Droplet forming device and method for producing gel particles

Abstract

To provide a droplet forming device and a method for producing gel particles, each enabling production of droplets having uniform size.SOLUTION: A droplet forming device comprises an ejection head that ejects droplets of liquid. The ejection head has a cylindrical liquid retention member that retains the liquid, and a vibration unit that covers one end of the liquid retention member and, together with the liquid retention member, forms a liquid chamber retaining the liquid, and ejects droplets based on an applied electrical signal. The liquid chamber has the other end of the liquid retention member that is open to the atmosphere. The vibration unit has a vibration member that vibrates based on the electrical signal, a film-like member with an ejection port through which droplets are ejected, and a resonating member that vibrates based on the vibration of the vibration member and transmits the vibration of the vibration member to the film-like member by vibrating itself. The resonating member is a plate-like member that overlaps and comes in contact with the film-like member in a planar manner.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a droplet forming device and a method for producing gel particles. [Background technology]

[0002] Gel particles are particles made of gel. Gels are polymers with a three-dimensional network structure or swollen polymers that can retain a large amount of water within the network structure. Such gel particles can encapsulate various functional molecules such as drugs, proteins, and dyes, and have recently attracted attention as functional materials in a wide range of fields, including medicine, food, cosmetics, optics, and printing.

[0003] Known methods for producing such gel particles include a method using emulsion polymerization and a method using polyion complex formation.

[0004] Patent Document 1 describes a method using emulsion polymerization in which a w / w emulsion is prepared, then emulsified with an oil phase to prepare a w / w / o emulsion, and the outer aqueous phase is gelled to produce gel particles of uniform size with a narrow particle size distribution.

[0005] Furthermore, Patent Document 2 describes a method of producing gel particles using polyion complex formation, in which a first polymer electrolyte solution containing a first polymer electrolyte is ejected from the tip of a needle and the formed microdroplets are brought into contact with a second polymer electrolyte solution containing a second polymer electrolyte. In the invention described in Patent Document 2, a voltage is applied to the needle when the first polymer electrolyte solution is ejected from the tip of the needle, thereby strongly charging the formed droplets and suppressing the coalescence of the droplets during flight, thereby producing gel particles of uniform size. Summary of the Invention [Problem to be solved by the invention]

[0006] From the viewpoint of physical stability and quality stability of the product, such gel particles are required to have small particle size variations. However, the methods of Patent Documents 1 and 2 above have room for improvement as follows.

[0007] When producing gel particles using the emulsion polymerization manufacturing method described in Patent Document 1, a washing process is required to remove the oil phase solvent and surfactant used during gel particle formation from the formed gel particles. This washing process requires repeated washing using organic solvents such as ethanol, hexane, and acetone, which has the disadvantage of requiring a lot of time and large amounts of water. Furthermore, if the formed gel particles undergo multiple washing steps, the mechanical stress during washing may damage the gel particles. Damage to the gel particles may result in the formation of gel particle fragments, which may lead to variations in particle size distribution.

[0008] Furthermore, in the manufacturing method of Patent Document 2, the liquid ejected from the manufacturing equipment is strongly charged to prevent droplets from coalescing. In this case, if the electrostatic repulsive force of the charged charge exceeds the surface tension of the droplets, the droplets may further break up into multiple smaller droplets in flight, forming gel particles with a varied particle size distribution. Therefore, in order to prevent excessive charging of the liquid, the manufacturing method of Patent Document 2 requires consideration of appropriately controlling the charging environment in which the droplets are placed and increasing the surface tension by appropriately selecting the droplet solvent and solute.

[0009] In the invention described in Patent Document 1, when the first polymer electrolyte solution is ejected from the tip of the needle, a voltage is applied to the needle to strongly charge the droplets that are formed. This causes the charged droplets to fly, suppresses the droplets from coalescing during flight, and produces microcapsules of uniform size.

[0010] When microcapsules encapsulating cells are produced by the method described in Patent Document 1, the cells are exposed to an electrically charged environment while droplets are being sprayed from the needle and inside the flying droplets. Therefore, with the method described in Patent Document 1, electrical stimulation is applied to the cells during the production of the microcapsules, which raises concerns about adverse effects on the cells.

[0011] The above-mentioned problems in the prior art can be solved if a droplet formation device capable of forming droplets of uniform size can be realized. Conventional device configurations have room for improvement in terms of forming droplets of uniform size.

[0012] The present invention has been made in view of the above circumstances, and has an object to provide a droplet forming device that can suitably produce droplets of uniform size, and a further object to provide a gel particle manufacturing method that can suitably produce gel particles of uniform size. [Means for solving the problem]

[0013] In order to solve the above problems, one aspect of the present invention provides a droplet forming device comprising an ejection head that ejects droplets of liquid, the ejection head having a cylindrical liquid holding member that holds the liquid, and a vibration unit that covers one end side of the liquid holding member and forms a liquid chamber that holds the liquid together with the liquid holding member, and ejects the droplets based on an applied electrical signal, the liquid chamber having the other end side of the liquid holding member open to the atmosphere, the vibration unit having a vibration member that vibrates based on the electrical signal, a film-like member having an ejection port through which the droplets are ejected, and a resonant member that vibrates based on the vibration of the vibration member and transmits the vibration of the vibration member to the film-like member by vibrating itself, the resonant member being a plate-like member that overlaps with the film-like member in a planar manner and is in contact with the film-like member. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide a droplet forming device that can suitably produce droplets of uniform size, and a method for producing gel particles that can suitably produce gel particles of uniform size. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of a droplet forming device 1 according to the first embodiment. [Figure 2] FIG. 2 is a partially enlarged view of the droplet forming device 1. As shown in FIG. [Figure 3] FIG. 3 is an exploded perspective view of the ejection head 110A. [Figure 4] FIG. 4 is an explanatory diagram of a method for measuring the resonant frequency of the structural vibration of the vibration means. [Figure 5] FIG. 5 is a diagram showing an example of a drive waveform supplied to ejection head 110A. [Figure 6] FIG. 6 is an explanatory diagram of a droplet forming device 2 according to the second embodiment. [Figure 7] FIG. 7 is an explanatory diagram of a droplet forming device 2 according to the second embodiment. [Figure 8] FIG. 8 is a graph showing the particle size distribution of the gel particles obtained in Example 1. [Figure 9] FIG. 9 is an image of the cells in the gel particles obtained in Example 3 observed under a phase contrast microscope. DETAILED DESCRIPTION OF THE INVENTION

[0016] [First embodiment] A droplet forming device and a method for producing gel particles according to a first embodiment will be described below with reference to Figures 1 to 5. In this embodiment, the droplet forming device is used to form droplets that are precursors of gel particles. In all of the following figures, the dimensions and proportions of each component have been appropriately changed to make the drawings easier to understand.

[0017] In the following explanation, an xyz Cartesian coordinate system is set, and the positional relationship of each component is explained with reference to this xyz Cartesian coordinate system. Here, a specific direction in a horizontal plane is defined as the x direction, a direction perpendicular to the x direction in the horizontal plane is defined as the y direction, and a direction perpendicular to both the x and y directions (i.e., the vertical direction) is defined as the z direction.

[0018] In addition, the vertically upward direction is the +z direction, and the vertically downward direction is the -z direction. In the following explanation, the "upper" in "upper" and "upper surface" and the "lower" in "lower" and "lower surface" have the same meaning.

[0019] Furthermore, in the following description, "planar view" refers to viewing an object from above (+z direction), and "planar shape" refers to the shape of an object viewed from above.

[0020] Gel particles In the droplet formation device and gel particle manufacturing method of this embodiment, droplets DR of a first solution L1 containing a first substrate are ejected and brought into contact with a second solution L2 containing a second substrate that reacts with the first substrate to form a hydrogel, thereby producing gel particles P in which a core C is covered with a hydrogel membrane HM (see FIG. 1).

[0021] As used herein, the term "gel" refers to a polymer having a three-dimensional network structure or a swollen body of such a polymer. A typical example of a gel is a hydrogel. As used herein, the term "hydrogel" refers to a gel that retains a large amount of water in the space within the three-dimensional network structure of the polymer. Hydrogels are also called "water-containing gels."

