JP5472844B2 - Method for producing gel - Google Patents

Description

  The present invention relates to a method for producing a gel, and more particularly, to a method for producing a microbead and a method for producing a gel having an arbitrary shape including the production method.

  It is known that cells are encapsulated in microbeads made of alginate gel and used for research of agricultural chemicals and immunology. These microbeads are formed by suspending cells to be encapsulated in a sodium alginate aqueous solution and spraying the cell suspension on a solution of calcium chloride or barium chloride from a nozzle (Patent Document 1, Non-Patent Document 1). ). However, these microbeads are also manufactured by the above-described spraying method in which a cell-suspended sodium alginate aqueous solution is sprayed from an air jet nozzle, and the particle size of the manufactured microbeads is not uniform.

  If the particle size of the microbeads is not uniform, for example, the amount of the substance enclosed in the microbeads differs for each microbead, making it difficult to use the microbeads quantitatively. In addition, when microbeads are used as a drug delivery system (DDS) or the like, it is difficult to function as a DDS other than microbeads having a size within a predetermined range. In addition, the conventional method requires a large-scale apparatus and cannot be easily produced at a normal laboratory level. Furthermore, it is convenient when producing a large amount, but becomes inefficient when producing a small amount. When it is made for the purpose of DDS, a large amount of drugs are required. Usually such drugs are often expensive or valuable samples.

  The present applicant can easily produce a microbead gel having a uniform particle size in a single step without performing a sorting operation after the generation of a microbead gel (microbead-like gel, the same applies hereinafter). A method for producing a microbead gel that can be easily produced in a small amount was invented and a patent application was filed (Patent Document 2). In this method, a microbead gel is generated by ejecting one of two types of liquids that form a gel upon contact into the other liquid by an inkjet method. Furthermore, the applicant uses this microbead gel manufacturing method to produce an arbitrary one-dimensional, two-dimensional, and three-dimensional gel by ejecting while changing the arrival position of the liquid ejected by the ink jet method. Invented a method to do this (Non-patent Document 1)

JP 2004-99465 A JP 2007-111591 A Japanese Patent Publication No. 2-51734 Japanese Unexamined Patent Publication No. 57-133076 Artificial Organ Vol.35 No.2 2006 S-127〜S-128

  The method described in Patent Document 2 has a problem that the salt concentration in the gel forming liquid is high. For example, in a method in which an aqueous solution of sodium alginate is jetted into an aqueous calcium chloride solution by an ink jet method, the concentration of the aqueous calcium chloride solution is high, especially when encapsulating cells in a gel to produce a three-dimensional shape, Since a stronger gel is required, it is necessary to increase the concentration of the aqueous calcium chloride solution. When encapsulating a biological component such as a cell in a gel, a high calcium chloride concentration is undesirable because it has a harmful effect on the cell. However, in this method, simply lowering the calcium chloride makes it difficult to form a gel.

  An object of the present invention is to provide a method for producing a microbead gel, which can produce a microbead gel even when the concentration of a salt necessary for gel formation such as calcium chloride is low. In addition, the object of the present invention is to use the microbead gel manufacturing method, to obtain gels of any one-dimensional, two-dimensional and three-dimensional shapes even if the concentration of active ingredients necessary for gel formation such as calcium chloride is low. It is providing the manufacturing method of the gel which can be manufactured.

  As a result of earnest research, the inventors of the present invention have a structure in which the gel structure is strengthened by including nanoparticles in at least one of the two liquids to be mixed, and the salt concentration of calcium chloride or the like is low. Even so, it was found that the gel was formed well, and the present invention was completed.

That is, the present invention is a method for producing microbeads by mixing a gel-forming liquid A and a gel-forming liquid B that forms a gel when contacted with the gel-forming liquid A. In the method for producing a microbead gel, including producing a microbead gel by ejecting droplets of the liquid A from the outside of the gel-forming B liquid to the gel-forming B liquid by an inkjet method. At least one of the gel-forming liquid A and the gel-forming liquid B contains nanoparticles having a diameter of 25 nm or more and 2000 nm or less, and cells that are desired substances to be enclosed in microbeads are designated as the liquid A and the liquid B. A method for producing a microbead gel, characterized in that microbeads are produced by suspending the cells in at least one of the above , thereby encapsulating the cells therein. Further, the present invention includes forming a microbead gel by the method of the present invention, and when forming the microbead gel by the method, from the outside of the B solution, the B solution is added to the B solution. Provided is a method for producing a gel having a one-dimensional, two-dimensional or three-dimensional structure, which comprises ejecting the liquid A while changing the reaching position.

