Method and apparatus for delivering fine bubbles to cells

Abstract

This method for delivering fine bubbles to cells is characterized by comprising a step for applying an electric field to a liquid containing fine bubbles, and thus delivering the fine bubbles to cells.

Description

Method and device for delivering fine bubbles to cells

[0001] The present invention relates to a method and device for delivering fine bubbles to cells.

[0002] Three-dimensional culture is becoming increasingly important in fields such as regenerative medicine, drug discovery, and cultured meat. However, one issue with three-dimensional culture is that it does not provide sufficient oxygen to the interior of the cell tissue, making it prone to necrosis.

[0003] Patent Document 1 discloses a cell culture method using a culture medium containing ultra-fine bubbles.

[0004] Japanese Patent Application Laid-Open No. 2021-073989

[0005] However, in the cell culture method disclosed in Patent Document 1, the cells are simply immersed in a culture solution containing ultrafine bubbles, and there are cases where fine bubbles are not sufficiently delivered to the cells.

[0006] The present invention has been made in consideration of the above problems, and aims to provide a method and device for more efficient delivery of fine bubbles to cells.

[0007] To solve the above problems, the method for delivering fine bubbles to cells of the present invention is characterized by comprising a step of delivering the fine bubbles to cells by applying an electric field to a liquid containing fine bubbles. Also, the device for delivering fine bubbles to cells is characterized by comprising an electric field application means for applying an electric field to a liquid containing fine bubbles, and delivering the fine bubbles to cells by the electric field application means.

[0008] According to the present invention, a method and device for more efficiently delivering fine bubbles to cells can be provided.

[0009] FIG. 1 is a schematic diagram showing an apparatus for delivering fine bubbles to cells in a first embodiment. FIG. 2 is a schematic diagram showing a cell sheet member in Example 1. FIG. 3 is a schematic diagram showing a cell sheet member in Example 1. FIG. 4 is a schematic diagram showing a apparatus for delivering fine bubbles to cells in Example 1. FIG. 5 is an evaluation result showing the delivery of fine bubbles to cells in Example 1. FIG. 6 is a schematic diagram showing an apparatus for delivering fine bubbles to cells in a second embodiment.

[0010] The method and device for delivering fine bubbles to cells according to the present invention are described in detail below, but the configuration, structure, materials, dimensions, settings, etc. may be modified as appropriate depending on the various conditions under which the invention is applied, and are not intended to limit the scope of the present invention.

[0011] In the present invention, the method for delivering fine bubbles to cells comprises a step of applying an electric field to a liquid containing fine bubbles to deliver the fine bubbles to cells. The device comprises an electric field application means for applying an electric field to the liquid containing fine bubbles, and the fine bubbles are delivered to cells by the electric field application means.

[0012] In recent years, fine bubbles, which are tiny bubbles with a diameter of less than 100 μm, have been attracting attention. Fine bubbles are classified into microbubbles with a diameter (number average particle size) of 1 μm or more but less than 100 μm, and ultrafine bubbles (hereinafter also referred to as UFB) with a diameter of less than 1 μm. While there is no particular limitation on the type of bubbles used in the present invention, it is more preferable to use ultrafine bubbles with a small diameter and high permeability. Ultrafine bubbles with a diameter of 200 nm or less are even more preferable. Since the pore size of a typical filtration sterilization filter is 220 nm, filtration sterilization can be performed with a diameter of 200 nm or less. Furthermore, the EPR effect (Enhanced Permeation and Retention Effect) can also be expected with a diameter of 200 nm or less. That is, in the case of ultrafine bubbles with a diameter of 200 nm or less, the EPR effect allows ultrafine bubbles to be efficiently supplied to tumor tissue. Furthermore, a diameter of 100 nm or less is preferable because it can penetrate between cells where tight junctions are not formed, and can be easily delivered to the interior of thick tissues.

[0013] In the present invention, it is preferable to include a step of preparing cells and a liquid containing fine bubbles prior to the step of delivering fine bubbles to cells. In this case, a liquid containing fine bubbles that has been prepared separately in advance may be used, or a step of producing a liquid containing fine bubbles prior to the step of preparing cells and a liquid containing fine bubbles may be included. Methods for producing fine bubbles include swirling flow liquid, ejector, Venturi, micropore, static mixer, pressurized dissolution deposition, heated precipitation, and direct vapor contact condensation. Methods for producing ultrafine bubbles include high-speed swirling flow, pressurized dissolution, surfactant-added micropore, ultrasonic cavitation, and film boiling using a heating element.

