JP2008054630A - Cell fusion device and cell fusion method using the same - Google Patents

Cell fusion device and cell fusion method using the same Download PDF

Info

Publication number
JP2008054630A
JP2008054630A JP2006237841A JP2006237841A JP2008054630A JP 2008054630 A JP2008054630 A JP 2008054630A JP 2006237841 A JP2006237841 A JP 2006237841A JP 2006237841 A JP2006237841 A JP 2006237841A JP 2008054630 A JP2008054630 A JP 2008054630A
Authority
JP
Japan
Prior art keywords
cell
cell fusion
voltage
cells
micropore
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP2006237841A
Other languages
Japanese (ja)
Inventor
Tatsu Futami
達 二見
Takahiro Maruyama
高廣 丸山
Atsushi Morimoto
篤史 森本
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tosoh Corp
Original Assignee
Tosoh Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tosoh Corp filed Critical Tosoh Corp
Priority to JP2006237841A priority Critical patent/JP2008054630A/en
Publication of JP2008054630A publication Critical patent/JP2008054630A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new cell fusion apparatus for more efficiently and more surely performing a cell fusion of a pair of two cells in an electric cell fusion and a cell fusion method using the same. <P>SOLUTION: The cell fusion apparatus is equipped with a pair of electrodes composed of conductive members arranged oppositely in a cell fusion area, a small cell fusion container which comprises a planar insulator arranged between a pair of the electrodes through a planar spacer and has one or a plurality of micropores passed through in the direction of the oppositely arranged electrodes and in which the insulator is arranged on the electrode surface of the cell fusion area side of one electrode of the two electrodes and the diameter of the micropore is smaller than the diameter of the cell to be fused and a power source for impressing an alternating voltage or a direct current pulse voltage to a pair of the electrodes. The cell fusion method using the same is provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、細胞融合を効率的に行うための細胞融合装置とそれを用いた細胞融合方法に関する。   The present invention relates to a cell fusion apparatus for efficiently performing cell fusion and a cell fusion method using the same.

従来、異なる細胞同士を融合させ１つの交雑細胞とする細胞融合技術として、主にポリエチレングリコール（ＰＥＧ）を用いる化学的融合法が用いられているが、この方法では（ｉ）ＰＥＧは細胞に対して強い毒性を持っている、（ｉｉ）融合するにあたりＰＥＧの重合度、添加量などの最適な諸条件を見出すのに手間がかかる、（ｉｉｉ）融合に際して高度な技術が要求され、特定の技術に習熟した人にしか使えない、（ｉｖ）２細胞の接触は偶発的であり、２細胞一対での細胞融合の制御が困難なため細胞融合確率が極めて低い、等の解決すべき課題があった。   Conventionally, a chemical fusion method mainly using polyethylene glycol (PEG) has been used as a cell fusion technique for fusing different cells into one hybrid cell. In this method, (i) PEG is attached to the cell. (Ii) It takes time and effort to find the optimum conditions such as the degree of polymerization of PEG and the amount added, (iii) Advanced technology is required for fusion, and specific technology (Iv) There are problems to be solved such as (iv) contact of two cells is accidental, and control of cell fusion with a pair of two cells is difficult, so that the probability of cell fusion is extremely low. It was.

これに対して、電気的細胞融合法は、高度な技術が不要で、簡単に効率よく融合させることができ、細胞に与える毒性がほとんどなく、高活性をもったままの状態で細胞を融合させることができるという利点がある。電気的細胞融合法は、１９８１年西ドイツのＺｉｍｍｅｒｍａｎｎが確立したものであり、その原理は次の通りである。すなわち、平行電極間に交流電圧を印加し、そこに細胞を導入すると、細胞は電流密度の高い方へ引き寄せられ数珠状にならぶ。なお、細胞が数珠状にならんだ状態を一般にパールチェーンと呼ぶ。この状態で数μｓｅｃ〜数十μｓｅｃ単位の直流パルス電圧を電極間に印加することにより細胞膜の電気伝導度が瞬間的に低下し、脂質二重層により構成される細胞膜の可逆的乱れとその再構成が行われ、その結果細胞融合が起こるものである。   In contrast, the electric cell fusion method does not require advanced technology, can be easily and efficiently fused, has little toxicity to cells, and fuses cells with high activity. There is an advantage that you can. The electric cell fusion method was established by Zimmermann in West Germany in 1981, and the principle is as follows. That is, when an alternating voltage is applied between parallel electrodes and cells are introduced therein, the cells are attracted toward the higher current density and form a bead shape. A state in which cells are arranged in a bead shape is generally called a pearl chain. In this state, when a DC pulse voltage of several μsec to several tens μsec is applied between the electrodes, the electric conductivity of the cell membrane is instantaneously reduced, and the reversible disturbance of the cell membrane constituted by the lipid bilayer and its reconstruction As a result, cell fusion occurs.

上記の電気的融合法には、主に微小電極法と平行電極法が用いられている。このうち微小電極法は、２細胞一対の融合を顕微鏡で観察しながらマイクロマニュピレーターで細胞を拾い集めては直流パルス電圧を印加する方法であり、極めて確実であり、微小電極法に用いる電極の例も報告されている（例えば、特許文献１参照）が、手間のかかる方法であり、その操作は熟練を要す上、大量の細胞を扱う上では実用的とはいえなかった。また平行電極法は、誘電泳動により複数の細胞を数珠状に配列形成させた後、直流パルス電圧を印加することによって融合させる方法であり、その取り扱いは簡単であるが、数珠状になった複数の細胞が融合するため化学的融合法と同様に２細胞の接触は偶発的であり、２細胞一対での細胞融合の確実な制御が難しいという課題があった。   For the electrical fusion method, a microelectrode method and a parallel electrode method are mainly used. Among these, the microelectrode method is a method of applying a DC pulse voltage by collecting cells with a micromanipulator while observing a fusion of a pair of two cells with a microscope, and is an extremely reliable example of an electrode used for the microelectrode method. (For example, refer to Patent Document 1) is a time-consuming method, which requires skill and is not practical in handling a large amount of cells. The parallel electrode method is a method in which a plurality of cells are arranged in a bead shape by dielectrophoresis, and then fused by applying a DC pulse voltage. As in the chemical fusion method, the contact of the two cells is accidental, and there is a problem that it is difficult to reliably control the cell fusion in a pair of two cells.

上記平行電極法の課題を解決するために、細胞融合用チャンバーの細胞融合領域に対向するように配置された導電部材よりなる一対の電極と、前記一対の電極間に配置され、且つ前記一対の電極方向に貫通した微細孔を有する絶縁体とよりなる細胞融合用チャンバーの例が報告されている（例えば、特許文献２参照）。   In order to solve the problem of the parallel electrode method, a pair of electrodes made of a conductive member disposed to face a cell fusion region of a cell fusion chamber, a pair of electrodes disposed between the pair of electrodes, and the pair of electrodes An example of a cell fusion chamber composed of an insulator having fine holes penetrating in the electrode direction has been reported (see, for example, Patent Document 2).

図１は上記例の細胞融合用チャンバーの断面図を示した概念図である。図１において、例えば樹脂材からなる細胞融合用チャンバーの細胞融合領域（１）の両側には、導電部材からなる電極（２）が配置され、これら電極は導電線（３）を介して外部に設けられた電源（４）と接続されている。外部に設けられた電源は電界の強さが約４００Ｖ／ｃｍ〜７００Ｖ／ｃｍ、周波数１ＭＨｚ程度の高周波交流電圧を出力する交流電源（５）と、約７ｋＶ／ｃｍ、パルス幅５０μｓｅｃ程度の直流パルス電圧を出力する直流パルス電源（６）と、電極と交流電源又は直流パルス電源の電気的接続を切り換える為の切換機構を有する切換スイッチ（７）とから構成されている。   FIG. 1 is a conceptual diagram showing a cross-sectional view of the cell fusion chamber of the above example. In FIG. 1, electrodes (2) made of conductive members are arranged on both sides of a cell fusion region (1) of a cell fusion chamber made of a resin material, for example, and these electrodes are connected to the outside via conductive wires (3). It is connected to a provided power supply (4). The power supply provided outside is an AC power supply (5) that outputs a high-frequency AC voltage with an electric field strength of about 400 V / cm to 700 V / cm and a frequency of about 1 MHz, and a DC pulse of about 7 kV / cm and a pulse width of about 50 μsec. A DC pulse power supply (6) for outputting a voltage and a changeover switch (7) having a switching mechanism for switching the electrical connection between the electrode and the AC power supply or the DC pulse power supply.

ここで、交流電源から出力する交流電圧の波形は、特に断りがない限りは一般に正弦波の波形を用いる。細胞融合用チャンバーは、電気的に絶縁な材料、例えばシリコーン樹脂からなる隔壁（３５）により２つの空間に区分けされている。ここで、絶縁体には最小口径が１μｍ〜数十μｍの微細孔（９）が設けられている。また、細胞Ａ（１０）及び細胞Ｂ（１１）はそれぞれ細胞融合用チャンバーの細胞融合領域内の細胞懸濁液内におかれている。   Here, as a waveform of the AC voltage output from the AC power supply, a sine wave waveform is generally used unless otherwise specified. The cell fusion chamber is divided into two spaces by a partition wall (35) made of an electrically insulating material such as silicone resin. Here, the insulator is provided with a fine hole (9) having a minimum diameter of 1 μm to several tens of μm. The cells A (10) and B (11) are each placed in a cell suspension in the cell fusion region of the cell fusion chamber.

上記例の動作を図２〜図４を用いて説明する。最初に、電源（４）の切換スイッチ（７）を電界の強さが約４００Ｖ／ｃｍ〜７００Ｖ／ｃｍ、周波数１ＭＨｚの高周波電圧を出力する交流電源（５）に接続させる。この状態において電気力線（１２）は、図２に示すように微細孔（９）に集中する。細胞Ａ（１０）および細胞Ｂ（１１）は、ここに集中する電気力線（１２）のため誘電泳動力を受け、図３に示すように微細孔（９）の中心付近に固定される。ここで細胞Ａ（１０）と細胞Ｂ（１１）は出会い接触する。次に、電源（４）の切換スイッチ（７）を直流パルス電源（６）に切り換える。図３に示した状態におかれた細胞Ａ（１０）及び細胞Ｂ（１１）は、直流パルス電圧により細胞Ａ（１０）および細胞Ｂ（１１）の接触点で細胞膜の可逆的破壊が起こり、図４に示すように融合が生ずる。このようにすることで、微細孔において細胞Ａと細胞Ｂを２細胞一対で細胞融合させることができる。   The operation of the above example will be described with reference to FIGS. First, the changeover switch (7) of the power source (4) is connected to an AC power source (5) that outputs a high-frequency voltage having an electric field strength of about 400 V / cm to 700 V / cm and a frequency of 1 MHz. In this state, the electric lines of force (12) are concentrated in the fine holes (9) as shown in FIG. The cells A (10) and B (11) receive a dielectrophoretic force due to the electric force lines (12) concentrated here, and are fixed near the center of the micropore (9) as shown in FIG. Here, cell A (10) and cell B (11) meet and come into contact. Next, the selector switch (7) of the power source (4) is switched to the DC pulse power source (6). The cells A (10) and B (11) placed in the state shown in FIG. 3 undergo reversible destruction of the cell membrane at the contact point of the cells A (10) and B (11) due to the DC pulse voltage, Fusion occurs as shown in FIG. By doing in this way, the cell A and the cell B can be cell-fused by a pair of cells in the micropore.

しかしながら、前記特許文献２に記載された細胞融合用チャンバーを用いて細胞融合を行う方法は、微細孔の直径が細胞Ａの直径より大きくかつ細胞Ｂの直径より大きい場合、それぞれの細胞が微細孔にトラップされ接触する際に、図２９に示すように、電気力線の向きと同一方向に細胞Ａと細胞Ｂが接触する確率が減るため、細胞融合の確率が低くなるという課題があった。また、逆に微細孔の直径が細胞Ａの直径より小さくかつ細胞Ｂの直径より小さい場合、両方の細胞は微細孔にトラップされ接触し融合されるが、融合後、図３０に示すように微細孔から細胞が外れなくなり、微細孔から細胞を取り出すことができなくなる上、無理に取り出そうとすると、融合した細胞が壊れてしまうという課題があった。   However, in the method of performing cell fusion using the cell fusion chamber described in Patent Document 2, when the diameter of the micropore is larger than the diameter of the cell A and larger than the diameter of the cell B, each cell is microporous. 29, there is a problem that the probability of cell fusion decreases because the probability that the cell A and the cell B contact in the same direction as the direction of the lines of electric force decreases as shown in FIG. On the contrary, when the diameter of the micropore is smaller than the diameter of the cell A and smaller than the diameter of the cell B, both cells are trapped and contacted and fused in the micropore, but after the fusion, as shown in FIG. There was a problem that the cells could not be removed from the pores, and the cells could not be taken out from the micropores, and the fused cells were broken when trying to forcibly remove them.

さらに、前記特許文献２に記載された細胞融合用チャンバーを用いて細胞融合を行う方法は、前記微細孔において２細胞一対を同時に固定することが難しいという課題があった。例えば、細胞Ａを微細孔に入れた後、さらに細胞Ｂを微細孔に入れるために細胞融合用チャンバー内に細胞Ｂを含有する細胞懸濁液を導入すると、導入する際の送液により、あらかじめ微細孔に固定しておいた細胞Ａが微細孔から脱離してしまう。また、細胞Ａと細胞Ｂを同時に微細孔に固定するには細胞懸濁液の送液に熟練を要し非常に困難であった。さらに、前記特許文献２に記載された方法により複数の細胞を同時に細胞融合させる場合、絶縁体上にアレイ状に形成した複数の微細孔に、２細胞を一対ずつ固定する必要がある。   Further, the method of performing cell fusion using the cell fusion chamber described in Patent Document 2 has a problem that it is difficult to fix two cell pairs simultaneously in the micropores. For example, after a cell A is put into a micropore and a cell suspension containing the cell B is introduced into a cell fusion chamber in order to further put a cell B into the micropore, The cells A fixed in the micropores are detached from the micropores. In addition, it is very difficult to fix the cells A and B to the micropores at the same time because it requires skill in feeding the cell suspension. Furthermore, when a plurality of cells are fused simultaneously by the method described in Patent Document 2, it is necessary to fix two cells one by one in a plurality of micropores formed in an array on an insulator.

ここでアレイ状とは、複数の微細孔の縦と横の間隔がほぼ等間隔に配置されていることを意味する。しかしながら、交流電源を接続して細胞を微細孔に固定する際に、複数の細胞が集中して固定される微細孔や、細胞が全く固定されない微細孔があり、複数のアレイ状に形成された微細孔で目的とする２細胞一対の細胞融合を行うことが非常に難しいという課題があった。   Here, the array shape means that the vertical and horizontal intervals of the plurality of micro holes are arranged at almost equal intervals. However, when an AC power supply is connected and cells are fixed in the micropores, there are micropores in which a plurality of cells are concentrated and fixed, or micropores in which cells are not fixed at all, and are formed in a plurality of arrays. There was a problem that it was very difficult to perform the desired cell fusion of a pair of two cells with micropores.

一方、アレイ状に形成した複数の微細孔に１つずつ細胞を固定する方法の例が報告されている（例えば、特許文献３参照）。特許文献３記載の方法は、微細孔（特許文献３では、マイクロウエルと記載されている）の内径と深さがそれぞれ細胞（特許文献３では、被検体リンパ球と記載されている）の直径の１〜２倍の大きさの複数の微細孔に、複数の細胞を含む液を微細孔を覆うように加え、微細孔内に細胞が沈むのを待つ過程と、微細孔外の細胞を洗い流す洗浄の過程を繰り返し行うことで１つの微細孔に１つの細胞を固定している。   On the other hand, an example of a method of fixing cells one by one in a plurality of micropores formed in an array has been reported (for example, see Patent Document 3). In the method described in Patent Document 3, the inner diameter and depth of a micropore (described as a microwell in Patent Document 3) are the diameters of cells (described as a subject lymphocyte in Patent Document 3), respectively. A process of adding a liquid containing a plurality of cells to a plurality of micropores 1 to 2 times larger than the size of the micropore so as to cover the micropores and waiting for the cells to sink into the micropores, and washing away the cells outside the micropores One cell is fixed to one micropore by repeating the washing process.

