cultured in a spherical cell tissue body, thereby promoting three-dimensional cell organization. The rocking culture conditions such as rocking and rotation acceleration added to the seed cells and their aggregates in this cell culture cell, and the addition time are the types of seed cells, the size of the desired spherical cell tissue and the biological function. The appropriate range can be determined experimentally according to the shape and size of the cell culture cell having a tapered portion or the like.

本発明の請求項９に記載の発明は、請求項８に記載の細胞培養方法であって、前記種細胞が肝細胞である構成を有している。
この構成によって、請求項８に記載の作用に加えて以下の作用を有する。
（ａ）種細胞としてヒトなどの肝臓由来の肝細胞を用いるので、種々の新薬の肝臓への影響調査等の機能チェックにこの肝細胞から培養された球状細胞組織体を適用して、広範囲の医療分野に適用することができる。
特に、肝臓から単離された初代肝細胞は、初代細胞の中でも高度に分化した機能を有しており、その肝臓に由来するタンパク質合成機能や解毒機能、薬物代謝機能などを保持している。制御された大きさを備えてチップ上に規則配列された担持位置を有する球状細胞組織体は産業上や医療上への利用価値が極めて高いものである。 The invention according to claim 9 of the present invention is the cell culture method according to claim 8, wherein the seed cell is a hepatocyte.
With this configuration, in addition to the operation of the eighth aspect, the following operation is provided.
(A) Since hepatocytes derived from the liver of humans or the like are used as seed cells, a spherical cell tissue cultured from this hepatocyte is applied to a function check such as an investigation of the effects of various new drugs on the liver. It can be applied to the medical field.
In particular, primary hepatocytes isolated from the liver have highly differentiated functions among the primary cells, and retain a protein synthesis function, a detoxification function, a drug metabolism function, and the like derived from the liver. A spherical cell tissue having a controlled size and regularly arranged on the chip has a very high industrial and medical utility value.

本発明の請求項１０に記載の細胞担持モジュールは、請求項８又は９に記載の細胞培養方法を用いて請求項１乃至７のいずれか１項に記載の細胞培養チップの前記細胞培養セル内に前記球状細胞組織体を担持させてなる細胞担持モジュールであって、複数個の前記細胞培養チップをそのモジュールケース内に平面状及び／又は積層状に規則配列させた構成を有している。
この構成によって以下の作用を有する。
（ａ）単位スペース内に配置される細胞培養チップの配置密度を高めることができるので、細胞担持モジュールが適用される医療機器などの小型化、高密度化が図られ、種々の用途に適用でき、汎用性に優れている。
（ｂ）同一モジュールケース内にそれぞれ種類の異なる球状細胞組織体を担持させた細胞培養チップを配置して、同時に複数の医薬品に対する応答性等を効率的にチェックでき、薬品検査や生体機能性チェック等の効率性に優れている。
（ｃ）モジュール内の球状細胞組織体の保持密度が高められ、検出感度に優れたバイオセンサシステムを構築できる。 The cell-carrying module according to claim 10 of the present invention uses the cell culture method according to claim 8 or 9, and uses the cell culture method according to any one of claims 1 to 7 in the cell culture cell. A cell-carrying module in which the spherical cell tissue is carried, and a plurality of the cell culture chips are regularly arranged in a planar shape and / or a laminated shape in the module case.
This configuration has the following effects.
(A) Since the arrangement density of the cell culture chips arranged in the unit space can be increased, the medical device to which the cell carrying module is applied can be miniaturized and densified, and can be applied to various applications. Excellent in versatility.
(B) By placing cell culture chips carrying different types of spherical cell tissues in the same module case, it is possible to efficiently check the responsiveness to multiple drugs at the same time, and to perform drug tests and biofunctionality checks It is excellent in efficiency.
(C) The retention density of the spherical cell tissue in the module is increased, and a biosensor system excellent in detection sensitivity can be constructed.

本発明の請求項１１に記載の発明は、請求項９に記載の細胞培養方法を用いて前記肝細胞から培養された球状細胞組織体であって、その全体サイズが５０〜３００μｍの範囲であり、そのアルブミン分泌速度が細胞１０6個当り２０〜５００μｇ／日の範囲である構成を有している。
この構成によって以下の作用を有する。
（ａ）その全体サイズとアルブミン分泌速度が所定範囲に規定されるので、常時一定の反応特性を維持して薬剤の機能性や種々の細胞応答性をチェックするバイオセンサとしての信頼性に優れた球状細胞組織体を提供できる。
（ｂ）このように形態及び機能が制御された球状細胞組織体を細胞担持モジュールなどに担持させたり、あるいは単独で用いたりして、これを再生医療や、ハイブリッド型人工臓器、器官臓器の機能調査用センサ、新薬のスクリーニング用センサデバイス、各種のバイオチップ等の各分野へ広く利用することができ、汎用性に優れている。
ここで、肝細胞から培養される球状細胞組織体の全体サイズが５０μｍより小さくなると、被検知体と接触して分泌されるアルブミン量が不足して検知感度が低下し、逆に３００μｍを超えて大きくなると、中心壊死などの弊害が生じて生体機能測定の安定性が損なわれるので好ましくない。
また、そのアルブミン分泌速度が細胞（細胞数：１０6個）当り２０μｇ／日より低下する場合は、細胞が有する本来の機能が低下し、細胞応答性などを良好に検出することができない。逆に、５００μｇ／日を超えると細胞の生理機能レベルが不安定化しているおそれがあるので好ましくない。 The invention according to claim 11 of the present invention is a spherical cell tissue cultured from the hepatocytes using the cell culture method according to claim 9, wherein the whole size is in the range of 50 to 300 μm. The albumin secretion rate is in the range of 20 to 500 μg / day per 10 6 cells.
This configuration has the following effects.
(A) Since its overall size and albumin secretion rate are defined within a predetermined range, it has excellent reliability as a biosensor for checking the functionality and various cell responsiveness of drugs while maintaining constant reaction characteristics at all times. A spherical cell tissue can be provided.
(B) A spherical cell tissue body whose form and function are controlled in this way is carried on a cell-carrying module or the like, or is used alone, and this is used for regenerative medicine, hybrid artificial organs, organ organ functions. It can be widely used in various fields such as research sensors, sensor devices for screening new drugs, various biochips, etc., and has excellent versatility.
Here, if the total size of the spherical cell tissue cultured from hepatocytes is smaller than 50 μm, the amount of albumin secreted in contact with the detection target is insufficient and the detection sensitivity is lowered, and conversely exceeds 300 μm. If it is increased, adverse effects such as central necrosis occur and the stability of biological function measurement is impaired, which is not preferable.
In addition, when the albumin secretion rate is lower than 20 μg / day per cell (cell number: 10 6 ), the original function of the cell is lowered and the cell responsiveness cannot be detected well. Conversely, if it exceeds 500 μg / day, the physiological function level of the cell may be unstable, which is not preferable.

請求項１記載の発明によれば、以下の効果を有する。
（ａ）細胞培養セルがチップ基板表面上に規則配列されているので、培養液中の種細胞を規則配列された細胞培養セル内に着床させ、この種細胞を均一な大きさの球状細胞組織体に培養してこれらをそれぞれ規則配列させた状態で担持させることができる。
（ｂ）このような球状細胞組織体を規則配列させた細胞培養チップを用いて信頼性に優れた自動分析システムなどを構築でき、新薬スクリーニングなどの検査処理を効率的に行うことができる。
（ｃ）細胞培養チップの細胞培養セル内で、バラツキのない所定大きさの球状細胞組織体を培養できるので、中心壊死などを生じさせることがなく、しかも細胞が本来有する生体機能を高いレベルで長期的に維持させることができる。
（ｄ）このような球状細胞組織体は、アルブミンなどの特定タンパク質の分泌特性に優れているので、再生医療や、ハイブリッド型人工臓器、有用物質生産、生物組織や器官、臓器の機能の調査、探索、新薬のスクリーニングや内分泌かく乱物質などの影響を評価するための動物実験代替法、センサ電極上に細胞を担持させて構築される細胞チップ関連技術などの各分野を開発するために好適に利用できる。 According to invention of Claim 1, it has the following effects.
(A) Since the cell culture cells are regularly arranged on the surface of the chip substrate, the seed cells in the culture solution are implanted in the regularly arranged cell culture cells, and the seed cells are uniformly spherical cells. These can be cultured in a tissue and supported in a state in which they are regularly arranged.
(B) A highly reliable automatic analysis system or the like can be constructed by using such a cell culture chip in which spherical cell tissues are regularly arranged, and examination processing such as new drug screening can be performed efficiently.
(C) Since a spherical cell tissue body of a predetermined size without variation can be cultured in the cell culture cell of the cell culture chip, it does not cause central necrosis and the biological function inherent in the cell is high. It can be maintained over the long term.
(D) Since such a spherical cell tissue body is excellent in the secretion characteristics of specific proteins such as albumin, regenerative medicine, hybrid artificial organs, production of useful substances, biological tissues and organs, investigation of organ functions, Suitable for use in various fields such as exploration, screening for new drugs, alternative animal experiments to evaluate the effects of endocrine disrupting substances, and cell chip-related technologies constructed by supporting cells on sensor electrodes it can.

