JP2007174901A - Apparatus for reversibly destroying cell membrane - Google Patents

Apparatus for reversibly destroying cell membrane Download PDF

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JP2007174901A
JP2007174901A JP2005373463A JP2005373463A JP2007174901A JP 2007174901 A JP2007174901 A JP 2007174901A JP 2005373463 A JP2005373463 A JP 2005373463A JP 2005373463 A JP2005373463 A JP 2005373463A JP 2007174901 A JP2007174901 A JP 2007174901A
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cell membrane
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Osamu Kurosawa
黒澤修
Masao Washizu
鷲津正夫
Hidehiro Oana
***英廣
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Abstract

<P>PROBLEM TO BE SOLVED: To achieve cell fusion or electroporation with high efficiency by preventing the application of an excessive voltage to a cell membrane in a part other than a pore and irreversible destruction of a cell when the pore is formed in one point of the cell membrane in an apparatus for destroying the cell membrane. <P>SOLUTION: The apparatus for reversibly destroying the cell membrane is designed to reversibly destroy the cell membrane by applying a pulse voltage. The apparatus is composed so as to apply modulation of 10 kHz to 1 MHz frequency wherein the average thereof is 0 to the applied voltage. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

電気的手段によって細胞融合を行う，または細胞内に遺伝子等の外来物質を導入するための装置に関する分野に関する。   The present invention relates to a field related to an apparatus for performing cell fusion by electric means or introducing a foreign substance such as a gene into a cell.

電気的手段によって細胞融合を行う電気細胞融合や，電気的手段により細胞内に遺伝子等の外来物質を導入するエレクトロポレーションにおいては，電圧の印加により細胞膜が過渡的・可逆的に破壊されて微小孔（ポア）が形成され，電圧の印加が終了した後にその微小孔が自復するという，「細胞膜の可逆的破壊」と呼ばれる現象が利用される。すなわち，細胞融合においては，膜の可逆的破壊により生ずるポアが起点となり2つの細胞の接触点で両者の膜が融合し，また，エレクトロポレーションにおいては，可逆的破壊の間にポアを通して周囲にある遺伝子等の外来物質が細胞内に取り込まれる。   In electrical cell fusion, in which cell fusion is performed by electrical means, and in electroporation in which foreign substances such as genes are introduced into cells by electrical means, the cell membrane is transiently and reversibly destroyed by the application of a voltage, resulting in a minute amount. A phenomenon called “reversible destruction of cell membranes” is used in which pores are formed and the micropores recover after the application of voltage is completed. That is, in cell fusion, the pore generated by the reversible disruption of the membrane is the starting point, and both membranes fuse at the contact point of the two cells. In electroporation, the pores are surrounded by the pores during the reversible disruption. A foreign substance such as a gene is taken up into the cell.

これらの過程においては，細胞膜全体を不可逆的に破壊してしまうことなく，細胞膜の一部分のみに可逆的な破壊を生じせしめることが必要である。可逆的破壊は，膜にかかる電圧が約1Vとなる時のみに生じ，これより高い膜電圧は膜を不可逆的に破壊してしまい，またこれより低い電圧ではポアが生じないことが知られている。従って，従来の手法においては，この条件を満たすために，下記の理論に基づいた適切なパルス電圧とパルス幅の選択を行っていた。   In these processes, it is necessary to cause reversible destruction of only part of the cell membrane without irreversibly destroying the entire cell membrane. Reversible breakdown occurs only when the voltage across the membrane is about 1V, higher membrane voltages irreversibly break the membrane, and lower voltages are known to not cause pores. Yes. Therefore, in the conventional method, in order to satisfy this condition, an appropriate pulse voltage and pulse width are selected based on the following theory.

