JP2619387B2 - Cell fusion method - Google Patents
Cell fusion methodInfo
- Publication number
- JP2619387B2 JP2619387B2 JP62104250A JP10425087A JP2619387B2 JP 2619387 B2 JP2619387 B2 JP 2619387B2 JP 62104250 A JP62104250 A JP 62104250A JP 10425087 A JP10425087 A JP 10425087A JP 2619387 B2 JP2619387 B2 JP 2619387B2
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- Prior art keywords
- cells
- chamber
- pair
- cell fusion
- fusion method
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- 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
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- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Microbiology (AREA)
- Sustainable Development (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Electromagnetism (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は細胞融合方法に係り、所望の細胞を融合させ
ることが可能な細胞融合方法に関する。Description: TECHNICAL FIELD The present invention relates to a cell fusion method, and more particularly to a cell fusion method capable of fusing desired cells.
異種細胞に電気パルスを加えて融合せしめる細胞融合
法に関しては、従来、ザ・ジヤーナル・オブ・メンブレ
ン・バイオロジー、67（1982年）第165頁から182頁（J.
of Membrane Biology,vol.67（1982）PP165−182）にお
いて論じられている。Regarding the cell fusion method of fusing heterogeneous cells by applying an electric pulse, conventionally, The Journal of Membrane Biology, 67 (1982), pp. 165 to 182 (J.
of Membrane Biology, vol. 67 (1982) PP165-182).
上記の従来技術では、異種の細胞群が分散している液
に交流電界を加えることによつて、細胞を鏡状に連結さ
せた状態（パールチエーン）を作り、しかる後に電気パ
ルスを加えることによつて、接触した細胞同士を融合せ
しめるものであつた。このため、融合した細胞は必ずし
も２種の細胞ＡおよびＢの組合せ、すなわちＡ−Ｂ、だ
けでなくＡ−A,あるいはＢ−Ｂ、またＡ−Ａ−A,A−Ａ
−Ｂなどの様々の組合せが混じり合つている。さらに液
中には融合されなかつた細胞群A,Bを混入しており、そ
の中から所望のＡ−Ｂの組合せで融合した細胞を選抜し
培養するには、大きな困難を伴つていた。In the above-mentioned conventional technique, by applying an alternating electric field to a liquid in which a heterogeneous group of cells is dispersed, a state in which the cells are connected in a mirror-like manner (pearl chain) and then an electric pulse is applied. Thus, the contacted cells were fused together. For this reason, the fused cells are not necessarily a combination of the two types of cells A and B, that is, not only AB, but also AA or BB, or AAA, AA
Various combinations such as -B are mixed. Furthermore, the cell groups A and B, which have not been fused, are mixed in the liquid, and it has been very difficult to select and culture the cells fused with a desired combination of AB from them.
本発明の目的は、所望の細胞を融合させることが可能
な細胞融合方法を提供することにある。An object of the present invention is to provide a cell fusion method capable of fusing desired cells.
上記目的は、融合すべぎ少なくとも一対の単位細胞を
収納する微小な容器（マイクロチヤンバと称する）を密
に配置した部材（チヤンバプレートと称する）を用意
し、それぞれの容器内に細胞を導入すること、および、
該部材の一部に電極を形成し、容器内の細胞対に電圧を
印加することによって達成される。The object is to prepare a member (referred to as a chamber plate) in which minute containers (referred to as microchambers) for storing at least a pair of unit cells for fusion are prepared, and cells are introduced into each container. To do, and
This is achieved by forming an electrode on a part of the member and applying a voltage to a cell pair in the container.
本発明の要旨は、マイクロチャンバ内に、一対の異種
細胞を収納するステップと、前記マイクロチャンバの底
面に設けられ、前記一対の異種細胞のそれぞれよりも寸
法の小さな開口部から吸引して前記底面に前記一対の異
種細胞を吸着するステップと、前記マイクロチャンバ内
で、前記一対の異種細胞には非接触となるように設けら
れた電極に電圧を印加し、前記一対の異種細胞間に電気
パルスを加えて前記一対の異種細胞を融合させるステッ
プとを有することを特徴とする細胞融合方法にある。The gist of the present invention is a step of accommodating a pair of heterogeneous cells in a microchamber, and provided on a bottom surface of the microchamber, wherein the bottom surface is sucked from an opening having a smaller size than each of the pair of heterogeneous cells. Adsorbing the pair of heterogeneous cells, applying a voltage to an electrode provided so as not to contact the pair of heterogeneous cells in the microchamber, and applying an electric pulse between the pair of heterogeneous cells. And fusing the pair of heterologous cells with each other.