請求項２記載の発明によれば、請求項１記載の効果に加えて以下の効果を有する。
（ａ）細胞培養セルが有底孔で形成されるので、基板が合成樹脂などからなる場合にエンボス加工やドリルを用いた穿孔加工など適用して、規則配列された細胞培養セルを精度よく基板上に形成させることができる。
（ｂ）細胞培養セルを有底孔としているので、その深さや孔径、内壁面の形状などを適正に設定することで内部に着床される種細胞の保持性を高め、凝集される球状細胞組織体の球形度を高めることができる。 According to invention of Claim 2, in addition to the effect of Claim 1, it has the following effects.
(A) Since the cell culture cell is formed with a bottomed hole, when the substrate is made of a synthetic resin or the like, the substrate can be accurately arranged by applying embossing or drilling using a drill. Can be formed on top.
(B) Since the cell culture cell has a bottomed hole, by appropriately setting the depth, hole diameter, shape of the inner wall surface, etc., the retention of the seed cells implanted inside is improved, and the spherical cells that are aggregated The sphericity of the tissue can be increased.

請求項３記載の発明によれば、請求項１記載の効果に加えて以下の効果を有する。
（ａ）透水膜を介して細胞培養セル内の種細胞凝集体の周囲に培養液などによる旋回流や下降流を形成することができるので、球状細胞組織体の形成を促進させることができる。
（ｂ）培養中における細胞培養セル内への培養液の循環流通を良好に維持させることができ、培養効率性に優れた細胞培養チップを提供できる。
（ｃ）培養液による上下方向への循環流を細胞培養チップが配置される培養器や細胞担持モジュール内に形成できるので、細胞培養チップを一定スペース内に多数積層させて配置したり、それぞれ異なる種類の細胞培養チップを高密度で平面配置させたりすることができる。
（ｄ）これによって種々の医療システムのデバイスとして適用できると共に、有用細胞の培養生産性やセンサ検知性能の向上をさらに図ることができる。 According to invention of Claim 3, in addition to the effect of Claim 1, it has the following effects.
(A) Since a swirling flow or a downward flow due to a culture solution or the like can be formed around the seed cell aggregate in the cell culture cell via the water permeable membrane, formation of a spherical cell tissue body can be promoted.
(B) It is possible to satisfactorily maintain the circulation of the culture solution into the cell culture cell during the culture, and to provide a cell culture chip excellent in culture efficiency.
(C) Since a circulating flow in the vertical direction by the culture solution can be formed in the incubator or the cell carrying module in which the cell culture chip is arranged, a large number of cell culture chips are stacked in a certain space, or different from each other. Various types of cell culture chips can be arranged in a plane at high density.
(D) As a result, it can be applied as a device for various medical systems, and the culture productivity of useful cells and sensor detection performance can be further improved.

請求項４記載の発明によれば、請求項１乃至３の内いずれか１項に記載の効果に加えて、以下の効果を有する。
（ａ）セル形状を特定範囲に設定しているので、各セル内で培養される種細胞同士が凝集して球状化するのをさらに促進させることができる。
（ｂ）各セル内にそれぞれ均一サイズの球状細胞組織体を効率的に培養することができ、常時一定の反応特性を有するバイオセンサなどとして機能させることができる。 According to invention of Claim 4, in addition to the effect of any one of Claims 1 thru | or 3, it has the following effects.
(A) Since the cell shape is set to a specific range, it is possible to further promote the aggregation and spheroidization of seed cells cultured in each cell.
(B) A spherical cell tissue of uniform size can be efficiently cultured in each cell, and can function as a biosensor having constant reaction characteristics at all times.

請求項５記載の発明によれば、請求項１乃至４の内いずれか１項に記載の効果に加えて、以下の効果を有する。
（ａ）細胞培養セルがその内壁部や底部にテーパ部やＵ字状などの湾曲部を備えるので、細胞培養セル内に種細胞を着床させて細胞培養チップを静置したり、又は同細胞培養チップに所定の水平回転や揺動を付与したりすることによって、そのセル内の種細胞をセル中心部に効率的に凝集させて担持させることができる。
（ｂ）球状細胞組織体に培養される担持位置をさらに正確に規定できるので、この球状細胞組織体が担持された細胞培養チップをバイオセンサなどに適用して、光ビームを所定のパターンで細胞培養チップ上のそれぞれの球状細胞組織体にあてるようにスキャンさせて、その反射光や透過光を検出してセル内の球状細胞組織体の薬剤に対する反応特性を取得する際の測定信頼性などを高めることができる。 According to invention of Claim 5, in addition to the effect of any one of Claims 1 thru | or 4, it has the following effects.
(A) Since the cell culture cell has a curved portion such as a taper portion or a U-shape on the inner wall portion or bottom portion thereof, seed cells are placed in the cell culture cell and the cell culture chip is left still, or the same By applying predetermined horizontal rotation and swinging to the cell culture chip, seed cells in the cell can be efficiently aggregated and supported on the center of the cell.
(B) Since the carrying position to be cultured in the spherical cell tissue can be more accurately defined, the cell culture chip carrying the spherical cell tissue is applied to a biosensor or the like, and the light beam is emitted in a predetermined pattern. The measurement reliability when acquiring the response characteristics to the drug of the spherical cell tissue in the cell by scanning each spherical cell tissue on the culture chip and detecting the reflected or transmitted light. Can be increased.

請求項６記載の発明によれば、請求項１乃至５の内いずれか１項に記載の効果に加えて以下の効果を有する。
（ａ）基板表面に着床した種細胞を付着させることなく、その近傍に穿設された細胞培養セルに落とし込むことができ、少ない種細胞濃度の培養液であっても所定の細胞数をセル内に確保することができる。
（ｂ）セルの内表面が親水性高分子などで形成されるので、種細胞の細胞培養セル壁への付着が抑制され、球状細胞組織体の形成を促進できる。 According to invention of Claim 6, in addition to the effect of any one of Claims 1 thru | or 5, it has the following effects.
(A) It is possible to drop seed cells that have been implanted on the surface of the substrate into a cell culture cell that has been drilled in the vicinity of the seed cells. Can be secured within.
(B) Since the inner surface of the cell is formed of a hydrophilic polymer or the like, the adhesion of seed cells to the cell culture cell wall is suppressed, and the formation of a spherical cell tissue body can be promoted.

請求項７記載の発明によれば、請求項１乃至６の内いずれか１項に記載の効果に加えて、以下の効果を有する。
（ａ）培養器本体と細胞培養チップとが一体に形成されるので、耐久性や取り扱い性に優れている。
（ｂ）培養器内に培養液を供給して、細胞培養セル内に種細胞を着床させたり、培養器を揺動させたりする際に、培養器の底部が平面状に形成されているので、培養液の流れが妨げられることがなく、その培養条件を常時適正に維持させることができる。 According to invention of Claim 7, in addition to the effect of any one of Claims 1 thru | or 6, it has the following effects.
(A) Since the incubator body and the cell culture chip are integrally formed, they are excellent in durability and handleability.
(B) When the culture solution is supplied into the incubator and the seed cells are implanted in the cell culture cell or the incubator is rocked, the bottom of the incubator is formed flat. Therefore, the flow of the culture solution is not hindered, and the culture conditions can be always properly maintained.

請求項８記載の発明によれば、以下の効果を有する。
（ａ）所定種細胞濃度の培養液中に細胞培養チップを保持させた培養器を静置する静置工程を有するので、種細胞が沈降して細胞培養セル内に所定数の種細胞を着床させることができる。
（ｂ）培養器を水平回転又は揺動させながら培養する細胞培養工程を有するので、細胞培養セル内に保持された種細胞を効率的にセル中心部分に凝集、担持させ、所定大きさで球形に整った形状の球状細胞組織体に培養できる。
（ｃ）培養される球状細胞組織体を細胞培養セルの中心に位置付けて培養できるので、球形度に優れた球状細胞組織体を生産することができる。 According to invention of Claim 8, it has the following effects.
(A) Since there is a stationary step in which a culture vessel holding a cell culture chip in a culture solution having a predetermined seed cell concentration is left, the seed cells settle and a predetermined number of seed cells are deposited in the cell culture cell. Can be floored.
(B) Since it has a cell culturing step of culturing while incubating the incubator horizontally or swinging, the seed cells held in the cell culture cell are efficiently aggregated and supported on the central part of the cell, and spherical with a predetermined size. Can be cultured in a spherical cell tissue with a well-defined shape.
(C) Since the cultured spherical cell tissue can be positioned and cultured at the center of the cell culture cell, a spherical cell tissue excellent in sphericity can be produced.

請求項９記載の発明によれば、請求項８記載の効果に加えて以下の効果を有する。
（ａ）種細胞としてヒトなどの肝臓由来の肝細胞を用いるので、種々の新薬の肝臓への影響調査等の機能チェックにこの肝細胞から培養された球状細胞組織体を適用して、広範囲の医療分野に適用することができる。 According to invention of Claim 9, in addition to the effect of Claim 8, it has the following effects.
(A) Since hepatocytes derived from the liver of humans or the like are used as seed cells, a spherical cell tissue cultured from this hepatocyte is applied to a function check such as an investigation of the effects of various new drugs on the liver. It can be applied to the medical field.