  The present invention provides for the first time a method for producing a microbead gel, which can produce a microbead gel satisfactorily even when the concentration of a salt for forming a gel such as calcium chloride is low. According to the method of the present invention, the concentration of a salt for forming a gel such as calcium chloride can be made lower than that of a known method. Therefore, when a biological component such as a cell is encapsulated inside a microbead gel, Toxicity to the biological component can be reduced. On the other hand, if the same concentration as in the known method is employed, a stronger gel structure can be obtained. In addition, according to the present invention, a method for producing an arbitrarily shaped gel having such advantages has been provided for the first time.

  As described above, the method of the present invention comprises a gel-forming liquid A (hereinafter sometimes simply referred to as “liquid A”) and a gel-forming liquid B (hereinafter simply referred to as “liquid“ A ”) that forms a gel upon contact with the liquid A. B liquid ”). Preferred examples of such combinations of solution A and solution B include (1) alginate aqueous solution and alkaline earth metal salt aqueous solution, (2) thrombin aqueous solution, fibrinogen aqueous solution and calcium salt aqueous solution, (3) peptide hydrogel formation Examples include, but are not limited to, one containing a salt in one gel-forming solution, such as a combination of an aqueous peptide solution and an aqueous sodium chloride solution. The method of the present invention has an important advantage that the concentration of the salt to be used can be lowered. However, when the gel is formed using the same components and component concentrations as the known method, Since there is an advantage that the structure becomes stronger, the combination of gel-forming liquids is not necessarily limited to those containing a salt, (4) boric acid aqueous solution and polyvinyl alcohol aqueous solution, and (5) when the temperature is raised A combination of a gelled thermoreversible hydrogel-forming hydrophilic polymer aqueous solution and warm water can also be employed.

  Here, regarding the combination of (1) above, examples of the alginate include sodium alginate, and examples of the alkaline earth metal salt include calcium chloride and barium chloride. The concentration of the alginate is not particularly limited, but is preferably about 0.5% to 1.5% by weight, and the concentration of the alkaline earth metal salt is not particularly limited, but the alkaline earth metal ion concentration is preferably about 30 mM to 180 mM, In particular, about 45 mM to 90 mM is preferable.

  In the combination (2), there are three components to be mixed, but an aqueous solution containing any two components and an aqueous solution containing the remaining one component are mixed. For example, an aqueous fibrinogen solution can be designated as solution A, and an aqueous solution containing thrombin and calcium salt can be designated as solution B. The concentration of thrombin is not particularly limited, but preferably about 200 units / mL to 800 units / mL, the concentration of fibrinogen is not particularly limited, but is preferably about 50 mg / mL to 150 mg / mL, and the calcium ion concentration is 30 mM to 180 mM. The degree is preferable, and about 45 mM to 90 mM is particularly preferable. As the calcium salt, calcium chloride or the like can be preferably used.

Examples of the peptide hydrogel-forming peptide of (3) above include Ac-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala- CONH ₂ (SEQ ID NO: 1) and amino acids represented by Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys-Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys (SEQ ID NO: 2) Like those having a sequence, a peptide having 12 to 20 amino acids, preferably about 16, in which neutral amino acids, acidic amino acids and / or basic amino acids are alternately arranged. It is commercially available under the trade name PuraMatrix from 3D Matrix. These peptide hydrogel-forming peptides react with sodium chloride in an aqueous sodium chloride solution to form a gel called peptide hydrogel. The concentration of sodium chloride may be a concentration that is contained in a normal cell culture medium, and is usually about 0.5 to 0.9 wt%.

  The above-mentioned (5) gelled thermoreversible hydrogel-forming hydrophilic polymer is a temperature sensitive polymer segment such as poly (N-isopropylacrylamide) or polypropylene oxide, and a hydrophilic polymer such as polyethylene oxide. A block copolymer composed of segments, for example, commercially available from Meviol Co., Ltd. under the trade name Meviol Gel. Meviol gel (trade name) is a sol at a low temperature and gels at 37 ° C. or higher. Therefore, as solution A, an aqueous meviol gel (trade name) solution of 36 ° C. or lower is used, and as liquid B, 37 ° C. or higher. By using warm water, if A liquid is injected in B liquid, A liquid will gelatinize in B liquid. In addition, since the meviol gel (trade name) aqueous solution has a relatively high viscosity, an ink jet printer capable of ejecting an ink having a relatively high viscosity is used.

