Micronozzle device

Abstract

To provide a micronozzle device allowing a lipid vesicle to act on a biological cell while controlling a culture environment of any local region of the biological cell.SOLUTION: A micronozzle device has: two capillaries for discharge whose one end is a discharge opening; one or two capillaries for suction whose one end is a suction opening; a discharge pump which discharges fluid from the capillary for discharge; and a suction pump which sucks fluid from the capillary for suction, where one capillary for discharge of the two capillaries for discharge discharges chemical solution controlling a culture environment of a biological cell, and the other capillary for discharge discharges liquid including a lipid vesicle.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a micronozzle device capable of discharging and sucking a fluid into a minute area. [Background technology]

[0002] Conventionally, when modifying the function of cells or measuring the cellular response, the method has been used to target a large number of whole cells in a system in which a large number of cells are cultured. However, for reasons such as (1) the possibility that drugs used as an auxiliary for functional modification may affect cells, (2) the need to examine the interaction between functionally modified cells and other cells, and (3) the high cost of the drugs used, it is desirable to locally manipulate the cellular environment of a single cell or a small number of cells.

[0003] In particular, it is known that the fusion of virus particles with cell membranes (i.e., infection of cells) generally occurs via proteins on the lipid membrane of the virus. If this mechanism can be elucidated and inhibitors of cell membrane fusion can be discovered, it will greatly contribute to the development of drugs that can suppress viral infections. For this reason, screening for inhibitors and promoters of cell membrane fusion is extremely important in infectious disease research.

[0004] However, changes in the cell culture environment caused by inhibitors or fusogens against cell membrane fusion may themselves be harmful to cells. Therefore, it is necessary to control the local culture environment of a single cell or a small number of cells, rather than performing virus fusion experiments with drugs added to the entire culture medium.

[0005] In the past, it was common to irradiate a caged compound with laser light in order to locally stimulate living cells using drugs. A caged compound is a compound in which a photodegradable protective group (cage) is bound to a biologically active substance, and the protective group is degraded by irradiation with visible or infrared light, resulting in the expression of the biological activity. Examples of biologically active substances that can be bound to the cage include neurotransmitters, low-molecular-weight ligands, and second messengers. By changing the molecular structure of such caged compounds with laser light or degrading the molecules (uncaging), the resulting molecules can be used to locally stimulate living cells.

[0006] However, while the method of irradiating a caged compound with a laser beam can stimulate a size range of several tens of micrometers by limiting the area where the laser beam is focused, there are problems in that the types of molecules that can be applied are limited, and that the caged compound before the reaction and the molecules generated (dissociated) after the reaction can affect living cells. In addition, it is necessary to use a costly and complex device equipped with a laser light source and a photodetector.

[0007] On the other hand, it is possible to locally stimulate biological cells by flowing a drug through a microchannel, but this requires that the biological cells be grown on the microchannel, and it is difficult to stimulate a desired location within the cell by flowing a drug.

[0008] Given the above background, there has been a demand for an ejection nozzle device that can eject a fluid into any minute area and limit the area in which the fluid acts to that minute area, thereby locally changing the cell culture environment.

[0009] As an example of such a discharge nozzle device, Non-Patent Document 1 describes the formation of nanowires using a nozzle chemical pen having two discharge nozzles and one suction nozzle. According to this, a solution of a silver compound (Ag(NH3)2OH) is discharged from one discharge nozzle, and a solution of a reducing agent that reduces the silver compound and precipitates silver is discharged from the other discharge nozzle, and the remaining liquid is sucked up by the suction nozzle, thereby forming silver nanowires directly on a substrate. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Lin HF, et al. Anal. Chem. 2019, 91, 7346-7352(2019). Summary of the Invention [Problem to be solved by the invention]

[0011] However, although the nozzle chemical pen disclosed in Non-Patent Document 1 describes the formation of metal nanowires, there is no description of a chemical pen that performs biological control, such as the manipulation of living cells.

[0012] The present invention has been made in consideration of the above circumstances, and aims to provide a micronozzle device that is capable of acting on lipid vesicles on biological cells while controlling the culture environment of any local region of the biological cells. [Means for solving the problem]

[0013] That is, in order to solve the above problems, the present invention proposes the following means. (1) A micronozzle device according to a first aspect of the present invention comprises two discharge capillaries each having a discharge opening at one end, one or two suction capillaries each having a suction opening at one end, a discharge pump for discharging a fluid from the discharge capillary, and a suction pump for sucking a fluid from the suction capillary, wherein one of the two discharge capillaries discharges a medicinal solution for controlling a culture environment of biological cells, and the other discharge capillary discharges a liquid containing lipid vesicles.

