JP5307489B2 - Method for producing three-dimensional cell structure - Google Patents
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本発明は、動物細胞の培養物に関する。 The present invention relates to animal cell cultures.
組織から単離した細胞を試験、検査に用いる手法は、バイオテクノロジー関連分野では欠かせない方法となっている。疾病、病態の診断、新薬の探索および薬効の判定、あるいは動物検査、植物検査、環境汚染物質の試験などに幅広く用いられている。単離した細胞は、直ちに試験に用いられる場合もあるが、多くは細胞培養の方法により培養 皿や試験管のなかで培養が行われている。この培養系のなかで種々の検査が行われる。 Techniques for testing and examining cells isolated from tissues are indispensable in biotechnology-related fields. It is widely used for diagnosing diseases and pathological conditions, searching for new drugs and determining their efficacy, animal testing, plant testing, and testing for environmental pollutants. The isolated cells may be used immediately for testing, but many are cultured in culture dishes or test tubes by the cell culture method. Various tests are performed in this culture system.
これらのアッセイは、通常均一な培養系を設定し、評価する薬物等の量、濃度などを変えてその効果を見るものである。その際に用いられる細胞は、生体から分離した初代細胞、また、セルバンクや細胞販売企業から入手される。この細胞は、種々の培地及び培養器内に播種された後、検査が行われる。 In these assays, a uniform culture system is usually set, and the effect is observed by changing the amount and concentration of the drug to be evaluated. The cells used at that time are obtained from primary cells separated from the living body, cell banks and cell sales companies. The cells are seeded in various media and incubators and then examined.
薬物の検査などに使用される培養法としては、非特許文献１に示した、コラーゲンをコートした細胞培養容器を用いて培養する方法、コラーゲンとマトリゲルを用いてサンドイッチ状に培養する単層培養方法が知られている。 Examples of the culture method used for drug inspection include the method of culturing using a cell culture vessel coated with collagen, as shown in Non-Patent Document 1, and the monolayer culture method of culturing in a sandwich form using collagen and Matrigel. It has been known.
この様に、広く利用されている細胞培養技術ではあるが、従来の細胞培養法（単層培養法）には、細胞が生体内で有している特異的な機能を長期間維持することができないという問題点があった。 As described above, although it is a widely used cell culture technique, the conventional cell culture method (monolayer culture method) can maintain a specific function of cells in a living body for a long period of time. There was a problem that it was not possible.
これらの問題点を解決するための培養方法として、三次元培養法が、近年、非常に注目を集めている。三次元培養によって得られた三次元細胞構造体は、単層培養法と比較して、細胞の特異的な機能を長期間維持でき、かつ、生体内と類似した構造体を作ることができる。このような特性を活かし、薬物等の検査への利用を目的に、三次元細胞構造体形状の三次元細胞構造体に関する研究開発が活発に行われている。例えば、特許文献１には均一な直径を有する三次元細胞構造体の作製方法が提案されている。また、特許文献２には均一な直径を有する三次元細胞構造体を培養する方法が提案されている。 As a culture method for solving these problems, a three-dimensional culture method has attracted much attention in recent years. Compared with the monolayer culture method, the three-dimensional cell structure obtained by three-dimensional culture can maintain a specific function of cells for a long period of time and can produce a structure similar to that in vivo. Taking advantage of these characteristics, research and development on three-dimensional cell structures in the shape of three-dimensional cell structures has been actively conducted for the purpose of use for examination of drugs and the like. For example, Patent Document 1 proposes a method for producing a three-dimensional cell structure having a uniform diameter. Patent Document 2 proposes a method for culturing a three-dimensional cell structure having a uniform diameter.
しかしながら、上記の方法によっても次のような課題があり、解決が望まれていた。
特許文献１に記載された方法は、実質細胞と、さらに１種類以上の細胞を用いて共培養し、三次元細胞構造体を得る方法であるが、培養に際し２種類以上の細胞を準備する必要があることから、多くの手間を要するだけでなく、肝細胞以外の細胞による薬物等の検査への影響が懸念される。
However, the above method has the following problems, and a solution has been desired.
The method described in Patent Document 1 is a method of obtaining a three-dimensional cell structure by co-culturing with a parenchymal cell and one or more types of cells, but it is necessary to prepare two or more types of cells for the culture Therefore, not only a lot of labor is required, but there is a concern about the influence of cells other than hepatocytes on the examination of drugs and the like.
特許文献２に記載された方法は、高粘度の培養液を使用し、回転培養法、還流培養法及び、浮遊培養法で培養する方法である。そのため、高粘度の培養液を使用するため、培地交換時の操作性に劣る、さらに、特殊な装置が必要になるという欠点があり、万人が用いることは困難である、 The method described in Patent Document 2 is a method of culturing by a rotary culture method, a reflux culture method, and a suspension culture method using a high-viscosity culture solution. Therefore, because it uses a high-viscosity culture solution, it is inferior in operability at the time of medium exchange, and further has a disadvantage that a special device is required, which is difficult for everyone to use.
したがって、本発明は、これら欠点を解消する簡便な方法で、均一な大きさを有する三次元細胞構造体を提供することを目的とする。 Therefore, an object of the present invention is to provide a three-dimensional cell structure having a uniform size by a simple method for eliminating these drawbacks.