[0022] (1st substrate, 2nd substrate) The first substrate and the second substrate are not particularly limited as long as they form a crosslinked structure and gel when mixed together, and can be appropriately selected depending on the purpose.

[0023] Specific examples of the first substrate include biopolymers such as collagen, elastin, gelatin, and fibroin; coagulation factors such as fibrinogen; adhesion factors such as fibronectin, laminin, and recombinant peptides; metal salts of polysaccharide compounds such as alginic acid and gellan gum; and synthetic polymers such as polylactic acid and polyethylene glycol. These may be used alone or in combination of two or more.

[0024] Specific examples of the second substrate include polysaccharides, polyvalent metal salts, fibrinogen, thrombin, fibronectin, laminin, recombinant peptides, chitosan, chitin, tetrafunctional polyethylene glycol (Tetra-PEG), etc. These may be used alone or in combination of two or more.

[0025] It is advisable to carry out a preliminary experiment in advance to determine the combination of the first substrate and the second substrate that will give a hydrogel with the desired physical properties.

[0026] The resulting hydrogel is preferably a material that functions as a scaffold for cell culture. For example, when sodium alginate is used as the first substrate and a calcium salt such as calcium chloride is used as the second substrate, the resulting hydrogel (calcium alginate) is a material that can be used as a cell scaffold for research purposes and is preferred.

[0027] The first solution L1 may also contain dispersed therein a substance (dispersoid) to be encapsulated in the resulting gel particles P. Such a substance contained in the first solution L1 and intended to be encapsulated in the gel particles P may hereinafter be referred to as an "encapsulated substance." As a result, the resulting gel particles P become particles that contain (encapsulate) the encapsulated substance.

[0028] The solvent (dispersion medium) of the first solution L1 is not particularly limited as long as it is an aqueous solution that can dissolve the first substrate and disperse the encapsulated substance in. Examples of the dispersion medium include known buffer solutions such as phosphate buffered saline and Hank's balanced salt solution, and culture media suitable for the cells used.

[0029] The solvent (dispersion medium) of the second solution L2 is not particularly limited as long as it is an aqueous solution that can dissolve the second substrate and disperse the encapsulated substance in. The solvent (dispersion medium) of the second solution L2 can be the same as the solvent (dispersion medium) of the first solution L1 described above.

[0030] The first solution L1 may contain either or both of cells and spheroids as encapsulated substances (dispersoids).

[0031] (cell) A representative example of the encapsulated substance contained in the first solution L1 is cells. By using cells as the encapsulated substance, the resulting gel particles P become hydrogel particles encapsulating cells.

[0032] There are no particular restrictions on the type of cells, and they can be selected appropriately depending on the purpose. All cells can be used, regardless of taxonomic classification, for example, karyotic cells, prokaryotic cells, multicellular organism cells, or unicellular organism cells.

[0033] Examples of eukaryotic cells include animal cells, insect cells, plant cells, and fungi. These may be used alone or in combination of two or more. Among these, animal cells are preferred, and when the cells form cell aggregates, adhesive cells that adhere to each other and have such cell adhesiveness that they cannot be isolated without physicochemical treatment are more preferred.

[0034] There are no particular limitations on the adhesive cells, and they can be appropriately selected depending on the purpose. Examples include differentiated cells and undifferentiated cells.

[0035] Examples of differentiated cells include hepatocytes, which are parenchymal cells of the liver; stellate cells; Kupffer cells; vascular endothelial cells; endothelial cells such as meatal endothelial cells and corneal endothelial cells; fibroblasts; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells such as epidermal keratinocytes; tracheal epithelial cells; digestive tract epithelial cells; cervical epithelial cells; epithelial cells such as corneal epithelial cells; mammary gland cells; pericytes; muscle cells such as smooth muscle cells and cardiac muscle cells; kidney cells; pancreatic islet cells of Langerhans; nerve cells such as peripheral nerve cells and optic nerve cells; chondrocytes; bone cells, etc. The adhesive cells may be primary cells directly collected from tissues or organs, or may be cells that have been passaged for several generations.

[0036] The undifferentiated cells are not particularly limited and can be appropriately selected depending on the purpose. Examples include undifferentiated cells such as embryonic stem cells and pluripotent stem cells such as mesenchymal stem cells that have the ability to differentiate into different types; unipotent stem cells such as vascular endothelial progenitor cells that have the ability to differentiate into a single type; and iPS cells.

[0037] The above-mentioned cells may form a cell mass (spheroid).

[0038] The gel particles P obtained in this manner may have a core-shell structure in which the first solution L1 is encapsulated as the core C and the core C is surrounded by a membrane HM. Furthermore, the gel particles P obtained may be gelled all the way to the center (core C) as long as at least the surface is covered with hydrogel. More specifically, since the core C contains the first substrate, the first substrate of the core C may also be gelled by reacting with the second substrate.

[0039] The average particle size of the gel particles P is not particularly limited, but is preferably 1 μm or more and 1000 μm or less. For example, when the encapsulated substances contained in the first solution L1 are cells, the average particle size of the gel particles P is preferably 5 μm or more.

[0040] From the viewpoint of physical stability and quality stability of the product, it is preferable that the gel particles P have little variation in particle size. Furthermore, if the gel particles P contain an encapsulated substance, the function and reactivity of the encapsulated substance will be affected, so it is preferable that the particle size distribution of the gel particles P is uniform and the particle size is uniform.

[0041] If the particle size of the gel particles P varies greatly, the following concerns arise. For example, if the encapsulated substance is a drug, the sustained release properties may differ for each gel particle P, which may result in variations in the efficacy of the drug. Also, if the encapsulated substance is a cell, the amount of oxygen and nutrients that reach the gel particle P may differ for each gel particle P, which may affect the survival rate of the cells.

[0042] The variation in particle size of the gel particles P can be expressed by the coefficient of variation (CV). The coefficient of variation of the gel particles P can be calculated using the formula (standard deviation) ÷ (average particle size) × 100. The smaller the coefficient of variation, the smaller the variation. The coefficient of variation is preferably in the range of 20% or less.

[0043] The average particle size and coefficient of variation of the gel particles P can be determined by taking a magnified image of the collection of gel particles P obtained using a phase contrast microscope (e.g., Olympus CKX53) and analyzing the obtained image. Image processing software (Image J) can be used to analyze the image and determine the average particle size and coefficient of variation of the gel particles P.

[0044] The average particle size of the gel particles P can be adjusted by appropriately using the ejection conditions, such as the opening diameter of the ejection port and the magnitude of the electrical signal supplied to the ejection section by the control device (described later), in the manufacturing apparatus and manufacturing method described below.

[0045] The droplet forming device of this embodiment described below can effectively form droplets DR from the above-mentioned first solution L1 and react the droplets DR with the second solution L2, thereby obtaining gel particles with a small coefficient of variation of 20% or less and a uniform particle size.

[0046] [Droplet formation device] Fig. 1 is a schematic diagram of a droplet forming device 1 of this embodiment. Fig. 2 is a partial enlarged view of the droplet forming device 1. As shown in Figs. 1 and 2, the droplet forming device 1 has a discharge unit 10 and a control device 50. The droplet forming device 1 may further have a storage unit 30 and a placement unit 40.

[0047] 《Discharge part》 As shown in FIG. 1, the ejection unit 10 includes an ejection head 110 and a transport unit 120.

[0048] <Discharge head> The ejection head 110 employs a so-called inkjet method, and ejects the first solution L1 held in the ejection head 110 to form droplets DR.

[0049] The "inkjet method" is a method of ejecting liquid, which is the material for droplets, in small amounts with high precision from the nozzles of an ejection head. In an ejection head that uses the inkjet method, stress such as pressure or inertial force is applied instantaneously to the liquid stored in the ejection head. As a result, the liquid in the ejection head near the nozzle separates into tiny liquid particles in response to the applied stress, forming droplets.

[0050] For example, when gel particles are produced using the emulsion polymerization method described in Patent Document 1, a washing process is required to remove the oil phase solvent and surfactant used during gel particle formation from the formed gel particles. This washing process requires repeated washing using organic solvents such as ethanol, hexane, and acetone, which has the disadvantage of requiring a lot of time and large amounts of water. Furthermore, if the formed gel particles undergo multiple washing processes, the mechanical stress during washing may damage the gel particles. Damage to the gel particles may result in the formation of gel particle fragments, which may lead to variations in particle size distribution.

