JP2018074982A - Method of exchanging cytoplasm, method of producing cells, and device - Google Patents
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本発明は、細胞質の交換方法、細胞の製造方法、及び装置に関する。 The present invention relates to a cytoplasm exchange method, a cell production method, and an apparatus.
ある細胞の細胞核を別の細胞の細胞質内へ移植したり、元の細胞核と交換したりすることで、細胞の性質を変える方法は、医療や産業に重要な役割を果たしている。 Methods that change the properties of cells by transplanting the nucleus of one cell into the cytoplasm of another cell or exchanging with the original nucleus play an important role in medicine and industry.
例えば、細胞核を除去した受精卵に、別個体の体細胞由来の核を移植した後、子宮に戻すことでクローン個体を産出する方法や、子宮に戻さず体外で胚盤胞へ発生させて胚性幹細胞 (Embryonic Stem cells: ES細胞) を樹立する体細胞由来胚性幹細胞(nuclear transfer Embryonic stem cells: NtES細胞) 樹立方法などがある。こうして得られたクローン個体やNtES細胞は、畜産や再生医療に発展する基礎研究に寄与している。 For example, after transplanting a nucleus derived from a separate somatic cell into a fertilized egg from which the cell nucleus has been removed, a cloned individual is produced by returning it to the uterus, or by developing it into a blastocyst outside the body without returning to the uterus. There are methods for establishing somatic cell-derived embryonic stem cells (NtES cells) that establish embryonic stem cells (ES cells). The cloned individuals and NtES cells obtained in this way contribute to basic research that develops into livestock and regenerative medicine.
細胞の性質を変える技術には、核移植以外にも、センダイウイルス法やプロトプラスト-ポリエチレングリコール法、電気的細胞融合法、電界集中細胞融合法などの細胞融合技
術がある。これは、２つ以上の細胞を融合させ、１つの細胞を形成する技術である。この方法では、細胞融合後の１つの細胞内には２つ以上の細胞に由来する細胞核と細胞質が存在する。
In addition to nuclear transfer, cell fusion technologies such as Sendai virus method, protoplast-polyethylene glycol method, electrical cell fusion method, and electric field concentration cell fusion method are available as techniques for changing cell properties. This is a technique in which two or more cells are fused to form one cell. In this method, a cell nucleus and cytoplasm derived from two or more cells are present in one cell after cell fusion.
特に、電界集中細胞融合法は、２つの細胞を、細胞核の直径以下の幅や径のスリットや孔 (オリフィス) を介して接触させ、スリットやオリフィスでパルス的に電界を集中させることで、２つの細胞膜の接点で電気的に細胞膜が部分的に破壊され、復元する過程で細胞を融合する方法である（例えば、特許文献１、２参照）。この方法では、オリフィスや流路の最大径や最大幅が細胞核径より小さいことにより、細胞核の混合を防ぐことが可能である（例えば、特許文献１、２参照）。近年、Polydimethyl siloxane (PDMS)を使用して作製された様々な流路を用いて細胞融合法が検討されている。例えば、PDMSで製造された２つの平行流路間をオリフィスのみで繋ぎ、各流路末端に細胞供給口と吸引口をそれぞれ設けた流路を用い、オリフィスで細胞対を形成後、細胞融合させる方法がある。例えば、細胞供給口にJurkat細胞または樹状細胞を導入し、吸引口方向へ流路上で細胞を移動させ、オリフィスでJurkat細胞と樹状細胞を融合させ、Jurkat細胞の細胞質を樹状細胞へ移動させた後、界面活性剤を添加し、Jurkat細胞側の流路に流れを生じさせて両細胞を切り離すことで、細胞質が移動できるようになった（例えば、非特許文献１参照）。 In particular, the electric field intensive cell fusion method brings two cells into contact with each other through slits or holes (orifices) having a width or diameter less than the diameter of the cell nucleus, and concentrates the electric field in a pulsed manner at the slits or orifices. This is a method in which cells are fused in the process of being partially destroyed and restored by electrical contact at the contact of two cell membranes (see, for example, Patent Documents 1 and 2). In this method, since the maximum diameter and the maximum width of the orifice and the flow path are smaller than the cell nucleus diameter, it is possible to prevent mixing of cell nuclei (for example, see Patent Documents 1 and 2). In recent years, cell fusion methods have been studied using various flow paths produced using Polydimethylsiloxane (PDMS). For example, two parallel flow paths manufactured by PDMS are connected only by an orifice, and a flow path in which a cell supply port and a suction port are provided at the end of each flow path is used to form a cell pair at the orifice and then fuse the cells. There is a way. For example, Jurkat cells or dendritic cells are introduced into the cell supply port, cells are moved on the flow path in the direction of the suction port, Jurkat cells and dendritic cells are fused at the orifice, and the Jurkat cell cytoplasm is moved to the dendritic cells. Then, a surfactant was added to cause flow in the flow path on the Jurkat cell side to separate both cells, so that the cytoplasm can be moved (for example, see Non-Patent Document 1).
本発明は、細胞質の交換方法、細胞の製造方法、及び細胞質を交換するための装置を提
供することを目的とする。
An object of the present invention is to provide a method for exchanging cytoplasm, a method for producing cells, and an apparatus for exchanging cytoplasm.
本発明の一実施態様は、第１の細胞の細胞核を有し、第１の細胞の細胞膜と第２の細胞の細胞質とを含む細胞の製造方法であって、第１の細胞の細胞質を除去する第１の工程と、第２の細胞の細胞質を、前記第１の細胞へ導入する第２の工程を含む、製造方法である。第１の工程の後で第２の工程が行われてもよい。その場合、第１の工程において、第１の細胞の細胞質が９０％以上除去され、第２の工程において、第２の細胞の細胞質が、９０％以上の細胞質が除去された前記第１の細胞へ導入されてもよい。また、第１の工程と第２の工程が同時に行われてもよい。その場合、第１の細胞の細胞質の４０％〜６０％と、第２の細胞の細胞質の４０％〜６０％とが交換される細胞質交換工程によって、第１の工程と第２の工程が同時に行われてもよく、前記細胞質交換工程が２回以上繰り返されてもよい。前記いずれかの細胞の製造方法において、前記製造された細胞が、９０％以上の第１の細胞の細胞膜と、９０％以上の第２の細胞の細胞質を含んでもよく、前記製造された細胞が、９５％以上の第１の細胞の細胞膜と、９５％以上の第２の細胞の細胞質を含んでもよい。 One embodiment of the present invention is a method for producing a cell having a cell nucleus of a first cell and comprising a cell membrane of the first cell and a cytoplasm of the second cell, wherein the cytoplasm of the first cell is removed And a second step of introducing the cytoplasm of the second cell into the first cell. The second step may be performed after the first step. In that case, in the first step, the cytoplasm of the first cell is removed by 90% or more, and in the second step, the cytoplasm of the second cell is removed by removing the cytoplasm of 90% or more. May be introduced. Further, the first step and the second step may be performed simultaneously. In that case, the first step and the second step are simultaneously performed by a cytoplasm exchange step in which 40% to 60% of the cytoplasm of the first cell and 40% to 60% of the cytoplasm of the second cell are exchanged. The cytoplasm exchange process may be repeated twice or more. In any one of the methods for producing a cell, the produced cell may contain 90% or more of the cell membrane of the first cell and 90% or more of the cytoplasm of the second cell. 95% or more of the cell membrane of the first cell and 95% or more of the cytoplasm of the second cell.
本発明のさらなる一実施態様は、第１の流路と第２の流路を備え、第１の流路の壁と第２の流路の壁は孔で連通している流路を用いて、第１の細胞の細胞核を有し、第１の細胞の細胞膜と第２の細胞の細胞質とを含む細胞の製造方法であって、第１の流路において第１の細胞を、第２の流路において中間体を、前記孔に誘導する第１の工程と、誘電泳動力により、前記孔を介して、第１の細胞と中間体との第１の対を形成させる第２の工程と、第１の細胞と中間体とを融合する第３の工程と、第１の細胞の細胞質を中間体へ移動させる第４の工程と、第１の細胞と中間体とを切り離す第５の工程と、第２の流路において第２の細胞を前記孔に誘導する第６の工程と、誘電泳動力により、前記孔を介して、第１の細胞と第２の細胞の第２の対を形成させる第７の工程と、第１の細胞と第２の細胞とを融合する第８の工程と、第２の細胞の細胞質を第１の細胞へ移動させる第９の工程と、第１の細胞と第２の細胞とを切り離す第１０の工程と、を含む細胞の製造方法である。第４の工程と、第９の工程とにおいて、第１の流路と第２の流路の流速差を利用して細胞質を移動させてもよい。第５の工程と、第１０の工程とにおいて、第１の流路と第２の流路の流速差を利用して細胞を切り離してもよい。第１の工程及び第６の工程において、第１の吸引口からポンプで吸引することによって細胞を前記孔へ誘導してもよい。第２の工程と第７の工程において、交流電界中の誘電泳動力により前記対を形成させてもよい。第３の工程と第８の工程において、前記対に電圧を印加することで融合させてもよい。 According to a further embodiment of the present invention, there is provided a flow path including a first flow path and a second flow path, wherein a wall of the first flow path and a wall of the second flow path are communicated by a hole. A method for producing a cell having a cell nucleus of a first cell and comprising a cell membrane of a first cell and a cytoplasm of a second cell, wherein the first cell is A first step of guiding an intermediate in the channel to the hole; a second step of forming a first pair of the first cell and the intermediate through the hole by dielectrophoretic force; A third step of fusing the first cell and the intermediate, a fourth step of moving the cytoplasm of the first cell to the intermediate, and a fifth step of separating the first cell and the intermediate. And a sixth step of guiding the second cell to the hole in the second flow path, and a second pair of the first cell and the second cell through the hole by dielectrophoretic force. A seventh step, an eighth step of fusing the first cell and the second cell, a ninth step of moving the cytoplasm of the second cell to the first cell, And a tenth step of separating the cells and the second cells. In the fourth step and the ninth step, the cytoplasm may be moved using the flow rate difference between the first flow path and the second flow path. In the fifth step and the tenth step, the cells may be separated using the flow rate difference between the first channel and the second channel. In the first step and the sixth step, cells may be guided to the hole by sucking with a pump from the first suction port. In the second step and the seventh step, the pair may be formed by a dielectrophoretic force in an alternating electric field. In the third step and the eighth step, fusion may be performed by applying a voltage to the pair.
