JP2010088347A - Method and container for spheroid culture - Google Patents
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- 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
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- C12M23/12—Well or multiwell plates
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Abstract
Description
本発明は、付着性細胞を用いてスフェロイドを培養する方法、及びスフェロイド培養に好適な培養容器に関する。 The present invention relates to a method for culturing spheroids using adherent cells, and a culture vessel suitable for spheroid culture.
細胞培養技術において、生体内と同等な三次元組織を構築できるスフェロイド培養が着目されている。スフェロイドは、細胞塊又は細胞凝集体とも称され、主に肝細胞や癌細胞などの付着性細胞から形成される。スフェロイド培養では、細胞が三次元に集合して塊となるので、細胞が二次元に集合する単層培養法と比べて、細胞の特異的な機能を長期間維持することができる。このような特性から、スフェロイド培養は、薬剤の毒性及び薬理活性評価のシミュレータや、ハイブリッド型人工臓器、バイオリアクタなどの分野において利用されることが期待されている。 In the cell culture technique, attention is focused on spheroid culture capable of constructing a three-dimensional tissue equivalent to that in a living body. Spheroids are also called cell clusters or cell aggregates, and are mainly formed from adherent cells such as hepatocytes and cancer cells. In spheroid culture, cells aggregate in three dimensions to form a lump, so that the specific functions of the cells can be maintained for a long period of time compared to a monolayer culture method in which cells aggregate in two dimensions. Because of these characteristics, spheroid culture is expected to be used in the fields of drug toxicity and pharmacological activity evaluation simulators, hybrid artificial organs, bioreactors, and the like.
スフェロイド培養においては、集合する細胞数を制御することが望まれる。スフェロイドは、生体の毛細血管に相当する管腔構造をもたないので、中心部の細胞と外表面の細胞とでは酸素及び栄養分の補給や老廃物の除去において差が生じる。例えば、スフェロイドが大きくなると、中心部の細胞が酸素不足や栄養不足となって細胞死が起こり得る。このような中心部の細胞の状態と外表面の細胞の状態との差が、例えば薬剤の毒性及び薬理活性を評価する上での差ともなり得る。仮に、スフェロイドにおける中心部の細胞の状態と外表面の細胞の状態とを同等にすることができれば、薬剤の毒性や薬理活性の評価において、スフェロイドの生死のみならず、スフェロイド内の細胞が有する特異的な機能の向上又は低下、スフェロイドにより形成された生体類似の三次元構造に対する組織学的な検査などが実現され得る。このようなスフェロイドによる薬剤の毒性や薬理活性の評価は、動物実験の一部を代替し得る。 In spheroid culture, it is desired to control the number of cells that gather. Since spheroids do not have a luminal structure corresponding to the capillaries of living bodies, differences occur in the supply of oxygen and nutrients and the removal of waste products between cells in the central part and cells on the outer surface. For example, when the spheroids become large, cells in the central part can become cell-dead due to oxygen deficiency or nutrient deficiency. Such a difference between the state of the cell in the central portion and the state of the cell on the outer surface can be a difference in, for example, evaluating the toxicity and pharmacological activity of the drug. If the state of the cell in the center of the spheroid and the state of the cell on the outer surface can be made equivalent, in the evaluation of the toxicity and pharmacological activity of the drug, not only the life and death of the spheroid but also the uniqueness of the cell in the spheroid Functional improvement or reduction, histological examination of a biologically similar three-dimensional structure formed by spheroids, and the like can be realized. Evaluation of drug toxicity and pharmacological activity by such spheroids can replace part of animal experiments.
従来より、スフェロイド培養として、ローラボトル培養、スピナーフラスコ培養、ハンギングドロップ培養などが知られている。また、スフェロイド培養のための96穴プレートが市販されている。 Conventionally, roller bottle culture, spinner flask culture, hanging drop culture, and the like are known as spheroid culture. A 96-well plate for spheroid culture is commercially available.
特許文献1には、基板表面上にアレイ状やハニカム状等に規則配列されて種細胞を凝集させて保持する細胞培養セルを備えた細胞培養チップが開示されている。この細胞培養セルに種細胞が分散された培養液を充填して細胞培養セル内に種細胞を着床させ、さらに、細胞培養チップを連続的又は間欠的に水平回転又は往復揺動させながら、細胞培養セル内の種細胞同士を凝集培養させてスフェロイドを形成させ得る。 Patent Document 1 discloses a cell culture chip provided with cell culture cells that are regularly arranged in an array, honeycomb, or the like on a substrate surface to aggregate and hold seed cells. The cell culture cell is filled with a culture solution in which seed cells are dispersed, and the seed cells are implanted in the cell culture cell. Further, while the cell culture chip is continuously or intermittently rotated horizontally or reciprocally swung, Spheroids can be formed by agglutination culture of seed cells in a cell culture cell.
特許文献2には、細胞接着性を示す第一領域と、その第一領域を囲み、第一領域に比べて低い細胞接着性を示す第二領域とを含む組織体形成領域を備えた細胞組織体マイクロデバイスが開示されている。細胞組織体マイクロデバイスの第一領域及び第二領域に播種された細胞は、互いに細胞間結合を形成し、この細胞間結合が維持されつつ第二領域から細胞が剥がれることにより、第一領域に細胞同士が三次元的に結合した細胞組織体が形成され得る。 Patent Document 2 discloses a cell tissue provided with a tissue body forming region including a first region exhibiting cell adhesion and a second region surrounding the first region and exhibiting lower cell adhesion than the first region. A body microdevice is disclosed. The cells seeded in the first region and the second region of the cell tissue microdevice form an intercellular bond with each other, and the cells are detached from the second region while maintaining the intercellular bond. A cell tissue in which cells are three-dimensionally connected can be formed.
例えば、前述された96穴プレートを用いたスフェロイド培養では、付着性細胞の懸濁液を96穴にそれぞれ注入する必要があり、操作が煩雑であり、また、各穴におけるスフェロイドの大きさを制御することが難しいという問題がある。 For example, in the spheroid culture using the above-described 96-well plate, it is necessary to inject the suspension of adherent cells into the 96-wells, and the operation is complicated, and the size of the spheroid in each hole is controlled. There is a problem that it is difficult to do.
特許文献1に記載の細胞培養チップを用いれば、各細胞培養セル内に着床させた種細胞を均一な大きさのスフェロイドに培養することができる。しかし、特許文献1によれば、細胞培養セル内において種細胞がスフェロイドに形成されるためには、細胞培養チップが連続的又は間欠的に水平回転又は往復揺動される必要がある。細胞培養チップが水平回転等されると、細胞培養セル内の種細胞が遠心力などによってセル外へ飛び出たり、さらに他のセルへ進入したりするおそれがあり、その結果、均一な大きさのスフェロイドが形成されないことがあり得る。このような問題が防止されるためには、種細胞と細胞培養セルとの親和性が考慮された上で、水平回転等の回転速度や継続時間が適切に設定されなければならないが、これら培養条件を設定するための実験が必要となり、また、商業的生産における品質管理などが煩雑になるという問題がある。 If the cell culture chip described in Patent Document 1 is used, seed cells implanted in each cell culture cell can be cultured into spheroids of uniform size. However, according to Patent Document 1, in order for the seed cells to be formed into spheroids in the cell culture cell, the cell culture chip needs to be horizontally rotated or reciprocated continuously or intermittently. If the cell culture chip is rotated horizontally, the seed cells in the cell culture cell may jump out of the cell due to centrifugal force, etc., or may enter another cell. Spheroids may not be formed. In order to prevent such problems, the rotational speed and duration such as horizontal rotation must be set appropriately after considering the affinity between the seed cell and the cell culture cell. There is a problem that an experiment for setting conditions is required, and quality control in commercial production becomes complicated.
特許文献2に記載の細胞組織体マイクロデバイスでは、組織体形成領域として、細胞接着性を示す第一領域と、その第一領域を囲み、第一領域に比べて低い細胞接着性を示す第二領域とが形成される必要がある。これら領域は基板の表面加工等によって形成され得るが、第二領域に付着した細胞が第一領域に付着した細胞と細胞間結合を維持しながら第二領域から剥がれるような第二領域の細胞接着性を設定することは困難である。また、そのような第二領域の細胞接着性が複数の組織体形成領域において一定に維持されることも困難である。したがって、細胞組織体マイクロデバイスは商業的な大量生産が難しく、また、組織体形成領域においてスフェロイドが形成されたとしても、スフェロイドの大きさを均一に制御することが難しい。 In the cell tissue microdevice described in Patent Document 2, as a tissue body forming region, a first region showing cell adhesiveness and a second region surrounding the first region and having lower cell adhesiveness than the first region are shown. A region needs to be formed. These regions can be formed by surface processing of the substrate, etc., but the cell adhesion of the second region such that cells attached to the second region are detached from the second region while maintaining cell-cell bonding with the cells attached to the first region. It is difficult to set gender. In addition, it is difficult to maintain the cell adhesiveness of such a second region constant in a plurality of tissue body formation regions. Therefore, commercial mass production of cell tissue microdevices is difficult, and even if spheroids are formed in the tissue formation region, it is difficult to uniformly control the size of the spheroids.
本発明は、これらの事情に鑑みてなされたものであり、その目的は、簡易な作業によって均一な大きさのスフェロイドを大量に培養するに適した手段を提供することにある。 The present invention has been made in view of these circumstances, and an object thereof is to provide means suitable for culturing a large amount of spheroids of a uniform size by a simple operation.