[0014] Although either method can be suitably used in the present invention, the film boiling method using a heating element is more preferable as a method for producing ultra-fine bubbles because it allows for longer storage periods. In other words, it is preferable to have a step of generating ultra-fine bubbles in a liquid by generating heat with a heating element.

[0015] Fine bubbles are known to have a zeta potential. Therefore, applying an electric field to a liquid containing fine bubbles can cause electrophoresis of the fine bubbles in the liquid. Furthermore, when the pH of the liquid is 7, cells dispersed in the liquid typically have a negative potential, and fine bubbles dispersed in the liquid also typically have a negative zeta potential. Therefore, to deliver fine bubbles to cells, it is necessary to overcome this potential barrier. In the present invention, an electric field is applied between the cells and the fine bubbles, causing the fine bubbles to move by electrophoresis and overcome the potential barrier, thereby efficiently delivering the fine bubbles to cells. Furthermore, ultrafine bubbles are preferred because they have a small diameter, making them easy to move in liquid and easily induce electrophoresis. Note that when an electric field is applied to a liquid containing fine bubbles by an electric field application means, the electric field needs to be applied to at least the liquid containing the fine bubbles; it may also be applied to other components, such as cells.

[0016] When fine bubbles are delivered to cells, the gas inside the fine bubbles, the substances that formed the shell of the fine bubbles, and the substances adsorbed to the fine bubbles are delivered to the cells. In other words, drug delivery in a broad sense, including gas delivery, can be achieved under the control of an electric field.

[0017] The present invention can be suitably used for in vitro culture and other applications. It can also be suitably used in in vivo treatments, such as administering a liquid containing fine bubbles and delivering medical gases or drugs to the affected area using an electric field. For example, cancer-affected areas are known to be hypoxic, reducing the effectiveness of anticancer drug therapy and radiation therapy. While it has been reported that administering a liquid containing oxygen fine bubbles can improve therapeutic efficacy, the present invention achieves even greater therapeutic efficacy by applying an electric field to the cancer-affected area. Furthermore, in organ preservation, ischemia-reperfusion injury is known to occur when blood flow is restored. While attempts to prevent ischemia-reperfusion injury using fine bubbles containing medical gases have been reported, the present invention enhances this effect by applying an electric field to the preserved organ.

[0018] The type of gas contained in the fine bubbles is not particularly limited. It may be oxygen, air, carbon dioxide, hydrogen, carbon monoxide, nitric oxide, xenon, hydrogen sulfide, ozone, or a mixture thereof. Neutral gases that do not ionize in water, such as oxygen, hydrogen, carbon monoxide, and nitric oxide, are particularly suitable for the present invention because they can only be electrically controlled by being converted into fine bubbles, thereby obtaining a zeta potential. Because the volume per ultrafine bubble is very small, when using ultrafine bubbles to deliver gas, the number concentration is preferably 1 billion bubbles / ml or more, and more preferably 10 billion bubbles / ml or more. At 10 billion bubbles / ml or more, assuming an internal pressure of approximately 30 atm (Laplace pressure) for a typical ultrafine bubble diameter of 100 nm, the amount of gas contained in the ultrafine bubbles exceeds 0.1 ppm, an amount comparable to the amount of dissolved gas.

[0019] The type of cells is not particularly limited. They may be adherent cells or suspension cells, cell lines or primary cells, eukaryotic cells or prokaryotic cells, or stem cells such as ES cells or iPS cells.

[0020] The cells may be in an aggregated form. For example, they may be three-dimensional tissues (three-dimensional cells) such as spheroids, organoids, cell sheets, tissues, organs, and cultured meat. A problem with thick three-dimensional tissues is the lack of oxygen supply to the interior, and the present invention is particularly suitable for culturing that solves this problem.

[0021] A scaffold material may be used for three-dimensional tissues. It is preferable that the scaffold material is a porous material with pores larger than the diameter of the fine bubbles. This makes it easier to deliver the fine bubbles to cells.