本発明のさらなる一実施態様は、第１の流路と第２の流路を備え、第１の流路の壁と第２の流路の壁は孔で連通している流路を用いて、第１の細胞の細胞核を有し、第１の細胞の細胞膜と第２の細胞の細胞質とを含む細胞の製造方法であって、第１の流路において第１の細胞を、第２の流路において中間体を、前記孔に誘導する第１の工程と、誘電泳動力により、前記孔を介して、第１の細胞と中間体との第１の対を形成させる第２の工程と、第１の細胞と中間体とを融合する第３の工程と、第１の細胞と中間体とを切り離す第５の工程と、第２の流路において新たな中間体を、前記孔に誘導する第１１の工程と、誘電泳動力により、前記孔を介して、第１の細胞と新たな中間体との新たな第１の対を形成させる第１２の工程と、第１の細胞と新たな中間体とを融合する第１３の工程と、第１の細胞と新たな中間体とを切り離す第１４の工程と、を含み、前記中間体として第２の細胞を用いる、細胞の製造方法である。第１１の工程から第１４の工程までがさらに１回以上繰り返されてもよい。前記いずれかの細胞の製造方法において、第４の工程及び第９の工程において、それぞれ第２の吸引口及び第３の吸引口から、ポンプで流速差発生部を経由して吸引することによって細胞質を移動させてもよい。第５の工程において、第１の細胞を誘電泳動力によって前記孔で保持しながら、第２の吸引口から、ポンプで流速差発生部を経
由して吸引することによって第１の細胞から中間体が切り離されてもよい。前記孔の最大幅または最大径は第１の細胞の核の径及び第２の細胞の核の径より小さくてもよい。前記孔の最大幅または最大径は６μｍ以下であってもよい。前記流速差は、細胞質が流入する側の細胞側の流路内の流速が、細胞質が流出する細胞側の流路内の流速より速くてもよい。第１の流路及び第２の流路が実質的に平行であってもよい。前記第１の細胞は体細胞であり、前記第２の細胞は幹細胞であってもよい。前記幹細胞は、ｉＰＳ細胞、ＥＳ細胞、神経幹細胞、上皮幹細胞、肝幹細胞、生殖幹細胞、造血幹細胞、間葉幹細胞、骨格筋幹細胞からなる群から選択されてもよく、前記体性細胞は、線維芽細胞または血球細胞であってもよい。
According to a further embodiment of the present invention, there is provided a flow path including a first flow path and a second flow path, wherein a wall of the first flow path and a wall of the second flow path are communicated by a hole. A method for producing a cell having a cell nucleus of a first cell and comprising a cell membrane of a first cell and a cytoplasm of a second cell, wherein the first cell is A first step of guiding an intermediate in the channel to the hole; a second step of forming a first pair of the first cell and the intermediate through the hole by dielectrophoretic force; , A third step of fusing the first cell and the intermediate, a fifth step of separating the first cell and the intermediate, and a new intermediate in the second channel is guided to the hole. An eleventh step, a twelfth step of forming a new first pair of the first cell and a new intermediate through the hole by dielectrophoretic force, and the first cell A method for producing a cell, comprising: a thirteenth step of fusing a new intermediate; and a fourteenth step of separating the first cell and the new intermediate, wherein the second cell is used as the intermediate. It is. The process from the 11th process to the 14th process may be repeated one more times. In any one of the above-described methods for producing cells, in the fourth step and the ninth step, the cytoplasm is obtained by sucking from the second suction port and the third suction port, respectively, with a pump through the flow rate difference generation unit. May be moved. In the fifth step, while the first cell is held in the hole by dielectrophoretic force, the intermediate from the first cell is sucked from the second suction port via the flow rate difference generation unit by a pump. May be cut off. The maximum width or maximum diameter of the pores may be smaller than the diameter of the first cell nucleus and the diameter of the second cell nucleus. The maximum width or maximum diameter of the holes may be 6 μm or less. The flow rate difference may be such that the flow rate in the flow channel on the cell side where the cytoplasm flows in is faster than that in the flow channel on the cell side where the cytoplasm flows out. The first channel and the second channel may be substantially parallel. The first cell may be a somatic cell, and the second cell may be a stem cell. The stem cells may be selected from the group consisting of iPS cells, ES cells, neural stem cells, epithelial stem cells, liver stem cells, reproductive stem cells, hematopoietic stem cells, mesenchymal stem cells, skeletal muscle stem cells, and the somatic cells are fibroblasts It may be a cell or a blood cell.
本発明のさらなる一実施態様は、細胞の細胞質を交換するための装置であって、第１の流路及び第２の流路と、第１の流路及び第２の流路のそれぞれに接続する細胞供給部と、第１の流路の壁と第２の流路の壁との間に設けられた孔と、孔を挟んで設けられた電極と、第１の流路及び第２の流路の流速を調節する流速調節部と、前記流速調節部に設けられた吸引口と、各吸引口に設けられたポンプと、直流交流信号発生装置と、を備えた装置である。前記孔の最大幅または最大径は、前記細胞の細胞核の径よりも小さくてもよい。前記孔の最大幅または最大径は６μｍ以下であってもよい。前記孔と前記吸引口間に圧力緩衝部が設けられてもよい。画像認識装置が設けられた顕微鏡を備え、前記画像認識装置と前記ポンプに、流速制御装置が設けられてもよい。第１の流路及び第２の流路が実質的に平行であってもよい。 A further embodiment of the present invention is an apparatus for exchanging the cytoplasm of a cell, which is connected to each of a first channel and a second channel, and a first channel and a second channel. A cell supply section, a hole provided between the wall of the first flow path and the wall of the second flow path, an electrode provided across the hole, the first flow path and the second flow path A flow rate adjusting unit that adjusts the flow rate of the flow path, a suction port provided in the flow rate adjusting unit, a pump provided in each suction port, and a DC / AC signal generator. The maximum width or maximum diameter of the pores may be smaller than the diameter of the cell nucleus of the cell. The maximum width or maximum diameter of the holes may be 6 μm or less. A pressure buffering part may be provided between the hole and the suction port. A microscope provided with an image recognition device may be provided, and a flow velocity control device may be provided in the image recognition device and the pump. The first channel and the second channel may be substantially parallel.
本発明のさらなる一実施態様は、第１の細胞の細胞質を除去する第１の工程と、第２の細胞の細胞質を第１の細胞へ導入する第２の工程を含む、細胞質の交換方法であって、第１の工程の後で第２の工程が行われる、第１の細胞の細胞質の交換方法である。 A further embodiment of the present invention is a cytoplasmic exchange method comprising a first step of removing the cytoplasm of the first cell and a second step of introducing the cytoplasm of the second cell into the first cell. A method for exchanging the cytoplasm of a first cell, wherein the second step is performed after the first step.
本発明のさらなる一実施態様は、第１の細胞の細胞質を除去する第１の工程と、第２の細胞の細胞質を第１の細胞へ導入する第２の工程を含む、細胞質の交換方法であって、第１の細胞の細胞質の４０％〜６０％と、第２の細胞の細胞質の４０％〜６０％とが交換される第１の細胞質交換工程によって、第１の工程と第２の工程が同時に行われ、細胞質交換工程後の第１の細胞の細胞質の４０％〜６０％と、新たな第２の細胞の細胞質の４０％〜６０％とが交換される第２の細胞質交換工程がさらに行われる、第１の細胞の細胞質の交換方法である。第２の細胞質交換工程がさらに１回以上行われてもよい。 A further embodiment of the present invention is a cytoplasmic exchange method comprising a first step of removing the cytoplasm of the first cell and a second step of introducing the cytoplasm of the second cell into the first cell. Wherein the first step and the second step are performed by a first cytoplasm exchange step in which 40% to 60% of the cytoplasm of the first cell and 40% to 60% of the cytoplasm of the second cell are exchanged. A second cytoplasm exchange step in which the steps are performed simultaneously and 40% to 60% of the cytoplasm of the first cell after the cytoplasm exchange step is exchanged with 40% to 60% of the cytoplasm of the new second cell Is a first cell cytoplasm exchange method. The second cytoplasm exchange step may be further performed once or more.
本発明によって、細胞質の交換方法、細胞の製造方法、及び細胞質を交換するための装置を提供することができるようになった。 According to the present invention, a cytoplasm exchange method, a cell production method, and an apparatus for exchanging cytoplasm can be provided.
以下、実施例を挙げながら、本発明の実施形態を詳細に述べる。 Hereinafter, embodiments of the present invention will be described in detail with reference to examples.
本発明の目的、特徴、利点、及びそのアイデアは、本明細書の記載により、当業者には明らかであり、本明細書の記載から、当業者であれば、容易に本発明を再現できる。以下
に記載された発明の実施の形態及び具体的に実施例などは、本発明の好ましい実施態様を示すものであり、例示又は説明のために示されているのであって、本発明をそれらに限定するものではない。本明細書で開示されている本発明の意図並びに範囲内で、本明細書の記載に基づき、様々な改変並びに修飾ができることは、当業者にとって明らかである。
The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.