[0014] (2) A micronozzle device according to a second aspect of the present invention is the micronozzle device of the first aspect, characterized in that the discharge opening and the suction opening each have an opening diameter of 1 mm or less.

[0015] (3) A micronozzle device according to a third aspect of the present invention is characterized in that, in the first or second aspect, the discharge capillary and the suction capillary are made of a material containing silicon.

[0016] (4) A fourth aspect of the present invention relates to a resin extrusion molding, and is characterized in that in any one of the first to third aspects, the discharge capillary is formed so that its inner diameter is larger than the opening diameter of the discharge opening upstream of the discharge opening, and the suction capillary is formed so that its inner diameter is larger than the opening diameter of the suction opening upstream of the suction opening.

[0017] (5) A resin extrusion molding according to a fifth aspect of the present invention is characterized in that, in any one of the first to fourth aspects, the sum of the flow rates of the fluids sucked in through the suction opening is greater than the sum of the flow rates of the fluids discharged from the discharge opening. Effect of the Invention

[0018] According to the present invention, it is possible to provide a micronozzle device capable of acting on a biological cell with a lipid vesicle while controlling the culture environment of an arbitrary local region of the biological cell. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is an enlarged perspective view of a main portion of a micronozzle device according to an embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing an arrangement of discharge openings and suction openings of a micronozzle device. [Diagram 3] FIG. 1 is a schematic diagram showing the state of control of a biological cell. [Figure 4] FIG. 13 is a schematic diagram showing another embodiment. [Diagram 5] 1 is a photograph showing the results of locally fusing a biological sample into a biological membrane using the micronozzle device of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Hereinafter, a micro nozzle device according to an embodiment of the present invention will be described with reference to the drawings. Each embodiment shown below is specifically described to better understand the gist of the invention, and does not limit the present invention unless otherwise specified. Furthermore, the drawings used in the following description may show essential parts enlarged for convenience in order to make the features of the present invention easier to understand, and the dimensional ratios of each component may not necessarily be the same as the actual ones.

[0021] Fig. 1 is an enlarged perspective view of a main portion of a micronozzle device according to one embodiment of the present invention, and Fig. 2 is a schematic diagram showing the arrangement of the discharge openings and suction openings of the micronozzle device. The micronozzle device 10 of this embodiment has a first discharge capillary 11 and a second discharge capillary 12, discharge pumps 21 and 22 which supply fluid to the first and second discharge capillaries 11 and 12, respectively, to discharge the fluid, a suction capillary 15, and a suction pump 25 which sucks the fluid from the suction capillary 15.

[0022] The first and second discharge capillaries 11 and 12 are, for example, hollow capillaries made of glass, with one end (opening end) serving as discharge openings 11a and 12a, respectively. The suction capillary 15 is, for example, a hollow capillary made of glass, with one end (opening end) serving as a suction opening 15a. The first and second discharge capillaries 11 and 12 and the suction capillary 15 are bundled together near one end to form a nozzle unit 16.

[0023] The first and second discharge capillaries 11, 12 and the suction capillary 15 may be made of a vitreous material containing silicon, such as fused silica, in addition to glass.

[0024] The discharge openings 11a, 12a and the suction opening 15a are open on the same plane. In this embodiment, the discharge openings 11a, 12a, and 13a are arranged so that their centers are located at the three vertices of a triangle T. That is, in the micronozzle device 10 of this embodiment, the suction opening 15a is located between the discharge opening 11a and the discharge opening 12a and at a position shifted from the line connecting the discharge opening 11a and the discharge opening 12a.

[0025] The discharge openings 11a, 12a of the first and second discharge capillaries 11, 12 may each have an opening diameter of 1 mm or less, for example, about 0.5 mm to 0.01 mm. The suction opening 15a of the suction capillary 15 may each have an opening diameter of 1 mm or less, for example, about 0.5 mm to 0.01 mm. The discharge opening 11a, discharge opening 12a, and suction opening 15a may each have a different opening diameter.

[0026] In this embodiment, the opening diameters of the discharge openings 11a, 12a and the suction opening 15a are each 0.32 mm. The outer diameters of the first and second discharge capillaries 11, 12 and the suction capillary 15 are each about 0.42 mm.

[0027] The discharge pumps 21, 22 and the suction pump 25 may be selected according to the type of fluid, and may be, for example, a syringe pump. Note that the discharge pumps 21, 22 and the suction pump 25 may be various pumps other than a syringe pump, such as a tube pump.

