JP2012170861A - Method of synthesizing non-spherical hydrogel particle and non-spherical hydrogel particle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new technique that makes it possible to synthesize non-spherical hydrogel particles with good reproducibility only through a simple operation, while eliminating the need for a complex manufacturing device and operation required heretofore to synthesize non-spherical hydrogel particles.SOLUTION: To a flow passage structure X having at least two entrances, at least one exit O, entrance flow passages connected to the entrances, and a flow passage part J which is formed as the entrance flow passages are combined at the same time or in steps and which is connected to the exit O, a sol solution Z serving as the raw material of hydrogel and a gelling agent solution G are continuously introduced from the entrance I1 and the entrance I2, respectively. Fibers where the sol solution Z is at least partially gelled at the flow passage part J are confined within liquid droplets and thereby non-spherical hydrogel particles are manufactured by cutting the fibers.

Description

  The present invention relates to a method for synthesizing non-spherical hydrogel particles and non-spherical hydrogel particles.

  Hydrogel particles are widely used as carriers for drug delivery in the body, cell culture materials for regenerative medicine, carriers for cell transplantation, immobilized carriers for bioreactors, and the like.

  As an existing technique for producing hydrogel particles, there is a technique in which an aqueous solution that forms a gel is dropped into an aqueous solution containing a gelling agent. In this method, relatively large hydrogel particles having a diameter of several millimeters are obtained, and the shape thereof is often a sphere or a shape similar to a sphere.

  On the other hand, in recent years, a method for synthesizing hydrogel particles having a diameter of several tens to several hundreds of μm by using a microchannel manufactured by using microfabrication technology has been proposed. Thus, a technique has been proposed in which droplets are formed using an oil phase as a continuous phase and an aqueous solution containing a hydrogel constituent as a dispersed phase, and the droplets are gelled. In this method, sodium alginate is used as the hydrogel constituent material, calcium carbonate mixed inside the droplet is used as the gelling agent, and the oil containing acetic acid is introduced downstream of the flow path, the calcium carbonate in the droplet dissolves. As a result, the droplets gel, and spherical calcium alginate hydrogel particles having a diameter of about 100 μm are obtained.

  In these existing methods, the shape of the obtained hydrogel particles is mainly spherical or similar, but for example, when the hydrogel particles are used as a carrier for cell transplantation, the shape does not inhibit blood flow. Things are desirable. In addition, when using hydrogel particles as an immobilization carrier for cells, enzymes, etc., a shape with a large specific surface area that enables efficient supply of oxygen and nutrients to the inside is desirable. Non-spherical hydrogel particles are preferred.

  As a technique for producing non-spherical hydrogel particles, a technique has also been proposed in which a photocurable monomer is irradiated with light in a microchannel for gelation. For example, a system called stopped flow lithography (SFL) is applied by irradiating an aqueous solution of poly (ethylene glycol) diacrylate (PEG-DA), which is a photocurable monomer, introduced into a microchannel, through a mask having an arbitrary shape. By periodically and automatically stopping the flow, the PEG-DA molecules in the UV-irradiated area are cross-linked into a polymer while the flow is stopped, and various shapes depending on the mask shape A technique for obtaining the hydrogel particles has also been reported.

JP 2007-186456 A

“Angewandte Chemie Int. Ed., 49, 87-90, 2010.

  However, although the technique described in Non-Patent Document 1 described above can synthesize non-spherical particles, it has a drawback of requiring a complicated production apparatus and operation. Moreover, since the shape of the obtained particles is limited to a planar shape (a uniform thickness in the depth direction), there is a problem that it is impossible to increase the surface area significantly.

  In addition, since the technique described in Non-Patent Document 1 cannot be applied to a polymer molecule having a photocrosslinkable reactive group, an inexpensive natural molecule having high biocompatibility and strength such as alginate gel is used as a material. It was impossible to produce non-spherical hydrogel particles.

  The present invention has been made in view of the above-described problems of the prior art, and the object of the present invention is a complicated production that has been conventionally required for synthesizing non-spherical hydrogel particles. It is an object of the present invention to provide a new technique that makes it possible to synthesize non-spherical hydrogel particles with simple reproducibility and with no reproducibility with no need for apparatus and operation.

  The present invention also provides a novel method for producing high-strength, non-spherical hydrogel particles composed of alginic acid, and for producing wool ball-shaped hydrogel particles having a larger specific surface area. It provides a new method.

  Furthermore, the present invention intends to provide a yarn ball-shaped hydrogel particle itself obtained by the novel method.