＝＝細胞質の交換方法＝＝
＜実施形態Ｉ＞
本発明の一実施形態に係る第１の細胞の細胞質の交換方法は、第１の細胞の細胞質を除去する第１の工程と、第２の細胞の細胞質を第１の細胞へ導入する第２の工程を含む、細胞質の交換方法であって、
（１）第１の工程の後で第２の工程が行われる、または
（２）第１の細胞の細胞質の４０％〜６０％と、第２の細胞の細胞質の４０％〜６０％とが交換される第１の細胞質交換工程によって、第１の工程と第２の工程が同時に行われる、方法である。
== Cytoplasm exchange method ==
<Embodiment I>
The first cell cytoplasm exchange method according to an embodiment of the present invention includes a first step of removing the cytoplasm of the first cell, and a second step of introducing the cytoplasm of the second cell into the first cell. A cytoplasmic exchange method comprising the steps of:
(1) the second step is performed after the first step, or (2) 40% -60% of the cytoplasm of the first cell and 40% -60% of the cytoplasm of the second cell In the method, the first step and the second step are simultaneously performed by the first cytoplasm exchange step to be exchanged.
（２）の場合、細胞質交換工程後の第１の細胞の細胞質の４０％〜６０％と、新たな第２の細胞の細胞質の４０％〜６０％とが交換される第２の細胞質交換工程がさらに行われてもよく、この第２の細胞質交換工程がさらに１回以上行われてもよい。この工程を繰り返すことによって、第１の細胞の細胞質が第２の細胞の細胞質に交換される割合が高くなる。本発明の一実施形態に係る細胞質の交換方法を用いて、第１の細胞の細胞核を有し、第２の細胞の細胞核を有さず、第１の細胞の細胞膜と第２の細胞の細胞質とを含む細胞を製造することができる。こうして製造された細胞において、第１の細胞の細胞膜の割合は特に限定されないが、５０％以上であることが好ましく、６０％以上であることがより好ましく、７０％以上であることがさらに好ましく、８０％以上であることがさらに好ましく、９０％以上であることがさらに好ましく、９５％以上であることがさらに好ましい。また、第２の細胞の細胞質の割合は特に限定されないが、５０％以上であることが好ましく、６０％以上であることがより好ましく、７０％以上であることがさらに好ましく、８０％以上であることがさらに好ましく、９０％以上であることがさらに好ましく、９５％以上であることがさらに好ましい。 In the case of (2), the second cytoplasm exchange step in which 40% to 60% of the cytoplasm of the first cell after the cytoplasm exchange step and 40% to 60% of the cytoplasm of the new second cell are exchanged. May be further performed, and the second cytoplasmic exchange step may be further performed one or more times. By repeating this process, the rate at which the cytoplasm of the first cell is exchanged for the cytoplasm of the second cell is increased. Using the cytoplasmic exchange method according to one embodiment of the present invention, the cell nuclei of the first cells, the cell nuclei of the second cells, the cell nuclei of the first cells, and the cytoplasm of the second cells Can be produced. In the cells thus produced, the ratio of the cell membrane of the first cell is not particularly limited, but is preferably 50% or more, more preferably 60% or more, and even more preferably 70% or more, It is more preferably 80% or more, further preferably 90% or more, and further preferably 95% or more. The cytoplasm ratio of the second cell is not particularly limited, but is preferably 50% or more, more preferably 60% or more, further preferably 70% or more, and 80% or more. More preferably, it is 90% or more, more preferably 95% or more.
以下、上記交換方法を用いて、細胞を製造する一実施態様を詳細に説明する。 Hereinafter, one embodiment for producing cells using the above exchange method will be described in detail.
＝＝細胞の製造方法＝＝
ここでは、図１を用いて、上記交換方法（１）を用いて行われる、本発明の一実施形態に係る細胞の製造方法を具体的に説明するが、本発明は本実施形態に限定されない。本実施形態では、第１の流路と第２の流路を備え、第１の流路の壁と第２の流路の壁は孔で連通している流路を用い、第１の細胞の細胞核を有し、第２の細胞の細胞核を有さず、第１の細胞の細胞膜と第２の細胞の細胞質とを含む細胞を製造する。
== Method for producing cells ==
Here, although the manufacturing method of the cell which concerns on one Embodiment of this invention performed using the said exchange method (1) using FIG. 1 is demonstrated concretely, this invention is not limited to this embodiment. . In the present embodiment, the first cell is provided with a first channel and a second channel, and the first cell wall and the second channel wall are in communication with each other through holes. And a cell containing the cell membrane of the first cell and the cytoplasm of the second cell without the cell nucleus of the second cell.
第１の工程として、まず、第１の流路にあって、残したい細胞核１０１０と、除去したい細胞質１０２０と、細胞膜１０２５と、を有する第１の細胞１０３０と、第２の流路にある中間体１０４０を、流路中の溶液の流れ１０５０によって、両方の流路の壁を貫通して設けられた孔１０８０の入り口を構成するそれぞれの孔近傍に誘導する（図１−１）。必要に応じて交流１０６０電界を発生させ、その中で細胞を誘導してもよい。流路の壁は絶縁体１０７０で構成されるのが好ましい。第１の流路内の流れ１０５０と第２の流路内の流れ１０５０の方向は特に限定されないが、実質的に平行であることが好ましい。溶液は、細胞を生きた状態で操作できるものであれば特に限定されず、例えば生理食塩水や細胞培養用培地などを例示できる。中間体は特に限定されず、対になった細胞と融合して細胞の細胞質を受け入れられるものであればよいが、具体的な例としては、細胞やリポソームが例示できる。細胞である場合、第１の細胞または第２の細胞と同じ種類であっても異
なる種類であってもよい。孔１０８０の形状は特に限定されず、スリット上、円状、長円上であってもよく、壁の厚みと同じ深さを有するため、厚い壁の場合は流路状であってもよい。
As the first step, first, in the first channel, the first cell 1030 having the cell nucleus 1010 to be left, the cytoplasm 1020 to be removed, and the cell membrane 1025, and the intermediate in the second channel. The body 1040 is guided by the solution flow 1050 in the flow path to the vicinity of each hole constituting the entrance of the hole 1080 provided through the walls of both flow paths (FIG. 1-1). If necessary, an AC 1060 electric field may be generated to induce cells therein. The wall of the flow path is preferably composed of an insulator 1070. The directions of the flow 1050 in the first flow path and the flow 1050 in the second flow path are not particularly limited, but are preferably substantially parallel. The solution is not particularly limited as long as cells can be manipulated in a live state, and examples thereof include physiological saline and a cell culture medium. The intermediate is not particularly limited as long as it is capable of accepting the cytoplasm of the cell by fusing with a pair of cells, and specific examples include cells and liposomes. When it is a cell, it may be the same type as the first cell or the second cell, or a different type. The shape of the hole 1080 is not particularly limited, and may be on a slit, a circle, or an ellipse, and since it has the same depth as the thickness of the wall, it may have a channel shape in the case of a thick wall.
第２の工程として、誘電泳動力により、孔１０８０を挟んで第１の細胞１０３０と中間体１０４０の第１の対を形成させる（図１−２）。誘電泳動力は、交流１０６０印加による電界集中により流路の壁で生じる。 As a second step, a first pair of the first cell 1030 and the intermediate 1040 is formed with the dielectrophoretic force across the hole 1080 (FIG. 1-2). The dielectrophoretic force is generated on the wall of the flow path due to electric field concentration caused by application of AC 1060.
第３の工程として、第１の対に電圧を印加１０９０することで、第１の細胞１０３０と中間体１０４０とを融合して第１の融合細胞１１００を作製する (図１−３)。この時印
加する電圧は、従来細胞融合に用いられることが知られている強さとすることができるが、０．１〜５０Ｖであることが好ましく、０．５Ｖ〜２０Ｖであることがより好ましく、１〜１０Ｖであることがさらに好ましく、１〜３Ｖであることがさらに好ましく、約２Ｖであることがさらに好ましい。また印加する時間は、特に限定されないが、１μｓｅｃ〜１ｍｓｅｃであることが好ましく、１μｓｅｃ〜５００μｓｅｃであることがより好ましく、１μｓｅｃ〜１００μｓｅｃであることがさらに好ましいが、可能な限り短い時間であることがより好ましい。中間体１０４０が細胞である場合、本工程では、細胞融合後、第１の細胞１０３０と中間体１０４０の細胞質がお互いに混入する。
As a third step, a voltage is applied 1090 to the first pair to fuse the first cell 1030 and the intermediate 1040 to produce the first fused cell 1100 (FIGS. 1-3). The voltage applied at this time can be a strength that is conventionally known for cell fusion, but is preferably 0.1 to 50 V, more preferably 0.5 V to 20 V, More preferably, it is 1-10V, It is more preferable that it is 1-3V, It is more preferable that it is about 2V. The application time is not particularly limited, but is preferably 1 μsec to 1 msec, more preferably 1 μsec to 500 μsec, further preferably 1 μsec to 100 μsec, but it should be as short as possible. More preferred. When the intermediate 1040 is a cell, the cytoplasm of the first cell 1030 and the intermediate 1040 is mixed with each other after cell fusion in this step.
第４の工程として、第１の細胞１０３０の細胞質１０２０を中間体１０４０へ移動させる（図１−４）。この細胞質１０２０の移動は、例えば、第２の流路内の流れ１１２０の速度を、第１の流路内の流れ１１１０の速度より大きくし、その流速差を利用することにより行うことができる。このとき、第１の流路内の流れ１１１０の流速は０、つまり第１の流路内に流れのない状態であってもよい。第１の流路内の流れ１１１０と第２の流路内の流れ１１２０の方向は特に限定されないが、実質的に平行であることが好ましい。ここで、孔１０８０の最大幅または最大径を、第１の細胞の核の径より小さくしておくことにより、第１の細胞１０３０の細胞核１０１０は、孔１０８０を通過できずに第１の流路内に残る。具体的には、孔１０８０の最大幅または最大径は、第１の細胞１０３０の種類によって適宜調整すればよいが、２０μｍ以下であることが好ましく、１０μｍ以下であることがより好ましく、６μｍ以下であることがさらに好ましい。なお、移動させる細胞質の割合は特に限定されないが、５０％以上であることが好ましく、６０％以上であることがより好ましく、７０％以上であることがさらに好ましく、８０％以上であることがさらに好ましく、９０％以上であることがさらに好ましく、９５％以上であることがさらに好ましい。 As a fourth step, the cytoplasm 1020 of the first cell 1030 is moved to the intermediate 1040 (FIGS. 1-4). The movement of the cytoplasm 1020 can be performed, for example, by making the velocity of the flow 1120 in the second flow channel larger than the velocity of the flow 1110 in the first flow channel and using the flow rate difference. At this time, the flow rate of the flow 1110 in the first flow path may be 0, that is, there may be no flow in the first flow path. The directions of the flow 1110 in the first flow path and the flow 1120 in the second flow path are not particularly limited, but are preferably substantially parallel. Here, by setting the maximum width or maximum diameter of the hole 1080 to be smaller than the diameter of the nucleus of the first cell, the cell nucleus 1010 of the first cell 1030 cannot pass through the hole 1080 and the first flow. Remain in the street. Specifically, the maximum width or maximum diameter of the hole 1080 may be appropriately adjusted depending on the type of the first cell 1030, but is preferably 20 μm or less, more preferably 10 μm or less, and 6 μm or less. More preferably it is. The ratio of cytoplasm to be moved is not particularly limited, but is preferably 50% or more, more preferably 60% or more, further preferably 70% or more, and further preferably 80% or more. Preferably, it is 90% or more, more preferably 95% or more.
