JPH0235071A - Time constant controlling type cell electrostimulator - Google Patents
Time constant controlling type cell electrostimulatorInfo
- Publication number
- JPH0235071A JPH0235071A JP63184057A JP18405788A JPH0235071A JP H0235071 A JPH0235071 A JP H0235071A JP 63184057 A JP63184057 A JP 63184057A JP 18405788 A JP18405788 A JP 18405788A JP H0235071 A JPH0235071 A JP H0235071A
- Authority
- JP
- Japan
- Prior art keywords
- chamber
- cell suspension
- resistance
- time constant
- cell
- 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.)
- Granted
Links
- 239000003990 capacitor Substances 0.000 claims abstract description 47
- 239000006285 cell suspension Substances 0.000 claims abstract description 41
- 230000005684 electric field Effects 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 16
- 230000007910 cell fusion Effects 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 230000004927 fusion Effects 0.000 abstract 2
- 238000001514 detection method Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- 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)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は遺伝子又は蛋白などの物質を細胞とともに混合
した細胞懸濁液に電気パルスを印加することによって遺
伝子や蛋白などを細胞内に取り込ませる遺伝子導入装置
や、細胞に電気刺激を与えて細胞どおしを電気的に融合
させる細胞融合装置などの細胞電気刺激装置に関するも
のである。[Detailed Description of the Invention] (Industrial Application Field) The present invention incorporates genes, proteins, etc. into cells by applying electric pulses to a cell suspension in which substances such as genes or proteins are mixed with cells. The present invention relates to cell electrical stimulation devices such as gene introduction devices and cell fusion devices that electrically fuse cells by applying electrical stimulation to cells.
遺伝子導入装置は細胞に与える電気刺激として直流電圧
パルスのみを与えるのに対して、細胞融合装置では交流
電圧を与えて誘電電気泳動により細胞とおしを接触させ
た後、直流電圧パルスを与えて融合させる。細胞融合装
置では交流電源がさらに必要であるが、両者はその他の
構成においては共通であるので、以下の説明では主とし
て遺伝子導入装置について説明するが、本発明は細胞融
合装置にも適用されるものである。Gene transfer devices apply only DC voltage pulses as electrical stimulation to cells, whereas cell fusion devices apply AC voltage to bring cells into contact with each other through dielectric electrophoresis, and then apply DC voltage pulses to fuse them. . Although the cell fusion device additionally requires an AC power source, other configurations of both devices are the same, so the following explanation will mainly be about the gene transfer device, but the present invention is also applicable to the cell fusion device. It is.
（従来の技術）
遺伝子導入装置の一例として、第４図に示されるように
直流電圧をコンデンサ（容量Ｃ（Ｆ） ）に充電した後
、細胞懸濁液（抵抗Ｒ（Ω））を負荷として時定数τ＝
ＣＲ（秒）で放電させる装置が提案されている（ ｒＰ
ｌａｎｔ Ｃｅ１ｌ Ｐｈｙｓｉｏｌ、Ｊ ２７（４）
：６１９−６２６（１９８６）参照）６２はチャンバ
であり、対向電極４，６を備え、対向電極４，６間には
細胞！＠濁液を収容して細胞に電界を印加する。８は直
流電源、９は俸抗である。チャンバ２と電源８に対して
容量の異なるコンデンサ１０−１〜１０−ｎが並列に設
けられている。コンデンサ１０−１〜１０−ｎの容量は
それぞれＣ，〜Ｃｎ（Ｆ）である。１２−１〜１２−ｎ
はコンデンサ１０−１〜１０−ｎのいずれかを選択する
スイッチ、１４は選択されたコンデンサを電源８に接続
するためのスイッチである。１５は充電されたコンデン
サをチャンバ２に接続するためのスイッチである。(Prior art) As an example of a gene transfer device, as shown in FIG. Time constant τ=
A device that discharges at CR (seconds) has been proposed (rP
lant Ce1l Physiol, J 27(4)
619-626 (1986)) 62 is a chamber, which is provided with opposing electrodes 4 and 6, and cells are placed between the opposing electrodes 4 and 6. @ Contain a suspension and apply an electric field to the cells. 8 is a DC power supply, and 9 is a resistor. Capacitors 10-1 to 10-n having different capacities are provided in parallel to the chamber 2 and the power source 8. The capacitances of the capacitors 10-1 to 10-n are C and -Cn(F), respectively. 12-1 to 12-n
14 is a switch for selecting one of the capacitors 10-1 to 10-n, and 14 is a switch for connecting the selected capacitor to the power supply 8. 15 is a switch for connecting the charged capacitor to the chamber 2;
遺伝子導入効率は細胞に印加されるコンデンサ放電波形
の初期電界強度Ｅ（Ｖ／ｃｍ）及び時定数τ＝ＣＲ（秒
）によって変化する。第４図の装置では時定数τを可変
にするために、容量の異なるコンデンサ１ｏ−１〜１０
−ｎをスイッチ１２−１〜１２−ｎで切り替えている。The gene transfer efficiency varies depending on the initial electric field strength E (V/cm) of the capacitor discharge waveform applied to the cells and the time constant τ=CR (seconds). In the device shown in Fig. 4, in order to make the time constant τ variable, capacitors 1o-1 to 10 with different capacities are used.