(1) 本発明に係るスフェロイド培養方法は、細胞非接着性の底面から重力方向における最下位置へ連続する斜面を有する細胞非接着性の凹陥部を複数有する培養容器に、付着性細胞の懸濁液を注入する第1ステップと、上記懸濁液が注入された培養容器を静置して、当該培養容器の凹陥部においてスフェロイドを形成させる第2ステップと、を含む。 (1) The spheroid culturing method according to the present invention is a method of suspending adherent cells in a culture container having a plurality of non-cell-adhesive depressions having a slope that continues from the cell-non-adhesive bottom surface to the lowest position in the gravity direction. A first step of injecting a suspension, and a second step of allowing the culture vessel into which the suspension has been injected to stand to form spheroids in the recessed portion of the culture vessel.
培養容器は、例えばシャーレのような皿形状であり、細胞懸濁液や培地などの液体を保持可能なものである。この培養容器の底面は細胞非接着性である。細胞非接着性とは、付着性細胞が付着し得ない表面性能をいう。ここで、付着性細胞が付着し得ないとは、付着性細胞がまったく付着しないことをいう趣旨ではなく、本発明が目的とするスフェロイドが形成されることを阻害しない程度に、付着性細胞が付着し得なければよいとの主旨である。このような細胞非接着性は、例えば、水接触角によって定義されてもよい。水接触角として好ましくは30度以下であり、さらに好ましくは10度以下である。 The culture container has a dish shape such as a petri dish, for example, and can hold a liquid such as a cell suspension or a culture medium. The bottom surface of this culture container is non-cell-adhesive. Cell non-adhesive refers to surface performance to which adherent cells cannot adhere. Here, the fact that the adherent cells cannot adhere does not mean that the adherent cells do not adhere at all, but the adherent cells do not inhibit the formation of the spheroid targeted by the present invention. The main point is that it should not adhere. Such cell non-adhesion may be defined, for example, by the water contact angle. The water contact angle is preferably 30 degrees or less, and more preferably 10 degrees or less.
前述された細胞非接着性は、例えば、細胞培養容器が、ポリエチレングリコールやポリヒドロキシエチルメタクリレート、エチレンビニルアルコール共重合体などの親水性の高い物質の成形品として実現されたり、細胞培養容器の底面が、界面活性剤やリン脂質などの表面性能を親水性にし得る物質でコーティングされたり、プラズマ処理などの表面処理によって親水性が付与されたりすることによって実現され得る。 The cell non-adhesiveness described above can be realized, for example, when the cell culture container is formed as a molded article of a highly hydrophilic substance such as polyethylene glycol, polyhydroxyethyl methacrylate, ethylene vinyl alcohol copolymer, or the bottom surface of the cell culture container. However, it can be realized by coating with a substance capable of making the surface performance hydrophilic, such as a surfactant or phospholipid, or by imparting hydrophilicity by surface treatment such as plasma treatment.
培養容器の底面には複数の凹陥部が設けられている。この凹陥部は、底面から重力方向へ凹む有底孔である。凹陥部の表面も底面と同様に細胞非接着性である。各凹陥部は、最下位置へ連続する斜面を有する。このような凹陥部は、例えば、底面及び凹陥部がプラスチックなどによって一体に成形されたり、底面に凹陥部が切削加工されたり、複数の孔が形成された平板形状の基材に薄膜が積層され、その薄膜が孔内に陥没するように変形されたりすることによって形成され得る。 A plurality of recesses are provided on the bottom surface of the culture vessel. The recessed portion is a bottomed hole that is recessed from the bottom surface in the direction of gravity. The surface of the recessed portion is also non-cell-adhesive like the bottom surface. Each recess has a slope that continues to the lowest position. For example, such a recess is formed by laminating a thin film on a flat base material in which the bottom surface and the recess are integrally formed of plastic or the like, the recess is cut on the bottom surface, or a plurality of holes are formed. The thin film can be formed by being deformed so as to sink into the hole.
第1ステップにおいて、付着性細胞の懸濁液が培養容器に注入される。付着性細胞として、例えば、ヒト骨肉腫細胞などのガン細胞や肝細胞などがあげられる。この付着性細胞が液体の培地に均一に拡散されて懸濁液が調整される。培地は、付着性細胞の培養に適した公知のものが用いられる。付着性細胞の個数は、培養容器の容量や凹陥部の大きさ、目的とするスフェロイドの大きさなどを考慮して適宜設定されるが、例えば、1mL当たり数万個〜数百万個程度の範囲で調整される。この懸濁液は、培養容器に対して1回のみ展開される。つまり、培養容器の底面が全て浸される量の懸濁液が培養容器に注入される。 In the first step, a suspension of adherent cells is injected into the culture vessel. Examples of adherent cells include cancer cells such as human osteosarcoma cells and hepatocytes. The adherent cells are uniformly diffused into the liquid medium to prepare a suspension. As the medium, a known medium suitable for culturing adherent cells is used. The number of adherent cells is appropriately set in consideration of the capacity of the culture vessel, the size of the recess, the size of the target spheroid, etc., for example, about tens of thousands to several million per mL Adjusted in range. This suspension is developed only once for the culture vessel. That is, an amount of the suspension in which the entire bottom surface of the culture vessel is immersed is injected into the culture vessel.
第2ステップにおいて、懸濁液が注入された培養容器が静置される。このとき培養容器はインキュベータなどによって所望の温度範囲や湿度範囲に保持されてもよい。静置される時間は、付着性細胞の種類や目的とするスフェロイドの大きさなどを考慮して適宜設定されるが、例えば、数時間から数日間程度である。この静置によって、懸濁液中の付着性細胞が重力により沈降して、培養容器の底面又は凹陥部へ到達する。 In the second step, the culture container into which the suspension has been injected is allowed to stand. At this time, the culture vessel may be maintained in a desired temperature range or humidity range by an incubator or the like. The standing time is appropriately set in consideration of the type of adherent cells, the target spheroid size, and the like, and is, for example, about several hours to several days. By this standing, adherent cells in the suspension settle by gravity and reach the bottom surface or the recessed portion of the culture vessel.
培養容器の凹陥部へ到達した付着性細胞は、斜面に沿って沈降して凹陥部の最下位置へ到達する。凹陥部の各面は細胞非接着性なので、付着性細胞は凹陥部内において壁面に付着することなく重力によって斜面に沿って沈降する。これにより、凹陥部へ到達した付着性細胞が最下位置へ集合され、その最下位置において付着性細胞同士が結合し、スフェロイドが形成される。静置された培養容器において、凹陥部へ到達する付着性細胞の数は、凹陥部の開口面積に比例するので、均等な凹陥部が複数設けられることによって、各凹陥部において均等な大きさのスフェロイドがそれぞれ形成される。 Adherent cells that have reached the recessed portion of the culture vessel settle along the slope and reach the lowest position of the recessed portion. Since each surface of the recessed portion is non-cell-adhesive, adherent cells settle along the slope by gravity without attaching to the wall surface in the recessed portion. As a result, the adherent cells that have reached the recess are gathered to the lowest position, and the adherent cells are bonded to each other at the lowest position to form a spheroid. In a stationary culture vessel, the number of adherent cells reaching the recess is proportional to the opening area of the recess, so that by providing a plurality of uniform recesses, the size of each of the recesses is equal. Each spheroid is formed.
(2) 上記培養容器に、当該培養容器内へ培地を流入させるための流入口と、当該培養容器内から培地を流出させるための流出口とが設けられ、上記懸濁液が注入されて静置されている培養容器に対して、上記流入口を通じて新たな培地を当該培養容器内へ注入し、かつ上記流出口を通じて当該培養容器内の培地を排出させる第3ステップをさらに含むものであってもよい。 (2) The culture container is provided with an inlet for allowing the culture medium to flow into the culture container and an outlet for allowing the culture medium to flow out of the culture container. And a third step of injecting a new medium into the culture container through the inlet and discharging the medium in the culture container through the outlet to the culture container placed. Also good.
前述されたような皿形状の培養容器に、培地の流入口及び流出口が設けられる。流入口を通じて外部から培養容器内に培地が流入され得る。流出口を通じて培養容器内から外部へ培地が流出され得る。 The culture medium inlet and outlet are provided in the dish-shaped culture container as described above. The medium can be flowed into the culture vessel from the outside through the inlet. The medium can be discharged from the inside of the culture vessel to the outside through the outlet.
第3ステップでは、前述された第2ステップにおいて培養容器が静置されて、懸濁液中の付着性細胞が凹陥部へ到達した後に、流入口を通じて新たな培地が外部から培養容器内へ流入され、かつ流出口を通じて培養容器内の培地が外部へ流出される。これにより、付着性細胞が凹陥部においてスフェロイドを形成している際に、培養容器内の培地交換が行われる。 In the third step, after the culture vessel is allowed to stand in the second step described above and the adherent cells in the suspension reach the recess, a new medium flows from the outside into the culture vessel through the inlet. And the culture medium in the culture vessel flows out to the outside through the outlet. Thereby, when the adherent cells form spheroids in the recessed portion, the medium in the culture vessel is exchanged.