  In order to achieve the above object, the invention according to one aspect of the present invention provides at least two inlets I1 to In (n ≧ 2), at least one outlet O, and inlet streams connected to the inlets I1 to In, respectively. For the channel structure X having the channel portion J connected to the outlet O, formed by the passages C1 to Cn and the inlet channels C1 to Cn joining at the same time or stepwise, The sol solution Z that is the raw material of the gel is continuously introduced with the gelling agent solution G from the inlet I2 to form a fiber in which the sol solution Z is at least partially gelled in the flow path portion J. A non-spherical hydrogel particle is obtained by cutting the fiber by confining the fiber in which the sol solution Z is at least partially gelled in the droplet inside or outside the channel X. The makes it possible to produce a synthesis method of a non-spherical hydrogel particles. This makes it possible to synthesize non-spherical hydrogel particles by simply introducing a plurality of types of solutions into a channel structure having a specific shape without using a special external device.

  In the invention according to this aspect, the droplet is preferably an oil phase or a water droplet dispersed in a gas phase, although not limited thereto. By doing so, it becomes possible to efficiently cut the fiber by confining the hydrogel fiber in the water droplet.

  Further, in the invention according to this aspect, although not limited, the flow path structure X has at least three inlets I1 to In (n ≧ 3). It is preferable that droplets are formed inside the channel X by introducing A. In this way, water droplets dispersed in the oil phase are formed inside the flow channel structure X, and the fibers can be cut by confining the hydrogel fibers in the water droplets.

  In the invention according to this aspect, although not limited, the flow path structure X has at least four inlets I1 to In (n ≧ 4), and at least one of the inlets I4 has a buffer. It is preferable to introduce the solution B. This prevents direct contact between the sol solution Z and the gelling agent solution G even when the speed of the gelation reaction caused by the contact between the sol solution Z and the gelling agent solution G is high. Thus, the blockage of the flow path can be prevented, and the gelation speed can be adjusted.

  In the invention according to this aspect, the channel structure X is preferably at least partially constituted by a planar microchannel, although not limited thereto. By doing so, it is possible to use a relatively inexpensive flow path structure formed on or in the polymer substrate, for example.

  In the invention according to this aspect, the flow path structure X may be at least partially configured with a structure using multiple tubes. By doing in this way, it becomes possible to produce a relatively inexpensive flow path structure constituted by a multiple tube in which capillary tubes such as glass, plastic, and metal are combined.

  Further, in the invention according to this aspect, although not limited, at least one of the inlet channels C1 to Cn has a branch in the middle thereof, and each branch is simultaneously with other inlet channels. Or it is preferable to join in steps. In this way, the number of inlets for introducing the solution can be reduced, the flow path structure X can be made more compact and simple, and improvement in operability is also expected. it can.

  Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable that any one value, such as the depth of the said flow path structure X, a width | variety, a diameter, is in the range of 1 micrometer-1 cm. By doing so, it is possible to produce non-spherical hydrogel particles having a small size (diameter of several μm to several cm).

  In the invention according to this aspect, although not limited, the sol solution Z is preferably a sodium alginate aqueous solution, and the gelling agent solution G is preferably a solution containing a polyvalent metal cation. . This makes it possible to produce non-spherical hydrogel particles composed of calcium alginate, barium alginate, magnesium alginate, etc. that have high strength and biocompatibility. It can be introduced and fixed.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable that the density | concentration of the gelatinizer in the gelatinizer solution G is previously adjusted to the appropriate value. In this way, at the time of cutting, the sol solution can be at least partially gelled.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable that the thickener is added to at least one of the gelatinizer solution G and the buffer solution B previously. By doing so, it is possible to stabilize the formation of droplets in the flow path X.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable that surfactant is previously added to at least one of the gelatinizer solution G, the oil A, and the buffer solution B. By doing in this way, it becomes possible to stabilize the formed droplet, and it becomes possible to make the size of the generated non-spherical hydrogel particles more uniform.

  In the invention according to this aspect, the channel structure X has at least two inlets I1 for introducing the sol solution Z, and the sol solution Z having the same or different composition may be introduced. In this way, it is possible to increase the volume ratio of the gel part in the non-spherical hydrogel particles, and to produce non-spherical hydrogel particles composed of two or more types of fibers having different compositions. Is possible.

  Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable that the said non-spherical shape is a yarn ball shape. As a result, the specific surface area is dramatically larger than the spherical hydrogel particles obtained by the prior art, and when the enzyme or microorganism is immobilized on the gel particles and used as a reactor, the reaction efficiency and metabolic activity are improved. Useful hydrogel materials can be provided as expected. In addition, this makes it possible to efficiently supply nutrients to the center of the particles because there are gaps in the particles when culturing animal cells or the like inside the gel particles. In addition, when using non-spherical hydrogel particles that encapsulate cells inside as a carrier in cell transplantation, which is one of the techniques of regenerative medicine, this also inhibits blood flow because there are gaps inside the particles. This is expected to be less likely.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable that the diameter of the said non-spherical hydrogel is 1 mm or less. This is expected to facilitate introduction into blood vessels when used as a carrier for cell transplantation.