本発明にかかる細胞培養方法は、（Ａ）生存率が７０％以上の細胞集団を密度勾配遠心法により濃縮、分離する工程から得られた細胞集団を、（Ｂ）有効底面積が０．０１〜０．１ｍｍ２の小領域を有する支持体に播種し、（Ｃ）培養する工程である。前記工程（Ｂ）において、密度勾配遠心法は遠心力が１００×ｇ以下であるのが好ましい。 In the cell culture method according to the present invention, a cell population obtained from the step of (A) concentrating and separating a cell population having a survival rate of 70% or more by density gradient centrifugation, (B) having an effective bottom area of 0.01 It is a step of seeding on a support having a small region of ˜0.1 mm 2 and culturing (C). In the step (B), the density gradient centrifugation method preferably has a centrifugal force of 100 × g or less.
本発明の工程（Ｃ）は、
（ｉ）；細胞を接着させるために培養する工程と、
（ii）；非接着性細胞を除去する工程と、
（iii）；三次元細胞構造体を形成させるために培養する工程とからなっていても良い。
ここで、「細胞を接着させるために培養する工程（ｉ）」の時間は、細胞を接着させることができればよく、用いられる細胞に応じて選択することができ、その時間は限定されるものではない。
Step (C) of the present invention comprises:
(I); culturing to attach the cells;
(Ii); removing non-adherent cells;
(Iii); a step of culturing in order to form a three-dimensional cell structure.
Here, the time of “the step of culturing for attaching cells (i)” is not limited as long as the cells can be attached and can be selected according to the cells used. Absent.
本発明の工程（Ｂ）においては、細胞の播種密度が１×１０４個から１×１０６個／ｃｍ２であるのが好ましい。また、前記小領域の表面の全体又は一部が、有機膜又は無機膜を備えたものであるのが好ましい。 In the step (B) of the present invention, the seeding density of the cells is preferably 1 × 10 4 to 1 × 10 6 cells / cm 2 . Moreover, it is preferable that the whole or part of the surface of the small region is provided with an organic film or an inorganic film.
さらに、本発明における前記細胞集団は、ヒト肝実質細胞であるのが好ましい。 Furthermore, the cell population in the present invention is preferably human hepatocytes.
本発明により、均一な大きさを有する高密度な三次元細胞構造体を簡便な方法で得ることができる。 According to the present invention, a high-density three-dimensional cell structure having a uniform size can be obtained by a simple method.
以下、本発明を適用した実施形態の一例について説明する。なお、本発明の趣旨に合致する限り、他の実施形態も本発明の範疇に属し得ることは言うまでもない。また、以降の図における各部材の材料、サイズや比率は、一例であり、これに限定されるものではない。 Hereinafter, an example of an embodiment to which the present invention is applied will be described. It goes without saying that other embodiments may also belong to the category of the present invention as long as they match the gist of the present invention. Moreover, the material, size, and ratio of each member in the following drawings are merely examples, and the present invention is not limited thereto.
本発明で培養する細胞集団は、三次元細胞構造体を形成するものであれば特に制限はないが、有用性の観点から、実質細胞であるのが好ましく、特に肝細胞であるのが好ましく、さらにはヒトの肝実質細胞であるのが好ましい。 The cell population cultured in the present invention is not particularly limited as long as it forms a three-dimensional cell structure, but from the viewpoint of usefulness, it is preferably a parenchymal cell, particularly preferably a hepatocyte, Furthermore, human hepatocytes are preferable.
生存率が７０％以上の細胞集団の由来は、生体から分離した初代細胞でもよく、生体から分離した後に凍結したものを融解して得られた初代細胞でもよく、これらを継代した培養細胞でもよく、市販されている株化細胞を継代した培養細胞でもよいが、生体外から分離された直後から、細胞の機能は低下または変化することが知られているため、より生体内に近い機能を有する三次元細胞構造体を得るには、生体から分離した直後の初代細胞または生体から分離した後凍結したものを融解して得られた初代細胞の方が好ましく、生体から分離した直後の初代細胞がより好ましい。 The origin of a cell population having a survival rate of 70% or more may be a primary cell isolated from a living body, a primary cell obtained by thawing a frozen product after being separated from a living body, or a cultured cell passaged from these cells. Well, it may be a cultured cell that has been subcultured from a cell line that is commercially available, but it is known that the function of the cell is reduced or changed immediately after it is isolated from outside the body. In order to obtain a three-dimensional cell structure having a primary cell, a primary cell immediately after separation from a living body or a primary cell obtained by thawing a frozen product after separation from a living body is preferable. Cells are more preferred.
本発明に用いられる細胞の生存率は７０％以下の細胞集団の場合、生存している細胞であっても、表面の膜タンパクなど損傷を受け、細胞播種後の細胞接着性に劣ることから少なくとも７０％以上必要であり、密度勾配遠心法による前処理による細胞回収効率の観点から、８０％以上がより好ましい。初期の細胞集団の生存率が７０％以下のときは、７０％以上の生存率に精製した後に本発明に供すればよい。細胞を精製する方法としては、細胞の比重を利用して分離する方法、または、例えば細胞を標識しマイクロビーズやセルソータなどを用いて分離する方法などが挙げられるが、これらに限定されるものではない。 In the case of a cell population of 70% or less, the viability of the cells used in the present invention is at least because even the surviving cells are damaged by surface membrane proteins and the like and inferior in cell adhesion after cell seeding. 70% or more is necessary, and 80% or more is more preferable from the viewpoint of cell recovery efficiency by pretreatment by density gradient centrifugation. When the survival rate of the initial cell population is 70% or less, it may be used for the present invention after purification to a survival rate of 70% or more. Examples of the method for purifying cells include a method of separating using the specific gravity of cells, or a method of labeling cells and separating them using microbeads or cell sorters, but is not limited thereto. Absent.