-n is switched by switches 12-1 to 12-n.
すなわち、スイッチ１２−１〜１２−ｎによってコンデ
ンサ１０−１〜１０−ｎを選択し、まずスイッチ１４を
オン、スイッチ１５をオフにしてその選択されたコンデ
ンサを充電し、その後スイッチ１４をオフ。That is, the capacitors 10-1 to 10-n are selected by the switches 12-1 to 12-n, the switch 14 is first turned on, the switch 15 is turned off to charge the selected capacitor, and then the switch 14 is turned off.
スイッチ１５をオンにして充電電荷をチャンバ２に放電
させる。The switch 15 is turned on to discharge the charged charge into the chamber 2.
（発明が解決しようとする課題）
第４図の装置ではコンデンサ１０−１〜１０−ｎの容量
の値Ｃ１〜Ｃｎは離散値であるために、時定数でも離散
値となり、時定数に関する細かな最適化ができない。も
し、時定数の最適化を十分に行なおうとすれば、コンデ
ンサ１０−１〜１〇−ｎの数を無限に増加させる必要が
あり、装置の規模から考えて実現しがたい。(Problem to be Solved by the Invention) In the device shown in FIG. 4, since the capacitance values C1 to Cn of the capacitors 10-1 to 10-n are discrete values, the time constants are also discrete values, and the detailed values regarding the time constants are Cannot be optimized. If the time constants were to be sufficiently optimized, it would be necessary to increase the number of capacitors 10-1 to 10-n infinitely, which is difficult to achieve considering the scale of the device.
また、細胞懸濁液の抵抗値は塩濃度、遺伝子濃度、細胞
密度などによって変化するので、時定数の制御は容易で
はない。Furthermore, since the resistance value of the cell suspension changes depending on the salt concentration, gene concentration, cell density, etc., it is not easy to control the time constant.
本発明はコンデンサを介して直流電圧パルスを印加する
場合の時定数を連続的に可変にすることのできる電気刺
激装置を提供することを目的とするものである。An object of the present invention is to provide an electrical stimulation device that can continuously vary the time constant when applying a DC voltage pulse via a capacitor.
（課題を解決するための手段） 第１図に本発明を示す。(Means for solving problems) The invention is illustrated in FIG.