請求項１０記載の発明によれば、以下の効果を有する。
（ａ）単位スペース内に配置される細胞培養チップの配置密度を高めることができるので、細胞担持モジュールが適用される医療機器などの小型化、高密度化が図られ、種々の用途に適用でき、汎用性に優れている。
（ｂ）同一モジュールケース内にそれぞれ種類の異なる球状細胞組織体を担持させた細胞培養チップを配置して、同時に複数の医薬品に対する応答性等を効率的にチェックでき、薬品検査や生体機能性チェック等の効率性に優れている。
（ｃ）モジュール内の球状細胞組織体の保持密度が高められ、検出感度に優れたバイオセンサシステムを構築できる。 According to invention of Claim 10, it has the following effects.
(A) Since the arrangement density of the cell culture chips arranged in the unit space can be increased, the medical device to which the cell carrying module is applied can be miniaturized and densified, and can be applied to various applications. Excellent in versatility.
(B) By placing cell culture chips carrying different types of spherical cell tissues in the same module case, it is possible to efficiently check the responsiveness to multiple drugs at the same time, and to perform drug tests and biofunctionality checks It is excellent in efficiency.
(C) The retention density of the spherical cell tissue in the module is increased, and a biosensor system excellent in detection sensitivity can be constructed.

請求項１１記載の発明によれば、以下の効果を有する。
（ａ）その全体サイズとアルブミン分泌速度が所定範囲に規定されるので、常時一定の反応特性を維持して薬剤の機能性や種々の細胞応答性をチェックするバイオセンサとしての信頼性に優れた球状細胞組織体を提供できる。
（ｂ）このように形態及び機能が制御された球状細胞組織体を細胞担持モジュールなどに担持させたり、あるいは単独で用いたりして、これを再生医療や、ハイブリッド型人工臓器、器官臓器の機能調査用センサ、新薬のスクリーニング用センサデバイス、各種のバイオチップ等の各分野へ広く利用することができ、汎用性に優れている。 According to invention of Claim 11, it has the following effects.
(A) Since its overall size and albumin secretion rate are defined within a predetermined range, it has excellent reliability as a biosensor for checking the functionality and various cell responsiveness of drugs while maintaining constant reaction characteristics at all times. A spherical cell tissue can be provided.
(B) A spherical cell tissue body whose form and function are controlled in this way is carried on a cell-carrying module or the like, or is used alone, and this is used for regenerative medicine, hybrid artificial organs, organ organ functions. It can be widely used in various fields such as research sensors, sensor devices for screening new drugs, various biochips, etc., and has excellent versatility.

（実施の形態１）
図１は本発明の実施の形態１に係る細胞培養チップが載置された培養器の斜視図であり、図２は細胞培養チップの拡大断面図である。但し、細胞培養チップの形状はこれに限定されるものではない。
図１及び図２において、１０はポリスチレンやポリカーボネート、ポリサルホン、ポリアクリレート、ポリエステル（ポリエチレンテレフタレート）、ポリウレタン等の合成樹脂を素材とする板状体を加工して載置したり又は合成樹脂製の容器底部を穿孔加工やエンボス加工等を施して培養容器と一体に形成されたりした実施の形態１の細胞培養チップ、１１は細胞培養チップ１０の基板表面１０ａに穿設されアレイ状やハニカム状などに規則配列された複数の細胞培養セル、１２は細胞培養チップ１０がその底部に載置又は形成され種細胞を含む培養液が入れられる円筒容器状の培養器である。
図２（ａ）に示すように細胞培養セル１１は円筒状の底部１１ａと壁部１１ｂとを備えた有底孔であって、その開口径が５０〜５００μｍ、その深さが２０〜２０００μｍに形成されている。
細胞培養チップ１０は、例えば一辺の長さが１５ｍｍ、厚さが０．１〜１０ｍｍの正方形板状や円盤状であって、その基板表面１０ａ上の各格子点に規則配列される細胞培養セル１１が４．０×１０2〜４．０×１０4個／ｃｍ2の配置密度となるようにマシニングセンタ等を用いた穿孔加工やエンボス加工等により形成される。 (Embodiment 1)
1 is a perspective view of an incubator on which a cell culture chip according to Embodiment 1 of the present invention is placed, and FIG. 2 is an enlarged cross-sectional view of the cell culture chip. However, the shape of the cell culture chip is not limited to this.
1 and 2, reference numeral 10 denotes a plate made of synthetic resin such as polystyrene, polycarbonate, polysulfone, polyacrylate, polyester (polyethylene terephthalate), polyurethane, or the like, or a synthetic resin container. The cell culture chip 11 according to the first embodiment, in which the bottom is subjected to drilling or embossing or the like and formed integrally with the culture vessel, 11 is drilled in the substrate surface 10a of the cell culture chip 10 to form an array or a honeycomb. A plurality of regularly arranged cell culture cells 12 are cylindrical vessel-shaped incubators in which a cell culture chip 10 is placed or formed on the bottom and into which a culture solution containing seed cells is placed.
As shown in FIG. 2A, the cell culture cell 11 is a bottomed hole having a cylindrical bottom portion 11a and a wall portion 11b, and has an opening diameter of 50 to 500 μm and a depth of 20 to 2000 μm. Is formed.
The cell culture chip 10 is, for example, a square plate or a disk having a side length of 15 mm and a thickness of 0.1 to 10 mm, and is regularly arranged at each lattice point on the substrate surface 10a. 11 is formed by drilling or embossing using a machining center or the like so that the arrangement density is 4.0 × 10 2 to 4.0 × 10 4 pieces / cm 2 .

なお、細胞培養セル１１は図２（ｂ）〜（ｇ）の変形例１〜６に示すようにその底面や壁面の一部又は全部にテーパ部１１ｃを設けることができる。
すなわち、図２（ｂ）は細胞培養セル１１の底面にすり鉢状のテーパ部１１ｃを設けた例を、図２（ｃ）は垂直壁面と底面との間にテーパ部１１ｃを設けた例を、図２（ｄ）は壁部全部をテーパ部１１ｃとした例を、図２（ｅ）は開口部近傍にのみテーパ部１１ｃを設けた例を示している。また図２（ｆ）は垂直壁を設けることなく、全体をすり鉢状に形成した例を、図２（ｇ）はセル底部を丸く湾曲させて形成した例を示している。
このようなテーパ部１１ｃが細胞培養セル１１に形成された細胞培養チップ１０を回転、揺動させたり、又は静置させたりすることによって、セル内で球状に凝集する種細胞をセル中心に位置付けることができ、細胞培養チップ１０上に配列される各球状細胞組織体の担持位置をより精密に規定することができる。こうして、細胞培養チップを多数のバイオ素子を有した複合センサに適用して自動化する際等の測定信頼性が高められる。 In addition, the cell culture cell 11 can provide the taper part 11c in the one part or all part of the bottom face and a wall surface, as shown in the modifications 1-6 of FIG.2 (b)-(g).
That is, FIG. 2 (b) shows an example in which a mortar-shaped tapered portion 11c is provided on the bottom surface of the cell culture cell 11, and FIG. 2 (c) shows an example in which a tapered portion 11c is provided between the vertical wall surface and the bottom surface. FIG. 2D shows an example in which the entire wall portion is a tapered portion 11c, and FIG. 2E shows an example in which the tapered portion 11c is provided only in the vicinity of the opening. FIG. 2 (f) shows an example in which the whole is formed in a mortar shape without providing a vertical wall, and FIG. 2 (g) shows an example in which the cell bottom is rounded and curved.
By such a taper portion 11c rotating, swinging, or leaving the cell culture chip 10 formed in the cell culture cell 11, the seed cells that aggregate in a spherical shape in the cell are positioned at the cell center. In addition, the carrying position of each spherical cell tissue array arranged on the cell culture chip 10 can be defined more precisely. In this way, measurement reliability such as when the cell culture chip is automated by applying it to a composite sensor having a large number of bioelements is improved.

以下、細胞培養チップ１０を用いて、細胞が本来有するタンパク質合成機能や解毒機能、薬物代謝機能を高レベルで有する球状細胞組織体を培養する細胞培養方法について説明する。
まず、種細胞着床工程においては、培養器１２に所定濃度のラット肝細胞などの種細胞が分散された培養液を充填して所定時間例えば、０．１〜２４時間静置する。これによって、培養液中の前記種細胞を沈降させ、その所定数の種細胞を細胞培養セル１１内に着床させる。
次の細胞培養工程では、培養器１２を水平回転又は揺動させることにより、回転や振動による所定の加速度を細胞培養セル１１内の種細胞に連続的又は間欠的に付与させる。こうして、種細胞同士の凝集を助長させながら培養器１２内を所定の温度、湿度、炭酸ガス濃度となる培養条件に維持することにより、種細胞を培養して所定大きさ、例えば、５０〜３００μｍで、しかも所定のタンパク質分泌能を備えた球状細胞組織体に成長させることができる。
このように大きさや形状が所定値に制御された組織構造を有する球状細胞組織体は、タンパク質合成機能や解毒機能、薬物代謝機能を高レベルで保持しており、しかもバイオセンサなどに必要なバラツキの少ない反応特性を備えている。
なお、以上のように培養された球状細胞組織体を担持させた細胞培養セル１１を備える細胞培養チップ１０を容器内に複数組み規則配列するとことにより細胞担持モジュールを構成することもできる。 Hereinafter, a cell culture method for culturing a spherical cell tissue having a high level of protein synthesis function, detoxification function, and drug metabolism function inherent to cells using the cell culture chip 10 will be described.
First, in the seed cell implantation step, the culture vessel 12 is filled with a culture solution in which seed cells such as rat hepatocytes having a predetermined concentration are dispersed and left to stand for a predetermined time, for example, 0.1 to 24 hours. As a result, the seed cells in the culture solution are sedimented, and the predetermined number of seed cells are implanted in the cell culture cell 11.
In the next cell culturing step, the incubator 12 is horizontally rotated or oscillated to apply a predetermined acceleration due to rotation or vibration to the seed cells in the cell culture cell 11 continuously or intermittently. In this way, by maintaining the inside of the incubator 12 at a predetermined temperature, humidity, and carbon dioxide gas concentration while promoting the aggregation of the seed cells, the seed cells are cultured to have a predetermined size, for example, 50 to 300 μm. In addition, it can be grown into a spherical cell tissue having a predetermined protein secretion ability.
The spherical cell tissue having a tissue structure whose size and shape are controlled to a predetermined value as described above retains a high level of protein synthesis function, detoxification function, and drug metabolism function, and is also necessary for biosensors and the like. It has less reaction characteristics.
In addition, a cell carrying module can also be comprised by arranging the cell culture chip | tip 10 provided with the cell culture cell 11 which carry | supported the spherical cell structure | tissue culture | cultivated as mentioned above regularly in a container.

