WO2012133804A1 - Cell modification method - Google Patents

Cell modification method Download PDF

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Publication number
WO2012133804A1
WO2012133804A1 PCT/JP2012/058638 JP2012058638W WO2012133804A1 WO 2012133804 A1 WO2012133804 A1 WO 2012133804A1 JP 2012058638 W JP2012058638 W JP 2012058638W WO 2012133804 A1 WO2012133804 A1 WO 2012133804A1
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cells
electric field
cell
pulse
yeast
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PCT/JP2012/058638
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French (fr)
Japanese (ja)
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勝木淳
秋山秀典
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国立大学法人熊本大学
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Publication of WO2012133804A1 publication Critical patent/WO2012133804A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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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  • the present invention relates to a cell modification method for applying a stimulus to a cell and modifying its properties.
  • Cells that have acquired particularly useful properties may improve production efficiency or bring about significant technological innovation for the purpose of culturing cells in various industries.
  • the present invention has been made in view of such circumstances, and provides a cell modification method capable of changing the properties of cells.
  • the cells are exposed to an electric field generated by a pulse voltage applied between a pair of electrodes, and the properties of the cells are changed.
  • the cell modification method according to claim 2 is characterized in that, in the cell modification method according to claim 1, the application time of the pulse voltage is 10 nsec to 300 nsec.
  • the cell modification method according to claim 3 is characterized in that, in the cell modification method according to claim 1 or 2, the electric field strength is 1 kV / cm to 30 kV / cm.
  • the cell modification method according to claim 4 is characterized in that, in the cell modification method according to any one of claims 1 to 3, the cell is yeast.
  • the cell modification method according to claim 5 is characterized in that in the cell modification method according to any one of claims 1 to 4, the property is a cell growth rate.
  • the cell modification that can change the properties of the cells A method can be provided.
  • the pulse voltage can be applied in an extremely short time, and the cell properties can be changed efficiently. it can.
  • the cells can be exposed to a strong electric field, and the properties of the cells can be changed efficiently. it can.
  • the cells are yeasts, yeasts having changed properties can be obtained.
  • the present invention provides a cell modification method in which cells are exposed to an electric field generated by a pulse voltage applied between a pair of electrodes to change the properties of the cells.
  • the electric field generated by the pulse voltage applied between the pair of electrodes described above is also called a pulse electric field
  • the process of exposing cells in the pulse electric field is also called a pulse electric field process.
  • cells have the relatively mild conditions in which constituent substances such as proteins and nucleic acids are not altered as the optimum conditions for growth and proliferation.
  • the cell when a large change occurs in the external environment, the cell may exhibit a trait that is not expressed in a normal state such as an optimum condition in order to adapt to the environmental change.
  • an impact force (pulse power) obtained by temporally compressing energy is applied to a cell, and a trait that is not normally expressed (hereinafter, also referred to as a non-normal trait) is expressed. It induces a phenomenon that is not observed in the cells of time.
  • the waveform of the pulse power applied to the cell is not particularly limited, and may be any one of a rectangular wave shape, a triangular wave shape, a sine wave shape, and the like.
  • the stimulus applied to the cell is within a predetermined preferred range, as shown in FIG. If the stimulation is smaller than this preferred range, the cells are less likely to develop abnormal characteristics (insensitivity), and if the stimulation is too large, the number of cells that die through processes such as apoptosis or necrosis increases, and the efficiency of cell modification is increased. It will fall (inactivation).
  • the preferred expression stimulus range even when a stimulus deviating from the preferred range of stimuli for expressing the unusual character (hereinafter also referred to as the preferred expression stimulus range) is given, the unusual character is not necessarily expressed. It doesn't mean.
  • a pulse voltage can be applied to the cell in a very short time.
  • the preferred range of expression stimulation at this time is that the application time of the pulse voltage applied to the cells is 10 nsec to 300 nsec, more preferably 70 nsec to 100 nsec.
  • the stimulation given to the cells can be made extremely short, and damage to the cells can be prevented as much as possible while the abnormal expression of the cells is expressed. Can be encouraged.
  • the electric field strength for exposing the cell is 1 kV / cm to 30 kV / cm, more preferably 5 kV / cm to 25 kV / cm.
  • the cells whose properties can be changed by the cell modification method according to the present embodiment are not particularly limited.
  • any of the cells comprising a multicellular organism such as animals including humans, plants, fruiting bodies of basidiomycetes, microorganisms composed of a plurality of cells, cells separated from the multicellular organism, and unicellular organisms. It may be.
  • the cell when stimulating a cell that constitutes a multicellular organism, the cell is stimulated by placing a multicellular organism between a pair of electrodes and applying a pulse voltage. be able to.
  • stimulation can be applied to the cell by placing the cell between a pair of electrodes and applying a pulse voltage.
  • yeast can be selected as the cell.
  • Yeast is widely used in the food and industrial fields, and can be modified to a more suitable yeast for the purpose of use of yeast in each field.
  • any cell may be used, but the property to be modified may be a growth rate.
  • the growth rate may be slower or faster than before the modification.
  • the production efficiency can be dramatically improved.
  • the cells are modified so that the growth rate is slower than before the modification, for example, it can be applied to the case where the growth of microorganisms is suppressed to prevent excessive fermentation in food production.
  • the amount of yeast produced can be increased.
  • a pulse power application device C is composed of a pulse power generation device A that generates pulse power to be applied to cells and a cue bed B that is electrically connected to the pulse power generation device A.
  • the pulse power generation device A includes a voltage adjusting unit 11, a pulse voltage generating unit 12, a control unit 13, and an input unit 14.
  • the voltage adjusting means 11 adjusts the voltage of the electric power supplied from the power supply 10 and increases or decreases the voltage supplied to the pulse voltage generating means 12.
  • the pulse voltage generation means 12 has a role of converting the power supplied from 11 into a pulse shape and outputting it from the terminal portions 12a and 12b.
  • the control means 13 is electrically connected to the voltage adjusting means 11 and the pulse voltage generating means 12 described above, and controls the voltage adjusting means 11 and the pulse voltage generating means 12.
  • control means 13 is connected to an input means 14 such as a control panel or a keyboard, and is configured such that an operation signal can be input to the control means 13 when the user operates the input means 14. ing.
  • the control means 13 11 transmits a signal for adjusting the voltage to the pulse voltage generator 12 and transmits a signal for adjusting the pulse waveform to the pulse voltage generating means 12.
  • the voltage adjusting means 11 adjusts the voltage of the electric power supplied to the pulse voltage generating means 12 based on the voltage adjustment signal transmitted from the control means 13.