しかしながら、前記特許文献３に記載された方法により１つの微細孔に１つの細胞を固定する方法は、重力により細胞が沈むのを待つ時間が５分程度と長いこと、微細孔内に細胞が沈むのを待つ過程と微細孔外の細胞を洗い流す洗浄過程を繰り返す必要があり操作が煩雑な上さらに時間を要すること、微細孔に入らなかった細胞を洗い流す過程で細胞が失われる可能性があるため、細胞すべてを有効に使用することが難しいという課題があった。一般に、細胞融合を行う場合は、細胞の活性を維持するためにその処理時間はできる限り短いことが好ましく、また、細胞１つ１つが持つ特異性を見出すためには、できる限り細胞の喪失がないことが好ましい。   However, the method of fixing one cell in one micropore by the method described in Patent Document 3 has a long waiting time of about 5 minutes for the cell to sink by gravity, and the cell sinks in the micropore. It is necessary to repeat the process of waiting for and washing the cells outside the micropores, which is cumbersome and requires more time, and cells may be lost in the process of washing cells that did not enter the micropores There is a problem that it is difficult to use all the cells effectively. In general, when performing cell fusion, it is preferable that the treatment time is as short as possible in order to maintain the activity of the cell, and in order to find the specificity of each cell, the loss of the cell is as much as possible. Preferably not.

特公平７−４０９１４号公報Japanese Patent Publication No. 7-40914 特公平７−４２１８号公報Japanese Patent Publication No. 7-4218 特許第３７２３８８２号公報Japanese Patent No. 3723882

本発明の目的は、かかる従来の実状に鑑みて提案されたものであり、電気的細胞融合において、２細胞一対での細胞融合をより効率的により確実に実施できる新規な細胞融合用チャンバーまたは細胞融合装置と、それらを用いた細胞融合方法を提供することを目的とする。   The object of the present invention has been proposed in view of the conventional situation, and a novel cell fusion chamber or cell capable of more efficiently and reliably performing two-cell cell fusion in electric cell fusion. It is an object of the present invention to provide a fusion device and a cell fusion method using them.

本発明は上記課題を解決する手段として、細胞融合領域内に対向して配置される導電部材からなる一対の電極と、前記一対の電極間に平板状のスペーサーを介して配置され、かつ前記対向して配置された電極の方向に貫通した１または複数の微細孔を有した平板状の絶縁体からなり、前記絶縁体が、前記電極の内いずれか一方の電極の細胞融合領域側の電極面上に配置されており、かつ前記微細孔の直径が、融合させる細胞の直径より小さいことを特徴とする細胞融合容器と、前記一対の電極に交流電圧及び直流パルス電圧を印加する電源と、を備えた細胞融合装置であって、前記交流電圧の波形が前記細胞の充電と放電を周期的に繰り返す波形であり、前記細胞融合領域内に第１の細胞を導入し、前記波形を有する交流電圧を印加することで前記微細孔内に前記第１の細胞を固定した後、前記細胞融合領域内に第２の細胞を導入して、前記波形を有する交流電圧を印加することで前記第１の細胞に前記第２の細胞を微細孔の位置において接触させ、前記直流パルス電圧を印加して細胞融合する細胞融合方法を用いることにより、上記の従来技術の課題を解決することができることを見出し、遂に本発明を完成するに至った。以下、本発明を詳細に説明する。   As a means for solving the above-mentioned problems, the present invention provides a pair of electrodes made of conductive members disposed opposite to each other in a cell fusion region, a flat spacer disposed between the pair of electrodes, and the opposite A flat insulator having one or a plurality of fine holes penetrating in the direction of the arranged electrode, and the insulator is an electrode surface on the cell fusion region side of one of the electrodes A cell fusion container, wherein the diameter of the micropores is smaller than the diameter of the cells to be fused, and a power source for applying an AC voltage and a DC pulse voltage to the pair of electrodes. An AC voltage having the waveform, wherein the waveform of the AC voltage is a waveform in which charging and discharging of the cells are periodically repeated, and the first cell is introduced into the cell fusion region. Applying After fixing the first cell in the micropore, the second cell is introduced into the cell fusion region, and the second voltage is applied to the first cell by applying an AC voltage having the waveform. It was found that the above-mentioned problems of the prior art can be solved by using a cell fusion method in which cells are brought into contact at the position of the micropore and the DC pulse voltage is applied to fuse the cells. Finally, the present invention has been completed. It came to do. Hereinafter, the present invention will be described in detail.

本発明の細胞融合装置は、細胞融合領域内に対向して配置される導電部材からなる一対の電極と、前記一対の電極間に平板状のスペーサーを介して配置され、かつ前記対向して配置された電極の方向に貫通した１または複数の微細孔を有した平板状の絶縁体からなる細胞融合容器と、前記一対の電極に交流電圧及び直流パルス電圧を印加する電源と、を備えた細胞融合装置であって、前記絶縁体が、前記電極の内いずれか一方の電極の細胞融合領域側の電極面上に配置されており、かつ前記微細孔の直径が、融合させる細胞の直径より小さいことを特徴とする細胞融合装置である。   The cell fusion device of the present invention includes a pair of electrodes made of a conductive member arranged to face each other in a cell fusion region, a flat spacer disposed between the pair of electrodes, and the face to face A cell fusion container comprising a flat insulator having one or a plurality of fine holes penetrating in the direction of the formed electrodes, and a power source for applying an AC voltage and a DC pulse voltage to the pair of electrodes In the fusion device, the insulator is disposed on the electrode surface on the cell fusion region side of one of the electrodes, and the diameter of the micropore is smaller than the diameter of the cell to be fused. This is a cell fusion device.

また本発明の細胞融合装置は、前記電源が、前記一対の電極に交流電圧を印加するための交流電源及び直流パルス電圧を印加するための直流パルス電源からなり、前記交流電源と前記直流パルス電源とを切り換える切換機構を有することを特徴とする細胞融合装置である。   In the cell fusion device of the present invention, the power source includes an AC power source for applying an AC voltage to the pair of electrodes and a DC pulse power source for applying a DC pulse voltage, the AC power source and the DC pulse power source. And a switching mechanism for switching between the two.

また本発明の細胞融合装置は、前記絶縁体に形成される微細孔が、１つの微細孔につき１つの細胞を固定できる形状であることを特徴とする細胞融合装置である。   The cell fusion device of the present invention is a cell fusion device characterized in that the micropores formed in the insulator have a shape capable of fixing one cell per micropore.

また本発明の細胞融合装置は、前記電源により、１つの微細孔につき１つの細胞を微細孔に固定する波形を有する交流電圧が前記電極間に印加されることを特徴とする細胞融合装置である。   The cell fusion device of the present invention is a cell fusion device characterized in that an alternating voltage having a waveform for fixing one cell to one micropore is applied between the electrodes by the power source. .

また本発明の細胞融合装置は、前記電源により、前記細胞の充電と放電を周期的に繰り返す波形を有する交流電圧が前記電極間に印加されることを特徴とする細胞融合装置である。   In the cell fusion device of the present invention, an AC voltage having a waveform that periodically repeats charging and discharging of the cells is applied between the electrodes by the power source.

また本発明の細胞融合装置は、前記交流電圧の波形が、０以外の値を有する電圧が一定時間変化しない時間を半周期内に１以上有する波形であることを特徴とする細胞融合装置である。   In the cell fusion device of the present invention, the waveform of the alternating voltage is a waveform having one or more times in a half cycle in which a voltage having a value other than 0 does not change for a certain period of time. .

また本発明の細胞融合装置は、前記交流電圧の波形が、矩形波、台形波、またはこれらを組み合わせた波形であることを特徴とする細胞融合装置である。   The cell fusion device of the present invention is a cell fusion device characterized in that the waveform of the AC voltage is a rectangular wave, a trapezoidal wave, or a combination thereof.

また本発明の細胞融合装置は、前記交流電圧の波形が、０以外の値を有する電圧が所定時間変化しない時間が細胞の静電容量と細胞を含有する細胞懸濁液の抵抗の積からなる時定数以上であることを特徴とする細胞融合装置である。   In the cell fusion device of the present invention, the time during which the voltage of the AC voltage has a value other than 0 does not change for a predetermined time is the product of the capacitance of the cell and the resistance of the cell suspension containing the cell. A cell fusion device characterized by having a time constant or more.

また本発明の細胞融合装置は、前記絶縁体に形成される微細孔の中心位置が、前記絶縁体の水平面において、隣り合ういずれの微細孔の中心位置からも、微細孔の中心位置が同じ位置に形成されていることを特徴とする細胞融合装置である。   Further, in the cell fusion device of the present invention, the center position of the micropore formed in the insulator is the same as the center position of the micropore from the center position of any adjacent micropore in the horizontal plane of the insulator. It is formed in the cell fusion device characterized by the above-mentioned.

また本発明の細胞融合装置は、前記絶縁体に形成される複数の微細孔が、絶縁体の面においてアレイ状に形成されていることを特徴とする細胞融合装置である。   The cell fusion device of the present invention is a cell fusion device characterized in that a plurality of micropores formed in the insulator are formed in an array on the surface of the insulator.

また本発明の細胞融合装置は、前記微細孔の隣合う間隔が、微細孔に入れる細胞の直径の０．５倍以上６倍以下であることを特徴とする細胞融合装置である。   The cell fusion device according to the present invention is a cell fusion device characterized in that an interval between adjacent micropores is not less than 0.5 times and not more than 6 times the diameter of cells to be inserted into the micropores.

また本発明の細胞融合装置は、前記スペーサーが、細胞融合領域を形成する貫通孔を有することを特徴とする細胞融合装置である。   The cell fusion device of the present invention is a cell fusion device characterized in that the spacer has a through-hole forming a cell fusion region.

また本発明の細胞融合装置は、前記スペーサーが、細胞を導入する導入流路および排出する排出流路を有することを特徴とする細胞融合装置である。   In the cell fusion device of the present invention, the spacer has an introduction flow channel for introducing cells and a discharge flow channel for discharging cells.

本発明の細胞融合装置を用いた細胞融合方法は、上記記載の細胞融合装置を用いた細胞融合方法であって、前記細胞融合領域内に第１の細胞を導入し、交流電圧を印加することで前記微細孔内に前記第１の細胞を固定した後、前記細胞融合領域内に第２の細胞を導入して、交流電圧を印加することで前記第１の細胞に前記第２の細胞を微細孔の位置において接触させ、直流パルス電圧を印加して細胞融合することを特徴とする細胞融合方法である。   The cell fusion method using the cell fusion device of the present invention is a cell fusion method using the cell fusion device described above, wherein the first cell is introduced into the cell fusion region and an alternating voltage is applied. After fixing the first cell in the micropore, the second cell is introduced into the cell fusion region, and the second cell is applied to the first cell by applying an alternating voltage. It is a cell fusion method characterized by contacting at the position of a micropore and applying a DC pulse voltage to fuse cells.

また本発明の細胞融合装置を用いた細胞融合方法は、前記交流電圧の波形が上記記載の波形を有する交流電圧であることを特徴とする上記記載の細胞融合方法である。   The cell fusion method using the cell fusion device of the present invention is the cell fusion method described above, wherein the waveform of the AC voltage is an AC voltage having the waveform described above.

また本発明の細胞融合装置を用いた細胞融合方法は、前記第１の細胞と前記第２の細胞が微細孔表面の近傍で細胞融合する上記記載の細胞融合方法である。   The cell fusion method using the cell fusion device of the present invention is the cell fusion method as described above, wherein the first cell and the second cell are fused in the vicinity of the micropore surface.

また本発明の細胞融合装置を用いた細胞融合方法は、前記第１の細胞の直径が、前記第２の細胞の直径よりも小さいことを特徴とする上記記載の細胞融合方法である。   The cell fusion method using the cell fusion device of the present invention is the cell fusion method as described above, wherein the diameter of the first cell is smaller than the diameter of the second cell.

以下では、図を用いて本発明の細胞融合装置及び、それを用いた細胞融合方法をさらに詳細に説明する。   Hereinafter, the cell fusion device of the present invention and the cell fusion method using the same will be described in more detail with reference to the drawings.

本発明の細胞融合装置は、細胞融合領域内に対向して配置される導電部材からなる一対の電極と、前記一対の電極間に平板状のスペーサーを介して配置され、かつ前記対向して配置された電極の方向に貫通した１または複数の微細孔を有した平板状の絶縁体からなる細胞融合容器と、前記一対の電極に交流電圧及び直流パルス電圧を印加する電源と、を備えた細胞融合装置であって、前記絶縁体が、前記電極の内いずれか一方の電極の細胞融合領域側の電極面上に配置されており、かつ前記微細孔の直径が、融合させる細胞の直径より小さいことを特徴とする細胞融合装置である。さらに、本発明の細胞融合装置に備える電源が、一対の電極に交流電圧を印加するための交流電源及び直流パルス電圧を印加するための直流パルス電源からなり、交流電源と直流パルス電源とを切り換える切換機構を有する細胞融合装置である。   The cell fusion device of the present invention includes a pair of electrodes made of a conductive member arranged to face each other in a cell fusion region, a flat spacer disposed between the pair of electrodes, and the face to face A cell fusion container comprising a flat insulator having one or a plurality of fine holes penetrating in the direction of the formed electrodes, and a power source for applying an AC voltage and a DC pulse voltage to the pair of electrodes In the fusion device, the insulator is disposed on the electrode surface on the cell fusion region side of one of the electrodes, and the diameter of the micropore is smaller than the diameter of the cell to be fused. This is a cell fusion device. Furthermore, the power source provided in the cell fusion device of the present invention comprises an AC power source for applying an AC voltage to a pair of electrodes and a DC pulse power source for applying a DC pulse voltage, and switches between the AC power source and the DC pulse power source. A cell fusion device having a switching mechanism.

図５に本発明の細胞融合装置の概念図を示す。本発明の細胞融合装置は大きく分けて、細胞融合容器（１３）と電源（４）から構成される。   FIG. 5 shows a conceptual diagram of the cell fusion device of the present invention. The cell fusion device of the present invention is roughly divided into a cell fusion container (13) and a power source (4).

細胞融合容器は、図５に示すように上部電極（１４）と下部電極（１５）の間に、スペーサー（１６）を配置し、微細孔（９）を形成した絶縁体（８）を下部電極の細胞融合領域側に配置した構造を有する。上部電極と下部電極の材質は導電部材であって化学的に安定な部材であればとくに制限はなく、白金、金、銅などの金属やステンレスなどの合金及び、ＩＴＯ（Ｉｎｄｉｕｍ Ｔｉｎ Ｏｘｉｄｅ：酸化インジウムスズ）等の透明導電性材料を成膜したガラス基板などでもよいが、細胞融合を観察するには、ＩＴＯなどの透明導電性材料を成膜したガラス基板を電極として用いることが好ましい。   As shown in FIG. 5, the cell fusion container has a spacer (16) disposed between an upper electrode (14) and a lower electrode (15), and an insulator (8) in which micropores (9) are formed. It has a structure arranged on the cell fusion region side. The material of the upper electrode and the lower electrode is not particularly limited as long as it is a conductive member and is a chemically stable member. An alloy such as platinum, gold, copper or an alloy such as stainless steel or ITO (Indium Tin Oxide) Although a glass substrate on which a transparent conductive material such as tin) is formed may be used, in order to observe cell fusion, it is preferable to use a glass substrate on which a transparent conductive material such as ITO is formed as an electrode.