[0051] Furthermore, in the manufacturing method of Patent Document 2, the liquid ejected from the manufacturing equipment is strongly charged to prevent droplets from coalescing. In this case, if the electrostatic repulsive force of the charged charge exceeds the surface tension of the droplets, the droplets may further break up into multiple smaller droplets in flight, forming gel particles with a varied particle size distribution. Therefore, in order to prevent excessive charging of the liquid, the manufacturing method of Patent Document 2 requires consideration of appropriately controlling the charging environment in which the droplets are placed and increasing the surface tension by appropriately selecting the droplet solvent and solute.

[0052] Furthermore, when encapsulating a desired substance in gel particles produced by the method described in Patent Document 1, it is necessary to set conditions in the step of emulsifying with an oil phase to prepare an emulsion, the step of UV-curing the external aqueous phase, and the washing step, taking into consideration the effects on the function, reactivity, etc. of the encapsulated substance.

[0053] Furthermore, when a desired substance is encapsulated in gel particles produced by the method described in Patent Document 2, the droplets are exposed to an electrically charged environment while being sprayed from the needle and inside the flying droplets, and therefore measures must be taken to prevent the adverse effects of electrical stimulation on the encapsulated substance.

[0054] In contrast, the inkjet discharge head 110 does not use an emulsion or a charged environment when forming the droplets DR. Therefore, by using the inkjet discharge head 110, there is no need to consider the adverse effects on particle size distribution caused by a cleaning process or the influence of overcharging. Furthermore, if an encapsulated substance is dispersed in the first solution L1, by using the inkjet discharge head 110, there is no need to take measures against the adverse effects on the function, reactivity, etc. of the encapsulated substance that may be caused by using an emulsion or a charged environment.

[0055] The discharge section 10 may have only one or more discharge heads 110. The discharge section 10 shown in Fig. 1 has three discharge heads 110a, 110b, and 110c. The three discharge heads 110a, 110b, and 110c are collectively referred to as a discharge unit 110L.

[0056] The ejection heads 110a, 110b, and 110c may have the same configuration or may have different configurations.

[0057] The three ejection heads 110a, 110b, and 110c are arranged in a direction (x direction in the drawing) that intersects with the ejection direction of the liquid ejected from the ejection head 110 (-z direction in the drawing).

[0058] As shown in FIG. 2, the ejection head 110A includes a liquid holding member 111, a vibration unit 115A, and a fixing member 117.

[0059] The space surrounded by the liquid holding member 111 and the vibration unit 115A is a liquid chamber 110S of the ejection head 110. The liquid chamber 110S holds a liquid (first solution L1) that is the source of droplets DR.

[0060] The amount of the first solution L1 held in the liquid chamber 110S is not particularly limited. For example, the amount of the first solution L1 held in the liquid chamber 110S can be approximately 1 μl to 1 ml. When an expensive liquid such as a drug or a cell suspension is ejected from the droplet forming device 1, the amount of the first solution L1 held in the liquid chamber 110S should be approximately 1 μl to 200 μl.

[0061] The ejection heads 110a, 110b, and 110c shown in FIG. 1 may each hold the same first solution L1, or may each hold a different first solution L1.

[0062] The configuration of ejection head 110A will be described in more detail below. Figure 3 is an exploded perspective view of ejection head 110A.

[0063] (liquid holding member) The liquid retention member 111 is a tubular member with both ends in the z direction open. The liquid retention member 111 may be, for example, a cylindrical member. Examples of materials for the liquid retention member 111 include metals such as stainless steel, nickel, and aluminum; plastics (resin materials) such as ABS, polycarbonate, and fluororesin; ceramics such as silicon dioxide, alumina, and zirconia; and silicon.

[0064] One end of the liquid holding member 111, that is, the lower end, is covered and blocked by the vibration unit 115A. The other end of the liquid holding member 111, that is, the upper end, is open to the atmosphere. When the upper part of the liquid holding member 111 is open to the atmosphere, the first solution L1 held in the liquid holding member 111 is less likely to be pressurized when droplets are ejected. Therefore, when cells are contained in the first solution L1, damage to the cells can be suppressed.

[0065] (vibration part) The excitation unit 115A has a nozzle plate (film-like member) 112, an excitation member 113, and a resonating member 114. The excitation unit 115A has the resonating member 114 positioned above and the nozzle plate 112 positioned below, but this is not limiting, and the nozzle plate 112 may be positioned above and the resonating member 114 below.

[0066] (Nozzle plate (film-like member)) The nozzle plate 112 is a film-like member having ejection ports 112x. The nozzle plate 112 closes the lower end of the liquid holding member 111 and, together with the liquid holding member 111, forms a liquid chamber 110S that holds the first solution L1. The ejection ports 112x are in communication with the liquid holding member 111.

[0067] There are no particular limitations on the planar shape, size in plan view, material, and structure of the nozzle plate 112, and they can be selected appropriately depending on the purpose.

[0068] Examples of the planar shape of the outer edge of the nozzle plate 112 include a circle, an ellipse, a rectangle, a square, a diamond, etc. For example, if the shape of the outer edge of the nozzle plate 112 is a circle, the nozzle plate 112 becomes an annular member.

[0069] If the nozzle plate 112 is too thick, it will be difficult to vibrate, and if it is too thin, the vibration will be difficult to stop, reducing the stability of droplet ejection. Therefore, the thickness of the nozzle plate 112 is preferably 5 μm to 500 μm, and more preferably 20 μm to 100 μm.

[0070] As an example, the nozzle plate 112 may be a circular member having a diameter of 20 mm and an average thickness of 0.05 mm (50 μm).

[0071] The nozzle plate 112 is not supported at its end on the ejection port 112x side in the surface direction (the end on the inner periphery of the nozzle plate 112). Therefore, the end of the nozzle plate 112 on the ejection port 112x side can vibrate up and down. When the end on the ejection port 112x side of the nozzle plate 112 vibrates, a downward force is applied to the first solution L1 near the ejection port 112x, causing it to be ejected as droplets DR from the ejection port 112x.

[0072] If the nozzle plate 112 is made of a material that is too soft, it will vibrate easily and it will be difficult to immediately stop the vibrations when no ink is being ejected. Therefore, it is preferable to use a material that has a certain degree of hardness.

[0073] Furthermore, a highly hydrophilic material is preferable for the material of the nozzle plate 112. Furthermore, when the first solution L1 contains cells, a material that has low cytotoxicity and to which cells do not easily adhere is preferable. As such a material, a highly hydrophilic material is preferable.

[0074] Such materials include, for example, metals, ceramics, and polymeric materials.

[0075] More specifically, examples of the material for the nozzle plate 112 include stainless steel, nickel, aluminum, silicon dioxide, alumina, zirconia, ABS, polycarbonate, fluororesin, etc. Furthermore, a composite material can be used in which the surface of the nozzle plate 112 formed from a material other than the above-mentioned materials is coated with the above-mentioned metal, ceramics, or synthetic phospholipid polymer that mimics a cell membrane (for example, Lipidure manufactured by NOF Corporation).

[0076] There are no particular limitations on the number of the discharge ports 112x arranged, the arrangement pattern, the interval (pitch), the opening shape, the opening size, etc., and these can be selected appropriately depending on the purpose.

[0077] The opening shape of the discharge port 112x can be selected appropriately depending on the purpose. Examples of the opening shape of the discharge port 112x include a circle, an ellipse, a rectangle, etc. Among these, a circle is preferable as the opening shape of the discharge port 112x.

[0078] The average opening diameter of the discharge port 112x is not particularly limited and can be appropriately selected depending on the purpose. When the first solution L1 to be discharged is a dispersion liquid, the opening shape of the discharge port 112x is preferably at least twice the maximum diameter of the dispersoids, in order to prevent the dispersoids, such as cells, dispersed in the first solution L1, from clogging the discharge port 112x.

[0079] When the dispersoids dispersed in the first solution L1 are animal cells, particularly human cells, the average opening diameter of the outlets 112x is preferably 10 μm or more and 1000 μm or less.