細胞を収納する容器に供給された細胞は、容器から飛
び出さないように、液体的手段等によつて容器内に保持
される。これによつて、近隣に配置された容器との間で
の細胞の移動、混入は生じない。一方、電極は容器内の
融合すべき少なくとも１対の細胞の間にのみ電圧を加え
ることができるように配置されている。これによつて、
容器内に形成した個々の単位細胞についてのみ融合が行
われる。The cells supplied to the container containing the cells are held in the container by liquid means or the like so as not to jump out of the container. As a result, there is no migration or contamination of cells with a container disposed in the vicinity. On the other hand, the electrodes are arranged such that a voltage can be applied only between at least one pair of cells to be fused in the container. By this,
Fusion is performed only for individual unit cells formed in the container.
以下、本発明を実施例を用いて詳細に説明する。 Hereinafter, the present invention will be described in detail with reference to examples.
第１図には、本発明を用いたチヤンバプレートの概観
と、その一部の断面を示している。プレート本体１に
は、細胞を収納するマイクロチヤンバ２が多数配置され
ている。マイクロチヤンバの底には、スリツト３が設け
てあり、液体が貫流する構造になつている。融合すべき
細胞は、蔗糖やマニトール溶液中に分散して、マイクロ
チヤンバに１個ずつ供給される。１個ずつ細胞を送り込
む方法としては、例えば特開昭62−32874で知られてい
るような流体的な方法がある。マイクロチヤンバに入つ
た細胞粒子４は、チヤンバの底に設けたスリツト３を介
してチヤンバ裏面から負圧で吸引されることにより、チ
ヤンバ底面に吸着されてチヤンバ外に出ることはない。
この目的から、チヤンバ底面に設けたスリットの幅は細
胞粒子の直径より小さくなくてはならない。FIG. 1 shows an overview of a chamber plate using the present invention and a partial cross section thereof. A large number of microchambers 2 for accommodating cells are arranged in the plate body 1. A slit 3 is provided at the bottom of the microchamber, and has a structure through which a liquid flows. The cells to be fused are dispersed in a sucrose or mannitol solution and supplied one by one to a microchamber. As a method for feeding cells one by one, there is a fluid method as disclosed in, for example, JP-A-62-32874. The cell particles 4 entering the micro-chamber are sucked at a negative pressure from the back surface of the chamber via the slit 3 provided at the bottom of the chamber, so that they are not adsorbed on the bottom surface of the chamber and leave the chamber.
For this purpose, the width of the slit provided in the bottom of the chamber must be smaller than the diameter of the cell particles.
上記のようにして２種の細胞群から１個ずつの細胞を
すべてのチヤンバ内に供給すれば、所望の細胞対がチヤ
ンバ内に形成される。By supplying one cell from each of the two cell groups into all the chambers as described above, a desired cell pair is formed in the chamber.
次いで、細胞間に融合を生じさせるために、各チヤン
バの中に電界を加える。このために本発明では、第１図
に示すように、Ptからなる電極5aおよび5bをその端部が
各チヤンバ内に配置されるようにチヤンバプレート上に
形成した。電極5a,5bの間に電気的パルスを加えること
により、チヤンバ内には電界が生じ、電気的な融合が可
能になる。この結果融合するのは、所望の組合せの細胞
対のみである。本例ではチヤンバプレート表面からチヤ
ンバ内に深く電極が伸長しており、融合しようとする細
胞のごく近くまで電極を近づけることが可能である。こ
の結果、融合に必要な電圧パルスの大きさは電極間隙に
比例して小さくなり、在来の電気融合用チヤンバに比べ
て の電圧で融合が可能になる。また、電極配置の適正化に
より、チヤンバプレート上の全チヤンバに同時に電圧を
印加すれば、逐次パルスを印加することができるから、
電極間の静電容量が実効的に小さくなる結果、融合効率
を左右する電圧パルスの波形が鈍ることなくチヤンバ内
に加わる。An electric field is then applied in each chamber to cause fusion between the cells. For this purpose, in the present invention, as shown in FIG. 1, the electrodes 5a and 5b made of Pt are formed on the chamber plate such that the ends thereof are arranged in the chambers. By applying an electric pulse between the electrodes 5a, 5b, an electric field is generated in the chamber, and electric fusion becomes possible. As a result, only the desired combination of cell pairs will fuse. In this example, the electrodes extend deep into the chamber from the chamber plate surface, and the electrodes can be brought very close to the cells to be fused. As a result, the magnitude of the voltage pulse required for fusion decreases in proportion to the electrode gap, and is smaller than that of a conventional electrofusion chamber. Fusion is possible with a voltage of Also, by optimizing the electrode arrangement, if a voltage is applied simultaneously to all chambers on the chamber plate, a pulse can be applied sequentially,
As a result of the effective reduction of the capacitance between the electrodes, the waveform of the voltage pulse that determines the fusion efficiency is applied to the chamber without dulling.