２はチャンバ、８は直流電圧電源、１０−１〜１０−ｎ
は電源８により充電され、チャンバ２に放電する並列接
続された互いに容量の異なるコンデンサである。２０は
チャンバ２に交流電流を流してチャンバ２の抵抗値を測
定する抵抗測定回路、２２は抵抗測定回路の測定値ｒと
チャンバ定数にとから細胞懸濁液の比抵抗ρを算出する
比抵抗算出手段、２４はチャンバ２の電極間間隔６１時
定数τ、コンデンサ１０−１〜１０−ｎの容量値Ｃ□〜
Ｃｎ及び算出された比抵抗ρから最適コンデンサ１０−
１を選定し、細胞懸濁液量ｖｏを算出するに件設定手段
、２６は抵抗測定回路２０の測定値Ｒ，チャンバ２の電
極間間隔ｄ及び算出された比抵抗ρから細胞懸濁液量Ｖ
を検出する細胞懸濁液量検出手段、２８は条件設定手段
２４で算出された細胞！！！！！濁液ｆｆ１Ｖｏを表示
する表示手段である。2 is a chamber, 8 is a DC voltage power supply, 10-1 to 10-n
are capacitors connected in parallel that are charged by the power source 8 and discharged into the chamber 2 and have different capacities. 20 is a resistance measuring circuit that measures the resistance value of the chamber 2 by passing an alternating current through the chamber 2, and 22 is a specific resistance that calculates the specific resistance ρ of the cell suspension from the measured value r of the resistance measuring circuit and the chamber constant. Calculation means 24 indicates the time constant τ of the inter-electrode interval 61 of the chamber 2, and the capacitance values C□~ of the capacitors 10-1 to 10-n.
Optimal capacitor 10- from Cn and calculated specific resistance ρ
1 is selected and the cell suspension volume vo is calculated by setting means; V
28 is the cell calculated by the condition setting means 24! ! ! ! ! This is a display means for displaying the cloudy liquid ff1Vo.
（作用） 遺伝子導入を行なう場合を例にして説明する。(effect) This will be explained using an example of gene introduction.
チャンバ２の電極間隔ｄと電極面積Ａで決まるチャンバ
定数ｋ （ｃｍ−１）＝ｄ／Ａは予めわかっており、比
抵抗算出手段２２に設定しておく。条件設定手段２４に
はチャンバ２の電極間隔ｄ、所望のパルス時定数τ（ｍ
ｓ）、コンデンサ１０−１〜１０−ｎの容量Ｃ工〜Ｃｎ
を設定しておく。The chamber constant k (cm-1)=d/A determined by the electrode spacing d and the electrode area A of the chamber 2 is known in advance and is set in the resistivity calculation means 22. The condition setting means 24 includes the electrode spacing d of the chamber 2 and the desired pulse time constant τ(m
s), capacitance C~Cn of capacitors 10-1~10-n
Set.
まず、細胞と遺伝子を懸濁させた細胞懸濁液の所定量を
チャンバ２に入れて、チャンバ２の電極を全て細胞懸濁
液で満たす。抵抗測定回路２０からチャンバ２へ交流電
流が流され、その電流値と交流電源の電圧とからチャン
バ２の抵抗ｒ （ＫΩ）が測定される。比抵抗算出手段
２２は測定された抵抗ｒとチャンバ定数にとから比抵抗
ρ（ＫΩ・ｃｍ）をρ＝ ｒ ／ ｋによって算出する
。First, a predetermined amount of a cell suspension in which cells and genes are suspended is placed in the chamber 2, and all electrodes in the chamber 2 are filled with the cell suspension. An alternating current is passed from the resistance measuring circuit 20 to the chamber 2, and the resistance r (KΩ) of the chamber 2 is measured from the current value and the voltage of the alternating current power supply. The resistivity calculation means 22 calculates the resistivity ρ (KΩ·cm) from the measured resistance r and the chamber constant by ρ=r/k.
条件設定手段２４はコンデンサ１０−１〜１０−ｎの各
容量Ｃ工〜Ｃｎに対して１時定数を設定された時定数τ
とするのに必要な細胞懸濁液の量Ｖを算出する。それら
の算出されたＶのうちチャンバ２の容量よりも小さく、
かつ、チャンバ２の容量に最も近い値をｖＯとして選定
し、そのｖｏを算出するのに使用された容量Ｃｉのコン
デンサ１０−ｉを最適値として選定する。選定されたｖ
Ｏは表示手段２８に表示される。The condition setting means 24 sets a time constant τ of 1 for each capacitance C to Cn of the capacitors 10-1 to 10-n.
Calculate the amount V of cell suspension required to achieve this. Of those calculated V, it is smaller than the capacity of chamber 2,
Then, the value closest to the capacitance of the chamber 2 is selected as vO, and the capacitor 10-i of the capacitance Ci used to calculate the vO is selected as the optimum value. selected v
O is displayed on the display means 28.
