JP6781363B2 - Cell patterning device and cell patterning method - Google Patents
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Description
本発明は、細胞パターニング装置及び細胞パターニング方法に関し、より詳細には細胞を任意の組織形態に形成する細胞パターニング装置及び細胞パターニング方法に関する。 The present invention relates to a cell patterning apparatus and a cell patterning method, and more particularly to a cell patterning apparatus and a cell patterning method for forming cells into an arbitrary tissue form.
再生医療，創薬スクリーニングやガンなどの病理研究においては、細胞を培養する技術はきわめて重要な基礎技術である。従来の細胞培養は、単純に培養容器の底面で平面的に行われてきたが、それらの細胞は、生体内の細胞と比べ機能発現が低く、振る舞いも異なることが分かってきている。そこで，細胞機能が高めるため、ゲル内での3次元培養や異種細胞との共培養などより生体内に近い状態で培養する技術が必要となる。
スフェロイドは細胞が多数個凝集して塊状となった細胞の凝集体であり、細胞のスフェロイド培養は，従来の平面での培養に比べ、細胞の機能発現を促進することが知られており、，新しい細胞培養方法のスタンダードとして注目されている。
本発明者は、培養液中において細胞を特定のパターンのスフェロイドの形態として作製する方法として、磁性化合物を添加した培養液に細胞を加え、培養液と細胞とを収容した容器に外部から磁場を作用させることにより特定のパターンのスフェロイドを作製する方法を提案した（特許文献１）。
In pathological research such as regenerative medicine, drug discovery screening and cancer, cell culture technology is an extremely important basic technology. Conventional cell culture has been performed simply on the bottom surface of the culture vessel, but it has been found that these cells have lower functional expression and different behaviors than cells in vivo. Therefore, in order to enhance cell function, a technique for culturing in a state closer to the living body, such as three-dimensional culture in a gel or co-culture with heterologous cells, is required.
Spheroids are aggregates of cells in which a large number of cells aggregate to form a mass, and it is known that cell spheroid culture promotes cell function expression as compared with conventional plane culture. It is attracting attention as a standard for new cell culture methods.
As a method for producing cells in a culture medium in the form of spheroids having a specific pattern, the present inventor adds cells to the culture medium to which a magnetic compound is added, and applies a magnetic field from the outside to a container containing the culture medium and the cells. We have proposed a method for producing a specific pattern of spheroids by allowing them to act (Patent Document 1).
特許文献１に示したスフェロイド作製方法は、磁性化合物を添加した培養液に細胞を加えることにより、細胞が磁性的に反磁性体として作用すること（磁気アルキメデス効果）を利用してパターンニングする方法である。溶媒中の粒子が受ける磁力は、溶媒と粒子との磁化率の差によって決定される。培養液に磁性化合物を添加したことにより、培養液中で細胞が反磁性体として作用する理由は、細胞の磁化率が溶媒の磁化率よりも小さくなり、外部磁界に対して細胞は相対的に反磁性体として作用する。 The spheroid production method shown in Patent Document 1 is a method of patterning by adding cells to a culture medium to which a magnetic compound is added, utilizing the fact that the cells act magnetically as diamagnetic substances (magnetic archimedes effect). Is. The magnetic force received by the particles in the solvent is determined by the difference in magnetic susceptibility between the solvent and the particles. The reason why the cells act as diamagnetic substances in the culture solution by adding the magnetic compound to the culture solution is that the magnetic susceptibility of the cells becomes smaller than the magnetic susceptibility of the solvent, and the cells are relative to the external magnetic field. Acts as a diamagnetic material.
特許文献１においては、培養液と細胞とを収容した容器を、平面形状が矩形で厚さ方向に着磁された永久磁石をＮ−Ｓ面が交互となるように格子状に配列した上に配置することにより、各々の永久磁石の頂点部分に細胞が凝集させ、スフェロイドを格子点配列とするスフェロイドの作製方法が示されている。 In Patent Document 1, a container containing a culture solution and cells is arranged in a grid pattern with permanent magnets having a rectangular planar shape and magnetized in the thickness direction so that the NS planes alternate. By arranging the cells, cells are aggregated at the apex portion of each permanent magnet, and a method for producing a spheroid having a spheroid as a lattice point arrangement is shown.
磁界を利用して細胞を非接触でハンドリングする従来方法は、細胞を磁性粒子により標識する方法である。磁性化合物を添加した培養液を使用し細胞を反磁性体として作用させてスフェロイドをパターニングする方法は、細胞から磁性粒子を取り除くといった必要がなく、完全にラベルフリーであり、非接触で大量の細胞を扱うことができるという利点がある。 A conventional method of non-contact handling of cells using a magnetic field is a method of labeling cells with magnetic particles. The method of patterning spheroids by allowing cells to act as diamagnetic substances using a culture medium containing a magnetic compound is completely label-free without the need to remove magnetic particles from the cells, and is non-contact and a large number of cells. Has the advantage of being able to handle.
しかしながら、細胞培養技術をより広範囲に適用できるようにするには、従来のような静的なパターニングではなく、様々な組織形態に対応して細胞をパターニングすることができる、いわば動的なパターニングを可能にする必要がある。
本発明は、３次元的なパターニングを含めた様々な形態に細胞をパターニングすることを可能にする細胞パターニング装置及び細胞パターニング方法を提供することを目的とする。
However, in order to make the cell culture technique more widely applicable, instead of the conventional static patterning, a dynamic patterning capable of patterning cells corresponding to various tissue morphologies is performed. Need to be possible.
An object of the present invention is to provide a cell patterning apparatus and a cell patterning method capable of patterning cells into various forms including three-dimensional patterning.