[0028] Of the ejection capillaries 11 and 12 constituting the micronozzle device 10 of this embodiment, the first ejection capillary 11 ejects a chemical liquid for controlling the culture environment of biological cells, and the second ejection capillary 12 ejects a liquid containing lipid vesicles.

[0029] Examples of the chemical liquid for controlling the culture environment of biological cells that is discharged from the first discharge capillary 11 include a low pH solution (such as citrate buffer, pH 3 to 6) as a fusion promoter, and fusion inhibitors such as metalloprotease inhibitors marimastat, prinomastat, and nafamostat, a TMPRSS2 inhibitor camostat, a cathepsin B inhibitor CA-074Me, epigallocatechin gallate (EGCG) derivatives, N-(4-hydroxyphenyl)retinamide (fenretinide), which reduces the fluidity of cell membranes, and Filipin III.

[0030] In addition, examples of the liquid containing lipid vesicles to be discharged from the second discharge capillary 12 include artificial lipid membrane vesicles (for example, vesicles containing the following lipid molecules: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolaminecholesterol (DOPE)), exosomes derived from living organisms, viruses, and virus budding particles derived from cells.

[0031] By placing the suction capillary 15 in the solution, the residual liquid after the reaction of the liquid discharged from the first and second discharge capillaries 11 and 12 described above is sucked in together with the surrounding solution. It is preferable that the total flow rate of the fluid sucked through the suction opening 15a is greater than the total flow rate of the fluid discharged through the discharge openings 11a and 12a.

[0032] The operation of the micronozzle device 10 having the above configuration will now be described. As shown in Figures 1 and 3, for example, a micronozzle device 10 is used to control the culture environment of one or several biological cells M, and lipid vesicles such as virus particles or extracellular vesicles are fused to the cell membrane of the biological cells M, and the changes are observed.

[0033] First, the nozzle unit 16 of the micronozzle device 10 is aligned with the position of the biological cell M to be observed. Then, the chemical solution Q1 for controlling the culture environment of the biological cell M is discharged from the first discharge capillary 11 toward the target biological cell M. This allows the chemical solution Q1 to selectively permeate only the target biological cell M, and the culture environment is controlled to a desired state.

[0034] Next, the liquid Q2 containing lipid vesicles to be fused with the target biological cells M is discharged from the second discharge capillary 12. This results in a state in which lipid vesicles are selectively present only in the biological cells M in a controlled culture environment. At the same time, the suction capillary 15 sucks in the discharged surplus drug solution Q1 and liquid Q2.

[0035] As a result, the medicinal liquid Q1 discharged from the discharge opening 11a and the liquid Q2 discharged from the discharge opening 12a are sucked in through the suction opening 15a, so that they do not spread to other biological cells present around the target biological cell M, and the medicinal liquid Q1 and the lipid vesicles contained in the liquid Q2 can be selectively acted on only the target biological cell M.

[0036] Then, by observing the action of the lipid vesicles on the target biological cells M, it is possible to determine whether or not the biological cells M are infected by viral particles or extracellular vesicles.

[0037] In this embodiment, the range of the biological cells M to be acted on can be controlled arbitrarily by making the amount of the liquid Q1 discharged from the discharge opening 11a, the amount of the liquid Q2 discharged from the discharge opening 12a, and the amount of the residual liquid sucked from the suction opening 15a different from one another. In particular, the range can be limited to a small range by making the total amount of the suction larger than the total amount of the discharge. For example, in this case, it is preferable to set the ratio of the amount of the suction from the suction opening 15a to the amount of the liquid Q1 or the liquid Q2 discharged to a range of 20 to 500.

[0038] Moreover, the range of the biological cells M to be acted on can be controlled arbitrarily by individually varying the viscosity of the medical fluid Q1 in the discharge opening 11a and the viscosity of the liquid Q2 in the discharge opening 12a.

[0039] Furthermore, by controlling the distance between the discharge opening 11a, the discharge opening 12a, and the substrate on which the biological cells M are placed, the range of the biological cells M to be acted on can be controlled arbitrarily.

[0040] In this embodiment, an example has been shown in which the micronozzle device is configured with a total of three capillaries, that is, two discharge capillaries and one suction capillary, but the number of suction capillaries can also be two. FIG. 4 is a schematic diagram showing a micronozzle device according to a modified example of the present embodiment described above.