コンデンサ１０−ｉが接続され、また、チャンバ２には
自動又は手動により細胞懸濁液が量ｖ。A capacitor 10-i is connected, and a volume v of the cell suspension is automatically or manually added to the chamber 2.
だけ注入される。only injected.
チャンバ２に細胞懸濁液が注入されると、抵抗測定回路
２０によって交流電流が流されてその時のチャンバ２の
抵抗Ｒが測定される。懸濁液量検出手段２６にもチャン
バ２の電極間間隔ｄを設定しておく。１ＩＩＩＩ濁液量
検出手段２６は電極間間隔ｄ、比抵抗ρ及び測定された
抵抗値Ｒとからチャンバ２に注入された細胞懸濁液量Ｖ
を
■＝ρｅｄ”／Ｒ
によって検出する。この検出されたＶと算出値ｖｏとの
比較によりチャンバ２に所定量の細胞懸濁液が注入され
たか否かを判断することができる。When the cell suspension is injected into the chamber 2, an alternating current is applied by the resistance measuring circuit 20, and the resistance R of the chamber 2 at that time is measured. The inter-electrode spacing d of the chamber 2 is also set in the suspension amount detection means 26. 1III The suspension amount detection means 26 determines the amount of cell suspension injected into the chamber 2 from the inter-electrode spacing d, the specific resistance ρ, and the measured resistance value R.
is detected by ■=ρed''/R. By comparing the detected V and the calculated value vo, it can be determined whether a predetermined amount of the cell suspension has been injected into the chamber 2.
所定量ｖｏの細胞懸濁液が注入されると１選定されたコ
ンデンサ１０−１に電ｇ８から充電され、その充電電荷
がチャンバ２に放電されることにより所望の時定数τを
もつパルス電界を細胞に印加することができる。When a predetermined amount vo of cell suspension is injected, one selected capacitor 10-1 is charged with an electric current g8, and the charged electric charge is discharged into the chamber 2, thereby creating a pulsed electric field with a desired time constant τ. can be applied to the cells.
時定数τを条件設定手段に設定すればその時定数τに応
じてコンデンサ１０−ｉが選定され、チャンバ２に注入
される細胞懸濁液量ｖｏが算出されるので、時定数τを
連続的に変化させて設定することができる。If the time constant τ is set in the condition setting means, the capacitor 10-i is selected according to the time constant τ, and the amount vo of cell suspension injected into the chamber 2 is calculated. It can be changed and set.
（実施例） 第２図は一実施例を表わす。(Example) FIG. 2 represents one embodiment.
チャンバ２は平行平板の対向電極４，６を備えており、
電極４，６は垂直方向に立っている。電極４，６間に細
胞懸濁液を注入することができ、注入量は任意の量とす
ることができる。電極６は接地され、電極４は切替えス
イッチ１５ａに接続されている。切替えスイッチ１５ａ
の一方の接点にはコンデンサを選択するスイッチ１２−
１〜１２−ｎが接続されている。スイッチ１２−１〜１
２−ｎにはそれぞれコンデンサ１０−１〜１〇−ｎの一
方の電極が接続され、コンデンサ１０−１〜１０−ｎの
他方の電極は接地されている。コンデンサの容量は０１
〜Ｃｎである。The chamber 2 is equipped with parallel plate facing electrodes 4 and 6,
Electrodes 4, 6 stand vertically. A cell suspension can be injected between the electrodes 4 and 6, and the injection amount can be arbitrary. Electrode 6 is grounded, and electrode 4 is connected to changeover switch 15a. Changeover switch 15a
One contact of the switch 12- for selecting the capacitor is connected to the switch 12-.
1 to 12-n are connected. Switch 12-1~1
One electrode of capacitors 10-1 to 10-n is connected to each of capacitors 2-n, and the other electrode of capacitors 10-1 to 10-n is grounded. Capacity of capacitor is 01
~Cn.
コンデンサ１０−１〜１０−ｎを充電するために、スイ
ッチ１２−１〜１２−ｎとコンデンサ１０−１〜１０−
ｎの回路にはスイッチ１４と抵抗９を介して直流電圧電
源８の十電極が接続され、電源８の一電極は接地されて
いる。In order to charge the capacitors 10-1 to 10-n, the switches 12-1 to 12-n and the capacitors 10-1 to 10-n
Ten electrodes of a DC voltage power source 8 are connected to the n circuit via a switch 14 and a resistor 9, and one electrode of the power source 8 is grounded.
