JP6176703B2 - Hepatocyte culture substrate and hepatocyte culture method - Google Patents
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本発明は、基板上に位置を制御して肝細胞を配列して、毛細胆管を構築する３次元肝培養細胞を簡便に作製することができる肝細胞培養用基板及びそれを用いた肝細胞の培養方法、該方法による毛細胆管を構築する３次元肝培養細胞の製造方法、該肝培養細胞の使用方法、及び該肝培養細胞を用いた装置に関する。 The present invention provides a hepatocyte culture substrate capable of easily producing a three-dimensional liver culture cell for constructing a capillary bile duct by controlling the position on a substrate and arranging hepatocytes, and a hepatocyte using the same. The present invention relates to a culture method, a method for producing a three-dimensional liver cultured cell for constructing a capillary bile duct by the method, a method for using the liver cultured cell, and an apparatus using the liver cultured cell.
創薬研究において、生体肝組織における薬物等の取り込み・代謝・排出（薬物輸送）を正しく評価することが極めて重要である。生体に近い構造である毛細胆管をもった肝組織（肝培養細胞）を基板上に作製するために、代表的な方法としてサンドイッチ培養法がある（非特許文献１）。サンドイッチ培養法は、コラーゲンと細胞外マトリクスゲルの間に肝細胞を培養して、毛細胆管を形成する肝細胞の培養方法である。しかしながら、この方法では擬似的な３次元環境を細胞に付与するためにコラーゲンやマトリゲル等の細胞外マトリクスゲルを加えているため、３次元組織化が十分でなく、毛細胆管の形成がランダムで安定して使用することができなかった。したがって、胆管代謝物の分析には、カルシウム不含溶液で細胞内容物を抽出させたものと、カルシウム含有溶液で細胞内容物を抽出させたものの、２つの培養系のものを差し引きする方法しかなかった（非特許文献２）。そのうえ、細胞外マトリクスゲルそのものが、培養やアッセイの障害になることが指摘されている。例えば、薬物等の取り込みを検討する際には、培養された肝細胞に薬物等を添加することになるが、細胞外マトリクスゲルで細胞が覆われているために、薬物等の浸透に差が生じてしまい、正確に評価することができないことがある。 In drug discovery research, it is extremely important to correctly evaluate the uptake, metabolism, and excretion (drug transport) of drugs in living liver tissue. As a typical method for producing a liver tissue (liver culture cell) having a capillary bile duct having a structure close to that of a living body on a substrate, there is a sandwich culture method (Non-patent Document 1). The sandwich culture method is a hepatocyte culture method in which hepatocytes are cultured between collagen and an extracellular matrix gel to form a capillary bile duct. However, in this method, since an extracellular matrix gel such as collagen or matrigel is added to give cells a pseudo three-dimensional environment, the three-dimensional organization is not sufficient, and the formation of capillary bile ducts is random and stable. And could not be used. Therefore, for the analysis of bile duct metabolites, there is only a method of subtracting the two culture systems, one in which the cell contents are extracted with a calcium-free solution and the other in which the cell contents are extracted with a calcium-containing solution. (Non-Patent Document 2). Moreover, it has been pointed out that the extracellular matrix gel itself becomes an obstacle to culture and assay. For example, when examining the uptake of drugs and the like, drugs and the like are added to cultured hepatocytes, but because the cells are covered with an extracellular matrix gel, there is a difference in the penetration of the drugs and the like. May occur and cannot be evaluated accurately.
そのため、細胞外マトリクスゲルを用いずに、毛細胆管を持った肝組織を作製するために、ピラー（非特許文献３）や細胞非接着基板（非特許文献４）、流路（非特許文献５）を用いて３次元細胞組織を作製する方法があるが、それらの毛細胆管の形成はランダムである（位置を制御できない）上、十分な毛細胆管形成には７日以上の長時間の培養期間を必要とするため、創薬研究などの実用化は進んでいない。 Therefore, in order to produce a liver tissue having a capillary bile duct without using an extracellular matrix gel, a pillar (Non-Patent Document 3), a cell non-adhesive substrate (Non-Patent Document 4), a flow path (Non-Patent Document 5) ), But the formation of the capillary bile ducts is random (the position cannot be controlled), and a long culture period of 7 days or longer is necessary for sufficient capillary bile duct formation. Therefore, the practical application of drug discovery research is not progressing.
また、特許文献１にはガス透過膜上に細胞外マトリクスで包埋された肝細胞を配置し、ガス透過性膜側から酸素を供給しつつ肝細胞を培養することを特徴とする肝細胞培養方法が開示されている。しかし、評価対象薬剤の毛細胆管への取り込み効率や代謝物の毛細胆管からの回収効率など、改善の余地があった。 Patent Document 1 discloses a hepatocyte culture characterized in that hepatocytes embedded with an extracellular matrix are disposed on a gas permeable membrane, and the hepatocytes are cultured while supplying oxygen from the gas permeable membrane side. A method is disclosed. However, there is room for improvement, such as the efficiency of taking the drug to be evaluated into the capillary bile duct and the efficiency of collecting the metabolite from the capillary bile duct.
本発明は、細胞外マトリクスゲルの重層を必要とせず、基板上に位置を制御して肝細胞を配列して、毛細胆管を構築する３次元肝培養細胞を簡便に作製させることができる肝細胞培養用基板およびそれを用いた培養方法を提供することを目的とするものである。更に、これにより、該肝細胞培養方法による毛細胆管を構築する３次元肝培養細胞の製造方法、該肝培養細胞の使用方法、及び該肝培養細胞を用いた装置など安定して簡便且つ高感度に薬物輸送検定などを行うことができる手段を提供することも目的とする。 The present invention does not require an overlay of an extracellular matrix gel, and allows hepatocytes to be easily produced by controlling the position on the substrate and arranging hepatocytes to construct a capillary bile duct. An object of the present invention is to provide a culture substrate and a culture method using the same. In addition, thereby, a method for producing a three-dimensional liver culture cell for constructing a capillary bile duct by the hepatocyte culture method, a method for using the liver culture cell, a device using the liver culture cell, and the like are stably and simply and highly sensitive. Another object of the present invention is to provide a means for conducting a drug transport assay.