工程（Ａ）における密度勾配遠心法に用いられる溶液は、例えば、Ｐｅｒｃｏｌｌ（Ｇ
Ｅヘルスケアバイオサイエンス社製）（ＰｅｒｃｏｌｌはＧＥヘルスケアバイオサイエン
ス社の登録商標）、Ｉｏｄｉｘａｎｏｌ溶液（Ａｘｉｓ−Ｓｈｉｅｌｄ社製）などが市販
されているがこれらに限定されるものではない。
また、工程（Ａ）において密度勾配遠心法は、細胞が受けるダメージの観点から遠心力
が１００×ｇ以下で行うことが好ましく、５０×ｇ以下で行うことがより好ましい。遠心
力の下限値は特に制限はないが、処理時間の観点から２０×ｇ以上で行うことが好ましい
。
The solution used for the density gradient centrifugation in the step ( A ) is, for example, Percoll (G
E Healthcare Bioscience (Percoll is a registered trademark of GE Healthcare Bioscience), Iodixanol solution (Axis-Shield) and the like are commercially available, but are not limited thereto.
In the step ( A ), the density gradient centrifugation is preferably performed at a centrifugal force of 100 × g or less, more preferably 50 × g or less, from the viewpoint of damage to the cells. The lower limit of the centrifugal force is not particularly limited, but is preferably 20 × g or more from the viewpoint of processing time.
ここで、工程（Ｃ）における（ｉ）細胞を接着させるために培養する工程について説明する。この工程は、播種した細胞が接着すればよく、３０分以下の場合、未接着の細胞が（ii）の工程で除かれてしまい、細胞数が減少する。２４時間以上の場合、細胞の内部液が培地中に漏出するため、正常な細胞の細胞死を招く恐れがあることから、３０分以上２４時間以内が好ましく、１時間から８時間がより好ましい。 Here, (i) the step of culturing to adhere the cells in step (C) will be described. In this step, the seeded cells only have to adhere, and in the case of 30 minutes or less, unattached cells are removed in the step (ii), and the number of cells decreases. In the case of 24 hours or more, the internal fluid of the cell leaks into the medium, which may lead to cell death of normal cells. Therefore, it is preferably 30 minutes or more and 24 hours or less, and more preferably 1 to 8 hours.
用いられる培地は、血清を含む血清を含んだ培地や、栄養分を添加した無血清培地等が挙げられるがこれらに限定されるものではない。 Examples of the medium used include, but are not limited to, a medium containing serum containing serum and a serum-free medium supplemented with nutrients.
本発明の（Ａ）、（Ｂ）、（Ｃ）の各工程で用いる培地は、全て同じ種類の培地を用いてもよく、例えば（Ａ）では無血清培地、（Ｂ）では血清入りの培地、（Ｃ）では無血清培地、といった、各工程で異なる培地を組合せてもよく、または、（Ａ）では無血清培地、（Ｂ）、（Ｃ）では無血清培地のように組合せてもよく、培地の組合せはこれらに限定されるものではない。 The medium used in each step (A), (B), or (C) of the present invention may be the same type of medium. For example, (A) is a serum-free medium, and (B) is a serum-containing medium. , (C) serum-free medium may be combined with different media in each step, or (A) serum-free medium, (B), (C) may be combined as serum-free medium. The combination of media is not limited to these.
次いで、小領域の形状について図１、２を用いて説明する。図１は、小領域を有する支持体の構成を示す平面図であり、図２は図１のＸ−Ｘ’断面図である。図１に示すように、小領域を有する支持体は、マイクロ容器１１、側壁１２、開口部１３を備える。小領域を有する支持体の１０の培養面には、複数の側壁１２が網目状に形成されており、この側壁１２に四方を囲われた空間がマイクロ容器１１となる。また、各マイクロ容器１１の四辺に形成された側壁１２の各辺の中央部に、開口部１３が形成されている。 Next, the shape of the small area will be described with reference to FIGS. FIG. 1 is a plan view showing a configuration of a support having a small region, and FIG. 2 is a cross-sectional view taken along the line X-X ′ of FIG. 1. As shown in FIG. 1, the support having a small region includes a micro container 11, a side wall 12, and an opening 13. A plurality of side walls 12 are formed in a mesh shape on the culture surface of the support 10 having a small region, and a space surrounded on all sides by the side walls 12 is a micro container 11. Moreover, the opening part 13 is formed in the center part of each edge | side of the side wall 12 formed in the four sides of each micro container 11.