切替えスイッチ１５ａの他方の接点には抵抗測定回路２
０が接続されている。抵抗測定回路２０では切替えスイ
ッチ１５ａの接点に抵抗３２を介して交流電源３４が接
続されている。抵抗３２の両端には電位差を測定するた
めに差動増幅器３６が接続され、差動増幅器３６の出力
は整流回路３８で整流され、Ａ／Ｄ変換器４０でデジタ
ル信号に変換されてマイクロコンピュータ３０に取り込
まれる。A resistance measuring circuit 2 is connected to the other contact of the changeover switch 15a.
0 is connected. In the resistance measuring circuit 20, an AC power source 34 is connected to a contact point of the changeover switch 15a via a resistor 32. A differential amplifier 36 is connected to both ends of the resistor 32 to measure the potential difference, and the output of the differential amplifier 36 is rectified by a rectifier circuit 38 and converted into a digital signal by an A/D converter 40, which is then sent to the microcomputer 30. be taken in.
コンデンサ１０−１〜１０−ｎを選択するスイッチ１２
−１〜１２−ｎ、スイッチ１４及び切替えスイッチ１５
ａはマイクロコンピュータ３０によりオン・オフ又は切
替えが制御される。Switch 12 for selecting capacitors 10-1 to 10-n
-1 to 12-n, switch 14 and changeover switch 15
On/off or switching of a is controlled by the microcomputer 30.
４２は本装置の動作を操作するとともに、チャンバ定数
ｋ、電極間間隔８１時定数τ、コンデンサの容ｆＣ工〜
Ｃｎを設定するために使用されるキーボードであり、２
８は表示手段としてのＣＲＴである。42 operates the operation of this device, and also controls the chamber constant k, the inter-electrode spacing 81, the time constant τ, and the capacitor capacity fC~
A keyboard used to set Cn, 2
8 is a CRT as a display means.
第１図における比抵抗算出手段２２、条件設定手段２４
及び細胞懸濁液量検出手段２６はマイクロコンピュータ
３０によって実現される。Specific resistance calculation means 22 and condition setting means 24 in FIG.
and the cell suspension amount detection means 26 are realized by a microcomputer 30.
次に、本実施例の動作を第３図のフローチャートを参照
して説明する。Next, the operation of this embodiment will be explained with reference to the flowchart of FIG.
まず、操作者はチャンバ２に細胞懸濁液の所定量を注入
する。細胞懸濁液を注入したことが入力されると（ステ
ップＳ１）、スイッチ１４．１５が第２図の状態から、
切替えスイッチ１５ａが抵抗測定回路２０側に切り替え
られる。これ）こより。First, the operator injects a predetermined amount of cell suspension into chamber 2. When it is input that the cell suspension has been injected (step S1), the switches 14 and 15 change from the state shown in FIG.
The changeover switch 15a is switched to the resistance measurement circuit 20 side. This) Koyori.
交流電源３４から抵抗３２を経てチャンバ２に交流電流
が流れ、このとき抵抗３２で発生する電圧が差動増幅器
３６により検出され、整流回路３８で整流され、Ａ／Ｄ
変換器４０でデジタル信号に変換されてマイクロコンピ
ュータ３０に取り込まれ、チャンバ２の抵抗ｒ （ＫΩ
）が算出される（ステップＳ２）。そして、チャンバ定
数ｋが取り込まれ、比抵抗ρが
ρ＝ ｒ ／ ｋ
として算出される（ステップＳ３．Ｓ４）。An alternating current flows from the alternating current power source 34 to the chamber 2 via the resistor 32, and the voltage generated at the resistor 32 at this time is detected by the differential amplifier 36, rectified by the rectifier circuit 38, and then connected to the A/D
It is converted into a digital signal by the converter 40 and taken into the microcomputer 30, and the resistance r (KΩ
) is calculated (step S2). Then, the chamber constant k is taken in, and the specific resistance ρ is calculated as ρ=r/k (steps S3 and S4).