  The non-spherical hydrogel particles according to another aspect of the present invention are obtained by using the method for synthesizing non-spherical hydrogel particles according to the first aspect. As a result, the specific surface area increases dramatically when compared with spherical hydrogel particles obtained by the prior art, improving reaction efficiency and metabolic activity when used as a reactor, and efficient cell culture. It is possible to provide non-spherical hydrogel particles having advantages such as a simple nutrient supply and a reduced possibility of inhibiting blood flow when used as a carrier for cell transplantation.

  Since the present invention is configured as described above, the solution is introduced into the channel structure without the need for complicated devices and operations required in the conventional method for synthesizing non-spherical hydrogel particles. Only it becomes possible to synthesize non-spherical hydrogel particles.

  In addition, since the present invention is configured as described above, the synthesis of non-spherical hydrogel particles can be performed using a hydrogel composed of alginic acid, which has hardly been reported in the past. Therefore, non-spherical hydrogel particles having high biocompatibility and strength can be provided.

  In addition, since the present invention is configured as described above, a hydrogel having a dramatically increased specific surface area, which has not been reported so far, can be produced in the form of yarn-ball shaped hydrogel particles. It is possible to provide materials, and it is possible to provide new materials that are very useful in various fields such as biological production, medical treatment, and substance production by bioprocesses.

It is the schematic which shows the principle of the synthesis | combining method of the nonspherical hydrogel particle in this invention. 1 is a schematic view of a device having the most basic channel structure for synthesizing non-spherical hydrogel particles. FIG. FIG. 2A is a schematic view showing a flow channel structure X configured in a plane and a device including the flow channel structure X as viewed from above, and B in FIGS. 2B to 2E. It is also an arrow view. 2 (b) to 2 (e) are cross-sectional views of the device including the channel structure X in the lines A0-A1, A2-A3, A4-A5, and A6-A7 in FIG. 2 (a), respectively. Represents. Schematic diagram of channel structure X, flow of each solution, and synthesis of non-spherical hydrogel particles used when forming droplets inside the channel by introducing oil into the channel according to the embodiment It is the schematic which showed the mode of. The flow channel structure X used when forming droplets by gravity outside the flow channel according to the embodiment, and the state in which each solution is introduced into the flow channel structure X and the non-spherical hydrogel particles are synthesized are shown. FIG. According to the embodiment, used to synthesize non-spherical hydrogel particles, a flow channel structure X constituted by multiple tubes, a state of each solution flowing in the flow channel structure X, and a state of non-spherical hydrogel particle synthesis It is the schematic which showed. FIG. 5 (a) is a view taken in the direction of arrow W4 in FIGS. 5 (b) and 5 (c), and FIGS. 5 (b) and 5 (c) are respectively the W0-W1 line and W2 in FIG. 5 (a). Sectional drawing of the solution which flows through the flow path structure X and the flow path structure X in the -W3 line | wire is represented. The inlet channels C2, C3, C4 used for synthesizing the non-spherical hydrogel particles according to the embodiment each have a branch in the middle, and each branch merges with other inlet channels in stages. It is the schematic which showed the mode of the flow path structure X which is carrying out, the mode of the flow of each solution in the flow path structure X, and the mode of non-spherical hydrogel particle synthesis. The flow of each solution in the flow channel structure X in the flow channel structure X having a form different from the flow channel structure shown in FIGS. 3 to 6 used for synthesizing the non-spherical hydrogel particles according to the embodiment. It is the schematic which showed the mode of this, and the mode of the synthesis | combination of a non-spherical hydrogel particle. It is the schematic of the device which has a flow-path structure for synthesize | combining the non-spherical hydrogel particle based on an Example. FIG. 8A is a schematic diagram showing a flow channel structure X configured in a plane and a device including the flow channel structure X as viewed from above, and an arrow B in FIGS. 8B to 8E. FIG. FIGS. 8B to 8E are cross-sectional views of the device including the channel structure X along the lines A0-A1, A2-A3, A4-A5, and A6-A7 in FIG. 8A, respectively. is there. In the flow path structure X for the Example of the synthesis | combining method of a non-spherical hydrogel particle, it is the microscope picture which observed the flow of the solution in the flow path structure X. FIG. 9A is a photomicrograph showing how the solutions merge at the junctions of the inlet channels C1 to C4. FIG. 9B shows a droplet formed in the channel portion J. FIG. 2 is a photomicrograph showing how the fiber is confined in the droplet and cut. It is a microscope picture of the synthetic | combination nonspherical hydrogel particle | grains using the flow-path structure X for the Example of the synthesis | combining method of nonspherical hydrogel particle | grains. FIGS. 10A and 10B are a micrograph of the same non-spherical hydrogel particles observed in a bright field, respectively, and (b) a micrograph of fluorescence observed.