図１において、マイクロ容器１１の底部の幅ａ、マイクロ容器１１を区画するための側壁１２の幅ｂ、高さｃ、隣接するマイクロ容器１１が互いに連通するための開口部１３の幅ｄを示した。本発明における底部面積とは、マイクロ容器開口水平面（側壁１２上面と同一面）に垂直方向に上方から容器底へ平行光を照射した際の投影面積のことをいう。例えば、マイクロ容器の底がＵ字形状である場合、その開口面に垂直方向な上方より底に入射した平行光が投影する形状が底部面積となる。投影底部の長径及び短径とは、円及び楕円の場合、その重心を通る長軸及び短軸と円周との交点の各軸上の距離を長径及び短径といい、多角形の場合、その多角形の面積との差が最小となり各頂点を通る外挿円又は外挿楕円の長径および短径をいい、各頂点を通る外挿円又は外挿楕円を描けない場合は、最も多くの頂点を通る近似円又は楕円の長径および短径をいう In FIG. 1, the width a of the bottom part of the micro container 11, the width b and the height c of the side wall 12 for partitioning the micro container 11, and the width d of the opening 13 for the adjacent micro containers 11 to communicate with each other are shown. It was. The bottom area in the present invention refers to a projected area when parallel light is irradiated from above to the bottom of the container in the direction perpendicular to the horizontal surface of the micro container opening (the same surface as the upper surface of the side wall 12). For example, when the bottom of the micro container is U-shaped, the shape projected by parallel light incident on the bottom from above in the direction perpendicular to the opening surface is the bottom area. The major axis and minor axis of the projected bottom are, in the case of circles and ellipses, the distance on each axis at the intersection of the major axis and minor axis and the circumference passing through the center of gravity, called the major axis and minor axis. The difference between the area of the polygon and the extrapolated circle or ellipse that passes through each vertex is the longest and shortest axis. The major and minor axes of an approximate circle or ellipse passing through the vertex
支持体の小領域の底面積及び高さは、所望する三次元細胞構造体の直径に応じて種々の大きさを選択することが可能であるが、細胞間相互作用観点から細胞少なくとも、１小領域当たり最小２個〜最大１０６個、三次元細胞構造体の大きさで５０μｍ〜１５０μｍの直径の範囲が好ましい。そのため、小領域の直径または一辺は、７０μｍ〜３００μｍの範囲で小領域の高さは、２０μｍ〜５０μｍが好ましい。 The bottom area and height of the small region of the support can be selected in various sizes depending on the desired diameter of the three-dimensional cell structure, but at least one cell is small from the viewpoint of cell-cell interaction. A minimum diameter of 2 to a maximum of 10 6 per region, with a three-dimensional cell structure size range of 50 μm to 150 μm in diameter is preferred. Therefore, the diameter or one side of the small region is preferably in the range of 70 μm to 300 μm, and the height of the small region is preferably 20 μm to 50 μm.
マイクロ容器１１の水平面と側壁１２とがなす角度は、細胞が乗り上げない角度でなければならないため、側面の上部から５０％以上の部分は８０〜９０°が好ましく、特に、８５°〜９０°であることが好ましい。 Since the angle formed between the horizontal surface of the micro container 11 and the side wall 12 must be an angle at which cells do not ride, the portion of 50% or more from the upper part of the side surface is preferably 80 to 90 °, particularly 85 to 90 °. Preferably there is.
図３において、隣接するマイクロ容器１１を互いに連通するための開口部１３の幅ｄは、培養細胞が最初に播種されたマイクロ容器１１から隣接するマイクロ容器１１に移動できない程度であればよい。１個の細胞の直径が１０〜２０μｍであることから、この直径より小さい５〜１５μｍであることが好ましい。なお、開口部１３は必須ではなく、図３及び図４に示すように、マイクロ容器１１の四辺が側壁１２により完全に囲まれていてもよい。ここで、図３は、本実施の形態に係る小領域を有する支持体の構成を示す平面図であり、図４は図１のＹ−Ｙ’断面図である。 In FIG. 3, the width d of the opening 13 for communicating adjacent microcontainers 11 with each other only needs to be such that the cultured cells cannot move from the microcontainer 11 seeded first to the adjacent microcontainer 11. Since the diameter of one cell is 10 to 20 μm, it is preferably 5 to 15 μm smaller than this diameter. The opening 13 is not essential, and the four sides of the micro container 11 may be completely surrounded by the side walls 12 as shown in FIGS. 3 and 4. Here, FIG. 3 is a plan view showing the configuration of the support having the small region according to the present embodiment, and FIG. 4 is a cross-sectional view taken along the line Y-Y ′ of FIG. 1.
小領域を有する支持体の作製方法は、モールドを用いた転写成形、３次元光造形、精密機械切削、ウェットエッチング、ドライエッチング、レーザー加工、放電加工等が挙げられるが、精度、コストの面から、モールドを用いた転写成型が好ましい。 Examples of the method for producing a support having a small region include transfer molding using a mold, three-dimensional stereolithography, precision machine cutting, wet etching, dry etching, laser processing, electric discharge machining, and the like, from the viewpoint of accuracy and cost. Transfer molding using a mold is preferred.
モールドを用いる転写成形方法の具体例としては、金属構造体を型として樹脂成形で小領域を形成する方法が挙げられる。この方法は金属構造体の形状を高い転写率で樹脂に小領域を再現することが可能であり、また汎用の樹脂材料を使用することにより材料コストを低くできるので好ましい。このような金属構造体の型を用いる方法は、低コストであり、高い寸法精度を満足できる点で優れている。 A specific example of the transfer molding method using a mold is a method of forming a small region by resin molding using a metal structure as a mold. This method is preferable because the shape of the metal structure can be reproduced in a small area on the resin with a high transfer rate, and the material cost can be reduced by using a general-purpose resin material. The method using such a metal structure mold is excellent in that it is low in cost and can satisfy high dimensional accuracy.
上記金属構造体の製造方法としては、例えば、フォトリソグラフィによって作製されたレジストパターンや３次元光造形によって作製された樹脂パターンへのメッキ処理、精密機械切削、ウェットエッチング、ドライエッチング、レーザー加工、放電加工等が挙げられる。用途、要求される加工精度、コスト等から、フォトリソグラフィによって作製されたレジストパターンへのメッキ処理が好ましい。 Examples of the method for manufacturing the metal structure include plating treatment on a resist pattern produced by photolithography and a resin pattern produced by three-dimensional stereolithography, precision mechanical cutting, wet etching, dry etching, laser processing, electric discharge. Processing etc. are mentioned. From the application, required processing accuracy, cost, etc., the plating process to the resist pattern produced by photolithography is preferable.