  • the pulse voltage generation means 12 converts the power output from the voltage adjustment means 11 into a pulse form based on the pulse waveform adjustment signal transmitted from the control means 13 and outputs it from the terminal portions 12a and 12b.
  • FIG. 4 is an example of the pulse power output from the pulse power generator A.
  • FIG. 4 shows a voltage waveform of the pulse power applied to the cuvette B, and the application time is 70 ns.
  • an application time of 10 nsec to 300 ns can be provided to the biological sample in the cuvette.
  • the cuvette B has a rectangular shape with a bottom, and the narrow wall 20 is formed by projecting the inner wall inward from the middle to the bottom. ing.
  • a pair of electrodes 23, 23 are arranged on both side walls of the narrow portion 20 so as to come into contact with the culture medium 21 accommodated in the cuvette B, and each of the electrodes 23, 23 is a pulse voltage generating means 12. Are connected to the terminal portions 12a and 12b. In addition, the distance between the electrodes 23 and 23 of the cuvette used in this embodiment is 4 mm.
  • the culture medium 21 is accommodated in the cuvette B, and the cells 24 are suspended in the culture medium 21.
  • the pulse power application device C configured as described above, when the pulse power is output from the pulse power generation device A as described above, a voltage having a predetermined pulse waveform is applied between the electrodes 23 and 23, and the cuvette B The cells 24 can be exposed to a pulsed electric field and stimulated.
  • yeast cells (hereinafter simply referred to as yeast) were used as an example of cells.
  • Saccharomyces cerevisiae BY4741a strain was used as yeast as shown in FIG.
  • yeast in the growing season shown by a dotted line
  • 80-120 minutes of shaking culture at 32 ° C. was used. .
  • YPD liquid medium 10 ml was dispensed into a 15 ml culture bottle.
  • This YPD liquid medium was prepared by dissolving 10 g of Bacto Yeast Extract, 20 g of Bacto Pepton and 20 g of D-(+)-Glucose in 1000 ml of distilled water and sterilizing at 120 ° C for 20 minutes in an autoclave. .
  • the above-mentioned yeast forming a single colony on a flat plate agar medium is inoculated with one platinum ear in the medium in the culture bottle, and 160 rpm for 24 hours at 32 ° C. using a shaking incubator manufactured by TAITEC. Incubated with shaking.
  • the electric field strength of the pulse power exposed to yeast was set to four types: 10 kV / cm, 15 kV / cm, 20 kV / cm, and 25 kV / cm.
  • the number of times of exposure to the electric field (hereinafter, “exposing the yeast n times to the electric field is expressed as“ n shot ””) was overlaid to change the energy applied to the cells.
  • FIG. 7 shows the number of live yeasts immediately after the pulse electric field treatment, with the number of live yeasts on the vertical axis and the input energy per unit volume on the horizontal axis. In addition, the number of live yeasts when the pulse electric field treatment is not performed is shown as a control.
  • a culture solution with 10 6 cfu / ml of live yeast is dispensed into cuvette B, and energy of 0.01 J / ml to 10 J / ml is applied at an electric field strength of 5 kV / cm to 25 kV / cm. Thereafter, the number of live yeasts when cultured for 4 hours was verified. The result is shown in FIG.
  • Fig. 8 (a) shows the number of live yeasts immediately after the pulse electric field treatment at each electric field strength
  • Fig. 8 (b) shows the number of live yeasts after 4 hours of culture.
  • shaft in Fig.8 (a) makes the number of living yeast when not performing a pulse electric field process 100%
  • shaft in FIG.8 (b) is cultured for 4 hours without performing a pulse electric field process.
  • the number of later live yeast is 100%.
  • the pulse electric field treatment is performed when the input energy is 0.01 J / ml to 10 J / ml at any electric field strength. A larger number of live yeasts was confirmed as compared to the number of live yeasts after culturing for 4 hours.
  • the preferred range of expression stimulation when stimulating yeast with a predetermined electric field strength is 5 to 15 kV / cm and input energy is 0.01 J / ml to 10 J / ml, or electric field strength. Is 15 to 25 kV / cm and the input energy is 0.01 J / ml to 1 J / ml.
  • 10 ml of YPD liquid medium was dispensed into a 15 ml culture bottle.
  • This YPD liquid medium is prepared by dissolving 10 g of Bacto Yeast Extract, 20 g of Bacto Yest Pepton, and 20 g of D-(+)-Glucose in 1000 ml of distilled water and sterilizing at 120 ° C for 20 minutes in an autoclave. It was.
  • the above-mentioned yeast forming a single colony on a flat plate agar medium is inoculated with one platinum ear in the medium in the culture bottle, and 160 rpm for 24 hours at 32 ° C. using a shaking incubator manufactured by TAITEC. Incubated with shaking.
  • the electric field strength in the pulse electric field treatment was fixed at 10 kV / cm, and six types of samples with different input energies were created: 0 shot (control), 3 shots, 10 shots, 30 shots, 100 shots, and 200 shots. .
  • 0.5 ml of yeast subjected to pulsed electric field treatment is taken out from the cuvette with a sterile pipette, inoculated into a 15 ml culture bottle into which 4.5 ml of YPD liquid medium has been dispensed, and then used with a shake incubator manufactured by TAITEC. While performing shaking culture at 32 ° C. for 24 hours at 160 rpm, the time course of the number of live yeasts was observed. The number of viable yeast was measured immediately after the pulse electric field treatment (0 h), after 4 hours of culture, after 12 hours of culture, and after 16 hours of culture. The result is shown in FIG.
  • the cells immediately after the pulse electric field treatment had the same number of live yeasts in all samples, but at the time of culturing for 4 hours, all the samples subjected to the pulse electric field treatment were The growth rate was improved compared to the control. Moreover, after 12 hours of culture, the difference in the number of live yeasts relative to the control was maintained. In addition, after 16 hours of culture, a tendency was observed that the difference in the number of live yeasts with respect to the control was slightly reduced.
  • cells (yeast) subjected to pulsed electric field treatment can increase the growth rate relatively early, 0 to 4 hours after treatment, compared to control cells (yeast) not subjected to pulsed electric field treatment. Indicated. That is, by applying a pulsed electric field treatment to yeast, an increase in the growth rate could be expressed as an unusual trait.
  • the cell modification method by exposing the cell to an electric field generated by a pulse voltage applied between a pair of electrodes, for example, changing a cell property such as a growth rate. Can do.