上記の課題を解決するために本発明者らは鋭意検討を行った結果、表面にすり鉢状の溝を有する肝細胞培養用基板を用いることにより、細胞外マトリクスゲルの重層を行わなくても、基板上に位置を制御して肝細胞を配列して、効率よく毛細胆管を構築する３次元肝培養細胞を簡便に作製できることを見出し、本発明を完成させた。 In order to solve the above-mentioned problems, the present inventors have conducted intensive studies, and as a result, by using a hepatocyte culture substrate having a mortar-shaped groove on the surface, without performing an overlay of an extracellular matrix gel, The inventors have found that a three-dimensional liver culture cell that efficiently constructs a capillary bile duct can be easily prepared by controlling the position on a substrate and arranging hepatocytes, thereby completing the present invention.
すなわち、本発明は以下を提供する。
［１］毛細胆管を形成する肝細胞を培養するための酸素透過性の基板であって、表面にすり鉢状の溝を有することを特徴とする、肝細胞培養用基板。
［２］前記すり鉢状の溝は、短手方向の幅が上部から底面に向かって狭くなっており、上部の幅は少なくとも１個の肝細胞が配置可能な幅であり、底面の幅は肝細胞が接着・伸展しない幅であり、溝の深さは少なくとも１個の肝細胞が積層可能な深さである、［１］に記載の肝細胞培養用基板。
［３］前記底面の幅が10μm以下である、［２］に記載の肝細胞培養用基板。
［４］前記上部の幅が20μm以上100μm以下である、［２］または３に記載の肝細胞培養用基板。
［５］前記深さは20μm以上1mm以下である、［２］〜［４］のいずれかに記載の肝細胞培養用基板。
［６］前記溝のパターンが線状である、［１］〜［５］のいずれかに記載の肝細胞培養用基板。
［７］前記すり鉢状の溝が基板上に複数配置された、［１］〜［６］のいずれかに記載の肝細胞培養用基板。
［８］複数のすり鉢状の溝の間隔が１個の肝細胞の幅より小さい、［７］に記載の肝細胞培養用基板。
［９］［１］〜［８］のいずれかに記載の肝細胞培養用基板を使用して肝細胞を培養する、肝細胞培養方法。
［１０］肝細胞の上に細胞外マトリクスゲルを重層しない、［９］に記載の肝細胞培養方法。
［１１］［９］に記載の方法により肝細胞を培養して毛細胆管を形成した培養肝細胞を製造し、得られた培養肝細胞を用いて化合物の代謝を評価する、化合物の代謝検定方法。
［１２］［９］に記載の方法により肝細胞を培養して毛細胆管を形成した培養肝細胞を製造する工程、該培養肝細胞の培養液に化合物を添加する工程、該化合物を除去する工程、および反応液中及び／又は毛細胆管液中の成分を分析する工程を含む、［１１］に記載の化合物の代謝検定方法。
［１３］化合物がビリルビン及び／又はビリルビン代謝物である［１１］または［１２］に記載の化合物の代謝検定方法。
［１４］［９］に記載の方法により肝細胞を培養して毛細胆管を形成した培養肝細胞を製造し、得られた培養肝細胞を用いて化合物の輸送を評価する、化合物の輸送検定方法。
［１５］［９］に記載の方法により肝細胞を培養して毛細胆管を形成した培養肝細胞を製造する工程、該培養肝細胞の培養液に化合物を添加する工程、該化合物を除去する工程、及び反応液中及び／又は毛細胆管液中の成分を分析する工程を含む、［１４］に記載の化
合物の輸送検定方法。
［１６］培養肝細胞を含む本体部と、本体部に化合物を供給する化合物供給部と、本体部から化合物またはその代謝物を回収する回収部とを有する培養肝細胞を用いた化合物検定装置であって、前記本体部は、［１］〜［８］のいずれかに記載の肝細胞培養用基板と、肝細胞と、を有することを特徴とする、化合物検定装置。
That is, the present invention provides the following.
[1] A substrate for culturing hepatocytes, which is an oxygen-permeable substrate for culturing hepatocytes that form capillary bile ducts and has a mortar-like groove on the surface.
[2] The mortar-shaped groove has a width in the short side direction that is narrowed from the top to the bottom, the width at the top is a width at which at least one hepatocyte can be arranged, and the width at the bottom is liver The substrate for culturing hepatocytes according to [1], which has a width in which cells do not adhere and extend, and the depth of the groove is a depth at which at least one hepatocyte can be stacked.
[3] The hepatocyte culture substrate according to [2], wherein the width of the bottom surface is 10 μm or less.
[4] The hepatocyte culture substrate according to [2] or 3, wherein the width of the upper part is 20 μm or more and 100 μm or less.
[5] The hepatocyte culture substrate according to any one of [2] to [4], wherein the depth is 20 μm or more and 1 mm or less.
[6] The hepatocyte culture substrate according to any one of [1] to [5], wherein the groove pattern is linear.
[7] The hepatocyte culture substrate according to any one of [1] to [6], wherein a plurality of the mortar-shaped grooves are arranged on the substrate.
[8] The hepatocyte culture substrate according to [7], wherein the interval between the plurality of mortar-shaped grooves is smaller than the width of one hepatocyte.
[9] A hepatocyte culture method for culturing hepatocytes using the hepatocyte culture substrate according to any one of [1] to [8].
[10] The method for culturing hepatocytes according to [9], wherein an extracellular matrix gel is not layered on the hepatocytes.
[11] A method for assaying metabolism of a compound, wherein cultured hepatocytes are formed by culturing hepatocytes by the method according to [9] to produce cultured hepatocytes, and metabolism of the compound is evaluated using the obtained cultured hepatocytes. .
[12] A step of producing a cultured hepatocyte formed by culturing hepatocytes by the method according to [9] to form a capillary bile duct, a step of adding a compound to the culture solution of the cultured hepatocyte, and a step of removing the compound And a method for assaying a compound according to [11], which comprises a step of analyzing a component in the reaction solution and / or capillary fluid.
[13] The metabolic assay method for a compound according to [11] or [12], wherein the compound is bilirubin and / or a bilirubin metabolite.
[14] Compound transport assay method for producing cultured hepatocytes by culturing hepatocytes by the method according to [9] to form capillary bile ducts, and evaluating the transport of the compounds using the obtained cultured hepatocytes .
