Method and apparatus for delivering fine bubbles to cells

Abstract

To provide a more efficient method and apparatus for delivering fine bubbles to cells.SOLUTION: A method for delivering fine bubbles to cells, characterized by comprising a step for applying an electric field to a liquid containing fine bubbles, and thus delivering the fine bubbles to cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[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. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-073989 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[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. [Effects of the Invention]

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

[0009] [Figure 1] FIG. 1 is a schematic diagram showing a device for delivering fine bubbles to cells in a first embodiment. [Figure 2] FIG. 1 is a schematic diagram showing a cell sheet member in Example 1. [Figure 3] FIG. 1 is a schematic diagram showing a device for delivering fine bubbles to cells in Example 1. [Figure 4] 1 shows the evaluation results showing the delivery of fine bubbles to cells in Example 1. [Figure 5] FIG. 10 is a schematic diagram showing a device for delivering fine bubbles to cells in a second embodiment. DETAILED DESCRIPTION OF THE INVENTION

[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 to 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, ultrafine bubbles with a small diameter and high penetrability are more preferable. Ultrafine bubbles with a diameter of 200 nm or less are even more preferable. Filter sterilization is possible with a diameter of 200 nm or less because the pore size of a typical sterilization filter is 220 nm. Furthermore, the EPR effect (Enhanced Permeation and Retention Effect) can be expected with diameters of 200 nm or less. In other words, ultrafine bubbles with a diameter of 200 nm or less can be efficiently delivered to tumor tissues due to the EPR effect. Furthermore, a diameter of 100 nm or less is preferable because they can penetrate between cells without tight junctions and are 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 the 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 is suitable for use in in vitro culture and other applications. It can also be used in vivo for treatments involving 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 can be oxygen, air, carbon dioxide, hydrogen, carbon monoxide, nitric oxide, xenon, hydrogen sulfide, ozone, or a mixture of these. 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 achieving a zeta potential. Because the volume of each ultrafine bubble is extremely 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, making it comparable to the amount of dissolved gas.

[0019] There are no particular limitations on the type of cells. 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 for the fine bubbles to be delivered to cells.

[0022] The type of liquid containing fine bubbles is not particularly limited. Suitable liquids include culture solutions for culturing cells, isotonic solutions such as physiological saline, and buffer solutions such as phosphate buffer. In other words, it is preferable that the liquid containing fine bubbles is at least one selected from the group consisting of culture solutions, isotonic solutions, and buffer solutions. Various additives can also be added as appropriate. To prevent the fine bubbles from running out, it is also preferable to generate fine bubbles continuously or to 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 constant in magnitude or direction; it 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 materials, arrangement, and electric field conditions, such as the magnitude and direction of the electric field applied between the pair of electrodes, are preferably determined so as to prevent electrochemical reactions, particularly electrolysis of the liquid 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 pair of electrodes) 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 to the lower limit of the applied voltage (potential difference between the pair of electrodes), but a value of 0.10 V or more is preferred. It is preferable to position the electrodes so that they do not come into direct contact with the cells. Even if an electrochemical reaction does occur, its effects can be mitigated.

[0025] The electrodes are preferably positioned so that an effective electric field is applied between the cells and the fine bubbles. For example, the electrodes may be positioned facing each other with the cells in between, positioned 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 position one electrode inside the three-dimensional tissue and the other outside the three-dimensional tissue to allow the fine bubbles to penetrate into the tissue. In this case, when positioning the electrodes inside the three-dimensional tissue, it is preferable to cover the electrodes placed inside the three-dimensional tissue with a PFA mesh to prevent them 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 to 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 placed inside the dish 1, one outside the cell culture insert 2 and one 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. [Example]

[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. The terms "parts" and "%" used to describe the amounts of components are based on 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, and Fig. 2 is a schematic diagram for explaining the cell sheet member in Fig. 3.