第５の工程として、第１の細胞１０３０と中間体１０４を切り離す（図１−５）。第１の細胞１０３５を孔１０８０の入り口で保持しながら、細胞質１０２０を受け取った中間体１０４５を切り離すのが好ましい。この切り離しは、例えば、第２の流路内の流れ１１２０の速度を、第１の流路内の流れ１１１０の速度より大きくし、その流速差を利用することにより行うことができる。第１の流路内の流れ１１１０の流速は０、つまり第１の流路内に流れのない状態であってもよい。 As a fifth step, the first cell 1030 and the intermediate 104 are separated (FIG. 1-5). The intermediate 1045 that received the cytoplasm 1020 is preferably detached while holding the first cell 1035 at the entrance of the hole 1080. This separation can be performed, for example, by making the speed of the flow 1120 in the second flow path larger than the speed of the flow 1110 in the first flow path and using the flow rate difference. The flow velocity of the flow 1110 in the first flow path may be 0, that is, there may be no flow in the first flow path.
このように、本実施形態では、第１の工程から第５の工程までの工程において、中間体を用いることにより、第１の細胞の細胞質を除去することができる。 Thus, in this embodiment, the cytoplasm of the first cell can be removed by using the intermediate in the steps from the first step to the fifth step.
第６の工程として、第１の細胞１０３０に移植したい細胞質１１３０と、必要のない細胞核１１４０を有する第２の細胞１１５０を、流れ１０５０により、孔１０８０の入り口を構成する、第２の流路側の孔近傍に誘導する (図１−６)。必要に応じて交流１０６０
電界を発生させ、その中で細胞を誘導してもよい。
As a sixth step, a cytoplasm 1130 to be transplanted into the first cell 1030 and a second cell 1150 having an unnecessary cell nucleus 1140 are formed by the flow 1050 to form the entrance of the hole 1080. Guide to the vicinity of the hole (Fig. 1-6). AC 1060 if necessary
An electric field may be generated to induce the cells therein.
第７の工程として、誘電泳動力により、孔１０８０を挟んで第１の細胞１０３０と第２の細胞１１５０の第２の対を形成させる (図１−７)。誘電泳動力は、交流１０６０印加
による電界集中により流路の壁で生じる。
As a seventh step, the second pair of the first cell 1030 and the second cell 1150 is formed by sandwiching the hole 1080 by the dielectrophoretic force (FIGS. 1-7). The dielectrophoretic force is generated on the wall of the flow path due to electric field concentration caused by application of AC 1060.
第８の工程として、第２の対に電圧を印加１０９０することで第１の細胞１０３０と第２の細胞１１５０を融合して第２の融合細胞１１６０を作製する (図１−８)。この時印
加する電圧は、従来細胞融合に用いられることが知られている強さであればよいが、１〜１０Ｖであることが好ましく、２Ｖであることがさらに好ましい。また印加する時間は、１００μｓｅｃであることが好ましく、１００μｓｅｃ以下で可能な限り短い時間であることがさらに好ましい。
As an eighth step, a voltage is applied 1090 to the second pair to fuse the first cell 1030 and the second cell 1150 to produce a second fused cell 1160 (FIGS. 1-8). The voltage to be applied at this time may be any strength that is conventionally used for cell fusion, but is preferably 1 to 10 V, and more preferably 2 V. The application time is preferably 100 μsec, more preferably 100 μsec or less and as short as possible.
第９の工程として、第２の細胞１１５０の細胞質１１３０を第１の細胞１０３５へ移動させる (図１−９)。この移動は、例えば、第１の流路内の流れ１１１０の速度を、第２
の流路内の流れ１１２０の速度より大きくし、その流速差を利用することにより行うことができる。第１の流路内の流れ１１２０の流速は０、つまり第２の流路内に流れのない状態であってもよい。ここで、孔１０８０の最大幅または最大径を、第２の細胞の核の径より小さくしておくことにより、第２の細胞１１５０の細胞核１１４０は、孔１０８０を通過できずに第２の流路内に残る。具体的には、孔１０８０の最大幅または最大径は、第２の細胞１１５０の種類によって適宜調整すればよいが、２０μｍ以下であることが好ましく、１０μｍ以下であることがより好ましく、６μｍ以下であることがさらに好ましい。なお、移動させる細胞質の割合は特に限定されないが、５０％以上であることが好ましく、６０％以上であることがより好ましく、７０％以上であることがさらに好ましく、８０％以上であることがさらに好ましく、９０％以上であることがさらに好ましく、９５％以上であることがさらに好ましい。
As a ninth step, the cytoplasm 1130 of the second cell 1150 is moved to the first cell 1035 (FIG. 1-9). For example, this movement may cause the velocity of the flow 1110 in the first flow path to be
It can be performed by making it larger than the speed of the flow 1120 in the flow path and utilizing the flow rate difference. The flow velocity of the flow 1120 in the first flow path may be 0, that is, there may be no flow in the second flow path. Here, by making the maximum width or maximum diameter of the hole 1080 smaller than the diameter of the nucleus of the second cell, the cell nucleus 1140 of the second cell 1150 cannot pass through the hole 1080 and the second flow. Remain in the street. Specifically, the maximum width or maximum diameter of the hole 1080 may be appropriately adjusted depending on the type of the second cell 1150, but is preferably 20 μm or less, more preferably 10 μm or less, and more preferably 6 μm or less. More preferably it is. The ratio of cytoplasm to be moved is not particularly limited, but is preferably 50% or more, more preferably 60% or more, further preferably 70% or more, and further preferably 80% or more. Preferably, it is 90% or more, more preferably 95% or more.
第１０の工程として、第１の細胞１０３０を、第２の細胞１１５０から切り離す (図１−１０)。こうして、第１の細胞１０３０の細胞質１０２０を、第２の細胞１１５０の細
胞質１１３０に交換することによって、第１の細胞１０３０の細胞核１０１０を有し、第２の細胞１１５０の細胞核１１４０を有さず、第１の細胞１０３０の細胞膜と第２の細胞１１５０の細胞質１１３０とを含む細胞１１７０の製造が完了する。
As a tenth step, the first cell 1030 is detached from the second cell 1150 (FIG. 1-10). Thus, by replacing the cytoplasm 1020 of the first cell 1030 with the cytoplasm 1130 of the second cell 1150, the cell nucleus 1010 of the first cell 1030 is obtained, and the cell nucleus 1140 of the second cell 1150 is not provided. The production of the cell 1170 including the cell membrane of the first cell 1030 and the cytoplasm 1130 of the second cell 1150 is completed.
このように、第６の工程から第１０の工程までの工程によって、第２の細胞の細胞質を第１の細胞へ導入することができる。 Thus, the cytoplasm of the second cell can be introduced into the first cell by the steps from the sixth step to the tenth step.
本方法において、第１の細胞１０３０と第２の細胞１１５０の細胞種は特に限定されないが、第１の細胞１０３０は体細胞であることが好ましく、線維芽細胞または血球細胞であることがさらに好ましい。また、第２の細胞１１５０は幹細胞であることが好ましく、ｉＰＳ細胞、ＥＳ細胞、神経幹細胞、上皮幹細胞、肝幹細胞、生殖幹細胞、造血幹細胞、間葉幹細胞、骨格筋幹細胞からなる群から選択されるのがさらに好ましい。第１の細胞１０３０は体細胞とし、第２の細胞１１５０を幹細胞とすることにより、例えば、幹細胞の細胞核を体細胞の細胞核に交換したことと同様の効果が得られる。 In this method, the cell types of the first cell 1030 and the second cell 1150 are not particularly limited, but the first cell 1030 is preferably a somatic cell, and more preferably a fibroblast or a blood cell. . The second cell 1150 is preferably a stem cell, and is selected from the group consisting of iPS cells, ES cells, neural stem cells, epithelial stem cells, hepatic stem cells, reproductive stem cells, hematopoietic stem cells, mesenchymal stem cells, and skeletal muscle stem cells. Is more preferable. By using the first cell 1030 as a somatic cell and the second cell 1150 as a stem cell, for example, an effect similar to that obtained by replacing the cell nucleus of the stem cell with the cell nucleus of the somatic cell can be obtained.