次に、設定された電極間間隔ｄ （ｃｍ）、時定数ｔ（
ｍｓ）及びコンデンサ１０−１−１０− ｎの容量Ｃ工
〜Ｃｎが取り込まれ（ステップＳ５）、その時定数τに
なるように、コンデンサ１ｏ−１〜１０−ｎの容量Ｃ工
〜Ｃｎに対する細胞懸濁液量ｖ （ｍＱ）が算出される
。この計算は次の式によって行なわれる。Next, set the inter-electrode spacing d (cm) and the time constant t (
ms) and the capacitances C~Cn of the capacitors 10-1-10-n are taken in (step S5), and the cell suspension for the capacitors C~Cn of the capacitors 1o-1~10-n is taken in so that the time constant τ is obtained. The volume of turbid liquid v (mQ) is calculated. This calculation is performed using the following formula.
■＝ρｄ ２Ｃｉ／τ
算出された細胞懸濁液量Ｖのうちチャンバ２の容量より
も小さく、かつチャンバ２の容量に最も近いものが最適
な細胞懸濁液量ｖｏとなり、そのときの容量Ｃｉととも
に選定され、コンデンサ選択用スイッチ１２−１〜１２
−ｎのうちその容量Ｃｉをもつコンデンサ１０−ｉのス
イッチ１２−１がオンとされ（ステップＳ６）、細胞懸
濁液量ｖｏはＣＲＴ２８に表示される（ステップＳ７）
。■=ρd 2Ci/τ Of the calculated cell suspension volume V, the one that is smaller than the volume of chamber 2 and closest to the volume of chamber 2 becomes the optimal cell suspension volume vo, and the volume at that time Ci The capacitor selection switches 12-1 to 12
-n, the switch 12-1 of the capacitor 10-i having the capacitance Ci is turned on (step S6), and the cell suspension volume vo is displayed on the CRT 28 (step S7).
.
比抵抗ρを測定するために注入した細胞懸濁液を取り出
した後、操作者は表示された細胞懸濁液量ｖＯになるよ
うにチャンバ２に細胞懸濁液を注入する。このとき細胞
懸濁液はチャンバ２内で均一な高さとなるように注入す
る。After taking out the cell suspension injected to measure the specific resistance ρ, the operator injects the cell suspension into the chamber 2 to the displayed cell suspension volume vO. At this time, the cell suspension is injected into the chamber 2 so that it has a uniform height.
細胞懸濁液が注入されたことが入力されると、マイクロ
コンピュータ３０はその時の抵抗測定回路２０の出力か
らチャンバ２の抵抗値Ｒを算出し、次式
％式％
によって実際に注入された細胞懸濁液量Ｖとその時の時
定数τを算出し、ＣＲＴ２８に表示する（ステップＳ８
）。マイクロコンピュータ３０は注入された細胞懸濁液
量Ｖと時定数τから算出された細胞懸濁液量ｖＯとを比
較し、Ｖがｖｏと一致すると判断される範囲内に入った
ことを確認すると（ステップＳ９）、スイッチ１４をオ
ンにして選定されたコンデンサ１０−ｉを電１１ｉＸ８
により所定の初期電圧まで充電させる（ステップ５１０
）。When it is input that the cell suspension has been injected, the microcomputer 30 calculates the resistance value R of the chamber 2 from the output of the resistance measurement circuit 20 at that time, and calculates the resistance value R of the chamber 2 using the following formula: The suspension volume V and the time constant τ are calculated and displayed on the CRT 28 (step S8
). The microcomputer 30 compares the injected cell suspension volume V with the cell suspension volume vO calculated from the time constant τ, and confirms that V is within a range that is determined to match vo. (Step S9), the switch 14 is turned on and the selected capacitor 10-i is connected to the capacitor 11iX8.
is charged to a predetermined initial voltage (step 510).
).
所定の初期電圧はマイクロコンピュータ３０に予め入力
されているものとする。It is assumed that the predetermined initial voltage has been input into the microcomputer 30 in advance.