上部電極と下部電極の面積等の寸法には特に制限はないが、取り扱いやすいサイズとして、例えば、縦７０ｍｍ×横４０ｍｍ×厚さ１ｍｍ程度のサイズが好ましい。   There are no particular limitations on the dimensions of the upper electrode and the lower electrode, but a size that is easy to handle is preferably, for example, a size of about 70 mm long × 40 mm wide × 1 mm thick.

スペーサーは、上部電極と下部電極が直接接触しないように設けられ、かつ細胞融合容器に細胞懸濁液を入れておくスペースを確保するための細胞融合領域を形成する貫通孔を有しているものであり、その材質は絶縁材料であればよく、例えばガラス、セラミック、樹脂等がある。またスペーサーには、細胞融合容器に細胞を導入、排出するため、細胞を導入する導入流路とそれに連通する導入口（１９）と、細胞を排出する排出流路とそれに連通する排出口（２０）が設けられていてもよい。スペーサーのサイズは上部電極と下部電極が接触しなければ特に制限はないが、前記電極に合わせたサイズが好ましい。例えば、電極サイズが縦７０ｍｍ×横４０ｍｍ程度であれば、スペーサーのサイズは例えば縦４０ｍｍ×横４０ｍｍ程度が好ましい。細胞融合領域（１）を形成するスペーサーの内側の空間と厚みも特に制限はないが、細胞懸濁液を例えば数μＬ〜数ｍＬ程度入れる容量があればよく、例えば、スペーサーのサイズが縦４０ｍｍ×横４０ｍｍ程度の場合、スペーサーの内側の空間は、縦２０ｍｍ×横２０ｍｍ程度であればよく、スペーサーの厚みは０．５〜２．０ｍｍ程度であればよい。   The spacer is provided so that the upper electrode and the lower electrode are not in direct contact with each other, and has a through-hole that forms a cell fusion region for securing a space for storing the cell suspension in the cell fusion container The material may be any insulating material, such as glass, ceramic, resin, and the like. In addition, in the spacer, in order to introduce and discharge cells to and from the cell fusion container, an introduction flow path for introducing cells, an introduction port (19) communicating with the cells, a discharge flow path for discharging cells and a discharge port (20) communicating therewith ) May be provided. The size of the spacer is not particularly limited as long as the upper electrode and the lower electrode are not in contact with each other, but a size matched to the electrode is preferable. For example, if the electrode size is about 70 mm long × 40 mm wide, the spacer size is preferably about 40 mm long × 40 mm wide, for example. The space and thickness inside the spacer forming the cell fusion region (1) are not particularly limited, but it is sufficient that the cell suspension has a capacity for storing several μL to several mL, for example, the spacer size is 40 mm in length. X In the case of about 40 mm in width, the space inside the spacer may be about 20 mm in length x about 20 mm in width, and the thickness of the spacer may be about 0.5 to 2.0 mm.

絶縁体（８）には微細孔（９）が形成されている。絶縁体（８）の材質は、例えばガラス、セラミック、樹脂等の絶縁材料であれば特に制限はないが、貫通した微細孔を形成させる必要があることから、樹脂等の比較的加工が容易な材料が好ましい。樹脂に貫通した微細孔を形成する手段としては、形成する微細孔の位置にレーザーを照射する方法や、微細孔の位置に貫通孔を形成するためのピンを有する金型を用いて成形する方法などの既知の方法を用いればよい。また、絶縁体にＵＶ硬化性樹脂などを用いる場合は、微細孔に相当するパターンを描画した露光用フォトマスクを用いて一般的なフォトリソグラフィー（露光）とエッチング（現像）により貫通した微細孔を形成することができる。絶縁体に複数の微細孔を形成する場合は、絶縁体にＵＶ硬化性樹脂を用いて、一般的なフォトリソグラフィーとエッチングによる方法で微細孔を形成することが好ましい。   A fine hole (9) is formed in the insulator (8). The material of the insulator (8) is not particularly limited as long as it is an insulating material such as glass, ceramic, resin, etc. However, since it is necessary to form through holes, it is relatively easy to process the resin or the like. Material is preferred. As a means for forming fine holes penetrating the resin, a method of irradiating a laser at the position of the fine hole to be formed, or a method of molding using a mold having a pin for forming the through hole at the position of the fine hole A known method such as the above may be used. In addition, when UV curable resin or the like is used for the insulator, a fine hole penetrating through general photolithography (exposure) and etching (development) using an exposure photomask on which a pattern corresponding to the fine hole is drawn is formed. Can be formed. When forming a plurality of fine holes in the insulator, it is preferable to form the fine holes by a general photolithography and etching method using a UV curable resin for the insulator.

なお図６は、図５の細胞融合容器のＡＡ’断面図を示した概略図である。上部電極（１４）、スペーサー（１６）、絶縁体（８）、下部電極（１５）を図６のように張り合わせる手段としては、それぞれを接着剤で張り合わせたり、加圧した状態で過熱して融着させる方法や、スペーサーを表面粘着性のあるＰＤＭＳ（ｐｏｌｙ−ｄｉｍｅｔｈｙｌｓｉｌｏｘａｎｅ）やシリコンシートのような樹脂を用いて作製することで圧着することにより張り合わせる方法など、既知の方法を用いればよい。このようにすることで図６に示した細胞融合領域（１）を形成することができる。   FIG. 6 is a schematic view showing an AA ′ cross-sectional view of the cell fusion container of FIG. 5. As a means for bonding the upper electrode (14), the spacer (16), the insulator (8), and the lower electrode (15) as shown in FIG. 6, each of them is bonded with an adhesive or heated in a pressurized state. A known method may be used such as a method of fusing or a method in which a spacer is bonded using a surface sticky PDMS (poly-dimethylsiloxane) or a resin such as a silicon sheet. In this way, the cell fusion region (1) shown in FIG. 6 can be formed.

細胞容器の上部電極と下部電極には導電線（３）を介して電源（４）が接続されている。電源（４）は交流電圧の波形を上部電極（１４）と下部電極（１５）の電極間に印加する交流電源と、細胞融合させるための直流パルス電圧を上部電極と下部電極の電極間に印加する直流パルス電源から構成されており、交流電源と直流パルス電源は、切換スイッチ（７）等の切換機構により適宜切り換えて使用することができる。   A power source (4) is connected to the upper and lower electrodes of the cell container via a conductive wire (3). The power source (4) applies an AC voltage waveform between the upper electrode (14) and the lower electrode (15), and a DC pulse voltage for cell fusion between the upper electrode and the lower electrode. The AC power source and the DC pulse power source can be appropriately switched and used by a switching mechanism such as a changeover switch (7).

また本発明の細胞融合装置は、前記した絶縁体に形成される複数の微細孔の中心位置が、前記絶縁体の水平面において、隣り合ういずれの微細孔の中心位置からも、微細孔の中心位置が同じ位置に形成されていること、例えば絶縁体の面においてアレイ状に形成されていることが好ましい。   Further, in the cell fusion device of the present invention, the center position of the plurality of micropores formed in the insulator is the center position of the micropore from the center position of any adjacent micropore in the horizontal plane of the insulator. Are preferably formed at the same position, for example, in the form of an array on the surface of the insulator.

図７には、絶縁体（８）に複数の微細孔（９）をアレイ状に形成した場合の本発明の細胞融合装置の概略図を示す。なお図８は、図７の細胞融合容器のＢＢ’断面図を示した概略図である。   FIG. 7 shows a schematic diagram of the cell fusion device of the present invention when a plurality of micropores (9) are formed in an array in the insulator (8). FIG. 8 is a schematic view showing a BB ′ cross-sectional view of the cell fusion container of FIG.

ここでアレイ状とは、微細孔の縦と横の間隔がほぼ等間隔に配置されていることを意味する。微細孔をアレイ状に配置することで、電極間に印加した電圧によって生じる電界がすべての微細孔にほぼ均等に生じる。また、１つの微細孔に１つの細胞を固定するためには、アレイ状に形成した微細孔の間隔が狭すぎても広すぎても不適当となることがある。微細孔の間隔が狭すぎる場合は、１つの微細孔に複数の細胞が固定される確率が高くなり結果として細胞の入らない微細孔が生じる確率が高くなる。また、微細孔の間隔が広すぎる場合には、微細孔と微細孔の間に細胞が残されてしまい、細胞の入らない微細孔が生じる確率が高くなる。従ってより具体的には、微細孔の隣り合う間隔が、微細孔に入れ固定する細胞の直径の０．５〜６倍の範囲であることが好ましく、さらには微細孔の間隔が固定する細胞の直径の１〜２倍程度であることがより好ましい。ここで意味する細胞の直径とは、融合させる細胞の大きさが異なる場合は、小さい細胞の直径を意味する。   Here, the array shape means that the vertical and horizontal intervals of the fine holes are arranged at substantially equal intervals. By arranging the fine holes in an array, an electric field generated by a voltage applied between the electrodes is generated almost uniformly in all the fine holes. In addition, in order to fix one cell in one micropore, it may be inappropriate if the interval between micropores formed in an array is too narrow or too wide. When the interval between the micropores is too narrow, the probability that a plurality of cells are fixed in one micropore is increased, and as a result, the probability that a micropore that does not contain cells is increased. In addition, when the interval between the micropores is too wide, cells are left between the micropores, and the probability that micropores that do not contain cells are increased. Therefore, more specifically, it is preferable that the interval between adjacent micropores is in the range of 0.5 to 6 times the diameter of the cells to be fixed in the micropores, and further, the interval between the micropores is fixed. More preferably, it is about 1 to 2 times the diameter. The cell diameter as used herein means the diameter of a small cell when the size of the cell to be fused is different.

以下では、１つの微細孔につき１つの細胞を固定するための交流電圧の波形と微細孔の形状に関して説明する。   Hereinafter, the waveform of the alternating voltage and the shape of the micropores for fixing one cell per micropore will be described.

本発明の細胞融合装置は、１つの微細孔につき１つの細胞を固定するため、前記交流電源により前記電極間に印加する交流電圧の波形が、電源により、前記細胞の充電と放電を周期的に繰り返す波形である細胞融合装置であって、好ましくは、前記交流電圧の波形が、０以外の値を有する電圧が所定時間変化しない時間を半周期内に少なくとも１以上有する波形であり、例えば、前記交流電圧の波形が、矩形波、台形波、またはこれらを組み合わせた波形である波形であり、さらに好ましくは、前記交流電圧の波形が、０以外の値を有する電圧が所定時間変化しない時間が細胞の静電容量と細胞を含有する細胞懸濁液の抵抗の積からなる時定数以上である波形の交流電圧を印加する電源からなる細胞融合装置である。   Since the cell fusion device of the present invention fixes one cell per micropore, the waveform of the AC voltage applied between the electrodes by the AC power source periodically charges and discharges the cell by the power source. A cell fusion device having a repeated waveform, preferably, the waveform of the alternating voltage is a waveform having at least one time within a half cycle in which a voltage having a value other than 0 does not change for a predetermined time, for example, The waveform of the AC voltage is a waveform that is a rectangular wave, a trapezoidal wave, or a combination thereof. More preferably, the AC voltage waveform has a time during which a voltage having a value other than 0 does not change for a predetermined time. A cell fusion device comprising a power supply for applying an alternating voltage having a waveform having a time constant equal to or greater than the product of the capacitance of the cell and the resistance of a cell suspension containing cells.

また、本発明の細胞融合装置は、前記微細孔の直径が融合させる細胞の直径より小さい細胞融合装置あり、さらに前記微細孔の隣り合う間隔が、固定する細胞の直径の０．５〜６倍の範囲である細胞融合装置である。   The cell fusion device of the present invention is a cell fusion device in which the diameter of the micropore is smaller than the diameter of the cell to be fused, and the interval between the micropores is 0.5 to 6 times the diameter of the cell to be fixed. It is a cell fusion device that is in the range.

上記のような交流電圧の波形を用い、微細孔の形状とすることで、１つの微細孔につき１つの細胞を固定することができ、固定した細胞のさらに上からもう１つの細胞を固定し、２細胞一対を複数の微細孔で接触させることで、一度に複数の微細孔において、２細胞一対での細胞融合を行うことができる。   By using the waveform of the alternating voltage as described above and forming a micropore shape, one cell can be fixed per one micropore, and another cell is fixed from above the fixed cell, By bringing a pair of two cells into contact with each other through a plurality of micropores, cell fusion with a pair of two cells can be performed in a plurality of micropores at a time.

本発明の細胞融合装置に用いる交流電圧の波形は、前記細胞の充電と放電を周期的に繰り返すことが可能であれば特に制限はなく、図９にこの態様の交流電圧の波形の一例を示す。図９の交流電圧の波形の半周期であるＴ／２ごとに前記細胞の充電と放電が繰り返される。なお図９の場合、半周期ごとに電圧の極性の正と負が反転するため、半周期ごとに前記細胞が充電されたときに電荷の極性が正と負に反転する。   The waveform of the alternating voltage used in the cell fusion device of the present invention is not particularly limited as long as the charging and discharging of the cells can be periodically repeated. FIG. 9 shows an example of the waveform of the alternating voltage in this mode. . The charging and discharging of the cells are repeated every T / 2, which is a half cycle of the waveform of the AC voltage in FIG. In the case of FIG. 9, since the polarity of the voltage is inverted every half cycle, the polarity of the charge is inverted between positive and negative when the cell is charged every half cycle.

なお、本発明の細胞融合装置に用いる交流電圧の波形は、直流成分を有しないことが好ましい。これは、直流成分により発生した静電気力により細胞が特定の方向に偏った力を受けて移動するため誘電泳動力により細胞を微細孔に固定することが困難になること、また細胞を含有する懸濁液に含まれるイオンが電極表面で電気反応を生じることで発熱が起こり、それにより細胞が熱運動を起こすため、誘電泳動力により細胞の動きを制御することができなくなり細胞を微細孔に引き寄せることが困難となるためである。   In addition, it is preferable that the waveform of the alternating voltage used for the cell fusion apparatus of this invention does not have a direct-current component. This is because the cells move by receiving a biased force in a specific direction due to the electrostatic force generated by the DC component, and it becomes difficult to fix the cells in the micropores due to the dielectrophoretic force. The ions contained in the suspension generate an electric reaction on the electrode surface, which generates heat, causing the cells to undergo thermal motion, and the cell movement cannot be controlled by the dielectrophoretic force, attracting the cells to the micropores. This is because it becomes difficult.

またより具体的には、本発明の細胞融合装置に用いる前記交流電圧の波形は、０以外の値を有する電圧が所定時間変化しない時間を半周期内に１以上有する波形である。図９におけるＳ［ｓ］は電圧が所定時間変化しない時間である。本発明では、０以外の値を有する電圧が所定時間変化しない時間Ｓ［ｓ］が細胞の静電容量Ｃ［Ｆ］と細胞を含有する細胞懸濁液の抵抗Ｒ［Ω］の積からなる時定数τ［ｓ］以上であることが好ましいことから、Ｓ≧τ（＝Ｃ×Ｒ）の関係であることが好ましい。   More specifically, the waveform of the AC voltage used in the cell fusion device of the present invention is a waveform having one or more times in a half cycle in which a voltage having a value other than 0 does not change for a predetermined time. S [s] in FIG. 9 is a time during which the voltage does not change for a predetermined time. In the present invention, the time S [s] during which the voltage having a value other than 0 does not change for a predetermined time is the product of the capacitance C [F] of the cell and the resistance R [Ω] of the cell suspension containing the cell. Since it is preferable that the time constant is equal to or greater than τ [s], a relationship of S ≧ τ (= C × R) is preferable.