[15] A step of producing a cultured hepatocyte obtained by culturing hepatocytes by the method described in [9] to form a capillary bile duct, a step of adding a compound to a culture solution of the cultured hepatocyte, and a step of removing the compound And a method for assaying a compound according to [14], comprising analyzing the components in the reaction solution and / or the capillary fluid.
[16] A compound test apparatus using cultured hepatocytes, comprising a main body containing cultured hepatocytes, a compound supply unit for supplying a compound to the main body, and a recovery unit for recovering the compound or its metabolite from the main body. And the said main-body part has the board | substrate for hepatocyte culture | cultivation in any one of [1]-[8], and a hepatocyte, The compound test | inspection apparatus characterized by the above-mentioned.
肝細胞を、酸素透過性の基板上に毛細胆管を形成する肝細胞を培養する肝細胞培養方法であって、すり鉢状の溝を有する基板に配置して培養することで、肝細胞が接着伸展せずに積層し、且つ、肝細胞に効率よく極性を誘導させることができる。その結果、肝細胞への更なる細胞外マトリクスの添加・重層なしに、毛細胆管などの微細構造を構築した３次元細胞組織を作製できる。よって、細胞外マトリクスの悪影響が無い、肝細胞培養方法が可能となる。 A hepatocyte culture method for culturing hepatocytes that form capillary bile ducts on an oxygen-permeable substrate, wherein the hepatocytes adhere to and extend by placing them on a substrate having a mortar-shaped groove. Without being laminated, and the liver cells can be efficiently induced in polarity. As a result, it is possible to produce a three-dimensional cellular tissue in which a fine structure such as a capillary bile duct is constructed without adding an extraneous matrix to the hepatocyte and without layering. Thus, a method for culturing hepatocytes that does not adversely affect the extracellular matrix is possible.
また、同一基板上の複数のすり鉢状の溝を設ける場合、溝の間隔を肝細胞が載らない程度に狭くすれば、余分な肝細胞の洗浄による除去無しに毛細胆管を形成した肝培養細胞をパターン化することができる。このようにして培養された肝細胞集団内では、毛細胆管を、位置、大きさ、形を制御して、容易に基板上に形成させることができる。そして、毛細胆管内の胆汁排泄物を容易に回収することが可能となり、多検体を同時に分析することも可能である。また、流路やインクジェット装置などの大がかりな機械を用いることなく、ヒト肝細胞のような高価な細胞を無駄にしない細胞配列制御が可能である。 In addition, when providing a plurality of mortar-shaped grooves on the same substrate, if the gap between the grooves is reduced to such an extent that no hepatocytes can be placed, hepatocyte culture cells that have formed capillary bile ducts without removal by washing excess hepatocytes can be obtained. Can be patterned. Within the cultured hepatocyte population, the capillary bile duct can be easily formed on the substrate by controlling the position, size and shape. And it becomes possible to collect | recover the bile excretion in a capillary bile duct easily, and it is also possible to analyze many samples simultaneously. Further, cell arrangement control without wasting expensive cells such as human hepatocytes is possible without using a large-scale machine such as a flow path or an ink jet device.
本発明の肝細胞培養用基板は、毛細胆管を形成する肝細胞を培養するための酸素透過性の基板であって、表面にすり鉢状の溝を有することを特徴とする。ここで、すり鉢状の溝とは、短手方向の幅が上部から底面に向かって狭くなっている溝を意味する。 The hepatocyte culture substrate of the present invention is an oxygen-permeable substrate for culturing hepatocytes forming capillaries, and has a mortar-like groove on the surface. Here, the mortar-shaped groove means a groove whose width in the short side direction becomes narrower from the top to the bottom.
すり鉢状溝の短手方向上部の幅は少なくとも１個の肝細胞が配置可能な幅であり、２個以上の肝細胞が配置可能な幅であることが好ましく、具体的には20μm以上100μm以下であることが好ましく、40μｍ以上80μｍ以下であることがより好ましい。 The width of the upper part of the mortar-shaped groove in the short direction is a width at which at least one hepatocyte can be arranged, and is preferably a width at which two or more hepatocytes can be arranged, specifically 20 μm or more and 100 μm or less. Preferably, it is 40 μm or more and 80 μm or less.
すり鉢状溝の短手方向下部（底面）の幅は肝細胞が接着・伸展しない幅であることが好ましく、具体的には、10μm以下であることが好ましく、鋭角（幅は0）であることがより好ましい。鋭角の場合、傾斜が３０°以上であれば、肝細胞が底面に接着しないため好ましく、４０°以上がより好ましい。 The width of the lower portion (bottom surface) of the mortar-shaped groove is preferably such that hepatocytes do not adhere or extend, and specifically, it is preferably 10 μm or less and has an acute angle (width is 0). Is more preferable. In the case of an acute angle, an inclination of 30 ° or more is preferable because hepatocytes do not adhere to the bottom surface, and 40 ° or more is more preferable.
すり鉢状溝の深さは少なくとも１個の肝細胞が積層可能な深さであり、２個以上の肝細胞が積層可能な深さが好ましく、具体的には、20μm以上1mm以下であることが好ましく、30μm以上200μm以下であることがより好ましく、40μm以上80μｍ以下であることがさら
に好ましい。
また、すり鉢状溝の短手方向上部の幅、あるいは、すり鉢状溝の深さの少なくとも一方が、２個以上の肝細胞が配置可能であることが好ましい。
The depth of the mortar-shaped groove is a depth at which at least one hepatocyte can be laminated, and is preferably a depth at which two or more hepatocytes can be laminated, specifically, 20 μm or more and 1 mm or less. Preferably, it is 30 μm or more and 200 μm or less, and more preferably 40 μm or more and 80 μm or less.
Moreover, it is preferable that two or more hepatocytes can be arranged in at least one of the width of the upper portion in the short direction of the mortar-shaped groove or the depth of the mortar-shaped groove.
