JP6481296B2 - Eukaryotic cell growth method and eukaryotic cell production method - Google Patents

Description

  The technical field of the present specification relates to a method for growing a eukaryotic cell and a method for producing a eukaryotic cell. More particularly, the present invention relates to a method for growing eukaryotic cells and a method for producing eukaryotic cells using plasma.

  Plasma technology is applied in the fields of electricity, chemistry, and materials. Inside the plasma, neutral particles such as atoms and molecules and ultraviolet rays are generated in addition to charged particles such as electrons and ions. Of these products generated in the plasma, particles having unpaired electrons (including atoms, molecules and ions) are called radicals. Such ultraviolet rays and radicals are known to have a bactericidal effect.

  For example, Patent Document 1 discloses a plasma sterilization apparatus and a plasma sterilization method for sterilizing an object to be sterilized by supplying plasma into a sterilization chamber. Thus, it is widely known that plasma products generated from plasma have a bactericidal action.

JP 2004-357888 A

  The present inventors have discovered that eukaryotic cells are proliferated by irradiating eukaryotic cells with plasma products generated inside non-equilibrium atmospheric pressure plasma for a short time. This is a result opposite to the conventional technique described above, and is an effect that cannot be predicted from the conventional technique.

  The technique of this specification has been made to solve the problems of the conventional techniques described above. That is, the subject is to provide a method for eukaryotic cell proliferation and a method for producing eukaryotic cells that efficiently proliferate eukaryotic cells using plasma.

The eukaryotic cell growth method according to the first aspect includes a radical irradiation step of irradiating eukaryotic cells with radicals. In the radical irradiation step, eukaryotic cells are proliferated by periodically irradiating eukaryotic cells with radicals in a first period , with the irradiation amount of radicals being equal to or less than a predetermined first irradiation amount. The first cycle is synchronized with the G1 phase in the cell cycle of eukaryotic cells.

  In this eukaryotic cell proliferation method, eukaryotic cells can be propagated by irradiating the eukaryotic cells with radicals generated from plasma generated in the plasma generation region by the plasma generator. In this eukaryotic cell growth method, the eukaryotic cell growth rate is faster than the normal eukaryotic cell culture method.

The method of proliferating eukaryotic cells in a second aspect includes a step of stopping while eukaryotic cells G1 phase, and a radical irradiation step of irradiating a radical eukaryotic cells, a. In the radical irradiation step, with the eukaryotic cells stopped in the G1 phase, the radical irradiation amount is set to a predetermined first irradiation amount or less to irradiate the eukaryotic cells to proliferate the eukaryotic cells. .

In the eukaryotic cell growth method according to the third aspect, in the radical irradiation step, at least one of a triplet oxygen atom and a singlet oxygen molecule is irradiated to the eukaryotic cell.

Here, the triplet oxygen atom is O ( ³ P _j ). As will be described later, the triplet oxygen atom irradiation amount includes an area irradiation amount and a volume irradiation amount. The area irradiation amount is the number of triplet oxygen atoms per unit area irradiated to the irradiated region. The volume irradiation amount is the number of triplet oxygen atoms supplied to the volume of the suspension containing eukaryotic cells. Therefore, when irradiating triplet oxygen atoms to eukaryotic cells arranged on a flat surface like an agar medium, the area irradiation amount is used. On the other hand, when irradiating triplet oxygen atoms to eukaryotic cells suspended in a solution having a volume such as a suspension, the volume irradiation amount is used. The singlet oxygen molecule is O ₂ ( ¹ Δ _g ).

The method for producing eukaryotic cells in the fourth aspect includes a suspension production step for producing a suspension in which eukaryotic cells are suspended. In the radical irradiation step, the suspension is irradiated with triplet oxygen atoms. The first irradiation dose is a first volume irradiation dose that is the number of triplet oxygen atoms supplied to the suspension volume. The first volume dose is 3.0 × 10 ¹⁷ cm ⁻³ .

In the method for producing eukaryotic cells in the fifth aspect, the number of eukaryotic cells is increased using the above-described eukaryotic cell growth method.

  In the present specification, there are provided a eukaryotic cell growth method and a eukaryotic cell production method for efficiently proliferating eukaryotic cells using plasma.

It is a figure which shows schematic structure of the plasma generator which concerns on 1st Embodiment. It is a perspective view which shows the irradiation part of the plasma generator which concerns on 1st Embodiment. It is a figure which shows the internal structure of the plasma generator which concerns on 1st Embodiment. It is a figure for demonstrating the cell cycle of a eukaryotic cell. It is a figure which shows schematic structure of the plasma generator used for experiment. It is a graph which shows the relationship between irradiation distance and the density of a triplet oxygen atom. It is a graph which shows the relationship between the irradiation distance of a radical, and radical density. It is a graph which shows the relationship between the mixing ratio of oxygen gas in plasma gas, and radical density. It is a graph which shows the relationship between the irradiation time of a radical, and the growth rate of budding yeast. It is a graph which shows the relationship between the irradiation time of a radical, and the growth rate of budding yeast in suspension. It is a graph which shows the relationship between the volume irradiation amount of a radical, and the change rate of the cell number of budding yeast in suspension. It is a graph which shows irradiation distance dependence about the relationship between the irradiation time of a radical, and the growth rate of budding yeast in suspension. It is a graph for comparing the survival number of the cell at the time of changing the cell cycle of yeast at the time of radical irradiation and radical irradiation time. It is a graph which shows the relationship between the volume irradiation amount of a radical, and the change rate of the cell number of a mouse | mouth fetal skin cell (NIH3T3).