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Abstract

The purpose of the invention is to provide a cell modification method with which a property of cells can be modified. The cell modification method of the invention involves exposing cells to an electrical field generated by pulsed voltage applied between a pair of electrodes and modifying a property of the same cells. The method is further characterized in that the pulsed voltage application time is between 10 and 300 nsec, the intensity of the electrical field is between 1 and 30 kV/cm, the cells are yeast, and the property is cell propagation strength.

Description

細胞改変方法Cell modification method
　本発明は、細胞に対して刺激を付与し、その性質を改変する細胞改変方法に関する。 The present invention relates to a cell modification method for applying a stimulus to a cell and modifying its properties.
　従来、医療や食品分野を中心に幅広い産業分野において、単細胞の微生物や多細胞生物を構成する細胞（以下、総称して細胞という。）の培養が頻繁に行われている。 Conventionally, in a wide range of industrial fields, mainly in the medical and food fields, single-cell microorganisms and cells constituting multicellular organisms (hereinafter collectively referred to as cells) have been frequently cultured.
　特に有用な性質を獲得した細胞は、各産業での細胞の培養目的において、生産効率を向上させたり、飛躍的な技術革新をもたらすことがある。 細胞 Cells that have acquired particularly useful properties may improve production efficiency or bring about significant technological innovation for the purpose of culturing cells in various industries.
　このような何らかの性質を獲得した細胞を得るためには、例えば、細胞を紫外線や放射線に曝露したり、遺伝物質を細胞内に取り込ませたりする方法が行われている（例えば、特許文献１参照。）。 In order to obtain cells that have acquired such a property, for example, a method in which the cells are exposed to ultraviolet rays or radiation, or a genetic material is taken into the cells has been performed (for example, see Patent Document 1). .)
特開２００３－０９３０５１号公報JP 2003-093051 A
　上述のように、細胞の性質を改変する方法は各種提案されているが、細胞は与える刺激の種類により異なる性質を示す場合もあり、産業分野における細胞の利便性の向上を図るためには、更なる細胞改変方法が求められている。 As described above, various methods for modifying the properties of cells have been proposed. However, cells may exhibit different properties depending on the type of stimulation applied. In order to improve the convenience of cells in the industrial field, There is a need for further cell modification methods.
　本発明は、斯かる事情に鑑みてなされたものであって、細胞の性質を変化させることのできる細胞改変方法を提供する。 The present invention has been made in view of such circumstances, and provides a cell modification method capable of changing the properties of cells.
　上記従来の課題を解決するために、請求項１に係る細胞改変方法では、一対の電極間に印加したパルス電圧により生じる電界中に細胞を暴露し、同細胞の性質を変化させることとした。 In order to solve the above-described conventional problems, in the cell modification method according to claim 1, the cells are exposed to an electric field generated by a pulse voltage applied between a pair of electrodes, and the properties of the cells are changed.
　また、請求項２に係る細胞改変方法では、請求項１に記載の細胞改変方法において、前記パルス電圧の印加時間は10nsec～300nsecであることに特徴を有する。 The cell modification method according to claim 2 is characterized in that, in the cell modification method according to claim 1, the application time of the pulse voltage is 10 nsec to 300 nsec.
　また、請求項３に係る細胞改変方法では、請求項１又は請求項２に記載の細胞改変方法において、前記電界の強度は、1kV/cm～30kV/cmであることに特徴を有する。 The cell modification method according to claim 3 is characterized in that, in the cell modification method according to claim 1 or 2, the electric field strength is 1 kV / cm to 30 kV / cm.
　また、請求項４に係る細胞改変方法では、請求項１～３いずれか1項に記載の細胞改変方法において、前記細胞は酵母であることに特徴を有する。 The cell modification method according to claim 4 is characterized in that, in the cell modification method according to any one of claims 1 to 3, the cell is yeast.
　また、請求項５に係る細胞改変方法では、請求項１～４いずれか1項に記載の細胞改変方法において、前記性質は、細胞の増殖速度であることに特徴を有する。 The cell modification method according to claim 5 is characterized in that in the cell modification method according to any one of claims 1 to 4, the property is a cell growth rate.
　請求項１に係る本発明では、一対の電極間に印加したパルス電圧により生じる電界中に細胞を暴露し、同細胞の性質を変化させることとしたため、細胞の性質を変化させることのできる細胞改変方法を提供することができる。 In the present invention according to claim 1, since the cells are exposed to the electric field generated by the pulse voltage applied between the pair of electrodes and the properties of the cells are changed, the cell modification that can change the properties of the cells A method can be provided.
　また、請求項２に係る本発明では、前記パルス電圧の印加時間は10nsec～300nsecであることとしたため、極短時間にパルス電圧を印加することができ、細胞の性質を効率よく変化させることができる。 Further, in the present invention according to claim 2, since the application time of the pulse voltage is 10 nsec to 300 nsec, the pulse voltage can be applied in an extremely short time, and the cell properties can be changed efficiently. it can.
　また、請求項３に係る本発明では、前記電界の強度は、1kV/cm～30kV/cmであることとしたため、細胞を強い電場に曝すことができ、細胞の性質を効率よく変化させることができる。 In the present invention according to claim 3, since the electric field strength is 1 kV / cm to 30 kV / cm, the cells can be exposed to a strong electric field, and the properties of the cells can be changed efficiently. it can.
　また、請求項４に係る本発明では、前記細胞は酵母であることとしたため、性質が変化した酵母を得ることができる。 Further, in the present invention according to claim 4, since the cells are yeasts, yeasts having changed properties can be obtained.
　また、請求項５に係る本発明では、前記性質は、細胞の増殖速度であることとしたため、増殖速度の大きな細胞を得ることができる。 In the present invention according to claim 5, since the property is the cell growth rate, cells having a high growth rate can be obtained.
細胞に対して付与する刺激の強さを概念的に示した説明図である。It is explanatory drawing which showed notionally the strength of the stimulus provided with respect to a cell. パルスパワー印加装置及びキューベットの外観を示した説明図である。It is explanatory drawing which showed the external appearance of the pulse power application apparatus and the cuvette. パルスパワー印加装置の構成を示したブロック図である。It is the block diagram which showed the structure of the pulse power application apparatus. 細胞に印加する電界の波形を示した説明図である。It is explanatory drawing which showed the waveform of the electric field applied to a cell. 酵母の形態及び形質を示した説明図である。It is explanatory drawing which showed the form and character of yeast. 酵母の増殖曲線を示した説明図である。It is explanatory drawing which showed the growth curve of yeast. パルス電界処理直後の生酵母数を示した説明図である。It is explanatory drawing which showed the number of living yeast immediately after a pulse electric field process. パルス電界処理を行った際の生酵母の比率を示した説明図である。It is explanatory drawing which showed the ratio of the live yeast at the time of performing a pulse electric field process. パルス電界処理を行った酵母の増殖曲線を示した説明図である。It is explanatory drawing which showed the growth curve of the yeast which performed the pulse electric field process.