本発明に係る細胞パターニング装置は、磁性化合物を添加した培養液と細胞とを収容する容器と、該容器に収容された前記細胞を含む培養液に磁力を作用させる磁場印加手段とを備え、前記磁場印加手段は、磁力を発生する手段と、該磁力を発生する手段により前記細胞を含む培養液に磁力を作用させる位置を制御する制御手段とを備え、前記容器は、前記培養液に上方から播種された前記細胞が、該培養液の表面側から重力の作用によって徐々に底面に向けて沈降するように収容されるものであり、前記磁力を発生する手段は、前記容器の底面の下方に、複数の板状の磁石が、Ｎ極とＳ極とが交互に配置されるように並列されると共に、該複数の板状の磁石の長手方向の長さが、該容器の底面を横切る長さに設けられることで、細胞が並列した列状に凝集されるように、前記容器内の平面領域内で磁力の強さの勾配を発生させるものであり、前記制御手段は、前記磁力を発生する手段を、前記容器に対し相対的にパターン移動させ、前記平面領域内での前記磁力の強さの勾配をパターン変化させることで、前記容器内を重力によって沈降する細胞の凝集に係るパターニングがなされるように、位置を制御するものであることを特徴とする。
The cell patterning apparatus according to the present invention includes a container for accommodating a culture solution containing a magnetic compound and cells, and a magnetic field applying means for applying a magnetic force to the culture solution containing the cells contained in the container. The magnetic field applying means includes a means for generating a magnetic force and a control means for controlling a position where the magnetic force is applied to the culture solution containing the cells by the means for generating the magnetic force, and the container is provided on the culture solution from above. The seeded cells are contained so as to gradually settle toward the bottom surface from the surface side of the culture solution by the action of gravity, and the means for generating the magnetic force is below the bottom surface of the container. , A plurality of plate-shaped magnets are arranged side by side so that N poles and S poles are alternately arranged, and the length of the plurality of plate-shaped magnets in the longitudinal direction crosses the bottom surface of the container. By being provided on the surface, a gradient of the strength of the magnetic force is generated in the plane region in the container so that the cells are aggregated in a parallel row , and the control means generates the magnetic force. patterning means for, is relatively pattern transfer to said container, said strength gradient of the magnetic force in a plane area be to pattern change, according to the aggregation of cells settle in the container by gravity It is characterized in that the position is controlled so that the position is controlled.
容器に収容されている細胞を含む培養液に対して磁力を作用させる位置を制御する制御手段とは、容器と磁力を発生する手段との間の相対的な位置を制御する手段であって、容器を固定した状態で永久磁石や電磁石といった磁力を発生する手段を移動させてもよいし、磁力を発生する手段を固定した状態として、容器を移動させてもよい。また、原理的には容器と磁力を発生する手段の双方を移動させることにより、細胞に作用する磁力の作用位置を移動させる（変化させる）ことも含む。 The control means for controlling the position where the magnetic force is applied to the culture solution containing the cells contained in the container is a means for controlling the relative position between the container and the means for generating the magnetic force. The means for generating magnetic force such as a permanent magnet or an electromagnet may be moved while the container is fixed, or the container may be moved with the means for generating magnetic force fixed. Further, in principle, it also includes moving (changing) the position of action of the magnetic force acting on the cell by moving both the container and the means for generating the magnetic force.
前記磁場印加手段としては、前記磁力を発生する手段を、単一または複数組からなる永久磁石または電磁石とし、前記制御手段を、前記永久磁石または電磁石を前記容器に対し相対的に移動させる移動手段とすることもでき、前記磁力を発生する手段を、複数個の電磁石を整列して配置して構成し、前記制御手段を、整列して配置した電磁石のうちの単数または複数の電磁石を選択的に通電する操作を制御することにより、電磁石が配置された領域内における磁力の発生位置を制御する構成とすることもできる。 As the magnetic field applying means, the means for generating the magnetic field is a permanent magnet or an electromagnet composed of a single or a plurality of sets, and the control means is a moving means for moving the permanent magnet or the electromagnet relative to the container. The means for generating the magnetic force may be configured by arranging and arranging a plurality of electromagnets, and the control means may selectively select one or a plurality of electromagnets among the electromagnets arranged in an aligned manner. By controlling the operation of energizing the electromagnet, the position where the magnetic force is generated in the region where the electromagnet is arranged can be controlled.
なお、細胞を含む培養液に磁力を作用させる磁力の発生手段を配置する位置は、培養液を収容する容器の外部と内部のいずれであってもよい。
容器の外側に磁力の発生手段を配置する場合には、容器の下方（底面側）や容器の側方、上方に配置して培養液に磁力を作用させる位置を制御することができる。
また、容器内に磁力の発生手段を配置する場合には、磁石の外面を保護するといった保護手段を設けて培養液や細胞に磁力以外の影響が及ばないようにし、磁力の発生手段を移動させる際に、培養液の流れによって細胞のパターニングが阻害されないように、十分に遅い速度で磁力の発生手段を移動させて細胞をパターニングする。
The position of arranging the means for generating the magnetic force that causes the magnetic force to act on the culture solution containing the cells may be either outside or inside the container that houses the culture solution.
When the means for generating the magnetic force is arranged on the outside of the container, the position where the magnetic force is applied to the culture solution can be controlled by arranging the means below the container (bottom side), on the side of the container, or above the container.
In addition, when arranging the means for generating magnetic force in the container, protective means such as protecting the outer surface of the magnet is provided so that the culture solution and cells are not affected by anything other than magnetic force, and the means for generating magnetic force is moved. At that time, the means for generating the magnetic force is moved at a sufficiently slow speed to pattern the cells so that the patterning of the cells is not hindered by the flow of the culture medium.
本発明に係る細胞パターニング方法は、磁性化合物を添加した培養液と細胞とを収容する容器と、該容器に収容された前記細胞を含む培養液に磁力を作用させる磁場印加手段とを備え、前記磁場印加手段は、磁力を発生する手段と、該磁力を発生する手段により前記細胞を含む培養液に磁力を作用させる位置を制御する制御手段とを備え、前記容器は、前記培養液に上方から播種された前記細胞が、該培養液の表面側から重力の作用によって徐々に底面に向けて沈降するように収容されるものであり、前記磁力を発生する手段は、前記容器の底面の下方に、複数の板状の磁石が、Ｎ極とＳ極とが交互に配置されるように並列されると共に、該複数の板状の磁石の長手方向の長さが、該容器の底面を横切る長さに設けられることで、細胞が並列した列状に凝集されるように、前記容器内の平面領域内で磁力の強さの勾配を発生させるものであり、前記制御手段は、前記磁力を発生する手段を、前記容器に対し相対的にパターン移動させ、前記平面領域内での前記磁力の強さの勾配をパターン変化させることで、前記容器内を重力によって沈降する細胞の凝集に係るパターニングがなされるように、位置を制御するものである細胞パターニング装置を用いる細胞パターニング方法であって、前記容器に細胞を播種する工程と、前記磁場印加手段により前記容器中で細胞をパターニングする工程と、パターニング後の細胞を培養する工程とを、細胞をパターニングする単位工程とし、この単位工程を繰り返して細胞の三次元組織を形成することを特徴とする。
この細胞パターニング方法によれば、単位工程をさまざまに組み合わせることにより、種々の３次元形態の細胞組織を作製することができる。
The cell patterning method according to the present invention includes a container for accommodating a culture solution containing a magnetic compound and cells, and a magnetic field applying means for applying a magnetic force to the culture solution containing the cells contained in the container. The magnetic field applying means includes a means for generating a magnetic force and a control means for controlling a position where the magnetic force is applied to the culture solution containing the cells by the means for generating the magnetic force, and the container is provided on the culture solution from above. The seeded cells are contained so as to gradually settle toward the bottom surface from the surface side of the culture solution by the action of gravity, and the means for generating the magnetic force is below the bottom surface of the container. , A plurality of plate-shaped magnets are arranged side by side so that N poles and S poles are alternately arranged, and the length of the plurality of plate-shaped magnets in the longitudinal direction crosses the bottom surface of the container. By being provided on the surface, a gradient of the strength of the magnetic force is generated in the plane region in the container so that the cells are aggregated in a parallel row , and the control means generates the magnetic force. patterning means for, is relatively pattern transfer to said container, said strength gradient of the magnetic force in a plane area be to pattern change, according to the aggregation of cells settle in the container by gravity as is made, a fine胞Pa Tanin grayed how to Ru with cell patterning device is for controlling the position, and a step of seeding cells on said vessel, cells in the vessel by the magnetic field applying means The step of patterning the magnet and the step of culturing the cell after patterning are defined as a unit step of patterning the cell, and the unit step is repeated to form a three-dimensional tissue of the cell.