  Hereinafter, specific embodiments will be described with reference to the drawings, taking eukaryotic cell growth methods and eukaryotic cell production methods as examples.

(First embodiment)
1. Plasma generator 1-1. Configuration of Entire Apparatus A plasma generator 100 used in the eukaryotic cell growth method and eukaryotic cell production method of the present embodiment will be described. The plasma generator 100 is a device that generates non-equilibrium atmospheric pressure plasma. FIG. 1 is a diagram showing a schematic configuration of a plasma generator 100. As shown in FIG. 1, the plasma generation apparatus 100 includes a chamber 110, a mounting table 120, a gas supply unit 130, a gas discharge unit 140, a plastic cover 150, and a radical irradiation unit 200. .

  The chamber 110 is for containing the radical irradiation unit 200 and for containing a gas cut off from the atmosphere. The mounting table 120 is a table for mounting a sample to be irradiated with plasma. The mounting table 120 can slide in a direction perpendicular to the plasma irradiation direction. Therefore, when the object is irradiated with the plasma product, the object can be irradiated with the plasma product evenly.

  The gas supply unit 130 is for supplying gas into the chamber 110. The gas discharge unit 140 is for discharging gas from the inside of the chamber 110. The plastic cover 150 is for preventing air from entering the inside of the plastic cover 150 during irradiation with radicals. Therefore, radicals can be suitably irradiated onto the suspension in a state where the influence of the external atmosphere is eliminated.

  The radical irradiation unit 200 is for irradiating radicals among plasma products generated in the plasma generation region. Here, the plasma product means chemical species generated in the plasma generation region. That is, examples of the plasma product include ions, electrons, radicals, and light. The radical irradiation unit 200 irradiates radicals among these plasma products. Specifically, the radical irradiation unit 200 irradiates triplet oxygen atoms and singlet oxygen molecules. Moreover, the radical irradiation part 200 may irradiate ozone. As will be described later, the radical irradiation unit 200 does not irradiate light such as ultraviolet rays. The radical irradiation unit 200 does not irradiate electrons or ions.

  Here, the radical is a neutral particle having unpaired electrons. Strictly speaking, the triplet oxygen atom has no unpaired electrons. However, in this specification, the radical includes a triplet oxygen atom and a singlet oxygen molecule.

  As shown in FIG. 1, the radical irradiation unit 200 includes an irradiation port 210, a plasma gas supply unit 220, a power supply unit 230, and a robot arm 240. The irradiation port 210 is for irradiating the sample with radicals. The plasma gas supply unit 220 is for supplying plasma gas to the radical irradiation unit 200. The power supply unit 230 is for supplying power to each part of the radical irradiation unit 200. The robot arm 240 is for moving the radical irradiation unit 200.

  FIG. 2 is a perspective view showing the irradiation port 210. The irradiation port 210 has two slits 211. The slit 211 is an opening having a length of 16 mm and a width of 0.5 mm. Radicals are irradiated from the slit 211. The irradiation port 210 can move in the width direction of the slit 211. This is because the sample is irradiated with radicals evenly.

1-2. Radiation Irradiation Unit FIG. 3 is a diagram showing the internal structure of the radical irradiation unit 200. The radical irradiation unit 200 has a discharge unit 250, an intermediate structure unit 260, and a nozzle unit 270 in addition to the irradiation port 210.

  The discharge unit 250 has a plasma generation region therein. Therefore, the discharge part 250 has an opposing electrode pair. Then, plasma is generated in the space between the electrode pair. The plasma contains ions, electrons, radicals, ultraviolet rays and the like.

  The intermediate structure 260 is a structure that removes ions, electrons, and ultraviolet rays from the plasma. Therefore, among the particles generated from the plasma, neutral particles containing radicals are supplied to the nozzle unit 270.

  The nozzle unit 270 is for sending neutral particles containing radicals to the slit 211 of the irradiation port 210. That is, the radical irradiation part 200 of this embodiment sprays neutral particles among plasma products on a sample. The neutral particles contain radicals and argon atoms.

2. Radiation dose 2-1. Area irradiation amount and volume irradiation amount Radiation irradiation amount includes area irradiation amount and volume irradiation amount. The area irradiation amount is used when eukaryotic cells arranged on a flat surface are directly irradiated with radicals. Volume irradiation is used when eukaryotic cells in a liquid are irradiated with radicals. The volume irradiation amount will be described later.