　本発明は、一対の電極間に印加したパルス電圧により生じる電界中に細胞を暴露し、同細胞の性質を変化させる細胞改変方法を提供するものである。以下の説明において、上述した一対の電極間に印加したパルス電圧により生じる電界をパルス電界ともいい、パルス電界中に細胞を暴露する処理をパルス電界処理ともいう。 The present invention provides a cell modification method in which cells are exposed to an electric field generated by a pulse voltage applied between a pair of electrodes to change the properties of the cells. In the following description, the electric field generated by the pulse voltage applied between the pair of electrodes described above is also called a pulse electric field, and the process of exposing cells in the pulse electric field is also called a pulse electric field process.
　一般に細胞は、タンパク質や核酸などの構成物質が変質しない比較的穏やかな条件を生育や増殖の至適条件としている場合が多い。 In general, in many cases, cells have the relatively mild conditions in which constituent substances such as proteins and nucleic acids are not altered as the optimum conditions for growth and proliferation.
　その一方で細胞は、外部環境に大きな変化が生じると、その環境変化に適応するために、至適条件下のような通常の状態では発現させていない形質を現すことがある。 On the other hand, when a large change occurs in the external environment, the cell may exhibit a trait that is not expressed in a normal state such as an optimum condition in order to adapt to the environmental change.
　そこで本発明では、エネルギーを時間的に圧縮した衝撃力（パルスパワー）を細胞に対して付与し、通常時には発現させていない形質（以下、非通常時形質ともいう。）を発現させて、通常時の細胞には観察されない現象を誘導するようにしている。 Therefore, in the present invention, an impact force (pulse power) obtained by temporally compressing energy is applied to a cell, and a trait that is not normally expressed (hereinafter, also referred to as a non-normal trait) is expressed. It induces a phenomenon that is not observed in the cells of time.
　細胞に対して付与するパルスパワーの波形は特に限定されるものではなく、例えば矩形波状、三角波状、正弦波状などのいずれであっても良い。 The waveform of the pulse power applied to the cell is not particularly limited, and may be any one of a rectangular wave shape, a triangular wave shape, a sine wave shape, and the like.
　細胞に非通常時形質を発現させるためには、図１に示すように、細胞に対して付与する刺激が所定の好適な範囲内であるのが望ましい。この好適な範囲よりも刺激が小さいと細胞は非通常時形質を発現しにくく（不感）、また、刺激が大きすぎるとアポトーシスまたはネクローシスなどの過程を経て死ぬ細胞が増加し、細胞の改変効率が低下してしまうこととなる（不活化）。なお、この非通常時形質を発現させる刺激の好適な範囲（以下、発現刺激好適範囲ともいう。）から外れた刺激を付与した場合であっても、必ずしも非通常時形質の発現が不可能となる訳ではない。 In order to cause a cell to develop an unusual character, it is desirable that the stimulus applied to the cell is within a predetermined preferred range, as shown in FIG. If the stimulation is smaller than this preferred range, the cells are less likely to develop abnormal characteristics (insensitivity), and if the stimulation is too large, the number of cells that die through processes such as apoptosis or necrosis increases, and the efficiency of cell modification is increased. It will fall (inactivation). In addition, even when a stimulus deviating from the preferred range of stimuli for expressing the unusual character (hereinafter also referred to as the preferred expression stimulus range) is given, the unusual character is not necessarily expressed. It doesn't mean.
　非通常時形質を発現させるためには、所定の刺激を細胞に対して付与する必要があるが、このような刺激の一例を挙げるとすると、パルス電圧の細胞への極短時間での印加を挙げることができる。また、この際の発現刺激好適範囲は、細胞に付与するパルス電圧の印加時間を10nsec～300nsec、より好ましくは70nsec～100nsecとすることが挙げられる。 In order to develop an unusual trait, it is necessary to apply a predetermined stimulus to the cell. To give an example of such a stimulus, a pulse voltage can be applied to the cell in a very short time. Can be mentioned. Further, the preferred range of expression stimulation at this time is that the application time of the pulse voltage applied to the cells is 10 nsec to 300 nsec, more preferably 70 nsec to 100 nsec.
　パルス電圧の印加時間をこのような条件とすることにより、細胞に対して与える刺激を極短時間とすることができ、細胞に対するダメージを可及的防止しながら、細胞の非通常時形質の発現を促すことができる。 By setting the pulse voltage application time to such a condition, the stimulation given to the cells can be made extremely short, and damage to the cells can be prevented as much as possible while the abnormal expression of the cells is expressed. Can be encouraged.
　また、他の所定の刺激としては、例えば、大きな電界強度への細胞の曝露を挙げることができ、この際の発現刺激好適範囲としては、例えば、細胞を曝露させる電界の強度を1kV/cm～30kV/cm、より好ましくは5kV/cm～25kV/cmとすることが挙げられる。 In addition, as the other predetermined stimulation, for example, exposure of the cell to a large electric field strength can be mentioned, and as a suitable range of the expression stimulation at this time, for example, the electric field strength for exposing the cell is 1 kV / cm to 30 kV / cm, more preferably 5 kV / cm to 25 kV / cm.
　電界強度をこのような条件とすることにより、細胞に対して大きな刺激を付与することができ、細胞に非通常時形質の発現を効果的に促すことができる。 By setting the electric field strength to such a condition, it is possible to give a large stimulus to the cell, and to effectively promote the expression of the trait in the non-normal time.
　ところで、本実施形態に係る細胞改変方法によって性質を変化させることのできる細胞は、特に限定されるものではない。 By the way, the cells whose properties can be changed by the cell modification method according to the present embodiment are not particularly limited.
　具体的には、ヒトを含む動物、植物、担子菌類の子実体、複数の細胞よりなる微生物などの多細胞生物を構成している細胞や、同多細胞生物から分離した細胞、単細胞生物のいずれであっても良い。 Specifically, any of the cells comprising a multicellular organism such as animals including humans, plants, fruiting bodies of basidiomycetes, microorganisms composed of a plurality of cells, cells separated from the multicellular organism, and unicellular organisms. It may be.
　例えば、多細胞生物を構成している細胞に対して刺激を付与する場合には、一対の電極間に多細胞生物を配置してパルス電圧を印加することで、細胞に対して刺激を付与することができる。 For example, when stimulating a cell that constitutes a multicellular organism, the cell is stimulated by placing a multicellular organism between a pair of electrodes and applying a pulse voltage. be able to.