[0030] (cell sheet) The cells used were mouse embryonic fibroblasts, NIH / 3T3. The culture medium was prepared by adding 10% FBS and 1% PS to high-glucose DMEM as the basal medium. Here, DMEM stands for Dulbecco's Modified Eagle Medium, FBS stands for Fetal Bovine Serum, and PS stands for Penicillin-Streptomycin. 1.5 × 10 cells were cultured in a temperature-responsive 6-cm dish (CellSeed, product name UpCell). 5 Cells were seeded at 1 cell / dish with 4 ml of medium per dish. They were incubated at 37°C with 5% CO2, and the medium was changed every 3 days for 7 days. At this time, 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 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] Four milliliters of medium was added, and the dish was again incubated at 37°C with a CO2 concentration of 5%, with the medium replaced every three days for seven days. At this time, the cells migrated and proliferated from the contracted cell sheet, spreading throughout the 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 to create a thick cell sheet.

[0034] The cell sheet was then pipetted and transferred to a U-shaped 96-well plate (Sumitomo Bakelite Co., Ltd., product name: PrimeSurface) for spheroid production. 100 μl of medium was added, followed by 10 μl of LOX-1 (Medical and Biological Laboratories Co., Ltd.), a hypoxia evaluation reagent diluted 50-fold with medium. The plate was then incubated for one day at 37°C with 5% CO2. The cell sheet formed a mass.

[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) Ultrafine bubble water was generated using an ultrafine bubble generator (IDEC, 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 the ultrafine 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, resulting in ultra-fine bubble water with a bubble concentration of 80 billion bubbles / ml and an average particle size of 98 nm.

[0038] (Assay medium) The basal medium was prepared by dissolving 8.3 g / L of phenol red-free powdered DMEM (Sigma-Aldrich) in ultrafine bubble water with 4.5 g / L of glucose and 3.7 g / L of sodium bicarbonate. The basal medium was measured using a zeta potential analyzer (Microtech Nichion, product name ZEECOM). The polarity of the zeta potential of the ultrafine bubbles was negative, and the mobility was 1 μm / s·cm / V. The basal medium was supplemented with 10% FBS to prepare the assay medium 13.

[0039] (jig) As shown in Figure 2, 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 communication opening 101 of 1 mm diameter. 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 fixture was set on a fluorescence microscope (Keyence Corporation, product name BZ-X810), and phosphorescence was observed from direction A in Figure 3. A filter cube for TRITC was used, with the excitation filter 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 barrier filter. The absorption maximum wavelength of LOX-1 is 483 nm, and the emission maximum wavelength of phosphorescence is 616 nm.

[0042] 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, using the image processing software ImageJ (developed by Wayne Rashand).

[0043] Using the transparent electrode 141 at the back 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. 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.

[0044] When the power was turned off, the phosphorescence intensity increased, indicating that oxygen was 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.

[0045] 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 advisable to perfuse the culture medium to prevent the fine bubbles from becoming depleted.

[0046] 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.

[0047] <Example 2> Ultrafine bubble water was obtained in the same manner as in Example 1, except that the UFB generator (ultrafine bubble generator) shown in Figure 1 of JP 2021-073989 A was used as the ultrafine bubble generator. This UFB generator is equipped with a film boiling type UFB generation unit that generates ultrafine bubbles in a liquid by heating a heating element.

[0048] Measurement using a nanoparticle size distribution analyzer SALD-7500nano (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.

[0049] 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, resulting in ultra-fine bubble water with a bubble concentration of 80 billion bubbles / ml and an average particle size of 98 nm.

[0050] As a result, similar to Example 1, when an electric field was applied between the cell sheet and the ultra-fine bubbles, the phosphorescence intensity decreased, indicating that the ultra-fine bubbles were efficiently delivered to the cell sheet, providing a sufficient supply of oxygen.

[0051] <Reference example 1> 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 ultra-pure 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 would not increase even if an electric field was applied.

[0052] [Second embodiment] FIG. 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 organism), is immersed in the medium 4 and skewered on a needle electrode 16. The portion of the needle electrode 16 not covered by the spheroid 17 is insulated to prevent current from flowing. The needle electrode 16 is also covered with a PFA mesh to prevent contact between the needle electrode 16 and the spheroid 17. A voltage is applied between the electrode 5 and the needle 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 electrode 16.

[0053] By using oxygen as the gas in the fine bubbles, spheroids can be cultured while reducing the oxygen deficiency inside them.

[0054] 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.

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. The 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. 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. 4. 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. 8. The 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 electrophoretically migrate 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. 8. 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. 2. 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. The method for delivering fine bubbles to cells 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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