＜実施形態ＩＩ＞
ここでは、上記交換方法（２）を用いて行われる、本発明の一実施形態に係る細胞の製造方法を具体的に説明するが、本発明は本実施形態に限定されない。本実施形態では、第１の流路と第２の流路を備え、第１の流路の壁と第２の流路の壁は孔で連通している流路を用い、第１の細胞の細胞核を有し、第２の細胞の細胞核を有さず、第１の細胞の細胞膜と第２の細胞の細胞質とを含む細胞を製造する。第１の工程から第３の工程は、実施形態Ｉに準じるが、中間体として、第２の細胞を用いる。第３の工程終了後、第１の細胞と第２の細胞の細胞質がお互いに混入する。このように、本実施形態では、第１の細胞の細胞
質を除去する第１の工程と、第２の細胞の細胞質を第１の細胞へ導入する第２の工程が同時に行われる。この際に、第１の対を融合させたままで、長時間放置すると、第１の細胞と第２の細胞の間で細胞質が均一になるが、交換される細胞質の割合は特に限定されず、第１の細胞の細胞質の１０％〜２５％と、第２の細胞の細胞質の１０％〜２５％とが交換されることが好ましく、第１の細胞の細胞質の２５％〜４０％と、第２の細胞の細胞質の２５％〜４０％とが交換されることがより好ましく、第１の細胞の細胞質の４０％〜６０％と、第２の細胞の細胞質の４０％〜６０％とが交換されることがさらに好ましい。そして、その後、第１の細胞と中間体とを切り離す第５の工程を行う。第５の工程の詳細は、実施形態ＩＩに準じる。
<Embodiment II>
Here, although the manufacturing method of the cell which concerns on one Embodiment of this invention performed using the said exchange method (2) is demonstrated concretely, this invention is not limited to this embodiment. In the present embodiment, the first cell is provided with a first channel and a second channel, and the first cell wall and the second channel wall are in communication with each other through holes. And a cell containing the cell membrane of the first cell and the cytoplasm of the second cell without the cell nucleus of the second cell. The first to third steps are in accordance with Embodiment I, but the second cell is used as an intermediate. After completion of the third step, the cytoplasm of the first cell and the second cell are mixed with each other. Thus, in the present embodiment, the first step of removing the cytoplasm of the first cell and the second step of introducing the cytoplasm of the second cell into the first cell are performed simultaneously. In this case, if the first pair is left fused for a long time, the cytoplasm becomes uniform between the first cell and the second cell, but the ratio of the exchanged cytoplasm is not particularly limited, Preferably, 10% to 25% of the cytoplasm of the first cell and 10% to 25% of the cytoplasm of the second cell are exchanged, 25% to 40% of the cytoplasm of the first cell, More preferably, 25% to 40% of the cytoplasm of the second cell is exchanged, and 40% to 60% of the cytoplasm of the first cell and 40% to 60% of the cytoplasm of the second cell are exchanged. More preferably. Thereafter, a fifth step of separating the first cell and the intermediate is performed. The details of the fifth step are in accordance with Embodiment II.
さらに、第２の流路において新たな第２の細胞を、前記孔に誘導する第１１の工程と、誘電泳動力により、前記孔を介して、第１の細胞と第２の細胞との新たな第１の対を形成させる第１２の工程と、第１の細胞と第２の細胞とを融合する第１３の工程と、第１の細胞と第２の細胞とを切り離す第１４の工程と、を行ってもよい。第１１〜１４の工程の詳細は、実施形態Ｉの第１〜３、５の工程に準じる。 Further, an eleventh step of guiding a new second cell in the second channel to the hole, and a new migration of the first cell and the second cell through the hole by dielectrophoretic force. A twelfth step of forming a first pair, a thirteenth step of fusing the first cell and the second cell, and a fourteenth step of separating the first cell and the second cell. , May be performed. The details of the 11th to 14th steps are in accordance with the 1st to 3rd and 5th steps of the embodiment I.
この後、第１１〜１４の工程をさらに１回以上、繰り返してもよいが、１回繰り返すことが好ましく、２回以上繰り返すことがより好ましく、５回以上繰り返すことがさらに好ましい。それによって、交換される細胞質の割合を高めることができるようになる。 Thereafter, the steps 11 to 14 may be repeated one or more times, but are preferably repeated once, more preferably two or more times, and even more preferably five or more times. Thereby, the percentage of cytoplasm exchanged can be increased.
＝＝細胞質を交換するための装置＝＝
以下、上記の方法を実行することができる本発明の装置の一実施態様を、図２（１）を用いて詳細に説明する。本発明の装置は、細胞の細胞質を交換するための装置であって、第１の流路２０３０及び第２の流路２０４０と、第１の流路２０３０及び第２の流路２０４０のそれぞれ接続する第１の細胞供給部２０１０と第２の細胞供給部２０２０と、第１の流路の壁と第２の流路の壁との間に設けられた孔２０５０と、孔２０５０を挟んで設けられた電極２０８０と、第１の流路２０３０及び第２の流路２０４０の流速を調節する流速調節部２０６０と、流速調節部２０６０に設けられた吸引路２０７０と、各吸引口２０７１，２０７２，２０７３に設けられたポンプ２２１１，２２１２，２２１３と、直流交流信号発生装置２０９０と、顕微鏡２１００とを備える。第１の流路２０３０及び第２の流路２０４０は、実質的に平行に走っていることが好ましい。
== Device for exchanging cytoplasm ==
Hereinafter, an embodiment of the apparatus of the present invention capable of executing the above method will be described in detail with reference to FIG. The apparatus of the present invention is an apparatus for exchanging cytoplasm of cells, and the first flow path 2030 and the second flow path 2040 are connected to the first flow path 2030 and the second flow path 2040, respectively. The first cell supply unit 2010 and the second cell supply unit 2020, the hole 2050 provided between the wall of the first channel and the wall of the second channel, and the hole 2050 provided therebetween The electrode 2080, the flow rate adjusting unit 2060 for adjusting the flow rate of the first channel 2030 and the second channel 2040, the suction path 2070 provided in the flow rate adjusting unit 2060, and the suction ports 2071 and 2072. Pumps 2211, 2122, 2213 provided in 2073, a DC / AC signal generator 2090, and a microscope 2100 are provided. It is preferable that the first flow path 2030 and the second flow path 2040 run substantially in parallel.
＜流路内を流れる溶液の流速の調節＞
図２（２）（３）に、流速調節部２０６０の拡大図を示し、第１の流路２０３０及び第２の流路２０４０内の溶液の流速の調節方法を以下で説明する。
<Adjustment of the flow rate of the solution flowing in the flow path>
2 (2) and 2 (3) are enlarged views of the flow rate adjusting unit 2060, and a method for adjusting the flow rate of the solution in the first channel 2030 and the second channel 2040 will be described below.
流路内を流れる溶液は、流速調節部２０６０によって、各吸引口２０７１，２０７２，２０７３への接続が調節される。そして、各吸引口２０７１，２０７２，２０７３に設けられたポンプ２２１１，２２１２，２２１３によって、独立に吸引される。吸引口２０７２，２０７３への溶液の流れを止め、吸引口２０７１で吸引すると、第１の流路２０３０と第２の流路２０４０の流れの方向と流速は同じになる。吸引口２０７１，２０７３への溶液の流れを止め、吸引口２０７２から吸引すると、流れの方向は同じだが、第２の流路２０４０の流れは流路壁とぶつかる事でわずかに流速は減少し、第１の流路２０３０の流れと比較して、流速差が生じる。逆に、吸引口２０７１，２０７２への溶液の流れを止め、吸引口２０７３から吸引すると、流れの方向は同じだが、第１の流路２０３０の流れは流路壁とぶつかる事でわずかに流速は減少し、第２の流路２０４０の流れと比較して、流速差が生じる。ここで、流速調節部２０６０に弁（例えば電磁弁など）を設けることによって、さらに流速の細かな制御が可能になる。 The flow rate of the solution flowing in the flow path is adjusted by the flow rate adjustment unit 2060 to each suction port 2071, 2072, 2073. And it is attracted | sucked independently by the pumps 2211, 2122, and 2213 provided in each suction port 2071, 2072, 2073. When the flow of the solution to the suction ports 2072 and 2073 is stopped and suctioned by the suction port 2071, the flow direction and the flow velocity of the first channel 2030 and the second channel 2040 become the same. When the flow of the solution to the suction ports 2071 and 2073 is stopped and sucked from the suction port 2072, the flow direction is the same, but the flow rate of the second flow path 2040 slightly decreases by colliding with the flow path wall, Compared with the flow of the first flow path 2030, a flow velocity difference is generated. Conversely, when the flow of the solution to the suction ports 2071 and 2072 is stopped and suctioned from the suction port 2073, the flow direction is the same, but the flow of the first flow path 2030 collides with the flow path wall so that the flow velocity is slightly As a result, the flow rate difference is generated as compared with the flow of the second flow path 2040. Here, by providing the flow rate adjusting unit 2060 with a valve (for example, an electromagnetic valve), finer control of the flow rate becomes possible.
＜追加の実施形態 弁の構成＞
弁の構成例を図２（１）を参照しながら、以下に説明する。
<Additional Embodiment Valve Configuration>
A configuration example of the valve will be described below with reference to FIG.
まず、弁を流速調節部２０６０内で、各吸引口２０７１、２０７２、２０７３に繋がる吸引路２０７０に設置することができる。それによって、各吸引口２０７１、２０７２、２０７３に向かう溶液の流れを独立に調節できるようになる。 First, a valve can be installed in the suction path 2070 connected to each suction port 2071, 2072, 2073 in the flow rate adjustment unit 2060. Thereby, the flow of the solution toward each suction port 2071, 2072, 2073 can be adjusted independently.
また、他の実施形態として、吸引路２０７０を流速調節部２０６０の下流で第１の吸引口２０７１の上流の吸引路２０７０に接続させた、新たな吸引路２０７４（２０７０）（図２（１）では点線で示した。）を設け、ポンプ２２１２，２２１３を除去する。この場合この実施例では、吸引口２０７１に接続されたポンプ２２１１のみで、第１の流路２０３０及び第２の流路２０４０内の溶液の流速を制御することが可能となる。 Further, as another embodiment, a new suction path 2074 (2070) in which the suction path 2070 is connected to the suction path 2070 upstream of the first suction port 2071 downstream of the flow rate adjustment unit 2060 (FIG. 2 (1)). Then, it is indicated by a dotted line), and the pumps 2212 and 2213 are removed. In this case, in this embodiment, the flow rate of the solution in the first channel 2030 and the second channel 2040 can be controlled only by the pump 2211 connected to the suction port 2071.