[0080] The size of human cells varies depending on the cell type, but is generally between 5 μm and 50 μm. Furthermore, when the dispersoid is a cell cluster (spheroid), the size of the cell cluster is several tens of μm to several mm. Therefore, by setting the size of the discharge port 112x as described above and providing the discharge port 112x with an average opening diameter appropriate for the cells to be discharged, clogging of the discharge port can be suppressed.

[0081] It should be noted that while a larger opening diameter of the discharge ports 112x allows relatively large cell aggregates to be discharged, the larger the opening diameter, the more difficult it is to achieve stable discharge. By setting the average opening diameter of the discharge ports 112x to 1000 μm or less, a large number of cell aggregates can be stably discharged. Furthermore, to achieve stable discharge, it is preferable that the upper limit of the average opening diameter of the discharge ports 112x be 200 μm or less.

[0082] Furthermore, the smaller the opening diameter of the discharge port 112x, the more likely shear stress is applied to the cells or cell aggregates passing through the discharge port 112x. Therefore, it is preferable that the average opening diameter of the discharge port 112x is large.

[0083] The position of the discharge ports 112x in the nozzle plate 112 is not particularly limited and can be appropriately selected depending on the purpose. For example, the discharge ports 112x may be located at the center of the nozzle plate 112 when viewed from above, or at a position other than the center of the nozzle plate 112 when viewed from above.

[0084] Furthermore, the number of ejection ports 112x in the nozzle plate 112 may be one or more. A nozzle plate 112 having multiple ejection ports 112x can be suitably employed in a cylindrical liquid retention member 111. In the nozzle plate 112 exposed to the internal space of the liquid retention member 111, the multiple ejection ports 112x are preferably arranged at an equal distance from the central axis of the liquid retention member 111. With such an arrangement, the vibration state of each ejection port 112x in the nozzle plate 112 becomes equivalent, that is, the amplitude and vibration mode of the multiple ejection ports 112x become equivalent, making it possible to equalize whether ejection occurs and the droplet size and ejection speed during ejection.

[0085] Note that the nozzle plate 112 having the plurality of ejection ports 112x can be used as a liquid retention member other than a cylindrical one. The nozzle plate 112 can be used as a liquid retention member of various shapes as long as the vibration state of the nozzle plate 112 at the plurality of ejection ports 112x is equivalent. For example, in the case of an elliptical cylindrical liquid retention member, if ejection ports are provided at positions that overlap with the focal points in the xy cross section (rectangle) of the liquid retention member, the vibration state at each ejection port becomes equivalent, and it becomes possible to equalize the presence or absence of ejection, and the droplet size and ejection speed when ejected.

[0086] Similarly, when the liquid holding member is a rectangular tube, by imagining an xy cross section of the liquid holding member, providing an outlet at an arbitrary point on the cross section, and providing an outlet at a position that is symmetrical (line symmetric, point symmetric) to the arbitrary point on the cross section, the vibration state at each outlet becomes equivalent, and it becomes possible to equalize whether or not ejection occurs, and the droplet size and ejection speed when ejected.

[0087] (Excitation member) The vibration member 113 vibrates the nozzle plate 112 based on an input electrical signal, causing droplets DR to be ejected from the ejection ports 112x. The vibration member 113 is installed on the upper surface of the nozzle plate 112 via a resonating member 114, which will be described later.

[0088] There are no particular limitations on the shape, size, material, and structure of the vibration member 113, and they can be selected appropriately depending on the purpose. There are no particular limitations on the shape and arrangement of the vibration member 113, as long as it can transmit vibration to the resonating member 114.

[0089] Examples of the vibrating member 113 include a piezoelectric element and an electromagnetic solenoid, with a piezoelectric element being preferred. The piezoelectric element may have a structure in which electrodes for applying a voltage are provided on the upper and lower surfaces of a piezoelectric material, for example. In this case, by applying a voltage between the upper and lower electrodes of the piezoelectric element from the control device 50, a compressive stress is applied in the lateral direction of the film, causing the resonating member 114 to vibrate in the vertical direction.

[0090] The piezoelectric material is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include lead zirconate titanate (PZT), bismuth iron oxide, metal niobate, barium titanate, or any of these materials to which a metal or a different oxide has been added. Among these, lead zirconate titanate (PZT) is preferred.

[0091] The vibration mode of a piezoelectric element can be a longitudinal mode that can provide vertical displacement. A longitudinal mode piezoelectric element can be, for example, a stacked type piezoelectric element stacked in the z direction, which expands in the vertical direction (z direction) and contracts in the horizontal direction (x and y directions) when a voltage is applied.

[0092] The shape of the piezoelectric element is not particularly limited, but a cylindrical or rectangular shape is preferable. In Fig. 3, a cylindrical laminated PZT is used as the vibration member 113.

[0093] (resonating member) The resonator member 114 is a plate-like member that contacts the nozzle plate 112 and transmits the vibration of the vibrator member 113 to the nozzle plate 112. More specifically, the resonator member 114 vibrates based on the vibration of the vibrator member 113, and transmits the vibration of the vibrator member 113 to the nozzle plate 112 by vibrating itself. The resonator member 114 is thicker and more rigid than the nozzle plate 112.

[0094] The resonator member 114 is a member having a circular ring shape in a planar view. The inner holes 114x of the resonator member 114 are arranged concentrically with the ejection ports 112x. When the nozzle plate 112 has a plurality of ejection ports 112x, the holes 114x are arranged at positions surrounding the plurality of ejection ports 112x in a planar view. The upper surface 114a of the resonator member 114 holds the liquid holding member 111 and the vibration member 113. The lower surface 114b of the resonator member 114 holds the nozzle plate 112.

[0095] The shape of the resonating member 114 is not limited to a circular shape when viewed from above, and various shapes can be adopted as long as it is in contact with the nozzle plate 112 and the vibrating member 113 and can transmit the vibration of the vibrating member 113 to the nozzle plate 112.

[0096] The resonating member 114 and the vibrating member 113 are bonded together with an adhesive (not shown). The elastic modulus of the adhesive is preferably 1 MPa to 1 GPa, and more preferably 10 MPa to 100 MPa, so as not to inhibit the displacement of the resonating member 114. The thickness of the adhesive layer is preferably 1 mm or less, and more preferably 200 μm or less, so as not to inhibit the displacement of the resonating member 114.

[0097] The inner peripheral end of the resonator member 114 is exposed to the liquid chamber 110S. Therefore, the resonator member 114 is preferably made of a material that does not affect the first solution L1. In particular, when the first solution L1 contains cells, it is preferable that the resonator member 114 be made of a material with low cytotoxicity, or that the inner peripheral end be coated with a resin material or the like with low cytotoxicity.

[0098] The material of the resonator member 114 is preferably a metal material with low brittleness, a synthetic resin, etc. The resonator member 114 made of such a material is less likely to be damaged even when vibrations from the vibrator member 113 are transmitted repeatedly.

[0099] When a predetermined electric signal (voltage pulse) is applied to the vibration member 113, the vibration member 113 vibrates the resonator member 114. The vibration of the resonator member 114 is further transmitted to the nozzle plate 112, and pressure is applied locally to the first solution L1 near the nozzle plate 112 in the liquid chamber 110S, causing a flow of the first solution L1 toward the discharge port 112x. A portion of the first solution L1 flowing in this manner is discharged from the discharge port 112x as droplets DR.

[0100] In the droplet forming device 1, the above operation prevents a large pressure from being applied to the entire liquid chamber, as is the case with known inkjet heads having closed liquid chambers. Therefore, when an encapsulated substance is dispersed in the first solution L1, mechanical stress is unlikely to be applied to the dispersion. Furthermore, when the dispersion is cells, this is preferable because the cells in the dispersion are unlikely to be damaged when the dispersion is ejected.

[0101] 2 and 3, the resonating member 114 is in direct contact with the exciting member 113, but this is not limiting. As long as the vibration of the exciting member 113 is transmitted to the resonating member 114, a spacer or an adjustment member used to adjust the resonance frequency of the resonating member 114 may be sandwiched between the exciting member 113 and the resonating member 114.

[0102] (fixing member) The fixing member 117 is provided on the upper end of the vibration member 113, and holds the discharge head 110A via the vibration member 113. The fixing member 117 is also used to attach the discharge head 110A to the transport section 120.