  Hereinafter, the synthesis method of the non-spherical hydrogel particles and the best mode of the non-spherical hydrogel particles according to the present invention will be described in detail. However, the present invention can be implemented in many different forms, and is not limited to the following embodiments and examples.

  FIG. 1 is a schematic view showing the principle of a method for synthesizing non-spherical hydrogel particles in the present invention.

  In FIG. 1, between two inlets I1 and I2, one outlet O, inlet channels C1 and C2 connected to the inlets I1 and I2, respectively, and between the junction of the inlet channels C1 and C2 and the outlet O A sol solution Z as a hydrogel raw material is continuously introduced from the inlet I1 and a gelling agent solution G is continuously introduced from the inlet I2 into the channel structure X having the existing channel portion J. In J, a hydrogel fiber in which the sol solution Z is at least partially gelled is formed, and further, outside the flow path X, the hydrogel fiber in which the sol solution Z is at least partially gelled is placed in the droplet. Non-spherical hydrogel particles are produced by cutting by confinement, and non-spherical hydrogel particles are obtained by collecting only non-spherical hydrogel particles from within the droplets. It is possible.

  FIG. 2 is a schematic view of a device having the most basic channel structure for synthesizing non-spherical hydrogel particles. FIG. 2A is a schematic view showing a flow channel structure X configured in a plane and a device including the flow channel structure X as viewed from above, and B in FIGS. 2B to 2E. It is also an arrow view. 2 (b)) to 2 (e) are cross-sectional views of the device including the channel structure X along the lines A0-A1, A2-A3, A4-A5, and A6-A7 in FIG. 2 (a), respectively. Represents.

  In FIG. 2, the flow path structure X has two inlets I1 and I2, one outlet O, inlet flow paths C1 and C2 connected to the inlets I1 and I2, respectively, and a junction of the inlet flow paths C1 and C2. And a channel portion J which is a portion between the outlet O and the outlet O.

  The flow path structure X in FIG. 2 is formed by adhering an upper substrate having a groove structure on the lower surface and a flat lower substrate, and the depth of the flow path is constant. However, the channel depths may be partially different, and the channel structure X may be a structure connected at least partially by circular tubes having the same or different diameters. However, since the flow path structure is at least partially configured in a planar manner, the flow path structure can be easily manufactured, and more precise flow path preparation is possible. More preferred.

  When producing a planar flow path structure, for example, a production technique using a mold such as molding or embossing is preferable in that the flow path structure can be easily produced. Manufacturing techniques such as wet etching, dry etching, laser processing, direct electron beam drawing, and machining can also be used.

  When producing a planar flow path structure, device materials include PDMS (polydimethylsiloxane), various polymer materials such as acrylic, glass, silicon, ceramics, various metals such as stainless steel, etc. It is also possible to use a combination of a plurality of types of substrates among these materials. However, it is preferable to use a polymer material at least partially in order to produce the flow path at low cost.

  In the flow channel structure X shown in FIG. 2A, the inlet flow channel Cn and the flow channel portion J are each linear, but this is not necessarily linear. In FIG. 2A, the flow path structure X is line symmetric with respect to the A6-A7 line as a symmetry axis, but it does not necessarily have a line symmetric form.

  FIG. 3 is a schematic diagram of the flow channel structure X used for forming droplets inside the flow channel by introducing oil into the flow channel in this embodiment, how each solution flows, and non-spherical hydro It is the schematic which showed the mode of the synthesis | combination of a gel particle.

  In FIG. 3, the channel structure X has seven inlets I1, I2, I2 ′, I3, I3 ′, I4, I4 ′, outlet O, and inlets I1, I2, I2 ′, I3, I3 ′, I4, I4. Inlet channels C1, C2, C2 ′, C3, C3 ′, C4, and C4 ′ connected to “′, respectively.

  In FIG. 3, by introducing the sol solution Z from the inlet I1 and the gelling agent solution G from the inlets I2 and I2 ', a partially gelled hydrogel fiber is formed in the channel portion J. By introducing the oil A from the inlets I3 and I3 ', it is possible to form droplets in the flow path portion J, using the continuous phase as oil and the dispersed phase as a mixed aqueous solution containing fibers. By introducing the buffer solution B containing no gelling agent from the inlets I4 and I4 ′, it is possible to prevent instantaneous gelation and blockage of the flow path due to contact between the sol solution Z and the gelling agent solution G. It becomes.