その後、マイクロコンピュータ３ｏはスイッチ１４をオ
フにし、スイッチ１５ａをコンデンサ側に切り替えてチ
ャンバ２にコンデンサ１０−１の電荷を放電させる（ス
テップ５１１）。これにより、チャンバ２の細胞懸濁液
に所定の初期電圧で、設定された時定数τのパルス電界
を与える。Thereafter, the microcomputer 3o turns off the switch 14, switches the switch 15a to the capacitor side, and discharges the charge of the capacitor 10-1 into the chamber 2 (step 511). As a result, a pulsed electric field with a set time constant τ is applied to the cell suspension in the chamber 2 at a predetermined initial voltage.
実施例は本発明を遺伝子導入装置に適用した例を示して
いるが、例えば第２図の装置にさらに交流電源装置を設
け、チャンバ２に交流電界と直流電界を切り替えて印加
するようにすれば、細胞融合装置として利用することが
できるようになる。Although the embodiment shows an example in which the present invention is applied to a gene introduction device, for example, if an AC power supply device is further provided in the device shown in FIG. , it can now be used as a cell fusion device.
（発明の効果）
本発明では複数のコンデンサのうちの最適なものに対し
、チャンバに注入する細胞懸濁液量を算出し、実際に注
入される細胞懸濁液量が算出された量になるように、抵
抗測定回路による抵抗値測定から監視し、その後にコン
デンサを経てチャンバの細胞懸濁液に直流電圧パルスを
印加するようにしたので、所望の時定数の直流電圧パル
スを印加することができ、実験の再現性がよくなる。(Effects of the Invention) In the present invention, the amount of cell suspension to be injected into the chamber is calculated for the optimal one of a plurality of capacitors, and the amount of cell suspension actually injected becomes the calculated amount. As shown in the figure, the resistance was monitored by measuring the resistance value using a resistance measurement circuit, and then a DC voltage pulse was applied to the cell suspension in the chamber via a capacitor, so it was possible to apply a DC voltage pulse with a desired time constant. This improves the reproducibility of experiments.
チャンバに注入する細胞懸濁液量は連続的に変えること
ができ、チャンバの容量以内であれば自由に変化させる
ことができるので、時定数を連続的に変化させることが
でき、きめの細かい実験を行なうことができる。The amount of cell suspension injected into the chamber can be changed continuously, and can be freely changed within the chamber capacity, so the time constant can be changed continuously, allowing for fine-grained experiments. can be done.
第１図は本発明を示すブロック図、第２図は一実施例を
示す回路図、第３図は一実施例の動作を示すフローチャ
ート図、第４図は従来の遺伝子導入装置を示す回路図で
ある。
２・・・・・・チャンバ、４，６・・・・・・電極、８
・・・・・・直流電圧電源、１０−１〜１０−ｎ・・・
・・・コンデンサ、２０・・・・・・抵抗測定回路、２
２・・・・・・比抵抗算出手段、２４・・・・・・条件
設定手段、２６・・・・・・細胞懸濁液量検出手段、２
８・・・・・・表示手段。
第
図
第４１．ＡFig. 1 is a block diagram showing the present invention, Fig. 2 is a circuit diagram showing one embodiment, Fig. 3 is a flow chart showing the operation of one embodiment, and Fig. 4 is a circuit diagram showing a conventional gene transfer device. It is. 2... Chamber, 4, 6... Electrode, 8
...DC voltage power supply, 10-1 to 10-n...