[0103] <Transportation section> The transport section 120 includes a first moving section 121 and a second moving section 122 .

[0104] (First moving part) The first moving section 121 has a support member 121a and a linear moving section 121b. The first moving section 121 is a pair of members provided at the end of the second moving section 122 on the +x side and the end of the second moving section 122 on the −x side.

[0105] The support member 121a is a rectangular member when viewed from the +y direction, and supports the second moving part 122.

[0106] The linear moving part 121b is a long member extending in the z direction. The linear moving part 121b moves the support member 121a up and down in the z direction. For example, a known linear actuator equipped with a stepping motor as a drive source can be used as the linear moving part 121b.

[0107] The first moving section 121 moves the support member 121a in the z direction, thereby moving the discharge unit 110L supported by the second moving section 122 in the z direction.

[0108] (Second moving part) The second moving portion 122 has a support member 122a and a linear moving portion 122b.

[0109] The support member 122a is a rectangular member when viewed from the +y direction, and supports the discharge unit 110L.

[0110] The linear moving portion 122b is a long member extending in the x direction. The linear moving portion 122b moves the support member 122a horizontally in the x direction. Both ends of the linear moving portion 122b are supported by the support members 121a of the first moving portion 121, respectively.

[0111] The linear movement portion 122b may be, for example, a known linear actuator equipped with a stepping motor as a drive source.

[0112] The second moving section 122 moves the support member 122a in the x direction, thereby moving the discharge unit 110L supported by the support member 122a in the x direction.

[0113] Reservoir The reservoir 30 is disposed in the ejection direction of the droplets DR and stores the second solution L2. The reservoir 30 is a container that is open upward (in the +z direction). The reservoir 30 may be, for example, a shallow container such as a petri dish or a deep container such as a beaker.

[0114] The material of the reservoir 30 is not particularly limited, and organic materials such as synthetic resins, inorganic materials such as glass, metal materials, and the like can be used as appropriate.

[0115] The storage unit 30 may have a stirring device that stirs the stored second solution L2. This can suppress precipitation and aggregation of gel particles P formed in the second solution L2. A known configuration can be used as the stirring device.

[0116] <<Placement section>> The storage unit 30 is placed on the placement unit 40. The placement unit 40 has an x-stage 41, a y-stage 42, and a base 43.

[0117] The x-stage 41 supports and fixes the storage unit 30. The x-stage 41 also moves the storage unit 30 horizontally in the x direction.

[0118] The y-stage 42 moves the x-stage 41 horizontally in the y direction. The base 43 supports the y-stage 42 .

[0119] The mounting unit 40 can employ a known structure known as an xy stage.

[0120] Control device The control device (first control device) 50 creates electrical signals to operate each part of the droplet forming device 1 and supplies them to each part to control them. The control device 50 creates drive signals to be supplied to, for example, the discharge unit 10 and the placement unit 40 and supplies them to each part to control the operation of each part. The control device 50 may be a dedicated product attached to the droplet forming device 1, or may be a general-purpose PC on which software for controlling the droplet forming device 1 is installed.

[0121] The control device 50 supplies an electric signal to control the operation of the ejection head 110A. More specifically, the control device 50 supplies an electric signal having a drive frequency of the following formula (1) to the vibration member 113 of the vibration unit 115A. That is, as a method of driving the ejection head, an electric signal having a drive frequency of the following formula (1) is supplied to the vibration member 113. [Driving frequency] = ([Resonant frequency of structural vibration of resonating member] / n) × a…(1) (where n is an integer between 1 and 100, and a is between 0.9 and 1.1.)

[0122] The above n is preferably an integer of 1 to 10.

[0123] The above formula (A) indicates that the driving frequency of the vibrating member 113 is approximately an integer fraction of the resonant frequency of the structural vibration of the resonant member 114. The driving frequency does not need to be an exact integer fraction of the resonant frequency, and a coefficient a is set as a tolerance.

[0124] Here, the "resonant frequency of the structural vibration of the resonant member" can be measured as follows: Figure 4 is an explanatory diagram of a method for measuring the resonant frequency of the structural vibration of the vibrating means.

[0125] As shown in Fig. 4, a white noise voltage is applied as an electrical signal S from the control device 50 to the vibrating member 113 of the ejection head 110A. In this state, the vibration of the nozzle plate 112 near the ejection orifices 112x is measured using a vibrometer 55. A known laser Doppler vibrometer can be used as the vibrometer 55. With the white noise voltage applied to the vibrating member 113, the nozzle plate 112 is irradiated with a measurement laser beam La from the vibrometer 55, and the vibration caused in the resonating member 114 by the white noise is measured.

[0126] The above measurements were performed (a) when the liquid chamber 110S was filled with water, and (b) when the liquid chamber 110S was empty. Comparing the two vibration spectra obtained, resonance points were found to appear at the same frequency as peaks in the vibration spectrum, regardless of the amount of water in the liquid chamber 110S. These peak frequencies can be understood as shared frequencies resulting from the structural vibration of the resonating member 114 used.

[0127] By supplying an electrical signal from the control device 50 to the vibration member 113 having a drive frequency that is approximately an integer fraction of the resonant frequency of the resonant member 114 (([resonant frequency of the structural vibration of the vibration means] / n)×a), droplets can be ejected in an appropriate manner.

[0128] Fig. 5 shows an example of a drive waveform supplied to the ejection head 110A. The drive waveform shown in Fig. 5 is a sine wave with a period set to 55.6 μs, which corresponds to a vibration period at a frequency of 18 kHz. The drive waveform does not necessarily have to be a continuous sine wave, but may be a composite wave formed by combining waveforms intended to stabilize the ejection of droplets, and may be a waveform whose frequency approximately matches the resonant frequency.

[0129] The effects obtained by the ejection head having such a configuration will be described below.

[0130] First, consider a case where the resonating member 114 is not included in the ejection head. In this case, droplets DR can be formed by vibrating the vibrating member of the ejection head in accordance with the resonant frequency of the first solution L1 stored in the liquid chamber 110S. However, in this case, the resonant frequency of the first solution L1 changes when the amount of the first solution L1 stored in the liquid chamber 110S changes. Therefore, in order to achieve stable ejection of droplets DR, it is considered necessary to change the driving conditions of the vibrating member 113 depending on the amount of the first solution L1 stored in the liquid chamber 110S, which makes control complicated.

[0131] Next, consider the case where a discharge head is used that has a vibrating member of the same shape as the resonating member 114 instead of the resonating member 114. In this case, it is thought that by supplying an electrical signal having a drive frequency that is approximately an integer fraction of the "resonant frequency of the structural vibration of the vibrating member" to the vibrating member of the discharge head, it is possible to stably discharge droplets regardless of the amount of first solution L1 stored in the liquid chamber 110S. In a discharge head configured in this way, the resonant frequency of the vibrating member can be measured using a method similar to that shown in FIG.

[0132] However, in a discharge head configured in this manner, the drive conditions of the vibration member are set based on discrete conditions, i.e., approximately one-integer fraction of the "resonance frequency of the structural vibration of the vibration member." When adjusting the discharge speed or size of the droplets DR, it is conceivable to adjust the vibration conditions of the vibration member and control the vibration state of the vibration member 113. However, if the settable drive conditions of the vibration member are limited, it may become necessary to change the vibration member itself in order to achieve the desired discharge state of the droplets DR. Changing the vibration member naturally changes the performance of the vibration member, which can make condition setting even more complicated.

[0133] On the other hand, in the case of a configuration in which the vibration of the vibrating member 113 is transmitted to the nozzle plate 112 via the resonating member 114, as in the above-described ejection head 110A, it is possible to arbitrarily create a resonating member 114 that exhibits a desired resonant frequency by appropriately adjusting the material and shape of the resonating member 114. Therefore, it is possible to manufacture an ejection head 110 that realizes a desired ejection state of droplets DR.

[0134] When vibrating the nozzle plate 112 in the ejection head 110A, the resonant vibration of the resonant member 114 is utilized, thereby enabling the droplets DR to be ejected continuously and stably regardless of the amount of the first solution L1 in the liquid chamber 110S. This enables the droplet forming device 1 to easily and stably form the droplets DR, and makes it possible to suitably produce gel particles of uniform size (particle size).