  In addition, when forming the droplet of the aqueous solution in the flow path, it is preferable that the surface inside the flow path portion J has an appropriate wettability so that the liquid droplet can be formed at least partially. . Therefore, it is also possible to perform an appropriate surface treatment as necessary.

  FIG. 4 shows a flow channel structure X used in the present embodiment when droplets are formed by gravity outside the flow channel, and each solution is introduced into the flow channel structure X to synthesize non-spherical hydrogel particles. It is the schematic which showed the mode.

  In FIG. 4, the channel structure X has five inlets I1, I2, I2 ′, I4, I4 ′, an outlet O, and inlet channels C1 connected to the inlets I1, I2, I2 ′, I4, I4 ′, respectively. , C2, C2 ′, C4, C4 ′.

  In FIG. 4, by introducing the sol solution Z from the inlet I1 and the gelling agent solution G from the inlets I2 and I2 ′, fibers in which the sol solution is partially gelled are formed in the channel portion J. . By introducing the buffer solution B from the inlets I4 and I4 ', it is possible to prevent instantaneous gelation and blockage of the flow path due to contact between the sol solution Z and the gelling agent solution G.

  Then, the fiber in the aqueous solution discharged from the outlet O to the outside of the flow channel structure X is cut and trapped in the droplet by dropping the aqueous solution as a droplet in the gas phase. Spherical hydrogel particles are synthesized.

  FIG. 5 is a schematic view showing a flow channel structure X constituted by multiple tubes, a state of each solution flowing in the flow channel structure X, and a state of synthesis of non-spherical hydrogel particles. FIG. 5 (a) is a view taken in the direction of arrow W4 in FIGS. 5 (b) and 5 (c), and FIGS. 5 (b) and 5 (c) are respectively W0-W1 lines and W2 in FIG. 5 (a). Sectional drawing of the solution which flows through the flow path structure X and the flow path structure X in the -W3 line | wire is represented.

  The flow path structure X shown in FIG. 5A includes four inlets I1, I2, I3, I4, an outlet O, and inlet flow paths C1, C2, C3, which are connected to I1, I2, I3, and I4, respectively. C4.

  The flow path structure X shown in FIG. 5 (a) is configured by combining circular tubes having partially different inner diameters. However, not all of the portions are necessarily circular tubes, such as glass tubes. It may be configured by a combination of a multiple tube formed of and a flow channel structure having a rectangular cross section formed of a polymer material or the like.

  By sequentially introducing a sol solution, a buffer solution, a gelling agent solution, and oil from the upstream side inlets I1, I4, I2, and I3 to the channel structure X shown in FIG. In J, as in the case of the flow path on a plane, it is possible to produce a droplet that contains non-spherical hydrogel particles.

  In FIG. 6, each of the inlet channels C2, C3, and C4 has a branch in the middle, and each branch merges with other inlet channels in stages, and the channel structure X It is the schematic which showed a mode that each solution flows through the inside, and the mode of non-spherical hydrogel particle synthesis | combination.

  6 has four inlets I1, I2, I3, I4, outlet O, and inlet passages C1, C2, C3, C4 connected to the inlets I1, I2, I3, I4, respectively. Have.

  In the channel structure X in FIG. 6, the inlet channels C2, C3, and C4 have branching / re-merging portions, and they merge stepwise at different junction points. May not have a branch, and all the inlet channels may join at one junction. Since the flow path structure X shown in FIG. 6 has a smaller number of inlets than the flow path structure shown in FIG. 3, the flow path structure becomes compact and the operability can be improved.

  FIG. 7 shows a flow channel structure X that enables synthesis of hydrogel particles partially composed of different components by introducing two types of sol solutions Z1 and Z2, and each of the flow channel structures X. It is the schematic which showed the mode of the flow of a solution, and the mode of the synthesis | combination of a non-spherical hydrogel particle.

  In FIG. 7, the channel structure X has nine inlets I1, I1′I2, I2 ′, I3, I3 ′, I4, I4 ′, I4 ″, an outlet O, and inlets I1, I1′I2, I2 ′, It has inlet channels C1, C1′C2, C2 ′, C3, C3 ′, C4, C4 ′, C4 ″ connected to I3, I3 ′, I4, I4 ′, I4 ″, respectively.

  In FIG. 7, non-spherical hydrogel particles composed of two fibers can be synthesized by introducing sol solutions Z having different compositions from the inlets I1 and I1 '.

  In FIG. 7, there are two inlets for introducing the sol solution Z, but this number may be two or more, and in that case, it is possible to synthesize non-spherical hydrogel particles composed of more types of parts. It becomes. In addition, by introducing the buffer solution B from the inlet I4 ″, the introduced sol solutions Z1 and Z2 can be independently gelled to form a fiber without contacting them.