　また、多細胞生物から分離した細胞や単細胞生物の場合も同様に、一対の電極間に細胞を配置してパルス電圧を印加することで細胞に対して刺激を付与することができる。 Similarly, in the case of a cell separated from a multicellular organism or a single cell organism, stimulation can be applied to the cell by placing the cell between a pair of electrodes and applying a pulse voltage.
　特に、細胞としては酵母を選択することができる。酵母は食品分野や工業分野で広く用いられており、各分野における酵母の使用目的においてより好適な酵母に改変することが可能となる。 In particular, yeast can be selected as the cell. Yeast is widely used in the food and industrial fields, and can be modified to a more suitable yeast for the purpose of use of yeast in each field.
　また、いずれの細胞であっても良いのであるが、改変する性質は増殖速度であっても良い。増殖速度は、改変前に比して遅くても良く、また、早くても良い。 In addition, any cell may be used, but the property to be modified may be a growth rate. The growth rate may be slower or faster than before the modification.
　増殖速度を改変前より早くなるように細胞を改変した際には、例えば、細胞を用いて所定の物質を生産しているような工業において、その生産効率を飛躍的に向上させることができる。また、増殖速度を改変前より遅くなるように細胞を改変した際には、例えば、微生物の発育を抑制して、食品製造等における過剰発酵を防止する場合などに応用することができる。 When cells are modified so that the growth rate is faster than before modification, for example, in an industry where cells are used to produce a predetermined substance, the production efficiency can be dramatically improved. Moreover, when the cells are modified so that the growth rate is slower than before the modification, for example, it can be applied to the case where the growth of microorganisms is suppressed to prevent excessive fermentation in food production.
　また、例えば、前述の酵母の場合、酵母の生産量を増加させることができる。 Also, for example, in the case of the yeast described above, the amount of yeast produced can be increased.
　以下、本実施形態に係る細胞改変方法について、図面を参照しながら更に具体的に説明する。 Hereinafter, the cell modification method according to the present embodiment will be described more specifically with reference to the drawings.
〔パルスパワー印加装置〕
　まず、後に説明する各試験に用いた機器類の構成について説明する。図２（ａ）にはパルスパワー発生装置Ａの外観を示し、図２（ｂ）にはキューベットＢの外観を示す。各試験では、細胞に付与するパルスパワーを発生させるパルスパワー発生装置Ａと、同パルスパワー発生装置Ａに電気的に接続したキューベットＢとにより、パルスパワー印加装置Ｃを構成している。 [Pulse power application device]
First, the structure of the equipment used for each test demonstrated later is demonstrated. 2A shows the appearance of the pulse power generator A, and FIG. 2B shows the appearance of the cue bed B. In each test, a pulse power application device C is composed of a pulse power generation device A that generates pulse power to be applied to cells and a cue bed B that is electrically connected to the pulse power generation device A.
　具体的に説明すると、図３のブロック図に示すように、パルスパワー発生装置Ａは、電圧調整手段１１と、パルス電圧発生手段１２と、制御手段１３と、入力手段１４とを備えている。 More specifically, as shown in the block diagram of FIG. 3, the pulse power generation device A includes a voltage adjusting unit 11, a pulse voltage generating unit 12, a control unit 13, and an input unit 14.
　電圧調整手段１１は、電源１０より供給される電力の電圧を調整するものであり、パルス電圧発生手段１２に供給する電圧の昇圧や降圧を行う。 The voltage adjusting means 11 adjusts the voltage of the electric power supplied from the power supply 10 and increases or decreases the voltage supplied to the pulse voltage generating means 12.
　パルス電圧発生手段１２は、１１より供給される電力をパルス状に変換して端子部１２ａ,１２ｂより出力する役割を有する。 The pulse voltage generation means 12 has a role of converting the power supplied from 11 into a pulse shape and outputting it from the terminal portions 12a and 12b.
　制御手段１３は、前述の電圧調整手段１１及びパルス電圧発生手段１２に電気的に接続されており、電圧調整手段１１及びパルス電圧発生手段１２の制御を行う。 The control means 13 is electrically connected to the voltage adjusting means 11 and the pulse voltage generating means 12 described above, and controls the voltage adjusting means 11 and the pulse voltage generating means 12.
　また、制御手段１３にはコントロールパネルやキーボード等よりなる入力手段１４が接続されており、使用者が同入力手段１４を操作することにより、制御手段１３に対して操作信号を入力可能に構成している。 Further, the control means 13 is connected to an input means 14 such as a control panel or a keyboard, and is configured such that an operation signal can be input to the control means 13 when the user operates the input means 14. ing.
　このような構成を有するパルスパワー発生装置Ａでは、使用者が制御手段１３に対し、入力手段１４を用いて所定の電圧や印加時間、パルス波形等について入力すると、制御手段１３は、電圧調整手段１１に対して電圧を調整する信号を送信するとともに、パルス電圧発生手段１２に対してパルス波形を調整する信号を送信する。 In the pulse power generator A having such a configuration, when a user inputs a predetermined voltage, application time, pulse waveform, etc. to the control means 13 using the input means 14, the control means 13 11 transmits a signal for adjusting the voltage to the pulse voltage generator 12 and transmits a signal for adjusting the pulse waveform to the pulse voltage generating means 12.
　電圧調整手段１１は、制御手段１３より送信された電圧調整信号に基づいてパルス電圧発生手段１２へ供給する電力の電圧を調整する。 The voltage adjusting means 11 adjusts the voltage of the electric power supplied to the pulse voltage generating means 12 based on the voltage adjustment signal transmitted from the control means 13.
　また、パルス電圧発生手段１２は、制御手段１３より送信されたパルス波形調整信号に基づいて、電圧調整手段１１より出力される電力をパルス状に変換し、端子部１２ａ,１２ｂより出力することとなる。図４は、パルスパワー発生装置Ａより出力したパルスパワーの一例である。図４では、キューベットＢに印加するパルスパワーの電圧波形を示しており、その印加時間は70nsとしている。ただし、パルスパワー発生装置の回路構成を変えることによって、10nsec～300nsの印加時間をキューベット内の生物試料に提供することができる。 Further, the pulse voltage generation means 12 converts the power output from the voltage adjustment means 11 into a pulse form based on the pulse waveform adjustment signal transmitted from the control means 13 and outputs it from the terminal portions 12a and 12b. Become. FIG. 4 is an example of the pulse power output from the pulse power generator A. FIG. 4 shows a voltage waveform of the pulse power applied to the cuvette B, and the application time is 70 ns. However, by changing the circuit configuration of the pulse power generator, an application time of 10 nsec to 300 ns can be provided to the biological sample in the cuvette.