2-2. Definition of area irradiation amount Here, the area irradiation amount of a radical is represented by the following equation.
DV = RD * V1 * ET * S1 / S2
DV: Radiation area dose (cm ^-2 )
RD: radical density (cm ⁻³ )
V1: Radical flow rate (m / sec)
ET: Radical irradiation time (sec)
S1: Area of irradiation port (cm ² )
S2: Area of the irradiated region (cm ² )
The area irradiation amount is the number of triplet oxygen atoms per unit area irradiated to the irradiated region. Here, the radical density RD is the density of triplet oxygen atoms.

The radical flux is expressed by the following equation.
F1 = RD x V1
F1: Flux (cm ^-2 / sec)

3. Method for Propagating Eukaryotic Cells by Radiation Irradiation The method for propagating eukaryotic cells in the present embodiment has a radical irradiation step of irradiating eukaryotes with radicals generated by the plasma generator 100. Then, in the radical irradiation step, the eukaryotic cell is propagated by irradiating the eukaryotic cell with the radical irradiation amount set to a predetermined first irradiation amount or less. In the radical irradiation step, eukaryotic cells are irradiated with at least one of triplet oxygen atoms and singlet oxygen molecules. In practice, eukaryotic cells are irradiated with both triplet oxygen atoms and singlet oxygen molecules. Moreover, it is good also as irradiating ozone.

3-1. Eukaryotic cell preparation process First, eukaryotic cells are prepared. For example, eukaryotic cells are cultured on an agar medium. Alternatively, eukaryotic cells are placed on other flat surfaces.

3-2. Radical irradiation process Next, the plasma generator 100 irradiates radicals toward the eukaryotic cells of the agar medium. In this embodiment, eukaryotic cells are irradiated with triplet oxygen atoms of 3.8 × 10 ¹⁷ cm ⁻² or less. Here, the first area irradiation dose is 3.8 × 10 ¹⁷ cm ⁻² . Thereby, eukaryotic cells proliferate. In this irradiation, eukaryotic cells are also irradiated with singlet oxygen molecules.

4). Effect of this Embodiment When a eukaryotic cell is irradiated with a radical containing a triplet oxygen atom and a singlet oxygen molecule by a first irradiation dose or less, the eukaryotic cell grows. Note that when eukaryotic cells are irradiated with radicals by an amount larger than the first irradiation amount, the eukaryotic cells decrease. The reason why eukaryotic cells grow or decrease depending on the amount of radical irradiation is not always clear. The experimental results will be described later.

5. Eukaryotic Cell Production Method Eukaryotic cells can be produced using the eukaryotic cell growth method described above. That is, it is possible to increase the number of eukaryotic cells and increase the production of eukaryotic cells in a short time.

6). Summary of this embodiment As described in detail above, the eukaryotic cell growth method of this embodiment irradiates eukaryotic cells with radicals of 3.8 × 10 ¹⁷ cm ⁻² or less as the first area irradiation dose. To proliferate eukaryotic cells.

  This embodiment is merely an example. Therefore, naturally, various improvements and modifications can be made without departing from the scope of the invention. For example, the gas that generates plasma is not limited to argon. Other rare gases may be used.

  Further, it is more preferable that the plasma conditions in the plasma irradiation apparatus are fed back by using vacuum ultraviolet absorption spectroscopy. Thereby, an electron density, gas temperature, and radical density can be adjusted.

(Second Embodiment)
A second embodiment will be described. In the present embodiment, similarly to the first embodiment, the eukaryotic cells are irradiated with radicals. However, in this embodiment, radicals are irradiated to a suspension in which eukaryotic cells are suspended. Therefore, in this embodiment, radicals supplied to the suspension act on eukaryotic cells in the liquid.

1. Radiation dose 1-1. Area irradiation amount and volume irradiation amount Radiation irradiation amount includes area irradiation amount and volume irradiation amount. Volume irradiation is used when eukaryotic cells in a liquid are irradiated with radicals.

1-2. Definition of Volume Irradiation Amount The volume irradiation amount of radicals supplied to a liquid is expressed by the following equation.
SV = RD × V1 × ET × S1 / C1
SV: Radical volume irradiation (cm ^-3 )
RD: radical density (cm ⁻³ )
V1: Radical flow rate (m / sec)
ET: Radical irradiation time (sec)
S1: Area of irradiation port (cm ² )
C1: Volume of suspension (cm ³ )
The volume irradiation amount is the number of triplet oxygen atoms supplied to the volume of the liquid (suspension) containing eukaryotic cells. Here, the radical density RD is the density of triplet oxygen atoms.

2. Method for Propagating Eukaryotic Cells by Radiation Irradiation The method for propagating eukaryotic cells in the present embodiment has a radical irradiation step of irradiating eukaryotes with radicals generated by the plasma generator 100. Then, in the radical irradiation step, the eukaryotic cell is propagated by irradiating the eukaryotic cell with the radical irradiation amount set to a predetermined first irradiation amount or less. In the radical irradiation step, eukaryotic cells are irradiated with at least one of triplet oxygen atoms and singlet oxygen molecules. In practice, eukaryotic cells are irradiated with both triplet oxygen atoms and singlet oxygen molecules. At that time, the eukaryotic cells are dispersed in the suspension.