  In addition, in the flow path structure X shown in FIGS. 3, 5, 6, and 7, droplets are formed inside the flow path structure X by introducing oil A. FIG. As shown, the droplets may be formed outside the channel structure X. That is, the flow channel structure X having a form in which the portions of the flow channel structure X shown in FIGS. 3 to 7 are arbitrarily combined may be used.

  The non-spherical hydrogel particles synthesized using the flow path structure X are yarn ball-shaped hydrogel particles.

  The size of the ball-shaped hydrogel particles and the fiber diameter in the particles can be arbitrarily controlled by adjusting the flow rate ratio of the solution to be introduced.

  In this embodiment, it is preferable to use an aqueous solution containing sodium alginate as the sol solution. Alginate hydrogel has high strength and biocompatibility, and can be applied as an immobilization carrier for cells and the like.

  Moreover, you may mix the solution which has arbitrary functions with the sodium alginate aqueous solution by the use of a fuzzy ball-shaped hydrogel particle. For example, in the case of using fuzzy ball-shaped hydrogel particles as a cell culture carrier, collagen, which is a preferred component as a cell culture material, may be added to a sodium alginate aqueous solution.

  By using the non-spherical hydrogel particle synthesizing method according to the present embodiment, complicated production apparatuses and operations necessary for the synthesis of conventional non-spherical hydrogel particles become unnecessary.

  Hereinafter, specific examples of the non-spherical hydrogel particle synthesis method and the non-spherical hydrogel particles according to the present invention will be described in detail.

  FIG. 8 is a schematic view of a device having a flow channel structure for synthesizing non-spherical hydrogel particles according to the present example. FIG. 8A is a schematic diagram showing a flow channel structure X configured in a plane and a device including the flow channel structure X as viewed from above, and an arrow B in FIGS. 8B to 8E. FIG. 8B to 8E are cross-sectional views of the device including the channel structure X, respectively, along the lines A0-A1, A2-A3, A4-A5, and A6-A7 in FIG. 8A. Represents.

  A device including a flow channel structure X for synthesizing non-spherical hydrogel particles according to the present embodiment does not have a flat polymer substrate (PDMS (polydimethylsiloxane)) having a fine groove structure and a groove structure. It is formed by bonding a flat polymer substrate (PDMS) up and down.

  Further, the device including the flow channel structure for synthesizing the non-spherical hydrogel particles according to the present example has a configuration similar to the flow channel structure shown in FIG. The overall size is about 2.5 × 7.5 cm.

  A channel structure X is formed on the lower surface of the upper polymer substrate, and its depth is about 110 μm. Larger values tend to form larger droplets, and smaller values tend to form smaller droplets. Therefore, it is possible to adopt a flow path structure having an arbitrary value of 1 μm minimum and 1 cm maximum depending on the size of the particles to be synthesized, and the same processing is applied to the upper surface of the lower substrate. The flow path structure may be partially different in depth.

  In FIG. 8, the channel structure X formed in the upper polymer substrate has four inlets I1 to I4 having an opening with a diameter of 2 mm, an outlet O having an opening with a diameter of 2 mm, a width of 100 μm, and a depth of about 110 μm. It is composed of a certain inlet channel C1 to C4, a channel portion J having a width of 400 μm and a depth of about 110 μm. Each of the inlet channels C2 to C4 is branched at a branch point in the middle, and 50% of the flow rate introduced from the inlet is distributed to each branch, and is designed to merge at the rejoining point. By doing in this way, it becomes possible to reduce the number of the inlets of the flow path structure X, and to make the flow path structure X into a more compact and simple structure.

  In FIG. 8, a silicone tube having an outer diameter of 2 mm and an inner diameter of 1 mm is connected to the through holes at the inlets I1 to I4 in order to continuously supply each solution from the inlet. Further, in order to continuously collect the synthesized non-spherical hydrogel particles, a silicone tube having an outer diameter of 2 mm and an inner diameter of 1 mm is connected to the outlet O. By immersing the tip of the tube in a container containing a 0.1 M calcium chloride aqueous solution for cleaning, non-spherical hydrogel particles can be automatically recovered.

  A method for synthesizing non-spherical hydrogel particles using the device having the above-described configuration and including a channel structure for synthesizing non-spherical hydrogel particles will be described.

  From inlet I1, a 2% (w / v) sodium alginate aqueous solution as a sol solution and a 0.01M calcium chloride aqueous solution containing 10% (w / v) dextran (molecular weight 500,000) as a gelling agent solution from inlet I2, Olive oil (containing 1% (w / w) lecithin) as an oil from the inlet I3 and 10% (w / v) dextran aqueous solution as a buffer solution from the I4 were successively introduced using a syringe pump.