[0022] The type of liquid containing fine bubbles is not particularly limited. Suitable liquids include culture medium for culturing cells, isotonic liquids such as physiological saline, and buffer solutions such as phosphate buffer. In other words, the liquid containing fine bubbles is preferably at least one selected from the group consisting of culture medium, isotonic liquid, and buffer solutions. Various additives can also be added as appropriate. To prevent the fine bubbles from being depleted, it is also preferable to continuously generate fine bubbles or perfuse the liquid. Furthermore, to minimize the effects of high and low pH on cells, the pH of the liquid containing fine bubbles is preferably between 6 and 8.

[0023] The direction of the electric field must be such that, at least at some point, the fine bubbles with zeta potential are electrophoresed toward the cells. However, the electric field does not need to be of constant magnitude or in the same direction at all times; they may change over time. If an electric field is applied in the same direction for a long period of time, electrolytes other than the fine bubbles may also move, changing the properties of the liquid. Therefore, it is preferable to change the magnitude and direction of the electric field over time. An intermittent electric field application, including periods when the electric field is not applied, is also acceptable. Furthermore, when cells form a three-dimensional tissue, in order to efficiently supply fine bubbles to the three-dimensional tissue, it is preferable that the direction of the electric field applied by the electric field application means be in a direction that minimizes the average thickness of the three-dimensional tissue.

[0024] Electrodes serving as an electric field application means are typically used as a pair of electrodes. The electrode material, configuration, and electric field conditions, such as the magnitude and direction of the electric field applied between the electrode pair, are preferably determined so as to prevent electrochemical reactions, particularly electrolysis of liquids containing fine bubbles. To this end, it is preferable to use a material with a wide potential window for the electrodes. Furthermore, when applying an AC or pulsed electric field, it is preferable to use electrodes with a large electric capacity, such as porous electrodes. When applying an electric field in the same direction for a long period of time, the applied voltage (potential difference between the electrode pair) is preferably 1.23 V or less, which is the voltage at which water electrolysis is unlikely to occur, more preferably 1.22 V or less, and particularly preferably 1.00 V or less. There is no particular limit on the lower limit of the applied voltage (potential difference between the electrode pair), but it is preferably 0.10 V or more. It is preferable to position the electrodes so that they do not come into direct contact with cells. Even if an electrochemical reaction does occur, its effects can be mitigated.

[0025] The electrodes are preferably arranged so that an effective electric field is applied between the cells and the fine bubbles. For example, the electrodes may be arranged facing each other so as to sandwich the cells, or may be arranged inside and outside the three-dimensional tissue, or one electrode may be inserted into the three-dimensional tissue. The electrodes need not necessarily be immersed in the liquid as long as they can apply an electric field to the liquid containing the fine bubbles. In particular, in the case of three-dimensional tissue, it is preferable to arrange one electrode inside the three-dimensional tissue and the other electrode outside the three-dimensional tissue in order to penetrate the fine bubbles into the tissue. In this case, when arranging the electrodes inside the three-dimensional tissue, it is preferable to cover the periphery of the electrode arranged inside the three-dimensional tissue with a PFA mesh to prevent the electrode from coming into direct contact with the three-dimensional tissue (cells).

[0026] Furthermore, to prevent electrophoresed fine bubbles from leaking and passing through areas other than the cells, it is preferable to position the cells so that the current path between the electrodes is blocked, or to restrict the current path with other components.

[0027] [First embodiment] Figure 1 is a schematic diagram showing an example of an apparatus for delivering fine bubbles to cells to which the present invention is applied. A cell culture insert 2 is inserted into a dish 1, and a multilayered cell sheet 3 is adhered onto a membrane 201 with open pores. The dish 1 and cell culture insert are filled with a medium (culture solution) 4 containing fine bubbles. Electrodes 5 are arranged inside the dish 1 at positions outside the cell culture insert 2 and inside the cell culture insert 2 so that they are immersed in the medium 4 containing fine bubbles. The electrodes 5 are connected to a power source 6. When a voltage is applied from the power source 6 to the electrodes 5, the fine bubbles in the medium 4 containing fine bubbles are delivered to the cell sheet 3.

[0028] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples as long as the gist of the invention is not exceeded. "Parts" and "%" used to describe the amounts of components are by mass unless otherwise specified.