2-1. Suspension preparation process Eukaryotic cells are cultured in an agar medium. Then, eukaryotic cells are taken out from the agar medium and suspended in phosphate buffered saline (PBS (−)). In this way, a suspension in which eukaryotic cells are suspended is prepared.

2-2. Radical irradiation process Next, the plasma generator 100 irradiates radicals toward the eukaryotic cells of the suspension. Here, the eucaryotic cell is irradiated with triplet oxygen atoms equal to or less than the first irradiation amount. The first irradiation dose is a first volume irradiation dose that is the number of triplet oxygen atoms supplied to the suspension volume. The first volume dose is 3.0 × 10 ¹⁷ cm ⁻³ . Thereby, eukaryotic cells proliferate. In this irradiation, eukaryotic cells are also irradiated with singlet oxygen molecules.

3. Effect of this Embodiment When a eukaryotic cell is irradiated with a radical containing a triplet oxygen atom and a singlet oxygen molecule by a first irradiation dose or less, the eukaryotic cell grows. Note that when eukaryotic cells are irradiated with radicals by an amount larger than the first irradiation amount, the eukaryotic cells decrease. The reason why eukaryotic cells grow or decrease depending on the amount of radical irradiation is not always clear. The experimental results will be described later.

4). Eukaryotic Cell Production Method Eukaryotic cells can be produced using the eukaryotic cell growth method described above. That is, it is possible to increase the number of eukaryotic cells and increase the production of eukaryotic cells in a short time.

5. Modified example 5-1. Suspension method Or collect cells from eukaryotic cells cultured in liquid using a centrifuge. Then, these eukaryotic cells may be suspended in 3 ml of phosphate buffered saline (PBS (−)).

6). Summary of the present embodiment As described in detail above, the eukaryotic cell growth method of the present embodiment includes eukaryotic cells with radicals of 3.0 × 10 ¹⁷ cm ⁻³ or less as the first volume dose. Eukaryotic cells are grown by irradiating the suspension.

  This embodiment is merely an example. Therefore, naturally, various improvements and modifications can be made without departing from the scope of the invention. For example, the gas that generates plasma is not limited to argon. Other rare gases may be used.

  Further, it is more preferable that the plasma conditions in the plasma irradiation apparatus are fed back by using vacuum ultraviolet absorption spectroscopy. Thereby, an electron density, gas temperature, and radical density can be adjusted.

(Third embodiment)
A third embodiment will be described. In the present embodiment, as in the second embodiment, eukaryotic cells in the suspension are irradiated with radicals. Differences from the second embodiment will be described.

1. Eukaryotic Cell and Cell Cycle FIG. 4 shows the cell cycle of eukaryotic cell division. All eukaryotic cells cause cell division by the cell cycle shown in FIG. That is, the cell cycle shown in FIG. 4 is an event common to all eukaryotic cells.

  As shown in FIG. 4, the cell cycle has a G1 phase, an S phase, a G2 phase, and an M phase in this order. The S phase is a period for replicating DNA. The M phase is a period for equally dividing the doubled DNA and intracellular organelles. The G1 period and the G2 period are periods located between them.

  If there are many eukaryotic cells in a container, those eukaryotic cells are generally in different cell cycles. For example, among eukaryotic cells in the container, one eukaryotic cell has reached the G1 phase, and another eukaryotic cell has reached the M phase.

2. Eukaryotic Cell Proliferation Method by Periodic Radiation Irradiation Here, the eukaryotic cell growth method in this embodiment will be described. In the radical irradiation process of this embodiment, radicals are periodically irradiated to eukaryotic cells in the first period. This first cycle is synchronized with the G1 phase in the cell cycle of eukaryotic cells. Thus, when the eukaryotic cell is in the G1 phase, the eukaryotic cell can be irradiated with radicals. As will be described later, among eukaryotic cells, those in the G1 phase proliferate, and those not in the G1 phase slightly decrease. As a result, as a whole, eukaryotic cells proliferate faster than usual.

2-1. Suspension preparation process Eukaryotic cells are cultured in an agar medium. Then, eukaryotic cells are taken out from the agar medium and suspended in phosphate buffered saline (PBS (−)). In this way, a suspension in which eukaryotic cells are suspended is prepared.

2-2. Radical irradiation process Next, the plasma generator 100 irradiates radicals toward the eukaryotic cells of the suspension. Here, the eucaryotic cell is irradiated with triplet oxygen atoms equal to or less than the first irradiation amount. The first irradiation dose is a first volume irradiation dose that is the number of triplet oxygen atoms supplied to the suspension volume. The first volume dose is 3.0 × 10 ¹⁷ cm ⁻³ . Thereby, eukaryotic cells proliferate. In this irradiation, eukaryotic cells are also irradiated with singlet oxygen molecules.