このように球状細胞組織体を担持させた細胞培養チップ１０や細胞培養モジュールは、再生医療、ハイブリッド型人工臓器、生体組織の機能探索、新薬のスクリーニング、内分泌かく乱物質等の影響評価、細胞チップ等の各技術分野に広く利用することができる。
以下に比較例を含む実施例を示して、本実施の形態１をさらに具体的に説明する。なお、本発明は以下の実施例のものに限定されるものではない。 Thus, the cell culture chip 10 and the cell culture module carrying the spherical cell tissue body are used for regenerative medicine, hybrid artificial organs, biological tissue function search, screening for new drugs, evaluation of effects of endocrine disrupting substances, cell chips, etc. It can be widely used in each technical field.
Hereinafter, the first embodiment will be described more specifically with reference to examples including comparative examples. The present invention is not limited to the following examples.

実施例で使用した肝細胞の調製および培養は以下のように行った。
まず、初代ラット肝細胞を調製するために０．５ｍｇ／ｍＬのコラゲナーゼ（和光純薬杜）溶液１５０ｍＬを用意した。７週齢の雄ウィスター系ラット（体重２５０ｇ）の門脈（肝臓に入る血管）にカニューレを導入し、脱血液を３０ｍＬ／ｍｉｎで５分間流した後、３７℃に加温したコラゲナーゼ溶液を１５ｍＬ／ｍｉｎで１０分間流した。コラゲナーゼによって処理された肝臓を培養液に入れ、メスとピペットを使って肝細胞を分散させた。得られた肝細胞懸濁液を３回洗浄し、肝細胞以外の細胞を取り除いた（９５%以上の純度）。このようにして単離した肝細胞を以下の培養実験の種細胞として使用した。 The hepatocytes used in the examples were prepared and cultured as follows.
First, in order to prepare primary rat hepatocytes, 150 mL of a 0.5 mg / mL collagenase (Wako Pure Chemical Industries Ltd.) solution was prepared. A cannula was introduced into the portal vein (blood vessel entering the liver) of a 7-week-old male Wistar rat (body weight 250 g), blood removal was allowed to flow at 30 mL / min for 5 minutes, and then 15 mL of collagenase solution heated to 37 ° C. was added. For 10 minutes. The liver treated with collagenase was placed in the culture medium, and hepatocytes were dispersed using a scalpel and a pipette. The obtained hepatocyte suspension was washed three times to remove cells other than hepatocytes (purity of 95% or more). The hepatocytes isolated in this way were used as seed cells for the following culture experiments.

肝細胞の培養には、ダルベッコ改変イーグル培地（ギブコ製：ｄｕ１ｂｅｃｃｏ'ｓ ｍｏｄｉｆｉｅｄ ｅａｇ１ｅ ｍｅｄｉｕｍ）１３．５ｇ／Ｌに、６０ｍｇ／Ｌプロリン、５０ｍｇ／ｍＬ上皮細胞成長因子（フナコシ製：ＥＧＦ）、１０ｍｇ／Ｌインシュリン（シグマ製）、７．５ｍｇ／Ｌヒドロコルチゾン（和光純薬製）、０．１μＭ硫酸銅・５水和物（和光純薬製）、３μｇ／Ｌセレン酸（和光純薬製）、５０ｐＭ硫酸亜鉛・７水和物（和光純薬製）、５０μｇ／Ｌリノール酸（シグマ製）、５８．８ｍｇ／Ｌペニシリン（明治製菓製）、１００ｍｇ／Ｌストレプトマイシン（明治製菓製）、１．０５ｇ／Ｌ炭酸水素ナトリウム（和光純薬製）、１．１９ｇ／Ｌの２−［４−（２−ヒドロキシエチル）−１−ピペラジニル］エタンスルホン酸（同人堂製：ＨＥＰＥＳ、２−［４−（２−ｈｙｄｒｏｘｙｅｔｈｙｌ）−１−ｐｉｐｅｒａｚｉｎｙｌ］ｅｔｈａｎｅｓｕｌｆｏｎｉｃ ａｃｉｄ）を加えた無血清培地を用い、５％炭酸ガス、９５％大気の雰囲気下で培養を行った。また、上記の２ｍＬの新鮮培地を用い、２日毎に培地交換を行った。   For the culture of hepatocytes, Dulbecco's modified eagle medium (manufactured by Gibco: du1becco's modified eagle medium) 13.5 g / L, 60 mg / L proline, 50 mg / mL epidermal growth factor (Funakoshi: EGF), 10 mg / L insulin (manufactured by Sigma), 7.5 mg / L hydrocortisone (manufactured by Wako Pure Chemical Industries), 0.1 μM copper sulfate pentahydrate (manufactured by Wako Pure Chemical Industries), 3 μg / L selenic acid (manufactured by Wako Pure Chemical Industries), 50 pM Zinc sulfate heptahydrate (manufactured by Wako Pure Chemical Industries), 50 μg / L linoleic acid (manufactured by Sigma), 58.8 mg / L penicillin (manufactured by Meiji Seika), 100 mg / L streptomycin (manufactured by Meiji Seika), 1.05 g / L sodium hydrogen carbonate (manufactured by Wako Pure Chemical Industries), 1.19 g / L of 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfur Using a serum-free medium supplemented with phonic acid (Doujindo: HEPES, 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid), the cells were cultured in an atmosphere of 5% carbon dioxide and 95% air. went. In addition, the medium was changed every 2 days using the above 2 mL of fresh medium.

マイクロマシン等を用いて、直径３５ｍｍのポリスチレン製の培養容器１２（ファルコン製）表面に１．５ｃｍ角、厚さ１．２ｍｍの細胞培養チップ１０を作製した。
すなわち、細胞培養チップ１０の基板表面１０ａに直径３００μｍ、深さ５０μｍの円柱状セル（細胞培養セル１１）を、例えばピッチ３３０μｍ、軸角６０度で形成された三角配置の格子点上に配列して設けた。この細胞培養チップ１０はその基板１０ａ上に合計約２４００個の細穴状の細胞培養セル１１を有する。このようにして作製された細胞培養チップ１０をアルコール滅菌及び６時間の紫外線滅菌処理をして、肝細胞の培養実験に使用した。
上記記載の方法によってラット肝細胞をそれぞれ調製し、細胞密度が５．０×１０5ｃｅ１１ｓ／ｍＬのラット細胞を含む懸濁液（種細胞を含む培養液）を得た。この培養液２ｍＬを以下の各条件（１）〜（４）の基板上に播種して培養する比較実験を行った。 Using a micromachine or the like, a cell culture chip 10 having a 1.5 cm square and a thickness of 1.2 mm was prepared on the surface of a polystyrene culture vessel 12 (manufactured by Falcon) having a diameter of 35 mm.
That is, cylindrical cells (cell culture cells 11) having a diameter of 300 μm and a depth of 50 μm are arranged on the substrate surface 10a of the cell culture chip 10 on triangular lattice points formed with a pitch of 330 μm and an axial angle of 60 degrees, for example. Provided. The cell culture chip 10 has a total of about 2400 fine cell culture cells 11 on the substrate 10a. The cell culture chip 10 thus prepared was sterilized with alcohol and subjected to ultraviolet sterilization for 6 hours, and used for a hepatocyte culture experiment.
Rat hepatocytes were prepared by the method described above, and a suspension (culture medium containing seed cells) containing rat cells having a cell density of 5.0 × 10 5 ce11 s / mL was obtained. A comparative experiment was conducted in which 2 mL of the culture solution was seeded on a substrate under the following conditions (1) to (4) and cultured.