[0029] Example 1 Fig. 3 is a schematic diagram showing a device for delivering fine bubbles to cells, which is a more specific embodiment of the first embodiment of the present invention. Figs. 2A to 2C are schematic diagrams for explaining the cell sheet member in Fig. 3.

[0030] (Cell Sheet) Mouse fetal fibroblast cells, NIH / 3T3, were used as the cells. The culture medium was prepared by adding 10% FBS and 1% PS to high-glucose DMEM as the basal medium. Here, DMEM means Dulbecco's Modified Eagle Medium, FBS means Fetal Bovine Serum, and PS means Penicillin-Streptomycin. 1.5 x 10 cells were cultured in a temperature-responsive 6 cm dish (CellSeed, product name UpCell). ５ The cells were seeded at 1000 cells / dish and 4 ml of medium per dish. ２ The cells were incubated in a 5% concentration environment, and the medium was changed every 3 days for 7 days, at which point the cells were overconfluent.

[0031] The dish was removed from the incubator, the medium was removed, and 100 μl of medium was added to prevent drying. The dish was left to stand in an environment at 20° C. for 20 minutes to form a cell sheet. At this point, the cell sheet had shrunk to a diameter of more than 6 cm.

[0032] 4 ml of medium was added, and the temperature was again 37°C and CO ２ The cells were incubated in a 5% aqueous solution, with the medium replaced every three days, for seven days. During this period, the cells migrated from the contracted cell sheet and proliferated, spreading throughout the entire dish.

[0033] The dish was removed from the incubator, the medium was removed, and 100 μl of medium was added to prevent drying. The dish was left to stand for 20 minutes in an environment at 20°C, and a cell sheet was again produced. The cell sheet production process was repeated twice in order to produce a thick cell sheet.

[0034] The cell sheet was sucked up with a pipette and transferred to a U-shaped 96-well plate for spheroid production (Sumitomo Bakelite Co., Ltd., trade name PrimeSurface). 100 μl of medium was added, followed by 10 μl of a hypoxia evaluation reagent LOX-1 (Medical and Biological Laboratories) diluted 50-fold with medium. The plate was then incubated at 37°C and CO ２ After incubation for one day in an environment with a concentration of 5%, the cell sheet became clumped.

[0035] LOX-1 emits phosphorescence, but this is quenched in the presence of oxygen due to a quenching effect. By evaluating the phosphorescence intensity of the cell sheet, the oxygen concentration within the cell sheet can be evaluated.

[0036] (Ultra-fine bubble water) Ultra-fine bubble water was generated using an ultra-fine bubble generator (IDEC Corporation, product name UltrafineGaLF, FZ1N-02-T). Ultrapure water was used as the liquid and oxygen as the gas, and the generator was operated for 9 hours with a liquid volume of 3 L. Measurements using a nanoparticle size distribution analyzer SALD-7500nano (Shimadzu Corporation) revealed that the number concentration of ultra-fine bubbles was 10 billion / ml and the number-average particle size was 98 nm.

[0037] This ultra-fine bubble water was evaporated in a water bath at 60°C and a pressure of 7.5 kPa to concentrate the liquid volume to 1 / 20, thereby obtaining ultra-fine bubble water with a bubble concentration of 80 billion bubbles / ml and a number-average particle size of 98 nm.

[0038] (Assay medium) A basal medium was prepared by dissolving 8.3 g / L of phenol red-free powdered DMEM (Sigma-Aldrich), 4.5 g / L of glucose, and 3.7 g / L of sodium bicarbonate in ultra-fine bubble water. The basal medium was measured using a zeta potential measuring device (Microtec Nition, trade name ZEECOM), and the polarity of the zeta potential of the ultra-fine bubbles was found to be negative, with a mobility of 1 (μm / s)·(cm / V). 10% FBS was added to the basal medium to prepare Assay Medium 13.

[0039] (Jig) As shown in Figures 2A to 2C, a block of cell sheet 8 was sandwiched and supported between a mesh 9 (150 mesh, 134 µm opening) made of PFA (perfluoroalkoxyalkane) (manufactured by Tantore). These were then sandwiched and supported between a current path limiting member 10 made of parafilm with a 1 mm diameter communication opening 101. The size of the communication opening 101 was smaller than the cell sheet, so that the cell sheet 8 was present across the entire communication opening. The current path limiting member 10 ensured that electrophoresed ultrafine bubbles reached the cell sheet 8 without leaking. The cell sheet 8 supported between the mesh 9 and the current path limiting member 10 had a diameter of approximately 2 mm and a thickness of approximately 150 µm. The combined assembly is referred to as the cell sheet member 11.