  Here, radical irradiation is periodically performed. That is, the eukaryotic cell is periodically irradiated with radicals in the first cycle. This first cycle is synchronized with the G1 phase in the cell cycle of eukaryotic cells. Therefore, the first cycle is an average value of the cell cycle of the eukaryotic cell. The first cycle may be in the range of 80% to 120% of the average value of the cell cycle of the eukaryotic cell. Preferably, it is in the range of 90% to 110% of the average value of the cell cycle of the eukaryotic cell. More preferably, it is in the range of 95% to 105% of the average value of the cell cycle of the eukaryotic cell. Further, the radical irradiation time may be within a period corresponding to the G1 period. That is, it may be at least a part of the G1 period.

3. Effects of this embodiment As will be described later, eukaryotic cells proliferate when irradiated with radicals having a dose equal to or less than the first dose in the G1 phase. Eukaryotic cells decrease when they are irradiated with an amount of radicals greater than the first dose during the G1 phase. In the S phase and M phase, eukaryotic cells slightly decrease when irradiated with radicals of the first dose or less.

  Therefore, eukaryotic cells proliferate if radicals are periodically irradiated for a time corresponding to the period of G1 phase. When a radical or the like in which a large number of eukaryotic cells are cultured is irradiated with radicals, the eukaryotic cells corresponding to the G1, S, G2, and M phases are irradiated with radicals. Therefore, proliferation of the eukaryotic cells in the G1 phase is promoted. S, G2 and M eukaryotic cells are slightly reduced. Combined with these proliferating and slightly decreasing effects, eukaryotic cells proliferate as a whole.

4). Eukaryotic Cell Production Method Eukaryotic cells can be produced using the eukaryotic cell growth method described above. That is, it is possible to increase the number of eukaryotic cells and increase the production of eukaryotic cells in a short time.

5. Summary of the present embodiment As described in detail above, in the eukaryotic cell growth method of the present embodiment, the eukaryotic cells are periodically irradiated with radicals in accordance with the G1 phase of the eukaryotic cells. Thereby, a eukaryotic cell can be propagated suitably.

(Effects from the first embodiment to the third embodiment)
As described in the above embodiment, eukaryotes can be grown by irradiating radicals. A eukaryote is an organism having eukaryotic cells. Eukaryotic cells include animal cells and plant cells.

  By using the eukaryotic cell growth method described in the above embodiment, it is possible to efficiently increase the production of useful organisms used in agriculture, food, medicine, medicine and the like.

A. Measurement of radicals A-1. Measurement Device Before describing the relationship between radicals and eukaryotic cells, the relationship between the plasma generator 100 and radicals irradiated from the plasma generator 100 will be described. FIG. 5 is a diagram showing a schematic configuration of the plasma generator 300 used in this experiment. The plasma generator 300 used in this experiment is obtained by adding a vacuum ultraviolet absorption spectrometer 350 for measuring neutral particles such as radicals to the plasma generator 100 described in FIG. The plastic cover 150 is omitted in FIG. Further, the description of the configuration already described in the plasma generator 100 is omitted.

The vacuum ultraviolet absorption spectrometer 350 includes a vacuum ultraviolet lamp 310, an MgF ₂ window 311, an exhaust port 312, a photomultiplier tube 320, an MgF ₂ window 321, and an exhaust port 322. The vacuum ultraviolet absorption spectrometer 350 absorbs the light emitted from the vacuum ultraviolet lamp 310 with the absorption length L between the MgF ₂ window 311 and the MgF ₂ window 321, and the absorption detected by the photomultiplier tube 320. This is for identifying the type of radical or the like by analyzing the spectrum. The radical density can be measured from the intensity of the absorption spectrum.

A-2. Plasma Conditions Here, conditions used in the experiment will be described. As shown in Table 1, the density of the plasma generated in the plasma generation region of the discharge unit 250 was 2 × 10 ¹⁶ cm ⁻³ . The irradiation distance, that is, the distance from the irradiation port 211 to the sample was 10 mm. The density of triplet oxygen atoms at the irradiation distance was 2.25 × 10 ¹⁴ cm ⁻³ . The radical flow rate at the irradiation distance was 10.4 m / s.

A mixed gas of Ar and oxygen gas was used as the plasma gas. The total gas flow rate in this plasma gas was 5.0 slm. The content of O ₂ contained in the plasma gas was 0.6%. In addition, Ar gas was supplied as atmospheric gas. Therefore, only radicals containing oxygen atoms are generated. However, Ar gas and the like are excluded. For example, those containing nitrogen atoms are not generated. That is, the generated radicals are a triplet oxygen atom radical (O ( ³ P _j )) and a singlet oxygen molecular radical (O ₂ ( ¹ Δ _g )). Ozone may also be generated.