  In the above combination, whichever may be the A liquid and which is the B liquid, it is preferable that the liquid having a low viscosity is the A liquid in order to stably discharge the ink jet. However, if both liquids are sufficiently low in viscosity and can be stably ejected by ink jet, either liquid is preferable. In the case of (1) above, an alginate aqueous solution is used as the liquid A. In the case of the above (5), it is preferable that the polymer aqueous solution is A liquid.

  In the method of the present invention, at least one of the liquid A and the liquid B contains nanoparticles having a diameter of 25 nm to 2000 nm μm, preferably 100 nm to 1200 nm (in general, when the diameter is 1000 nm or more, May not be called “nanoparticles”, but in the present invention, particles of 25 nm or more and 2000 nm are conveniently called “nanoparticles.” If the diameter of the nanoparticles exceeds 2000 nm, gel formation is inhibited, If the diameter of the nanoparticles is less than 25 nm, some of the nanoparticles may be detached from the gel after gel formation.Normally, either liquid A or liquid B contains nanoparticles, and the nanoparticles are wasted during production. It is preferable to include in the liquid A. The nanoparticle may be any material as long as it has the above-mentioned diameter, is stable in water, and does not react with other components or substances to be encapsulated. Siri with a diameter in the above range Since the particles, latex particles, etc. are widely available on the market, they can be used.The diameter of the commercially available nanoparticles is very uniform, and the diameter is displayed on the market. The diameter of the nanoparticles can be preferably used, and the diameter of the nanoparticles can be easily measured using a commercially available particle size distribution measuring apparatus using a He / Ne laser. The concentration of the nanoparticles is usually 1 mg / ml (0.1 wt%) to 50 mg / ml (5 wt%), preferably 0.3 wt% to 2.5 wt%. The concentration of the particles contained in these nanoparticles is, as desired, biological substances such as various proteins and nucleic acids exemplified as substances to be encapsulated in the gel, as described later, and labels such as fluorescent labels and chemiluminescent labels. Etc., for example FITC etc. Nanoparticles such as conjugated labels are commercially available.

  In the method of the present invention, microbeads are generated by ejecting droplets of the liquid A from the outside of the gel-forming B liquid onto the gel-forming B liquid by an inkjet method. It is preferable to stir or shake the B liquid or to change the spray position while the A liquid is being jetted. Stirring and shaking can be performed by shaking the petri dish containing the B liquid using a magnetic stirrer or a petri dish shaker. When changing the spray position, install it in a device that moves the nozzle mechanically. The mechanically moving device can be a two-axis stage or a dispenser robot that is also used in general inkjet printers and plotters. The “inkjet method” is a method in which a pressure in an ink cavity communicating with a nozzle (hereinafter referred to as “inkjet nozzle”) is increased to eject droplets from the ink jet nozzle. The ink jet method is a method employed in so-called ink jet printers, and is well known per se, since the term “ink jet” has been established in the field of printers. In the description and claims, the term “inkjet” is also used, and the ejected liquid is sometimes referred to as “ink”. However, in the present invention, the ejected liquid is the above-mentioned A liquid, and in most cases the ink The pressure in the ink cavity is increased by an electrical pulse signal. As a method for forming the ink cavity, a part of the ink cavity is formed by a piezoelectric element, and an electric pulse signal is applied to the piezoelectric element to deform the piezoelectric element, thereby deforming the ink cavity and reducing the volume of the ink cavity, thereby reducing the ink cavity. The pressure in the ink cavity is instantaneously increased (see Patent Document 3), or the ink in the ink cavity is heated by an electric pulse signal to vaporize part of the ink, and the pressure in the ink cavity is instantaneously increased by the ink vapor. There is a method (see Patent Document 4), etc., but any of these methods can be adopted in the present invention, although biological materials such as cells and proteins are encapsulated in microbeads as described later. If this is the case, heat may be adversely affected. A method of increasing the pressure in the ink cavity is preferable, and the ink cavity communicates with the ink tank, and ink is replenished from the ink tank when the ink in the ink cavity decreases due to the ejection of ink. For example, since the size of the droplet is controlled by the electric pulse signal, a droplet having a uniform size is ejected.