According to this cell patterning method, various three-dimensional morphological cell tissues can be produced by combining various unit steps.
本発明に係る細胞パターニング装置及び細胞パターニング方法においては、細胞を培養する培養液に磁性化合物を添加し、この磁性化合物を添加した培養液に細胞を播種することにより、磁場を印加した環境下において細胞が反磁性体として作用すること（磁気アルキメデス効果）を利用している。
磁気アルキメデス効果は次式の溶媒中の粒子に作用する磁力Fｍ（ベクトル）によって説明される。
The magnetic Archimedes effect is explained by the magnetic force F m (vector) acting on the particles in the solvent of the following equation.
上式で、通常の培養液中に細胞が存在する場合を考えると、細胞と培養液はともに磁化率はゼロに近く（χp≒0、χm≒0）細胞に作用する磁力は無視することができる。しかし、培養液に磁性化合物を添加してχm＞0となるように設定すると、細胞の磁化率χpはほぼ0であるから、χp−χm＜0となり、係数がマイナスとなって、細胞は反磁性体として振る舞うことになる。
磁性化合物を添加した培養液に播種した細胞に磁力を作用させると、細胞は磁力の作用によって反発され、磁力が弱い領域に集まるようになる。すなわち、磁力の作用を利用して細胞を特定の領域に集めたり、特定のパターンに配置する（パターニング）することが可能となる。
In the above equation, considering the case where cells are present in a normal culture medium, the magnetic susceptibility of both the cells and the culture medium is close to zero (χ p ≈ 0, χ m ≈ 0), and the magnetic force acting on the cells is ignored. be able to. However, when a magnetic compound is added to the culture solution and set so that χ m > 0, the magnetic susceptibility χ p of the cells is almost 0, so χ p − χ m <0, and the coefficient becomes negative. , The cell will behave as a diamagnetic substance.
When a magnetic force is applied to the cells seeded in the culture medium to which the magnetic compound is added, the cells are repelled by the action of the magnetic force and gather in the region where the magnetic force is weak. That is, it is possible to collect cells in a specific region or arrange them in a specific pattern (patterning) by utilizing the action of magnetic force.
培養液に添加する磁性化合物は、使用上の安全性（細胞に対する毒性等）を考慮して選択する。培養液に添加する磁性化合物としては、例えばガドリニウム化合物を用いることができる。 The magnetic compound to be added to the culture solution is selected in consideration of safety in use (toxicity to cells, etc.). As the magnetic compound added to the culture solution, for example, a gadolinium compound can be used.
本発明に係る細胞パターニング装置及び細胞パターニング方法によれば、さまざまな形態に細胞をパターニングすることができ、細胞を培養する種々の用途に利用することができる。 According to the cell patterning apparatus and cell patterning method according to the present invention, cells can be patterned into various forms and can be used for various purposes for culturing cells.
（第１の実施の形態）
図１は、本発明に係る細胞パターニング方法の第１の実施の形態の構成例を示す。図１に示す細胞パターニング装置は、磁性化合物を含む培養液と細胞とを収容する容器１０と、容器１０の底部の下方に配置される磁場印加手段とを備える。磁場印加手段は永久磁石のような磁力を発生するための手段と、容器１０の底部の全面にわたって磁場を印加する位置を制御する制御手段を備える。
(First Embodiment)
FIG. 1 shows a configuration example of the first embodiment of the cell patterning method according to the present invention. The cell patterning apparatus shown in FIG. 1 includes a container 10 for accommodating a culture solution containing a magnetic compound and cells, and a magnetic field applying means arranged below the bottom of the container 10. The magnetic field application means includes means for generating a magnetic force such as a permanent magnet, and control means for controlling the position where the magnetic field is applied over the entire bottom surface of the container 10.
細胞パターニング装置の磁場印加手段としては、たとえば磁力を発生する手段として永久磁石を使用し、永久磁石をX-Yステージ等の移動手段に支持することにより、容器の下方で磁場を印加する位置を任意に制御することができる。
図１（b）は、磁場印加手段として棒状の永久磁石１２を使用し、移動手段により容器１０の底部の下方で永久磁石１２を移動させることにより磁場を印加する位置を変える例を示す。図１(b)では、永久磁石１２の容器１０の底面に対向する磁極をN極としているが、永久磁石１２のS極を容器１０の底面に対向させる配置としてもよい。
As the magnetic field application means of the cell patterning apparatus, for example, a permanent magnet is used as a means for generating a magnetic force, and by supporting the permanent magnet with a moving means such as an XY stage, the position where the magnetic field is applied can be arbitrarily set below the container. Can be controlled.
FIG. 1B shows an example in which a rod-shaped permanent magnet 12 is used as the magnetic field applying means, and the position where the magnetic field is applied is changed by moving the permanent magnet 12 below the bottom of the container 10 by the moving means. In FIG. 1B, the magnetic pole facing the bottom surface of the container 10 of the permanent magnet 12 is the north pole, but the south pole of the permanent magnet 12 may be arranged to face the bottom surface of the container 10.