なお、すり鉢状溝は、底面側にすり鉢状の部分を有する限り、必ずしも全体がすり鉢状である必要はない。すなわち、肝細胞を培養する部分がすり鉢状になっていればよく、その上部は幅が同じであってもよい。このように、すり鉢状部分の上に、更に、肝細胞を収納しない空間があっても良く、その上面は開口していても非開口でも良い。少なくとも、肝細胞を配置する際や培養液等を交換する際等には開口される。 In addition, as long as the mortar-shaped groove has a mortar-shaped part on the bottom surface side, the whole does not necessarily have a mortar shape. That is, the portion for culturing hepatocytes may be mortar-shaped, and the upper portion thereof may have the same width. Thus, there may be a space that does not contain hepatocytes on the mortar-shaped portion, and the upper surface may be open or non-open. At least when opening hepatocytes or exchanging the culture medium or the like.
また、基板上でのすり鉢状溝のパターンは、肝細胞をサンドイッチ培養しなくても効率良く毛細胆管が形成可能なものであれば特に限定されないが、好ましくは線状であり、より好ましくは直線状である。 The pattern of the mortar-shaped groove on the substrate is not particularly limited as long as it can efficiently form a capillary bile duct without sandwich culture of hepatocytes, but is preferably linear, more preferably linear. Is.
本発明に使用可能な基板は、肝細胞を培養できる酸素透過性のものであれば限定しないが、公知の酸素透過性膜基板を用いることができる。 The substrate that can be used in the present invention is not limited as long as it is an oxygen-permeable substrate capable of culturing hepatocytes, and a known oxygen-permeable membrane substrate can be used.
酸素透過性基板の素材は酸素ガスを透過できるものであれば良いが、多孔質で疎水性の高いものが適している。例えば、ポリジメチルシロキサン（PDMS）、フルオロカーボン、ポリテトラフルオロエチレン(４フッ化)、ポリウレタンが挙げられ、これらの誘導体や類似物質も含まれる。 The material of the oxygen permeable substrate may be any material that can permeate oxygen gas, but a porous and highly hydrophobic material is suitable. Examples include polydimethylsiloxane (PDMS), fluorocarbon, polytetrafluoroethylene (tetrafluoride), and polyurethane, and derivatives and similar substances thereof are also included.
ガス透過性基板はコラーゲンで表面をコートしてもよい。ガス透過性基板の表面をコラーゲンでコートする場合、コラーゲンは、公知の方法で調製されたものが使用できるが、市販のコラーゲン溶液(例えば、ベクトンディッキンソン社製のラット尾コラーゲン)を用いて酸素を透過できる厚さに被覆することができる。また、コラーゲンで酸素透過性基板をコートする方法は、公知方法を使用することができる。例えば、酸素プラズマ処理をしてコラーゲンをガス透過性基板に吸着させる方法や化学的に反応する官能基を使用して共有結合させる方法が挙げられる。共有結合を使用したPDMS膜へのコラーゲンの結合方法としては、例えば、非特許文献６に述べられている方法が挙げられる。効率良く、安定かつ長期的に、毛細胆管を形成した肝細胞を調製することができるため、共有結合によりコラーゲンでガス透過性基板を覆うことが好ましい。 The gas permeable substrate may be coated on the surface with collagen. When the surface of the gas permeable substrate is coated with collagen, collagen prepared by a known method can be used, but oxygen can be added using a commercially available collagen solution (for example, rat tail collagen manufactured by Becton Dickinson). It can be coated to a thickness that can penetrate. Moreover, a well-known method can be used for the method of coating an oxygen-permeable substrate with collagen. For example, a method of adsorbing collagen on a gas-permeable substrate by oxygen plasma treatment or a method of covalently bonding using a chemically reactive functional group can be mentioned. Examples of the method for binding collagen to the PDMS membrane using a covalent bond include the method described in Non-Patent Document 6. Since hepatocytes in which capillaries are formed can be prepared efficiently, stably and for a long period of time, it is preferable to cover the gas-permeable substrate with collagen by covalent bonding.
なお、短期的な毛細胆管の形成効率は共有結合でも吸着結合でも同様であることから、短期的な測定に使用する場合には、いずれのコート方法も使用することができる。試験に必要な毛細胆管を形成できるかぎり、肝細胞の培養条件に合わせて、適宜、最適な結合方法を選択することができる。 In addition, since the formation efficiency of a short-term capillary bile duct is the same whether it is a covalent bond or an adsorptive bond, when using for a short-term measurement, any coating method can be used. As long as the capillary bile duct necessary for the test can be formed, an optimal binding method can be appropriately selected according to the culture conditions of hepatocytes.
本発明の肝細胞培養用基板に形成されるすり鉢状の溝は単数でも複数でも良い。複数であれば、多数の条件を容易に評価することが可能となり好ましい。 The mortar-shaped groove formed on the hepatocyte culture substrate of the present invention may be singular or plural. If it is plural, it is possible because a large number of conditions can be easily evaluated.
複数のすり鉢状の溝の間隔は、肝細胞が載らない幅、すなわち肝細胞１個の幅より小さいことが好ましい。具体的には、５μｍ以下、好ましくは３μｍ以下、より好ましくは１μｍ以下である。このような幅にすれば基板に肝細胞を配置する際に、洗浄等の労力をかけず、また、配置されなかった無駄になる肝細胞を生じることもなく、簡便に行うことができるので好ましい。下限は隣り合う溝同士が判別できる幅であればよいが、例えば０．１μｍ以上である。 The interval between the plurality of mortar-shaped grooves is preferably smaller than the width where hepatocytes do not rest, that is, the width of one hepatocyte. Specifically, it is 5 μm or less, preferably 3 μm or less, more preferably 1 μm or less. Such a width is preferable because it can be carried out easily without placing any labor such as washing when placing hepatocytes on the substrate, and without generating wasted hepatocytes that have not been placed. . The lower limit may be any width as long as adjacent grooves can be distinguished, and is, for example, 0.1 μm or more.
酸素透過性の基板にすり鉢状の溝を形成する方法は特に限定されないが、鋳型を利用するなど公知の手法が使用できる。 A method for forming a mortar-like groove on an oxygen permeable substrate is not particularly limited, but a known method such as using a mold can be used.
以下、本発明の基板を用いた肝細胞の培養方法の一態様を説明する。好ましくはすり鉢状の溝を有する酸素透過性基板を適当な厚さになるようにコラーゲンで覆い、その上に肝細胞を播種し、培養する。 Hereinafter, an embodiment of a method for culturing hepatocytes using the substrate of the present invention will be described. Preferably, an oxygen-permeable substrate having a mortar-like groove is covered with collagen so as to have an appropriate thickness, and hepatocytes are seeded thereon and cultured.