なお、本発明における交流電圧の波形は図９に示す波形のみに限定されるものではなく、発明の要旨を逸脱しない範囲で、任意に変更が可能であることは言うまでもない。例えば、交流電圧の波形が、矩形波（図１０）、台形波（図１１）、またはこれらを組み合わせた波形（図１２）であってもよい。また、印加する電圧値や周波数は、細胞融合容器の電極間距離や、融合対象となる細胞の種類や大きさ、細胞を含有する細胞懸濁液の種類によって適切な値を設定すればよい。例えば、細胞融合容器の面積が２ｃｍ×２ｃｍ程度、電極間距離が１ｍｍ程度、融合対象の細胞が直径１０μｍ程度の細胞、懸濁腋の成分が３００ｍＭのマンニトール水溶液の場合、直径１０μｍ程度の細胞の静電容量は一般に１ｐＦ程度、面積２ｃｍ×２ｃｍ程度、電極間距離１ｍｍ程度の細胞融合容器に３００ｍＭのマンニトール水溶液を入れたときの抵抗値が５ｋΩ程度であることから、細胞の静電容量Ｃ［Ｆ］と細胞を含有する細胞懸濁液の抵抗Ｒ［Ω］の積からなる時定数τ［ｓ］は５ｎｓとなる。従って電極間に印加する交流電圧の波形が、０以外の値を有する電圧が少なくとも５ｎｓだけ変化しない時間を半周期内に１以上有する交流電圧波形であることが好ましい。例えば、図１０に示した矩形波交流電圧波形を用いた場合、Ｓの時間が５ｎｓより長くなる、すなわち、周波数が１００ＭＨｚ（＝１／（２×５ｎｓ））未満であることが好ましく、さらには電気的な取り扱いのしやすさや市販の信号発生器で容易に扱うことができることを考慮すると、周波数は１〜３ＭＨｚ程度の矩形波交流電圧波形が好ましい。またこの場合の矩形波交流電圧波形の電圧は、微細孔に細胞を引き寄せるのに十分な誘電泳動力を発生させるため、１０〜２０Ｖｐｐ程度であることが好ましい。なお、この例の条件の場合、微細孔に細胞が引き寄せられる時間は１〜５秒程度であり、瞬時に細胞を微細孔に固定することができる。   It should be noted that the waveform of the AC voltage in the present invention is not limited to the waveform shown in FIG. 9 and can be arbitrarily changed without departing from the gist of the invention. For example, the waveform of the AC voltage may be a rectangular wave (FIG. 10), a trapezoidal wave (FIG. 11), or a combination of these (FIG. 12). The voltage value and frequency to be applied may be set appropriately depending on the distance between the electrodes of the cell fusion container, the type and size of cells to be fused, and the type of cell suspension containing cells. For example, when the area of the cell fusion container is about 2 cm × 2 cm, the distance between the electrodes is about 1 mm, the cell to be fused is a cell having a diameter of about 10 μm, and the suspension cage component is a 300 mM mannitol aqueous solution, the cell having a diameter of about 10 μm The capacitance is generally about 1 pF, the area is about 2 cm × 2 cm, and the resistance value when a 300 mM mannitol aqueous solution is put into a cell fusion container having a distance between electrodes of about 1 mm is about 5 kΩ. The time constant τ [s] consisting of the product of F] and the resistance R [Ω] of the cell suspension containing the cells is 5 ns. Therefore, the waveform of the AC voltage applied between the electrodes is preferably an AC voltage waveform having one or more times in a half cycle in which a voltage having a value other than 0 does not change by at least 5 ns. For example, when the rectangular wave AC voltage waveform shown in FIG. 10 is used, it is preferable that the time S is longer than 5 ns, that is, the frequency is less than 100 MHz (= 1 / (2 × 5 ns), Considering the ease of electrical handling and the ease of handling with a commercially available signal generator, a rectangular wave AC voltage waveform with a frequency of about 1 to 3 MHz is preferable. In this case, the voltage of the rectangular AC voltage waveform is preferably about 10 to 20 Vpp in order to generate a dielectrophoretic force sufficient to attract the cells to the micropores. In the case of the conditions of this example, the time for attracting cells to the micropores is about 1 to 5 seconds, and the cells can be fixed in the micropores instantaneously.

次に、上記態様の交流電圧の波形と上記態様の微細孔を用いた場合に、１つの微細孔につき１つの細胞が固定される理由を図１３〜図２３を用いて説明する。   Next, the reason why one cell is fixed per one minute hole when the waveform of the alternating voltage in the above aspect and the minute hole in the above aspect is used will be described with reference to FIGS.

図１３〜図１５には本発明の細胞融合装置において、微細孔に細胞が入る過程の概念図を示した。絶縁体（８）の厚みは細胞Ａ′（４４）及び細胞Ａ′′（４５）の直径より小さく、微細孔の内径も細胞Ａ′及び細胞Ａ′′の直径よりも小さい。また、図１４に示すように微細孔Ａ′（１７）に細胞Ａ′が入った後、図１５に示すように微細孔Ａ′′（２１）に細胞Ａ′′が入る場合を想定している。図１６〜図１８には、それぞれ図１３〜図１５を電気的な等価回路で表現した図を示した。細胞を含有する細胞懸濁液は抵抗（抵抗値：５ｋΩ）、細胞はコンデンサー（容量：１ｐＦ）で表現することができる。   FIG. 13 to FIG. 15 show conceptual diagrams of processes in which cells enter micropores in the cell fusion device of the present invention. The thickness of the insulator (8) is smaller than the diameters of the cells A ′ (44) and A ″ (45), and the inner diameter of the micropore is also smaller than the diameters of the cells A ′ and A ″. Further, it is assumed that the cell A ′ enters the micropore A ′ (17) as shown in FIG. 14 and then the cell A ″ enters the micropore A ″ (21) as shown in FIG. Yes. FIGS. 16 to 18 show diagrams in which FIGS. 13 to 15 are expressed by electrical equivalent circuits, respectively. A cell suspension containing cells can be expressed by resistance (resistance value: 5 kΩ), and a cell can be expressed by a capacitor (capacity: 1 pF).

図１９に示す、周波数ｆ［Ｈｚ］の矩形波形の交流電圧を印加すると、図２〜図３に示した場合と同様に微細孔において電気力線の集中が生じることで細胞に対して誘電泳動力が発生し、細胞が微細孔に引き寄せられ、微微細孔Ａ′に細胞Ａ′が固定され細胞Ａ′が微細孔Ａ′を塞ぐ。なお細胞は誘電泳動力以外にも重力及び電極からの静電気力によっても微細孔に誘導される。細胞Ａ′で塞がれた微細孔Ａ′の部分は、図１７に示すようにコンデンサーＡ（２９）と電気的に等価となる。図１９に示す電圧波形を印加した場合、図１７のコンデンサーＡにおける電圧波形を図２０、電流波形を図２１に示す。図２０のようにコンデンサーＡは細胞Ａ′の容量Ｃ［Ｆ］と細胞を含む細胞懸濁液の抵抗値Ｒ［Ω］の積で求められる時定数τ［ｓ］（＝Ｃ×Ｒ）の時間を要して充電される。   When an AC voltage having a rectangular waveform with a frequency f [Hz] shown in FIG. 19 is applied, the lines of electric force are concentrated in the micropores as in the case shown in FIGS. A force is generated, the cell is attracted to the micropore, the cell A ′ is fixed in the micropore A ′, and the cell A ′ blocks the micropore A ′. In addition to the dielectrophoretic force, the cells are induced into the micropores by gravity and electrostatic force from the electrodes. The portion of the micropore A ′ blocked by the cell A ′ is electrically equivalent to the capacitor A (29) as shown in FIG. When the voltage waveform shown in FIG. 19 is applied, the voltage waveform in the capacitor A of FIG. 17 is shown in FIG. 20, and the current waveform is shown in FIG. As shown in FIG. 20, the capacitor A has a time constant τ [s] (= C × R) obtained by the product of the capacity C [F] of the cell A ′ and the resistance value R [Ω] of the cell suspension containing the cell. It takes time to charge.

なお、０以外の値を有する電圧が所定時間変化しない時間が細胞の静電容量と細胞を含有する細胞懸濁液の抵抗の積からなる時定数以上が好ましいことから、τ＜１／２ｆを満たす周波数の矩形波交流電圧を用いる事が好ましい。コンデンサーＡが充電されると電流は流れなくなるため、図２１に示すように、コンデンサーＡに流れる電流は、時定数τの時間だけパルス状に電流が流れるものの、その後は電流が流れなくなり絶縁体と電気的に等価になる。このため細胞Ａ′の入った微細孔Ａ′では、電気力線の集中が生じなくなり、微細孔Ａ′が新たに細胞を引き寄せる確率は低くなる。一方、微細孔Ａ′′には電気力線の集中が生じているため、細胞Ａ′′が誘電泳動力により引き寄せられ微細孔Ａ′′に細胞Ａ′′が固定され細胞Ａ′′が微細孔Ａ′′を塞ぐ。これを繰り返すことにより、空の微細孔につぎつぎと細胞が入っていくことで、１つの微細孔に１つの細胞を固定することができる。   Since the time during which the voltage having a value other than 0 does not change for a predetermined time is preferably a time constant consisting of the product of the capacitance of the cell and the resistance of the cell suspension containing the cell, τ <1 / 2f is set. It is preferable to use a rectangular wave AC voltage having a frequency to satisfy. Since the current does not flow when the capacitor A is charged, as shown in FIG. 21, the current flowing in the capacitor A flows in a pulse shape for the time of the time constant τ. It becomes electrically equivalent. For this reason, in the micropore A ′ containing the cell A ′, the concentration of electric field lines does not occur, and the probability that the micropore A ′ newly attracts cells becomes low. On the other hand, since the electric lines of force are concentrated in the micropore A ″, the cell A ″ is attracted by the dielectrophoretic force, the cell A ″ is fixed in the micropore A ″, and the cell A ″ is fine. Close the hole A ″. By repeating this, one cell can be fixed to one minute hole by successively entering cells into empty minute holes.

微細孔の直径は細胞の直径より小さいことがより好ましい態様であるが、微細孔の直径が細胞の直径の１倍以上２倍未満であっても同等の効果は得られる。しかしながら、微細孔の直径が細胞の直径の２倍以上大きいと、細胞は微細孔を十分塞ぐことができず、電気力線の集中が発生し細胞が誘電泳動力により引き寄せられるため、１つの微細孔に２以上の細胞が入る確率が高くなる。従って、２以上の細胞を微細孔に固定した状態で細胞融合を行う場合は、微細孔の直径が微細孔に固定する細胞の直径よりも大きくてもよいが、１つの微細孔に１つの細胞を固定した状態で細胞融合を行う場合は、微細孔の平面形状の直径が細胞の直径より小さいことがより好ましい。ここで、融合させる２つの細胞の直径が異なる場合は、微細孔の平面形状の直径が細胞の直径より小さいとは、微細孔の直径が、小さい細胞の直径よりも小さいことを意味する。また、微細孔の最小径は、微細孔が前記絶縁体を貫通していれば特に制限はない。また、微細孔の深さにも特に制限はない。   In a more preferred embodiment, the diameter of the micropore is smaller than the diameter of the cell, but the same effect can be obtained even if the diameter of the micropore is 1 to 2 times the diameter of the cell. However, if the diameter of the micropores is more than twice the diameter of the cells, the cells cannot sufficiently close the micropores, the electric lines of force are concentrated and the cells are attracted by the dielectrophoretic force. The probability that two or more cells enter the hole increases. Accordingly, when cell fusion is performed in a state where two or more cells are fixed in the micropore, the diameter of the micropore may be larger than the diameter of the cell fixed in the micropore, but one cell per micropore. When cell fusion is performed in a state in which is fixed, it is more preferable that the diameter of the planar shape of the micropore is smaller than the diameter of the cell. Here, when the diameters of the two cells to be fused are different, the diameter of the planar shape of the micropore being smaller than the cell diameter means that the diameter of the micropore is smaller than the diameter of the small cell. The minimum diameter of the fine hole is not particularly limited as long as the fine hole penetrates the insulator. Moreover, there is no restriction | limiting in particular also in the depth of a micropore.

また、１つの微細孔に１つの細胞を固定するためには、微細孔の間隔が狭すぎても広すぎても不適当である。微細孔の間隔が狭すぎる場合は、１つの微細孔に複数の細胞が固定される確率が高くなり結果として細胞の入らない微細孔が生じる確率が高くなる。また、微細孔の間隔が広すぎる場合には、微細孔と微細孔の間に細胞が残されてしまい、細胞の入らない微細孔が生じる確率が高くなる。従って具体的には、微細孔の間隔は、固定する細胞の直径の０．５倍から６倍程度の範囲であることが好ましく、さらには微細孔の間隔が固定する細胞の直径の１〜２倍程度であることがより好ましい。ここで意味する細胞の直径とは、融合させる細胞の大きさが異なる場合は、小さい細胞の直径を意味する。   In addition, in order to fix one cell in one micropore, it is inappropriate that the interval between micropores is too narrow or too wide. When the interval between the micropores is too narrow, the probability that a plurality of cells are fixed in one micropore is increased, and as a result, the probability that a micropore that does not contain cells is increased. In addition, when the interval between the micropores is too wide, cells are left between the micropores, and the probability that micropores that do not contain cells are increased. Therefore, specifically, the interval between the micropores is preferably in the range of about 0.5 to 6 times the diameter of the cells to be fixed, and moreover, the interval between the micropores is 1-2 times the diameter of the cells to be fixed. More preferably, it is about double. The cell diameter as used herein means the diameter of a small cell when the size of the cell to be fused is different.

なお、０以外の値を有する電圧が所定時間変化しない時間が細胞の静電容量と細胞を含有する細胞懸濁液の抵抗の積からなる時定数以下である場合、図１７におけるコンデンサーＡが十分充電されないため、コンデンサーＡに電流が流れ続け、細胞Ａ′が入った微細孔Ａ′において引き続き電流が流れ電気力線の集中が生じる。よって、細胞Ａ′′は細胞Ａ′の入った微細孔Ａ′に引き寄せられる可能性があるため、１つの微細孔に２以上の細胞が固定される確率が高くなる。   If the time during which the voltage having a value other than 0 does not change for a predetermined time is equal to or less than the time constant formed by the product of the capacitance of the cell and the resistance of the cell suspension containing the cell, the capacitor A in FIG. Since the battery A is not charged, the current continues to flow through the capacitor A, and the current continues to flow in the micropore A ′ containing the cell A ′, causing concentration of electric lines of force. Therefore, since the cell A ″ may be attracted to the micropore A ′ containing the cell A ′, the probability that two or more cells are fixed in one micropore increases.

次に、図２２に示す周波数ｆ［Ｈｚ］の正弦波形の交流電圧を印加すると、図２〜図３に示した場合と同様に微細孔において電気力線の集中が生じることで細胞に対して誘電泳動力が発生し、細胞が誘電泳動力により微細孔に引き寄せられ、細胞Ａ′が微細孔Ａ′を塞ぐ。細胞Ａ′が塞いだ微細孔Ａ′の部分は、図１７に示すようにコンデンサーＡと等価となる。図２２に示す電圧波形を印加した場合、コンデンサーＡにおける電圧波形と電流波形を図２３に示す。図２３に示すように印加する交流電圧の波形が正弦波の場合は、正弦波の位相が９０度すすむだけで、正弦波の波形は変化しないため、細胞Ａ′が入った微細孔Ａ′において引き続き電流が流れ、電気力線の集中が生じる。このため、細胞Ａ′′は細胞Ａ′の入った微細孔Ａ′に引き寄せられる可能性があるため、１つの微細孔に２以上の細胞が固定される確率が高くなる。従って、正弦波や三角波のように常に電圧が連続的に変化する交流電圧の波形では、複数の細胞が集中して固定される微細孔と、細胞が全く固定されない微細孔があり、１つの微細孔につき１つの細胞を固定することが難しい。なお、印加する電圧が直流の場合は、細胞を含有する細胞懸濁液に含まれるイオンが電極表面で電気反応を生じることで発熱が起こり、それにより細胞が熱運動を起こすため、誘電泳動力により細胞の動きを制御することができなくなり細胞を微細孔に引き寄せることが困難となる。   Next, when an alternating voltage having a sine waveform having a frequency f [Hz] shown in FIG. 22 is applied, the electric lines of force are concentrated in the micropores as in the case shown in FIGS. Dielectrophoretic force is generated, cells are attracted to the micropores by the dielectrophoretic force, and the cells A ′ block the micropores A ′. The portion of the micropore A ′ closed by the cell A ′ is equivalent to the capacitor A as shown in FIG. When the voltage waveform shown in FIG. 22 is applied, the voltage waveform and current waveform in the capacitor A are shown in FIG. As shown in FIG. 23, when the waveform of the applied AC voltage is a sine wave, the phase of the sine wave only advances 90 degrees, and the waveform of the sine wave does not change. Therefore, in the microhole A 'containing the cell A'. Electric current continues to flow, causing concentration of electric field lines. For this reason, since the cell A ″ may be attracted to the micropore A ′ containing the cell A ′, there is a high probability that two or more cells are fixed in one micropore. Therefore, in the waveform of the AC voltage in which the voltage continuously changes like a sine wave or a triangular wave, there are fine holes in which a plurality of cells are concentrated and fixed, and fine holes in which the cells are not fixed at all. It is difficult to fix one cell per hole. When the applied voltage is direct current, the ions contained in the cell suspension containing the cells generate an electric reaction on the electrode surface, which generates heat, causing the cells to undergo thermal motion. This makes it impossible to control the movement of the cells, making it difficult to draw the cells into the micropores.