条件（１）：細胞培養セルの部分がない市販の平面基板を備えた培養容器（ファルコン社製、ポリスチレンディッシュ、３５ｍｍ径）に、種細胞を分散させた培養液を入れて静置し、その平面基板上で種細胞を培養したもの（平面基板−静置培養）。
条件（２）：前記市販の培養器をその平面基板の振幅２５ｍｍ、回転速度５０ｒｐｍで旋回させながら培養したもの（平面基板−旋回培養）。
条件（３）：細胞培養セルを格子点上に規則配列させて形成させた円柱セルアレイ化基板（細胞培養チップ１０）が形成された培養器１２に種細胞を分散させた培養液を入れ、これを静置状態で培養したもの（円柱セルアレイ化基板−静置培養）。
条件（４）：細胞培養チップ１０の振幅２５ｍｍ、回転速度５０ｒｐｍで、回転円盤上に載置された培養器１２を旋回させながら種細胞を培養したもの（円柱セルアレイ化基板−旋回培養）。 Condition (1): A culture solution in which seed cells are dispersed is placed in a culture container (Falcon, polystyrene dish, 35 mm diameter) equipped with a commercially available flat substrate having no cell culture cell portion, and left to stand. A seed cell cultured on a flat substrate (planar substrate-stationary culture).
Condition (2): Cultured while rotating the commercially available incubator with an amplitude of 25 mm of the flat substrate and a rotation speed of 50 rpm (planar substrate-rotating culture).
Condition (3): A culture solution in which seed cells are dispersed is placed in a culture vessel 12 on which a cylindrical cell array substrate (cell culture chip 10) formed by regularly arranging cell culture cells on lattice points is formed. Is cultured in a stationary state (cylindrical cell array substrate-stationary culture).
Condition (4): Cell culture chip 10 was cultured with seed cells cultured while rotating incubator 12 placed on a rotating disk at an amplitude of 25 mm and a rotational speed of 50 rpm (cylindrical cell array substrate-rotating culture).

なお、得られた肝細胞の培養状態を評価するために、培養の各期間において、位相差顕微鏡による細胞形態を観察した。また、スフェロイドが形成された条件においてはその粒径を測定し、スフェロイド粒径分布を評価した。ここで、スフェロイド粒径の測定は、デジタルマイクロスコープ（キーエンス社）を用い、１条件当り１００個のスフェロイド粒径を測定し、粒径分布を求めた。
細胞播種直後（培養０日目）、培養３日目、培養７日目の各条件の肝細胞形態を図３〜６に示す。条件（３）の円柱セルアレイ化基板−静置培養ではスフェロイドの形成は見られなかったが、その他の条件では、培養初期に分散状態であった細胞が、徐々に集合化し、細胞同士が結合、凝集化して三次元的な細胞集合体であるスフェロイドを多数形成した。
条件（１）の平面基板−静置培養および条件（２）の平面基板−旋回培養では、基板上におけるスフェロイドの形成がランダムであったが、条件（４）の円柱セルアレイ化基板−旋回培養では、１つの細胞培養セル１１に１個のスフェロイドが形成され、かつ全体的にみると基板上でアレイ化された状態であった。
スフェロイドが形成された三つの条件、すなわち、条件（１）の平面基板−静置培養、条件（２）の平面基板−旋回培養、条件（４）の円柱セルアレイ化基板−旋回培養のスフェロイド粒径分布を図７および表１に示す。この結果、条件（１）の平面基放−静置培養と条件（２）の平面基板−旋回培養では様々な粒径のスフェロイドが存在するが、条件（４）の円柱セルアレイ化基板−旋回培養では均一な粒径のスフェロイドが形成されることが示された。
In addition, in order to evaluate the culture state of the obtained hepatocytes, the cell morphology with a phase contrast microscope was observed in each period of culture. Moreover, the particle size was measured on the conditions in which the spheroid was formed, and the spheroid particle size distribution was evaluated. Here, the spheroid particle size was measured by measuring 100 spheroid particle sizes per condition using a digital microscope (Keyence Corporation) to obtain the particle size distribution.
The hepatocyte morphology of each condition immediately after cell seeding (culture day 0), culture day 3 and culture day 7 is shown in FIGS. Spheroid formation was not observed in the cylindrical cell array substrate-stationary culture of the condition (3), but in other conditions, the cells that were dispersed in the initial stage of the culture gradually gathered and the cells joined together. Aggregates to form many spheroids, which are three-dimensional cell aggregates.
In the flat substrate of the condition (1)-stationary culture and the flat substrate of the condition (2)-swirl culture, the formation of spheroids on the substrate was random, but in the cylindrical cell array substrate-swirl culture of the condition (4) One spheroid was formed in one cell culture cell 11, and as a whole, it was in an arrayed state on the substrate.
Three conditions under which spheroids were formed, that is, a flat substrate of condition (1)-stationary culture, a flat substrate of condition (2)-swirl culture, and a cylindrical cell array substrate of condition (4)-spheroid particle size of swirl culture The distribution is shown in FIG. As a result, spheroids of various particle sizes exist in the flat group release-stationary culture of the condition (1) and the flat substrate-spinning culture of the condition (2), but the cylindrical cell array substrate-spinning culture of the condition (4). Shows that spheroids having a uniform particle size are formed.

これらの結果により、細胞培養チップ１０の基板上で細胞を旋回培養することによって、均一な所定粒径のスフェロイドが形成され、かつ多数のスフェロイドが基板上にアレイ配列されたセル内に固定化できることが示された。   Based on these results, spheroids having a uniform predetermined particle diameter can be formed by rotating and culturing cells on the substrate of the cell culture chip 10, and a large number of spheroids can be immobilized in cells arrayed on the substrate. It has been shown.

実施例２の条件（４）と同様の培養を行い、肝細胞の機能であるアルブミン分泌能を評価した。比較例として、２．５×１０5ｃｅ１１ｓ／ｍＬのラット細胞懸濁液２ｍＬを直径３５ｍｍのコラーゲンコートディッシュ（岩城硝子製）に播種することによって単層培養を行って比較した。単層培養の培養培地および条件は、上記と同様の条件を用いた。
培養１４日目に培養培地中に分泌されたアルブミンを酵素標識免疫測定法により定量し、単位細胞数あたりのアルブミン分泌速度（μｇ／１０6ｃｅ１１ｓ／ｄａｙ）に換算した。細胞数の測定は、クリスタルバイオレットにより細胞核を染色し、核数を計数する方法を用いた。すなわち、細胞数Ｘと、核数Ｙとの割合（Ｘ／Ｙ）は１：１．４５になることが実験的に知られており、この関係をもとに計数された核数を１．４５で除算して細胞数を算出した。この細胞数をもとに、上記の細胞（１０6ｃｅ１１ｓ）当りのアルブミン分泌速度を算出した。
図８に培養１４日目の肝細胞形態の比較を示す。
コラーゲンコートディッシュでは、ラット肝細胞は基板上に伸展した単層状態であったが、円柱セルアレイ化基板−旋回培養ではラット肝細胞はスフェロイドを形成し、その形態は良好に維持された。
図９にアルブミン分泌能の比較を示す。この結果、円柱セルアレイ化基板−旋回培養で形成されたスフェロイドは、単層培養の肝細胞よりも高機能発現ができることが示された。 Culture was performed in the same manner as in condition (4) of Example 2, and the albumin secretion ability, which is a function of hepatocytes, was evaluated. As a comparative example, 2 mL of 2.5 × 10 5 ce11 s / mL rat cell suspension was seeded on a collagen coated dish (manufactured by Iwaki Glass Co., Ltd.) having a diameter of 35 mm and compared by performing monolayer culture. The culture medium and conditions for monolayer culture were the same as described above.
Albumin secreted into the culture medium on the 14th day of culture was quantified by enzyme-labeled immunoassay, and converted to albumin secretion rate (μg / 10 6 ce11s / day) per unit cell number. The number of cells was measured by staining the cell nucleus with crystal violet and counting the number of nuclei. That is, it has been experimentally known that the ratio (X / Y) of the number X of cells to the number Y of nuclei is 1: 1.45, and the number of nuclei counted based on this relationship is 1. The number of cells was calculated by dividing by 45. Based on this number of cells, the albumin secretion rate per the above cells (10 6 ce11s) was calculated.
FIG. 8 shows a comparison of hepatocyte morphology on day 14 of culture.
In the collagen-coated dish, the rat hepatocytes were in a monolayer state extending on the substrate. However, in the columnar cell array substrate-rotating culture, the rat hepatocytes formed spheroids and their morphology was maintained well.
FIG. 9 shows a comparison of albumin secretion ability. As a result, it was shown that spheroids formed by columnar cell array substrate-swirl culture can express higher functions than hepatocytes in monolayer culture.