[0040] As shown in Figure 3, the cell sheet member 11 was fixed by sandwiching it between electrodes 14 (manufactured by Metrohm, product name ITO10) via a cylindrical resin spacer 12. The distance between the electrodes was 2 mm. The electrode 14 consisted of a transparent ITO electrode 141 and a wire 142 connected to it formed on a transparent resin. When viewed from the direction A in Figure 3, the cell sheet 8 could be optically observed through the electrode 14. The space between the cell sheet member 11 and the electrode 14 was filled with an assay medium 13. A voltage source 15 was connected to the wire 142, allowing an electric field to be applied to the assay medium 13 containing ultra-fine bubbles.

[0041] (Evaluation) The jig was set on a fluorescence microscope (Keyence Corporation, product name BZ-X810), and phosphorescence was observed from direction A in FIG. 3 . A filter cube for TRITC was used, in which the excitation filter was changed to GFP (EX: 470 / 40 nm, DM: 565 nm, BA: 605 / 70 nm). Here, in the filter cube, EX stands for excitation filter, DM stands for dichroic mirror, and BA stands for absorption filter (barrier filter). The absorption maximum wavelength of LOX-1 is 483 nm, and the emission maximum wavelength of phosphorescence is 616 nm. When observing phosphorescence, the bleaching reduction mode was used to minimize the irradiation time of excitation light to prevent bleaching. The phosphorescence intensity was evaluated by analyzing the brightness of the cell sheet 8 excluding the mesh 9 portion using image processing software ImageJ (developed by Wayne Rashand).

[0042] With the transparent electrode 141 at the back side as viewed from direction A as the reference, a voltage of -1.00 V was applied intermittently at 10-minute intervals to the transparent electrode 141 at the front side. No gas generation due to electrolysis or deposition on the electrode surface was observed. Figure 4 shows the evaluation results showing the change in phosphorescence intensity over time.

[0043] When the power was off, the phosphorescence intensity increased, indicating that oxygen was being consumed and the oxygen concentration in the cell sheet was decreasing. In other words, simply immersing the cells in a culture medium containing ultra-fine bubbles did not provide an adequate supply of oxygen to the cells. In contrast, when an electric field was applied between the cell sheet and the ultra-fine bubbles, the phosphorescence intensity decreased. This indicated that the ultra-fine bubbles were efficiently delivered to the cell sheet, providing a sufficient supply of oxygen.

[0044] Because it can supply a large amount of oxygen to cells, cells can be cultured efficiently. In particular, thick, three-dimensional tissues can be cultured while reducing internal oxygen deficiency. For long-term culture, it is sufficient to perfuse the culture medium to prevent the fine bubbles from becoming depleted.

[0045] In this example, gas was delivered to cells using fine bubbles, but it is clear that substances that formed the shell of the fine bubbles or substances that were adsorbed to the fine bubbles can also be delivered in a similar manner.

[0046] Example 2 Ultra-fine bubble water was obtained in the same manner as in Example 1, except that the UFB generator (ultra-fine bubble generator) shown in Figure 1 of JP 2021-073989 A was used as the ultra-fine bubble generator. This UFB generator is equipped with a film boiling type UFB generation unit that generates ultra-fine bubbles in a liquid by generating heat from a heating element.

[0047] Measurement using a nanoparticle size distribution analyzer SALD-7500 nano (manufactured by Shimadzu Corporation) revealed that the number concentration of the obtained ultrafine bubbles was 10 billion / ml and the number-average particle size was 98 nm.

[0048] This ultra-fine bubble water was evaporated in a water bath at 60°C and a pressure of 7.5 kPa to concentrate the liquid volume to 1 / 20, thereby obtaining ultra-fine bubble water with a bubble concentration of 80 billion bubbles / ml and a number-average particle size of 98 nm.

[0049] As a result, when an electric field was applied between the cell sheet and the ultra-fine bubbles, the phosphorescence intensity decreased, as in Example 1. This indicates that the ultra-fine bubbles were efficiently delivered to the cell sheet, providing a sufficient supply of oxygen.