[Table 1]
Plasma density (generation area) 2 × 10 ¹⁶ cm ^-3
Irradiation distance 10mm
Triplet oxygen atom density (irradiation region) 2.25 × 10 ¹⁴ cm ⁻³
Flow velocity (irradiation area) 10.4 m / s
Plasma gas Ar + O ₂
Total flow rate 5.0 / min
O ₂ content 0.6%
Atmospheric gas Ar gas

A-3. Radical FIG. 6 is a graph showing the relationship between the irradiation distance from the slit 211 to the sample and the density of triplet oxygen atoms. As shown in FIG. 6, the density of triplet oxygen atoms decreases exponentially as the irradiation distance increases. When the irradiation distance from the slit 211 to the sample is 10 mm, the density of triplet oxygen atoms is 2.25 × 10 ¹⁴ cm ⁻³ .

FIG. 7 is a graph showing the relationship between radical irradiation distance and radical density. The horizontal axis in FIG. 7 is the radical irradiation distance. One of the vertical axes in FIG. 7 is the radical density.
The other of the vertical axis | shafts of FIG. 7 is D value. The D value will be described later. The density of the triplet oxygen atom decreases exponentially as the radical irradiation distance increases. On the other hand, the density of singlet oxygen molecules remains almost constant even when the irradiation distance of radicals is increased.

FIG. 8 is a graph showing the relationship between the O ₂ content in the plasma gas of Table 1 and the radical density. The horizontal axis in FIG. 8 represents the oxygen concentration in the plasma gas. One of the vertical axes in FIG. 8 is the radical density. The other of the vertical axis | shafts of FIG. 8 is D value. The D value will be described later. As shown in FIG. 8, the triplet oxygen atom concentration is the highest when the oxygen concentration is around 0.6%. Therefore, as shown in Table 1, when the oxygen concentration is about 0.6%, the sample can be irradiated with neutral particles having a high density of triplet oxygen atoms. Also, the higher the triplet oxygen atom concentration, the lower the D value.

  Here, the D value represents the time during which the number of bacteria is 10% or less of the initial value by continuing to irradiate plasma. The smaller this value, the stronger the bactericidal effect. In FIG. 7, the higher the triplet oxygen atom density, the smaller the D value. Thus, it is thought that the bactericidal effect of budding yeast originates from triplet oxygen atoms. As shown in FIG. 8, the higher the density of triplet oxygen atoms, the smaller the D value. That is, the bactericidal effect is high.

B. Budding yeast 1
Hereinafter, the relationship between radicals and eukaryotic cells will be described.

B-1. Experimental Procedure First, a method for producing a sample will be described. An agar medium was prepared in a petri dish having a diameter of 90 mm. This agar medium contained yeast extract, peptone, and glucose. Then, budding yeast was cultured on this agar medium. Saccharomyces cerevisiae is one of eukaryotic cells. The colonies thus formed were taken out with a toothpick and suspended in 3 ml of phosphate buffered saline (PBS (−)). The concentration of the cultured budding yeast was in the range of 1000000 cells / ml or more and 3000000 cells / ml or less.

  Alternatively, cells were collected from budding yeast that had been cultured in liquid using a centrifuge. These budding yeasts were suspended in 3 ml of phosphate buffered saline (PBS (−)).

Next, these samples are irradiated with plasma under the above conditions for a predetermined time. Then, the number of budding yeast was counted. At that time, a hemocytometer was used. The viable cell count of the budding yeast was evaluated by the amount of change in the viable cell rate represented by the following formula (1).
Amount of change in viability rate (%) = (N−N ₀ ) / N ₀ × 100 (1)
N: Viable count of the sample irradiated with radicals
N ₀ : Number of viable bacteria in the sample not irradiated with radicals

  Therefore, if the variation | change_quantity of the viable cell rate of Formula (1) is a positive value, it has shown that the number of budding yeast increased by irradiating a radical. Conversely, if the amount of change in the viable cell rate is a negative value, it indicates that the number of budding yeast cells has decreased by irradiation with radicals.

B-2. Experimental results The experimental results are shown in FIG. The horizontal axis in FIG. 9 represents the time when the sample was irradiated with plasma. The vertical axis | shaft of FIG. 9 is the variation | change_quantity of the viable cell rate of Formula (1) in that case. For example, when the amount of change in the viable cell rate is 10%, it is suggested that the number of budding yeast increased by 10% by irradiation with radicals.

  As shown in FIG. 9, the number of budding yeasts increases as the radical irradiation time increases from 30 seconds to 90 seconds. On the other hand, the number of budding yeasts decreases as the radical irradiation time increases from 120 seconds to 180 seconds. Thus, the number of budding yeast increases when the area irradiation amount of radicals is small, and the number of budding yeast decreases when the area irradiation amount of radicals is large. To do.

From FIG. 9, the budding yeast is proliferating when the radical irradiation time is 90 seconds or less. The density of triplet oxygen atoms at this time is 2.25 × 10 ¹⁴ cm ⁻³ . The flow rate is 10.4 m / sec. Therefore, the budding yeast is proliferating when the area irradiation amount of triplet oxygen atoms is 3.8 × 10 ¹⁷ cm ⁻² or less when these are multiplied. Within this range, the growth rate of budding yeast is about 10%.