上記で得られた金属構造体を型として用いて樹脂へ小領域を成形する方法として例えば、射出成形、プレス成形、モノマーキャスト成形、溶剤キャスト成形、ホットエンボス成形、押出成形によるロール転写等の方法を挙げることができる。生産性及び型転写性の観点から射出成形を採用することが好ましい。 Examples of methods for molding a small region into a resin using the metal structure obtained above as a mold include, for example, injection molding, press molding, monomer cast molding, solvent cast molding, hot emboss molding, roll transfer by extrusion molding, etc. Can be mentioned. It is preferable to employ injection molding from the viewpoint of productivity and mold transferability.
小領域を有する支持体を構成する材料としては、コスト面から合成樹脂のほうが好ましく、培養時の細胞形態観察の面から、透過性の高い合成樹脂が好ましい。このような合成樹脂としては、ポリメタクリル酸メチル、メタクリル酸メチル−スチレン共重合体等のアクリル系樹脂、ポリスチレン等のスチレン系樹脂、シクロオレフィン等のオレフィン系樹脂、ポリエチレンテレフタレート、ポリ乳酸等のエステル系樹脂、ポリジメチルシロキサン等のシリコン系樹脂、ポリカーボネート樹脂等が挙げられるが、コスト、透明性の面から、アクリル系樹脂またはスチレン系樹脂がより好ましい。 As a material constituting the support having a small region, a synthetic resin is preferable from the viewpoint of cost, and a synthetic resin having high permeability is preferable from the viewpoint of observing cell morphology during culture. Examples of such synthetic resins include acrylic resins such as polymethyl methacrylate and methyl methacrylate-styrene copolymers, styrene resins such as polystyrene, olefin resins such as cycloolefin, and esters such as polyethylene terephthalate and polylactic acid. Examples of the resin include silicone resins such as polydimethylsiloxane, polycarbonate resins, and the like. From the viewpoints of cost and transparency, acrylic resins or styrene resins are more preferable.
小領域を有する支持体の表面は、細胞親和性の観点から、改質やコーティング処理がなされている方が好ましい。これらに用いられる物質としては、ガラスなどの無機物質、ポリ−Ｌ−リシンやポリエチレングリコール（ＰＥＧ）などの合成物質、コラーゲンやフィブロネクチンといった細胞外マトリクス等が挙げられるが、コスト、操作性の観点から、ポリ−Ｌ−リシンやＰＥＧなどの合成物質がより好ましい。より好ましくは、合成樹脂の場合、細胞親和性物質を均一にコートするために、ガラスなどにより表面を親水化した方がよく、コート物質としては、コストの観点から、ポリ−Ｌ−リシンやＰＥＧなどの親水性の合成物質が良い。 The surface of the support having a small region is preferably modified or coated from the viewpoint of cell affinity. Substances used for these include inorganic substances such as glass, synthetic substances such as poly-L-lysine and polyethylene glycol (PEG), and extracellular matrices such as collagen and fibronectin. From the viewpoint of cost and operability. Synthetic substances such as poly-L-lysine and PEG are more preferred. More preferably, in the case of a synthetic resin, it is better to hydrophilize the surface with glass or the like in order to uniformly coat the cytophilic substance. As the coating substance, poly-L-lysine or PEG is preferable from the viewpoint of cost. A hydrophilic synthetic substance such as is preferable.
本発明者らは、鋭意研究した結果、(Ａ)生存率が７０％以上の細胞集団を密度勾配遠心法により濃縮、分離する工程から得られた細胞集団を（Ｂ）有効底面積が０．０１〜０．１ｍｍ２の小領域を有する支持体に播種する工程と、（Ｃ）培養する工程を含む、三次元細胞構造体の作製方法を提供するに至った。以下に本発明の実施例を示す。 As a result of diligent research, the present inventors have found that (A) a cell population obtained by concentrating and separating a cell population having a survival rate of 70% or more by density gradient centrifugation (B) has an effective bottom area of 0. and a step of seeding the support having a small area 01~0.1mm 2, the step of culturing (C), led to provide a method for manufacturing a three-dimensional cell structure. Examples of the present invention are shown below.
＜小領域を有する支持体の作製＞
図３に示す凹凸パターン形状であって、ａ＝２００μｍ、ｂ＝２０μｍ、ｃ＝５０μｍ
のパターンをフォトリソグラフィにより作製し、Ｎｉ電解メッキを行い、対応する凹凸形状を有する金型を得た。その金型を用い、ホットエンボス成形によりポリスチレン上に凹凸パターン形状の転写を行い、前記寸法の樹脂基材を作製した。その樹脂基材表面へ真空蒸着により二酸化ケイ素膜を１００ｎｍ形成させ、γ線滅菌を行い、小領域を有する支持体を得た。この支持体を２４ウェルプレート（ＦＡＬＣＯＮ社製）の各ウェルに、培養表面を上にしてセットした。０．０１％のポリ−Ｌ−リシン（ＳＩＧＭＡ社製）を３００μＬづつ添加した後、４℃で２４時間インキュベートする。２４時間後、リン酸緩衝液（ＧＩＢＣＯ社製）で３回洗浄した。
<Preparation of a support having a small region>
FIG. 3 shows a concavo-convex pattern shape in which a = 200 μm, b = 20 μm, and c = 50 μm.