以上の理由から、本発明の細胞融合装置は、前記交流電源により前記電極間に印加する交流電圧の波形が、前記細胞の充電と放電を周期的に繰り返す波形であり、またより具体意的には、前記交流電圧の波形が、０以外の値を有する電圧が所定時間変化しない時間を半周期内に少なくとも１以上有する波形であり、例えば矩形波、台形波、またはこれらを組み合わせた波形であって、さらに好ましくは、０以外の値を有する電圧が所定時間変化しない時間が細胞の静電容量と細胞を含有する細胞懸濁液の抵抗の積からなる時定数以上である細胞融合装置であって、あって、絶縁体を貫通する微細孔の平面形状の直径は、微細孔に固定する細胞の直径より小さく、さらに複数の微細孔の隣り合う間隔が、固定する細胞の直径の０．５〜６倍の範囲であることで、絶縁体上にアレイ状に形成した複数の微細孔において、１つの微細孔につき１つの細胞を固定することが可能となる。   For the above reasons, in the cell fusion device of the present invention, the waveform of the AC voltage applied between the electrodes by the AC power supply is a waveform that periodically repeats charging and discharging of the cells, and more specifically. The waveform of the AC voltage is a waveform having at least one time in a half cycle in which a voltage having a value other than 0 does not change for a predetermined time, such as a rectangular wave, a trapezoidal wave, or a combination of these. More preferably, the cell fusion device is such that the time during which the voltage having a value other than 0 does not change for a predetermined time is not less than the time constant consisting of the product of the capacitance of the cell and the resistance of the cell suspension containing the cell. Thus, the diameter of the planar shape of the micropore penetrating the insulator is smaller than the diameter of the cell fixed in the micropore, and the interval between the plurality of micropores is 0.5 of the diameter of the cell to be fixed. ~ 6 times the range By it, in a plurality of fine holes formed in an array on the insulator, it is possible to fix the one cell per micropore.

ところで、本発明の細胞融合方法では、１つの微細孔に第１の細胞を固定した後、固定した第１の細胞のさらに上から第２の細胞を固定する。第２の細胞には誘電泳動力、重力、及び第１の細胞の静電気力が作用し第１の細胞と接触する。しかしながら、前述した理由により、微細孔を第１の細胞が塞いでしまうため電流が流れにくくなることで電気力線の発生が抑制され、第２の細胞に作用する誘電泳動力が弱くなる。従って、第２の細胞を、微細孔に固定した第１の細胞に１つずつ接触させる確率が低下する。しかしながら、第２の細胞の濃度を第１の細胞の濃度よりも高くし、細胞融合領域に過剰に導入することで、第１の細胞と第２の細胞の接触確率を上げることが可能である。   By the way, in the cell fusion method of the present invention, after fixing the first cell in one micropore, the second cell is fixed further from above the fixed first cell. Dielectric migration force, gravity, and electrostatic force of the first cell act on the second cell and come into contact with the first cell. However, for the reasons described above, the first cells block the micropores, making it difficult for the current to flow, thereby suppressing the generation of electric lines of force and weakening the dielectrophoretic force acting on the second cells. Therefore, the probability of bringing the second cells into contact with the first cells fixed in the micropores one by one is reduced. However, it is possible to increase the contact probability between the first cell and the second cell by making the concentration of the second cell higher than that of the first cell and introducing it excessively into the cell fusion region. .

次に、図を用いて本発明における細胞融合方法に関してさらに詳細に説明する。   Next, the cell fusion method in the present invention will be described in more detail with reference to the drawings.

本発明の細胞融合方法は、前記細胞融合装置を用いた細胞融合方法であって、細胞融合領域内に第１の細胞を導入し、交流電圧を印加することで微細孔内に第１の細胞を固定した後、細胞融合領域内に第２の細胞を導入して、交流電圧を印加することで第１の細胞に第２の細胞を微細孔の位置において接触させ、直流パルス電圧を印加して細胞融合する細胞融合方法である。なお、前記交流電圧の波形は、上記記載の波形を有する交流電圧であることがより好ましい。   The cell fusion method of the present invention is a cell fusion method using the cell fusion device, wherein the first cell is introduced into the cell fusion region, and an alternating voltage is applied to the first cell in the micropore. After fixing the cell, the second cell is introduced into the cell fusion region, and an AC voltage is applied to bring the second cell into contact with the first cell at the position of the micropore, and a DC pulse voltage is applied. Cell fusion method for cell fusion. The AC voltage waveform is more preferably an AC voltage having the waveform described above.

ここで、本発明の細胞融合方法の概略図を図２４に示す。図２４には、いずれも第１の細胞の直径が第２の細胞の直径よりも小さい例を示している。本発明の細胞融合方法は、第１の細胞の直径が第２の細胞の直径よりも小さいことが好ましく、さらには、微細孔の直径が、細胞融合させる直径の異なる２細胞のうち、直径の小さい細胞の直径より小さいことが好ましい。このようにすることで、微細孔に細胞をより確実に固定することが可能となり、その後、第２の細胞を導入することで、２細胞一対での接触および細胞融合を効率的に行うことが可能となる。しかしながら、本質的には第１の細胞の直径が第２の細胞の直径と等しいか大きくてもよい。   Here, a schematic diagram of the cell fusion method of the present invention is shown in FIG. FIG. 24 shows an example in which the diameter of the first cell is smaller than the diameter of the second cell. In the cell fusion method of the present invention, it is preferable that the diameter of the first cell is smaller than the diameter of the second cell, and further, the diameter of the micropore is a diameter of two cells having different diameters for cell fusion. It is preferably smaller than the diameter of the small cells. By doing in this way, it becomes possible to fix a cell to a micropore more reliably, and the contact and cell fusion by 2 cell pairs can be performed efficiently by introduce | transducing a 2nd cell after that. It becomes possible. However, in essence, the diameter of the first cell may be equal to or greater than the diameter of the second cell.

以下に本発明の細胞融合方法が好ましい理由を説明する。まず、第１の細胞（１８）として、直径の小さい細胞を細胞懸濁液（３４）とともに細胞融合領域（１）に導入し、前述した波形を有する交流電圧を印加して１つの微細孔（９）につき第１の細胞１つを固定する。この場合、第１の細胞は主に誘電泳動力、重力、電極からの静電気力によって微細孔に誘導され第１の細胞が微細孔に固定される。第１の細胞の数に特に制限はないが、第１の細胞を有効に使用することを考慮すると、微細孔の数と同等であることが好ましい。   The reason why the cell fusion method of the present invention is preferred will be described below. First, as the first cell (18), a cell having a small diameter is introduced into the cell fusion region (1) together with the cell suspension (34), and an alternating voltage having the waveform described above is applied to form one micropore ( Fix one first cell per 9). In this case, the first cell is guided to the micropore mainly by the dielectrophoretic force, gravity, and electrostatic force from the electrode, and the first cell is fixed to the micropore. Although there is no restriction | limiting in particular in the number of 1st cells, When considering using the 1st cell effectively, it is preferable that it is equivalent to the number of micropores.

次に、第２の細胞（２２）として、直径の大きい細胞を細胞懸濁液（３４）とともに細胞融合領域（１）に導入する。このとき、第１の細胞（１８）は誘電泳動力により微細孔に固定されているため、第２の細胞を送液することにより第１の細胞が微細孔から離脱することはほとんどない。導入された第２の細胞は、前述した波形を有する交流電圧（３４）を印加することにより、微細孔に固定された第１の細胞の上から接触し固定される。この場合、第２の細胞には、微細孔での誘電泳動力も作用するが、主に重力、第１の細胞からの静電気力によって微細孔に固定された第１の細胞に誘導される。第２の細胞の数に特に制限はないが、第２の細胞を有効に使用することを考慮すると、微細孔の数と同等であることが好ましいが、微細孔の数よりも過剰に導入することで、第１の細胞と第２の細胞の接触確率を上げることが可能である。   Next, as the second cell (22), a cell having a large diameter is introduced into the cell fusion region (1) together with the cell suspension (34). At this time, since the first cells (18) are fixed in the micropores by the dielectrophoretic force, the first cells are hardly detached from the micropores by feeding the second cells. The introduced second cell is contacted and fixed from above the first cell fixed in the micropore by applying the AC voltage (34) having the waveform described above. In this case, the dielectrophoretic force in the micropores also acts on the second cells, but is induced by the first cells fixed in the micropores mainly by gravity and electrostatic force from the first cells. The number of the second cells is not particularly limited, but considering the effective use of the second cells, it is preferably equal to the number of micropores, but introduced in excess of the number of micropores. Thus, it is possible to increase the contact probability between the first cell and the second cell.

次に、電源を直流パルス電源（６）に切り換え、細胞融合を行うための直流パルス電圧を印加することで、微細孔において接触した第１の細胞と第２の細胞を２細胞一対で細胞融合させ、融合細胞（３２）を得ることができ、効率的な２細胞一対での細胞融合が可能となる。   Next, by switching the power source to the DC pulse power source (6) and applying a DC pulse voltage for cell fusion, the first cell and the second cell in contact with each other in the micropore are fused in pairs. Thus, a fused cell (32) can be obtained, and efficient cell fusion in a pair of two cells becomes possible.

ところで、微細孔では電気力線の集中が生じるため、微細孔付近の電界強度は、図２５に示すように微細孔内の電極面の電界強度が最も高く、絶縁膜面からもう一方の電極に向けて次第に電界強度が弱くなる。図２５は、一方の電極に任意の膜厚の絶縁膜に任意の直径と深さを有する微細孔を１個配置し、電極間に任意の電圧を印加した場合の電界強度を有限要素法を用いて計算した。縦軸が電界強度を最大の電界強度で正規化した値であり、横軸は電極間の位置である。横軸の原点に絶縁膜を配置した電極が存在している。絶縁膜面は図中の点線で示した位置に相当し、横軸の原点から点線までの範囲が絶縁膜厚に相当する。今回行った計算では、絶縁膜の材質や厚み、微細孔の大きさや深さにあまり大きく依存せず、図２５に示すように微細孔表面の電界強度は、微細孔表面から離れるに従って小さくなる結果となった。   By the way, since the electric field lines are concentrated in the fine hole, the electric field strength in the vicinity of the fine hole has the highest electric field strength on the electrode surface in the fine hole as shown in FIG. 25, and from the insulating film surface to the other electrode. The electric field strength gradually decreases. FIG. 25 shows a finite element method for calculating the electric field strength when an arbitrary voltage is applied between electrodes by arranging one minute hole having an arbitrary diameter and depth in an insulating film of an arbitrary thickness on one electrode. Used to calculate. The vertical axis is the value obtained by normalizing the electric field intensity with the maximum electric field intensity, and the horizontal axis is the position between the electrodes. There is an electrode in which an insulating film is arranged at the origin of the horizontal axis. The insulating film surface corresponds to the position indicated by the dotted line in the figure, and the range from the origin of the horizontal axis to the dotted line corresponds to the insulating film thickness. The calculation performed this time does not depend greatly on the material and thickness of the insulating film and the size and depth of the fine holes, and the electric field strength on the surface of the fine holes decreases as the distance from the surface of the fine holes decreases, as shown in FIG. It became.

一般に電気的細胞融合法は、前述したように、細胞融合させるための直流パルス電圧を印加することで細胞膜の電気伝導度が瞬間的に低下し、脂質二重層から構成される細胞膜の可逆的乱れとその再構成が行われることで細胞融合させる。一般に細胞の直径が小さいほど細胞膜の可逆的乱れを生じさせる直流パルス電圧は高くなる。従って、直径の小さい細胞を第１の細胞として微細孔に入れ、直径の大きい細胞を第２の細胞として微細孔に固定された第１の細胞の上から固定すれば、印加する直流パルス電圧は同じでも、図２５に示すように、電界強度は微細孔の表面から次第に小さくなるために、微細孔表面に固定された直径の小さい第１の細胞にはより高い電圧が印加され、第１の細胞の上に固定された直径の大きい第２の細胞には第１の細胞に印加される電圧よりも低い電圧が印加される。このようにすることで、直径の異なる細胞を細胞融合させる場合の細胞膜の可逆的乱れを生じさせる電圧の違いをある程度キャンセルすることが可能となる。   In general, as described above, in the electric cell fusion method, by applying a DC pulse voltage for cell fusion, the electric conductivity of the cell membrane is instantaneously reduced, and the reversible disturbance of the cell membrane composed of lipid bilayers. And the cell is fused by reconfiguration. In general, the smaller the cell diameter, the higher the DC pulse voltage that causes reversible disturbance of the cell membrane. Therefore, if a cell having a small diameter is put in the micropore as a first cell and a cell having a large diameter is fixed as a second cell from above the first cell fixed in the micropore, the DC pulse voltage to be applied is Even in the same case, as shown in FIG. 25, since the electric field strength gradually decreases from the surface of the micropore, a higher voltage is applied to the first cell having a small diameter fixed to the surface of the micropore, A voltage lower than the voltage applied to the first cell is applied to the second cell having a large diameter fixed on the cell. By doing so, it becomes possible to cancel to some extent the voltage difference that causes reversible disturbance of the cell membrane when cells of different diameters are fused.

また、前述したように微細孔に固定した第１の細胞と第２の細胞の接触確率を上げるため、第２の細胞の濃度を第１の細胞の濃度より高くし、細胞融合領域に過剰に導入した場合でも、図２５に示すように、電界強度は微細孔近傍で最も高く、微細孔から離れるに従って小さくなっていくため、直流パルス電圧を適切に調整することで、微細孔近傍で接触した第１の細胞と第２の細胞のみの細胞膜が可逆的乱れを生じ細胞融合する。従って、微細孔近傍で接触した第１の細胞と第２の細胞のみを選択的に細胞融合させることが可能となる。   In addition, as described above, in order to increase the contact probability between the first cells fixed in the micropores and the second cells, the concentration of the second cells is set higher than the concentration of the first cells, and excessively in the cell fusion region. Even when it is introduced, as shown in FIG. 25, the electric field strength is the highest in the vicinity of the fine hole and becomes smaller as the distance from the fine hole becomes smaller, so that the contact is made in the vicinity of the fine hole by appropriately adjusting the DC pulse voltage. The cell membranes of only the first cell and the second cell cause reversible disturbance and cell fusion. Therefore, it is possible to selectively fuse only the first cell and the second cell that are in contact with each other in the vicinity of the micropore.