On the other hand, when the radical irradiation time is 120 seconds or more, budding yeast is decreased. The density of triplet oxygen atoms at this time is 2.25 × 10 ¹⁴ cm ⁻³ . The flow rate is 10.4 m / sec. Therefore, when the area irradiation amount of triplet oxygen atoms is 2.8 × 10 ¹⁹ cm ⁻² or more when multiplied by these, budding yeast is decreased.

C. Budding yeast 2
C-1. Experimental Procedure The experimental conditions are almost the same as the experimental conditions used in Experiment B.

  First, yeast was cultured in YPD medium. Thereafter, the cells were collected, and the budding yeast was suspended in 3 ml of phosphate buffered saline (PBS (−)). The plastic cover 150 was used to irradiate the suspension with plasma after eliminating the influence of the surrounding atmosphere. Thereafter, the yeast was cultured again in YPD medium. Thereafter, the number of viable yeast was counted every 24 hours.

C-2. Experimental Results FIG. 10 is a graph showing the number of viable yeast cells with respect to the plasma irradiation time. The horizontal axis in FIG. 10 is the radical irradiation time. The vertical axis | shaft of FIG. 10 is the variation | change_quantity of the viable cell rate of Formula (1). Here, the viable cell rate is calculated from the viable cell count after 48 hours from the irradiation of radicals. Region L1 in FIG. 10 shows a case where the number of viable yeast is larger than usual. Region L2 shows the case where the number of viable yeast is less than usual.

  In FIG. 10, the same tendency as in FIG. 9 is observed. That is, when the radical irradiation is small, the number of viable yeast is large, and when the radical irradiation is large, the number of live yeast is small.

  FIG. 11 is a graph showing the change rate of the number of cells with respect to the volume irradiation amount of radicals. In drawing this graph, the data of FIG. 10 was used. The horizontal axis in FIG. 11 represents the volume irradiation amount of radicals. The vertical axis | shaft of FIG. 11 is the viable cell rate of the yeast compared with the case where a radical is not irradiated.

As shown in FIG. 11, in the region R1, the growth of yeast is promoted. In the region R2, the growth of yeast is suppressed. In region R3, the yeast is inactivated. The boundary between the region R1 and the region R2 is 2.7 × 10 ¹⁷ cm ⁻³ . Therefore, the first volume irradiation amount is 2.7 × 10 ¹⁷ cm ⁻³ . That is, the first volume irradiation amount is about 3.0 × 10 ¹⁷ cm ⁻³ . The boundary between the region R2 and the region R3 is 1.5 × 10 ¹⁸ cm ⁻³ . Therefore, the second volume irradiation amount is 1.5 × 10 ¹⁸ cm ⁻³ .

  FIG. 12 is a graph showing the irradiation distance dependence on the relationship between radical irradiation time and the growth rate of budding yeast in suspension. As shown in FIG. 12, the peak for growing budding yeast is located closer to the shorter irradiation time as the distance from the irradiation port 210 to the sample is shorter. On the other hand, in FIG. 6, the shorter the distance from the irradiation port 210 to the sample, the higher the density of triplet oxygen atoms. From these facts, it is considered that the growth of budding yeast is promoted when the supply amount of triplet oxygen atoms is near a certain value. Therefore, it is speculated that it is the triplet oxygen atom of the oxygen radical that plays a major role in the increase and decrease of budding yeast.

D. Budding yeast 3 (cell cycle)
D-1. Experimental Procedure First, yeast was cultured in YPD medium. Thereafter, the cells were collected, and the budding yeast was suspended in 3 ml of phosphate buffered saline (PBS (−)). Each of α-factor, Hydroxyurea and Nocodeazole was treated in a yeast medium. Yeast treated with α-factor stops in the G1 phase. Yeast treated with Hydroxyrea stops in S phase. Yeast treated with Nocodeazole stops in the M phase. Thereby, yeast only in G1 phase, yeast only in S phase, and yeast only in M phase were produced.

  Then, radicals were irradiated to the yeast only in the G1 phase, the yeast only in the S phase, and the yeast only in the M phase. Thereafter, the yeast was cultured again in YPD medium. Thereafter, after 60 hours, the number of viable yeast was counted.

D-2. Experimental results The experimental results are shown in FIG. As shown in FIG. 13, in the G1 phase, when the radical was irradiated for 10 seconds, the yeast grew. The increase rate was about 10%. In the G1 phase, the yeast decreased when the radicals were irradiated for 20 seconds. Compared to the case where radicals were not irradiated, the viable cell count of yeast was about 90%. In the G1 phase, when the radical was irradiated for 30 seconds, the yeast decreased. The number of viable yeast was about 60% as compared with the case where radicals were not irradiated.

  As shown in FIG. 13, in the S phase, when radicals were irradiated for 10 seconds, yeast decreased slightly. Compared to the case where radicals were not irradiated, the number of viable yeast was about 97%. When the radicals were irradiated for 20 seconds, the number of viable yeast was about 96% compared to when the radicals were not irradiated. When the radicals were irradiated for 30 seconds, the number of viable yeast was about 90% compared to when the radicals were not irradiated.