磁場印加手段は永久磁石のような磁力を発生する手段と、磁場を印加する位置を制御する制御手段とを備える。
磁力を発生する手段としては永久磁石のように固定した磁力を備えるものの他に、電磁石等の磁力の強さが可変となる磁力の発生手段を利用して、作用する磁力の強さを制御することもできる。
磁場を印加する位置を制御する制御手段としては、単一の永久磁石等の磁力を発生する手段そのものを移動させる方法を利用するものの他に、複数個の永久磁石をN、S極を交互配置した組み合わせた形態のものを移動させる方法も可能である。
また、容器１０の下方に多数個の小さな電磁石をマトリックス状に整列して配置し、通電する電磁石を選択することにより、磁場を印加する位置を選択するといった方法を利用することもできる。
The magnetic field applying means includes means for generating a magnetic force such as a permanent magnet and control means for controlling the position where the magnetic field is applied.
As a means for generating a magnetic force, in addition to a means having a fixed magnetic force such as a permanent magnet, a means for generating a magnetic force such as an electromagnet having a variable strength of the magnetic force is used to control the strength of the acting magnetic force. You can also do it.
As the control means for controlling the position where the magnetic field is applied, in addition to the method of moving the means itself for generating magnetic force such as a single permanent magnet, a plurality of permanent magnets are arranged alternately with N and S poles. It is also possible to move the combined form.
Further, it is also possible to use a method of selecting a position to apply a magnetic field by arranging a large number of small electromagnets in a matrix below the container 10 and selecting an electromagnet to be energized.
磁場を印加する位置を制御する制御手段としては、容器の下方の平面内における位置を制御する他に、容器と磁力を発生する手段との離間距離（Z方向距離）を制御する手段を備えることも可能である。容器と磁力を発生する手段との離間距離を制御することにより、容器（培養液、細胞）に作用する磁力の強さを制御することができる。 As the control means for controlling the position where the magnetic field is applied, in addition to controlling the position in the plane below the container, a means for controlling the separation distance (distance in the Z direction) between the container and the means for generating the magnetic force is provided. Is also possible. By controlling the separation distance between the container and the means for generating the magnetic force, the strength of the magnetic force acting on the container (culture solution, cells) can be controlled.
図１（a）、（b）、(c)は、磁場印加手段である永久磁石１２を用いて培養液中の細胞をパターニングする方法を示す。
図１(a)は、容器１０に磁性化合物を含む培養液２０を収容し、容器１０の開口側である容器１０の上方から細胞２２を播種している状態である。容器１０の開口部から磁性化合物を含む培養液２０に播種された細胞２２は、培養液２０の表面側から徐々に容器１０の底面に向けて沈降する。細胞２２の沈降は重力の作用による。
FIGS. 1 (a), 1 (b), and 1 (c) show a method of patterning cells in a culture medium using a permanent magnet 12 which is a magnetic field application means.
FIG. 1A shows a state in which the culture solution 20 containing the magnetic compound is contained in the container 10 and the cells 22 are seeded from above the container 10 which is the opening side of the container 10. The cells 22 seeded in the culture solution 20 containing the magnetic compound from the opening of the container 10 gradually settle from the surface side of the culture solution 20 toward the bottom surface of the container 10. The sedimentation of cells 22 is due to the action of gravity.
磁性化合物を含む培養液２０に播種された細胞２２は外部から作用する磁場に対しては反磁性体として作用する（磁気アルキメデス効果）。図１(b)は、容器１０の底部に磁場印加手段として永久磁石１２を近づけることにより、永久磁石１２の磁力によって細胞２２を反発させ、永久磁石１２から遠ざけるようにしている状態を示す。
容器１０の上方から培養液２０中を沈降した細胞２２は、永久磁石１２の磁力によって反発され、容器１０の底側から上向きに押し除けられる（弾き飛ばされる）。
The cells 22 seeded in the culture medium 20 containing the magnetic compound act as diamagnetic substances against a magnetic field acting from the outside (magnetic Archimedes effect). FIG. 1B shows a state in which the permanent magnet 12 is brought close to the bottom of the container 10 as a magnetic field applying means, so that the cells 22 are repelled by the magnetic force of the permanent magnet 12 and kept away from the permanent magnet 12.
The cells 22 that have settled in the culture solution 20 from above the container 10 are repelled by the magnetic force of the permanent magnet 12 and are pushed upward (flicked off) from the bottom side of the container 10.
図１(b)は、容器１０の下方で永久磁石１２を左右に動かすことで、容器１０の左側の壁面側に沈降してきた細胞２２を永久磁石１２の磁力で弾き飛ばし、底に沈降させないようにしながら、右側へ向けて細胞２２を徐々に押しやるようにする操作を行っている状態である。
このように、容器１０の下方で永久磁石１２を操作することにより、培養液２０に播種した細胞２２を徐々に容器１０の右側に集めることができる。
図１(c)は、培養液２０に播種した細胞２２を、永久磁石１２の磁力を利用して、最終的に、容器２０の右側の壁面の近くに集めた（パターニングした）状態を示す。
In FIG. 1 (b), by moving the permanent magnet 12 to the left and right below the container 10, the cells 22 that have settled on the left wall side of the container 10 are repelled by the magnetic force of the permanent magnet 12 so as not to settle to the bottom. This is a state in which the cell 22 is gradually pushed toward the right side.
By operating the permanent magnet 12 below the container 10 in this way, the cells 22 seeded in the culture solution 20 can be gradually collected on the right side of the container 10.
FIG. 1 (c) shows a state in which the cells 22 seeded in the culture solution 20 are finally collected (patterned) near the wall surface on the right side of the container 20 by using the magnetic force of the permanent magnet 12.
図１(a)、(b)、(c)は、永久磁石１２の磁力により、培養液２０中で反磁性体として作用する細胞２２が反発される作用を利用して、容器１０の一方側に細胞２２を凝集させた例である。
このように、培養液２０中で細胞２２が反磁性体として作用し、磁力で反発される作用を利用して細胞をパターニングする操作は、培養液２０中で、磁力が強い領域から磁力が弱い領域に細胞２２が移動するという作用による。したがって、容器１０の平面内において磁力の強さが強弱となるように設定することで、磁力がより弱い領域に細胞２２を誘導して細胞２２を集める位置を設定することができる。言い換えれば、容器１０の平面領域内で磁力の強さの勾配を設け、磁力の勾配を動的に操作することにより、細胞２２を所望の配置に移動させ所定配置に細胞をパターニングすることができる。
1 (a), (b), and (c) show one side of the container 10 by utilizing the action of repelling cells 22 acting as diamagnetic substances in the culture solution 20 by the magnetic force of the permanent magnet 12. This is an example in which cells 22 are aggregated in a magnet.