(3) 本発明は、細胞非接着性の底面と、当該底面を囲んで設けられた壁とを具備するスフェロイド培養容器であって、上記底面に細胞非接着性の表面で構成された複数の凹陥部が設けられ、上記各凹陥部が、重力方向における最下位置へ連続する斜面を有するものであるスフェロイド培養容器として捉えられてもよい。 (3) The present invention is a spheroid culture vessel comprising a non-cell-adhesive bottom surface and a wall provided surrounding the bottom surface, and a plurality of non-cell-adhesive surfaces formed on the bottom surface. A recessed part is provided, and each said recessed part may be grasped | ascertained as a spheroid culture container which has a slope which continues to the lowest position in a gravitational direction.
(4) また、上記各凹陥部は、上記底面に規則的に配置されてもよい。これにより、懸濁液中を沈降して凹陥部へ到達する付着性細胞が、各凹陥部に対して均等となる。 (4) Moreover, each said recessed part may be regularly arrange | positioned at the said bottom face. As a result, the adherent cells that settle in the suspension and reach the recesses are even for the recesses.
(5) 上記斜面として、上記最下位置の周囲に配置された半球形状又は円錐形状のものがあげられる。 (5) Examples of the inclined surface include hemispherical or conical shapes arranged around the lowermost position.
本発明によれば、付着性細胞の懸濁液が培養容器に一回展開されて培養容器が静置されると、懸濁液中の付着性細胞が重力により沈降して培養容器の凹陥部へ到達し、凹陥部内においては、斜面に沿って付着性細胞が沈降して最下位置へ集合され、その最下位置において付着性細胞同士が結合し、スフェロイドが形成される。これにより、簡易な作業によって均一な大きさのスフェロイドが大量に培養される。 According to the present invention, when the suspension of adherent cells is developed once in the culture container and the culture container is left still, the adherent cells in the suspension settle by gravity and are recessed in the culture container. In the recessed part, adherent cells settle along the slope and gather to the lowest position, and adherent cells bind to each other at the lowest position to form spheroids. Thereby, spheroids of a uniform size are cultured in large quantities by a simple operation.
以下、本発明の好ましい実施形態を説明する。なお、本実施形態は本発明の一実施態様にすぎず、本発明の要旨を変更しない範囲で実施態様を変更できることは言うまでもない。 Hereinafter, preferred embodiments of the present invention will be described. In addition, this embodiment is only one embodiment of this invention, and it cannot be overemphasized that an embodiment can be changed in the range which does not change the summary of this invention.
[図面の説明]
図1は、本発明の第1実施形態にかかるスフェロイド培養容器10の正面図である。なお、スフェロイド培養容器10の右側面図、左側面図及び背面図は、正面図と同じなのでここでは省略されている。図2は、スフェロイド培養容器10の平面図である。図3は、スフェロイド培養容器10の底面図である。なお、図3において、スフェロイド培養容器10が透明な素材からなるものであれば、図2と同様に底12に凹陥部20が透けて現れる。図4は、図1におけるIV−IV切断線における断面を示す断面図である。図5は、図2における領域Vを示す拡大平面図である。図6は、図4における領域VIを示す拡大断面図である。図7は、スフェロイド培養容器10を用いたスフェロイド培養方法の第1ステップを示す断面図である。図8は、スフェロイド培養容器10を用いたスフェロイド培養方法の第2ステップを示す拡大断面図である。図9は、第2実施形態に係るスフェロイド培養方法の第3ステップを示す断面図である。図10は、第3実施形態に係るスフェロイド培養容器10を示す平面図である。図11は、図10におけるXI−XI切断線における断面を示す断面図である。図12は、図10における領域XIIを示す拡大平面図である。図13は、図11における領域XIIIを示す拡大断面図である。図14は、第4実施形態に係るスフェロイド培養容器10を示す断面図である。図15は、図14における領域XVを示す拡大断面図である。図16は、第5実施形態に係るスフェロイド培養容器10を示す断面図である。図17は、図16における領域XVIIを示す拡大断面図である。図18は、スフェロイド培養容器10の製造方法を示す模式図である。図19及び図20は、第1実施例の観察結果を示すものである。図21は、第2実施例に係るスフェロイド培養容器10を示す断面図である。図22は、第2実施例の観察結果を示すものである。なお、図19,図20及び図22において示されるスケールバーは、いずれも100μmである。
[Explanation of drawings]
FIG. 1 is a front view of a spheroid culture vessel 10 according to the first embodiment of the present invention. The right side view, left side view, and rear view of the spheroid culture vessel 10 are the same as the front view, and are omitted here. FIG. 2 is a plan view of the spheroid culture vessel 10. FIG. 3 is a bottom view of the spheroid culture vessel 10. In FIG. 3, if the spheroid culture vessel 10 is made of a transparent material, the recessed portion 20 appears through the bottom 12 as in FIG. 4 is a cross-sectional view showing a cross section taken along the line IV-IV in FIG. FIG. 5 is an enlarged plan view showing a region V in FIG. FIG. 6 is an enlarged cross-sectional view showing a region VI in FIG. FIG. 7 is a cross-sectional view showing the first step of the spheroid culture method using the spheroid culture vessel 10. FIG. 8 is an enlarged cross-sectional view showing the second step of the spheroid culture method using the spheroid culture vessel 10. FIG. 9 is a cross-sectional view showing a third step of the spheroid culture method according to the second embodiment. FIG. 10 is a plan view showing the spheroid culture vessel 10 according to the third embodiment. 11 is a cross-sectional view showing a cross section taken along the line XI-XI in FIG. FIG. 12 is an enlarged plan view showing a region XII in FIG. FIG. 13 is an enlarged cross-sectional view showing a region XIII in FIG. FIG. 14 is a cross-sectional view showing a spheroid culture vessel 10 according to the fourth embodiment. FIG. 15 is an enlarged cross-sectional view showing a region XV in FIG. FIG. 16 is a cross-sectional view showing a spheroid culture vessel 10 according to the fifth embodiment. FIG. 17 is an enlarged cross-sectional view showing a region XVII in FIG. FIG. 18 is a schematic diagram showing a method for manufacturing the spheroid culture vessel 10. 19 and 20 show the observation results of the first example. FIG. 21 is a cross-sectional view showing the spheroid culture vessel 10 according to the second embodiment. FIG. 22 shows the observation results of the second example. The scale bars shown in FIGS. 19, 20, and 22 are all 100 μm.
[第1実施形態]
以下に、本発明の第1実施形態が説明される。
[First Embodiment]
The first embodiment of the present invention will be described below.
[スフェロイド培養容器10]
図1から図4に示されるように、スフェロイド培養容器10は、シャーレの如き皿形状であり、側壁11が円筒形状となって円盤形状の底12を囲み、かつ底12からほぼ垂直方向の上向きに起立されている。この側壁11及び底12によって囲まれるスフェロイド培養容器10の内部空間に、細胞懸濁液や培地などの液体が保持される。
[Spheroid culture vessel 10]
As shown in FIGS. 1 to 4, the spheroid culture vessel 10 has a dish shape such as a petri dish, the side wall 11 has a cylindrical shape and surrounds a disk-shaped bottom 12, and faces upward in a substantially vertical direction from the bottom 12. Is standing. A liquid such as a cell suspension or a medium is held in the internal space of the spheroid culture vessel 10 surrounded by the side wall 11 and the bottom 12.
側壁11において容器内側となる内面13、及び底12において容器内側となる底面14は、細胞非接着性である。この細胞非接着性は、疎水性のプラスチックからなる内面13及び底面14に対して、両親媒性の高分子がコーティングされることにより付与されている。両新媒性の高分子のうち、疎水性の部分が内面12及び底面14に吸着し、親水性の部分が容器内側へ露出される。これにより内面13及び底面14に親水性のコーティングが施され、付着性細胞が内面13及び底面14に付着しない。 The inner surface 13 that is the inner side of the container in the side wall 11 and the bottom surface 14 that is the inner side of the container in the bottom 12 are non-cell-adhesive. This cell non-adhesiveness is imparted by coating the inner surface 13 and the bottom surface 14 made of hydrophobic plastic with an amphiphilic polymer. Of both the amphiphilic polymers, the hydrophobic portion is adsorbed on the inner surface 12 and the bottom surface 14, and the hydrophilic portion is exposed to the inside of the container. As a result, hydrophilic coating is applied to the inner surface 13 and the bottom surface 14, and adherent cells do not adhere to the inner surface 13 and the bottom surface 14.
[凹陥部20]
図2及び図4に示されるように、底面14には、重力方向(図4において下向き)へ凹む複数の凹陥部20が設けられている。凹陥部20は、後述されるように半球形状をなした有底孔である。図2に示されるように、各凹陥部20は、平面視において、底面14との境界21が円形をなしている。複数の凹陥部20は、底面14の全領域において、規則的且つ密に配置されている。
[Concave part 20]
As shown in FIGS. 2 and 4, the bottom surface 14 is provided with a plurality of recessed portions 20 that are recessed in the direction of gravity (downward in FIG. 4). The recessed portion 20 is a bottomed hole having a hemispherical shape as will be described later. As shown in FIG. 2, each recess 20 has a circular boundary 21 with the bottom surface 14 in plan view. The plurality of recessed portions 20 are regularly and densely arranged in the entire area of the bottom surface 14.