  A sodium alginate solution having a concentration of 2% was used, but it should be in the range of 0.1 to 10%. The higher the concentration, the stronger non-spherical hydrogel particles can be produced. The molecular weight of alginic acid may be a high molecular weight or a low molecular weight. In addition, as the proportion of guluronic acid and mannuronic acid, which are constituents of alginic acid, the higher the proportion of guluronic acid, the higher the hardness of the non-spherical hydrogel particles produced, the optimal one is selected according to the application. Is possible.

  Dextran was used as a thickener to increase the viscosity of the buffer solution and gelling agent solution and stabilize the liquid transfer. However, depending on the conditions such as the scale of the flow path, dextran increases the viscosity if liquid transfer is possible. It is not necessary to use an agent, and it is possible to use dextran having a different molecular weight as long as it is a molecule that has an effect as a thickener. Furthermore, a water-soluble polymer other than dextran can be used as a thickener. It is also possible.

  As the gelling agent solution, a solution containing calcium chloride as a gelling agent was used, but instead of calcium chloride, a gelling agent such as barium chloride or magnesium chloride may be contained, Any combination of these may be included.

  The concentration of the gelling agent contained in the gelling agent solution was 0.01M. This value depends on the scale of the flow path, the types and concentrations of the gelling agent and sol solution, the physical properties of the solution, etc. It is desirable to set the optimal value.

  As the oil, other vegetable oils, mineral oils and the like can be used instead of olive oil. In addition, lecithin was used as a surfactant to prevent the droplets from coalescing, but even when no surfactant is used, it is possible to produce yarn-ball shaped hydrogel particles. It is also possible to use a molecule having a surface-active effect of this kind, and it is also possible to use a surfactant dissolved in a buffer solution or a gelling agent solution.

  Furthermore, when the cells are embedded in the hydrogel part of the non-spherical hydrogel particles, it is preferable to select a solution that does not damage the cells. For example, when animal cells are used, it is desirable that the sol solution, gelling agent solution, and buffer solution are made isotonic in advance.

  The introduction flow rates from the inlets I1 to I4 were, for example, 100 μL / h, 10 μL / h, 1 μL / h, and 3 μL / h, respectively. With respect to this flow rate, it is sufficient that the sol solution is partially gelled, droplets are formed in the flow path, and the partially gelled fiber is cut. An appropriate value can be set according to the size of the non-spherical hydrogel particles to be produced.

  FIG. 9 shows a flow state in the channel structure X under the above-described solution and flow rate conditions, and (a) shows a state in which the solutions merge at the junction points of the inlet channels C1 to C4. (B) is a photomicrograph showing a state in which a droplet is formed in the flow path portion J and the fiber is confined in the droplet and cut. It was observed that about 2 mm downstream from the meeting point, droplets were formed and the fibers were cut.

  In FIG. 10, green fluorescent fine particles having a diameter of 1 μm are added to the gelled solution, prepared under the above-described solution and flow rate conditions, recovered from the outlet O, and further washed with a 0.1 M calcium chloride aqueous solution. Later, a photograph of non-spherical hydrogel particles when observed with a microscope is shown. (A) is the microscope picture observed in the bright field, (b) is the microscope picture observed by fluorescence. It was confirmed that wool ball-shaped hydrogel particles having an average diameter of about 220 μm were synthesized.

  In addition, by changing the channel size, flow rate condition, and solution condition, a non-spherical hydrogel having a minimum of about 30 μm and a maximum of about 800 μm has been successfully produced.

  It is also possible to form a film on the outside of the non-spherical hydrogel particles by immersing the produced non-spherical hydrogel particles in a polycation solution such as poly-L-lysine or polyethyleneimine. By coating with a membrane in this way, non-spherical hydrogel particles are produced using calcium chloride as a gelling agent, and swelling occurs when immersed in a solution containing phosphoric acid such as animal cell culture medium. It is possible to suppress. In addition, when barium chloride is used as a gelling agent, it is possible to suppress swelling even when coating with a film is not performed.

  In addition, when cultured animal cells were suspended in an isotonic sol solution, and an isotonic gelling agent solution and non-spherical hydrogel particles were prepared and cultured, cells such as 3T3 Proliferation was confirmed, and it was demonstrated that application as a culture carrier is possible.

  The technique for synthesizing the hydrogel having the same form as the non-spherical hydrogel particles obtained in the present invention has not been reported so far. Such yarn ball-like gel particles have a dramatically larger specific surface area than spherical hydrogel particles obtained by the prior art. Therefore, when an enzyme or microorganism is immobilized in a gel material and used as a reactor, improvement in reaction efficiency, metabolic activity, etc. is expected.