以下にチヤンバプレートの製造プロセスの１例を第２
図を用いて説明する。プレートの素材はSi単結晶の（10
0）ウエハ６であり、板厚390μm,直径は76mmである。チ
ヤンバを形成するにはKOH水溶液による異方性エツチン
グを利用し、サイドエツチを防いで精密なエツチプロフ
イルを得る。まず、第２図（ａ）に示すようにウエハの
裏面からチヤンバのスリツトに相当する部分７をエツチ
ングし、次いで第２図（ｂ）に示すような裏面からチヤ
ンバ本体の部分８をエツチングする。いずれもエツチン
グのマスクとしてはSiO2膜を使い、エツチングする裏面
もSiO2膜で保護しておく。この結果第２図（ｃ）に示す
ようにチヤンバ底厚約25μｍ、スリツト幅約15μｍのチ
ヤンバができる。この大きさのチヤンバは約600μｍの
間隔でSiウエハの全面に形成することができるから、１
ウエハ上に約5000個程度のチヤンバを作ることができ
る。The following is an example of the manufacturing process of the chamber plate in the second section.
This will be described with reference to the drawings. The plate is made of Si single crystal (10
0) The wafer 6 has a thickness of 390 μm and a diameter of 76 mm. To form a chamber, anisotropic etching with a KOH aqueous solution is used to prevent side etching and obtain a precise etching profile. First, a portion 7 corresponding to the slit of the chamber is etched from the back surface of the wafer as shown in FIG. 2 (a), and then a portion 8 of the chamber body is etched from the back surface as shown in FIG. 2 (b). In each case, an SiO 2 film is used as an etching mask, and the back surface to be etched is protected by the SiO 2 film. As a result, as shown in FIG. 2C, a chamber having a chamber bottom thickness of about 25 μm and a slit width of about 15 μm is formed. Since chambers of this size can be formed on the entire surface of the Si wafer at intervals of about 600 μm,
About 5000 chambers can be made on a wafer.
このチヤンバに電極を形成するため、Siの全面をSiO2
膜で覆い絶縁した後、第２図（ｄ）に示すようにポジレ
ジスト10を塗布する。このような凹凸の激しい表面には
レジスト膜が一定の厚さに塗布できないから、このまま
では電極パターンを形成できない。そこで第２図（ｅ）
に示すように遮光膜11としてCrを全面に蒸着し、これに
電極パターンの反転像12をホトリソグラフイで形成す
る。次いで上面から平行光を当てながら、厚いレジスト
層を現像していくと、第２図（ｇ）に示すように電極パ
ターンが形成できる。ここに電極材としてPt13を全面に
蒸着し、第２図（ｈ）リフトオフの手法で不要部を除去
すれば、第２図（ｉ）に示すように電極がチヤンバ内に
形成される。To form the electrodes on the Chiyanba, SiO 2 on the entire surface of the Si
After covering with a film and insulating, a positive resist 10 is applied as shown in FIG. 2 (d). Since a resist film cannot be applied to such a highly uneven surface at a constant thickness, an electrode pattern cannot be formed as it is. Therefore, FIG. 2 (e)
As shown in (1), Cr is vapor-deposited on the entire surface as a light shielding film 11, and an inverted image 12 of the electrode pattern is formed thereon by photolithography. Next, by developing the thick resist layer while applying parallel light from the upper surface, an electrode pattern can be formed as shown in FIG. 2 (g). If Pt13 is vapor-deposited on the entire surface as an electrode material and unnecessary portions are removed by a lift-off method in FIG. 2 (h), an electrode is formed in the chamber as shown in FIG. 2 (i).