肝細胞の培養は通常、酸素を供給しつつ行う。当業者であれば、形成したい毛細胆管のサイズや種類に合わせて、基板の種類や酸素供給方法を適宜選択することができる。 Hepatocyte culture is usually performed while supplying oxygen. A person skilled in the art can appropriately select the type of substrate and the oxygen supply method according to the size and type of the capillary bile duct to be formed.
播種する細胞密度は、肝細胞が正常に生存可能であれば良い。肝細胞の細胞密度は通常は1.0〜2.0 x 105cells/cm2の密度で撒かれるが、培養条件や使用する培養器具などに合わせて、適宜、好ましい細胞密度を設定することができる。 The cell density to be seeded is not limited as long as hepatocytes can survive normally. The cell density of hepatocytes is usually set at a density of 1.0 to 2.0 × 10 5 cells / cm 2 , but a preferable cell density can be appropriately set according to the culture conditions and the culture instrument used.
培養条件は公知の肝細胞を培養する方法に準じて行えば良く、培地としては、例えば、ダルベッコ修飾イーグル培地やウイリアムズE培地に血清、インスリン・トランスフェリン・セレニウム塩、デキサメタゾンを添加した培地を用いることができる。
そして、一般的な細胞培養と同じく、通常37℃、5%CO2の条件で培養を行う。ただし、特殊な肝細胞や条件で培養を行う場合は、温度やCO2濃度は適宜変更すればよい。培養条件を調節することによって、毛細胆管の数を調節すること等ができる。
このようにして培養を行うことにより、肝細胞に3次元的位置情報を付与することができる。
The culture conditions may be in accordance with a known method for culturing hepatocytes. As the medium, for example, a medium in which serum, insulin / transferrin / selenium salt, dexamethasone is added to Dulbecco's modified Eagle medium or Williams E medium is used. Can do.
Then, as in general cell culture, the culture is usually performed under conditions of 37 ° C. and 5% CO 2 . However, when culturing with special hepatocytes and conditions, the temperature and CO 2 concentration may be appropriately changed. By adjusting the culture conditions, the number of capillaries can be adjusted.
By culturing in this way, three-dimensional position information can be given to hepatocytes.
本発明で使用可能な培養可能な肝細胞は、いずれの動物由来でも良く、ヒト、サル、イヌ、ネコ、ウシ、ブタ、ミニブタ、ハムスター、フェレット、ウサギ、ラット、マウス等に由来する肝細胞が挙げられる。また、該動物から肝細胞を単離する方法は、公知の方法に従って行うことができる。肝細胞の由来は胎児、新生児、成体のいずれであってもよい。また、胚性幹（ES）細胞および誘導多能性(iPS)幹細胞、または臍帯血、骨髄、脂肪、血液由来組織性幹細胞から分化誘導される肝細胞を用いることもできる。これらの細胞から肝細胞を誘導する方法は公知の方法に従って行うことができる。 The culturable hepatocytes that can be used in the present invention may be derived from any animal, such as hepatocytes derived from humans, monkeys, dogs, cats, cows, pigs, minipigs, hamsters, ferrets, rabbits, rats, mice, and the like. Can be mentioned. The method for isolating hepatocytes from the animal can be performed according to a known method. The origin of hepatocytes may be any of fetus, newborn, and adult. In addition, embryonic stem (ES) cells and induced pluripotent (iPS) stem cells, or hepatocytes induced to differentiate from cord blood, bone marrow, fat, or blood-derived tissue stem cells can also be used. A method for inducing hepatocytes from these cells can be performed according to a known method.
本発明の基板を用いて培養することにより、基底膜や毛細胆管ネットワークを有する３次元肝培養細胞を得ることができる。
基底膜には化合物を取り込むための有機アニオン輸送体群、およびナトリウム/タウロコール酸共輸送体群が発現している。代表的なものはOATP（Organic anion transportingpolypeptide）1a1, OATP1b2, OATP1b3, OAT2, OATP4, OATP8であり、これらに特異的な抗体を用いた細胞抗体染色によって存在を確認することができる。
By culturing using the substrate of the present invention, three-dimensional liver cultured cells having a basement membrane or a capillary bile duct network can be obtained.
In the basement membrane, an organic anion transporter group for taking in the compound and a sodium / taurocholic acid cotransporter group are expressed. Representative examples are OATP (Organic anion transporting polypeptide) 1a1, OATP1b2, OATP1b3, OAT2, OATP4, and OATP8, and their presence can be confirmed by cell antibody staining using antibodies specific to these.
毛細胆管ネットワークには主要なATP binding cassette (ABC)トランスポータータンパク質が発現する。代表的なものはMRP2（Multidrug-Resistance Protein 2）, MDR1（Multidrug-Resistance 1）, BCRP（breast cancer resistance protein）であり、それぞれestradiol-17β-glucuronide、Digoxin、Taurocholateの輸送活性から存在の有無を判別できる。また、MRP2, MDR1, BCRPに特異的な抗体を用いた細胞抗体染色によっても確認することができる。 A major ATP binding cassette (ABC) transporter protein is expressed in the capillary network. Typical ones are MRP2 (Multidrug-Resistance Protein 2), MDR1 (Multidrug-Resistance 1), and BCRP (breast cancer resistance protein). Can be determined. It can also be confirmed by cell antibody staining using antibodies specific for MRP2, MDR1, and BCRP.
本発明の肝細胞培養方法により、細胞外マトリクスゲルの重層を必要とせずに（ただし、細胞外マトリクスゲルを重層する態様を除外するものではない）、毛細胆管を形成した肝細胞を位置を制御して作製できる。これにより、化合物を直接的に肝細胞に添加し、毛細胆管に排出される代謝物を解析することが容易であり、精度良く化合物の代謝特性を検定することができる。 According to the method for culturing hepatocytes of the present invention, the position of hepatocytes in which capillary bile ducts are formed is controlled without requiring an extra layer of extracellular matrix gel (however, this does not exclude the mode of overlaying extracellular matrix gel). Can be produced. Thus, it is easy to add a compound directly to hepatocytes and analyze a metabolite excreted into the capillary bile duct, and the metabolic characteristics of the compound can be assayed with high accuracy.