本発明によれば、以下の効果を奏することができる。
（１）本発明の細胞融合装置は、微細孔を形成した絶縁体を細胞融合領域側のどちらか一方の電極面上に配置することで、微細孔に細胞を確実に固定し、微細孔近傍にある２細胞一対を選択的に細胞融合させることができ、２細胞一対での細胞融合を効率的に行うことが可能となる。
（３）本発明の細胞融合装置は、微細孔の平面形状の直径が、微細孔に固定する細胞の直径より小さく、このようにすることで、微細孔に確実に細胞を固定することができる。
（４）本発明の細胞融合装置は、微細孔が絶縁体上に複数個、アレイ状に形成されており、このようにすることで、複数の微細孔に固定した２細胞一対の細胞を同時に細胞融合させることが可能となり、２細胞一対での細胞融合を効率的に行うことが可能となる。
（５）本発明の細胞融合装置においては、微細孔の隣り合う間隔が、微細孔に入れる細胞の直径の０．５倍以上６倍以下の範囲であり、このようにすることで、１つの微細孔に１つの細胞を固定する確率を高めることができ、微細孔において２細胞を一対で接触させる確率を上げることが可能になる。
（６）本発明の細胞融合装置においては、交流電源により電極間に印加する交流電圧の波形を制御することで、１つの微細孔に１つの細胞を固定することが可能となる。
（７）本発明の細胞融合方法においては、上記記載の細胞融合装置を用いた細胞融合方法であって、細胞融合領域内に第１の細胞を導入し、交流電圧を印加することで微細孔内に前記第１の細胞を固定した後、細胞融合領域内に第２の細胞を導入して、交流電圧を印加することで第１の細胞に第２の細胞を微細孔の位置において接触させ、直流パルス電圧を印加して細胞融合する細胞融合方法であり、前記交流電圧は、前述した波形を有する交流電圧であることが好ましく、また、第１の細胞と第２の細胞が微細孔表面の近傍で細胞融合するものであり、このようにすることで、微細孔近傍にある２細胞一対を選択的に細胞融合させることができ、２細胞一対での細胞融合を効率的に行うことが可能となる。
（８）本発明の細胞融合方法においては、第１の細胞の直径が、第２の細胞の直径よりも小さいものを用いるものであり、このようにすることで、直径の異なる細胞を細胞融合させる場合の細胞膜の可逆的乱れを生じさせる電圧の違いをある程度キャンセルすることが可能となる。 According to the present invention, the following effects can be obtained.
(1) In the cell fusion device of the present invention, the insulator in which the micropores are formed is arranged on one of the electrode surfaces on the cell fusion region side, so that the cells are securely fixed in the micropores, and the vicinity of the micropores The two cell pairs in the cell can be selectively fused with each other, and the cell fusion with the two cell pairs can be performed efficiently.
(3) In the cell fusion device of the present invention, the diameter of the planar shape of the micropore is smaller than the diameter of the cell to be fixed in the micropore, and in this way, the cell can be fixed securely in the micropore. .
(4) In the cell fusion device of the present invention, a plurality of micropores are formed in an array on an insulator. By doing so, a pair of two cells fixed in the plurality of micropores can be simultaneously obtained. Cell fusion is possible, and cell fusion in a pair of two cells can be performed efficiently.
(5) In the cell fusion device of the present invention, the interval between adjacent micropores is in the range of 0.5 to 6 times the diameter of the cells to be inserted into the micropore. The probability of fixing one cell in the micropore can be increased, and the probability of bringing two cells into contact with each other in the micropore can be increased.
(6) In the cell fusion device of the present invention, it is possible to fix one cell in one minute hole by controlling the waveform of the AC voltage applied between the electrodes by the AC power source.
(7) The cell fusion method of the present invention is a cell fusion method using the above-described cell fusion device, wherein the first cell is introduced into the cell fusion region and an alternating voltage is applied to thereby micropores. After fixing the first cell in the cell, the second cell is introduced into the cell fusion region, and an AC voltage is applied to bring the second cell into contact with the first cell at the position of the micropore. , A cell fusion method in which a DC pulse voltage is applied to fuse the cells, and the AC voltage is preferably an AC voltage having the waveform described above, and the first cell and the second cell have a microporous surface. In this way, two cell pairs in the vicinity of the micropores can be selectively fused, and cell fusion with two cell pairs can be efficiently performed. It becomes possible.
(8) In the cell fusion method of the present invention, a cell having a diameter of the first cell smaller than that of the second cell is used. By doing so, cells having different diameters are fused. It is possible to cancel to some extent the voltage difference that causes reversible disturbance of the cell membrane.

以下、本発明の実施の形態について詳細に説明する。なお本発明は、これらの実施例のみに限定されるものではなく、発明の要旨を逸脱しない範囲で、任意に変更が可能であることは言うまでもない。
（実施例１）
図７に実施例１に用いた細胞融合装置の概念図を示す。細胞融合装置は大きく分けて、細胞融合容器（１３）と電源（４）から構成される。細胞融合容器は、図７に示すように上部電極（１４）と下部電極（１５）の間に、スペーサー（１６）を配置し、複数の微細孔をアレイ状に形成した絶縁体（８）をスペーサーと下部電極で挟んだ構造を有する。なお、実施例２では、後述するように一般的なフォトリソグラフィーとエッチングにより、下部電極（１５）と複数の微細孔をアレイ状に形成した絶縁体を一体形成した微細孔付き絶縁体一体型下部電極（２８）を用いた。 Hereinafter, embodiments of the present invention will be described in detail. Needless to say, the present invention is not limited to these examples, and can be arbitrarily changed without departing from the scope of the invention.
(Example 1)
FIG. 7 shows a conceptual diagram of the cell fusion device used in Example 1. The cell fusion device is roughly divided into a cell fusion container (13) and a power source (4). As shown in FIG. 7, the cell fusion container has an insulator (8) in which a spacer (16) is arranged between an upper electrode (14) and a lower electrode (15), and a plurality of micropores are formed in an array. It has a structure sandwiched between a spacer and a lower electrode. In Example 2, as will be described later, an insulator-integrated lower portion with a fine hole in which a lower electrode (15) and an insulator in which a plurality of fine holes are formed in an array are integrally formed by general photolithography and etching. An electrode (28) was used.

上部電極と下部電極は、縦７０ｍｍ×横４０ｍｍ×厚さ１ｍｍのパイレックス（登録商標）基板に、ＩＴＯを成膜（膜厚１５０ｎｍ）したものを用いた。スペーサーは、縦４０ｍｍ×横４０ｍｍ×厚さ１．５ｍｍのシリコンシートの中央を縦２０ｍｍ×横２０ｍｍにくりぬいた形状にして用いた。また、図７に示すように、細胞が含有した細胞懸濁液を導入、排出するための導入口（１９）と排出口（２０）を設けた。複数の微細孔を有する絶縁体（８）は、図２６に示すフォトリソグラフィーとエッチングによる方法により下部電極に一体形成することで作製した。   The upper electrode and the lower electrode were formed by depositing ITO (film thickness 150 nm) on a Pyrex (registered trademark) substrate 70 mm long × 40 mm wide × 1 mm thick. The spacer was used by hollowing out the center of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm into a length of 20 mm and a width of 20 mm. Moreover, as shown in FIG. 7, the introduction port (19) and the discharge port (20) for introducing and discharging the cell suspension containing the cells were provided. The insulator (8) having a plurality of fine holes was produced by integrally forming the lower electrode by a photolithography and etching method shown in FIG.

まずはじめに、ＩＴＯ（２３）を成膜したパイレックス（登録商標）ガラス（２４）のＩＴＯ成膜面にレジスト（２５）を２．５μｍの膜厚になるようスピンコーターを用いて塗布し、４５分自然乾燥後、ホットプレートを用いてプリベーク（８０℃、１５分）を行った。レジストにはキシレン系のネガタイプレジストを用いた。次に、縦３０ｍｍ×横３０ｍｍのエリアに、微細孔と微細孔の縦と横の間隔が３０μｍで、縦１０００個×横１０００個のアレイ状に並べた直径φ４．５μｍの微細孔パターンを描いた露光用フォトマスク（２６）を用いて、ＵＶ露光機にてレジストを露光（２７）し、現像液（３３）で現像した。露光時間と現像時間は、微細孔の深さがレジストの膜厚と等しい２．５μｍになるように調整し、微細孔の底面にＩＴＯが露出するようにした。現像後、ホットプレートを用いてポストベーク（１１５℃、３０分）を行いレジストを固めた。このようにして作製した上部電極（１４）、スペーサー（１６）、微細孔付き絶縁体一体型下部電極（２８）を図８のように積層し圧着した。図８は、図７に示した細胞融合容器のＢＢ’断面図である。シリコンシートの表面は粘着性があり、圧着することで各部品は密着し、細胞を含有した細胞懸濁液を漏れなく細胞融合容器の中に入れることができた。スペーサーをくりぬいた面積が縦２０ｍｍ×横２０ｍｍであることから、この空間に存在する微細孔の数は約４０万個である。また、電極間に電圧を印加する電源は、交流電源として信号発生器（エヌエフ回路設計ブロック製、ＷＦ１９６６）、直流パルス電源として細胞融合用電源（ネッパジーン製、ＬＦ１０１）を導電線（３）を介して接続し、交流電源と直流パルス電源は切換スイッチにより電極への接続を切り換えられるようにした。   First, a resist (25) was applied to the ITO film-forming surface of Pyrex (registered trademark) glass (24) on which ITO (23) was formed using a spin coater so as to have a film thickness of 2.5 μm. After natural drying, pre-baking (80 ° C., 15 minutes) was performed using a hot plate. A xylene negative resist was used as the resist. Next, in the area of 30 mm length × 30 mm width, a microhole pattern with a diameter of 4.5 μm is arranged in an array of 1000 vertical x 1000 horizontal, with the vertical and horizontal spacing of the micropores being 30 μm. The resist was exposed (27) with a UV exposure machine using the exposure photomask (26), and developed with a developer (33). The exposure time and development time were adjusted so that the depth of the micropores was 2.5 μm, which is equal to the film thickness of the resist, so that the ITO was exposed on the bottom surfaces of the micropores. After development, the resist was hardened by post-baking (115 ° C., 30 minutes) using a hot plate. The upper electrode (14), the spacer (16), and the insulator-integrated lower electrode (28) with fine holes were laminated and pressure-bonded as shown in FIG. FIG. 8 is a BB ′ sectional view of the cell fusion container shown in FIG. 7. The surface of the silicon sheet was sticky, and the parts were brought into close contact with each other by pressure bonding, and the cell suspension containing the cells could be put into the cell fusion container without leakage. Since the area where the spacer is hollowed is 20 mm long × 20 mm wide, the number of micropores existing in this space is about 400,000. The power source for applying a voltage between the electrodes is a signal generator (NF circuit design block, WF1966) as an AC power source, and a cell fusion power source (Neppagene, LF101) as a DC pulse power source via a conductive wire (3). The AC power supply and DC pulse power supply can be switched to the electrode by a changeover switch.

細胞は、マウス抗体産生細胞（φ５μｍ）とマウスミエローマ細胞（φ１０μｍ）を用いた。両方の細胞を３００ｍＭの濃度のマンニトール水溶液に懸濁させ、０．７×１０６個／ｍＬの密度になるように細胞懸濁液を調整した。両細胞懸濁液には、細胞融合での細胞膜の再生を促進するために、０．１ｍＭの塩化カルシウム、０．１ｍＭの塩化マグネシウムを添加した。 As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Both cells were suspended in an aqueous mannitol solution having a concentration of 300 mM, and the cell suspension was adjusted to a density of 0.7 × 10 6 cells / mL. To both cell suspensions, 0.1 mM calcium chloride and 0.1 mM magnesium chloride were added in order to promote cell membrane regeneration by cell fusion.

まずはじめに、上記マウス抗体産生細胞の細胞懸濁液６００μＬ（マウス抗体産生細胞数：約４０万個）をスペーサーの導入口よりシリンジを用いて注入し、交流電源により電圧１０Ｖｐｐ、周波数３ＭＨｚの矩形波交流電圧を電極間に印加したところ、２〜３秒程度の極めて短い時間でアレイ状に形成した複数の微細孔に１つずつ、１つのマウス抗体産生細胞を固定することができ、複数の細胞をアレイ状に配置させることができた。このときの、１つの微細孔に１つのマウス抗体産生細胞が入る確率は４５％であった。   First, 600 μL of the mouse antibody-producing cell suspension (number of mouse antibody-producing cells: about 400,000) was injected from the introduction port of the spacer using a syringe, and a rectangular wave with a voltage of 10 Vpp and a frequency of 3 MHz was supplied from an AC power source. When an alternating voltage is applied between the electrodes, one mouse antibody-producing cell can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. Could be arranged in an array. At this time, the probability of one mouse antibody-producing cell entering one micropore was 45%.

続いて、交流電源により電圧１０Ｖｐｐ、周波数３ＭＨｚの矩形波交流電圧を電極間に印加したまま、上記マウスミエローマ細胞の細胞懸濁液６００μＬ（マウスミエローマ細胞数：約４０万個）をスペーサーの導入口よりシリンジを用いて注入したところ、２〜３秒程度の極めて短い時間でアレイ状に形成した複数の微細孔に１つずつ、１つのマウスミエローマ細胞を固定することができ、複数の細胞をアレイ状に配置させることができた。このときの、１つの微細孔に１つのマウスミエローマ細胞が入る確率は５５％であった。マウスミエローマ細胞を導入する際に、先に入れたマウス抗体産生細胞が微細孔から脱離する様子はほとんど観察されなかったことから、微細孔においてマウス抗体産生細胞とマウスミエローマ細胞が２細胞一対で接触している確立は、約２５％（＝４５％×５５％）であると推定される。   Subsequently, 600 μL of the cell suspension of mouse myeloma cells (number of mouse myeloma cells: about 400,000 cells) was applied to the spacer inlet while a rectangular wave AC voltage having a voltage of 10 Vpp and a frequency of 3 MHz was applied between the electrodes by an AC power source. When injected with a syringe, one mouse myeloma cell can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. It was possible to arrange in the shape. At this time, the probability of one mouse myeloma cell entering one micropore was 55%. When the mouse myeloma cells were introduced, it was hardly observed that the mouse antibody-producing cells put in the micropores were detached from the micropores. Therefore, the mouse antibody-producing cells and the mouse myeloma cells were paired in pairs in the micropores. Estimated contact is estimated to be about 25% (= 45% × 55%).

次に、電源を直流パルス電源（ネッパジーン株式会社製、ＬＦ１０１）に切り換えて、電極間に電圧６０Ｖ、パルス幅３０μｓの直流パルス電圧を印加し細胞融合を行い、そのまま１０分静置したあと細胞融合容器内の細胞懸濁液をＨＡＴ培地（Ｈ：ヒポキサンチン（ｈｙｐｏｘａｎｔｈｉｎｅ）、Ａ：アミノプテリン（ａｍｉｎｏｐｔｅｒｉｎｅ）、Ｔ：チミジン（ｔｈｙｍｉｄｉｎｅ）を成分とする培地）に入れ、融合細胞の培養を行った。なお、ＨＡＴ培地は、融合細胞のみを選択的に増殖させる培地である。細胞懸濁液を入れたＨＡＴ培地をＣＯ２インキュベータに入れて細胞培養を行い６日後に融合細胞をカウントした結果、４０個の融合細胞を確認することができ、全マウス抗体産生細胞４０万個に対して１．６／１００００の融合確率を得られた。これは、比較例１に示した通常の電気的細胞融合における融合確率０．２／１００００の８倍の融合確率であり、効率的な２細胞一対での細胞融合を確認することができた。 Next, the power source is switched to a DC pulse power source (LF101, manufactured by Nepagene Co., Ltd.), a cell fusion is performed by applying a DC pulse voltage with a voltage of 60 V and a pulse width of 30 μs between the electrodes. The cell suspension in the container was placed in a HAT medium (H: hypoxanthine, A: medium containing aminopterine, T: thymidine), and the fused cells were cultured. The HAT medium is a medium for selectively growing only fused cells. The HAT medium containing the cell suspension was placed in a CO 2 incubator, and the cells were cultured. After 6 days, the number of fused cells was counted. A fusion probability of 1.6 / 10000 was obtained. This is a fusion probability that is 8 times the fusion probability of 0.2 / 10000 in the normal electric cell fusion shown in Comparative Example 1, and an efficient cell fusion in a pair of two cells could be confirmed.