[0041] The micronozzle device 30 of this embodiment has a first discharge capillary 11, a second discharge capillary 12, discharge pumps 21, 22 which supply fluid to the first and second discharge capillaries 11, 12, respectively, to cause them to discharge, a first suction capillary 31, a second suction capillary 32, and suction pumps 26, 27 which suck in fluid from the first and second suction capillaries 31, 32.

[0042] 5 is a photograph showing the result of locally fusing a biological sample into a biological membrane using the micronozzle device of this embodiment. The conditions were as follows: the opening diameter of the discharge openings 11a, 12a and the suction opening 15a was 95 micrometers, the ratio of the suction amount of liquid to the discharge amount was 25, and the distance from each opening end to the substrate on which the biological cell M was placed was 40 micrometers.

[0043] Figure 5(A) is a fluorescent image of nitrobenzoxadiazole (NBD), and Figure 5(B) is a fluorescent image of rhodamine B (Rho-B). In addition, the outer and inner circumferences of the multiple circles in Figure 5 are superimposed on the fluorescent image so that the positions of the respective opening ends can be seen.

[0044] In this example, a lipid support membrane (DOPC) containing NBD was formed on a glass substrate, and a vesicle, which is a biological sample, was fused to the lipid support membrane using the micronozzle device of this embodiment. The vesicle was 18:1 DGS (1,2-dioleoyl-sn-glycero-3-succinate) containing Rho-B. This vesicle has the property of fusing with a lipid membrane at low pH.

[0045] The micronozzle device of this embodiment was placed 40 micrometers above the glass substrate on which the lipid support membrane was formed and which contained a pH 7.0 buffer solution. From above, 18:1 DGS containing Rho-B was discharged into Q1, and citrate buffer (pH 4.0) was discharged into Q2, and the solution was then sucked in through the suction opening 15a. After 20 minutes, the lipid membrane was observed with a fluorescent microscope. By using this single suction opening 15a, the two solutions, Q1 and Q2, were mixed locally.

[0046] In Fig. 5(A), it can be seen that the lipid support membrane is shown as a circle that exhibits fluorescence. Meanwhile, the area where the fluorescence is weak, indicated by the arrow, is shown as a line. Also, in Fig. 5(B), it can be seen that there is a linear area that exhibits fluorescence, as indicated by the arrow. This linear fluorescent area coincides with the area indicated by the arrow in Fig. 5(A). This linear area is formed in the area where the solutions ejected from the two ejection openings 11a, 12a are mixed.

[0047] The above results show that the micronozzle device of this embodiment can be used to locally fuse vesicles to a lipid support membrane. The local decrease in fluorescence intensity seen in Figure 5(A) is due to the decrease in NBD fluorescence caused by intermolecular energy transfer called FRET.

[0048] By providing the first and second suction capillaries 31, 32 as in this embodiment, it becomes possible to lay out fine regions in a greater variety of shapes in which a liquid containing a drug solution or lipid vesicles that controls the culture environment of biological cells can be applied.

[0049] Although the embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope of the invention and its equivalents as described in the claims, as well as in the scope and spirit of the invention. [Explanation of symbols]

[0050] 10. Micro nozzle device 11...First discharge capillary 12...Second discharge capillary tube 16…Nozzle unit 11a, 12a...Discharge opening 15...Capillary tube for suction 15a...Suction opening 21, 22...Discharge pump 25…Suction pump

Claims

1. The fluid supply device includes two discharge capillaries each having a discharge opening at one end, one or two suction capillaries each having a suction opening at one end, a discharge pump for discharging a fluid from the discharge capillaries, and a suction pump for sucking a fluid from the suction capillary, A micronozzle device, characterized in that of the two ejection capillaries, one of the ejection capillaries ejects a chemical solution for controlling the culture environment of biological cells, and the other of the ejection capillaries ejects a liquid containing lipid vesicles.

2. 2. The micronozzle device according to claim 1, wherein the discharge opening and the suction opening each have a diameter of 1 mm or less.

3. 3. The micronozzle device according to claim 1, wherein the discharge capillary and the suction capillary are made of a material containing silicon.

4. 3. The micronozzle device according to claim 1, wherein the discharge capillary is formed so that an inner diameter thereof is larger than an opening diameter of the discharge opening on an upstream side of the discharge opening, and the suction capillary is formed so that an inner diameter thereof is larger than an opening diameter of the suction opening on an upstream side of the suction opening.

5. 3. The micronozzle device according to claim 1, wherein a flow rate of the fluid sucked through the suction opening is greater than a sum of flow rates of the fluid discharged from the discharge opening.

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