　キューベットＢは、図２（ｂ）にも示したように有底の矩形筒状としており、その内部には、内壁を中途部から底部にかけて内方にせり出させて狭隘部２０を形成している。 As shown in FIG. 2B, the cuvette B has a rectangular shape with a bottom, and the narrow wall 20 is formed by projecting the inner wall inward from the middle to the bottom. ing.
　この狭隘部２０の両側壁には、一対の電極２３,２３が、キューベットＢ内に収容した培地２１に接触するように配置されており、各電極２３,２３は、それぞれパルス電圧発生手段１２の端子部１２ａ,１２ｂに接続されている。なお、本実施形態にて使用するキューベットの電極２３,２３間の距離は4mmである。 A pair of electrodes 23, 23 are arranged on both side walls of the narrow portion 20 so as to come into contact with the culture medium 21 accommodated in the cuvette B, and each of the electrodes 23, 23 is a pulse voltage generating means 12. Are connected to the terminal portions 12a and 12b. In addition, the distance between the electrodes 23 and 23 of the cuvette used in this embodiment is 4 mm.
　また、キューベットＢ内には培地２１が収容されており、この培地２１には細胞２４が懸濁されている。 Further, the culture medium 21 is accommodated in the cuvette B, and the cells 24 are suspended in the culture medium 21.
　このような構成としたパルスパワー印加装置Ｃにおいて、前述のようにパルスパワー発生装置Ａからパルスパワーを出力させると、所定のパルス波形を有する電圧が電極２３,２３間に印加され、キューベットＢ内の細胞２４をパルス電界中に曝露させて刺激付与することができる。 In the pulse power application device C configured as described above, when the pulse power is output from the pulse power generation device A as described above, a voltage having a predetermined pulse waveform is applied between the electrodes 23 and 23, and the cuvette B The cells 24 can be exposed to a pulsed electric field and stimulated.
〔細胞について〕
　次に、後述の試験にて使用する細胞について説明する。以下の試験では、細胞の一例として酵母細胞（以下、単に酵母という。）を用いることとした。 [About cells]
Next, cells used in the test described below will be described. In the following tests, yeast cells (hereinafter simply referred to as yeast) were used as an example of cells.
　酵母は、図５にも示すように、Saccharomyces cerevisiae BY4741a株を使用した。また酵母は、パルスパワーを付与するに際し、図６の増殖曲線中に示すように、３２℃にて振盪培養し８０～１２０分経過した生育期の酵母（点線で示す）を使用することとした。 Saccharomyces cerevisiae BY4741a strain was used as yeast as shown in FIG. In addition, when applying pulse power, as shown in the growth curve of FIG. 6, yeast in the growing season (shown by a dotted line) after 80-120 minutes of shaking culture at 32 ° C. was used. .
〔上限電界強度調査試験〕
　次に、酵母に対して付与する刺激としての電界強度の発現刺激好適範囲の上限値、換言すれば、酵母に対してどの程度の刺激を与えると生酵母数が減少するかを調べるための試験を行った。 [Upper field strength investigation test]
Next, the upper limit of the preferred range of stimulation of the expression of electric field strength as a stimulus to be applied to yeast, in other words, a test for investigating how much stimulus is applied to yeast to reduce the number of live yeast Went.
　15ml容量の培養瓶に10mlのYPD液体培地を分注した。このYPD液体培地は、1000mlの蒸留水に、10ｇのBacto Yeast Extract、20ｇのBacto Pepton、20ｇのD-(+)-Glucoseを溶解し、オートクレーブにて120℃20分間殺菌処理したものを用いた。 10 ml of YPD liquid medium was dispensed into a 15 ml culture bottle. This YPD liquid medium was prepared by dissolving 10 g of Bacto Yeast Extract, 20 g of Bacto Pepton and 20 g of D-(+)-Glucose in 1000 ml of distilled water and sterilizing at 120 ° C for 20 minutes in an autoclave. .
　次に、平板寒天培地上にて単一コロニーを形成している前述の酵母を、培養瓶中の培地に一白金耳植菌し、TAITEC社製振盪培養器を用い32℃にて24時間160rpmで振盪培養を行った。 Next, the above-mentioned yeast forming a single colony on a flat plate agar medium is inoculated with one platinum ear in the medium in the culture bottle, and 160 rpm for 24 hours at 32 ° C. using a shaking incubator manufactured by TAITEC. Incubated with shaking.
　このようにして得られた酵母培養液（1×105cfu/ml）を滅菌済みのマイクロピペットで0.8ml採取し、キューベットに分注してパルス電界処理に供した。 0.8 ml of the thus obtained yeast culture solution (1 × 10 5 cfu / ml) was collected with a sterilized micropipette, dispensed into a cuvette, and subjected to pulsed electric field treatment.
　本試験で、酵母を曝したパルスパワーの電界強度は、10kV/cm, 15kV/cm, 20kV/cm, 25kV/cmの４種類とした。また、電界に曝露する回数（以下、酵母をｎ回電界に曝すことを「ｎショット」というように表現する。）を重ねて、細胞に付与するエネルギーをそれぞれ違えることとした。複数ショットのパルス電界を細胞に対して付与することにより、細胞に与えるエネルギー（投入エネルギー）を増加させることができる。その結果を図７に示す。 In this test, the electric field strength of the pulse power exposed to yeast was set to four types: 10 kV / cm, 15 kV / cm, 20 kV / cm, and 25 kV / cm. In addition, the number of times of exposure to the electric field (hereinafter, “exposing the yeast n times to the electric field is expressed as“ n shot ””) was overlaid to change the energy applied to the cells. By applying a pulse electric field of a plurality of shots to a cell, energy (input energy) given to the cell can be increased. The result is shown in FIG.
　図７はパルス電界処理直後の生酵母数を示しており、縦軸に生酵母数、横軸は単位体積あたりの投入エネルギーを示している。また、パルス電界処理を行っていない時の生酵母数をコントロールとして示している。 FIG. 7 shows the number of live yeasts immediately after the pulse electric field treatment, with the number of live yeasts on the vertical axis and the input energy per unit volume on the horizontal axis. In addition, the number of live yeasts when the pulse electric field treatment is not performed is shown as a control.
　図７からも分かるように、10kV/cm及び15kV/cmにおいては、約20J/mlのパルスエネルギーを付与した場合であっても、パルス電界処理直後の生酵母数はコントロールに対して顕著な差異は見られなかった。 As can be seen from FIG. 7, at 10 kV / cm and 15 kV / cm, even when pulse energy of about 20 J / ml was applied, the number of live yeast immediately after the pulse electric field treatment was significantly different from the control. Was not seen.