直径３５ｍｍのポリスチレンディッシュ（ファルコン製）表面の１．５ｃｍ角に、セル深さが５０μｍと一定で、セル直径とピッチが異なる三つの条件のハニカム構造を有する細胞培養チップを作製し、セル直径がスフェロイド形成に与える効果を比較した。
条件（５）：直径１００μｍ、深さ５０μｍの円柱状セルをピッチ１１０μｍの三角配置で設けた基板（１００μｍ直径−円柱セルアレイ化基板）。
条件（６）：直径３００μｍ、深さ５０μｍの円柱状セルをピッチ３３０μｍの三角配置で設けた基板（３００μｍ直径−円柱セルアレイ化基板）。
条件（７）：直径５００μｍ、深さ５０μｍの円柱状セルをピッチ５５０μｍの三角配置で設けた基板（５００μｍ直径−円柱セルアレイ化基板）。
このようにして作製された各円柱セルアレイ化基板をアルコール滅菌および６時間の紫外線滅菌し、肝細胞の培養実験に使用した。
上記記載の方法によってラット肝細胞を調製し、細胞密度が５．０×１０5ｃｅ１１ｓ／ｍＬのラット細胞懸濁液を得た。この懸濁液２ｍＬを各条件（５）〜（７）の培養基板上に播種し、基板の振幅２５ｍｍ、回転速度５０ｒｐｍの条件で旋回培養を行った。培養の各期間において、位相差顕微鏡による観察を行い、各セル内の細胞形態を評価した。 A cell culture chip having a honeycomb structure of three conditions with a cell depth of 50 μm and a different cell diameter and pitch on a 1.5 cm square of a 35 mm diameter polystyrene dish (manufactured by Falcon) is prepared. The effect on spheroid formation was compared.
Condition (5): A substrate (100 μm diameter-cylindrical cell array substrate) in which cylindrical cells having a diameter of 100 μm and a depth of 50 μm are provided in a triangular arrangement with a pitch of 110 μm.
Condition (6): A substrate (300 μm diameter-cylindrical cell array substrate) in which cylindrical cells having a diameter of 300 μm and a depth of 50 μm are provided in a triangular arrangement with a pitch of 330 μm.
Condition (7): A substrate (500 μm diameter-columnar cell array substrate) in which cylindrical cells having a diameter of 500 μm and a depth of 50 μm are provided in a triangular arrangement with a pitch of 550 μm.
Each columnar cell array substrate thus prepared was sterilized with alcohol and UV-sterilized for 6 hours, and used for a hepatocyte culture experiment.
Rat hepatocytes were prepared by the method described above to obtain a rat cell suspension having a cell density of 5.0 × 10 5 ce11 s / mL. 2 mL of this suspension was seeded on the culture substrate of each of the conditions (5) to (7), and swirl culture was performed under the conditions of the substrate amplitude of 25 mm and the rotation speed of 50 rpm. In each period of culture, observation with a phase contrast microscope was performed to evaluate the cell morphology in each cell.

図１０に培養７日目における各条件のラット肝細胞の形態を示す。
条件（５）の１００μｍ直径−円柱セルアレイ化基板では、細胞培養セル内に細胞が導入されない部分も存在したが、細胞が導入された細胞培養セル内ではスフェロイドが形成された。
条件（７）の５００μｍ直径−円柱セルアレイ化基板では、各細胞培養セル内においてスフェロイドの形成は見られるものの、１つの細胞培養セル内に２個以上のスフェロイドが形成される部分も存在した。
条件（６）の３００μｍ直径−円柱セルアレイ化基板では、１つの細胞培養セル内に１個のスフェロイドが形成された。
また、細胞培養セルの直径が５００μｍを超えるようになると、培養されるスフェロイドが大きくなりすぎてその中心部に壊死が生じたり、同一セル内に複数のスフェロイドが生じたりするなどの傾向が現れ、これらの結果より、スフェロイドを基板上で良好に培養させるためには、細胞培養セルの直径は５００μｍ以下が適していることが示された。 FIG. 10 shows the morphology of rat hepatocytes under various conditions on day 7 of culture.
In the 100 μm diameter-cylindrical cell array substrate of the condition (5), there was a portion where cells were not introduced into the cell culture cell, but spheroids were formed in the cell culture cell into which the cells were introduced.
In the 500 μm diameter-cylindrical cell array substrate of the condition (7), spheroids were formed in each cell culture cell, but there were also portions where two or more spheroids were formed in one cell culture cell.
In the 300 μm diameter-cylindrical cell array substrate of the condition (6), one spheroid was formed in one cell culture cell.
In addition, when the diameter of the cell culture cell exceeds 500 μm, the spheroids to be cultured become too large and necrosis occurs in the center, or multiple spheroids occur in the same cell, etc. From these results, it was shown that a cell culture cell having a diameter of 500 μm or less is suitable for culturing spheroids well on a substrate.

直径３５ｍｍのポリスチレンディッシュ（ファルコン製）表面の１．５ｃｍ角に、セル直径が３００μｍ、ピッチが３３０μｍと一定で、セル深さが異なる三つの条件のアレイ構造を有する培養基板を作製し、セル深さがスフェロイドの形成に与える効果を比較した。
条件（８）：直径３００μｍ、深さ５０μｍの円柱セルをピッチ３３０μｍの三角配置で設けた基板（５０μｍ深さ−円柱セルアレイ化基板）。
条件（９）：直径３００μｍ、深さ１００μｍの円柱セルをピッチ３３０μｍの三角配置で設けた基板（１００μｍ深さ−円柱セルアレイ化基板）。
条件（１０）：直径３００μｍ、深さ２００μｍの円柱セルをピッチ３３０μｍの三角配置で設けた基板（２００μｍ深さ−円柱セルアレイ化基板）。
このようにして作製した円柱セルアレイ化基板をアルコール滅菌および６時間の紫外線滅菌し、肝細胞の培養実験に使用した。
上記記載の方法によってラット肝細胞を調製し、細胞密度が５．０×１０5ｃｅｌｌｓ／ｍＬのラット細胞懸濁液を得た。この懸濁液２ｍＬを各条件の培養基板上に播種し、基板の振幅２５ｍｍ、回転速度５０ｒｐｍの旋回培養条件で旋回培養を行った。培養の各期間において、位相差顕微鏡による観察を行い、細胞形態を評価した。
図１１に培養７日目における各条件のラット肝細胞の形態を示す。いずれの条件においても、１つの細胞培養セル内に１個のスフェロイドが形成され、かつ形成されるスフェロイドの粒径はほぼ均一であった。
また、有底孔を有した細胞培養セルの場合はセルの深さが２０００μｍを超えるとスフェロイドへの酸素や栄養分の供給が阻害される場合があり、これらの結果より、細胞の生存に必要な酸素や栄養素などを十分に供給できる範囲のセル深さであれば、均一な粒径のスフェロイドを基板上でアレイ化することができることが示された。 A culture substrate having an array structure of three conditions with a cell diameter of 300 μm, a pitch of 330 μm constant, and different cell depths on a 1.5 cm square of a 35 mm diameter polystyrene dish (manufactured by Falcon) is prepared. Compared the effects of Saga on the formation of spheroids.
Condition (8): A substrate (50 μm depth-columnar cell array substrate) in which cylindrical cells having a diameter of 300 μm and a depth of 50 μm are arranged in a triangular arrangement with a pitch of 330 μm.
Condition (9): A substrate (100 μm depth-columnar cell array substrate) in which cylindrical cells having a diameter of 300 μm and a depth of 100 μm are arranged in a triangular arrangement with a pitch of 330 μm.
Condition (10): A substrate (200 μm depth−cylindrical cell array substrate) in which cylindrical cells having a diameter of 300 μm and a depth of 200 μm are arranged in a triangular arrangement with a pitch of 330 μm.
The cylindrical cell array substrate thus prepared was sterilized with alcohol and UV-sterilized for 6 hours, and used for a hepatocyte culture experiment.
Rat hepatocytes were prepared by the method described above to obtain a rat cell suspension having a cell density of 5.0 × 10 5 cells / mL. 2 mL of this suspension was seeded on a culture substrate under each condition, and swirl culture was performed under swirl culture conditions with a substrate amplitude of 25 mm and a rotation speed of 50 rpm. In each period of culture, observation with a phase contrast microscope was performed to evaluate the cell morphology.
FIG. 11 shows the morphology of rat hepatocytes under various conditions on the seventh day of culture. Under any condition, one spheroid was formed in one cell culture cell, and the formed spheroid had a substantially uniform particle size.
In addition, in the case of a cell culture cell having a bottomed hole, if the cell depth exceeds 2000 μm, supply of oxygen and nutrients to the spheroids may be inhibited. From these results, it is necessary for cell survival. It has been shown that spheroids having a uniform particle size can be arrayed on a substrate if the cell depth is within a range where oxygen, nutrients, and the like can be sufficiently supplied.

直径３５ｍｍのポリスチレンディッシュ（ファルコン製）表面の１．５ｃｍ角に、Ｕ字部またはテーパ部を備えた培養セルを有する細胞培養チップを作製し、セル形状がスフェロイドの配置に与える効果を検討した。
条件（１１）：直径３００μｍ、深さ１５０μｍの半球状セルをピッチ３３０μｍの三角配置で設けた基板（３００μｍ直径−半球セルアレイ化基板）。
条件（１２）：直径３００μｍ、深さ１５０μｍの円すい状セルをピッチ３３０μｍの三角配置で設けた基板（３００μｍ直径−円すいセルアレイ化基板）。
このようにして作製された各アレイ化基板をアルコール滅菌および６時間の紫外線滅菌し、肝細胞の培養実験に使用した。
上記記載の方法によってラット肝細胞を調製し、細胞密度が２．５×１０5ｃｅｌｌｓ／ｍＬのラット肝細胞懸濁液を得た。この懸濁液２ｍＬを条件（１１）、（１２）の培養基板上に播種し、振幅２５ｍｍ、回転速度５０ｒｐｍの条件で旋回培養を行った。培養の各期間において、位相差顕微鏡による観察を行い、各セル内の細胞形態を評価した。
図１２に培養７日目における各条件のラット肝細胞の形態を示す。いずれの条件においても、１つの細胞培養セル内にほぼ均一な粒径のスフェロイドが形成された。これらの結果より、培養セルが湾曲部やテーパ部を備えることで、均一な粒径のスフェロイドを基板上でアレイ化できることが示された。 A cell culture chip having a culture cell with a U-shaped portion or a tapered portion on a 1.5 cm square surface of a 35 mm diameter polystyrene dish (manufactured by Falcon) was prepared, and the effect of the cell shape on the arrangement of spheroids was examined.
Condition (11): A substrate (300 μm diameter-hemispheric cell array substrate) in which hemispherical cells having a diameter of 300 μm and a depth of 150 μm are provided in a triangular arrangement with a pitch of 330 μm.
Condition (12): A substrate (300 μm diameter-conical cell array substrate) in which conical cells having a diameter of 300 μm and a depth of 150 μm are arranged in a triangular arrangement with a pitch of 330 μm.
Each arrayed substrate thus prepared was sterilized with alcohol and UV-sterilized for 6 hours, and used for a hepatocyte culture experiment.
Rat hepatocytes were prepared by the method described above to obtain a rat hepatocyte suspension having a cell density of 2.5 × 10 5 cells / mL. 2 mL of this suspension was seeded on the culture substrate of conditions (11) and (12), and swirl culture was performed under the conditions of an amplitude of 25 mm and a rotation speed of 50 rpm. In each period of culture, observation with a phase contrast microscope was performed to evaluate the cell morphology in each cell.
FIG. 12 shows the morphology of rat hepatocytes under various conditions on day 7 of culture. Under any condition, spheroids having a substantially uniform particle size were formed in one cell culture cell. From these results, it was shown that spheroids having a uniform particle diameter can be arrayed on the substrate by providing the culture cell with a curved portion or a tapered portion.