The pattern was prepared by photolithography and Ni electrolytic plating was performed to obtain a mold having a corresponding uneven shape. The mold was used to transfer the concavo-convex pattern shape onto polystyrene by hot embossing to produce a resin substrate having the above dimensions. A silicon dioxide film having a thickness of 100 nm was formed on the surface of the resin substrate by vacuum deposition, and γ-ray sterilization was performed to obtain a support having a small region. This support was set in each well of a 24-well plate (manufactured by FALCON) with the culture surface facing up. After adding 300 μL of 0.01% poly-L-lysine (manufactured by SIGMA), incubation is performed at 4 ° C. for 24 hours. After 24 hours, the plate was washed 3 times with a phosphate buffer (GIBCO).
＜細胞数及び生存率の測定＞
トリパンブルー（ＧＩＢＣＯ社製）を用いて死細胞を染色し、総細胞数（死細胞数と生細胞数）および死細胞の数を計数して、総細胞数に占める生細胞数の割合を生存率とした。生細胞数の数を細胞数とした。
<Measurement of cell number and viability>
Stain dead cells using trypan blue (GIBCO), count the total number of cells (number of dead cells and number of living cells) and the number of dead cells, and survive the ratio of the number of living cells to the total number of cells. Rate. The number of living cells was taken as the number of cells.
＜細胞の採取＞
６週例、オスのウィスターラットから、２段階コラゲナーゼ灌流法により肝細胞を分離した。具体的には次のようにして行なった。ウィスターラットは麻酔下で開腹し、門脈よりカニューレを入れ、３７度に加温した前還流液（０．０１Ｍ ＨＥＰＥＳ（ＤＯＪＩＮＤＯ社製）、０．５ｍＭ ＥＧＴＡ（ＤＯＪＩＮＤＯ社製）、ペニシリン−ストレプトマイシン（ＧＩＢＣＯ社製）、１ｇ／Ｌ グルコース（和光純薬社製）を含むカリウム及びカルシウム不含リン酸緩衝液）を、２５ｍL／分の流速で流しはじめると同時に、肝動脈を切断した。前還流液１５０ｍＬを流す。次に、３７度に加温したコラゲナーゼ溶液（０．５ｇ／Ｌ コラゲナーゼ（和光純薬社製）、０．０５ｇ／Ｌ トリプシンインヒビター（ＧＩＢＣＯ社製）、０．０１Ｍ ＨＥＰＥＳ（ＤＯＪＩＮＤＯ社製）、ペニシリン−ストレプトマイシン（ＧＩＢＣＯ社製）を含むカルシウム含有リン酸緩衝液）を３０ｍＬ／分で５０ｍＬ流した後、３０ｍＬ／分で１００ｍＬ流す。ラットから肝臓を切除し、１０ｍＬのＤＭＥＭ（ＧＩＢＣＯ社製）を入れた直径１０ｃｍのシャーレに入れた後、メスで肝臓を細かく切断した後、ガーゼで濾過した後、濾液と９０ｍＬのＤＭＥＭを４５μｍの金属製メッシュで濾過した。濾液を５０ｍＬの遠心チューブ（ＦＡＬＣＯＮ社製）２本に分注し、３０×ｇで９０秒間遠心処理をした。上澄みを廃棄し、再度ＤＭＥＭ溶液をそれぞれ４０ｍＬ加え、攪拌した後、３０×ｇで９０秒間遠心処理をした。上澄みを廃棄し、ＤＭＥＭ／Ｆ１２溶液をいれて、０．１×１０７個／ｍＬの濃度の細胞懸濁液を作製した。このときの生存率は９０±５％であった。
<Collection of cells>
Six weeks, hepatocytes were isolated from male Wistar rats by a two-stage collagenase perfusion method. Specifically, it was performed as follows. Wistar rats were laparotomized under anesthesia, cannulated from the portal vein, pre-refluxed solution (0.01 M HEPES (manufactured by DOJINDO), 0.5 mM EGTA (manufactured by DOJINDO), penicillin-streptomycin ( Gibco) and potassium and calcium-free phosphate buffer containing 1 g / L glucose (manufactured by Wako Pure Chemical Industries) were started to flow at a flow rate of 25 mL / min, and at the same time, the hepatic artery was cut. Flow 150 mL of pre-reflux liquid. Next, a collagenase solution (0.5 g / L collagenase (manufactured by Wako Pure Chemical Industries), 0.05 g / L trypsin inhibitor (manufactured by GIBCO), 0.01 M HEPES (manufactured by DOJINDO), penicillin, heated to 37 ° C. -After flowing 50 mL of streptomycin (a calcium-containing phosphate buffer solution containing GIBCO) at 30 mL / min, flow 100 mL at 30 mL / min. The liver was excised from the rat, placed in a petri dish with a diameter of 10 cm containing 10 mL of DMEM (GIBCO), and then the liver was finely cut with a scalpel and filtered with gauze, and then the filtrate and 90 mL of DMEM were added to 45 μm. Filter through a metal mesh. The filtrate was dispensed into two 50 mL centrifuge tubes (manufactured by FALCON) and centrifuged at 30 × g for 90 seconds. The supernatant was discarded, and 40 mL of each DMEM solution was added again and stirred, followed by centrifugation at 30 × g for 90 seconds. The supernatant was discarded and a DMEM / F12 solution was added to prepare a cell suspension having a concentration of 0.1 × 10 7 cells / mL. The survival rate at this time was 90 ± 5%.