  In the method of the present invention, an ink jet mechanism (including an ink jet nozzle, an ink cavity, an ink tank, an ink passage connecting these, an electric control circuit, etc.) of a commercially available ink jet printer can be used as it is. In a commercially available ink jet printer, the ink jet head usually includes a plurality of ink jet nozzles. In the present invention, it is preferable from the viewpoint of improving productivity that the liquid A is simultaneously ejected from the plurality of ink jet nozzles. In a commercially available color inkjet printer, at least four colors of ink of black, cyan, magenta, and yellow are respectively stored in different ink tanks and ejected from different inkjet nozzles. An ejection mechanism of an ink jet printer can be preferably used. When using an ink jet mechanism of a commercially available ink jet printer, the liquid A may be about 0.3 mL or more, which is convenient when handling a small amount of sample.

  The inner diameter (diameter) of the inkjet nozzle is not particularly limited, and is usually about 5 μm to 100 μm. However, when encapsulating the cells in the gel, about 20 μm to 100 μm that can discharge the liquid A containing cells is preferable. . Ink jet printers having various ink jet nozzle inner diameters are commercially available depending on the resolution, and therefore commercially available products having desired ink jet nozzle diameters can be selected.

The ink jet printer has the following characteristics.
1) A fine droplet can be ejected by an ink jet mechanism.
2) The injection signal is controlled by the position of injection and the position of the head, and the appropriate material is injected in the right place. (On-demand injection)
3) High-resolution images can be printed with extremely small droplet size and position control. 4) Thousands of ink droplets are ejected from one nozzle per second.
5) Draw and draw droplets without touching the object to be printed.
6) Accumulation of ink jet heads enables drawing with a large number of nozzles.
7) Select any object. Ink droplets can be ejected, whether paper or water.
8) Computer control is established.

  The size (diameter) of the microbeads to be manufactured can be appropriately selected depending on the application, but is usually about 5 μm to 100 μm, preferably about 10 μm to 40 μm. The size of the microbead can be controlled by controlling the volume of the droplet ejected from the inkjet nozzle. The volume of the droplet can be controlled by adjusting the hole diameter of the ink jet nozzle or controlling the pressure applied to the ink cavity, the pressurization time, the pressurization frequency, and the like with an electric signal. For example, in the case of a microbead having a diameter of 40 μm produced in the following example, the volume of a droplet to be ejected is about 30 to 40 pL when regarded as a spherical shape. Inkjet printers with various resolutions are commercially available, so select a printer that gives microbeads of the desired size (the larger the resolution, the smaller the volume of the droplets), and use the ejection mechanism as it is You can also

  According to the method of the present invention, when the thickness (depth) of the liquid B is sufficient, spherical microbeads are generated. By reducing the thickness of the B liquid (that is, by reducing the depth of the B liquid), disk-shaped microbeads can be formed. Disc-shaped microbeads have not been known so far and have been provided for the first time by the method of the present invention. Disk-shaped microbeads are more advantageous in the case of being injected into blood vessels as carriers for intravascular administration type DDS because they are less likely to occlude blood vessels than spherical microbeads. The diameter of the disk-shaped microbeads is not particularly limited, and is usually about 5 μm to 100 μm, preferably about 10 μm to 40 μm, like the diameter of the spherical microbeads. The thickness of the disk-shaped microbead is not particularly limited, but is usually about 10 to 50% of the diameter. When manufacturing such disc-shaped microbeads, the thickness of the liquid B is suitably about 1 mm to 1 cm in normal ink jet discharge. The term “disc shape” is used in a broad sense, and includes a contact lens shape (a bottom surface is flat or concave, and a top surface is raised at the center), a substantially short cylinder shape, and the like. In the case of a contact lens shape, the above-described “thickness” is the thickness of the thickest portion near the center. A ring-shaped gel ring with a hole in it is also possible depending on how it is made.

A desired substance can be encapsulated inside the microbeads produced by the method of the present invention. Desired material to be sealed in the microbeads are various cells. More specifically, vascular endothelial cells, fibroblasts, smooth muscle cells, erythrocytes, leukocytes, platelets, cancer cells, and bacteria (single cells) such as Escherichia coli and lactic acid bacteria can be exemplified. Microbeads with encapsulated cells can be used to protect cells from various damage stimuli such as drying, and as cell / bacterial carriers for therapeutic equipment such as cell transplant beads and diagnostic equipment such as biochips. it can.