すなわち、本発明の化合物検定装置（図６に一例を示す）は、本体部と、本体部に化合
物を供給する化合物供給部と、本体部から化合物またはその代謝物を回収する化合物またはその代謝物の回収部とを有する本発明の肝細胞培養方法で培養された培養肝細胞を用いた化合物検定装置であって、前記本体部は、本発明の基板と、該基板上に培養された肝細胞とを有する。位置を制御して毛細胆管が形成されるため、ガラス管などの微細チューブを毛細胆管に挿入し、毛細胆管内に排出される代謝物を回収し、解析することが容易となる。
That is, the compound test apparatus of the present invention (an example is shown in FIG. 6) includes a main body part, a compound supply part that supplies a compound to the main body part, and a compound or metabolite thereof that recovers the compound or its metabolite from the main body part. A compound assay apparatus using cultured hepatocytes cultured by the method for culturing hepatocytes of the present invention, wherein the main body comprises the substrate of the present invention and hepatocytes cultured on the substrate. And have. Since the capillary bile duct is formed by controlling the position, it becomes easy to insert a fine tube such as a glass tube into the capillary bile duct and collect and analyze the metabolite discharged into the capillary bile duct.
また、微細チューブなどの流出先に蛍光光度計や質量分析装置（LC-MSやLC/MS/MS等）や高速液体クロマトグラフィ(HPLC)などの分析装置へ直結させることで、解析を同じデバイス内で同時に行うこともできる。分析装置は、解析する化合物に併せて適宜選択することができる。 In addition, the analysis can be performed within the same device by connecting directly to an analyzer such as a fluorimeter, mass spectrometer (LC-MS, LC / MS / MS, etc.) or high performance liquid chromatography (HPLC) at the outflow destination such as a micro tube. Can be done at the same time. The analyzer can be appropriately selected according to the compound to be analyzed.
本発明によれば、位置を制御して効率的に毛細胆管を形成した肝細胞を作製できるため、自動分析装置などへの応用が容易である。 According to the present invention, hepatocytes in which capillaries were efficiently formed by controlling the position can be easily applied to an automatic analyzer or the like.
化合物の代謝検定の方法としては、当業者であれば、公知の方法を適宜変更して実施できるが、例えば、本発明に従って毛細胆管を形成した培養肝細胞を製造し、該培養肝細胞の培養液に化合物を添加して、該毛細胆管への取り込みを行った後、洗浄液で該培養肝細胞を洗って該化合物を除去し、化合物を含まない反応液中で代謝反応に供した後、反応液（培養肝細胞外液）中及び／又は毛細胆管液中の成分を分析して、化合物の代謝特性を検定することができる。反応液中は生体における血液成分の代替え、毛細胆管液中は生体における毛細胆管代謝物の代替えと見なすことができる。 As a method for assaying the metabolism of the compound, those skilled in the art can carry out the method by appropriately changing known methods. For example, according to the present invention, cultured hepatocytes in which capillary bile ducts are formed are produced, and the cultured hepatocytes are cultured. After the compound is added to the solution and taken into the capillary bile duct, the cultured hepatocytes are washed with a washing solution to remove the compound and subjected to a metabolic reaction in a reaction solution not containing the compound. Components in the fluid (cultured extrahepatic fluid) and / or capillary bile fluid can be analyzed to test the metabolic properties of the compound. The reaction solution can be regarded as a substitute for blood components in the living body, and the capillary fluid can be regarded as a substitute for capillary biliary metabolites in the living body.
毛細胆管中に排泄される物質であれば好適に利用でき、蛍光やＲＩ等の標識ビリルビンやビリルビン代謝物も利用可能である。ビリルビン代謝物としては、非抱合（直接）ビリルビンや抱合(間接)ビリルビンが挙げられ、更に抱合ビリルビンとしては、グルクロン酸抱合体や硫酸抱合体などが挙げられる。また、他の肝細胞で代謝され、毛細胆管中に排泄される生体成分や化合物についても同様に利用可能なことは容易に理解できる。生体成分としては、Taurocholic acid、glycocholic acid、taurochenodeoxycholic acid、glycochenodeoxycolic acid、a- and b-tauromuricholic acidなどの胆汁酸などが挙げられ、化合物としては、Cephradine、d8-taurocholic acid、Digoxin、enkepharine hydrate、iodocyanine green、Indomethacine、Ouabain、Pravastatin、Rosuvastatin、Methotrexate、Vincristine、Doxorubicinなどが挙げられる。 Any substance excreted in the capillary bile duct can be suitably used, and labeled bilirubin and bilirubin metabolites such as fluorescence and RI can also be used. Examples of bilirubin metabolites include unconjugated (direct) bilirubin and conjugated (indirect) bilirubin, and examples of conjugated bilirubin include glucuronic acid conjugates and sulfate conjugates. In addition, it can be easily understood that biological components and compounds that are metabolized in other hepatocytes and excreted in the capillary bile duct can be used in the same manner. Biological acids include bile acids such as Taurocholic acid, glycocholic acid, taurochenodeoxycholic acid, glycochenodeoxycolic acid, a- and b-tauromuricholic acid, and compounds include Cephradine, d8-taurocholic acid, Digoxin, enkepharine hydrate, iodocyanine Examples include green, Indomethacine, Ouabain, Pravastatin, Rosuvastatin, Methotrexate, Vincristine, and Doxorubicin.
本発明の肝細胞培養用基板を使用して培養した肝培養細胞は、薬物輸送検定や医薬候補物質のハイスループットなスクリーニングにも使用することができる。
薬物輸送検定としては、薬物がどのくらいの量と速度で肝細胞に取り込まれ、胆汁に排出されるかどうかの検定が例示される。または、ある化合物Aが化合物Bの輸送を阻害・促進するかどうかの検定が例示される。
薬物輸送検定の方法としては、例えば、非特許文献７の方法が挙げられる。また、上述した代謝検定の方法を適宜改変して行うこともできる。
Liver culture cells cultured using the hepatocyte culture substrate of the present invention can also be used for drug transport assays and high-throughput screening of drug candidates.