図5に示されるように、平面視において、各凹陥部20の中心Oと、隣り合う凹陥部20の中心Oとの距離Lは、各々の凹陥部20において一定である。また、距離Lは、平面視において、各凹陥部20の中心Oから底面14との境界21までの距離R(平面視において円形の境界21の半径に相当する。)の3倍以下である。つまり、各凹陥部20は、隣り合う凹陥部20と距離Rより接近して配置されている。 As shown in FIG. 5, the distance L between the center O of each recessed portion 20 and the center O of the adjacent recessed portion 20 is constant in each recessed portion 20 in plan view. Further, the distance L is not more than three times the distance R (corresponding to the radius of the circular boundary 21 in plan view) from the center O of each recessed portion 20 to the boundary 21 with the bottom surface 14 in plan view. That is, each recessed part 20 is disposed closer to the adjacent recessed part 20 than the distance R.
各凹陥部20は、底面14から半球形状に陥没しており、図6に示されるように、縦断面視において、各凹陥部20は半円形状の輪郭をなす。この半円形状の輪郭における最下位置22が各凹陥部20において最も深い位置である。各凹陥部20における最下位置22までの深さは、前述された距離Rと同等である。 Each recessed part 20 is depressed in a hemispherical shape from the bottom surface 14, and as shown in FIG. 6, each recessed part 20 has a semicircular outline in a longitudinal sectional view. The lowest position 22 in the semicircular contour is the deepest position in each recess 20. The depth to the lowest position 22 in each recess 20 is equal to the distance R described above.
各凹陥部20において、境界21から最下位置22までは、球面状に傾斜する斜面23である。この斜面23は、境界21から最下位置22へ連続する滑らかな面であり、最下位置22を中心として境界21まで点対称な形状をなしている。最下位置22及び斜面23も、前述された内面13及び底面14と同様のコーティングがなされた細胞非接着性である。 In each recessed portion 20, a slope 23 inclined in a spherical shape is formed from the boundary 21 to the lowest position 22. The slope 23 is a smooth surface that continues from the boundary 21 to the lowest position 22, and has a point-symmetric shape from the lowest position 22 to the boundary 21. The lowermost position 22 and the slope 23 are also non-cell-adhesive with the same coating as the inner surface 13 and the bottom surface 14 described above.
[スフェロイド培養方法]
以下に、前述されたスフェロイド培養容器10を用いたスフェロイド培養方法が説明される。
[Spheroid culture method]
Below, the spheroid culture | cultivation method using the spheroid culture container 10 mentioned above is demonstrated.
本実施形態におけるスフェロイド培養方法は、主として以下の2つのステップに大別される。
(1)スフェロイド培養容器10に、付着性細胞の懸濁液を注入する第1ステップ。
(2)懸濁液が注入されたスフェロイド培養容器10を静置して、各凹陥部20においてスフェロイドを形成させる第2ステップ。
The spheroid culture method in this embodiment is roughly divided into the following two steps.
(1) A first step of injecting a suspension of adherent cells into the spheroid culture vessel 10.
(2) A second step in which the spheroid culture vessel 10 into which the suspension has been injected is allowed to stand and spheroids are formed in each recess 20.
図7に示されるように、第1ステップにおいて、付着性細胞の懸濁液30がスフェロイド培養容器10に注入される。付着性細胞としては、例えば、ヒト骨肉腫細胞などのガン細胞や肝細胞などが目的に応じて用いられる。この付着性細胞が液体の培地に均一に拡散されて懸濁液30が調整される。付着性細胞の個数は、例えば、培地1mL当たり数万個〜数百万個程度の範囲で調整される。懸濁液30は、スフェロイド培養容器10に対して1回のみ展開される。 As shown in FIG. 7, in the first step, a suspension 30 of adherent cells is injected into the spheroid culture vessel 10. As the adherent cells, for example, cancer cells such as human osteosarcoma cells or hepatocytes are used depending on the purpose. The adherent cells are uniformly diffused into the liquid medium, and the suspension 30 is adjusted. The number of adherent cells is adjusted, for example, in the range of about tens of thousands to several million per 1 mL of the medium. The suspension 30 is developed only once with respect to the spheroid culture vessel 10.
第2ステップにおいて、懸濁液30が注入されたスフェロイド培養容器10が静置される。このとき、スフェロイド培養容器10はインキュベータなどによって所望の温度範囲や湿度範囲に保持される。静置される時間は、例えば、1日間から数日間程度である。 In the second step, the spheroid culture vessel 10 into which the suspension 30 has been injected is allowed to stand. At this time, the spheroid culture vessel 10 is maintained in a desired temperature range or humidity range by an incubator or the like. The standing time is, for example, about 1 day to several days.
図8(A)に示されるように、静置されたスフェロイド培養容器10において、懸濁液30中の付着性細胞31が重力により沈降して、スフェロイド培養容器10の底面14又は凹陥部20へ到達する。なお、図8において下向きが重力方向である。前述されたように、複数の凹陥部20が底面14の全領域に密に配置されているので、付着性細胞31の大半が凹陥部20へ到達する。また、付着性細胞31は、懸濁液30において均一に分散されており、また、平面視における各凹陥部20の境界21により囲まれる面積(凹陥部20の垂直投影面積に相当する。)は同じなので、各凹陥部20へ到達する付着性細胞の個数は均等になる。 As shown in FIG. 8 (A), in the spheroid culture container 10 that has been allowed to stand, the adherent cells 31 in the suspension 30 settle down due to gravity, and reach the bottom surface 14 or the recessed portion 20 of the spheroid culture container 10. To reach. In FIG. 8, the downward direction is the direction of gravity. As described above, since the plurality of recessed portions 20 are densely arranged in the entire area of the bottom surface 14, most of the adherent cells 31 reach the recessed portion 20. In addition, the adherent cells 31 are uniformly dispersed in the suspension 30 and the area surrounded by the boundary 21 of each recess 20 in a plan view (corresponding to the vertical projection area of the recess 20). Since they are the same, the number of adherent cells reaching each recess 20 is equal.
各凹陥部20へ到達した付着性細胞31は、重力によって各凹陥部20の斜面23に沿って沈降して最下位置22へ向かう。各凹陥部20の最下位置22及び斜面23は細胞非接着性なので、付着性細胞31は各凹陥部20内において斜面23に付着することなく、重力によって斜面23に沿って沈降する。これにより、図8(B)に示されるように、各凹陥部20へ到達した付着性細胞31が最下位置22へ集合され、最下位置22において付着性細胞31同士が結合し、スフェロイド32が形成される。静置されたスフェロイド培養容器10において、各凹陥部20へ均等な数の付着性細胞が到達することによって、各凹陥部20において均等な大きさのスフェロイド32がそれぞれ形成される。 Adherent cells 31 that have reached the respective recessed portions 20 settle down along the slopes 23 of the respective recessed portions 20 and move toward the lowest position 22 due to gravity. Since the lowest position 22 and the slope 23 of each recess 20 are non-cell-adhesive, the adherent cells 31 do not adhere to the slope 23 in each recess 20 but settle along the slope 23 by gravity. As a result, as shown in FIG. 8B, the adherent cells 31 that have reached the respective recessed portions 20 are gathered to the lowest position 22, and the adherent cells 31 are bonded to each other at the lowest position 22, and the spheroid 32. Is formed. In the stationary spheroid culture vessel 10, when an equal number of adherent cells reach each recess 20, spheroids 32 having an equal size are formed in each recess 20.
[第1実施形態の作用効果]
前述されたように、付着性細胞31の懸濁液30がスフェロイド培養容器10に一回展開されてスフェロイド培養容器10が静置されると、懸濁液30中の付着性細胞31が重力により沈降して各凹陥部20へ到達し、各凹陥部20内においては、斜面23に沿って付着性細胞31が沈降して最下位置22へ集合され、最下位置22において付着性細胞31同士が結合し、スフェロイド32が形成される。これにより、簡易な作業によって均一な大きさのスフェロイド32が大量に培養される。
[Effects of First Embodiment]
As described above, when the suspension 30 of the adherent cells 31 is developed once in the spheroid culture vessel 10 and the spheroid culture vessel 10 is allowed to stand, the adherent cells 31 in the suspension 30 are separated by gravity. It sinks and reaches each concave part 20, and in each concave part 20, the adherent cells 31 settle along the slope 23 and gather to the lowest position 22. Are combined to form a spheroid 32. As a result, a large amount of spheroids 32 having a uniform size is cultured by a simple operation.
[第2実施形態]
以下に、本発明の第2実施形態が説明される。第2実施形態では、培地交換のための流入口15及び流出口16がスフェロイド培養容器10に設けられ、前述された第2ステップにおいて、スフェロイド培養容器10内の培地交換(第3ステップ)が行われる点において第1実施形態と異なる他は、第1実施形態と同様のスフェロイド培養容器10が用いられてスフェロイド培養が行われる。以下には、第1実施形態と異なる点についてのみ詳細な説明がなされ、第1実施形態と同様の点については詳細な説明が省略される。なお、各図において第1実施形態と同様の参照符号が付された部材などは、第1実施形態と同様のものである。
[Second Embodiment]
The second embodiment of the present invention will be described below. In the second embodiment, an inlet 15 and an outlet 16 for medium exchange are provided in the spheroid culture container 10, and medium exchange (third step) in the spheroid culture container 10 is performed in the second step described above. In other respects, the spheroid culture vessel 10 is used to perform spheroid culture except that it is different from the first embodiment. In the following, detailed description will be made only for points different from the first embodiment, and detailed description of points similar to those of the first embodiment will be omitted. In addition, in each figure, the member etc. which were attached with the same referential mark as 1st Embodiment are the same as that of 1st Embodiment.