[0135] <Detection unit, supply unit> The droplet forming device 1 may further include a detection unit 60 and a supply unit 65. The detection unit 60 detects the amount of the first solution L1 in the liquid chamber 110S of the ejection head 110.

[0136] The detection unit 60 may be, for example, a level gauge that emits measurement light L from above the liquid chamber 110S and measures the height of the liquid level of the first solution L1. Furthermore, if at least a part of the liquid holding member 111 is optically transparent, the detection unit 60 may be a level gauge that measures the liquid level of the first solution L1 in the liquid chamber 110S from the outside of the liquid holding member 111.

[0137] The supply unit 65 supplies the first solution L1 to the liquid chamber 110S based on the detection result by the detection unit 60. The supply unit 65 can be any device that can supply the first solution L1 to the liquid chamber 110S without any particular restrictions.

[0138] The control device 50 controls the supply unit 65 based on the detection result of the detection unit 60, and maintains the amount of the first solution L1 in the liquid chamber 110S at or above a predetermined specified amount. The control device 50 that performs such control corresponds to the second control device of the present invention.

[0139] Specifically, when the detection unit 60 obtains a measurement result indicating that the amount of the first solution L1 in the liquid chamber 110S (the liquid level of the first solution L1) is below a preset threshold (specified value), the control device 50 drives the supply unit 65 to supply the first solution L1 to the liquid chamber 110S. At this time, by setting the liquid level position after supplying the first solution L1 to a position (set value) higher than the specified value, the liquid level height of the first solution L1 can be maintained at or above the specified amount and managed between the set value and the specified value.

[0140] Alternatively, the amount of the first solution L1 stored in the liquid chamber 110S may be roughly calculated based on the amount at the start of ejection (initial value) and the total amount of ejected droplets DR, and the timing when the amount of the first solution L1 in the liquid chamber 110S (the liquid level of the first solution L1) will fall below a specified value may be predicted. In this case, the supply unit 65 supplies the specified amount of the first solution L1 to the liquid chamber 110S at the timing predicted in this way, and can adjust the liquid level of the first solution L1 to a position higher than the specified value.

[0141] The droplet forming device configured as described above can form droplets of uniform size (particle diameter), and therefore, by using the droplet forming device, it is possible to suitably produce gel particles of uniform size (particle diameter).

[0142] In this embodiment, the control device 50 is described as serving both as a control device (first control device) that controls the operation of the ejection head and as a control device (second control device) that controls the operation of the supply unit 65 based on the detection result of the detection unit 60, but this is not limiting. The droplet forming device may have the first control device and the second control device independently.

[0143] [Second embodiment] 6 and 7 are explanatory diagrams of a droplet forming device according to the second embodiment. In this embodiment, components common to those in the first embodiment are given the same reference numerals, and detailed descriptions thereof will be omitted.

[0144] In the discharge head 110B of the droplet forming device 2, the vibration generating unit 115B has a detachable member 118. The detachable member 118 is provided between the resonating member 114 and the vibration generating member 113. The detachable member 118 has a first detachable member 118a and a second detachable member 118b. The first detachable member 118a is detachable from the second detachable member 118b.

[0145] The first detachable member 118a is a member having a circular ring shape in a plan view and is provided on the upper surface 114a of the resonance member 114. The first detachable member 118a and the resonance member 114, which also has a circular ring shape in a plan view, are concentrically arranged with the same central axis in a plan view. The outer edge of the first detachable member 118a is located outside the outer edge of the resonance member 114.

[0146] In the discharge head 110B, a configuration including the liquid holding member 111, the nozzle plate 112, and the resonator member 114 is referred to as the discharge member 150. The first detachable member 118a and the resonator member 114 are bonded together with an adhesive (not shown). This makes the first detachable member 118a integrated with the discharge member 150, and the discharge member 150 can be removed from the droplet forming device 1 by removing the first detachable member 118a from the second detachable member 118b.

[0147] The elastic modulus of the adhesive is preferably 1 MPa or more and 1 GPa or less, and more preferably 10 MPa or more and 100 MPa or less, so as not to inhibit the displacement of the resonator member 114. The thickness of the adhesive layer is preferably set to 1 mm or less, and more preferably 200 μm or less, so as not to inhibit the displacement of the resonator member 114.

[0148] The material of the first detachable member 118a is not particularly limited.

[0149] The resonance frequency of the first detachable member 118a is preferably different from the resonance frequency of the resonance member 114. This prevents the resonance of the resonance member 114 from being hindered.

[0150] The second detachable member 118b is provided at the lower end of the vibration member 113. The second detachable member 118b may be screwed to the first detachable member 118a, or may be fixed in a luer lock manner.

[0151] The material of the second detachable member 118b is not particularly limited.

[0152] The resonant frequency of the second detachable member 118b is preferably different from the resonant frequency of the resonant member 114. This prevents the resonance of the resonant member 114 from being disturbed.

[0153] As shown in FIG. 7, the droplet forming device 2 has a plurality of discharge members 150 (a first discharge member 150A and a second discharge member 150B in FIG. 7), which are replaceable. This makes it possible to prevent the resulting gel particles P from being contaminated with different samples by replacing the discharge members 150 when different types of first solutions L1 are discharged from the droplet forming device 2. Furthermore, the discharge members 150 can be easily replaced if they are damaged or contaminated. Although FIG. 7 shows two discharge members 150, three or more may be used.

[0154] Furthermore, the resonant member 114A of the first discharge member 150A and the resonant member 114B of the second discharge member 150B can have different resonance frequencies, which makes it easier to adjust the discharge conditions of the droplets DR and to achieve a desired discharge state of the droplets DR.

[0155] [Method of manufacturing gel particles] The method for producing gel particles can be suitably carried out and gel particles of uniform size can be produced by using the droplet forming devices 1 and 2. The method for producing gel particles includes a step of dropping droplets DR of a first solution L1 discharged from a discharge head 110 onto a second solution L2 stored in the direction of discharge of the droplets DR, thereby forming gel particles P having at least the surface covered with hydrogel.

[0156] As described above, the first solution L1 contains a first substrate. The first solution L1 may have dispersed therein a substance to be encapsulated in the resulting gel particles P. For example, the first solution L1 may contain either or both of cells and spheroids as the encapsulated substance (dispersoid). The second solution L2 contains a second substrate that reacts with the first substrate to form a hydrogel.

[0157] In this manufacturing method, by using droplet forming devices 1 and 2 that have a resonant member 114 and vibrate a nozzle plate 112 via the resonated resonant member 114 to eject droplets DR, stable ejection of droplets DR becomes possible, and gel particles P of uniform size can be stably produced.

[0158] According to the droplet forming device configured as above, the droplets DR of the first solution L1 can be formed simply and stably, and gel particles of uniform size can be suitably produced.

[0159] Furthermore, according to the method for producing gel particles as described above, gel particles having a uniform size can be suitably produced.

[0160] While the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to these examples. The shapes and combinations of the components shown in the above examples are merely examples, and various modifications can be made based on design requirements, etc., without departing from the spirit of the present invention.

[0161] For example, in the above embodiment, a configuration for forming gel particles using a droplet forming device (gel particle manufacturing device) has been described, but the use of the droplet forming device is not limited to this. The droplet forming device described above has the effect of being able to eject droplets of uniform size, so it can also be suitably used, for example, when dispensing liquid in equal volumes.

[0162] In addition, in the droplet forming device, the control device 50 may be connected to a server via a wired or wireless line. The server may be installed in the facility where the droplet forming device is installed, or in a facility managed by the user of the droplet forming device, or may be set up on a cloud network.

[0163] Furthermore, it is preferable that the control device 50 be configured so that the molding results of droplets or gel particles under the manufacturing conditions set at the time of use can be input in association with the manufacturing conditions.

[0164] In the configuration of the above embodiment, it is preferable that the droplet formation devices 1 and 2 are capable of uploading to a server the manufacturing conditions set during use and the manufacturing results of gel particles under those manufacturing conditions. It is also preferable that the droplet formation devices 1 and 2 are capable of downloading from the server the manufacturing conditions set by other users when they use the droplet formation devices 1 and 2 in other environments and the manufacturing results under those manufacturing conditions. The manufacturing conditions that can be uploaded and downloaded include not only the conditions under which suitable gel particles were obtained, but also the conditions under which suitable gel particles were not obtained.