＜細胞培養＞
培地は、播種培地（１０％ＦＢＳ、２ｍＭ Ｌ−グルタミン、１０ｍＭ ニコチンアミド、５０μＭ ２−メルカプトエタノール（ＳＩＧＭＡ社製）、０．１μＭ デキサメタゾン（ＳＩＧＭＡ社製）、２６ｍＭ アスコルビン酸（ＳＩＧＭＡ社製）、５ｍＭ ＨＥＰＥＳ（ＤＯＪＩＮＤＯ社製）、０．１μＭ インスリン（和光純薬社製）を含むＤＭＥＭ／Ｆ１２（ＳＩＧＭＡ社製）溶液）と、維持培地（２０ｎｇ／ｍＬ ＥＧＦ（ＳＩＧＭＡ社製）を含む播種培地）を使用した。細胞を含む播種培地を、それぞれのウェルに３００μＬづつ入れ、３７度、二酸化炭素濃度が５％に制御されたインキュベータ内に入れ４時間インキュベートした後、非接着細胞を播種培地と共に吸い取り、新たに、維持培地をそれぞれ３００μＬ入れ、３７度、二酸化炭素濃度が５％に制御されたインキュベータ内に入れ５日間培養した。培地は２日に１回行い、５日間培養した。
<Cell culture>
The medium is a seeding medium (10% FBS, 2 mM L-glutamine, 10 mM nicotinamide, 50 μM 2-mercaptoethanol (manufactured by SIGMA), 0.1 μM dexamethasone (manufactured by SIGMA), 26 mM ascorbic acid (manufactured by SIGMA), 5 mM. HEPES (manufactured by DOJINDO), DMEM / F12 (manufactured by SIGMA) containing 0.1 μM insulin (manufactured by Wako Pure Chemical Industries), and maintenance medium (seeding medium containing 20 ng / mL EGF (manufactured by SIGMA)). used. Place 300 μL of the seeding medium containing the cells in each well, and incubate for 4 hours in an incubator at 37 ° C. with a carbon dioxide concentration controlled to 5%. Then, suck the non-adherent cells with the seeding medium, Each 300 μL of the maintenance medium was placed in an incubator controlled at 37 ° C. and 5% carbon dioxide concentration, and cultured for 5 days. The medium was performed once every two days and cultured for 5 days.
＜観察＞
透過型倒立顕微鏡（オリンパス社製）で、支持体の培養表面の全体を観察し、（ａ）５０〜１００μｍの直径をもつ三次元細胞構造体と（ｂ）５０μｍ未満、１５０μｍ以上の直径をもつ三次元細胞構造体の数を測定した。１ｍｍ２の三次元細胞構造体の個数を三次元細胞構造体密度とした。
<Observation>
The entire culture surface of the support was observed with a transmission inverted microscope (Olympus), and (a) a three-dimensional cell structure having a diameter of 50 to 100 μm and (b) a diameter of less than 50 μm and a diameter of 150 μm or more. The number of three-dimensional cell structures was measured. The number of 1-mm 2 three-dimensional cell structures was defined as the three-dimensional cell structure density.
［実施例］ 密度勾配遠心法による処理群
ＤＭＥＭ／Ｆ１２溶液をいれて、細胞懸濁液を２５ｍＬ、９０％Ｐｅｒｃｏｌｌを含むＨＢＳＳ溶液（９０％ Ｐｅｒｃｏｌｌ（ＧＥヘルスケアバイオサイエンス株式会社製）、５．３３ｍＭ ＫＣｌ（和光純薬社製）、０．４４１ｍＭ ＫＨ２ＰＯ４（和光純薬社製）、４．１７ｍＭ ＮａＨＣＯ３（和光純薬社製）、１３７．９ｍＭ ＮaＣｌ（和光純薬社製）、０．３３８ｍＭ ＮａＨＰＯ４（和光純薬社製）、５．５６ｍＭ Ｄ−Ｇｌｕｃｏｓｅ（和光純薬社製）、０．０２６６ｍＭ フェノールレッド（和光純薬社製））２４ｍＬを混合し密度勾配遠心法用の溶液を作製した。得られた密度勾配遠心法用の溶液に対し転倒混和を５回行なった後、５０×ｇで１０分間遠心処理をし、上澄みを廃棄した（密度勾配遠心操作）。次に、ＤＭＥＭ／Ｆ１２溶液を３０ｍＬ加え、攪拌した後、５０×ｇで１分間遠心処理をし、上澄みを廃棄した（洗浄操作）。この洗浄操作を合計２回行い、播種培地を用いて細胞数が、４×１０５個になるようにした後、培養を行なった。
[Example] Treatment Group by Density Gradient Centrifugation Method A DMEM / F12 solution is added, and the cell suspension is 25 mL, and an HBSS solution containing 90% Percoll (90% Percoll (manufactured by GE Healthcare Biosciences)). 33 mM KCl (manufactured by Wako Pure Chemical Industries, Ltd.), 0.441 mM KH 2 PO 4 (manufactured by Wako Pure Chemical Industries, Ltd.), 4.17 mM NaHCO 3 (manufactured by Wako Pure Chemical Industries, Ltd.), 137.9 mM NaCl (manufactured by Wako Pure Chemical Industries, Ltd.), 0.338 mM NaHPO 4 (manufactured by Wako Pure Chemical Industries, Ltd.), 5.56 mM D-Glucose (manufactured by Wako Pure Chemical Industries, Ltd.), 0.0266 mM phenol red (manufactured by Wako Pure Chemical Industries, Ltd.)) 24 mL were mixed for density gradient centrifugation. A solution was made. The resulting solution for density gradient centrifugation was mixed by inversion 5 times, and then centrifuged at 50 × g for 10 minutes, and the supernatant was discarded (density gradient centrifugation operation). Next, 30 mL of the DMEM / F12 solution was added and stirred, and then centrifuged at 50 × g for 1 minute, and the supernatant was discarded (washing operation). This washing operation was performed twice in total, and the number of cells was adjusted to 4 × 10 5 using a seeding medium, followed by culturing.