In this way, the operation of patterning cells by using the action of the cells 22 acting as antimagnetic substances in the culture medium 20 and being repelled by the magnetic force is performed in the culture medium 20 from the region where the magnetic force is strong to the weak magnetic force. This is due to the action of cells 22 moving to the region. Therefore, by setting the strength of the magnetic force to be strong or weak in the plane of the container 10, it is possible to set the position where the cells 22 are guided to the region where the magnetic force is weaker and the cells 22 are collected. In other words, by providing a gradient of the strength of the magnetic force in the plane region of the container 10 and dynamically manipulating the gradient of the magnetic force, the cells 22 can be moved to a desired arrangement and the cells can be patterned in a predetermined arrangement. ..
細胞２２を含む培養液２０に対して磁力を印加する位置を自在に選択して制御することができる磁場印加手段を利用すれば、図１に示すように、単に容器１０の一方側に細胞２２を凝集させる例に限らず、培養液中における細胞の位置を自由にコントロールすることができる。言い換えれば、磁場印加手段を利用することで、細胞の配置を自在にコントロールして、スフェロイドを自在にパターニングすることが可能となる。
培養液中の細胞を磁気的に反発させてパターニングする方法におけるパターニングの精度は磁力を発生する手段（磁石）のサイズに依存すると考えられる。したがって、細胞を高精度に（精細）にパターニングできるようにするには、磁力を発生する手段を出来るだけ微小サイズとする必要がある。しかし、重力により細胞にかかる沈降力は1pN弱であり、細胞を操作しパターニングするためには、それを越える磁力を与える必要があることから、少なくとも磁石は500μm程度のサイズとする必要がある。また、磁力を作用させる位置を位置制御する制御手段のコントロール方法についても高精度に設定する必要がある。
If a magnetic field applying means capable of freely selecting and controlling the position where the magnetic force is applied to the culture solution 20 containing the cells 22 is used, as shown in FIG. 1, the cells 22 are simply placed on one side of the container 10. The position of the cells in the culture medium can be freely controlled, not limited to the example of agglutinating the cells. In other words, by using the magnetic field application means, it is possible to freely control the arrangement of cells and freely pattern the spheroids.
It is considered that the accuracy of patterning in the method of magnetically repelling cells in the culture medium to pattern them depends on the size of the means (magnet) that generates the magnetic force. Therefore, in order to enable high-precision (fine) patterning of cells, it is necessary to make the means for generating magnetic force as small as possible. However, the sedimentation force applied to cells by gravity is less than 1 pN, and in order to manipulate and pattern cells, it is necessary to apply a magnetic force that exceeds that, so at least the magnet needs to be about 500 μm in size. Further, it is necessary to set the control method of the control means for controlling the position where the magnetic force is applied with high accuracy.
（第２の実施の形態）
磁性化合物を添加した培養液と細胞とを容器に収容し、磁場印加手段により細胞を含む培養液に対して磁力を作用させることにより細胞の凝集位置を制御する方法を利用すれば、容器内で種々の形態に細胞をパターニングすることができる。
(Second Embodiment)
If a method is used in which the culture solution to which the magnetic compound is added and the cells are housed in a container and the aggregation position of the cells is controlled by applying a magnetic force to the culture solution containing the cells by a magnetic field application means, the cells are contained in the container. The cells can be patterned into various forms.
図２は、第１層目として、容器１０の底面の全面に細胞を均一に配置されるように形成し（図２（a））、第２層目については、細胞を並列した列状に配置し（図２（b））、第３層目については、列方向と第２層目とは90度変えた向きに配置する操作を行った例（図２(c))を示す。 In FIG. 2, as the first layer, cells are formed so as to be uniformly arranged on the entire bottom surface of the container 10 (FIG. 2 (a)), and in the second layer, cells are arranged in a row. An example (FIG. 2 (c)) is shown in which the cells are arranged (FIG. 2 (b)), and the third layer is arranged in a direction in which the column direction and the second layer are different by 90 degrees (FIG. 2 (c)).
容器１０の底面に均一に細胞２２を配置する第１層目を形成するには、容器１０に磁性化合物を添加した培養液を収容し、容器１０の上部の開口面から細胞２２を播種すればよい。培養液に播種された細胞２２は重力の作用によって容器１０の底面に向けて沈降し、容器１０の底面に均一に配列される。図２（a）は、容器１０の底面に均一に細胞２２aが配列された状態を示す。 In order to form the first layer in which the cells 22 are uniformly arranged on the bottom surface of the container 10, the culture solution containing the magnetic compound is contained in the container 10 and the cells 22 are seeded from the opening surface at the upper part of the container 10. Good. The cells 22 seeded in the culture medium settle toward the bottom surface of the container 10 by the action of gravity, and are uniformly arranged on the bottom surface of the container 10. FIG. 2A shows a state in which cells 22a are uniformly arranged on the bottom surface of the container 10.
容器１０の底面に細胞２２aが均一に配置された状態で一定時間、細胞２２aを培養すると、細胞２２aは容器１０の底面に接着する。細胞２２aが容器１０の底面に接着した後、第２層目の細胞をパターニングする操作を行う。
細胞を並列した列状（帯状）に配置する方法としては、図２（b）に示すように、容器１０の底面の下方に、長手方向の長さが容器１０の底面を横切る長さとした板状の永久磁石を、厚さ方向に着磁し、異極の面を対向させる配置（吸着し合う配置）として永久磁石を連結する配置とし、この状態で容器１０の開口面から細胞２２を播種すればよい。
容器１０に作用させる磁力の発生手段を板状の永久磁石をN極とS極が交互に配置した構成としたことにより、N極とS極の磁極端面の長手方向に沿って細胞２２ｂが配列される。
When the cells 22a are cultured for a certain period of time with the cells 22a uniformly arranged on the bottom surface of the container 10, the cells 22a adhere to the bottom surface of the container 10. After the cells 22a adhere to the bottom surface of the container 10, an operation of patterning the cells in the second layer is performed.
As a method of arranging the cells in a row (strip shape) in parallel, as shown in FIG. 2 (b), a plate having a length in the longitudinal direction crossing the bottom surface of the container 10 below the bottom surface of the container 10. The permanent magnets in the shape are magnetized in the thickness direction, and the permanent magnets are connected in an arrangement in which the surfaces of different poles face each other (arrangement of attracting each other), and in this state, the cells 22 are seeded from the opening surface of the container 10. do it.