  When a desired substance is to be encapsulated in the microbeads, the desired substance is dissolved or suspended in at least one of liquid A and liquid B. It is preferable that the substance is uniformly dissolved or uniformly suspended in the A liquid or the B liquid so that a substance of as uniform a size as possible is encapsulated in each microbead. When dissolved or suspended in solution A, the concentration of the substance is determined by the amount to be enclosed in the microbeads, the concentration and viscosity of the material constituting the microbeads, and the ability of the inkjet nozzle to be used. As long as the viscosity can be discharged by the inkjet nozzle used, it is sufficient. With a normal inkjet nozzle, if it has a viscosity of 10 centipoise or less, it can be stably ejected. One type of microgel beads can be produced even if the viscosity becomes higher by dissolving or suspending a desired substance in the solution B.

  As described above, the ink ejection mechanism of the color inkjet printer includes a plurality of ink tanks and ejects different color inks from different inkjet nozzles. In the method of the present invention, each ink tank is filled with liquid A in which different types of desired encapsulated substances are dissolved or suspended, and simultaneously ejected from different nozzles, so that a plurality of types of microbeads in which different substances are encapsulated. The mixture can be prepared in a single step. For example, microbeads encapsulating vascular endothelial cells and microbeads encapsulating vascular endothelial growth factor (VEGF) can be prepared in a single step, and such a microbead mixture can be used for angiogenesis for injection. It can be suitably used for gel beads and the like. By adopting this method, the complicated process of preparing different types of microbeads enclosing different types of substances separately, quantifying the microbeads and mixing them in a predetermined amount can be performed in a single step. Is advantageous.

  When forming the microbead gel by the above-described method for producing the microbead gel of the present invention, the A liquid is changed from the outside of the B liquid to the B liquid while changing the arrival position of the A liquid. By injecting the liquid, a gel having a desired one-dimensional, two-dimensional or three-dimensional structure can be produced. Hereinafter, this method will be described.

  In this method, the A liquid is ejected while changing the arrival position of the A liquid. Here, the “arrival position” means a position on the liquid surface of the B liquid when the ejected droplet of the A liquid reaches the liquid surface of the B liquid. Therefore, ejecting the A liquid while changing the arrival position of the A liquid means that the A liquid is drawn on the B liquid as if it were ink. This can be easily performed by injecting the liquid A while moving the nozzle. At this time, it is preferable to adjust the ejection frequency of the liquid A and the moving speed of the nozzle so that two continuous droplets ejected continuously from the nozzle come into contact with each other. Thousands or more droplets are ejected from one nozzle of the ink jet per second. It is also possible to accumulate 10 times and 100 times nozzles. In the apparatus used in the following examples, 12 nozzles are provided in one head. Some commercial printers have hundreds of nozzles. When ejection is performed while moving the ink jet nozzle, the arrival position of each droplet on the B liquid level is slightly shifted. A single droplet of liquid A gels in liquid B to form microbeads. When a droplet and a droplet ejected one after that are ejected at an ejection frequency and a nozzle moving speed in contact with the B liquid, the two gel microbeads are fused. . Further, when the arrival position of the next ejected liquid droplet is a position in contact with the second ejected liquid droplet, the third gel microbead is also fused, and the three microbeads are connected. It becomes a shape. When the gel microbeads are successively adhered in this manner, a linear gel is formed. A linear gel having an arbitrary shape (straight line or arbitrary curve) can be obtained by jetting under the above-mentioned conditions while arbitrarily moving the inkjet nozzle.

  In addition, after a single linear gel is formed by this method, a second linear gel is formed at a position in contact with the linear gel. A gel that is wider than a linear gel can be formed. Further, by forming the third linear gel at a position in contact with the second linear gel, the third linear gel can also be adhered to the second linear gel. Thus, a sheet-like gel can be formed by forming a linear gel one after another at a position in contact with the previous linear gel. In this case, when the lengths of the respective linear gels are the same and the linear gel to be formed is arranged directly beside the previous linear gel, a rectangular sheet is formed. A sheet having an arbitrary shape can be formed by arbitrarily setting the length and position of the gel.