［比較例］ 密度勾配遠心未処理群
細胞懸濁液１０ｍＬを５０ｍＬの遠心チューブに入れた後、５０×ｇで１分間遠心処理をした後、上澄みを廃棄し（洗浄操作）、播種培地で、４×１０５個／ｍＬになるように調製した後培養した。このときの生存率は９０±５％であった。
[Comparative Example] Density gradient centrifugation untreated group After 10 mL of cell suspension was put into a 50 mL centrifuge tube, centrifuged at 50 × g for 1 minute, and then the supernatant was discarded (washing operation). The cells were cultured after being adjusted to 4 × 10 5 cells / mL. The survival rate at this time was 90 ± 5%.
表１に実施例と比較例の５日後三次元細胞構造体の密度を示した。実施例は比較例の約２倍の密度を示した。また、実施例では、三次元細胞構造体のほとんどが、直径５０〜１００μｍの三次元細胞構造体であった。一方、比較例では、直径５０μｍ未満、１００μｍ以上の三次元細胞構造体が多く存在しておりその密度は、実施例の約２倍であった。 Table 1 shows the densities of the three-dimensional cell structures after 5 days of the examples and comparative examples. The example showed about twice the density of the comparative example. In the examples, most of the three-dimensional cell structures were three-dimensional cell structures having a diameter of 50 to 100 μm. On the other hand, in the comparative example, there are many three-dimensional cell structures having a diameter of less than 50 μm and 100 μm or more, and the density was about twice that of the example.
図５に実施例と比較例の三次元細胞構造体密度の経時変化を示した。実施例では、培養２日後から三次元細胞構造体の形成が確認されたが、比較例では、ほとんど観察されなかった。一方、比較例では、２日後ではほとんど三次元細胞構造体が形成されていなかった。３日後〜５日後にかけて実施例は比較例の２倍以上の三次元細胞構造体密度となった。 FIG. 5 shows changes with time in the density of the three-dimensional cell structures of the example and the comparative example. In the examples, formation of a three-dimensional cell structure was confirmed after 2 days of culture, but was hardly observed in the comparative examples. On the other hand, in the comparative example, almost no three-dimensional cell structure was formed after 2 days. From 3 days to 5 days later, the example had a three-dimensional cell structure density more than twice that of the comparative example.
図６に実施例と比較例の３日後及び５日後の形態写真を示した。三次元細胞構造体を矢印で示した。実施例では、培養３日後にきれいな球状の三次元細胞構造体が確認され、５日目には、ほとんどの小領域できれいな球状の三次元細胞構造体が確認できた。比較例では、３日後及びに三次元細胞構造体が確認されるものの、三次元細胞構造体形状のものは、実施例より格段に少なかった。 FIG. 6 shows morphological photographs of the example and the comparative example after 3 days and after 5 days. Three-dimensional cell structures are indicated by arrows. In Examples, clean spherical three-dimensional cell structures were confirmed after 3 days of culture, and clean spherical three-dimensional cell structures were confirmed in almost all small areas on the fifth day. In the comparative example, the three-dimensional cell structure was confirmed after 3 days, but the number of the three-dimensional cell structure was much less than that of the example.
三次元細胞構造体の密度が、２倍以上であるため、比較例で約２ｃｍ２の底面積を有する２４ウェルで試験を実施した場合、実施例では、その約半分の面積である１ｃｍ２の底面積を有する４８ウェルでの試験が可能となる。すなわち、試験可能なサンプル数が２倍となり、本発明は、薬物試験の効率を大幅に向上させることができる。 Since the density of the three-dimensional cell structure is more than twice, when the test is performed in 24 wells having a bottom area of about 2 cm 2 in the comparative example, in the example, about 1 cm 2 , which is about half of the area, Tests in 48 wells with a bottom area are possible. That is, the number of testable samples is doubled, and the present invention can greatly improve the efficiency of drug tests.
１０ 肝細胞培養容器
１１ マイクロ容器
１２ 側壁
１３ 開口部
10 Hepatocyte culture vessel 11 Micro vessel 12 Side wall 13 Opening
Claims (8)
（Ｂ）；工程（Ａ）で得られた細胞集団を有効底面積が０．０１〜０．１ｍｍ２の小領域を有する支持体に播種する工程と、
（Ｃ）；工程（Ｂ）で播種された細胞を培養する工程と
を含む、三次元細胞構造体の作製方法。 (A); a step of concentrating and separating a cell population having a survival rate of 70% or more by density gradient centrifugation;
(B); seeding the cell population obtained in step (A) on a support having a small region having an effective bottom area of 0.01 to 0.1 mm 2 ;
(C); a method for producing a three-dimensional cell structure, comprising a step of culturing the cells seeded in the step (B).
（ｉ）；細胞を接着させるために培養する工程と、
（ii）；非接着性細胞を除去する工程と、
（iii）；三次元細胞構造体を形成させるために培養する工程と
からなる請求項１に記載の三次元細胞構造体の作製方法。 The culture step of (C)
(I); culturing to attach the cells;
(Ii); removing non-adherent cells;
(Iii): The method for producing a three-dimensional cell structure according to claim 1, comprising a step of culturing to form the three-dimensional cell structure.
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