[0165] By allowing multiple droplet forming devices 1, 2 to share information on manufacturing conditions and manufacturing results via a server, users of the droplet forming devices 1, 2 can easily obtain suitable manufacturing conditions for the desired gel particles, and can easily manufacture the desired gel particles without extensive trial and error. [Example]

[0166] The present invention will be specifically described below by showing examples, but the following description is not intended to limit the scope of the present invention to the scope of the examples.

[0167] [Example 1] In the droplet forming device described above, an ejection head with an opening diameter of 200 μm was used, and droplets were formed by driving the vibration member by supplying an electrical signal having a driving frequency of the following formula (1), thereby producing gel particles. [Driving frequency] = ([Resonant frequency of structural vibration of resonating member] / n) × a…(1) (n=1, a=1)

[0168] (1) Preparation of the first solution 100 mg of sodium alginate (Kimica Algin SKAT-ONE, manufactured by Kimica) was dissolved in 5 mL of PBS(-) (Gibco, Dulbecco's Phosphate Buffered Saline: DPBS(1x)) and filtered through a 0.2 μm average pore size filter (Minisart S6534, manufactured by Sartorius) to prepare a 2.0 (w / v)% sodium alginate solution. 1.5 g of the 2.0 (w / v)% sodium alginate solution was then mixed with 0.5 g of PBS(-) to prepare a 1.5 (w / v)% sodium alginate solution. The term "(w / v)%" refers to a mass-to-volume ratio concentration that defines the ratio of the weight of a solute contained in a solvent per unit volume.

[0169] (2) Preparation of the second solution 605 mg of calcium chloride (model number: 039-00475, manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as "CaCl") was dissolved in 50 mL of ultrapure water, and then filtered through the above-mentioned filter to prepare a CaCl aqueous solution (second solution) with a concentration of 109 mmol / L.

[0170] (3) Preparation of gel particles Using a discharge head filled with the first solution, droplets were dropped into a 35 mm dish containing 3 mL of the second solution to produce gel particles.

[0171] [Gel particle size and coefficient of variation] Magnified images of the resulting collection of gel particles were taken using a phase-contrast microscope (Olympus CKX53), and the acquired images were analyzed to determine the particle size distribution and coefficient of variation. Figure 8 is a histogram showing the particle size distribution of the gel particles determined based on the analysis results. In Figure 8, the horizontal axis represents the particle size (µm) of the gel particles, the left vertical axis represents the frequency (count), and the right vertical axis represents the percentage (%). The average particle size of the gel particles in Example 1 was 218 µm, and the coefficient of variation (CV) was 6.5%. As can be seen from Figure 8, the particle size distribution had a high kurtosis, confirming that the particle sizes were uniform.

[0172] [Example 2] (1) C2C12 cell culture C2C12 cells were cultured in a 150 mm dish in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific, DMEM (1x), hereafter referred to as "DMEM") containing 10% by mass fetal bovine serum (hereafter referred to as "FBS") and 1% by mass antibiotic (Nacalai Tesque, Antibiotic-Antimycotic Mixed Stock Solution (100x)) in an incubator (Panasonic, CO2 incubator KM-CC17RU2) for 72 hours.

[0173] (2) Preparation of C2C12 cell suspension 15 mL of PBS(-) was added to the dish, and the surface was washed by aspirating the PBS(-). After washing with PBS(-), 6 mL of 0.05% trypsin-0.05% EDTA solution (Life Technologies) was added to the dish and heated in an incubator for 5 minutes to detach the cells from the dish.

[0174] After confirming cell detachment under a phase-contrast microscope, 6 mL of FBS-supplemented DMEM was added to the dish to inactivate the trypsin. The cell suspension in the dish was transferred to a 50 mL centrifuge tube and centrifuged (KOKUSAN H-19FM, 200 × g, 3 minutes). The supernatant was then removed using an aspirator. After removal, 6 mL of FBS-supplemented DMEM was added to the centrifuge tube and gently pipetted to disperse the cells, yielding a cell suspension.

[0175] 100 μL of the obtained cell suspension was taken into an Eppendorf tube, the sample was aspirated into a cassette (Via1-Cassette, manufactured by Chemometec), and the number of cells in the liquid was counted using a cell counter (Nucleo Counter NC-3000, manufactured by Chemometec). A portion of the cell suspension was transferred to a 50 mL centrifuge tube and centrifuged (200 × g, 3 minutes), and the supernatant was removed using a pipette. PBS(-) was added to the remaining portion after the supernatant was removed, and the cell concentration was adjusted to 4 × 10 7 A cell suspension of 100 cells / mL was obtained.

[0176] (3) Preparation of gel particles The same procedure as in Example 1 was repeated except that 0.5 mL of the cell suspension was mixed with the sodium alginate aqueous solution instead of 0.5 g of PBS(-), to obtain a solution of 1 × 10 7 A 1.5 (w / v)% aqueous solution of sodium alginate containing cells / mL was prepared. Gel particles encapsulating cells were prepared in the same manner as in Example 1, except that the obtained aqueous sodium alginate solution was used.

[0177] [Gel particle size and coefficient of variation] Measurements were performed in the same manner as in Example 1, and the average particle size of the resulting gel particles was 270 μm, with a coefficient of variation (CV) of 16%. Even though cells were encapsulated, the coefficient of variation of the gel particles was 20% or less, indicating that gel particles of uniform size were formed.

[0178] [Example 3] Gel particles were prepared in the same manner as in Example 2. After removing the PBS(-), DMEM containing 10% by mass of FBS and 1% by mass of antibiotics was added. PI (Thermo Fisher Scientific, P1304MP) and Hoechst (Thermo Fisher Scientific, H3570) were then added to prepare a 2000-fold dilution to prepare a dispersion of gel particles.

[0179] The resulting dispersion was dispensed into five randomly selected wells of a 96-well plate, and the number of gel particles in each well was counted using a phase-contrast microscope. Figure 9 shows a magnified photograph of cells encapsulated in gel particles.

[0180] The gel particles were dissolved in a 55 mmol / L aqueous solution of sodium citrate (product number: 191-01785, manufactured by Wako Pure Chemical Industries, Ltd.) for 2 minutes to dissolve the cells. The cells were then observed under a phase-contrast microscope, and fluorescent images of PI and Hoechst were obtained.

[0181] Based on the fluorescence observation images, cells stained with PI were considered dead cells, and cells stained with Hoechst were considered total cells, and the viability (%) was calculated as (total cell count - dead cell count) / (total cell count) × 100. The results are shown in Table 1.

[0182] [Table 1]

[0183] As shown in Table 1, high cell viability values ​​of 80% or more were obtained in all wells. It was confirmed that even when gel particles containing cells were produced using the droplet formation device and production method of the present invention, the production process of the gel particles did not adversely affect cell viability.

[0184] [Comparative Example 1] Gel particles were prepared in the same manner as in Example 1, except that an electrical signal having a drive frequency determined by n = 180 and a = 1 in equation (1) described in Example 1 was supplied to drive an ejection head with an opening diameter of 160 μm.

[0185] The obtained gel particles were measured in the same manner as in Example 1, and the average particle size of the gel particles was 87 μm, with a coefficient of variation (CV) of 66%. The coefficient of variation was large and the particle size distribution was broad, confirming that the particle sizes were varied.

[0186] Comparative Example 2 Gel particles were produced in the same manner as in Example 1, except that an electrical signal having the same drive frequency as in Comparative Example 1 was supplied to drive a discharge head having an opening diameter of 500 μm.

[0187] The obtained gel particles were measured in the same manner as in Example 1, and the average particle size of the gel particles was 84 μm, and the coefficient of variation (CV) was 28%. The coefficient of variation was greater than 20%, indicating a broad particle size distribution, and it was confirmed that the particle sizes were varied.

[0188] The present invention includes the following aspects.