図9に示されるように、第2実施形態に係るスフェロイド培養容器10には、側壁11を厚み方向(水平方向)へ貫通する孔が設けられて、流入口15及び流出口16が形成されている。流入口15及び流出口16は、側壁11をそれぞれ貫通してスフェロイド培養容器10の内外に通ずる流路を形成している。なお、同図には、一対の流入口15及び流出口16のみが示されているが、1つのスフェロイド培養容器10に対して、複数の流入口15及び流出口16が設けられてもよい。また、流入口15と流出口16とは、スフェロイド培養容器10の中央に対して対称位置に設けられていることが好ましい。 As shown in FIG. 9, the spheroid culture vessel 10 according to the second embodiment is provided with a hole penetrating the side wall 11 in the thickness direction (horizontal direction), and an inlet 15 and an outlet 16 are formed. Yes. The inflow port 15 and the outflow port 16 form a flow path that passes through the side wall 11 and communicates with the inside and outside of the spheroid culture vessel 10. Although only a pair of inlets 15 and outlets 16 are shown in the figure, a plurality of inlets 15 and outlets 16 may be provided for one spheroid culture vessel 10. In addition, the inflow port 15 and the outflow port 16 are preferably provided at symmetrical positions with respect to the center of the spheroid culture vessel 10.
図9には現れていないが、流入口15には、側壁11の外側においてチューブなどが連結されて、新たな培地が流入口15を通じてスフェロイド培養容器10内部へ流入する流路が形成される。流出口16には、側壁11の外側においてチューブなどが連結されて、スフェロイド培養容器10内の培地が流出口16を通じて外部へ流出する流路が形成される。また、必要に応じて、流入口15側又は流出口16側に培地を流動させるためのポンプが設けられてもよい。 Although not shown in FIG. 9, a tube or the like is connected to the inflow port 15 on the outside of the side wall 11 to form a flow path through which a new medium flows into the spheroid culture vessel 10 through the inflow port 15. A tube or the like is connected to the outflow port 16 on the outside of the side wall 11 to form a flow path through which the medium in the spheroid culture vessel 10 flows out through the outflow port 16. Moreover, the pump for making a culture medium flow to the inflow port 15 side or the outflow port 16 side may be provided as needed.
第2実施形態では、第1実施形態における第2ステップにおいて、流入口15を通じて新たな培地をスフェロイド培養容器10内へ注入し、かつ流出口16を通じてスフェロイド培養容器10内の培地を排出させる第3ステップが行われる。 In the second embodiment, in the second step in the first embodiment, a new medium is injected into the spheroid culture container 10 through the inlet 15 and the medium in the spheroid culture container 10 is discharged through the outlet 16. Steps are performed.
第3ステップでは、前述された第2ステップにおいてスフェロイド培養容器10にが静置されて、懸濁液30中の付着性細胞31が各凹陥部20へ到達してスフェロイドを形成した後に、流入口15を通じて新たな培地が外部からスフェロイド培養容器10内へ流入され、かつ流出口16を通じてスフェロイド培養容器10内の培地が外部へ流出される。これにより、付着性細胞31が凹陥部20においてスフェロイド32を形成している際に、スフェロイド培養容器10内の培地交換が行われる。 In the third step, the spheroid culture vessel 10 is allowed to stand in the second step described above, and the adherent cells 31 in the suspension 30 reach the respective recessed portions 20 to form spheroids. A new medium is introduced into the spheroid culture vessel 10 from the outside through 15, and the medium in the spheroid culture vessel 10 is flowed out through the outlet 16. Thereby, when the adherent cell 31 forms the spheroid 32 in the recessed part 20, the medium exchange in the spheroid culture vessel 10 is performed.
前述された培地交換において、スフェロイド培養容器10内において培地の流れが生じるが、スフェロイド32が凹陥部20から飛び出さない程度に培地の流れを制御することにより、培地と共にスフェロイド32が流出することが防止される。 In the medium exchange described above, a medium flow occurs in the spheroid culture vessel 10, but the spheroid 32 flows out together with the medium by controlling the medium flow to such an extent that the spheroid 32 does not jump out of the recessed portion 20. Is prevented.
前述された培地交換によって、比較的長い培養期間を要する大きなスフェロイド32を形成したり、形成されたスフェロイド32を長期間保存することが可能となる。 By the medium exchange described above, it is possible to form a large spheroid 32 that requires a relatively long culture period, or to store the formed spheroid 32 for a long period of time.
[第3実施形態]
以下に、本発明の第3実施形態が説明される。第3実施形態では、凹陥部40の形状が第1実施形態の凹陥部20と異なる他は、第1実施形態と同様である。また、凹陥部40が異なってもスフェロイド培養方法は第1実施形態及び第2実施形態と同様に行われる。以下には、第1実施形態と異なる凹陥部40についてのみ詳細な説明がなされ、第1実施形態と同様の点については詳細な説明が省略される。なお、各図において第1実施形態と同様の参照符号が付された部材などは、第1実施形態と同様のものである。
[Third Embodiment]
The third embodiment of the present invention will be described below. The third embodiment is the same as the first embodiment except that the shape of the recessed portion 40 is different from the recessed portion 20 of the first embodiment. Moreover, even if the recessed part 40 differs, the spheroid culture | cultivation method is performed similarly to 1st Embodiment and 2nd Embodiment. Below, detailed description is given only about the recessed part 40 different from 1st Embodiment, and detailed description is abbreviate | omitted about the point similar to 1st Embodiment. In addition, in each figure, the member etc. which were attached with the same referential mark as 1st Embodiment are the same as that of 1st Embodiment.
[凹陥部40]
図10及び図11に示されるように、底面14には、重力方向(図11において下向き)へ凹む複数の凹陥部40が設けられている。凹陥部40は、後述されるように概ね逆円錐形状をなした有底孔である。図10及び図12に示されるように、各凹陥部40は、平面視において、底面14との境界41が円形をなしている。複数の凹陥部40は、底面14の全領域において、規則的且つ密に配置されている。
[Recess 40]
As shown in FIGS. 10 and 11, the bottom surface 14 is provided with a plurality of recessed portions 40 that are recessed in the direction of gravity (downward in FIG. 11). The recessed portion 40 is a bottomed hole having a generally inverted conical shape as will be described later. As shown in FIGS. 10 and 12, each recess 40 has a circular boundary 41 with the bottom surface 14 in plan view. The plurality of recessed portions 40 are regularly and densely arranged in the entire area of the bottom surface 14.
図12に示されるように、平面視において、各凹陥部40の中心Oと、隣り合う凹陥部40の中心Oとの距離Lは、各々の凹陥部40において一定である。また、距離Lは、平面視において、各凹陥部40の中心Oから底面14との境界41までの距離R(平面視が円形の境界41の半径に相当する。)の3倍以下である。つまり、各凹陥部40は、隣り合う凹陥部40と距離Rより接近して配置されている。 As shown in FIG. 12, the distance L between the center O of each recessed portion 40 and the center O of the adjacent recessed portion 40 is constant in each recessed portion 40 in plan view. Further, the distance L is not more than three times the distance R from the center O of each recessed portion 40 to the boundary 41 with the bottom surface 14 in plan view (the plan view corresponds to the radius of the circular boundary 41). That is, each recess 40 is arranged closer to the adjacent recess 40 than the distance R.
各凹陥部40は、底面14から概ね逆円錐形状に陥没しており、図13に示されるように、縦断面視において、各凹陥部40は底辺側が短い台形状の輪郭をなす。この台形状の輪郭における最下位置42が各凹陥部40において最も深い位置である。各凹陥部40における最下位置42までの深さは、前述された距離Rと同等である。 Each recessed portion 40 is recessed in a generally inverted conical shape from the bottom surface 14, and as shown in FIG. 13, each recessed portion 40 has a trapezoidal outline whose bottom side is short in a longitudinal sectional view. The lowest position 42 in the trapezoidal contour is the deepest position in each recessed portion 40. The depth to the lowest position 42 in each recess 40 is equal to the distance R described above.
各凹陥部40において、境界41から最下位置42までは、縦断面視において直線状に傾斜する斜面43である。最下位置42は、平面視において円形状であり、斜面43は、最下位置42を囲む曲面として境界41から最下位置42へ連続する滑らかな面であり、最下位置42を中心として境界41まで点対称な形状をなしている。最下位置42及び斜面43も、前述された内面13及び底面14と同様のコーティングがなされた細胞非接着性である。 In each recessed portion 40, the boundary 41 to the lowest position 42 is a slope 43 that is inclined linearly in a longitudinal sectional view. The lowermost position 42 is circular in plan view, and the slope 43 is a smooth surface that continues from the boundary 41 to the lowermost position 42 as a curved surface that surrounds the lowermost position 42. It has a point-symmetric shape up to 41. The lowest position 42 and the slope 43 are also non-cell-adhesive with the same coating as the inner surface 13 and the bottom surface 14 described above.
本実施形態においても、前述された第1実施形態と同様のスフェロイド培養方法が行われる。第1ステップにおいて、付着性細胞の懸濁液30が、スフェロイド培養容器10に対して1回のみ展開され、第2ステップにおいて、懸濁液30が注入されたスフェロイド培養容器10が静置される。 Also in this embodiment, the same spheroid culture method as in the first embodiment described above is performed. In the first step, the adherent cell suspension 30 is developed only once with respect to the spheroid culture vessel 10, and in the second step, the spheroid culture vessel 10 into which the suspension 30 has been injected is allowed to stand. .