  In addition, when culturing cells such as animal cells inside the gel particles, when conventional spherical hydrogel particles are used, oxygen and nutrients penetrate into the interior by diffusion from the outside, so that the cells near the particle surface Although the nutrients are distributed, there is a problem that the cells near the center are difficult to reach and the cells are killed. On the other hand, when wool ball-shaped particles are used, oxygen and nutrients can be efficiently supplied to the center of the particle because there is a gap in the particle. Can do. Therefore, it becomes possible to provide materials that are very useful in applications in tissue engineering and in substance production processes using animal cells.

  Furthermore, in cell transplantation, which is one technique of regenerative medicine, research has been conducted in which cells are encapsulated in a hydrogel and used as a carrier. In the case of conventional spherical hydrogel particles, because of their spherical shape, There is a possibility of obstructing blood flow at the time of transplantation and inhibiting blood flow. On the other hand, when the yarn ball-shaped hydrogel particles are used, it is considered that there is a low possibility that blood flow is inhibited because there are gaps inside the particles.

In addition to the application to the medical field, the hydrogel particles themselves are widely used in various other fields. Therefore, the yarn ball-shaped particles of the present invention are used in many fields such as food, pharmaceuticals, cosmetics, and regenerative medicine. It is considered useful.

Claims (16)

At least two inlets I1-In (n ≧ 2);
At least one exit O;
Inlet channels C1 to Cn respectively connected to the inlets I1 to In;
For the flow channel structure X having the flow channel portion J formed by joining the inlet flow channels C1 to Cn simultaneously or stepwise and connected to the outlet O,
The sol solution Z, which is a raw material of the hydrogel, is continuously introduced from the inlet I1, and the gelling agent solution G is continuously introduced from the inlet I2, and the sol solution Z is at least partially gelled in the flow path portion J. Forming fibers,
Furthermore, it is possible to produce non-spherical hydrogel particles by cutting the fiber by confining the fiber in which the sol solution Z is at least partially gelled in the droplet inside or outside the channel X. A method for synthesizing non-spherical hydrogel particles.   The method for synthesizing non-spherical hydrogel particles according to claim 1, wherein the droplets are water droplets dispersed in an oil phase or a gas phase.   The channel structure X has at least three inlets I1 to In (n ≧ 3), and by introducing oil A from at least one of the inlets I3, droplets are formed inside the channel X. The method for synthesizing non-spherical hydrogel particles according to claim 1, wherein:   The flow path structure X has at least four inlets I1 to In (n ≧ 4), and the buffer solution B is introduced from at least one of the inlets I4. A method for synthesizing the described non-spherical hydrogel particles.   The method for synthesizing non-spherical hydrogel particles according to any one of claims 1 to 4, wherein the channel structure X is constituted by at least a partially planar microchannel.   The method for synthesizing non-spherical hydrogel particles according to any one of claims 1 to 5, wherein the flow path structure X is at least partially configured by a structure using a multi-pipe.   7. At least one of the inlet channels C1 to Cn has a branch in the middle thereof, and each branch merges with other inlet channels at the same time or stepwise. 2. A method for synthesizing non-spherical hydrogel particles according to item 1.   The method for synthesizing non-spherical hydrogel particles according to any one of claims 1 to 7, wherein any one of the depth, width, diameter and the like of the flow channel structure X is in a range of 1 µm to 1 cm.   The non-spherical hydrogel particles according to any one of claims 1 to 8, wherein the sol solution Z is a sodium alginate aqueous solution, and the gelling agent solution G is a solution containing a polyvalent metal cation. Synthesis method.   The method for synthesizing non-spherical hydrogel particles according to any one of claims 1 to 9, wherein the concentration of the gelling agent in the gelling agent solution G is adjusted to an appropriate value in advance.   The method for synthesizing non-spherical hydrogel particles according to any one of claims 1 to 10, wherein a thickener is added in advance to at least one of the gelling agent solution G and the buffer solution B.   The method for synthesizing non-spherical hydrogel particles according to any one of claims 1 to 11, wherein a surfactant is previously added to at least one of the gelling agent solution G, the oil A, and the buffer solution B. .   The flow path structure X has at least two inlets I1 for introducing the sol solution Z, and introduces the sol solution Z having the same or different composition. A method for synthesizing non-spherical hydrogel particles.   The method for synthesizing non-spherical hydrogel particles according to any one of claims 1 to 13, wherein the non-spherical shape is a yarn ball shape.   The method for synthesizing nonspherical hydrogel particles according to any one of claims 1 to 14, wherein the diameter of the nonspherical hydrogel particles is 1 mm or less. A non-spherical hydrogel particle produced using the method according to any one of claims 1 to 15.