（実施の形態２）
図１３は実施の形態２の細胞担持モジュールの模式断面図である。
図１３において、２０は実施の形態２の細胞担持モジュール、２１はその底部に配設されたメンブレンフィルム２１ａを有して所定間隔を有して積層配置された細胞培養チップ、２２は細胞培養チップ２１の基板表面２１ｂ上に円形又は多角形状断面の壁部２２ａを有して穿設された無底孔の底部をメンブレンフィルム２１ａで封止して形成されその内部に球状細胞組織体Ｓを保持する細胞培養セルである。 (Embodiment 2)
FIG. 13 is a schematic cross-sectional view of the cell carrying module of the second embodiment.
In FIG. 13, 20 is a cell-carrying module according to the second embodiment, 21 is a cell culture chip having a membrane film 21a disposed at the bottom thereof and being stacked with a predetermined interval, and 22 is a cell culture chip. The bottom part of the bottomless hole drilled with the wall part 22a having a circular or polygonal cross section on the substrate surface 21b is formed by sealing with a membrane film 21a, and the spherical cell tissue S is held therein. It is a cell culture cell.

細胞培養チップ２１は前記実施の形態１の細胞培養チップ１０と同様にその基板表面２１ｂに細胞培養セル２２が規則配列されて穿設されたポリアセタール等の親水性高分子からなる板状体である。
なお、細胞培養チップ２１の素材としては、ポリアセタール等の親水性高分子の他に、例えば、ポリスチレン、スチレン−ブタジエン共重合体、スチレン−エチレン−ブチレン共重合体、イソプレン重合体、ブタジエン−スチレン共重合体、ポリブタジエン、１，２−ポリブタジエン、イソプレン−イソブチレン共重合体、クロロプレン重合体等のジエン系ポリマー等の疎水性の基板表面２１ｂを穿孔加工した後、コラーゲンやホスファチジルコリン、ホスファチジルセリン、レシチンなどやこれらの不飽和脂肪酸を水添したもの等で被覆したりプラズマ処理等を行って、表面及びセル内壁面を親水性表面にすることもできる。
このようにして、細胞培養チップ２１上に種細胞を所定濃度で分散させた培養液を入れて種細胞を着床させる際の基板表面２１ｂへの種細胞の付着を防止すると共に、細胞培養チップ２１を回転又は揺動させてセル内の種細胞を球状細胞組織体に培養保持させる際に、球状細胞組織体Ｓが側壁面に付着しないようにして、球状細胞組織体Ｓの担持位置をセル中心位置に位置付ける位置制御性を高めるようにしてもよい。 Similar to the cell culture chip 10 of the first embodiment, the cell culture chip 21 is a plate-like body made of a hydrophilic polymer such as polyacetal in which cell culture cells 22 are regularly arranged on the substrate surface 21b. .
Examples of the material for the cell culture chip 21 include, in addition to hydrophilic polymers such as polyacetal, polystyrene, styrene-butadiene copolymer, styrene-ethylene-butylene copolymer, isoprene polymer, and butadiene-styrene copolymer. After perforating a hydrophobic substrate surface 21b such as a polymer, polybutadiene, 1,2-polybutadiene, isoprene-isobutylene copolymer, diene polymer such as chloroprene polymer, collagen, phosphatidylcholine, phosphatidylserine, lecithin, etc. The surface and the inner wall surface of the cell can be made hydrophilic by coating with a hydrogenated one of these unsaturated fatty acids or performing plasma treatment.
In this way, while preventing the seed cells from adhering to the substrate surface 21b when the seed cells are implanted by putting a culture solution in which the seed cells are dispersed at a predetermined concentration on the cell culture chip 21, the cell culture chip is used. When the seed cell in the cell is cultured and held in the spherical cell tissue body by rotating or swinging the cell 21, the spherical cell tissue S is not attached to the side wall surface, and You may make it improve the position controllability located in a center position.

細胞培養セル２２を形成する無底孔は、機械加工又はレーザービーム加工などにより形成することができる。なお、細胞培養チップ２１をスポンジ状などの三次元網目状組織体で構成してこの網目間を通液孔として、細胞培養セル２２の壁面や底面側から栄養液や培養液が流通できるようにしたものを用いることも可能である。   The bottomless hole forming the cell culture cell 22 can be formed by machining or laser beam processing. The cell culture chip 21 is composed of a sponge-like three-dimensional network structure, and the nutrient solution and the culture solution can be circulated from the wall surface and the bottom surface side of the cell culture cell 22 through the mesh holes. It is also possible to use what is done.

メンブレンフィルム２１ａなどの透水膜は、供給される栄養液などを透過させると共に、細胞培養セル２２内に球状細胞組織体Ｓを保持させる機能を担っている。
メンブレンフィルム２１ａの素材としてはセルロース系や合成高分子系、セルロース−合成高分子複合系等からなる中空糸型や平膜型の透析膜が適用できる。合成高分子からなるメンブレンフィルムには、ポリテトラフルオロエチレン膜やポリフッ化ビニリデン膜，ポリカーボネート膜が用いられる。なお、膜面のタンパク質吸着性や疎水性、親水性などを調整する場合は、フィブロネクチンやラミニン、コラーゲン、マトリゲルなどの表面性状調整成分を必要に応じてコーティングすることができる。 The water permeable membrane such as the membrane film 21 a has a function of permeating the nutrient solution supplied and the like and holding the spherical cell tissue S in the cell culture cell 22.
As the material of the membrane film 21a, a hollow fiber type or flat membrane type dialysis membrane made of cellulose, synthetic polymer, cellulose-synthetic polymer composite or the like can be applied. As the membrane film made of a synthetic polymer, a polytetrafluoroethylene film, a polyvinylidene fluoride film, or a polycarbonate film is used. In addition, when adjusting protein adsorption property, hydrophobicity, hydrophilicity, etc. on the membrane surface, a surface property adjusting component such as fibronectin, laminin, collagen, or matrigel can be coated as necessary.

細胞担持モジュール２０は、実施の形態１に記載の細胞培養方法を用いて、球状細胞組織体Ｓを細胞培養チップ２１の細胞培養セル２２内で培養して、これを担持させた細胞培養チップ２１を平面状又は図１３に示すように積層状に配列させて全体を構成することができる。   The cell-carrying module 20 uses the cell culture method described in Embodiment 1 to culture the spherical cell tissue S in the cell culture cell 22 of the cell culture chip 21 and to carry the cell culture chip 21 that carries this. As shown in FIG. 13, the entire structure can be configured by arranging them in a planar shape.

実施の形態２の細胞担持モジュール２０は以上のように構成されているので、以下の作用を有する。
（ａ）細胞担持モジュール２０は、球状細胞組織体Ｓをそれぞれ担持させた細胞培養チップ２１の高密度集積化が可能であり、このような細胞担持モジュール２０を用いて化学、生化学関連装置を微小化でき、試料の節減、操作の自動化、高速化などが図られる。
（ｂ）各細胞培養セル２２内にそれぞれ種類やタンパク質分泌機能などが異なる球状細胞組織体Ｓを配置することもでき、試験体に対する種々の官能性等を同時にチェックして、チェック項目が多岐にわたる新薬スクリーニング等の処理を迅速かつ的確に行うことができる。
（ｃ）各セル内に栄養分や酸素などを補給循環させるメンブレンフィルム２１ａを有し、球状細胞組織体Ｓを細胞培養セル２２内に保持させているので、三次元状に立体配置される各セル内の環境を球状細胞組織体が所定の生体機能を発現するような環境条件に設定でき、信頼性に優れた分析システムなどを構築することができる。 Since the cell carrying module 20 of Embodiment 2 is configured as described above, it has the following operations.
(A) The cell-carrying module 20 is capable of high-density integration of the cell culture chip 21 carrying the spherical cell tissue S. The cell-carrying module 20 can be used for chemical and biochemical related devices. Miniaturization is possible, and the sample can be saved, the operation is automated, and the speed is increased.
(B) It is also possible to arrange spherical cell tissue bodies S of different types and protein secretion functions in each cell culture cell 22, and simultaneously check various functionalities etc. with respect to the test specimen, and there are a wide variety of check items Processes such as new drug screening can be performed quickly and accurately.
(C) Since each cell has a membrane film 21a that replenishes and circulates nutrients, oxygen, and the like and holds the spherical cell tissue S in the cell culture cell 22, each cell that is three-dimensionally arranged in three dimensions The internal environment can be set to environmental conditions such that the spherical cell tissue body exhibits a predetermined biological function, and an analysis system having excellent reliability can be constructed.