[0189] [1] A droplet forming device comprising an ejection head that ejects droplets of liquid, the ejection head having a cylindrical liquid holding member that holds the liquid, and a vibration unit that covers one end side of the liquid holding member and forms a liquid chamber that holds the liquid together with the liquid holding member, and ejects the droplets based on an applied electrical signal, the liquid chamber having the other end side of the liquid holding member open to the atmosphere, the vibration unit having a vibration member that vibrates based on the electrical signal, a film-like member having an ejection port through which the droplets are ejected, and a resonant member that vibrates based on the vibration of the vibration member and transmits the vibration of the vibration member to the film-like member by vibrating itself, the resonant member being a plate-like member that overlaps with the film-like member in a planar manner and is in contact with the film-like member.

[0190] [2] A droplet forming device as described in [1], comprising a first control device that supplies the electrical signal and controls the operation of the ejection head, wherein the first control device supplies the electrical signal having a drive frequency of the following formula (1) to the vibration member. [Driving frequency] = ([Resonant frequency of the structural vibration of the resonating member] / n) × a... (1) (where n is an integer between 1 and 100, and a is between 0.9 and 1.1.)

[0191] [3] The vibration unit has a detachable member provided between the resonating member and the vibration member, and the detachable member allows the ejection member including the liquid holding member, the film-like member, and the vibration member to be detachably attached to or detached from the vibration member. [1] or [2]

[0192] [4] The droplet forming device according to [3], which has a plurality of the discharge members.

[0193] [5] The droplet forming device described in [4], wherein the plurality of discharge members include a first discharge member and a second discharge member, and the second discharge member includes a second resonant member having a different resonant frequency from the resonant member of the first discharge member.

[0194] [6] A droplet forming device described in any one of [1] to [5], comprising a detection unit that detects the amount of liquid in the liquid chamber, a supply unit that supplies the liquid to the liquid chamber, and a second control device that controls the supply unit based on the detection result of the detection unit and maintains the amount of liquid in the liquid chamber at or above a predetermined specified amount.

[0195] [7] The droplet forming device according to any one of [1] to [6], further comprising a storage section arranged in the ejection direction of the droplets, wherein the liquid is a first solution containing a first substrate, the storage section stores a second solution containing a second substrate, and the first substrate reacts with the second substrate to form a hydrogel.

[0196] [8] The droplet forming apparatus according to [7], wherein the first solution contains a dispersoid that is either one or both of cells and spheroids.

[0197] [9] A method for producing gel particles, comprising the steps of: using the droplet forming device described in [7] or [8], dropping the droplets ejected from the ejection head into the second solution stored in the ejection direction of the droplets, and forming gel particles having at least the surface covered with hydrogel.

[0198] Furthermore, in order to solve the problem of suitably producing microcapsules (gel particles) with uniform sizes, the present invention includes the following aspects.

[0199] [A1] A method for manufacturing a liquid ejection head, the method comprising: ejecting droplets of a first solution containing a first substrate and a dispersoid that is either one or both of cells and spheroids; a storage unit disposed in the ejection direction of the droplets and storing a second solution containing a second substrate; and a control unit that supplies an electric signal to control the operation of the ejection head, wherein the first substrate reacts with the second substrate to form a hydrogel; the ejection head having a tubular liquid holding unit that holds the first solution; and a vibration unit that ejects the droplets based on the electric signal, a film-like member having an outlet for ejecting the droplets, a vibrating member that vibrates based on the electric signal, and a resonating member that is in contact with the film-like member and the vibrating member and transmits the vibration of the vibrating member to the film-like member, wherein the vibrating member covers one end of the liquid holding member and, together with the liquid holding member, forms a liquid chamber that holds the first solution, the other end of the liquid holding member of the liquid chamber is open to the atmosphere, and the control unit supplies the vibrating member with the electric signal having a drive frequency expressed by the following formula (1). [Driving frequency] = ([Resonant frequency of structural vibration of resonating member] / n) × a…(1) (where n is an integer between 1 and 100, and a is between 0.9 and 1.1.)

[0200] [A2] The vibrating unit has a detachable member provided between the resonating member and the vibrating member, and the detachable member allows the vibrating member to be freely attached and detached to and from the ejection member including the liquid holding unit, the film-like member, and the resonating member.

[0201] [A3] The microcapsule manufacturing apparatus according to [A2], which has a plurality of the discharge members.

[0202] [A4] The microcapsule manufacturing apparatus described in [A3], wherein the plurality of discharge members include a first discharge member and a second discharge member, and the second discharge member includes a second resonant member having a different resonant frequency from the resonant member of the first discharge member.

[0203] [A5] A microcapsule manufacturing device described in any one of [A1] to [A4], comprising a detection unit that detects the amount of liquid of the first solution in the liquid chamber, a supply unit that supplies the first solution to the liquid chamber, and a control unit that controls the detection unit and the supply unit, wherein the control unit controls the supply unit based on the detection result of the detection unit to maintain the amount of liquid of the first solution in the liquid chamber at or above a predetermined specified amount.

[0204] [A6] A method for manufacturing a microcapsule, comprising the steps of: using the microcapsule manufacturing apparatus described in any one of [A1] to [A5]; dropping the droplets ejected from the ejection head into the second solution stored in the ejection direction of the droplets; and forming a microcapsule covered with a hydrogel membrane. [Explanation of symbols]

[0205] 1, 2... droplet forming device, 30... storage section, 50... control device, 60... detection section, 65... supply section, 110, 110a to 110c, 110A, 110B... discharge head, 110S... liquid chamber, 110L... discharge unit, 111... liquid holding member, 112... nozzle plate (film-shaped member), 112x... discharge port, 113... vibration member, 114, 114A, 114B... resonance member, 115A, 115B... vibration section, 118... detachable member, 150, 150A, 150B... discharge member, DR... droplet, L... liquid, L1... first solution, L2... second solution, HM... hydrogel film, P... gel particle, S... electrical signal [Prior art documents] [Patent documents]

[0206] [Patent Document 1] Patent No. 5858451 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-201350

Claims

1. a discharge head for discharging droplets of liquid; The ejection head includes a cylindrical liquid holding member that holds the liquid; a vibration unit that covers one end side of the liquid holding member, forms a liquid chamber that holds the liquid together with the liquid holding member, and ejects the droplets based on an applied electric signal; The liquid chamber is open to the atmosphere at the other end side of the liquid holding member, The vibration unit includes a vibration member that vibrates based on the electrical signal; a film-like member having a discharge port for discharging the droplets; a resonating member that vibrates based on the vibration of the vibrating member and transmits the vibration of the vibrating member to the film member by vibrating itself, The resonator member is a plate-like member that overlaps with the film-like member in a planar manner and is in contact with the film-like member.

2. a first control device that supplies the electrical signal and controls the operation of the ejection head; The droplet forming device according to claim 1 , wherein the first control device supplies the electric signal having a drive frequency of the following formula (1) to the vibration applying member: [Driving frequency] = ([Resonant frequency of the structural vibration of the resonating member] / n) × a (1) (where n is an integer from 1 to 100, and a is an integer from 0.9 to 1.1.)

3. the excitation unit has a detachable member provided between the resonating member and the excitation member, 3. The droplet forming device according to claim 1, wherein the detachable member detachably connects the discharge member including the liquid holding member, the film-like member, and the resonating member to the vibration member.

4. The droplet forming device according to claim 3 , comprising a plurality of the discharge members.

5. the plurality of discharge members include a first discharge member and a second discharge member, The droplet forming device according to claim 4 , wherein the second discharge member has a second resonant member having a different resonance frequency from the resonant member of the first discharge member.

6. a detection unit that detects the amount of the liquid in the liquid chamber; a supply unit that supplies the liquid to the liquid chamber; 3. The droplet forming device according to claim 1, further comprising: a second control device that controls the supply unit based on the detection result of the detection unit and maintains the amount of liquid in the liquid chamber at or above a predetermined specified amount.

7. a reservoir disposed in the ejection direction of the droplets, the liquid is a first solution containing a first substrate; the reservoir stores a second solution containing a second substrate; The droplet forming device according to claim 1 or 2, wherein the first substrate reacts with the second substrate to form a hydrogel.

8. The droplet forming apparatus according to claim 7 , wherein the first solution contains dispersoids that are either or both of cells and spheroids.

9. A method for producing gel particles, comprising the steps of: using the droplet forming device described in claim 7, dropping the droplets ejected from the ejection head into the second solution stored in the ejection direction of the droplets, and forming gel particles having at least the surface covered with hydrogel.

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