＜追加の実施形態 その他＞
孔２０５０は、図１で詳細に説明した物に準じる。
<Additional embodiments>
The holes 2050 conform to those described in detail in FIG.
図３（１）に示すように、孔２０５０と吸引口２０７１、２０７２、２０７３間に圧力緩衝部２１１０を設けてもよい。これによって、吸引による急激な流速変化を緩和することができ、安定的に細胞質の移動と細胞の分離が可能になる。 As shown in FIG. 3A, a pressure buffer 2110 may be provided between the hole 2050 and the suction ports 2071, 2072, 2073. As a result, a rapid change in flow rate due to suction can be mitigated, and cytoplasm movement and cell separation can be stably performed.
図３（２）に示すように、顕微鏡２１００に画像認識部２２００を設け、画像認識部２２００とポンプ２２１０に、流速制御装置２２２０を設けてもよい。これによって、効率的に細胞質の移動と細胞の分離が可能になる。この流速制御装置２２２０は、上述した弁（電磁弁）弁を調節してもよい。 As illustrated in FIG. 3B, an image recognition unit 2200 may be provided in the microscope 2100, and a flow velocity control device 2220 may be provided in the image recognition unit 2200 and the pump 2210. This enables efficient cytoplasmic movement and cell separation. The flow rate control device 2220 may adjust the above-described valve (solenoid valve) valve.
＝＝細胞質を交換するための装置の使用方法＝＝
以下、本発明の一実施態様にかかる上記装置を用い、細胞質を交換するための使用方法と、それによる新規細胞の製造方法を詳細に説明する。
== How to use the device to exchange cytoplasm ==
Hereinafter, a method of use for exchanging cytoplasm using the above-described apparatus according to one embodiment of the present invention and a method for producing novel cells thereby will be described in detail.
第１の工程として、第２の吸引口２０７２及び第３の吸引口２０７３を閉止し、第１の細胞供給部２０１０内の第１の細胞と、第２の細胞供給部２０２０内の中間体を、第１の吸引口２０７１からポンプ２２１１で吸引することで、細胞を孔２０５０の入り口を構成するそれぞれの孔近傍へ誘導する。 As the first step, the second suction port 2072 and the third suction port 2073 are closed, and the first cell in the first cell supply unit 2010 and the intermediate in the second cell supply unit 2020 are used. The cells are sucked from the first suction port 2071 by the pump 2211 to guide the cells to the vicinity of each hole constituting the entrance of the hole 2050.
第２の工程として、直流交流信号発生制御装置２０９０で発生させた交流電界中の誘電泳動力により、孔２０５０を介して、第１の細胞と中間体との第１の対を形成させる。 As a second step, the first pair of the first cell and the intermediate is formed through the hole 2050 by the dielectrophoretic force in the AC electric field generated by the DC / AC signal generation control device 2090.
第３の工程として、直流交流信号発生制御装置２０９０で、第１の対へ電圧を印加することで第１の細胞と中間体とを融合する。 As a third step, the DC / AC signal generation controller 2090 fuses the first cell and the intermediate by applying a voltage to the first pair.
第４の工程として、第１の細胞と中間体との融合の完了後、第１の吸引口２０７１及び第３の吸引口２０７３を閉止し、ポンプ２２１２で第２の吸引口２０７２から吸引して、第１の細胞の細胞質を中間体へ移動させる。 As the fourth step, after the fusion of the first cell and the intermediate is completed, the first suction port 2071 and the third suction port 2073 are closed, and the pump 2212 sucks the second cell from the second suction port 2072. , Move the cytoplasm of the first cell to the intermediate.
第５の工程として、直流交流信号発生制御装置２０９０で発生させた誘電泳動力で第１の細胞を孔２０５０で保持しながら、第１の吸引口２０７１及び第３の吸引口２０７３を閉止し、ポンプ２２１２で第２の吸引口２０７２から吸引して、第１の細胞から中間体を切り離す。 As a fifth step, the first suction port 2071 and the third suction port 2073 are closed while holding the first cell in the hole 2050 by the dielectrophoretic force generated by the DC / AC signal generation control device 2090. The pump 2212 sucks from the second suction port 2072 to separate the intermediate from the first cell.
第６の工程として、第２の吸引口２０７２及び第３の吸引口２０７３を閉止し、第１の吸引口２０７１からポンプ２２１１で吸引することで、第２の細胞供給部２０２０内の第
２の細胞を孔２０５０の入り口を構成する孔近傍へ誘導する。ここで、第３の細胞供給部を設け、第２の細胞供給部２０２０を中間体用にし、第３の細胞供給部を第２の細胞用にしてもよい。
As a sixth step, the second suction port 2072 and the third suction port 2073 are closed, and suction is performed by the pump 2211 from the first suction port 2071, so that the second inside the second cell supply unit 2020 The cells are guided to the vicinity of the hole constituting the entrance of the hole 2050. Here, a third cell supply unit may be provided, the second cell supply unit 2020 may be used for an intermediate, and the third cell supply unit may be used for a second cell.
第７の工程として、直流交流信号発生制御装置２０９０で発生させた交流電界中の誘電泳動力により、孔２０５０を介して、第１の細胞と第２の細胞の第２の対を形成させる。 As a seventh step, the second pair of the first cell and the second cell is formed through the hole 2050 by the dielectrophoretic force in the AC electric field generated by the DC / AC signal generation control device 2090.
第８の工程として、直流交流信号発生制御装置２０９０で、第２の対へ電圧を印加することで、第１の細胞と第２の細胞を融合する。 As an eighth step, the DC / AC signal generation controller 2090 applies a voltage to the second pair to fuse the first cell and the second cell.
第９の工程として、第１の細胞と第２の細胞の融合の完了後、第１の吸引口２０７１及び第２の吸引口２０７２を閉止し、ポンプ２２１３で、第３の吸引口２０７３から吸引して、第１の細胞へ第２の細胞の細胞質を移動させる。 As the ninth step, after the fusion of the first cell and the second cell is completed, the first suction port 2071 and the second suction port 2072 are closed, and suction is performed from the third suction port 2073 by the pump 2213. Then, the cytoplasm of the second cell is moved to the first cell.
第１０の工程として、第１の吸引口２０７１及び第２の吸引口２０７２を閉止し、ポンプ２２１３で、第３の吸引口２０７３から吸引して、第１の細胞を第２の細胞から切り離す。 As a tenth step, the first suction port 2071 and the second suction port 2072 are closed, and the pump 2213 suctions the third suction port 2073 to separate the first cells from the second cells.
装置構成において述べたように、顕微鏡２１００に画像認識部２２００を設け、流速調節部２０６０に弁を設け、細胞を画像認識部２２００で分析しながら、流速制御装置２２２０によって弁及びポンプを調節することによって溶液の流速を調節することで、効率よく本法を実施することが可能になる。 As described in the apparatus configuration, the microscope 2100 is provided with the image recognition unit 2200, the flow rate adjustment unit 2060 is provided with a valve, and the cell and the image recognition unit 2200 are used to adjust the valve and pump by the flow rate control unit 2220. By adjusting the flow rate of the solution by the method, the present method can be carried out efficiently.
このように、本発明は、２つの流路における流速差を制御することで、細胞と中間体あるいは細胞同士の融合後の細胞質の移動方向を制御でき、かつ、界面活性剤を使わず細胞の分離・除去が可能となるため、従来の電界集中細胞融合法では達成されなかった細胞核移植が可能になる。 Thus, the present invention can control the movement direction of the cytoplasm after fusion of cells and intermediates or cells by controlling the flow rate difference between the two flow paths, and without using a surfactant. Since separation / removal is possible, cell nucleus transplantation that has not been achieved by the conventional electric field concentration cell fusion method becomes possible.
（実施例１）
本実施例では、Ｊｕｒｋａｔ細胞の細胞質を、細胞質を除去した別のＪｕｒｋａｔ細胞内へ移植する。用いた装置は、以下の通りである。
Example 1
In this example, the cytoplasm of Jurkat cells is transplanted into another Jurkat cell from which the cytoplasm has been removed. The apparatus used is as follows.
・ファンクションジェネレーター
交流印加条件：周波数１ ＭＨｚ、電圧５ Ｖ
パルス印加条件：時間１００ μｓ、電圧１０ Ｖ
・デジタルオシロスコープ
・バイポーラ電力増幅装置
・位相差・蛍光顕微鏡
・ Function generator AC application condition: Frequency 1 MHz, voltage 5 V
Pulse application condition: time 100 μs, voltage 10 V
・ Digital oscilloscope ・ Bipolar power amplifier ・ Phase difference ・ Fluorescence microscope
また、蛍光染色試薬は、以下のものを用いた。 Further, the following fluorescent staining reagents were used.
・使用蛍光染色試薬：
・Calcein, AM - Special Packaging (Excitation/Emission (nm): 494/514 C3100MP、サーモフィッシャーサイエンティフィック社、マサチューセッツ・米国)、細胞質を緑色染
色する
・CellTraceTM Calcein Red-Orange, AM - Special Packaging (Excitation/Emission (nm): 577/590、C34851、サーモフィッシャーサイエンティフィック社)、細胞質を赤色染色する
・CellstainR Hoechst 33342 solution (Excitation/Emission (nm): 350/461、346-0795
1、(株)同仁化学研究所、東京・日本)、細胞核を青色染色する
Ｊｕｒｋａｔ細胞懸濁液（１ｘ１０６個／ｍＬ、ＤＭＥＭ＋１０％ＦＢＳ）１ｍＬずつ２本の１５ｍＬ遠心管に加え、一方の遠心管ＡにCalcein AM １μＬ、もう一方の遠心管ＢにはCalcein red-orange １１μＬを添加し、その後Hoechst33342 １μＬを両方の遠心管に加えて、３７℃で３分間インキュベーションして細胞質と細胞核を染色した。余分な染色試薬を除くため、遠心管を６００ｒｐｍで２分間遠心した後、上清を除去した。
・ Fluorescent staining reagent used:
· Calcein, AM - Special Packaging ( Excitation / Emission (nm): 494/514 C3100MP, Thermo Fisher Scientific, the United States Massachusetts), - the green stain the cytoplasm CellTrace TM Calcein Red-Orange, AM - Special Packaging ( Excitation / Emission (nm): 577/590, C34851, Thermo Fisher Scientific), staining the cytoplasm in red ・ Cellstain R Hoechst 33342 solution (Excitation / Emission (nm): 350/461, 346-0795
1. Dojindo Laboratories Co., Ltd., Tokyo, Japan) Jurkat cell suspension (1 × 10 6 cells / mL, DMEM + 10% FBS) 1 mL each is added to two 15 mL centrifuge tubes. Add 1 μL of Calcein AM to tube A and 11 μL of Calcein red-orange to the other centrifuge tube B, then add 1 μL of Hoechst33342 to both tubes and incubate at 37 ° C. for 3 minutes to stain the cytoplasm and nucleus. . To remove excess staining reagent, the centrifuge tube was centrifuged at 600 rpm for 2 minutes, and then the supernatant was removed.