An example of a drug transport assay is an assay of how much and how much drug is taken up by hepatocytes and excreted into bile. Alternatively, an assay of whether a certain compound A inhibits or promotes the transport of compound B is exemplified.
Examples of the drug transport assay include the method of Non-Patent Document 7. In addition, the metabolic assay method described above can be appropriately modified.
ハイスループットなスクリーニングの具体的な方法としては、例えば、複数（好ましくは多数）のすり鉢状の溝を有する肝細胞培養用基板を使用して培養した肝培養細胞において、微細チューブと高感度な検出器でごく微量の化合物を解析でき、さらにこれを自動あるいは半自動で行うような以下の方法などが挙げられる。
ハイスループットなスクリーニングのためには、毛細胆管を形成した肝細胞が培養されている培養部分それぞれに、試験化合物暴露させる注入口と、それぞれの区画の肝細胞で代謝され毛細胆管に排出される代謝物を回収するための口が設けられたマイクロ流路デバイスを構築することで可能となる。
上記マイクロ流路デバイスの注入口には化合物ライブラリから化合物溶液を分取し送液するポンプ、出口には代謝物を高速液体クロマトグラフィ(HPLC)や質量分析装置（LC/MSやLC/MS/MS等）や蛍光光度計等に送って代謝物の定量や組成分析結果を導く流路が接続され、これらの動作をコンピューターなどで操作するようなものが例示される。
Specific methods for high-throughput screening include, for example, microtubes and high-sensitivity detection in cultured hepatocyte cells using a hepatocyte culture substrate having multiple (preferably many) mortar-shaped grooves. Examples include the following methods that can analyze a very small amount of a compound with a vessel, and further perform this automatically or semi-automatically.
For high-throughput screening, each portion of the culture where hepatocytes that have formed capillaries are cultured, and an injection port that is exposed to the test compound, and metabolism that is metabolized by hepatocytes in each compartment and excreted into the capillaries. This is possible by constructing a micro-channel device provided with a port for collecting objects.
The microchannel device has an injection port that pumps the compound solution from the compound library and delivers it, and the metabolite at the outlet is a high performance liquid chromatography (HPLC) or mass spectrometer (LC / MS or LC / MS / MS). Etc.) or a flowmeter that is sent to a fluorimeter or the like to guide the quantification of metabolites and the result of composition analysis is connected, and these operations are exemplified by a computer or the like.
本発明の方法で作製した毛細胆管を形成した肝細胞が、生体を反映し、様々な用途に使用できることは、当業者であれば公知の手法を用いて容易に確認することができる。例えば、毛細胆管の形成を視覚的に確認したり、既知の化合物を利用して化合物検定を行ったり、薬物代謝関連遺伝子群の遺伝子発現量を測定したりして、生体肝と同様な機能を有するか確認することができる。 Those skilled in the art can easily confirm that hepatocytes formed with capillaries produced by the method of the present invention reflect the living body and can be used for various purposes, using a known method. For example, it is possible to visually confirm the formation of capillaries, conduct compound tests using known compounds, and measure the gene expression level of drug metabolism-related genes. You can check if you have it.
以下に、実施例を挙げて本発明を更に詳細に説明するが、本発明はこれらの実施例により制限されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
＜実施例１＞
＜実施例１−１：マイクロパターン化基板の作製＞
マイクロパターン化基板は、モールディング（型取）により、すり鉢状の溝を複数備えたパターン（線状のこぎり歯パターン）、あるいは上部の幅と底面の幅が同じ線状凹型パターンを有する基板をポリジメチルシロキサン（PDMS）で製作した。図１に示すように、線状のこぎり歯パターンのモールドは、異方性ウェットエッチングを利用してシリコンウェハーで作製した。モールドによりPDMS薄膜表面に作製された各溝のサイズは、深さ約７０μｍ、底面は鋭角であり、短手方向上部の幅は約６０μｍ、斜面の傾斜は約５４℃である。また、比較用として、PDMS薄膜表面に幅約40μm深さ約50μmの線状凹型パターンを同じくモールディングにより形成した基板（線状凹型パターン）と、表面に溝を形成されていない厚さ1mm程度のPDMS薄膜（パターンなし基板）を作製した。
<Example 1>
<Example 1-1: Production of micropatterned substrate>
A micropatterned substrate can be obtained by molding a pattern having a plurality of mortar-shaped grooves (linear sawtooth pattern) or a substrate having a linear concave pattern with the same width at the top and bottom by molding. Made of siloxane (PDMS). As shown in FIG. 1, a linear sawtooth pattern mold was fabricated with a silicon wafer using anisotropic wet etching. The size of each groove formed on the surface of the PDMS thin film by the mold is about 70 μm deep, the bottom is an acute angle, the width of the upper part in the short direction is about 60 μm, and the slope of the slope is about 54 ° C. Also, for comparison, a substrate (linear concave pattern) in which a linear concave pattern with a width of about 40 μm and a depth of about 50 μm is formed on the surface of a PDMS thin film, and a thickness of about 1 mm with no groove formed on the surface. PDMS thin film (unpatterned substrate) was prepared.
＜実施例１−２：肝細胞培養＞
実施例１で作製した線状のこぎり歯パターンのPDMS基板、線状凹型パターンのPDMS基板及びパターン無しのPDMS基板を70％エタノール溶液に浸すことで滅菌した。その後、プラズマエッチングを行い親水化した後、酸性10％コラーゲンI（ベクトン・ディッキンソン社製）溶液に浸し、乾かすことで表面にコラーゲンコート処理を施した。播種培地で1回基板表面を洗浄したのち、ラットの肝臓およびヒトの肝臓から調製した肝細胞を1.5×105
cells/cm2で全体に均一に広がるように播種した。培養は37℃、5％CO2/95％空気のCO2インキュベータを用いて行った。播種後、約4時間後に播種培地を交換し、非接着肝細胞を除き、培養を継続した。播種後、24時間後に培養培地に交換し、培養を継続した。その後24時間ごとに培養培地を交換した。図２に培養の模式図を示した。
<Example 1-2: Hepatocyte culture>
The PDMS substrate having a linear sawtooth pattern, the PDMS substrate having a linear concave pattern, and the PDMS substrate having no pattern prepared in Example 1 were sterilized by being immersed in a 70% ethanol solution. Then, after plasma-etching to make it hydrophilic, it was immersed in an acidic 10% collagen I (Becton Dickinson) solution and dried to give a collagen coat treatment on the surface. After washing the substrate surface once with the seeding medium, 1.5 × 10 5 hepatocytes prepared from rat liver and human liver
The cells were seeded so as to spread evenly at cells / cm 2 . Culturing was performed using a CO 2 incubator at 37 ° C. and 5% CO 2 /95% air. About 4 hours after seeding, the seeding medium was changed, non-adherent hepatocytes were removed, and the culture was continued. After seeding, the culture medium was replaced 24 hours later, and the culture was continued. Thereafter, the culture medium was changed every 24 hours. FIG. 2 shows a schematic diagram of culture.