　一方、20kV/cm及び25kV/cmにおいては、約5J/ml以上のパルスエネルギーを付与することにより、105程度の生酵母数が最大で103程度まで減少する結果となった。 On the other hand, at 20 kV / cm and 25 kV / cm, by applying pulse energy of about 5 J / ml or more, the number of live yeasts of about 10 5 decreased to a maximum of about 10 3 .
　これらの結果を総合的に勘案し、最大で25kV/cmの電界強度を付与する場合には、投入エネルギーを約2Ｊ/ml以下とすることにより、生酵母の減少割合を１/10以下に抑えることが可能であることが示唆された。
〔パルス電界処理後に一定時間培養した際の生酵母数の検証〕 Considering these results comprehensively, when applying an electric field strength of 25 kV / cm at the maximum, the rate of decrease in live yeast is suppressed to 1/10 or less by making the input energy about 2 J / ml or less. It was suggested that it was possible.
[Verification of the number of live yeasts when cultured for a certain time after pulse electric field treatment]
　次に、生酵母数が106cfu/mlの培養液をキューベットＢ内に分注し、5kV/cm～25kV/cmの電界強度にて0.01J/ml～10J/mlのエネルギーを付与し、その後４時間培養したときの生酵母数の検証を行った。その結果を図８に示す。 Next, a culture solution with 10 6 cfu / ml of live yeast is dispensed into cuvette B, and energy of 0.01 J / ml to 10 J / ml is applied at an electric field strength of 5 kV / cm to 25 kV / cm. Thereafter, the number of live yeasts when cultured for 4 hours was verified. The result is shown in FIG.
　図８（ａ）は各電界強度でのパルス電界処理直後の生酵母数を示し、図８（ｂ）はその後４時間培養を行った際の生酵母数を示している。なお、図８（ａ）における縦軸はパルス電界処理を行わなかった際の生酵母数を100％としており、また、図８（ｂ）における縦軸はパルス電界処理を行わず４時間培養した後の生酵母数を100％としている。 Fig. 8 (a) shows the number of live yeasts immediately after the pulse electric field treatment at each electric field strength, and Fig. 8 (b) shows the number of live yeasts after 4 hours of culture. In addition, the vertical axis | shaft in Fig.8 (a) makes the number of living yeast when not performing a pulse electric field process 100%, and the vertical axis | shaft in FIG.8 (b) is cultured for 4 hours without performing a pulse electric field process. The number of later live yeast is 100%.
　図８（ａ）からも分かるように、電界強度5～25kV/cmの範囲で投入エネルギーを0.01～10J/mlの範囲とした場合には、いずれの電界強度においても、パルス電界処理を行わなかった際の生酵母数に比して0％～30％程度の減少に留まっていた。 As can be seen from FIG. 8 (a), when the input energy is in the range of 0.01 to 10 J / ml in the electric field strength range of 5 to 25 kV / cm, no pulse electric field treatment is performed at any electric field strength. Compared to the number of live yeast at the time, the decrease was only about 0% to 30%.
　一方、図８（ｂ）に示すように、パルス電界処理後に４時間培養を行うと、いずれの電界強度においても、投入エネルギーを0.01J/ml～10J/mlとした場合には、パルス電界処理を行わず４時間培養した後の生酵母数に比して、より多くの生酵母数が確認された。 On the other hand, as shown in FIG. 8 (b), when the culture is performed for 4 hours after the pulse electric field treatment, the pulse electric field treatment is performed when the input energy is 0.01 J / ml to 10 J / ml at any electric field strength. A larger number of live yeasts was confirmed as compared to the number of live yeasts after culturing for 4 hours.
　また、電界強度を5～15kV/cmの範囲とし、投入エネルギーを1J/ml～10J/mlとした場合においても、パルス電界処理を行わず４時間培養した後の生酵母数に比して、より多くの生酵母数が確認された。 In addition, even when the electric field strength is in the range of 5 to 15 kV / cm and the input energy is 1 J / ml to 10 J / ml, compared to the number of live yeasts after culturing for 4 hours without pulse electric field treatment, More live yeast numbers were confirmed.
　しかしながら、電界強度を20～25kV/cmとし、投入エネルギーを1J/ml～10J/mlとした場合には、パルス電界処理を行わず４時間培養した後の生酵母数に比して、同等かそれ以下の生酵母数しか確認されなかった。 However, when the electric field strength is 20 to 25 kV / cm and the input energy is 1 J / ml to 10 J / ml, is it equivalent to the number of live yeasts after 4 hours of culture without pulsed electric field treatment? Only live yeast counts below that were confirmed.
　これらの結果から、酵母に対して所定の電界強度にて刺激を与える際の発現刺激好適範囲は、電界強度が5～15kV/cmで投入エネルギーが0.01J/ml～10J/ml、又は電界強度が15～25kV/cmで投入エネルギーが0.01J/ml～1J/mlであることが示唆された。 From these results, the preferred range of expression stimulation when stimulating yeast with a predetermined electric field strength is 5 to 15 kV / cm and input energy is 0.01 J / ml to 10 J / ml, or electric field strength. Is 15 to 25 kV / cm and the input energy is 0.01 J / ml to 1 J / ml.
〔パルス電界処理後の生酵母数の経時変化〕
　次に、パルス電界処理を行った酵母が培養中において、生酵母数がどのように経時変化するかについて検証を行った。
　まず、パルス電界処理を施すための酵母の培養を行った。15ml容量の培養瓶に10mlのYPD液体培地を分注した。このYPD液体培地は、1000mlの蒸留水に、10ｇのBacto Yeast Extract、20ｇのBacto Yest Pepton、20ｇのD-(+)-Glucoseを溶解し、オートクレーブにて120℃20分間殺菌処理したものを用いた。 [Changes in the number of live yeasts after pulse electric field treatment]
Next, it was verified how the number of viable yeast changed with time during the cultivation of the yeast subjected to the pulse electric field treatment.
First, yeast was cultured for pulse electric field treatment. 10 ml of YPD liquid medium was dispensed into a 15 ml culture bottle. This YPD liquid medium is prepared by dissolving 10 g of Bacto Yeast Extract, 20 g of Bacto Yest Pepton, and 20 g of D-(+)-Glucose in 1000 ml of distilled water and sterilizing at 120 ° C for 20 minutes in an autoclave. It was.