上記の実施例では、電極をすべてチヤンバ内に配置し
たが、第３図に示すようにチヤンバプレート14の表と裏
に電極15aおよび15bを形成しても細胞16を融合させるこ
とができる。またこれらの電極の一方をチヤンバプレー
ト上に形成し、他方を外部電極19として接近させても同
様の効果が得られることは当然である。In the above embodiment, the electrodes are all arranged in the chamber. However, the cells 16 can be fused by forming electrodes 15a and 15b on the front and back of the chamber plate 14 as shown in FIG. It is natural that the same effect can be obtained even if one of these electrodes is formed on the chamber plate and the other is brought close to the external electrode 19.
さらに、上記実施例ではチヤンバプレートをSiで形成
したが、他の材料、例えば感光性ガラスを用いても、チ
ヤンバプレート本体を形成することができる。また、細
胞を供給するのに液滴中に１個の細胞を入れてチヤンバ
内に供給すれば、チヤンバ底面のスリツトは必ずしも必
要でない。この場合には、第４図に示すように電極17a
および17bによつて、細胞18を融合することができる。Furthermore, although the chamber plate is formed of Si in the above embodiment, the chamber plate body can be formed by using other materials, for example, photosensitive glass. In addition, if one cell is put in a droplet and supplied into the chamber to supply the cells, the slit on the bottom of the chamber is not necessarily required. In this case, as shown in FIG.
And 17b allow cell 18 to be fused.
また、さきの実施例では細胞をチヤンバ内に留めてお
く手段として、流体的な手段を用いたが、本発明におけ
るチヤンバ内配線を利用して、静電的に細胞を電極部分
に吸着させておくことも可能である。Further, in the above embodiment, fluid means is used as a means for retaining cells in the chamber, but cells are electrostatically adsorbed to the electrode portion by using wiring in the chamber in the present invention. It is also possible to put.
電極材料としてはPt以外にもAuなど、イオン化しにく
く、生体に害を及ぼしにくい導電体が使用可能である。As the electrode material, other than Pt, a conductor such as Au which is hardly ionized and which is hardly harmful to the living body can be used.
また、上記実施例では細胞対を融合するものについて
示したが、例えば３個の細胞を融合する場合にはチヤン
バ内に融合すべき３個の細胞を入れて融合させることも
可能である。Further, in the above-described embodiment, the case where the cell pair is fused is described. However, for example, when three cells are fused, it is also possible to put three cells to be fused in the chamber and perform the fusion.
これらの実施例によれば、細胞を電気的な手段によっ
て大量に融合させるプロセスにおいて、所望の組合せの
少なくとも一対の細胞を融合させることが可能になる。
従来は集団的に融合した細胞群の中から、所望の組合せ
の細胞を選抜できないか、又は選抜法が極めて煩雑であ
るなどの障害があったが、これらの実施例によれば、こ
の困難さを軽減することができる。According to these embodiments, it is possible to fuse at least a pair of cells in a desired combination in a process of fusing cells in a large amount by electrical means.
Conventionally, there were obstacles such as the inability to select a desired combination of cells from a group of cells fused collectively, or the selection method was extremely complicated. Can be reduced.
本発明によれば、所望の細胞を融合させることができ
る。According to the present invention, desired cells can be fused.
第１図は本発明の一実施例を示す細胞融合装置のチヤン
バプレートの一部を切断した概観図、第２図は第１図の
チヤンバプレートの製造工程の一実施例を示す断面図、
第３図はチヤンバプレートの他の実施例を示す断面図、
第４図はチヤンバプレートのさらに他の実施例を示す断
面図である。 １……チヤンバプレート本体、２……マイクロチヤン
バ、３……スリツト、４……細胞、5a……電極、5b……
電極。FIG. 1 is a schematic view of a part of a chamber plate of a cell fusion device showing one embodiment of the present invention, and FIG. 2 is a cross-sectional view showing one embodiment of a manufacturing process of the chamber plate of FIG. ,
FIG. 3 is a sectional view showing another embodiment of the chamber plate,
FIG. 4 is a sectional view showing still another embodiment of the chamber plate. 1 ... Chamber plate body, 2 ... Micro chamber, 3 ... Slit, 4 ... Cell, 5a ... Electrode, 5b ...