図8(A)で示されたようにして、静置されたスフェロイド培養容器10において、懸濁液30中の付着性細胞31が重力により沈降して、スフェロイド培養容器10の底面14又は凹陥部40へ到達する。前述されたように、複数の凹陥部40が底面14の全領域に密に配置されているので、付着性細胞31の大半が凹陥部40へ到達する。また、付着性細胞31は、懸濁液30において均一に分散されており、また、平面視における各凹陥部40の境界41により囲まれる面積(凹陥部40の垂直投影面積に相当する。)は同じなので、各凹陥部40へ到達する付着性細胞の個数は均等になる。 As shown in FIG. 8 (A), in the spheroid culture vessel 10 that has been allowed to stand, the adherent cells 31 in the suspension 30 settle by gravity, and the bottom surface 14 or the recessed portion of the spheroid culture vessel 10. Reach 40. As described above, since the plurality of recessed portions 40 are densely arranged in the entire area of the bottom surface 14, most of the adherent cells 31 reach the recessed portion 40. In addition, the adherent cells 31 are uniformly dispersed in the suspension 30 and the area surrounded by the boundary 41 of each recessed portion 40 in a plan view (corresponding to the vertical projection area of the recessed portion 40). Since they are the same, the number of adherent cells reaching each recess 40 is equal.
図8(B)に示されたようにして、各凹陥部40へ到達した付着性細胞31は、重力により各凹陥部40の斜面43に沿って沈降して最下位置42へ向かう。各凹陥部40の最下位置42及び斜面43は細胞非接着性なので、付着性細胞31は各凹陥部40内において斜面43に付着することなく、重力によって斜面43に沿って沈降する。これにより、各凹陥部40へ到達した付着性細胞31が最下位置42へ集合され、最下位置42において付着性細胞31同士が結合し、スフェロイド32が形成される。静置されたスフェロイド培養容器10において、各凹陥部40へ均等な数の付着性細胞が到達することによって、各凹陥部40において均等な大きさのスフェロイド32がそれぞれ形成される。 As shown in FIG. 8B, the adherent cells 31 that have reached the respective recessed portions 40 settle down along the slopes 43 of the respective recessed portions 40 by gravity and travel toward the lowest position 42. Since the lowest position 42 and the slope 43 of each recess 40 are non-cell-adhesive, the adherent cells 31 do not adhere to the slope 43 in each recess 40, but settle along the slope 43 by gravity. As a result, the adherent cells 31 that have reached the respective recesses 40 are gathered to the lowest position 42, and the adherent cells 31 are bonded to each other at the lowest position 42, thereby forming a spheroid 32. In the stationary spheroid culture vessel 10, when an equal number of adherent cells reach each recess 40, spheroids 32 having an equal size are formed in each recess 40.
前述されたように、第3実施形態にかかる凹陥部40においても、第1実施形態と同様の作用効果が奏される。また、第3実施形態においても、第2実施形態と同様に、スフェロイド培養容器10に流入口15及び流出口16が設けられて、前述された第3ステップのように、スフェロイド培養容器10の培地交換が行われてもよい。 As described above, also in the recessed portion 40 according to the third embodiment, the same effect as the first embodiment is exhibited. Moreover, also in 3rd Embodiment, the inlet 15 and the outlet 16 are provided in the spheroid culture container 10 similarly to 2nd Embodiment, and the culture medium of the spheroid culture container 10 is carried out like the 3rd step mentioned above. Exchanges may be made.
[第4実施形態]
以下に、本発明の第4実施形態が説明される。第4実施形態では、凹陥部50の形状が第1実施形態の凹陥部20と異なる他は、第1実施形態と同様である。また、凹陥部20が異なってもスフェロイド培養方法は第1実施形態及び第2実施形態と同様に行われる。以下には、第1実施形態と異なる凹陥部50についてのみ詳細な説明がなされ、第1実施形態と同様の点については詳細な説明が省略される。なお、各図において第1実施形態と同様の参照符号が付された部材などは、第1実施形態と同様のものである。
[Fourth Embodiment]
Below, 4th Embodiment of this invention is described. The fourth embodiment is the same as the first embodiment except that the shape of the recessed portion 50 is different from the recessed portion 20 of the first embodiment. Moreover, even if the recessed part 20 differs, the spheroid culture | cultivation method is performed similarly to 1st Embodiment and 2nd Embodiment. Below, detailed description is given only about the recessed part 50 different from 1st Embodiment, and detailed description is abbreviate | omitted about the point similar to 1st Embodiment. In addition, in each figure, the member etc. which were attached with the same referential mark as 1st Embodiment are the same as that of 1st Embodiment.
[凹陥部50]
図14及び図15に示されるように、底面14には、重力方向(図14における下向き)へ凹む複数の凹陥部50が設けられている。凹陥部50は、後述されるように逆円錐形状をなした有底孔である。図2に示された凹陥部20と同様に、各凹陥部50は、平面視において、底面14との境界51が円形をなしている。複数の凹陥部50は、底面14の全領域において、規則的且つ密に配置されている。
[Recess 50]
As shown in FIGS. 14 and 15, the bottom surface 14 is provided with a plurality of recessed portions 50 that are recessed in the direction of gravity (downward in FIG. 14). The recessed portion 50 is a bottomed hole having an inverted conical shape as will be described later. Similarly to the recessed portion 20 shown in FIG. 2, each recessed portion 50 has a circular boundary 51 with the bottom surface 14 in plan view. The plurality of recessed portions 50 are regularly and densely arranged in the entire area of the bottom surface 14.
なお、各凹陥部50の中心Oと、隣り合う凹陥部50の中心Oとの距離Lが、各凹陥部50の中心Oから底面14との境界51までの距離R(平面視が円形の凹陥部50の半径に相当する。)の3倍以下であることなど、底面14における各凹陥部50の配列は、第1実施形態と同様であるので、ここでは詳細な説明が省略される。 The distance L between the center O of each recessed portion 50 and the center O of the adjacent recessed portion 50 is the distance R from the center O of each recessed portion 50 to the boundary 51 with the bottom surface 14 (the recessed portion having a circular shape in plan view). The arrangement of the recessed portions 50 on the bottom surface 14 is the same as that of the first embodiment, such as being equal to or less than 3 times the radius of the portion 50. Therefore, detailed description thereof is omitted here.
各凹陥部50は、底面14から逆円錐形状に陥没しており、図15に示されるように、縦断面視において、各凹陥部50は逆三角形状の輪郭をなす。この逆三角形状の輪郭における最下位置52が各凹陥部50において最も深い位置である。各凹陥部50における最下位置52までの深さは、前述された距離Rと同等である。 Each recessed portion 50 is recessed in an inverted conical shape from the bottom surface 14, and as shown in FIG. 15, each recessed portion 50 has an inverted triangular outline in a longitudinal sectional view. The lowest position 52 in the inverted triangular contour is the deepest position in each recessed portion 50. The depth to the lowest position 52 in each recess 50 is equal to the distance R described above.
各凹陥部50において、境界51から最下位置52までは、縦断面視において直線状に傾斜する斜面53である。斜面53は、最下位置52を囲む曲面として境界51から最下位置52へ連続する滑らかな面であり、最下位置52を中心として境界51まで点対称な形状をなしている。最下位置52及び斜面53も、前述された内面13及び底面14と同様のコーティングがなされた細胞非接着性である。 In each recessed part 50, the boundary 51 to the lowest position 52 is a slope 53 that is inclined linearly in a longitudinal sectional view. The slope 53 is a smooth surface that continues from the boundary 51 to the lowest position 52 as a curved surface surrounding the lowest position 52, and has a point-symmetric shape from the lowest position 52 to the boundary 51. The lowest position 52 and the slope 53 are also non-cell-adhesive with the same coating as the inner surface 13 and the bottom surface 14 described above.
本実施形態においても、前述された第1実施形態と同様のスフェロイド培養方法が行われる。第1ステップにおいて、付着性細胞の懸濁液30が、スフェロイド培養容器10に対して1回のみ展開され、第2ステップにおいて、懸濁液30が注入されたスフェロイド培養容器10が静置される。 Also in this embodiment, the same spheroid culture method as in the first embodiment described above is performed. In the first step, the adherent cell suspension 30 is developed only once with respect to the spheroid culture vessel 10, and in the second step, the spheroid culture vessel 10 into which the suspension 30 has been injected is allowed to stand. .
図8(A)で示されたようにして、静置されたスフェロイド培養容器10において、懸濁液30中の付着性細胞31が重力により沈降して、スフェロイド培養容器10の底面14又は凹陥部50へ到達する。前述されたように、複数の凹陥部50が底面14の全領域に密に配置されているので、付着性細胞31の大半が凹陥部50へ到達する。また、付着性細胞31は、懸濁液30において均一に分散されており、また、平面視における各凹陥部50の境界51により囲まれる面積(凹陥部50の垂直投影面積に相当する。)は同じなので、各凹陥部50へ到達する付着性細胞の個数は均等になる。 As shown in FIG. 8 (A), in the spheroid culture vessel 10 that has been allowed to stand, the adherent cells 31 in the suspension 30 settle by gravity, and the bottom surface 14 or the recessed portion of the spheroid culture vessel 10. Reach 50. As described above, since the plurality of recessed portions 50 are densely arranged in the entire area of the bottom surface 14, most of the adherent cells 31 reach the recessed portion 50. In addition, the adherent cells 31 are uniformly dispersed in the suspension 30 and the area surrounded by the boundary 51 of each recessed portion 50 in a plan view (corresponding to the vertical projection area of the recessed portion 50). Since they are the same, the number of adherent cells reaching each recess 50 is equal.