沈殿した細胞に対して、細胞融合用緩衝液（２００ｍＭソルビトール、１ｍＭ酢酸カルシウム、０．５ｍＭ酢酸マグネシウム、１ｍｇ／ｍＬ牛血清アルブミン）１ｍＬを加えて混合した。再び遠心管を上記と同条件で遠心して、生細胞を沈殿させ、上清を１ｍＬ除去した。この細胞洗浄工程をさらに２回繰り返した。最後に、細胞融合緩衝液を１５０μＬ加えて混合して、細胞融合用細胞懸濁液とした。以下、図４の写真を参考にしながら、各工程を説明する。 To the precipitated cells, 1 mL of a cell fusion buffer (200 mM sorbitol, 1 mM calcium acetate, 0.5 mM magnesium acetate, 1 mg / mL bovine serum albumin) was added and mixed. The centrifuge tube was centrifuged again under the same conditions as described above to precipitate live cells, and 1 mL of the supernatant was removed. This cell washing step was repeated two more times. Finally, 150 μL of cell fusion buffer was added and mixed to obtain a cell suspension for cell fusion. Hereafter, each process is demonstrated, referring the photograph of FIG.
＜細胞融合１回目＞
図２に示したようなＰＤＭＳデバイス上の細胞供給口Ａへ遠心管Ａの細胞懸濁液（細胞Ａ含有）を加え、細胞供給口であるサンプリングポートＢへ遠心管Ｂの細胞懸濁液（細胞Ｂ含有）を加えた。ピペットマン１で細胞懸濁液を吸引し、各細胞を細胞融合路上へ導いた（図４−１）。交流印加による誘電泳動力により細胞Ａと細胞Ｂを細胞融合路内へ導き、細胞融合路を挟んで細胞ＡとＢの対を形成させた（図４−２〜４、２：位相差；３〜４：蛍光視野）。パルスを１回以上印加しながら、パルス印加過程を位相差と蛍光視野で観察して、細胞融合による蛍光色素の移動を確認した（図４−５）。
<First cell fusion>
The cell suspension (containing cell A) of the centrifuge tube A is added to the cell supply port A on the PDMS device as shown in FIG. 2, and the cell suspension (centrifuge tube B) is added to the sampling port B which is the cell supply port ( Cell B containing) was added. The cell suspension was aspirated with Pipetteman 1, and each cell was guided onto the cell fusion pathway (FIG. 4A). The cells A and B are guided into the cell fusion path by the dielectrophoretic force by application of alternating current, and a pair of cells A and B is formed across the cell fusion path (FIGS. 4-2 to 4, 2: phase difference; 3 ~ 4: fluorescence field). While applying the pulse once or more, the pulse application process was observed with the phase difference and the fluorescence field to confirm the movement of the fluorescent dye due to cell fusion (FIGS. 4-5).
＜細胞核の回収＞
交流印加を停止して、ピペットマン２で吸引することで、細胞Ｂの細胞質を細胞Ａへ移動させた（図４−６〜７、６：蛍光視野；７：位相差）。引き続きピペットマン２で吸引することで、細胞Ｂから細胞Ａを分離した（図４−８〜９）。細胞Ｂを細胞融合路で保持するため再び交流を印加することで、細胞Ｂの核を孔に維持した（図４−１０）。
<Recovering cell nuclei>
The application of alternating current was stopped and suctioned with the pipetteman 2 to move the cytoplasm of the cell B to the cell A (FIGS. 4-6 to 7, 6: fluorescence field; 7: phase difference). Subsequently, the cell A was separated from the cell B by aspiration with the pipetteman 2 (FIGS. 4-8 to 9). The nucleus of the cell B was maintained in the pore by applying alternating current again to hold the cell B in the cell fusion pathway (FIGS. 4-10).
＜細胞融合２回目＞
再びピペットマン１で細胞懸濁液を吸引し、別の細胞Ａを細胞融合路上へ導いた（図４−１１〜１２）。交流印加による誘電泳動力により細胞Ａを細胞融合路内へ導いて、細胞融合路を挟んで細胞ＡとＢの対を形成させた（図４−１３〜１５、１３：位相差；１４〜１５：蛍光視野）。パルスを１回以上印加しながら、パルス印加過程を位相差と蛍光視野で観察し、細胞融合による蛍光色素の移動を確認した（図４−１６）。
<Second cell fusion>
The cell suspension was again aspirated with Pipetteman 1, and another cell A was guided onto the cell fusion pathway (FIGS. 4-11 to 12). The cell A was guided into the cell fusion path by the dielectrophoretic force by the application of alternating current, and a pair of cells A and B was formed across the cell fusion path (FIGS. 4-13 to 15, 13: phase difference; 14 to 15 : Fluorescence field). While applying the pulse once or more, the pulse application process was observed with the phase difference and the fluorescence field, and the movement of the fluorescent dye due to cell fusion was confirmed (FIG. 4-16).
＜細胞質の移植＞
交流印加を停止して、ピペットマン３で吸引することで、細胞Ｂへ細胞Ａの細胞質を移動させた（図４−１７〜１８、１７：位相差；１８：蛍光視野）。引き続きピペットマン３で吸引することで、細胞Ｂを細胞Ａから分離した（図４−１９〜２０）。これにより、細胞Ｂの細胞核は、細胞質との関係において、細胞Ａの細胞質内へ移植したのと同様の効果が得られた。
<Transplantation of cytoplasm>
The application of alternating current was stopped, and the cytoplasm of cell A was moved to cell B by suctioning with pipetteman 3 (FIGS. 4-17 to 18, 17: phase difference; 18: fluorescence field). Subsequently, the cell B was separated from the cell A by aspiration with the pipetteman 3 (FIGS. 4-19 to 20). As a result, the cell nucleus of cell B had the same effect as transplanted into the cytoplasm of cell A in relation to the cytoplasm.
（実施例２）
本実施例２では、ｉＰＳ細胞の細胞質を、細胞質を除去した線維芽細胞へ移植した。すなわち、ｉＰＳ細胞(Calcein AMとHoechst33342で染色)を実施例１の細胞Ａの代わりに、線維芽細胞 (Calcein red-orangeとHoechs33342で染色)を実施例１の細胞Ｂの代わりに用い、同じ実験条件で、実験を行ったところ、実験１と同様に、ｉＰＳ細胞の細胞質を、細胞質を除去した線維芽細胞へ移植することができ、ｉＰＳ細胞の細胞核を線維芽細胞の細胞質に移植したのと同様の効果が得られた。
(Example 2)
In Example 2, the cytoplasm of iPS cells was transplanted into fibroblasts from which the cytoplasm had been removed. That is, iPS cells (stained with Calcein AM and Hoechst33342) were used in place of cell A in Example 1, and fibroblasts (stained with Calcein red-orange and Hoechs33342) were used in place of cell B in Example 1. When the experiment was performed under the same conditions as in Experiment 1, the cytoplasm of iPS cells could be transplanted into fibroblasts from which cytoplasm had been removed, and the nucleus of iPS cells was transplanted into the cytoplasm of fibroblasts. Similar effects were obtained.
１０１０ 第１の細胞の細胞核
１０２０ 第１の細胞の細胞質
１０２５ 第１の細胞の細胞膜
１０３０ 第１の細胞
１０３５ 操作後の第１の細胞
１０４０ 中間体
１０４５ 操作後の中間体側
１０５０ 溶液の流れ（第１の流路と第２の流路で流速が同じ場合）
１０６０ 交流
１０７０ 絶縁体の壁
１０８０ 孔
１０９０ 電圧印加
１１００ 第１の融合細胞
１１１０ 第１の流路における溶液の流れ
１１２０ 第２の流路における溶液の流れ
１１３０ 第２の細胞の細胞質
１１４０ 第２の細胞の細胞核
１１５０ 第２の細胞
１１６０ 第２の融合細胞
１１７０ 細胞質を交換した細胞
２０１０ 第１の細胞の細胞供給部
２０２０ 第２の細胞及び中間体の細胞供給部
２０３０ 第１の流路
２０４０ 第２の流路
２０５０ 孔
２０６０ 流速調節部
２０７０ 吸引路
２０７１ 第１の吸引口
２０７２ 第２の吸引口
２０７３ 第３の吸引口
２０８０ 電極
２０９０ 直流交流信号発生制御装置
２１００ 顕微鏡
２１１０ 圧力緩衝部
２２００ 画像認識部
２２１０ ポンプ
２２１１ ポンプ
２２１２ ポンプ
２２１３ ポンプ
２２２０ 流速制御装置
1010 first cell nucleus 1020 first cell cytoplasm 1025 first cell cell membrane 1030 first cell 1035 first cell 1040 after operation intermediate 1045 intermediate side after operation 1050 flow of solution (first When the flow velocity is the same in the second flow path and the second flow path)
1060 AC 1070 Insulator wall 1080 Hole 1090 Voltage application 1100 First fused cell 1110 Solution flow in first flow channel 1120 Solution flow in second flow channel 1130 Second cell cytoplasm 1140 Second cell Cell nucleus 1150 second cell 1160 second fused cell 1170 cytoplasm-exchanged cell 2010 first cell cell supply unit 2020 second cell and intermediate cell supply unit 2030 first channel 2040 second Flow path 2050 Hole 2060 Flow rate adjustment unit 2070 Suction path 2071 First suction port 2072 Second suction port 2073 Third suction port 2080 Electrode 2090 DC / AC signal generation control device 2100 Microscope 2110 Pressure buffer unit 2200 Image recognition unit 2210 Pump 2211 Pump 2212 Pump 2213 Pump 2220 Flow rate control Location
Claims (34)
第１の細胞の細胞質を除去する第１の工程と、
第２の細胞の細胞質を、前記第１の細胞へ導入する第２の工程を含む、製造方法。 A method for producing a cell having a cell nucleus of a first cell, comprising a cell membrane of a first cell and a cytoplasm of a second cell,
A first step of removing the cytoplasm of the first cell;
A production method comprising a second step of introducing a cytoplasm of a second cell into the first cell.