本発明は、細胞を培養して担持させるための細胞培養チップ、培養器及びそれらを用いた球状細胞組織体の細胞培養方法、球状細胞組織体を担持させた細胞担持モジュール、球状細胞組織体などの細胞培養技術に関し、培養液中の種細胞を規則配列された細胞培養セル内に着床させ、この種細胞を均一な大きさの球状細胞組織体に培養してこれらをそれぞれ規則配列させた状態で担持させることができるので、再生医療や、ハイブリッド型人工臓器、生体有用物質生産、生物組織や器官・臓器の機能の調査・探索、新薬のスクリーニング、内分泌かく乱物質等の影響を評価する動物実験代替法、センサ機能を有する細胞チップ等の各分野へ適用することができる。   The present invention relates to a cell culture chip for culturing and supporting cells, an incubator, a cell culture method of a spherical cell tissue body using the same, a cell support module supporting a spherical cell tissue body, a spherical cell tissue body, etc. In the cell culture technique, seed cells in the culture solution are implanted in regularly arranged cell culture cells, and these seed cells are cultured in a spherical cell tissue of uniform size, and these are regularly arranged. Animals that are evaluated for the effects of regenerative medicine, hybrid artificial organs, production of biologically useful substances, investigation / search for functions of biological tissues and organs / organs, screening for new drugs, endocrine disruptors, etc. It can be applied to various fields such as an experimental alternative and a cell chip having a sensor function.

実施の形態１の細胞培養チップが載置された培養器の斜視図The perspective view of the incubator in which the cell culture chip of Embodiment 1 was mounted. 細胞培養チップの拡大断面図Expanded cross section of cell culture chip 条件（１）平面基板−静置培養におけるラット肝細胞の形態比較図Condition (1) Comparison of morphology of rat hepatocytes in flat substrate-stationary culture 条件（２）平面基板−旋回培養におけるラット肝細胞の形態比較図Condition (2) Comparison of morphology of rat hepatocytes in planar substrate-rotating culture 条件（３）円柱セルアレイ化基板−静置培養におけるラット肝細胞の形態比較図Condition (3) Cylindrical cell array substrate-Comparison of morphology of rat hepatocytes in stationary culture 条件（４）円柱セルアレイ化基板−旋回培養におけるラット肝細胞の形態比較図Condition (4) Cylindrical cell array substrate-Comparison of morphology of rat hepatocytes in swirling culture ラット肝細胞スフェロイドの培養７日目における粒径分布図Size distribution map of rat hepatocyte spheroids on day 7 of culture ラット肝細胞の形態比較図Comparison of rat hepatocyte morphology アルブミン分泌能の比較図Comparison of albumin secretion capacity 条件（５）〜（７）における培養７日目のラット肝細胞の形態比較図Comparison of morphology of rat hepatocytes on day 7 of culture under conditions (5) to (7) 条件（８）〜（１０）における培養７日目のラット肝細胞の形態比較図Morphological comparison chart of rat hepatocytes on day 7 of culture under conditions (8) to (10) 条件（１１）、（１２）における培養７日目のラット肝細胞の形態比較図Comparison of morphology of rat hepatocytes on day 7 of culture under conditions (11) and (12) 実施の形態２の細胞担持モジュールの模式断面図Schematic sectional view of the cell-carrying module of the second embodiment

符号の説明Explanation of symbols

１０ 細胞培養チップ
１０ａ 基板表面
１１ 細胞培養セル
１１ａ 底部
１１ｂ 壁部
１１ｃ テーパ部
１２ 培養器
２０ 細胞担持モジュール
２１ 細胞培養チップ
２１ａ メンブレンフィルム
２１ｂ 基板表面
２２ 細胞培養セル
２２ａ 壁部
DESCRIPTION OF SYMBOLS 10 Cell culture chip 10a Substrate surface 11 Cell culture cell 11a Bottom part 11b Wall part 11c Taper part 12 Incubator 20 Cell carrying module 21 Cell culture chip 21a Membrane film 21b Substrate surface 22 Cell culture cell 22a Wall part

Claims (11)

基板表面上に種細胞を着床させる細胞培養チップであって、前記基板表面上にアレイ状やハニカム状等に規則配列され前記種細胞を凝集させて保持する細胞培養セルを備えたことを特徴とする細胞培養チップ。   A cell culture chip for implanting seed cells on a substrate surface, comprising a cell culture cell regularly arranged in an array or a honeycomb on the substrate surface to hold the seed cells in an aggregated state. A cell culture chip. 前記細胞培養セルが、前記基板に窪み状に形成された有底孔であることを特徴とする請求項１に記載の細胞培養チップ。   The cell culture chip according to claim 1, wherein the cell culture cell is a bottomed hole formed in a recess shape in the substrate. 前記細胞培養セルが、前記基板に形成された無底孔と、前記無底孔の底部を形成して前記基板の一面に積層されたメンブレンフィルム等の透水膜と、を有していることを特徴とする請求項１に記載の細胞培養チップ。   The cell culture cell has a bottomless hole formed in the substrate, and a water permeable film such as a membrane film that forms a bottom portion of the bottomless hole and is laminated on one surface of the substrate. The cell culture chip according to claim 1, wherein 前記細胞培養セルが円形又は多角形状の開口部を有し、その開口径が培養される前記種細胞の大きさに対して２〜５０倍の範囲であることを特徴とする請求項１乃至３の内いずれか１項に記載の細胞培養チップ。   The cell culture cell has a circular or polygonal opening, and the diameter of the opening is in the range of 2 to 50 times the size of the seed cell to be cultured. The cell culture chip according to any one of the above. 前記細胞培養セルの底面や壁面の一部又は全部が下方中心に向けて傾斜状に形成されたテーパ部や湾曲部を有していることを特徴とする請求項１乃至４の内いずれか１項に記載の細胞培養チップ。   5. The cell culture cell according to claim 1, wherein a part or all of a bottom surface or a wall surface of the cell culture cell has a tapered portion or a curved portion formed in an inclined shape toward the lower center. The cell culture chip according to Item. 前記基板表面や前記細胞培養セルの内表面が親水性であることを特徴とする請求項１乃至５の内いずれか１項に記載の細胞培養チップ。   The cell culture chip according to any one of claims 1 to 5, wherein the substrate surface and the inner surface of the cell culture cell are hydrophilic. 請求項１乃至６の内いずれか１項に記載の細胞培養チップの前記基板の外周部の一方向に立設された側壁を備えていることを特徴とする培養器。   An incubator comprising a side wall provided upright in one direction of the outer periphery of the substrate of the cell culture chip according to any one of claims 1 to 6. 請求項１乃至６の内いずれか１項に記載の細胞培養チップを培養器の底部に固定して、又は、請求項７に記載の培養器を用いて、前記培養器に所定濃度の種細胞が分散された培養液を充填して静置し、前記種細胞を前記細胞培養セル内に着床させる種細胞着床工程と、
前記培養器を連続的又は間欠的に所定加速度で水平回転又は往復揺動させながら前記細胞培養セル内の前記種細胞同士を凝集培養させて球状細胞組織体に成長させる球状細胞培養工程と、を有することを特徴とする細胞培養方法。 A cell culture chip according to any one of claims 1 to 6 is fixed to a bottom of an incubator, or a seed cell having a predetermined concentration is used in the incubator using the incubator according to claim 7. Filled with a culture medium in which is dispersed, and seeded, and a seed cell implantation step of implanting the seed cells in the cell culture cell;
A spherical cell culturing step in which the seed cells in the cell culture cell are coagulated and cultured to grow into a spherical cell tissue while the incubator is rotated horizontally or reciprocally swings at a predetermined acceleration continuously or intermittently. A cell culture method characterized by comprising. 前記種細胞が肝細胞であることを特徴とする請求項８に記載の細胞培養方法。   The cell culture method according to claim 8, wherein the seed cells are hepatocytes. 請求項８又は９記載の細胞培養方法を用いて請求項１乃至７のいずれか１項に記載の細胞培養チップの前記細胞培養セル内に球状細胞組織体を担持させてなる細胞担持モジュールであって、複数個の前記細胞培養チップをそのモジュールケース内に平面状及び／又は積層状に規則配列させたことを特徴とする球状細胞組織体を担持させた細胞担持モジュール。   A cell-carrying module in which a spherical cell tissue is carried in the cell culture cell of the cell culture chip according to any one of claims 1 to 7, using the cell culture method according to claim 8 or 9. A cell-carrying module carrying a spherical cell tissue, wherein a plurality of the cell culture chips are regularly arranged in a planar shape and / or a laminated shape in the module case. 請求項９記載の細胞培養方法を用いて前記肝細胞から培養された球状細胞組織体であって、その全体サイズが５０〜３００μｍの範囲であり、そのアルブミン分泌速度が細胞１０6個当り２０〜５００μｇ／日の範囲であることを特徴とする球状細胞組織体。
A spherical cell tissue cultivated from the hepatocytes using the cell culture method according to claim 9, wherein the whole size is in the range of 50 to 300 μm, and the albumin secretion rate is 20 to 10 per 10 6 cells. A spherical cell tissue characterized by being in a range of 500 μg / day.
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