（比較例１）
比較例１として、通常の電気的細胞融合を行った。電気的細胞融合を行う電極には、電極間１ｍｍの金製のワイヤー電極（ネッパジーン株式会社製、ＭＳゴールドワイヤー電極）を用い、この電極に細胞融合用電源（ネッパジーン製、ＬＦ１０１）を接続した。 (Comparative Example 1)
As Comparative Example 1, normal electric cell fusion was performed. A gold wire electrode (MS gold wire electrode, manufactured by Nepagene Co., Ltd.) having a distance between electrodes of 1 mm was used as an electrode for performing electric cell fusion, and a cell fusion power source (manufactured by Nepagene, LF101) was connected to this electrode.

細胞は、マウス抗体産生細胞（φ５μｍ）とマウスミエローマ細胞（φ１０μｍ）を用いた。マウス抗体産生細胞とマウスミエローマ細胞を４：１で混合し、両方の細胞を３００ｍＭの濃度のマンニトール水溶液に懸濁させ、１．７×１０７個／ｍＬの密度になるように細胞懸濁液を調整した。両細胞懸濁液には、細胞融合での細胞膜の再生を促進するために、０．１ｍＭの塩化カルシウム、０．１ｍＭの塩化マグネシウムを添加した。 As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Mouse antibody-producing cells and mouse myeloma cells are mixed at a ratio of 4: 1. Both cells are suspended in an aqueous mannitol solution having a concentration of 300 mM, and the cell suspension is adjusted to a density of 1.7 × 10 7 cells / mL. Adjusted. To both cell suspensions, 0.1 mM calcium chloride and 0.1 mM magnesium chloride were added in order to promote cell membrane regeneration by cell fusion.

上記細胞懸濁液４０μＬ（マウス抗体産生細胞数：約６０万個、マウスミエローマ細胞数：１５万個）を電極間に注入し、細胞融合用電源を用いて、電圧２０Ｖｐｐ、周波数３ＭＨｚの正弦波交流電圧を電極間に印加し細胞パールチェーンの形成を確認後、細胞融合を行うため、電圧値２００Ｖ、パルス幅３０μｓの直流パルス電圧を印加した。１０分静置したあと細胞融合容器内の細胞懸濁液をＨＡＴ培地に入れた。細胞懸濁液を入れたＨＡＴ培地をＣＯ２インキュベータに入れて細胞培養を行い６日後に融合細胞をカウントした結果、１２個の融合細胞を確認することができ、全マウス抗体産生細胞６０万個に対して０．２／１００００の融合確率を得られた。 40 μL of the above cell suspension (number of mouse antibody producing cells: about 600,000, number of mouse myeloma cells: 150,000) was injected between the electrodes, and using a cell fusion power source, a sine wave with a voltage of 20 Vpp and a frequency of 3 MHz. After confirming the formation of the cell pearl chain by applying an AC voltage between the electrodes, a DC pulse voltage having a voltage value of 200 V and a pulse width of 30 μs was applied to perform cell fusion. After standing for 10 minutes, the cell suspension in the cell fusion container was placed in HAT medium. The HAT medium containing the cell suspension was placed in a CO 2 incubator and cultured. The number of fused cells was counted after 6 days. As a result, 12 fused cells could be confirmed, and 600,000 total mouse antibody-producing cells were obtained. A fusion probability of 0.2 / 10000 was obtained.

（比較例２）
図２７に比較例２に用いた細胞融合装置の概念図を示す。細胞融合装置は大きく分けて、細胞融合容器（１３）と電源（４）から構成される。細胞融合容器は、図２７に示すように上部電極（１４）と下部電極（１５）の間に、スペーサー（１６）を２枚配置し、複数の微細孔をアレイ状に形成した絶縁体（８）を２枚のスペーサー間に挟んだ構造を有する。上部電極と下部電極は、縦７０ｍｍ×横４０ｍｍ×厚さ１ｍｍのパイレックス（登録商標）基板に、ＩＴＯを成膜（膜厚１５０ｎｍ）したものを用いた。スペーサーは、縦４０ｍｍ×横４０ｍｍ×厚さ１ｍｍのシリコンシートの中央を縦２０ｍｍ×横２０ｍｍにくりぬいた形状にして用いた。また、図２７に示すように、細胞が含有した細胞懸濁液を導入、排出するための導入口（１９）と排出口（２０）を設けた。複数の微細孔を有する絶縁体（８）は、縦４０ｍｍ×横４０ｍｍ×膜厚２５μｍのポリイミドフィルムに、フォトリソグラフィーとエッチングによる方法により、中央の縦３０ｍｍ×横３０ｍｍのエリアに、微細孔と微細孔の縦と横の間隔が５０μｍで、縦３００個×横３００個のアレイ状に並べた直径φ２０μｍの微細孔を形成し製作した。 (Comparative Example 2)
FIG. 27 shows a conceptual diagram of the cell fusion device used in Comparative Example 2. The cell fusion device is roughly divided into a cell fusion container (13) and a power source (4). As shown in FIG. 27, the cell fusion container has an insulator (8) in which two spacers (16) are arranged between the upper electrode (14) and the lower electrode (15), and a plurality of micropores are formed in an array. ) Is sandwiched between two spacers. The upper electrode and the lower electrode were formed by depositing ITO (film thickness 150 nm) on a Pyrex (registered trademark) substrate 70 mm long × 40 mm wide × 1 mm thick. The spacer was used by hollowing out the center of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1 mm into a length of 20 mm and a width of 20 mm. Moreover, as shown in FIG. 27, the introduction port (19) and the discharge port (20) for introducing and discharging the cell suspension containing the cells were provided. Insulator (8) having a plurality of fine holes is formed in a polyimide film having a length of 40 mm, a width of 40 mm, and a film thickness of 25 μm, by a method using photolithography and etching. Fine holes having a diameter of 20 μm arranged in an array of 300 vertical × 300 horizontal were formed with a space between the vertical and horizontal holes of 50 μm.

このようにして作製した上部電極（１４）、スペーサー（１６）、複数の微細孔を形成した絶縁体（８）、下部電極（１５）を図２７のように積層し圧着した。図２９は、図２７に示した細胞融合容器のＣＣ’断面図である。シリコンシートの表面は粘着性があり、圧着することで各部品は密着し、細胞を含有した細胞懸濁液を漏れなく細胞融合容器の中に入れることができた。スペーサーをくりぬいた面積が縦２０ｍｍ×横２０ｍｍであることから、この空間に存在する微細孔の数は約４万個である。また、電極間に電圧を印加する電源は、交流電源として信号発生器（エヌエフ回路設計ブロック製、ＷＦ１９６６）、直流パルス電源として細胞融合用電源（ネッパジーン製、ＬＦ１０１）を導電線（３）を介して接続し、交流電源と直流パルス電源は切換スイッチにより電極への接続を切り換えられるようにした。   The thus prepared upper electrode (14), spacer (16), insulator (8) having a plurality of fine holes, and lower electrode (15) were laminated and pressure-bonded as shown in FIG. FIG. 29 is a CC ′ cross-sectional view of the cell fusion container shown in FIG. The surface of the silicon sheet was sticky, and the parts were brought into close contact with each other by pressure bonding, and the cell suspension containing the cells could be put into the cell fusion container without leakage. Since the area formed by hollowing out the spacer is 20 mm long × 20 mm wide, the number of micropores existing in this space is about 40,000. The power source for applying a voltage between the electrodes is a signal generator (NF circuit design block, WF1966) as an AC power source, and a cell fusion power source (Neppagene, LF101) as a DC pulse power source via a conductive wire (3). The AC power supply and DC pulse power supply can be switched to the electrode by a changeover switch.

細胞は、マウス抗体産生細胞（φ５μｍ）とマウスミエローマ細胞（φ１０μｍ）を用いた。両方の細胞を３００ｍＭの濃度のマンニトール水溶液に懸濁させ、０．７×１０６個／ｍＬの密度になるように細胞懸濁液を調整した。両細胞懸濁液には、細胞融合での細胞膜の再生を促進するために、０．１ｍＭの塩化カルシウム、０．１ｍＭの塩化マグネシウムを添加した。 As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Both cells were suspended in an aqueous mannitol solution having a concentration of 300 mM, and the cell suspension was adjusted to a density of 0.7 × 10 6 cells / mL. To both cell suspensions, 0.1 mM calcium chloride and 0.1 mM magnesium chloride were added in order to promote cell membrane regeneration by cell fusion.

まずはじめに、上記マウス抗体産生細胞の細胞懸濁液６００μＬ（マウス抗体産生細胞数：約４０万個）をスペーサーの導入口よりシリンジを用いて注入し、信号発生器により電圧１５Ｖｐｐ、周波数３ＭＨｚの矩形波交流電圧を電極間に印加したところ、２〜３秒程度の極めて短い時間でアレイ状に形成した微細孔にマウス抗体産生細胞を固定することがでた。ただし、１つの微細孔に複数のマウス抗体産生細胞が固定されている微細孔や、マウス抗体産生細胞が全く固定されていない微細孔があり、複数の微細孔１つずつに１つのマウス抗体産生細胞が固定されている微細孔はほとんど確認できなかった。   First, 600 μL of the cell suspension of mouse antibody-producing cells (number of mouse antibody-producing cells: about 400,000) was injected from the introduction port of the spacer using a syringe, and a rectangular shape with a voltage of 15 Vpp and a frequency of 3 MHz by a signal generator. When a wave AC voltage was applied between the electrodes, mouse antibody-producing cells could be fixed in the micropores formed in an array in an extremely short time of about 2 to 3 seconds. However, there are micropores in which a plurality of mouse antibody-producing cells are fixed in one micropore and micropores in which no mouse antibody-producing cells are fixed at all, and one mouse antibody is produced for each of the plurality of micropores. Micropores in which cells were fixed could hardly be confirmed.

続いて、交流電源により電圧１５Ｖｐｐ、周波数３ＭＨｚの矩形波交流電圧を電極間に印加したまま、上記マウスミエローマ細胞の細胞懸濁液６００μＬ（マウスミエローマ細胞数：約４０万個）をスペーサーの導入口よりシリンジを用いて注入したところ、先に微細孔に固定されていたマウス抗体産生細胞が微細孔からほとんど脱離してしまい、マウス抗体産生細胞とマウスミエローマ細胞を微細孔において細胞融合させることはできなかった。   Subsequently, 600 μL of the mouse myeloma cell suspension (number of mouse myeloma cells: about 400,000) was applied to the spacer inlet while a rectangular wave AC voltage having a voltage of 15 Vpp and a frequency of 3 MHz was applied between the electrodes by an AC power supply. When injected with a syringe more, mouse antibody-producing cells previously fixed in the micropores are almost detached from the micropores, and the mouse antibody-producing cells and mouse myeloma cells cannot be fused in the micropores. There wasn't.

特許文献１記載の細胞融合用チャンバーの断面図を示した概念図である。It is the conceptual diagram which showed sectional drawing of the chamber for cell fusion of patent document 1. FIG. 特許文献１記載の細胞融合用チャンバーの動作を説明する第１の図である。FIG. 3 is a first diagram illustrating the operation of the cell fusion chamber described in Patent Document 1. 特許文献１記載の細胞融合用チャンバーの動作を説明する第２の図である。FIG. 10 is a second diagram illustrating the operation of the cell fusion chamber described in Patent Document 1. 特許文献１記載の細胞融合用チャンバーの動作を説明する第３の図である。FIG. 10 is a third diagram illustrating the operation of the cell fusion chamber described in Patent Document 1. 本発明の細胞融合装置の一例を示す概念図である。It is a conceptual diagram which shows an example of the cell fusion apparatus of this invention. 図５に示した細胞融合容器のＡＡ’断面図である。FIG. 6 is an AA ′ cross-sectional view of the cell fusion container shown in FIG. 5. 本発明の細胞融合装置の一例及び、実施例２で用いた細胞融合装置の概念図である。It is a conceptual diagram of an example of the cell fusion apparatus of this invention, and the cell fusion apparatus used in Example 2. FIG. 図７に示した細胞融合容器のＢＢ’断面図である。FIG. 8 is a BB ′ sectional view of the cell fusion container shown in FIG. 7. 本発明に用いる交流電圧の波形の一例である。It is an example of the waveform of the alternating voltage used for this invention. 本発明に用いる交流電圧の波形の一例として、矩形波を示した図である。It is the figure which showed the rectangular wave as an example of the waveform of the alternating voltage used for this invention. 本発明に用いる交流電圧の波形の一例として、台形波を示した図である。It is the figure which showed the trapezoid wave as an example of the waveform of the alternating voltage used for this invention. 本発明に用いる交流電圧の波形の一例として、矩形波と台形波を組み合わせた波形を示した図である。It is the figure which showed the waveform which combined the square wave and the trapezoid wave as an example of the waveform of the alternating voltage used for this invention. 本発明の細胞操作方法を説明する第１の図である。It is a 1st figure explaining the cell operation method of this invention. 本発明の細胞操作方法を説明する第２の図である。It is a 2nd figure explaining the cell operation method of this invention. 本発明の細胞操作方法を説明する第３の図である。It is a 3rd figure explaining the cell operation method of this invention. 図１３を電気的な等価回路で表した図である。It is the figure which represented FIG. 13 with the electrical equivalent circuit. 図１４を電気的な等価回路で表した図である。It is the figure which represented FIG. 14 with the electrical equivalent circuit. 図１５を電気的な等価回路で表した図である。FIG. 16 is a diagram illustrating FIG. 15 with an electrical equivalent circuit. 周波数ｆ［Ｈｚ］の矩形波交流電圧の波形を示す図である。It is a figure which shows the waveform of the rectangular wave alternating voltage of frequency f [Hz]. 図１９に示した周波数ｆ［Ｈｚ］の矩形波交流電圧の波形を電極間に印加した場合の、図１７のコンデンサーＡにおける電圧波形を示す図である。It is a figure which shows the voltage waveform in the capacitor | condenser A of FIG. 17 at the time of applying the waveform of the rectangular wave alternating voltage of the frequency f [Hz] shown in FIG. 19 between electrodes. 図１９に示した周波数ｆ［Ｈｚ］の矩形波交流電圧の波形を電極間に印加した場合の、図１７のコンデンサーＡにおける電流波形を示す図である。It is a figure which shows the electric current waveform in the capacitor | condenser A of FIG. 17 at the time of applying the waveform of the rectangular wave alternating voltage of the frequency f [Hz] shown in FIG. 19 between electrodes. 周波数ｆ［Ｈｚ］の正弦波交流電圧の波形を示す図である。It is a figure which shows the waveform of the sine wave alternating voltage of frequency f [Hz]. 図２２に示した周波数ｆ［Ｈｚ］の正弦波交流電圧の波形を電極間に印加した場合の、図１７のコンデンサーＡにおける電圧波形または電流波形を示す図である。It is a figure which shows the voltage waveform or current waveform in the capacitor | condenser A of FIG. 17 at the time of applying the waveform of the sine wave alternating voltage of the frequency f [Hz] shown in FIG. 22 between electrodes. 本発明の細胞融合方法の例を示す図である。It is a figure which shows the example of the cell fusion method of this invention. 微細孔近傍の電界強度を示した図である。It is the figure which showed the electric field strength of a micropore vicinity. 一般的なフォトリソグラフィーとエッチング方法の概略図である。It is the schematic of general photolithography and the etching method. 本発明の比較例２で用いた細胞融合装置の概念図である。It is a conceptual diagram of the cell fusion apparatus used in the comparative example 2 of this invention. 図２７に示した細胞融合容器のＣＣ’断面図である。It is CC 'sectional drawing of the cell fusion container shown in FIG. 微細孔の直径が２つの細胞よりの直径よりも大きい場合の例を示す概念図である。It is a conceptual diagram which shows an example in case the diameter of a micropore is larger than the diameter from two cells. 微細孔の直径が２つの細胞よりの直径よりも小さい場合の例を示す概念図である。It is a conceptual diagram which shows an example in case the diameter of a micropore is smaller than the diameter from two cells.