  As shown in FIG. 13, in the M phase, the yeast decreased slightly when the radical was irradiated for 10 seconds. The number of viable yeast was about 98% compared to the case where no radical was irradiated. When the radicals were irradiated for 20 seconds, the number of viable yeast was about 93% compared to when the radicals were not irradiated. When the radicals were irradiated for 30 seconds, the number of viable yeast was about 80% compared to when the radicals were not irradiated.

  Thus, when the yeast was irradiated with radicals having a dose equal to or lower than the first irradiation amount in the G1 phase, the yeast grew. For this reason, as described in the third embodiment, it is considered that if the radicals are irradiated in a cycle synchronized with the G1 phase in accordance with the cell cycle of the yeast, the yeast suitably grows. Even if the S-phase or M-phase yeast is irradiated with radicals, it decreases only slightly. That is, the influence of radicals is sufficiently small for S-phase and M-phase yeasts.

E. Mouse fetal skin cells (NIH3T3)
E-1. Cultured cells In this experiment, animal eukaryotic cells were used. That is, mouse fetal skin cells (NIH3T3).

E-2. Experimental Procedure First, mouse fetal skin cells (NIH3T3) are cultured on a dish. The cells were collected and suspended in 3 ml of phosphate buffered saline (PBS (−)). The plastic cover 150 was used to irradiate the suspension with plasma after eliminating the influence of the surrounding atmosphere. Thereafter, cultured cells were collected with a centrifuge, and NIH3T3 was cultured in a serum-containing culture solution. Then, after 60 hours, the number of surviving cells was counted.

E-3. Experimental Results FIG. 14 is a graph showing the relationship between the dose of triplet oxygen atom radicals and the cell growth rate of mouse fetal skin cells (NIH3T3). As shown in FIG. 14, NIH3T3 cells proliferate within a range where the irradiation dose of radicals is 3.0 × 10 ¹⁷ cm ⁻³ or less. When the irradiation dose of radicals is larger than 3.0 × 10 ¹⁷ cm ⁻³ , NIH3T3 cells are considered to decrease due to consistency with the above experiment.

That is, NIH3T3 cells proliferate when the first volume irradiation dose is 3.0 × 10 ¹⁷ cm ⁻³ or less. In particular, when the first volume irradiation dose was 2.0 × 10 ¹⁷ cm ⁻³ or more and 2.7 × 10 ¹⁷ cm ⁻³ or less, NIH3T3 cells proliferated 30% or more. Compared to those not irradiated with radicals, the number of NIH3T3 cells was about 135%.

  Thus, NIH3T3 cells, which are animal cells, proliferated by irradiation with radicals. From Experiment B-E, it is considered that cells can be proliferated by irradiating radicals to plant cells and animal cells as well as the experimental cells.

DESCRIPTION OF SYMBOLS 100 ... Plasma generator 110 ... Chamber 120 ... Mounting part 130 ... Gas supply part 140 ... Gas discharge part 200 ... Radical irradiation part 210 ... Irradiation port 211 ... Slit 250 ... Discharge part 260 ... Intermediate structure part 270 ... Nozzle part

Claims (5)

Having a radical irradiation step of irradiating eukaryotic cells with radicals;
In the radical irradiation step,
As following the first dose which predetermined irradiation amount of the radical, radicals and periodically irradiated in eukaryotic cells grown eukaryotic cell in a first period,
The first period is:
A method for proliferating eukaryotic cells, which is synchronized with the G1 phase in the cell cycle of eukaryotic cells. Stopping eukaryotic cells in the G1 phase;
Radiation irradiation process to irradiate eukaryotic cells with radicals ;
Have
In the radical irradiation step,
In a state where the eukaryotic cell is stopped in the G1 phase, the irradiation amount of the radical is set to a predetermined first irradiation amount or less and the radical is irradiated to the eukaryotic cell,
A method of proliferating eukaryotic cells, characterized by growing eukaryotic cells. In the eukaryotic cell growth method according to claim 1 or 2 ,
In the radical irradiation step,
A method for growing a eukaryotic cell, comprising irradiating the eukaryotic cell with at least one of a triplet oxygen atom and a singlet oxygen molecule. The eukaryotic cell growth method according to any one of claims 1 to 3 ,
A suspension preparation step of preparing a suspension in which the eukaryotic cells are suspended;
In the radical irradiation step,
Irradiating the suspension with triplet oxygen atoms;
The first dose is
A first volume dose that is the number of triplet oxygen atoms supplied to the volume of the suspension;
The first volume dose is
A method for proliferating eukaryotic cells, which is 3.0 × 10 ¹⁷ cm ⁻³ . A method for producing a eukaryotic cell, wherein the number of eukaryotic cells is increased using the method for growing a eukaryotic cell according to any one of claims 1 to 4 .