図8(B)に示されたようにして、各凹陥部50へ到達した付着性細胞31は、重力により各凹陥部50の斜面53に沿って沈降して最下位置52へ向かう。各凹陥部50の最下位置52及び斜面53は細胞非接着性なので、付着性細胞31は各凹陥部50内において斜面53に付着することなく、重力によって斜面53に沿って沈降する。これにより、各凹陥部50へ到達した付着性細胞31が最下位置52へ集合され、最下位置52において付着性細胞31同士が結合し、スフェロイド32が形成される。静置されたスフェロイド培養容器10において、各凹陥部50へ均等な数の付着性細胞が到達することによって、各凹陥部50において均等な大きさのスフェロイド32がそれぞれ形成される。 As shown in FIG. 8B, the adherent cells 31 that have reached the respective recessed portions 50 settle down along the slopes 53 of the respective recessed portions 50 and move toward the lowest position 52 due to gravity. Since the lowest position 52 and the slope 53 of each recess 50 are non-cell-adhesive, the adherent cells 31 do not adhere to the slope 53 in each recess 50, but settle along the slope 53 by gravity. As a result, the adherent cells 31 that have reached the respective recessed portions 50 are gathered to the lowermost position 52, and the adherent cells 31 are bonded to each other at the lowermost position 52 to form the spheroid 32. In the stationary spheroid culture vessel 10, when an equal number of adherent cells reach each recess 50, spheroids 32 having an equal size are formed in each recess 50.
前述されたように、第4実施形態にかかる凹陥部50においても、第1実施形態と同様の作用効果が奏される。また、第4実施形態においても、第2実施形態と同様に、スフェロイド培養容器10に流入口15及び流出口16が設けられて、前述された第3ステップのように、スフェロイド培養容器10の培地交換が行われてもよい。 As described above, also in the recessed portion 50 according to the fourth embodiment, the same effect as the first embodiment is exhibited. Moreover, also in 4th Embodiment, the inlet 15 and the outlet 16 are provided in the spheroid culture container 10 similarly to 2nd Embodiment, and the culture medium of the spheroid culture container 10 is carried out like the 3rd step mentioned above. Exchanges may be made.
[第5実施形態]
以下に、本発明の第5実施形態が説明される。第5実施形態では、凹陥部60の形状が第1実施形態の凹陥部20と異なる他は、第1実施形態と同様である。また、凹陥部20が異なってもスフェロイド培養方法は第1実施形態及び第2実施形態と同様に行われる。以下には、第1実施形態と異なる凹陥部60についてのみ詳細な説明がなされ、第1実施形態と同様の点については詳細な説明が省略される。なお、各図において第1実施形態と同様の参照符号が付された部材などは、第1実施形態と同様のものである。
[Fifth Embodiment]
Hereinafter, a fifth embodiment of the present invention will be described. The fifth embodiment is the same as the first embodiment except that the shape of the recessed portion 60 is different from the recessed portion 20 of the first embodiment. Moreover, even if the recessed part 20 differs, the spheroid culture | cultivation method is performed similarly to 1st Embodiment and 2nd Embodiment. Below, detailed description is given only about the recessed part 60 different from 1st Embodiment, and detailed description is abbreviate | omitted about the point similar to 1st Embodiment. In addition, in each figure, the member etc. which were attached with the same referential mark as 1st Embodiment are the same as that of 1st Embodiment.
[凹陥部60]
図16及び図17に示されるように、凹陥部60は、最下位置62までの深さが距離Rより大きい点で第1実施形態の凹陥部20と異なる。底面14に設けられた複数の凹陥部60の配置は、第1実施形態の凹陥部20と同様であるので、ここでは詳細な説明が省略される。
[Recess 60]
As shown in FIGS. 16 and 17, the recessed portion 60 is different from the recessed portion 20 of the first embodiment in that the depth to the lowest position 62 is larger than the distance R. Since the arrangement of the plurality of recessed portions 60 provided on the bottom surface 14 is the same as that of the recessed portion 20 of the first embodiment, detailed description thereof is omitted here.
各凹陥部60は、底面14から概ね半球形状に陥没しており、図17に示されるように、縦断面視において、各凹陥部60はドーム形状の輪郭をなす。このドーム形状の輪郭における最下位置62が各凹陥部60において最も深い位置である。各凹陥部60における最下位置62までの深さは、底面14と凹陥部60との境界61が平面視においてなす円形の半径、つまり距離Rより大きい。 Each recessed portion 60 is recessed in a substantially hemispherical shape from the bottom surface 14, and as shown in FIG. 17, each recessed portion 60 has a dome-shaped outline in a longitudinal sectional view. The lowest position 62 in the dome-shaped contour is the deepest position in each recess 60. The depth to the lowest position 62 in each recessed portion 60 is larger than the circular radius formed by the boundary 61 between the bottom surface 14 and the recessed portion 60, that is, the distance R.
各凹陥部60において、境界21から重力方向(図17における下向き)へ垂直壁63が形成されており、その垂直壁63から最下位置62までは、球面状に傾斜する斜面64が形成されている。この垂直壁63及び斜面64は、境界61から最下位置62へ連続する滑らかな面であり、最下位置62を中心として境界61まで点対称な形状をなしている。最下位置62、垂直壁63及び斜面64も、前述された内面13及び底面14と同様のコーティングがなされた細胞非接着性である。 In each recessed portion 60, a vertical wall 63 is formed from the boundary 21 in the direction of gravity (downward in FIG. 17), and from the vertical wall 63 to the lowest position 62, a slope 64 inclined in a spherical shape is formed. Yes. The vertical wall 63 and the inclined surface 64 are smooth surfaces that continue from the boundary 61 to the lowest position 62, and have a point-symmetric shape from the lowest position 62 to the boundary 61. The lowest position 62, the vertical wall 63, and the inclined surface 64 are also non-cell-adhesive with the same coating as the inner surface 13 and the bottom surface 14 described above.
本実施形態においても、前述された第1実施形態と同様のスフェロイド培養方法が行われることにより、各凹陥部60へ到達した付着性細胞31が、重力により斜面64に沿って沈降して最下位置62へ集合され、最下位置62において付着性細胞31同士が結合し、スフェロイド32が形成される。静置されたスフェロイド培養容器10において、各凹陥部60へ均等な数の付着性細胞が到達することによって、各凹陥部60において均等な大きさのスフェロイド32がそれぞれ形成される。 Also in this embodiment, the same spheroid culturing method as that in the first embodiment described above is performed, so that the adherent cells 31 that have reached the respective recessed portions 60 settle down along the slope 64 due to gravity and reach the bottom. At the lowest position 62, the adherent cells 31 are joined together to form a spheroid 32. In the stationary spheroid culture vessel 10, when an equal number of adherent cells reach the respective recessed portions 60, spheroids 32 having an equal size are formed in each recessed portion 60.
前述されたように、第5実施形態にかかる凹陥部60においても、第1実施形態と同様の作用効果が奏される。また、第5実施形態においても、第2実施形態と同様に、スフェロイド培養容器10に流入口15及び流出口16が設けられて、前述された第3ステップのように、スフェロイド培養容器10の培地交換が行われてもよい。凹陥部60の深さが深くなることにより、培地交換においてスフェロイド32が凹陥部60から流出することが抑制される。 As described above, also in the recessed portion 60 according to the fifth embodiment, the same effect as that of the first embodiment is exhibited. Also in the fifth embodiment, the inlet 15 and the outlet 16 are provided in the spheroid culture vessel 10 as in the second embodiment, and the medium of the spheroid culture vessel 10 is provided as in the third step described above. Exchanges may be made. By increasing the depth of the recessed portion 60, the spheroid 32 is prevented from flowing out of the recessed portion 60 in the medium exchange.
[スフェロイド培養容器10の製造方法]
以下に、前述されたスフェロイド培養容器10の製造方法の一例が示される。
[Method for Producing Spheroid Culture Container 10]
Below, an example of the manufacturing method of the spheroid culture container 10 mentioned above is shown.
図18(A)に示されるように、アクリルなどから型枠80を作製する。この型枠80は、その内部空間81がスフェロイド培養容器10の内面に対応した形状に加工されている。型枠80の内部空間81にポリジメチルシロキサン溶液(以下、「PDMS溶液」とも称される。)を充填して固化させる。 As shown in FIG. 18A, a formwork 80 is made from acrylic or the like. The mold 80 has an internal space 81 processed into a shape corresponding to the inner surface of the spheroid culture vessel 10. The interior space 81 of the mold 80 is filled with a polydimethylsiloxane solution (hereinafter also referred to as “PDMS solution”) and solidified.
PDMS溶液の固化によって型枠80内に鋳型82が形成される。図18(B)に示されるように、この鋳型82を型枠80から取り外す。そして、鋳型82の表面をプラズマ処理する。 A mold 82 is formed in the mold 80 by solidification of the PDMS solution. As shown in FIG. 18B, the mold 82 is removed from the formwork 80. Then, the surface of the mold 82 is plasma treated.