  Furthermore, a sheet-like gel can be laminated | stacked on the sheet-like gel formed as mentioned above by forming a sheet-like gel similarly. A three-dimensional gel can be formed by laminating sheet gels one after another. At this time, if the shape of each sheet-like gel to be laminated is the same, a columnar gel such as a rectangular parallelepiped is formed, but by arbitrarily setting the shape of each sheet-like gel and the position to be laminated, A gel having an arbitrary three-dimensional shape can be formed.

  A gel having a three-dimensional shape can also be formed by sequentially laminating a curvilinear gel on a curvilinear gel. For example, a gel on a tube can be formed by forming a circular linear gel and sequentially laminating the circular linear gel thereon.

  Thus, according to the method of the present invention, a gel having an arbitrary one-dimensional, two-dimensional or three-dimensional structure can be formed. Here, the “one-dimensional, two-dimensional or three-dimensional structure” means a structure in which gel microbeads formed by ejecting one drop of liquid A are considered as one point. Accordingly, the one-dimensional structure means a straight line, the two-dimensional structure means a sheet or curve, and the three-dimensional structure means a three-dimensional structure.

  As described above, it is preferable to adjust the ejection frequency of the liquid A and the moving speed of the nozzle so that two continuous droplets ejected continuously from the nozzle come into contact with each other. The gel in a shape can be produced by ejecting the liquid A at random until each droplet finally comes into contact within a predetermined plane, and thus is not essential.

  In a commercially available inkjet printer, printing is performed while an inkjet head including nozzles moves in a straight line and paper to be printed is moved. However, when the liquid B is moved, it is difficult to form a fine gel structure due to the liquid surface being rippled. Therefore, it is preferable to perform the ejection while moving the ink jet head without moving the liquid B. In a commercially available ink jet printer, the ink jet head can move only in a straight line, so that free drawing cannot be performed. Therefore, in the method of the present invention, it is preferable to use an ink jet ejecting apparatus in which the ink jet nozzle moves in at least a biaxial (XY axis) direction, preferably a triaxial direction (XYZ axis). Such an apparatus can be easily manufactured by mounting an inkjet head of a commercially available inkjet printer on a biaxial or triaxial actuator. Since biaxial or triaxial actuators are widely marketed, commercially available products can be used. Therefore, the ejection device can be easily installed by mounting an inkjet head of a commercially available inkjet printer on a commercially available 2-axis or 3-axis actuator (using all the mechanisms included in the inkjet printer for inkjet ejection as it is). Can be produced. When forming a gel having a three-dimensional structure, it is possible to use a triaxial actuator in order to make it possible to make the distance between the nozzle and the position where the jetted A droplet gels constant. preferable.

  The liquid A can be ejected at a frequency and a nozzle moving speed at which the liquid A droplet ejected from the inkjet nozzle and the liquid A droplet ejected one after that contact in the liquid B. The jetting speed and nozzle moving speed that can be used vary depending on the types of A liquid and B liquid used, the nozzle diameter, etc., but the jetting frequency and the nozzle moving speed are changed as specifically described in the following examples. It can be easily set by routine experiments. The ejection frequency can be changed by changing the frequency (launch frequency) of the electric pulse signal applied to the ink jet cavity.

  When forming a gel having a predetermined structure, a thickener may be included in the B liquid in order to prevent the gel from moving in the B liquid under construction. As the thickener, a water-soluble polymer such as polyvinyl alcohol (PVA), hyaluronic acid or a salt thereof can be preferably used. The concentration of the thickener is appropriately selected, but is usually about 0.2% by weight to 1% by weight.

  When a desired substance is to be encapsulated in the gel, the desired substance is dissolved or suspended in at least one of liquid A and liquid B. It is preferable that the substance is uniformly dissolved or uniformly suspended in the liquid A or the liquid B so that a uniform amount of the substance is encapsulated in each gel. When dissolved or suspended in liquid A, the concentration of the substance is determined by the amount to be enclosed in the gel, the concentration and viscosity of the material constituting the gel, and the ability of the inkjet nozzle to be used. It is sufficient that the viscosity be ejected by the inkjet nozzle. With a normal inkjet nozzle, if it has a viscosity of 10 centipoise or less, it can be stably ejected. One type of microgel beads can be produced even if the viscosity becomes higher by dissolving or suspending a desired substance in the solution B.