[0050] Reference Example 1 is the same as Example 1, except that the solvent in the assay medium in Example 1 was changed from ultra-fine bubble water to ultrapure water. The evaluation results of Reference Example 1 showed that the phosphorescence intensity increased in the same way both when the power was off and when it was on. In other words, it was found that without fine bubbles, the supply of oxygen to cells did not increase even when an electric field was applied.

[0051] [Second Embodiment] Figure 5 is a schematic diagram showing an example of an apparatus for delivering fine bubbles to cells according to the present invention. An electrode 5 is placed in a dish 1, which is filled with a medium 4 (culture solution) containing fine bubbles. A spheroid 17, a three-dimensional tissue (three-dimensional multicellular structure), is immersed in the medium 4 while being skewered by a needle-shaped electrode 16. The portion of the needle-shaped electrode 16 not covered by the spheroid 17 is insulated to prevent current from flowing. The needle-shaped electrode 16 is also covered with a PFA mesh to prevent contact between the needle-shaped electrode 16 and the spheroid 17. A voltage is applied between the electrode 5 and the needle-shaped electrode 16 from a power source 6. The electric field is oriented so that the fine bubbles are electrophoresed from the outside to the inside of the spheroid 17. The fine bubbles are delivered to the spheroid 17 present in the uninsulated portion of the needle-shaped electrode 16.

[0052] By using oxygen as the gas in the fine bubbles, spheroids can be cultured while reducing the oxygen deficiency inside them.

[0053] From the above, we have been able to provide a device for delivering fine bubbles to cells efficiently, which is characterized by having cells, a liquid containing fine bubbles, and an electric field application means for applying an electric field to the liquid.

[0054] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0055] This application claims priority based on Japanese Patent Application No. 2024-051066 filed on March 27, 2024 and Japanese Patent Application No. 2025-044143 filed on March 18, 2025, the entire contents of which are incorporated herein by reference.

Claims

1. A method for delivering fine bubbles to cells, comprising the step of applying an electric field to a liquid containing fine bubbles to deliver the fine bubbles to cells.

2. A method for delivering fine bubbles to cells according to claim 1, comprising the step of preparing the cells and a liquid containing the fine bubbles.

3. The method for delivering fine bubbles to cells according to claim 1, wherein the fine bubbles are ultrafine bubbles.

4. The method for delivering fine bubbles to cells according to claim 3, wherein the number-average particle size of the ultra-fine bubbles is 200 nm or less.

5. The method for delivering fine bubbles to cells according to claim 3, wherein the concentration of the ultrafine bubbles in the liquid is 10 billion bubbles / ml or more.

6. The method for delivering fine bubbles to cells according to claim 3, further comprising the step of generating the ultra-fine bubbles in the liquid by heating a heating element.

7. The method for delivering fine bubbles to cells according to claim 1, further comprising an electric field application means for applying an electric field to the liquid.

8. A method for delivering fine bubbles to cells according to claim 7, wherein the electric field application means is controlled to apply the electric field in a direction that causes the fine bubbles to electrophorese toward the cells.

9. The method for delivering fine bubbles to cells according to claim 7, wherein the electric field application means is controlled to change the magnitude and direction of the electric field over time.

10. The method for delivering fine bubbles to cells according to claim 7, wherein the electric field application means has an electrode pair, and the potential difference between the electrode pair is 1.23 V or less.

11. The method for delivering fine bubbles to cells according to claim 10, wherein the electrodes of the electrode pair are positioned so as not to come into direct contact with the cells.

12. The method for delivering fine bubbles to cells according to claim 1, wherein the fine bubbles contain a gas that does not ionize in water.

13. The method for delivering fine bubbles to cells according to claim 1, wherein the liquid containing the fine bubbles is at least one selected from the group consisting of a culture medium, an isotonic solution, and a buffer solution.

14. The method for delivering fine bubbles to cells according to claim 1, wherein the cells form a three-dimensional tissue.

15. A method for delivering fine bubbles to cells as described in claim 7, wherein the electric field application means has an electrode pair, one electrode of the electrode pair being placed inside the three-dimensional tissue and the other electrode being placed outside the three-dimensional tissue.

16. An apparatus for delivering fine bubbles to cells, comprising an electric field application means for applying an electric field to a liquid containing fine bubbles, and delivering the fine bubbles to the cells by the electric field application means.

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