符号の説明Explanation of symbols

１：細胞融合領域
２：電極
３：導電線
４：電源
５：交流電源
６：直流パルス電源
７：切換スイッチ
８：絶縁体
９：微細孔
１０：細胞Ａ
１１：細胞Ｂ
１２：電気力線
１３：細胞融合容器
１４：上部電極
１５：下部電極
１６：スペーサー
１７：微細孔Ａ′
１８：第１の細胞
１９：導入口
２０：排出口
２１：微細孔Ａ′′
２２：第２の細胞
２３：ＩＴＯ
２４：パイレックス（登録商標）ガラス
２５：レジスト
２６：露光用フォトマスク
２７：露光
２８：微細孔付き絶縁体一体型下部電極
２９：コンデンサーＡ
３０：コンデンサーＢ
３１：抵抗
３２：融合細胞
３３：現像液
３４：細胞懸濁液
３５：隔壁
３６：導電線
３７：電源
３８：絶縁体
３９：微細孔
４０：細胞融合容器
４１：上部電極
４２：下部電極
４３：スペーサー
４４：細胞Ａ′
４５：細胞Ａ′′
４６：導入口
４７：排出口
４８：細胞融合領域
1: Cell fusion region 2: Electrode 3: Conductive wire 4: Power supply 5: AC power supply 6: DC pulse power supply 7: Changeover switch 8: Insulator 9: Micropore 10: Cell A
11: Cell B
12: Electric field lines 13: Cell fusion container 14: Upper electrode 15: Lower electrode 16: Spacer 17: Micropore A '
18: first cell 19: introduction port 20: discharge port 21: micropore A ″
22: Second cell 23: ITO
24: Pyrex (registered trademark) glass 25: Resist 26: Photomask for exposure 27: Exposure 28: Insulator-integrated lower electrode with fine holes 29: Capacitor A
30: Capacitor B
31: Resistance 32: Fusion cell 33: Developer 34: Cell suspension 35: Partition 36: Conductive wire 37: Power supply 38: Insulator 39: Micropore 40: Cell fusion container 41: Upper electrode 42: Lower electrode 43: Spacer 44: Cell A ′
45: Cell A ″
46: Inlet 47: Outlet 48: Cell fusion region

Claims (17)

細胞融合領域内に対向して配置される導電部材からなる一対の電極と、前記一対の電極間に平板状のスペーサーを介して配置され、かつ前記対向して配置された電極の方向に貫通した１または複数の微細孔を有した平板状の絶縁体からなる細胞融合容器と、前記一対の電極に交流電圧及び直流パルス電圧を印加する電源と、を備えた細胞融合装置であって、前記絶縁体が、前記電極の内いずれか一方の電極の細胞融合領域側の電極面上に配置されており、かつ前記微細孔の直径が、融合させる細胞の直径より小さいことを特徴とする細胞融合装置。 A pair of electrodes made of conductive members arranged opposite to each other in the cell fusion region, and a flat spacer disposed between the pair of electrodes and penetrating in the direction of the oppositely arranged electrodes A cell fusion device comprising: a cell fusion container made of a flat insulator having one or a plurality of micropores; and a power source for applying an AC voltage and a DC pulse voltage to the pair of electrodes. A cell fusion device, wherein a body is disposed on an electrode surface on the cell fusion region side of any one of the electrodes, and the diameter of the micropore is smaller than the diameter of the cells to be fused . 前記電源が、前記一対の電極に交流電圧を印加するための交流電源及び直流パルス電圧を印加するための直流パルス電源からなり、前記交流電源と前記直流パルス電源とを切り換える切換機構を有することを特徴とする請求項１記載の細胞融合装置。 The power source includes an AC power source for applying an AC voltage to the pair of electrodes and a DC pulse power source for applying a DC pulse voltage, and has a switching mechanism for switching between the AC power source and the DC pulse power source. The cell fusion device according to claim 1, wherein: 前記絶縁体に形成される微細孔が、１つの微細孔につき１つの細胞を固定できる形状であることを特徴とする請求項１または請求項２に記載の細胞融合装置。 The cell fusion device according to claim 1 or 2, wherein the micropore formed in the insulator has a shape capable of fixing one cell per micropore. 前記電源により、１つの微細孔につき１つの細胞を微細孔に固定する波形を有する交流電圧が前記電極間に印加されることを特徴とする請求項１〜３のいずれかに記載の細胞融合装置。 The cell fusion device according to any one of claims 1 to 3, wherein an AC voltage having a waveform for fixing one cell to one micropore is applied between the electrodes by the power source. . 前記電源により、前記細胞の充電と放電を周期的に繰り返す波形を有する交流電圧が前記電極間に印加されることを特徴とする請求項１〜４のいずれかに記載の細胞融合装置。 The cell fusion device according to any one of claims 1 to 4, wherein an AC voltage having a waveform that periodically repeats charging and discharging of the cells is applied between the electrodes by the power source. 前記交流電圧の波形が、０以外の値を有する電圧が一定時間変化しない時間を半周期内に１以上有する波形であることを特徴とする請求項１〜５のいずれかに記載の細胞融合装置。 The cell fusion device according to any one of claims 1 to 5, wherein the waveform of the AC voltage is a waveform having one or more times in a half cycle in which a voltage having a value other than 0 does not change for a certain period of time. . 前記交流電圧の波形が、矩形波、台形波、またはこれらを組み合わせた波形であることを特徴とする請求項１〜６のいずれかに記載の細胞融合装置。 The cell fusion device according to any one of claims 1 to 6, wherein the waveform of the AC voltage is a rectangular wave, a trapezoidal wave, or a combination thereof. 前記交流電圧の波形が、０以外の値を有する電圧が所定時間変化しない時間が細胞の静電容量と細胞を含有する細胞懸濁液の抵抗の積からなる時定数以上であることを特徴とする請求項１〜７のいずれかに記載の細胞融合装置。 The waveform of the alternating voltage is characterized in that the time during which a voltage having a value other than 0 does not change for a predetermined time is not less than a time constant consisting of the product of the capacitance of the cell and the resistance of the cell suspension containing the cell The cell fusion device according to any one of claims 1 to 7. 前記絶縁体に形成される微細孔の中心位置が、前記絶縁体の水平面において、隣り合ういずれの微細孔の中心位置からも、微細孔の中心位置が同じ位置に形成されていることを特徴とする請求項１〜８のいずれかに記載の細胞融合装置。 The center position of the micro hole formed in the insulator is formed at the same position from the center position of any adjacent micro hole in the horizontal plane of the insulator. The cell fusion device according to any one of claims 1 to 8. 前記絶縁体に形成される複数の微細孔が、絶縁体の面においてアレイ状に形成されていることを特徴とする請求項１〜９のいずれかに記載の細胞融合装置。 The cell fusion device according to any one of claims 1 to 9, wherein a plurality of micropores formed in the insulator are formed in an array on the surface of the insulator. 前記微細孔の隣合う間隔が、微細孔に入れる細胞の直径の０．５倍以上６倍以下であることを特徴とする請求項１〜１０のいずれかに記載の細胞融合装置。 The cell fusion device according to any one of claims 1 to 10, wherein an interval between adjacent micropores is not less than 0.5 times and not more than 6 times the diameter of cells to be inserted into the micropores. 前記スペーサーが、細胞融合領域を形成する貫通孔を有することを特徴とする請求項１〜１１のいずれかに記載の細胞融合装置。 The cell fusion device according to any one of claims 1 to 11, wherein the spacer has a through-hole forming a cell fusion region. 前記スペーサーが、細胞を導入する導入流路および排出する排出流路を有することを特徴とする請求項１〜１２のいずれかに記載の細胞融合装置。 The cell fusion device according to any one of claims 1 to 12, wherein the spacer has an introduction channel for introducing cells and a discharge channel for discharging cells. 請求項１〜１３のいずれかに記載の細胞融合装置を用いた細胞融合方法であって、前記細胞融合領域内に第１の細胞を導入し、交流電圧を印加することで前記微細孔内に前記第１の細胞を固定した後、前記細胞融合領域内に第２の細胞を導入して、交流電圧を印加することで前記第１の細胞に前記第２の細胞を微細孔の位置において接触させ、直流パルス電圧を印加して細胞融合することを特徴とする細胞融合方法。 It is a cell fusion method using the cell fusion apparatus in any one of Claims 1-13, Comprising: A 1st cell is introduce | transduced in the said cell fusion area | region, and an alternating voltage is applied, In said micropore After fixing the first cell, the second cell is introduced into the cell fusion region, and an AC voltage is applied to bring the second cell into contact with the first cell at the position of the micropore. And cell fusion by applying a direct-current pulse voltage. 請求項１４記載の交流電圧が、請求項４〜８のいずれかに記載の波形を有する交流電圧であることを特徴とする請求項１４記載の細胞融合方法。 The cell fusion method according to claim 14, wherein the AC voltage according to claim 14 is an AC voltage having the waveform according to any one of claims 4 to 8. 前記第１の細胞と前記第２の細胞が微細孔表面の近傍で細胞融合することを特徴とする請求項１４または請求項１５に記載の細胞融合方法。 The cell fusion method according to claim 14 or 15, wherein the first cell and the second cell are fused in the vicinity of a micropore surface. 前記第１の細胞の直径が、前記第２の細胞の直径よりも小さいことを特徴とする請求項１４〜１６のいずれかに記載の細胞融合方法。

The cell fusion method according to any one of claims 14 to 16, wherein a diameter of the first cell is smaller than a diameter of the second cell.

JP2006237841A 2006-09-01 2006-09-01 Cell fusion device and cell fusion method using the same Pending JP2008054630A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006237841A JP2008054630A (en) 2006-09-01 2006-09-01 Cell fusion device and cell fusion method using the same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP2006237841A JP2008054630A (en) 2006-09-01 2006-09-01 Cell fusion device and cell fusion method using the same

Publications (1)

Publication Number Publication Date
JP2008054630A true JP2008054630A (en) 2008-03-13

Family

ID=39238119

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2006237841A Pending JP2008054630A (en) 2006-09-01 2006-09-01 Cell fusion device and cell fusion method using the same

Country Status (1)

Country Link
JP (1) JP2008054630A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009128483A1 (en) * 2008-04-15 2009-10-22 東ソー株式会社 Cell selection apparatus, and cell selection method using the same
JP2009254292A (en) * 2008-04-17 2009-11-05 Tosoh Corp Cell fusion apparatus and cell fusion method
US8697446B2 (en) 2005-06-13 2014-04-15 Tosoh Corporation Cell fusion chamber, cell fusion device, and method for cell fusion using the same
US9249385B1 (en) 2014-12-18 2016-02-02 City University Of Hong Kong System and method for fusing cells

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60251871A (en) * 1984-05-30 1985-12-12 Hitachi Ltd Apparatus for cell fusion
JPS6349065A (en) * 1986-08-14 1988-03-01 Norin Suisansyo Nogyo Seibutsu Shigen Kenkyusho Electrode for electrical cell operation apparatus and operation chamber
JPS63152971A (en) * 1986-12-15 1988-06-25 Nogyo Seibutsu Shigen Kenkyusho Electrical cell operation method and flow chamber therefor
JPS63152972A (en) * 1986-12-15 1988-06-25 Nogyo Seibutsu Shigen Kenkyusho High speed electrical cell manipulating apparatus for electrical cell manipulation
JPS63173574A (en) * 1987-01-13 1988-07-18 Nok Corp Electrode for cell fusion
JPH01141582A (en) * 1987-11-27 1989-06-02 Shimadzu Corp Gene-introducing device
JPH0740914B2 (en) * 1986-08-20 1995-05-10 株式会社アドバンス Electrode for electric cell fusion

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60251871A (en) * 1984-05-30 1985-12-12 Hitachi Ltd Apparatus for cell fusion
JPS6349065A (en) * 1986-08-14 1988-03-01 Norin Suisansyo Nogyo Seibutsu Shigen Kenkyusho Electrode for electrical cell operation apparatus and operation chamber
JPH0740914B2 (en) * 1986-08-20 1995-05-10 株式会社アドバンス Electrode for electric cell fusion
JPS63152971A (en) * 1986-12-15 1988-06-25 Nogyo Seibutsu Shigen Kenkyusho Electrical cell operation method and flow chamber therefor
JPS63152972A (en) * 1986-12-15 1988-06-25 Nogyo Seibutsu Shigen Kenkyusho High speed electrical cell manipulating apparatus for electrical cell manipulation
JPS63173574A (en) * 1987-01-13 1988-07-18 Nok Corp Electrode for cell fusion
JPH01141582A (en) * 1987-11-27 1989-06-02 Shimadzu Corp Gene-introducing device

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8697446B2 (en) 2005-06-13 2014-04-15 Tosoh Corporation Cell fusion chamber, cell fusion device, and method for cell fusion using the same
WO2009128483A1 (en) * 2008-04-15 2009-10-22 東ソー株式会社 Cell selection apparatus, and cell selection method using the same
JP2009254292A (en) * 2008-04-17 2009-11-05 Tosoh Corp Cell fusion apparatus and cell fusion method
US9249385B1 (en) 2014-12-18 2016-02-02 City University Of Hong Kong System and method for fusing cells

Similar Documents

Publication Publication Date Title
US8697446B2 (en) Cell fusion chamber, cell fusion device, and method for cell fusion using the same
Masuda et al. Novel method of cell fusion in field constriction area in fluid integration circuit
Şen et al. Cell pairing using a dielectrophoresis-based device with interdigitated array electrodes
Hu et al. Cell electrofusion in microfluidic devices: A review
US20170306317A1 (en) Method and device for uniformly treating adherent cells
WO2015021270A2 (en) Microfluidic vortex-assisted electroporation system and method
WO2012048230A2 (en) Dielectrophoresis devices and methods therefor
JP4677832B2 (en) Microfluidic substrate for cell fusion, microfluidic structure for cell fusion using the same, and cell fusion method
JP2010011824A (en) Cell fusion vessel, cell fusion apparatus, and method for cell fusion using the same
WO2008018390A1 (en) Cell patterning method
JP2008054630A (en) Cell fusion device and cell fusion method using the same
JP4910716B2 (en) Cell fusion device and cell fusion method using the same
JP2007296510A (en) Fine particle operation apparatus and fine particle operation method
JP4918811B2 (en) Cell fusion chamber, cell fusion device, and cell fusion method using them
Hu et al. A cell electrofusion microfluidic chip with micro-cavity microelectrode array
Yang et al. Chip‐Based Cell Electrofusion
KR101336555B1 (en) Microdevice for Fusing Cells
JPH0687782B2 (en) Cell electroporation method and device
US20130089931A1 (en) Microdevice for fusing cells
US20130089929A1 (en) Microdevice for fusing cells
JP2008194029A (en) Cell fusion apparatus and cell fusion method using the same
JP2007325586A (en) Device for cell fusion and method of cell fusion by using the same
JP2013126381A (en) Cell fusion method
Pan et al. Cell membrane damage and cargo delivery in nano-electroporation
Clow et al. Coplanar film electrodes facilitate bovine nuclear transfer cloning

Legal Events

Date Code Title Description
A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20090810

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20120104

A02 Decision of refusal

Free format text: JAPANESE INTERMEDIATE CODE: A02

Effective date: 20120508