図18(C)に示されるように、鋳型82をシャーレ形状の容器83の中央に配置して、鋳型82が浸るまで容器83にPDMS溶液84を注入する。そして、容器83内においてPDMS溶液84を固化させる。 As shown in FIG. 18C, the mold 82 is placed in the center of the petri dish-shaped container 83, and the PDMS solution 84 is poured into the container 83 until the mold 82 is immersed. Then, the PDMS solution 84 is solidified in the container 83.
PDMS溶液84の固化によって容器83内にスフェロイド培養容器10が形成される。図18(D)に示されるように、このスフェロイド培養容器10を容器83から取り出し、さらに鋳型82を取り外す。これにより、スフェロイド容器10の量産が可能となる。 As the PDMS solution 84 is solidified, the spheroid culture vessel 10 is formed in the vessel 83. As shown in FIG. 18D, the spheroid culture vessel 10 is taken out from the vessel 83, and the mold 82 is further removed. Thereby, mass production of the spheroid container 10 becomes possible.
以下に、本発明の実施例が説明される。 In the following, examples of the present invention are described.
(第1実施例)
[スフェロイド培養容器]
前述された第1実施形態に係るスフェロイド培養容器10を作製した。詳細には、ポリエチレンを用いて凹陥部20が形成されたスフェロイド培養容器10を成形し、そのスフェロイド培養容器10を70%エタノールに1時間浸漬して滅菌した。滅菌されたスフェロイド培養容器10をクリーンベンチ内で乾燥した後、4%プルロニックF−127水溶液2mLを注入して、37℃に保持されたCO2インキュベータ内で一晩放置した。その後、スフェロイド培養容器10をリン酸緩衝液で2回洗浄した。
(First embodiment)
[Spheroid culture vessel]
The spheroid culture container 10 according to the first embodiment described above was produced. In detail, the spheroid culture container 10 in which the recessed part 20 was formed was shape | molded using polyethylene, and the spheroid culture container 10 was immersed in 70% ethanol for 1 hour, and sterilized. After the sterilized spheroid culture vessel 10 was dried in a clean bench, 2 mL of 4% pluronic F-127 aqueous solution was injected and left overnight in a CO 2 incubator maintained at 37 ° C. Thereafter, the spheroid culture vessel 10 was washed twice with a phosphate buffer.
[付着性細胞の懸濁液]
付着性細胞としてヒト骨肉腫細胞MG63を用いて、10%FBSを含むαMEM培地を用いて100万個/2mLとなるように懸濁液を調整した。
[Suspension of adherent cells]
Using human osteosarcoma cells MG63 as adherent cells, the suspension was adjusted to 1 million cells / 2 mL using αMEM medium containing 10% FBS.
[スフェロイド培養]
スフェロイド培養容器10にヒト骨肉腫細胞MG63の懸濁液を2mL注入し、インキュベータ内(37℃、5%CO2)にて、3日間培養した。培養後に、スフェロイド培養容器10の凹陥部20に形成されたスフェロイドを光学顕微鏡(Leica DM14000)を用いて観察した。スフェロイド培養容器10の凹陥部20における観察結果((A)50倍、(B)100倍)が図19に示され、スフェロイド培養容器10から回収されたスフェロイドの観察結果((A)50倍、(B)100倍)が図20に示される。
[Spheroid culture]
2 mL of a suspension of human osteosarcoma cells MG63 was injected into the spheroid culture vessel 10 and cultured in an incubator (37 ° C., 5% CO 2 ) for 3 days. After the culture, the spheroids formed in the recess 20 of the spheroid culture vessel 10 were observed using an optical microscope (Leica DM14000). The observation results ((A) 50 times, (B) 100 times) in the recessed portion 20 of the spheroid culture vessel 10 are shown in FIG. 19, and the observation results of the spheroids collected from the spheroid culture vessel 10 ((A) 50 times, (B) 100 times) is shown in FIG.
図19に示されるように、1つの凹陥部20に対して1つのスフェロイドが形成されていることが観察された。また、図20に示されるように、ほぼ均一な大きさのスフェロイドが多量に形成されていることが観察された。なお、回収されたスフェロイドの平均直径(N=35)は231.3±7.9μmであった。 As shown in FIG. 19, it was observed that one spheroid was formed for one recess 20. Further, as shown in FIG. 20, it was observed that a large amount of spheroids of almost uniform size were formed. The average diameter (N = 35) of the collected spheroids was 231.3 ± 7.9 μm.
(第2実施例)
[スフェロイド培養容器]
前述された第1実施形態に係るスフェロイド培養容器10を作製した。詳細には、図21に示されるように、円孔71が複数形成されたアクリル板70にポリジメチルシロキサンの薄膜72を積層した。薄膜72を、アクリル板70と、ポリジメチルシロキサンからなる円筒形状の培養槽73とで挟み込み、薄膜72と培養槽73とで囲まれる空間に細胞懸濁液が保持されるようにした。アクリル板70に形成された流路74を通じて各円孔71の内部を減圧することによって、薄膜72を各円孔71内へ吸い込んで変形させ、変形された薄膜72を凹陥部20とした。薄膜72及び培養槽73の表面を、前述された第1実施形態と同様にして、4%プルロニックF−127水溶液でコーティングした。
(Second embodiment)
[Spheroid culture vessel]
The spheroid culture container 10 according to the first embodiment described above was produced. Specifically, as shown in FIG. 21, a polydimethylsiloxane thin film 72 was laminated on an acrylic plate 70 in which a plurality of circular holes 71 were formed. The thin film 72 was sandwiched between an acrylic plate 70 and a cylindrical culture tank 73 made of polydimethylsiloxane so that the cell suspension was held in a space surrounded by the thin film 72 and the culture tank 73. By decompressing the inside of each circular hole 71 through the flow path 74 formed in the acrylic plate 70, the thin film 72 was sucked into the circular hole 71 and deformed, and the deformed thin film 72 was used as the recessed portion 20. The surfaces of the thin film 72 and the culture tank 73 were coated with a 4% pluronic F-127 aqueous solution in the same manner as in the first embodiment described above.
[付着性細胞の懸濁液]
付着性細胞としてヒト骨肉腫細胞MG63及びヒト肝癌由来細胞HepG2をそれぞれ用い、10%FBSを含むαMEM培地を用いて、スフェロイド培養容器10に対して1展開当たり400万個,200万個,100万個,50万個,25万個,12.5万個となるように懸濁液をそれぞれ調整した。
[Suspension of adherent cells]
Human osteosarcoma cells MG63 and human liver cancer-derived cells HepG2 are used as adherent cells, respectively, and 4 million cells, 2 million cells, and 1 million cells per spheroid culture vessel 10 using αMEM medium containing 10% FBS. The suspensions were adjusted so that there were 1, 500,000, 250,000, and 125,000, respectively.
[スフェロイド培養]
第1実施例と同様にしてスフェロイド培養を行い、スフェロイド培養容器10の凹陥部20に形成されたスフェロイドを光学顕微鏡(200倍)を用いて観察した。各細胞数におけるヒト骨肉腫細胞MG63の結果が図22(A)に示され、各細胞数におけるヒト肝癌由来細胞HepG2の結果が図22(B)に示される。
[Spheroid culture]
Spheroid culture was performed in the same manner as in the first example, and the spheroids formed in the recessed portion 20 of the spheroid culture vessel 10 were observed using an optical microscope (200 times). The result of human osteosarcoma cell MG63 in each cell number is shown in FIG. 22 (A), and the result of human liver cancer-derived cell HepG2 in each cell number is shown in FIG. 22 (B).
図22に示されるように、懸濁液におけるヒト骨肉腫細胞MG63又はヒト肝癌由来細胞HepG2の個数に応じた大きさのスフェロイドが、各凹陥部20においてそれぞれ形成されることが観察された。 As shown in FIG. 22, it was observed that spheroids having a size corresponding to the number of human osteosarcoma cells MG63 or human hepatoma-derived cells HepG2 in the suspension were formed in the respective recessed portions 20.
10・・・培養容器
11・・・側壁(壁)
14・・・底面
15・・・流入口
16・・・流出口
20,40,50,60・・・凹陥部
22,42,52,62・・・最下位置
23,43,53,64・・・斜面
30・・・懸濁液
31・・・付着性細胞
32・・・スフェロイド
10 ... Culture container 11 ... Side wall (wall)
14 ... Bottom 15 ... Inlet 16 ... Outlet 20, 40, 50, 60 ... Depressed part 22, 42, 52, 62 ... Lowermost position 23, 43, 53, 64 ..Slope 30 ... Suspension 31 ... Adherent cells 32 ... Spheroid
Claims (5)
上記懸濁液が注入された培養容器を静置して、当該培養容器の凹陥部においてスフェロイドを形成させる第2ステップと、を含むスフェロイド培養方法。 A first step of injecting a suspension of adherent cells into a culture vessel having a plurality of non-cell-adhesive depressions having a slope that continues from the non-cell-adhesive bottom surface to the lowest position in the direction of gravity;
A second step of allowing the culture vessel into which the suspension is injected to stand to form spheroids in the recessed portion of the culture vessel.
上記底面に細胞非接着性の表面で構成された複数の凹陥部が設けられ、
上記各凹陥部が、重力方向における最下位置へ連続する斜面を有するものであるスフェロイド培養容器。 A spheroid culture vessel comprising a non-cell-adhesive bottom surface and a wall provided surrounding the bottom surface,
The bottom surface is provided with a plurality of recesses composed of a non-cell-adhesive surface,
A spheroid culture vessel in which each of the recesses has a slope that continues to the lowest position in the direction of gravity.
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