半径aの細胞に一様な電界Ｅ0を時刻 t＝０ においてステップ状に印加した時，細胞膜にかかる膜電圧Ｖm（θ） の時間変化は

Figure 2007174901



Figure 2007174901

ただし、θ：電界方向を θ= ０ととった時の極座標の天頂角，τ：時定数，
Ｃm：細胞膜の単位面積あたりの静電容量，
ρin 、ρout：細胞内液および外液の抵抗率
U. Zimmermann and W. M. Arnold: "Biophysics of electroinjection and electrofusion",Journal of Electrostatics, 21, p.309-345 (1988)
で与えられる（図1参照）。しかしながら，式(1)は，球形の完全な膜が存在する時の膜電圧を与えるもので，たとえば図１で示すパルス電圧の印加により膜の一点にポアが形成された場合は，膜電圧の分布がこの式から大きく異なることになる。
特表２００２−５１６０８８号公報 When a uniform electric field E 0 is applied to cells of radius a stepwise at time t = 0, the time change of the membrane voltage V m (θ) applied to the cell membrane is
Figure 2007174901


Figure 2007174901

Where θ is the zenith angle in polar coordinates when the electric field direction is θ = 0, τ is the time constant,
C m : capacitance per unit area of the cell membrane,
ρ in, ρ out : resistivity of intracellular and external fluids
U. Zimmermann and WM Arnold: "Biophysics of electroinjection and electrofusion", Journal of Electrostatics, 21, p.309-345 (1988)
(See Figure 1). However, equation (1) gives the membrane voltage when a spherical complete membrane exists. For example, when a pore is formed at one point of the membrane by applying the pulse voltage shown in FIG. The distribution will be very different from this equation.
Japanese translation of PCT publication No. 2002-516088

すなわち，式(1)は，細胞膜にかかる電圧の大きさは，電気力線の最上流側（北極：θ＝０）の位置および最下流側（南極：θ＝π）の位置で最大となることを示しているので，電圧を印加した場合，まずこのいずれかの位置にポアが形成されることが予想される。すると，ここを通して電流が流れ，他方の極の充電がさらにすすみ，ここには，式(1)で与えられる以上の過大な電圧がかかることになり，この位置が不可逆的に破壊されてしまう。このような，いわば将棋倒しのように細胞膜の別の位置に過電圧が伝搬する現象は，細胞を破壊してしまうため，細胞融合やエレクトロポレーションの効率の低下をまねく。   That is, in Equation (1), the voltage applied to the cell membrane is maximized at the position of the most upstream side (north pole: θ = 0) and the most downstream side (south pole: θ = π) of the electric field lines. Therefore, when a voltage is applied, it is expected that a pore is first formed at any one of these positions. Then, current flows through this, and charging of the other pole proceeds further, and an excessive voltage higher than that given by Equation (1) is applied to this position, and this position is irreversibly destroyed. The phenomenon of overvoltage propagating to another position on the cell membrane, like so-called shogi, destroys cells, leading to a decrease in cell fusion and electroporation efficiency.

本発明は，細胞膜の1点にポアが生じた時に，それ以外の部分の膜に過大な膜電圧がかかって細胞が不可逆的に破壊されてしまうことを防止することにより，高い効率での細胞融合やエレクトロポレーションを実現する。
In the present invention, when a pore occurs at one point of the cell membrane, an excessive membrane voltage is applied to the other portion of the membrane to prevent the cell from being irreversibly destroyed. Realize fusion and electroporation.

本発明においては，印加する電圧に，適切に選択された周波数で，かつ平均値が0となるような高周波の変調をかけることにより，ポア形成時の細胞膜の不可逆的破壊を防ぐ。
In the present invention, irreversible destruction of the cell membrane during pore formation is prevented by subjecting the applied voltage to high-frequency modulation with an appropriately selected frequency and an average value of 0.

本装置によれば，膜上の1点にポアが形成された時にも，膜の他の場所での膜電圧分布はポア形成前と同じであるので，細胞膜の不可逆的破壊が生じず，高い効率での細胞融合やエレクトロポレーションが実現される。   According to this device, even when a pore is formed at one point on the membrane, the membrane voltage distribution at the other location of the membrane is the same as before the pore formation, so that irreversible destruction of the cell membrane does not occur and is high Efficient cell fusion and electroporation are realized.

本発明によれば，膜上の1点にポアが形成された時にも，膜の他の場所での膜電圧分布はポア形成前と同じであるので，細胞膜の不可逆的破壊が生じず，高い効率での細胞融合やエレクトロポレーションが実現される。   According to the present invention, even when a pore is formed at one point on the membrane, the membrane voltage distribution at the other location of the membrane is the same as before the pore formation, so that irreversible destruction of the cell membrane does not occur and is high Efficient cell fusion and electroporation are realized.