... Capacitor, 20 ... Resistance measurement circuit, 2
2... Specific resistance calculation means, 24... Condition setting means, 26... Cell suspension amount detection means, 2
8...Display means. Figure 41. A
Claims (1)
るチャンバと、直流電圧電源と、前記電源により充電さ
れ、前記チャンバに放電する並列接続された互いに容量
の異なる複数個のコンデンサと、前記チャンバに交流電
流を流して前記チャンバの抵抗値を測定する抵抗測定回
路と、この抵抗測定回路の測定値とチャンバ定数とから
細胞懸濁液の比抵抗を算出する比抵抗算出手段と、前記
チャンバの電極間間隔、時定数、前記コンデンサの容量
値及び算出された比抵抗から最適コンデンサを選定し、
細胞懸濁液量を算出する条件設定手段と、前記抵抗測定
回路の測定値、前記チャンバの電極間間隔及び算出され
た比抵抗から細胞懸濁液量を検出する細胞懸濁液量検出
手段と、前記条件設定手段で算出された細胞懸濁液量を
表示する表示手段とを備えた時定数制御型細胞電気刺激
装置。(1) A chamber that accommodates a cell suspension between opposing electrodes and applies an electric field, a DC voltage power source, and a plurality of parallel-connected capacitors with different capacities that are charged by the power source and discharged into the chamber. a resistance measuring circuit for measuring the resistance value of the chamber by passing an alternating current through the chamber; and a resistivity calculating means for calculating the specific resistance of the cell suspension from the measured value of the resistance measuring circuit and a chamber constant. , selecting an optimal capacitor from the inter-electrode spacing of the chamber, the time constant, the capacitance value of the capacitor, and the calculated resistivity;
a condition setting means for calculating the amount of cell suspension; and a cell suspension amount detecting means for detecting the amount of cell suspension from the measured value of the resistance measuring circuit, the interval between the electrodes of the chamber, and the calculated specific resistance. and display means for displaying the amount of cell suspension calculated by the condition setting means.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63184057A JP2621384B2 (en) | 1988-07-22 | 1988-07-22 | Time constant controlled cell electrical stimulator |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63184057A JP2621384B2 (en) | 1988-07-22 | 1988-07-22 | Time constant controlled cell electrical stimulator |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0235071A true JPH0235071A (en) | 1990-02-05 |
| JP2621384B2 JP2621384B2 (en) | 1997-06-18 |
Family
ID=16146619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63184057A Expired - Fee Related JP2621384B2 (en) | 1988-07-22 | 1988-07-22 | Time constant controlled cell electrical stimulator |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2621384B2 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100458662B1 (en) * | 2002-03-13 | 2004-12-03 | 문영훈 | Embryonic cell fusion machine have voltage regulator and environment memory circuit. |
| JP2008260008A (en) * | 2007-03-19 | 2008-10-30 | Tosoh Corp | Fine-particle operation apparatus and fine-particle operation method using it |
| JP2010512151A (en) * | 2006-12-06 | 2010-04-22 | バイオ−ラッド ラボラトリーズ，インコーポレイティド | Multi-channel electroporation system |
| US7732175B2 (en) | 2004-06-14 | 2010-06-08 | Lonza Cologne Ag | Method and circuit arrangement for treating biomaterial |
| US8173416B2 (en) | 2001-04-23 | 2012-05-08 | Lonza Cologne Gmbh | Circuit arrangement for injecting nucleic acids and other biologically active molecules into the nucleus of higher eucaryotic cells using electrical current |
| CN110437999A (en) * | 2019-07-15 | 2019-11-12 | 广州蛟龙细胞医药科技有限公司 | A kind of electroporation |
-
1988
- 1988-07-22 JP JP63184057A patent/JP2621384B2/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8173416B2 (en) | 2001-04-23 | 2012-05-08 | Lonza Cologne Gmbh | Circuit arrangement for injecting nucleic acids and other biologically active molecules into the nucleus of higher eucaryotic cells using electrical current |
| KR100458662B1 (en) * | 2002-03-13 | 2004-12-03 | 문영훈 | Embryonic cell fusion machine have voltage regulator and environment memory circuit. |
| US7732175B2 (en) | 2004-06-14 | 2010-06-08 | Lonza Cologne Ag | Method and circuit arrangement for treating biomaterial |
| US8058042B2 (en) | 2004-06-14 | 2011-11-15 | Lonza Cologne Gmbh | Method and circuit arrangement for treating biomaterial |
| JP2010512151A (en) * | 2006-12-06 | 2010-04-22 | バイオ−ラッド ラボラトリーズ，インコーポレイティド | Multi-channel electroporation system |
| JP2008260008A (en) * | 2007-03-19 | 2008-10-30 | Tosoh Corp | Fine-particle operation apparatus and fine-particle operation method using it |
| CN110437999A (en) * | 2019-07-15 | 2019-11-12 | 广州蛟龙细胞医药科技有限公司 | A kind of electroporation |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2621384B2 (en) | 1997-06-18 |
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