The means for generating the magnetic force acting on the container 10 is such that plate-shaped permanent magnets are arranged alternately with the north and south poles, so that the cells 22b are arranged along the longitudinal direction of the magnetic pole end faces of the north and south poles. Will be done.
図３に、第１層目と第２層目の細胞を配列した状態を正面方向から見た状態を示す。
図３（a）は、容器１０の底面に細胞２２aが均一に配列されて培養された状態を示す。
図３（b）は、容器１０の底面に細胞２２aを均一に配列した状態で培養した後、第２層目の細胞２２ｂを配列した状態を示す。容器１０の下方に、厚さ方向に着磁した（隣り合った永久磁石の対向面が磁極となる）永久磁石が並列に配置されている。
FIG. 3 shows a state in which the cells of the first layer and the second layer are arranged as viewed from the front direction.
FIG. 3A shows a state in which cells 22a are uniformly arranged and cultured on the bottom surface of the container 10.
FIG. 3B shows a state in which cells 22a in the second layer are arranged after culturing in a state where cells 22a are uniformly arranged on the bottom surface of the container 10. Permanent magnets magnetized in the thickness direction (opposite surfaces of adjacent permanent magnets serve as magnetic poles) are arranged in parallel below the container 10.
図３（b）に示すように、永久磁石を並列に配置すると、隣り合った永久磁石の境界面付近の磁力が強くなり、各々の永久磁石の中央部付近の磁力が相対的に弱くなって、培養液２０に播種した細胞は、各々の永久磁石の中央部に集まり、永久磁石の中央部で長手方向に沿って細胞２２ｂがパターニングされる。すなわち、図２（b）に示すように、永久磁石の長手方向に沿って第２層目の細胞２２ｂが配列される。細胞２２ｂは、各々の永久磁石のN極とS極の中間位置付近で凝集する。 As shown in FIG. 3B, when the permanent magnets are arranged in parallel, the magnetic force near the boundary surface of the adjacent permanent magnets becomes stronger, and the magnetic force near the center of each permanent magnet becomes relatively weak. , The cells seeded in the culture solution 20 gather at the central portion of each permanent magnet, and the cells 22b are patterned along the longitudinal direction at the central portion of the permanent magnets. That is, as shown in FIG. 2B, the cells 22b of the second layer are arranged along the longitudinal direction of the permanent magnet. The cells 22b aggregate near the intermediate position between the north and south poles of each permanent magnet.
図２(c)は、第３層目の細胞をパターニングする状態を示す。第３層目の細胞のパターニングは、図２(b)での永久磁石の配置方向を平面内で90度回転することで行っている。図２(b)における永久磁石の向きを90度変換して培養液２０に細胞を播種することにより、第３層目では、第２層目での細胞２２ｂが配列される方向とは90度、配列方向が異なる配置で細胞２２ｃがパターニングされる。すなわち、第２の層目の細胞２２ｂと第３層目の細胞２２ｃとで、平面方向からみて格子状に細胞２２ｂと細胞２２ｃがパターニングされる。 FIG. 2C shows a state in which cells in the third layer are patterned. The patterning of the cells in the third layer is performed by rotating the arrangement direction of the permanent magnets in FIG. 2B by 90 degrees in a plane. By changing the orientation of the permanent magnets in FIG. 2 (b) by 90 degrees and seeding the cells in the culture medium 20, the direction in which the cells 22b in the second layer are arranged in the third layer is 90 degrees. , The cells 22c are patterned in different arrangement directions. That is, the cells 22b in the second layer and the cells 22c in the third layer are patterned in a grid pattern when viewed from the plane direction.
第３層目の細胞２２ｃを配列する場合も、第２層目の細胞２２ｂをパターニングした後、一定時間、細胞２２ｂを培養することにより細胞２２ｂを相互に接着させた後、パターニングする。
細胞を培養することによって得られる細胞間の接着力は数μNである。これに対して、本発明における磁場印加手段によって細胞を操作するときに細胞に作用する磁力は１nN以下であり、細胞の接着力に対して磁力による操作力ははるかに小さい。したがって、既に接着している細胞を剥離させるといった影響を及ぼすことなく細胞をパターニングすることができる。
When arranging the cells 22c in the third layer, the cells 22b in the second layer are patterned, and then the cells 22b are adhered to each other by culturing the cells 22b for a certain period of time, and then the cells 22b are patterned.
The adhesive force between cells obtained by culturing the cells is several μN. On the other hand, the magnetic force acting on the cells when the cells are manipulated by the magnetic field application means in the present invention is 1 nN or less, and the operating force by the magnetic force is much smaller than the adhesive force of the cells. Therefore, the cells can be patterned without having the effect of exfoliating the cells that have already adhered.
図２、３に示す細胞のパターニング方法は、細胞を形成した面上に、細胞をパターニングする操作を繰り返すことにより、細胞を３次元的にパターニングする例を示す。
この例のように、容器に細胞を播種する工程と、磁場印加手段により容器中で細胞をパターニングする工程と、パターニング後の細胞を培養する工程とを、細胞をパターニングするための単位工程とし、この単位工程を繰り返すことにより、細胞の三次元組織を形成することができる。
磁場印加手段を利用して細胞をパターニングする方法によれば、磁力の発生手段や、磁力の発生手段を制御する制御手段を用途に応じて適宜設計することにより、さまざまな形態の細胞の三次元組織（スフェロイド）を作製することができる。
The cell patterning method shown in FIGS. 2 and 3 shows an example in which cells are three-dimensionally patterned by repeating an operation of patterning cells on a surface on which cells are formed.
As in this example, the step of seeding the cells in the container, the step of patterning the cells in the container by the magnetic field application means, and the step of culturing the cells after patterning are set as unit steps for patterning the cells. By repeating this unit process, a three-dimensional tissue of cells can be formed.
According to the method of patterning cells using a magnetic field applying means, by appropriately designing a means for generating a magnetic force and a control means for controlling the means for generating a magnetic force according to an application, three-dimensional cells of various forms are formed. Tissues (spheroids) can be made.