本発明の実施例を図2を用いて説明する。
細胞膜にポアが生じる前の膜電圧分布は，図1および式(1)-(2)で説明した通りである。一般的に，細胞膜の単位面積あたりの静電容量Cmは，細胞種によらず 1 μＦ／cm2 程度であることが知られているので，式(2)の細胞膜充電の時定数は，半径 a = 5 μm の細胞に対して，周囲の媒質が生理塩濃度の溶液である場合にはτ = 40 ns，周囲の媒質が1mM程度の低濃度の塩溶液である場合には τ = 2 μs 程度となる。従って，膜に不可逆的破壊を起こすためには，これより長いパルス電圧を用いることになる。現実的には，ポアの形成を確実にするため，数十μs以上，数十ms程度までの長いパルスが用いられることが多い。 An embodiment of the present invention will be described with reference to FIG.
The membrane voltage distribution before pores are generated in the cell membrane is as explained in Fig. 1 and equations (1)-(2). In general, the capacitance C m per unit area of the cell membrane is known to be about 1 μF / cm 2 regardless of the cell type, so the time constant for charging the cell membrane in equation (2) is For cells of radius a = 5 μm, τ = 40 ns if the surrounding medium is a physiological salt solution, and τ = 2 if the surrounding medium is a low concentration salt solution of about 1 mM. It is about μs. Therefore, a longer pulse voltage is used to cause irreversible breakdown in the film. In practice, in order to ensure the formation of pores, a long pulse of several tens of μs or more and several tens of ms is often used.

一方，図2に示したように，膜の1点にポア3が生ずると，この等価抵抗4を通して，ここから電流5が流れ込み，これが膜の等価容量6を充電することになる。これにより，膜は，式(1)で与えられる膜電圧を越えてさらに充電されることになり，結果として，ある確率で膜の不可逆的破壊が生じ，これが細胞融合やエレクトロポレーションの効率低下につながる。 On the other hand, as shown in FIG. 2, when a pore 3 is generated at one point of the membrane, current 5 flows from this through the equivalent resistance 4, which charges the equivalent capacitance 6 of the membrane. This causes the membrane to be further charged beyond the membrane voltage given by Equation (1), resulting in irreversible destruction of the membrane with some probability, which reduces cell fusion and electroporation efficiency. Leads to.

ところで，このポア形成後の膜の充電の時定数τcは，等価抵抗4の値をR，等価容量6の値をCとすると、
τc= C R （３）
で与えられる。Rは，薄膜に小孔のある場合の縮流抵抗，
R＝ρ／δ （４）
ただし，ρは媒質の導電率，δは小孔の直径
で，Cは膜全体の静電容量
C = ４π a2 Cm （５）
で近似できるので，これらの値を用いれば
τc= 4 π a2 Cm ρ/ δ （６）
となる。ちなみに，半径 a = ５ μm，ポアの直径 δ = １００ nmとし，生理塩濃度の溶液を仮定すれば τc= 160 μs，１mM程度の塩溶液を仮定すればτc= ２４ ms 程度である。 By the way, the time constant τ c for charging the film after the pore formation is represented by R as the value of the equivalent resistance 4 and C as the value of the equivalent capacitance 6.
τ c = CR (3)
Given in. R is the contraction resistance when there are small holes in the thin film,
R = ρ / δ (4)
Where ρ is the conductivity of the medium, δ is the diameter of the small hole, and C is the capacitance of the entire film.
C = 4π a 2 C m (5)
If these values are used,
τ c = 4 π a 2 C m ρ / δ (6)
It becomes. By the way, radius a = 5 μm, pore diameter δ = 100 nm, τ c = 160 μs if a physiological salt concentration solution is assumed, and τ c = 24 ms if a salt solution of about 1 mM is assumed.

図2（a)の等価回路によれば，印加される電圧の周期Tが
T <τc （７）
であれば，ポア形成後の膜充電が進まないことがわかる。上記の数値例からもわかるように τ<<τcであるので、
τ<T <τc （８）
となるような印加電圧の周期Tの選択が容易に可能で，そのようにとれば，ポアの形成はされるが，ポア形成後の膜充電は生じないことになる。 According to the equivalent circuit of FIG. 2 (a), the period T of the applied voltage is
T <τ c (7)
If so, it can be seen that the membrane charging after the pore formation does not proceed. As you can see from the numerical example above, τ << τ c .
τ <T <τ c (8)
It is possible to easily select the period T of the applied voltage so that the pore is formed, but the membrane charging after the pore formation does not occur.