　次に、平板寒天培地上にて単一コロニーを形成している前述の酵母を、培養瓶中の培地に一白金耳植菌し、TAITEC社製振盪培養器を用い32℃にて24時間160rpmで振盪培養を行った。 Next, the above-mentioned yeast forming a single colony on a flat plate agar medium is inoculated with one platinum ear in the medium in the culture bottle, and 160 rpm for 24 hours at 32 ° C. using a shaking incubator manufactured by TAITEC. Incubated with shaking.
　このようにして得られた酵母培養液（1×105 cfu/ml）を滅菌済みのマイクロピペットで0.8ml採取し、キューベットに分注してパルス電界処理に供した。 0.8 ml of the thus obtained yeast culture solution (1 × 10 5 cfu / ml) was collected with a sterilized micropipette, dispensed into a cuvette, and subjected to pulsed electric field treatment.
　本試験では、パルス電界処理における電界強度は10kV/cmに固定し、０ショット（コントロール）、３ショット、１０ショット、３０ショット、１００ショット、２００ショットの６種類の投入エネルギーの異なるサンプルを作成した。 In this test, the electric field strength in the pulse electric field treatment was fixed at 10 kV / cm, and six types of samples with different input energies were created: 0 shot (control), 3 shots, 10 shots, 30 shots, 100 shots, and 200 shots. .
　次いで、パルス電界処理を施した酵母を、キューベット中から滅菌ピペットにて0.5ml取りだし、4.5mlのYPD液体培地を分注した15ml容量の培養瓶に接種し、TAITEC社製振盪培養器を用い32℃にて24時間160rpmでそれぞれ振盪培養を行いつつ、生酵母数の経時変化を観察した。生酵母数の測定は、パルス電界処理直後（０ｈ）、４時間培養後、１２時間培養後、１６時間培養後において行った。その結果を図９に示す。 Next, 0.5 ml of yeast subjected to pulsed electric field treatment is taken out from the cuvette with a sterile pipette, inoculated into a 15 ml culture bottle into which 4.5 ml of YPD liquid medium has been dispensed, and then used with a shake incubator manufactured by TAITEC. While performing shaking culture at 32 ° C. for 24 hours at 160 rpm, the time course of the number of live yeasts was observed. The number of viable yeast was measured immediately after the pulse electric field treatment (0 h), after 4 hours of culture, after 12 hours of culture, and after 16 hours of culture. The result is shown in FIG.
　図９からも分かるように、パルス電界処理直後の細胞はいずれのサンプルも同程度の生酵母数であったが、４時間培養を行った時点では、パルス電界処理を行ったサンプルはいずれも、コントロールに比して増殖速度の向上が見られた。また培養１２時間後では、コントロールに対する生酵母数の差が維持されていた。また、培養１６時間後では、コントロールに対する生酵母数の差がやや縮小する傾向が観察された。 As can be seen from FIG. 9, the cells immediately after the pulse electric field treatment had the same number of live yeasts in all samples, but at the time of culturing for 4 hours, all the samples subjected to the pulse electric field treatment were The growth rate was improved compared to the control. Moreover, after 12 hours of culture, the difference in the number of live yeasts relative to the control was maintained. In addition, after 16 hours of culture, a tendency was observed that the difference in the number of live yeasts with respect to the control was slightly reduced.
　これらの結果から、パルス電界処理を施した細胞（酵母）は、パルス電界処理を施していないコントロールの細胞（酵母）に比べ、処理後０～４時間という比較的早期に増殖速度を増加できることが示された。すなわち、酵母に対してパルス電界処理を施すことにより、非通常時形質として、増殖速度の増加を発現させることができた。 From these results, it can be seen that cells (yeast) subjected to pulsed electric field treatment can increase the growth rate relatively early, 0 to 4 hours after treatment, compared to control cells (yeast) not subjected to pulsed electric field treatment. Indicated. That is, by applying a pulsed electric field treatment to yeast, an increase in the growth rate could be expressed as an unusual trait.
　上述してきたように、本実施形態に係る細胞改変方法によれば、一対の電極間に印加したパルス電圧により生じる電界中に細胞を暴露することにより、例えば増殖速度など細胞の性質を変化させることができる。 As described above, according to the cell modification method according to the present embodiment, by exposing the cell to an electric field generated by a pulse voltage applied between a pair of electrodes, for example, changing a cell property such as a growth rate. Can do.
　最後に、上述した各実施の形態の説明は本発明の一例であり、本発明は上述の実施の形態に限定されることはない。このため、上述した各実施の形態以外であっても、本発明に係る技術的思想を逸脱しない範囲であれば、設計等に応じて種々の変更が可能であることは勿論である。 Finally, the description of each embodiment described above is an example of the present invention, and the present invention is not limited to the above-described embodiment. For this reason, it is a matter of course that various modifications can be made in accordance with the design and the like as long as they do not depart from the technical idea according to the present invention other than the embodiments described above.
１０　電源
１１　電圧調整手段
１２　パルス電圧発生手段
１３　制御手段
１４　入力手段
２０　狭隘部
２１　培地
２３　電極
２４　細胞
Ａ　パルスパワー発生装置
Ｂ　キューベット
Ｃ　パルスパワー印加装置 DESCRIPTION OF SYMBOLS 10 Power supply 11 Voltage adjustment means 12 Pulse voltage generation means 13 Control means 14 Input means 20 Narrow part 21 Medium 23 Electrode 24 Cell A Pulse power generator B Cuvette C Pulse power application apparatus

Claims (5)

  1. 　一対の電極間に印加したパルス電圧により生じる電界中に細胞を暴露し、同細胞の性質を変化させる細胞改変方法。 A cell modification method in which cells are exposed to an electric field generated by a pulse voltage applied between a pair of electrodes to change the properties of the cells.
  2. 　前記パルス電圧の印加時間は10nsec～300nsecであることを特徴とする請求項１に記載の細胞改変方法。 2. The cell modification method according to claim 1, wherein the application time of the pulse voltage is 10 nsec to 300 nsec.
  3. 　前記電界の強度は、1kV/cm～30kV/cmであることを特徴とする請求項１又は請求項２に記載の細胞改変方法。 3. The cell modification method according to claim 1, wherein the intensity of the electric field is 1 kV / cm to 30 kV / cm.
  4. 　前記細胞は酵母であることを特徴とする請求項１～３いずれか1項に記載の細胞改変方法。 The cell modification method according to any one of claims 1 to 3, wherein the cell is yeast.
  5. 　前記性質は、細胞の増殖速度であることを特徴とする請求項１～４いずれか1項に記載の細胞改変方法。 The cell modification method according to any one of claims 1 to 4, wherein the property is a cell growth rate.
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