細胞の培養に用いられる培養液には、一般に動物細胞の培養に用いられる培地、たとえば、ダルベッコ改変イーグル培地（DMEM）、Mem−α、RPMI1640等を使用することができる。培養液には、細胞の増殖を促進するための血清を添加するか、あるいは血清に代替するものとして、例えばFGF、EGF、PDGＦ等の細胞増殖因子やトランスフェリン等の既知の血清成分を添加してもよい。なお、血清を添加する場合の濃度は、そのときの培養状態によって適宜変更することができるが、通常10v／v％とすることができる。細胞の培養には、通常の培養条件、例えば37℃の温度で5% CO2濃度のインキュベーター内での培養が適用される。 As the culture medium used for culturing cells, a medium generally used for culturing animal cells, for example, Dalveco modified Eagle's medium (DMEM), Mem-α, RPMI1640 and the like can be used. Serum for promoting cell proliferation is added to the culture medium, or as a substitute for serum, cell growth factors such as FGF, EGF, PDGF and known serum components such as transferrin are added. May be good. The concentration at which serum is added can be appropriately changed depending on the culture state at that time, but is usually 10 v / v%. For cell culture, normal culture conditions, for example, culture in an incubator at a temperature of 37 ° C. and a 5% CO 2 concentration are applied.
培養液に添加する磁性化合物としては、塩化第一鉄、塩化マンガン、ガドリニウム錯体、テルビウム錯体などを挙げることができる。この他に、培養液に溶解しないが分散状態となっている磁性ナノ粒子のコロイド溶液等を用いることも可能である。これらのうち、細胞毒性等の観点からガドリニウム化合物を用いることが好ましく、ガドリニウムとキレート化合物との錯体、例えば、DTPAでキレート化された３価ガドリニウム（Gd（III））（Gd−DTPA）またはDOTAでキレート化された３価ガドリニウム（Gd（III））（Gd−DOTA）、HP-DO3Aでキレート化された３価ガドリニウム（Gd（III））Gd-HP-DO3Aが好適に用いられる。これらの磁性化合物の使用濃度は、使用する磁性化合物によって異なるが、Gd−DTPAを用いる場合は、10ｍＭ以上とすることが好ましく、細胞毒性の観点から1ｍＭ〜40ｍＭとするのがよい。 Examples of the magnetic compound added to the culture solution include ferrous chloride, manganese chloride, gadolinium complex, and terbium complex. In addition to this, it is also possible to use a colloidal solution of magnetic nanoparticles that is not dissolved in the culture solution but is in a dispersed state. Of these, it is preferable to use a gadolinium compound from the viewpoint of cytotoxicity and the like, and a complex of gadolinium and a chelating compound, for example, trivalent gadolinium (Gd (III)) (Gd-DTPA) chelated with DTPA or DOTA Trivalent gadolinium (Gd (III)) (Gd-DOTA) chelated with HP-DO3A and trivalent gadolinium (Gd (III)) Gd-HP-DO3A chelated with HP-DO3A are preferably used. The concentration of these magnetic compounds used varies depending on the magnetic compound used, but when Gd-DTPA is used, it is preferably 10 mM or more, and from the viewpoint of cytotoxicity, it is preferably 1 mM to 40 mM.
本発明に係る細胞パターニング方法が適用可能な細胞としては、特に制限はなく、肝臓、膵臓、腎臓、神経、皮膚、血管，脂肪等から採取される初代細胞や、各種の因子を導入して得られた初期化したｉＰＳ細胞（induced pluripotent stem cells、人工多能性幹細胞）、未分化な幹細胞、胚由来のＥＳ細胞（Embryonic Stem Cell）、樹立されている株化細胞、またはこれらに遺伝子操作等を施した細胞等である。 The cells to which the cell patterning method according to the present invention can be applied are not particularly limited, and can be obtained by introducing primary cells collected from liver, pancreas, kidney, nerve, skin, blood vessels, fat, etc., and various factors. Reprogrammed iPS cells (induced pluripotent stem cells), undifferentiated stem cells, embryonic stem cells (Embryonic Stem Cells), established cell lines, or genetic manipulation on these cells, etc. These are cells that have been subjected to.
（実験例）
板状の磁石を並列に配置した磁力の発生手段（磁石アレイ）を用いて細胞をパターニングする例として、下記の実験を行った。
40mMのGd-DOTAを含むD-PBS（Dulbecco's Phosphate-Buffered Saline）に、密度および磁化率が細胞に近い蛍光ポリスチレン微粒子を分散した液を、内寸10×10×10mmのガラス容器（底面の厚みは、0.2mm程度）に注入した。
この液を注入したガラス容器を、磁石アレイ上に設置して蛍光ポリスチレン微粒子のパターニングを行った。
磁石アレイには，ネオジム磁石（30mm×5mm、厚さ1mm、厚さ方向に磁化）15枚を、図３と同様にNS極が向かい合うように並べたものを用いた。
図４は、磁石アレイの上にガラス容器を置いて１時間経過した後の状態である。蛍光ポリスチレン微粒子がライン状に配列している。図４で明るいライン状に見える部分が蛍光ポリスチレン微粒子が配列されている部位である。
この実験結果は、磁力の発生手段を用いて培養液中で細胞をパターニングすることができることを示している。
(Experimental example)
The following experiment was conducted as an example of patterning cells using a magnetic force generating means (magnet array) in which plate-shaped magnets are arranged in parallel.
D-PBS (Dulbecco's Phosphate-Buffered Saline) containing 40 mM Gd-DOTA is filled with a solution of fluorescent polystyrene fine particles whose density and magnetic susceptibility are close to those of cells, in a glass container with an internal size of 10 x 10 x 10 mm (bottom thickness). Was injected into (about 0.2 mm).
A glass container into which this liquid was injected was placed on a magnet array to pattern fluorescent polystyrene fine particles.
For the magnet array, 15 neodymium magnets (30 mm × 5 mm, thickness 1 mm, magnetized in the thickness direction) were arranged so that the NS poles faced each other as in FIG.
FIG. 4 shows a state after the glass container is placed on the magnet array and one hour has passed. Fluorescent polystyrene fine particles are arranged in a line. The portion that looks like a bright line in FIG. 4 is the portion where the polystyrene fine particles are arranged.
The results of this experiment show that cells can be patterned in culture using a means of generating magnetic force.