この条件は，式（８）を満たすような単一パルスによっても実現可能であるが，ポアの形成をより確実にするためには，前述のように，ある程度の時間（１msec〜50msec）にわたり電圧（1Ｖ〜1.5Ｖ）を印加し続けることが望ましい。このためには，図2に示すような高周波バースト波形において，電圧が正である周期T+と電圧が負である周期T-のいずれもが式（７）を満たすようにとった波形が最も適切である。周期T+は例えば0.5〜25msec、周期T-は、例えば、0.5〜25msecが示されるが、これらの数値に限られるものではない。 This condition can be realized by a single pulse that satisfies Equation (8), but in order to make pore formation more reliable, as described above, the voltage is applied over a certain period of time (1 msec to 50 msec). It is desirable to continue applying (1 V to 1.5 V). For this purpose, in the high-frequency burst waveform as shown in FIG. 2, the waveform in which both the cycle T + in which the voltage is positive and the cycle T − in which the voltage is negative satisfies Equation (7) is the most. Is appropriate. The period T + is 0.5 to 25 msec, for example, and the period T − is 0.5 to 25 msec, for example, but is not limited to these numerical values.

なお，このような波形において，直流分を含んでいると，その直流分はポア形成後の膜充電を生ずることになる。従って，印加電圧波形は、直流分を含まない，すなわちその平均値が0となる波形であることが必要になる。ただし、この波形に対する条件は，図2において T+=T-であることに限定されるものではなく，いかなるT+とT-の組み合わせに対しても電圧の時間平均が０であればよい。
In addition, if such a waveform includes a direct current component, the direct current component causes membrane charging after pore formation. Therefore, it is necessary that the applied voltage waveform does not include a direct current component, that is, a waveform whose average value is zero. However, the condition for this waveform is not limited to T + = T − in FIG. 2, and the time average of the voltage may be 0 for any combination of T + and T − .

本発明は、細胞の薬剤応答計測や薬剤スクリーニング，動植物の品種改良、生育調整、形質転換等の動植物の細胞操作等様々な生化学的分野において有効に用いられる。
INDUSTRIAL APPLICABILITY The present invention can be effectively used in various biochemical fields such as cell drug response measurement and drug screening, animal and plant variety improvement, growth control, and cell manipulation of animals and plants such as transformation.

従来例を示す図である。It is a figure which shows a prior art example. 本発明の一実施例を示す図である。It is a figure which shows one Example of this invention.

符号の説明Explanation of symbols

１ 細胞
２ 細胞膜
３ ポア
４ ポアの等価抵抗，
５ 充電電流
６ 細胞膜の等価容量 1 cell 2 cell membrane 3 pore 4 pore equivalent resistance,
5 Charging current 6 Cell membrane equivalent capacity

Claims (4)

パルス電圧を印加することにより細胞膜を可逆的に破壊する装置において，印加する電圧にその平均値が0であるような周波数10kHz-1MHzの変調をかけることを特徴とする細胞膜の可逆破壊装置。 An apparatus for reversibly destroying a cell membrane by applying a pulse voltage, wherein the applied voltage is modulated at a frequency of 10 kHz-1 MHz such that the average value is zero. 前記パルス電圧の周期Ｔが、膜の充電の時定数 τcより小さい請求項１に記載の細胞膜の可逆破壊装置。 The cell membrane reversible disruption device according to claim 1, wherein a period T of the pulse voltage is smaller than a time constant τ c for charging the membrane. 前記パルス電圧が交番パルスである請求項１に記載の細胞膜の可逆破壊装置。 The reversible disruption device for a cell membrane according to claim 1, wherein the pulse voltage is an alternating pulse. 前記パルス電圧がバースト状である請求項１に記載の細胞膜の可逆破壊装置。 The device for reversible cell membrane destruction according to claim 1, wherein the pulse voltage is in a burst form.
JP2005373463A 2005-12-26 2005-12-26 Apparatus for reversibly destroying cell membrane Pending JP2007174901A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018074982A (en) * 2016-11-11 2018-05-17 株式会社日立製作所 Method of exchanging cytoplasm, method of producing cells, and device

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018074982A (en) * 2016-11-11 2018-05-17 株式会社日立製作所 Method of exchanging cytoplasm, method of producing cells, and device

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