＜実施例１−３：毛細胆管形成評価＞
（１）5 (and 6)-carboxy-2’,7’-dichlorofluorescein diacetate CDFDAによる毛細胆管染色
ラット(三協ラボサービス、Slc/Wister)肝細胞をHBSSバッファ(0.35 g/L KCl, 0.25g/L
MgSO4, 0.18 g/L CaCl2, 0.16 g/L KH2PO4, 4.8 g/L HEPES,7.9 g/L NaCl, and 0.9 g/L
glucose, pH 7.4)で2回洗浄したのち、10μmol/L CDFDAを含むHBSSで15分間、37℃,5%CO2で培養した。
<Example 1-3: Capillary bile duct formation evaluation>
(1) Capillary bile duct staining with 5 (and 6) -carboxy-2 ', 7'-dichlorofluorescein diacetate CDFDA Rats (Sankyo Lab Service, Slc / Wister) Hepatocytes in HBSS buffer (0.35 g / L KCl, 0.25g / L
MgSO 4 , 0.18 g / L CaCl 2 , 0.16 g / L KH 2 PO 4 , 4.8 g / L HEPES, 7.9 g / L NaCl, and 0.9 g / L
After washing twice with glucose, pH 7.4), the cells were cultured in HBSS containing 10 μmol / L CDFDA for 15 minutes at 37 ° C. and 5% CO 2.
（２）画像解析
（１）で染色した肝細胞を、Zeiss社製LSM710共焦点顕微鏡で顕鏡し、CDFDA染色の3次元画像を取得した。Zeiss社製画像解析ソフトAxioVisionでCDFDAにより染色された領域の面積を計算した。
(2) Image analysis The hepatocytes stained in (1) were examined with a Zeiss LSM710 confocal microscope to obtain a three-dimensional image of CDFDA staining. The area of the area stained with CDFDA was calculated with Zex image analysis software AxioVision.
（３）結果
培養2日目（図３）に毛細胆管形成をCDFDA染色により、評価した。図４左に線状のこぎり歯パターンPDMS基板、図４右にパターン無しPDMS基板の結果を示す。線状凹型パターンPDMS基板は、パターン無しPDMS基板とほぼ同じ結果だったため、図示を省略する。また、図５に取り込みを評価したものを示す。毛細胆管に蓄積するCDFDAの蛍光面積から、１画像あたりの毛細胆管の面積を算出した（図５）。線状のこぎり歯パターンPDMS基板を用いた場合、培養培地に細胞外マトリクスゲルを含まなくても、線状凹型パターンPDMS基板およびパターン無しPDMS基板と比較して、毛細胆管形成が有意に促進された。すなわち、サンドイッチ培養の必要が無いことが示された。
(3) Results Capillary bile duct formation was evaluated by CDFDA staining on the second day of culture (FIG. 3). The results for the linear sawtooth pattern PDMS substrate are shown on the left of FIG. 4, and the pattern-free PDMS substrate is shown on the right of FIG. Since the linear concave pattern PDMS substrate has almost the same result as the non-patterned PDMS substrate, the illustration is omitted. FIG. 5 shows an evaluation of uptake. The area of the capillary bile duct per image was calculated from the fluorescence area of CDFDA accumulated in the capillary bile duct (FIG. 5). When a linear sawtooth pattern PDMS substrate was used, capillary bile duct formation was significantly promoted even when the culture medium did not contain extracellular matrix gel, compared to the linear concave pattern PDMS substrate and unpatterned PDMS substrate. . That is, it was shown that there is no need for sandwich culture.
本発明によって得られる効率的に毛細胆管を形成する３次元肝培養細胞は、肝細胞を用いた医薬品候補化合物のハイスループットスクリーニングや薬物輸送検定などに利用できる。また、生体を用いた試験の代替え法とできることは、倫理的にも意義が高い。そして、それは医薬品候補化合物等の肝細胞における取り込み・代謝・排泄の解析精度と効率の上昇に寄与し、創薬プロセスの高効率化につながる。 The three-dimensional liver culture cells that efficiently form capillary bile ducts obtained by the present invention can be used for high-throughput screening of drug candidate compounds using hepatocytes and drug transport assays. In addition, it is ethically significant to be able to be an alternative to tests using living organisms. This contributes to an increase in analysis accuracy and efficiency of uptake, metabolism, and excretion of drug candidate compounds and the like in hepatocytes, leading to higher efficiency of the drug discovery process.
A: 化合物検定装置、１：培養容器、２：マニュピレーター、３：オイルインジェクター１（化合物供給部）、３’：オイルインジェクター２（化合物または代謝物回収部）、４：顕微鏡 A: Compound test apparatus, 1: culture vessel, 2: manipulator, 3: oil injector 1 (compound supply unit), 3 ': oil injector 2 (compound or metabolite recovery unit), 4: microscope
Claims (16)
細胞培養用基板。 The hepatocyte culture substrate according to any one of claims 1 to 3 , wherein the width of the upper part is 20 µm or more and 100 µm or less.
請求項１〜８のいずれか一項に記載の肝細胞培養用基板と、
肝細胞と、
を有することを特徴とする、化合物検定装置。 A compound test apparatus using cultured hepatocytes having a main body part containing cultured hepatocytes, a compound supply part for supplying a compound to the main body part, and a recovery part for recovering the compound or its metabolite from the main body part, The main body is
A substrate for culturing hepatocytes according to any one of claims 1 to 8,
Hepatocytes,
A compound assay device comprising:
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