  As described above, the ink ejection mechanism of the color inkjet printer includes a plurality of ink tanks and ejects different color inks from different inkjet nozzles. In the method of the present invention, a plurality of types of gels in which different substances are encapsulated are formed by putting liquid A in which different types of desired encapsulated substances are dissolved or suspended in each ink tank and simultaneously ejecting them from different nozzles. A gel can be formed that includes elements in place. For example, liquid A in which organ cells are suspended and liquid B in which vascular cells are suspended are sprayed from different nozzles, and gels of a desired shape are formed by the above-described methods (the vascular cell-containing gel is, for example, It is possible to form a tissue having a three-dimensional structure having blood vessels in the organ. Thus, according to the present invention, it is possible to form a tissue having an arbitrary structure composed of a plurality of types of cells.

In addition, the following effect is acquired by embedding a cell in a gel by the method of this invention.
1) By embedding in a gel, damage due to cell drying can be prevented.
2) Enables three-dimensional layered modeling by embedding in a gel.
3) The gel does not bleed even in an aqueous solution. Do not mix.
4) This method using gelation enables 3D modeling by ink jetting in an aqueous solution such as a culture solution. Thereby, the produced three-dimensional structure is protected from drying even after production. In addition, it can be cultured and grown as it is.
5) Due to the buoyancy caused by water, it is difficult to be crushed by gravity even if it is a structure made of soft gel.
6) It also works to alleviate the physical impact that the cells receive.

  After the gel is formed by the method of the present invention, it can be dissolved. For example, in the case of an alginate gel, it is known that when a calcium chelating agent such as citric acid or EDTA is added, calcium ions are deprived and the alginate gel is dissolved again (see Reference Example 2 below). It is also known that in the case of a gel that melts depending on the temperature (the above-described (5) gelled thermoreversible hydrogel at elevated temperature), it can be melted by changing the temperature. Therefore, if the gel is melted after forming a gel having a one-dimensional, two-dimensional or three-dimensional structure by the method of the present invention, it is possible to leave only the cells or contents that have been struck out in the space.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples. Note that “%” relating to the concentration means wt% unless otherwise indicated.

Comparative Examples 1 and 2
An ink jet printer in which the hole diameter of the ink jet nozzle was 60 μm and the head itself moved triaxially was used. A 0.8% sodium alginate solution was jetted from an ink jet printer onto a 10% (comparative example 1) or 5% (comparative example 2) calcium chloride aqueous solution (containing 0.5% sodium hyaluronate). As shown in FIG. 1, the spraying was performed while moving the nozzle so that the formed microbead gel drawn a circle having a diameter of 1 mm (nozzle moving speed 20 mm / sec). When the circle of the formed microbead gel is drawn, the nozzle is moved so that the second stage microbead gel is laminated on the drawn circle, and this is repeated to make a circle. The arranged microbead gels were laminated one after another to form a three-dimensional tube. In FIG. 1, 10 is an inkjet nozzle, 12 is a three-dimensional tube made of microbead gel, and 14 is a calcium chloride aqueous solution (containing 0.5% sodium hyaluronate).

As a result, when an alginate aqueous solution was injected into a 10% concentration calcium chloride aqueous solution, a three-dimensional tube made of microbead gel was formed, but when an alginate aqueous solution was injected into a 5% calcium chloride aqueous solution, The gel formation was insufficient, and a three-dimensional tube was not formed.

Reference Examples 1-4
A suspension of commercially available FITC-labeled nanoparticles (diameter 250 nm, polystyrene latex particles, trade name micromer-green F, manufactured by Corefront) in a 0.8% sodium alginate solution at a final concentration of 3.25 mg / ml (0.325)% Was used as solution A, and the concentration of calcium chloride aqueous solution (including 0.5% sodium hyaluronate) was 10% ( Reference Example 1), 2% ( Reference Example 2), 1% ( Reference Example 3) or 0.5% ( Reference Example) The same operation as in Comparative Example 1 was performed except that 4).

  As a result, a three-dimensional tube composed of a microbead gel was formed not only when the concentration of the calcium chloride aqueous solution was 10% but also when the concentration was 2%, 1%, or 0.5%. In addition, the nanoparticles were retained in the gel and never separated out of the gel.