第２の工程において、第２の細胞の細胞質が、９０％以上の細胞質が除去された前記第１の細胞へ導入される、請求項２に記載の製造方法。 In the first step, the cytoplasm of the first cell is removed by 90% or more,
The production method according to claim 2, wherein in the second step, the cytoplasm of the second cell is introduced into the first cell from which 90% or more of the cytoplasm has been removed.
第１の流路において第１の細胞を、第２の流路において中間体を、前記孔に誘導する第１の工程と、
誘電泳動力により、前記孔を介して、第１の細胞と中間体との第１の対を形成させる第２の工程と、
第１の細胞と中間体とを融合する第３の工程と、
第１の細胞の細胞質を中間体へ移動させる第４の工程と、
第１の細胞と中間体とを切り離す第５の工程と、
第２の流路において第２の細胞を前記孔に誘導する第６の工程と、
誘電泳動力により、前記孔を介して、第１の細胞と第２の細胞の第２の対を形成させる第７の工程と、
第１の細胞と第２の細胞とを融合する第８の工程と、
第２の細胞の細胞質を第１の細胞へ移動させる第９の工程と、
第１の細胞と第２の細胞とを切り離す第１０の工程と、
を含む細胞の製造方法。 A first channel and a second channel are provided, and a wall of the first channel and a wall of the second channel are communicated with each other by a hole, and the cell nucleus of the first cell is provided. A method for producing a cell comprising a cell membrane of a first cell and a cytoplasm of a second cell,
A first step of guiding the first cell in the first channel and the intermediate in the second channel into the hole;
A second step of forming a first pair of a first cell and an intermediate through the pores by dielectrophoretic force;
A third step of fusing the first cell and the intermediate;
A fourth step of transferring the cytoplasm of the first cell to the intermediate;
A fifth step of separating the first cell and the intermediate;
A sixth step of guiding second cells into the pores in the second flow path;
A seventh step of forming a second pair of a first cell and a second cell through the hole by dielectrophoretic force;
An eighth step of fusing the first cell and the second cell;
A ninth step of moving the cytoplasm of the second cell to the first cell;
A tenth step of separating the first cell and the second cell;
A method for producing a cell comprising:
第１の流路において第１の細胞を、第２の流路において中間体を、前記孔に誘導する第１の工程と、
誘電泳動力により、前記孔を介して、第１の細胞と中間体との第１の対を形成させる第２の工程と、
第１の細胞と中間体とを融合する第３の工程と、
第１の細胞と中間体とを切り離す第５の工程と、
第２の流路において新たな中間体を、前記孔に誘導する第１１の工程と、
誘電泳動力により、前記孔を介して、第１の細胞と新たな中間体との新たな第１の対を形成させる第１２の工程と、
第１の細胞と新たな中間体とを融合する第１３の工程と、
第１の細胞と新たな中間体とを切り離す第１４の工程と、
を含み、前記中間体として第２の細胞を用いる、細胞の製造方法。 A first channel and a second channel are provided, and a wall of the first channel and a wall of the second channel are communicated with each other by a hole, and the cell nucleus of the first cell is provided. A method for producing a cell comprising a cell membrane of a first cell and a cytoplasm of a second cell,
A first step of guiding the first cell in the first channel and the intermediate in the second channel into the hole;
A second step of forming a first pair of a first cell and an intermediate through the pores by dielectrophoretic force;
A third step of fusing the first cell and the intermediate;
A fifth step of separating the first cell and the intermediate;
An eleventh step of guiding a new intermediate to the hole in the second flow path;
A twelfth step of forming a new first pair of the first cell and a new intermediate through the hole by dielectrophoretic force;
A thirteenth step of fusing the first cell with a new intermediate;
A fourteenth step of separating the first cell from the new intermediate;
And using a second cell as the intermediate.
載の細胞の製造方法。 The method for producing cells according to any one of claims 1 to 21, wherein the first flow path and the second flow path are substantially parallel.
第１の流路及び第２の流路と、
第１の流路及び第２の流路のそれぞれに接続する細胞供給部と、
第１の流路の壁と第２の流路の壁との間に設けられた孔と、
孔を挟んで設けられた電極と、
第１の流路及び第２の流路の流速を調節する流速調節部と、
前記流速調節部に設けられた吸引口と、
各吸引口に設けられたポンプと、
直流交流信号発生装置と、
を備えた装置。 A device for exchanging cytoplasm of cells,
A first channel and a second channel;
A cell supply unit connected to each of the first channel and the second channel;
A hole provided between the wall of the first channel and the wall of the second channel;
An electrode provided across the hole;
A flow rate adjusting unit that adjusts the flow rates of the first channel and the second channel;
A suction port provided in the flow rate adjusting unit;
A pump provided at each suction port;
A DC / AC signal generator;
With a device.
前記画像認識装置と前記ポンプに、流速制御装置が設けられた、請求項２６〜２８のいずれか１項に記載の装置。 A microscope equipped with an image recognition device is provided,
The apparatus according to any one of claims 26 to 28, wherein a flow rate control device is provided in the image recognition device and the pump.
第２の細胞の細胞質を第１の細胞へ導入する第２の工程を含む、細胞質の交換方法であって、
第１の工程の後で第２の工程が行われる、第１の細胞の細胞質の交換方法。 A first step of removing the cytoplasm of the first cell;
A cytoplasm exchange method comprising a second step of introducing the cytoplasm of a second cell into the first cell,
A method for exchanging the cytoplasm of a first cell, wherein a second step is performed after the first step.
第２の細胞の細胞質を第１の細胞へ導入する第２の工程を含む、細胞質の交換方法であって、
第１の細胞の細胞質の４０％〜６０％と、第２の細胞の細胞質の４０％〜６０％とが交換される第１の細胞質交換工程によって、第１の工程と第２の工程が同時に行われ、
細胞質交換工程後の第１の細胞の細胞質の４０％〜６０％と、新たな第２の細胞の細胞質の４０％〜６０％とが交換される第２の細胞質交換工程がさらに行われる、第１の細胞
の細胞質の交換方法。 A first step of removing the cytoplasm of the first cell;
A cytoplasm exchange method comprising a second step of introducing the cytoplasm of a second cell into the first cell,
The first step and the second step are simultaneously performed by the first cytoplasm exchange step in which 40% to 60% of the cytoplasm of the first cell and 40% to 60% of the cytoplasm of the second cell are exchanged. Done,
A second cytoplasm exchange step is further performed in which 40% to 60% of the cytoplasm of the first cell after the cytoplasm exchange step and 40% to 60% of the cytoplasm of the new second cell are exchanged. A method for exchanging cytoplasm of one cell.
34. The method for exchanging the cytoplasm of a first cell according to claim 33, wherein the second cytoplasm exchange step is further performed once or more.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019159558A1 (en) * | 2018-02-13 | 2019-08-22 | 株式会社日立製作所 | Cytoplasm exchange apparatus |
| JP7450871B2 (en) | 2019-08-28 | 2024-03-18 | 株式会社日立製作所 | Cell manufacturing equipment |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007174901A (en) * | 2005-12-26 | 2007-07-12 | Advance Co Ltd | Apparatus for reversibly destroying cell membrane |
| JP2009178105A (en) * | 2008-01-31 | 2009-08-13 | Advance Co Ltd | Electric cell fusion apparatus |
-
2016
- 2016-11-11 JP JP2016220953A patent/JP6864287B2/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007174901A (en) * | 2005-12-26 | 2007-07-12 | Advance Co Ltd | Apparatus for reversibly destroying cell membrane |
| JP2009178105A (en) * | 2008-01-31 | 2009-08-13 | Advance Co Ltd | Electric cell fusion apparatus |
Non-Patent Citations (4)
| Title |
|---|
| ITAGAKI Y., ET AL., CYTOPLASMIC TRANSFER WITHOUT NUCLEI MIXING BETWEEN DENDRITIC CELLS AND TUMOR CEL, JPN7020002350, ISSN: 0004322089 * |
| NAT METHODS (2009) VOL.6, NO.2, PP.147-152 (PMC 2012 AUTHOR MANUSCRIPT PP.1-15), JPN6020029037, ISSN: 0004322091 * |
| PROC. SPIE (2011) VOL.8204, NO.82041G, PP.1-6, JPN7020002351, ISSN: 0004322090 * |
| 板垣雄太郎他, マイクロ流体デバイスを用いた樹状細胞と癌細胞の１：１電気細胞融合による細胞質移植法の開, JPN6020029034, ISSN: 0004322088 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019159558A1 (en) * | 2018-02-13 | 2019-08-22 | 株式会社日立製作所 | Cytoplasm exchange apparatus |
| JP7450871B2 (en) | 2019-08-28 | 2024-03-18 | 株式会社日立製作所 | Cell manufacturing equipment |
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