前述した第１の実施形態、第２の実施形態においては、培養液を収容した容器の下方に永久磁石を配置して細胞をパターニングしたが、磁力の発生手段は必ずしも容器の外側に配置しなければならないものではなく、容器内に磁力の発生手段を配置して細胞をパターニングすることも可能である。
ただし、容器内に磁力の発生手段を配置する場合は、永久磁石に錆が生じないように防錆処理を施した磁石を使用したり、永久磁石の移動によって生じる培養液の流れにより細胞をパターニングする操作が影響を受けないように、十分に遅い速度で永久磁石を移動させるといった必要がある。
磁力の発生手段を利用して細胞をパターニングする方法では、磁力が作用する範囲が数ｍｍ程度に限定されるから、高さ方向に10ｍｍを超えるような細胞のアセンブリが困難である。このような大きな細胞の凝集体を作製する場合や、高さのある構造体として細胞をパターニングする場合には、細胞に近接した位置で磁力を作用させることが可能な、容器内において、磁力の発生手段の移動を制御しもしくは細胞に作用させる磁力を制御することが有効である。
In the first embodiment and the second embodiment described above, the permanent magnets are arranged below the container containing the culture solution to pattern the cells, but the means for generating the magnetic force must not necessarily be arranged outside the container. It is not necessary, and it is also possible to arrange a means for generating magnetic force in the container to pattern the cells.
However, when arranging the means for generating magnetic force in the container, use a magnet that has been rust-proofed so that the permanent magnet does not rust, or pattern the cells by the flow of the culture solution generated by the movement of the permanent magnet. It is necessary to move the permanent magnet at a sufficiently slow speed so that the operation to be performed is not affected.
In the method of patterning cells using a means for generating magnetic force, the range in which the magnetic force acts is limited to about several mm, so that it is difficult to assemble cells that exceed 10 mm in the height direction. When making such a large cell aggregate or when patterning a cell as a tall structure, the magnetic force can be applied in a container close to the cell. It is effective to control the movement of the generating means or the magnetic force acting on the cells.
１０ 容器
１２ 永久磁石
２０ 培養液
２２、２２a、２２ｂ、２２ｃ 細胞
10 Container 12 Permanent magnet 20 Culture solution 22, 22a, 22b, 22c cells
Claims (4)
該容器に収容された前記細胞を含む培養液に磁力を作用させる磁場印加手段とを備え、
前記磁場印加手段は、磁力を発生する手段と、
該磁力を発生する手段により前記細胞を含む培養液に磁力を作用させる位置を制御する制御手段とを備え、
前記容器は、前記培養液に上方から播種された前記細胞が、該培養液の表面側から重力の作用によって徐々に底面に向けて沈降するように収容されるものであり、
前記磁力を発生する手段は、前記容器の底面の下方に、複数の板状の磁石が、Ｎ極とＳ極とが交互に配置されるように並列されると共に、該複数の板状の磁石の長手方向の長さが、該容器の底面を横切る長さに設けられることで、細胞が並列した列状に凝集されるように、前記容器内の平面領域内で磁力の強さの勾配を発生させるものであり、
前記制御手段は、前記磁力を発生する手段を、前記容器に対し相対的にパターン移動させ、前記平面領域内での前記磁力の強さの勾配をパターン変化させることで、前記容器内を重力によって沈降する細胞の凝集に係るパターニングがなされるように、位置を制御するものであることを特徴とする細胞パターニング装置。 A container for containing the culture solution to which the magnetic compound was added and the cells,
A magnetic field applying means for applying a magnetic force to the culture solution containing the cells contained in the container is provided.
The magnetic field applying means includes means for generating magnetic force and
It is provided with a control means for controlling the position where the magnetic force is applied to the culture solution containing the cells by the means for generating the magnetic force.
The container contains the cells seeded in the culture solution from above so as to gradually settle from the surface side of the culture solution toward the bottom surface by the action of gravity.
The means for generating the magnetic force is such that a plurality of plate-shaped magnets are arranged in parallel below the bottom surface of the container so that the north and south poles are alternately arranged, and the plurality of plate-shaped magnets are arranged. By providing the length in the longitudinal direction across the bottom surface of the container, the gradient of the strength of the magnetic force is set in the planar region in the container so that the cells are aggregated in a parallel row. It is something to generate
Wherein the control means, the means for generating the magnetic force, the moving relatively pattern to the container, the slope of the intensity of the magnetic force in the planar region that is pattern changes, gravity the vessel A cell patterning apparatus characterized in that the position is controlled so that patterning related to aggregation of cells settled by the cell is performed.
前記制御手段が、整列して配置された電磁石のうちの単数または複数の電磁石を選択的に通電する操作を制御して、前記電磁石が配置された領域内における磁力の発生位置を制御するものであることを特徴とする請求項１記載の細胞パターニング装置。 The means for generating the magnetic force is configured by arranging a plurality of electromagnets in an aligned manner.
The control means controls an operation of selectively energizing one or a plurality of electromagnets arranged in an aligned manner to control a position where a magnetic force is generated in a region in which the electromagnets are arranged. The cell patterning apparatus according to claim 1, wherein the cell patterning apparatus is provided.
前記容器に細胞を播種する工程と、前記磁場印加手段により前記容器中で細胞をパターニングする工程と、パターニング後の細胞を培養する工程とを、細胞をパターニングする単位工程とし、
この単位工程を繰り返して細胞の三次元組織を形成することを特徴とする細胞パターニング方法。
A container for accommodating a culture solution to which a magnetic compound is added and cells, and a magnetic field applying means for applying a magnetic force to the culture solution containing the cells contained in the container are provided, and the magnetic field applying means generates a magnetic force. The container includes means and a control means for controlling the position where the magnetic force is applied to the culture solution containing the cells by the means for generating the magnetic force, and the container is such that the cells seeded from above in the culture solution are cultured. The liquid is accommodated so as to gradually settle toward the bottom surface by the action of gravity from the surface side of the liquid, and the means for generating the magnetic force is that a plurality of plate-shaped magnets are provided below the bottom surface of the container. The north pole and the south pole are arranged side by side so as to be arranged alternately, and the length of the plurality of plate-shaped magnets in the longitudinal direction is provided so as to cross the bottom surface of the container, so that the cells can be formed. A gradient of the strength of the magnetic force is generated in the plane region in the container so as to be aggregated in a parallel row , and the control means makes the means for generating the magnetic force relative to the container. manner by pattern transfer, the intensity gradient of the magnetic force in a plane area be to pattern change, the container so that the patterning of the aggregation of cells to settle by gravity is made, controlling the position a fine胞Pa turning method using cell patterning device is intended to,
The step of seeding the cells in the container, the step of patterning the cells in the container by the magnetic field application means, and the step of culturing the cells after patterning are set as unit steps for patterning the cells.
A cell patterning method characterized by repeating this unit process to form a three-dimensional tissue of cells.
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