Comparative Example 3
3.5 x 10 in 0.8% aqueous sodium alginate solution ⁶ A tube made of microbead gel encapsulating HeLa cells was formed by performing the same operation as in Comparative Example 1 except that a cell suspension in which HeLa cells were suspended at a density of cells / ml was used as solution A. After forming the tube, the encapsulated HeLa cells were recovered by adding EDTA and re-dissolving the gel, and the survival rate of the recovered HeLa cells was measured. Viability was measured by staining dead cells with trypan blue staining, counting the total number of cells and the number of dead cells, and calculating the percentage of living cells. The experiment was performed 5 times at each concentration, and the average value of survival rate ± standard deviation was calculated. The average survival rate ± standard deviation was 69 ± 6.7%.

Example 5
Similar to Reference Example 3 (calcium chloride concentration 1%) except that a cell suspension obtained by suspending HeLa cells in a 0.8% sodium alginate aqueous solution at a density of 3.5 × 10 ⁶ cells / ml is used as solution A. The operation was performed to form a tube made of microbead gel encapsulating HeLa cells. In the same manner as in Comparative Example 3, the survival rate of the cells encapsulated in the gel was measured. The mean value ± standard deviation of the survival rate was 85 ± 1.6%.

Comparing Comparative Example 3 and Example 5, it can be seen that the viability of the cells increased from 69% to 85% by adding the nanoparticles. Therefore, according to the method of the present invention, damage to cells to be encapsulated can be made smaller than that of a known method.

Reference examples 5 and 6
Reference Example 1 except that polystyrene latex particles having a diameter of 100 nm (trade name micromer100, manufactured by Corefront, Reference Example 5 ) or polystyrene latex particles having a diameter of 800 nm (trade name micromer800, manufactured by Corefront, Reference Example 6 ) are used. The same operation was performed to form a tube made of microbead gel.

As a result, it was possible to form a tube at any of the four calcium chloride concentrations, regardless of which nanoparticle was used. When nanoparticles with a diameter of 800 nm were used, a more complete clean tube could be formed.

Reference Example 7
The same operation as in Reference Example 2 was performed, except that the liquid A contained FITC-labeled polystyrene latex particles (trade name: micromer25, manufactured by Corefront) having a diameter of 25 nm at a concentration of 2.5 mg / ml.

  As a result, a tube made of microbead gel was formed. When the tube was observed 1 hour after the tube was formed, a small amount of nanoparticles leaked out of the tube, but most of the nanoparticles were retained in the tube-shaped gel.

It is a figure which shows typically the method of forming the tube which consists of microbead gel by the method of this invention.

Explanation of symbols

10 Inkjet nozzle
Three-dimensional tube consisting of 12 microbead gels 14 Calcium chloride aqueous solution (containing 0.5% sodium hyaluronate)

Claims (7)

A method of producing a microbead by mixing a gel-forming liquid A and a gel-forming liquid B that forms a gel when contacted with the gel-forming liquid A. In the method for producing a microbead gel, the method includes producing the microbead gel by spraying the gel-forming B liquid from the outside of the gel-forming B liquid by an ink jet method. At least one of the gel-forming B liquid contains nanoparticles having a diameter of 25 nm or more and 2000 nm or less, and a cell as a desired substance to be encapsulated in microbeads is suspended in at least one of the A liquid and B liquid. turbid; then, thereby characterized in that to produce a micro-beads encapsulating the cells in the interior, the manufacturing method of the microbeads gel.   The method according to claim 1, wherein the concentration of the nanoparticles in the liquid A and / or liquid B containing the nanoparticles is 0.1 wt% to 5 wt%.   The method according to claim 1 or 2, wherein the nanoparticles have a particle size of 100 nm to 1200 nm.   The combination of the A liquid and the B liquid is (1) an alginate aqueous solution and an alkaline earth metal salt aqueous solution, and the concentration of the alkaline earth metal salt aqueous solution is 30 mM to 180 mM. The method described in 1.   The method according to claim 4, wherein the alkaline earth metal salt is calcium chloride.   Forming a microbead gel by the method according to any one of claims 1 to 5, and when forming the microbead gel by the method, from the outside of the B solution to the B solution, A method for producing a gel having a one-dimensional, two-dimensional, or three-dimensional structure, comprising ejecting the liquid A while changing the position where the liquid A reaches.   The method according to claim 6, wherein a gel having a three-dimensional structure is produced.

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