electrode.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 小野里 陽正 国分寺市東恋ヶ窪１丁目280番地 株式 会社日立製作所中央研究所内 (56)参考文献 特開 昭61−111680（ＪＰ，Ａ) 特開 昭61−219386（ＪＰ，Ａ) 特開 昭63−84481（ＪＰ，Ａ) 特開 昭63−173573（ＪＰ，Ａ) ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Yomasa Onori 1-280 Higashi-Koigabo, Kokubunji-shi Inside the Central Research Laboratory, Hitachi, Ltd. (56) References JP-A-61-111680 (JP, A) JP-A-61 JP-A-219386 (JP, A) JP-A-63-84481 (JP, A) JP-A-63-173573 (JP, A)
Claims (4)
収納するステップと、 前記マイクロチャンバの底面に設けられ、前記一対の異
種細胞のそれぞれよりも寸法の小さな開口部から吸引し
て前記底面に前記一対の異種細胞を吸着するステップ
と、 前記マイクロチャンバ内で、前記一対の異種細胞には非
接触となるように設けられた電極に電圧を印加し、前記
一対の異種細胞間に電気パルスを加えて前記一対の異種
細胞を融合させるステップとを有することを特徴とする
細胞融合方法。1. A step of accommodating a pair of heterogeneous cells in a micro-chamber, provided on a bottom surface of the micro-chamber, and suctioned from an opening smaller in size than each of the pair of heterogeneous cells to attach the same to the bottom surface. Adsorbing the pair of heterogeneous cells, applying a voltage to an electrode provided so as not to contact the pair of heterogeneous cells in the microchamber, and applying an electric pulse between the pair of heterogeneous cells. Additionally, the step of fusing the pair of heterologous cells.
上部に設けられていることを特徴とする特許請求の範囲
第１項記載の細胞融合方法。2. The cell fusion method according to claim 1, wherein said electrode is provided on an upper portion of a side wall of said microchamber.
数設けられていることを特徴とすることを特徴とする特
許請求の範囲第１項または第２項記載の細胞融合方法。3. The cell fusion method according to claim 1, wherein a plurality of said microchambers are provided on one substrate.
た一対の異種細胞に同時に電気パルスを印加することを
特徴とする特許請求の範囲第３項記載の細胞融合方法。4. The cell fusion method according to claim 3, wherein an electric pulse is simultaneously applied to a pair of heterogeneous cells housed in the plurality of microchambers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62104250A JP2619387B2 (en) | 1987-04-30 | 1987-04-30 | Cell fusion method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62104250A JP2619387B2 (en) | 1987-04-30 | 1987-04-30 | Cell fusion method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63269977A JPS63269977A (en) | 1988-11-08 |
| JP2619387B2 true JP2619387B2 (en) | 1997-06-11 |
Family
ID=14375687
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62104250A Expired - Lifetime JP2619387B2 (en) | 1987-04-30 | 1987-04-30 | Cell fusion method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2619387B2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2829005B2 (en) * | 1988-11-11 | 1998-11-25 | 株式会社日立製作所 | Micro-chamber plate, cell detection method, treatment method and apparatus using the same, and cell |
| FR2659347B1 (en) * | 1990-03-12 | 1994-09-02 | Agronomique Inst Nat Rech | DEVICE FOR CULTURING CELLS PROVIDING THEIR IMMOBILIZATION. |
| US5173158A (en) * | 1991-07-22 | 1992-12-22 | Schmukler Robert E | Apparatus and methods for electroporation and electrofusion |
| EP0962524B1 (en) * | 1998-05-27 | 2004-11-03 | Micronas GmbH | Apparatus and process for the intracellular manipulation of a biological cell |
| US6355485B1 (en) * | 1998-06-10 | 2002-03-12 | University Of South Florida | Electrofusion chamber |
| DE69943044D1 (en) * | 1998-06-10 | 2011-01-27 | Univ South Florida | ELECTRIC FUSION CHAMBER |
| DE19917571B4 (en) * | 1999-04-19 | 2014-03-20 | Robert Poppe | Electrode grid for electroporation reaction batches |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0658B2 (en) * | 1984-11-07 | 1994-01-05 | 株式会社日立製作所 | Cell handling equipment |
| JPS61219386A (en) * | 1985-03-26 | 1986-09-29 | Sanyo Electric Co Ltd | Method for agglomerating polarizable fine particle and apparatus therefor |
| JPS6384481A (en) * | 1986-09-29 | 1988-04-15 | Shimadzu Corp | Cell-fusion apparatus |
| JPS63173573A (en) * | 1987-01-13 | 1988-07-18 | Nok Corp | Electrode for cell fusion |
-
1987
- 1987-04-30 JP JP62104250A patent/JP2619387B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63269977A (en) | 1988-11-08 |
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