JP2017079722A - Anisotropic hydrogels, microbeads, production method of microbeads, scaffolds and production methods of scaffolds - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide anisotropic hydrogels, microbeads, production methods of microbeads, scaffolds and production methods of scaffolds which can perform cell cultures which was difficult by conventional techniques, such as the differentiation induction of each stem cell and co-culture between different types of cells.SOLUTION: Microbeads having an anisotropy by containing a plurality of varieties of components which are compartmented in a single particle is produced, wherein at least one of the plurality of varieties components is a biogenic component. For example, as a plurality of varieties of components, the microbeads containing a plurality of collagens 1a and 1b which have different properties each other are produced. At this time, each of the plurality of collagens 1a and 1b is different in at least one of molecular modification, concentration and type.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an anisotropic hydrogel, microbeads using the anisotropic hydrogel, a method for producing microbeads, a scaffold using the anisotropic hydrogel, and a method for producing the scaffold.

Collagen is a kind of extracellular matrix (ECM) and is widely known as a substance that supports activities such as cell function and differentiation. Microbeads made of collagen (hereinafter also referred to as “collagen beads”) are one of the most important materials for cell culture that reproduces the in vivo environment in vitro. It has become. In recent years, it has been widely used not only for cell culture but also in the field of tissue engineering such as construction of a three-dimensional tissue because of its excellent operability.
As a method for producing collagen beads, for example, there is a method of forming collagen beads by dropping a collagen solution into a tannin solution (see, for example, Patent Document 1). A method of forming a collagen bead by dropping a collagen solution into oil is also known.

Japanese Patent Laid-Open No. 06-141822

The inventors of the present invention can implement a cell culture method that has been difficult with conventional collagen beads, such as inducing differentiation of individual stem cells or co-culturing between different cells in a single particle. Therefore, it came to consider producing anisotropy collagen beads.
However, with the technique described in Patent Document 1, it has been difficult to produce collagen beads having anisotropy. The reason is that collagen transitions from sol to gel and hardens, but it takes a very long time for gelation (it takes several minutes to 30 minutes at the earliest). For example, by placing collagen solutions in tannin solution or oil at different concentrations on the left and right sides, even when trying to produce collagen beads with different properties in the right and left hemispheres, the collagen solution (collagen) diffuses before it hardens Therefore, the right hemisphere collagen and the left hemisphere collagen cannot be clearly separated. In addition, for example, even when trying to produce collagen beads with different properties in the right and left hemispheres by discharging the collagen solution from the inkjet nozzle, the gelation of the collagen is still in time and the collagen solution (collagen) is mixed. Therefore, the collagen in the right hemisphere and the collagen in the left hemisphere cannot be clearly separated.

Therefore, conventionally, an anisotropic collagen bead cannot be produced, and an anisotropic microenvironment in a living body (in vivo) cannot be reproduced in a test tube (in vitro).
The present invention pays attention to the above points, and an anisotropic hydrogel capable of performing a cell culture method that has been difficult with conventional techniques such as differentiation induction of individual stem cells and co-culture between different cells, It aims at providing the manufacturing method of the microbead using this anisotropic hydrogel, the manufacturing method of a microbead, the scaffold using an anisotropic hydrogel, and a scaffold.

In order to solve the above problems, one embodiment of the present invention has anisotropy by containing a plurality of types of components partitioned in a single particle, and at least one of the plurality of types of components is It is a biological component. For example, the gist of the microbead according to one embodiment of the present invention is a microbead containing a plurality of collagens having different properties as a plurality of types of components.
In another aspect of the present invention, (a) a mixed solution in which collagen and alginic acid are mixed is generated, and (b) a mixture containing ungelatinized collagen by gelation of alginic acid contained in the mixed solution. The gist is that the method is a microbead manufacturing method in which a bead is produced, (c) a thin film is formed on the surface of the mixed bead, and (d) the gel of alginic acid and the gelation of collagen are performed inside the mixed bead. To do.

Still another embodiment of the present invention includes (a) a plurality of types of components partitioned in the microchamber, (b) at least one of the plurality of types of components is a biological component, and (c) a plurality of components. The gist is that the normal direction of the boundary surface between the types of components is a scaffold (culture environment) intersecting the normal direction of the bottom surface of the microchamber.
Still another embodiment of the present invention provides (a) a mixed solution in which collagen and alginic acid are mixed, and (b) mixed beads containing ungelatinized collagen by gelation of alginic acid contained in the mixed solution. The gist of the present invention is a method of manufacturing a scaffold, in which (c) mixed beads are housed in a microchamber, and (d) the gel of alginic acid and the gelation of collagen are performed inside the microchamber.

  Still another aspect of the present invention has an occupied area as a three-dimensional shape surrounded by a boundary surface, and the occupied area is partitioned into a plurality of areas each containing a plurality of types of components, The gist of the invention is an anisotropic hydrogel characterized in that at least one of the components is a biological component.

  According to the present invention, an anisotropic hydrogel capable of inducing differentiation of individual stem cells and co-culturing cells in a single particle with high survival rate, and using this anisotropic hydrogel Microbeads, microbead manufacturing methods, scaffolds using anisotropic hydrogels, and scaffold manufacturing methods can be provided.

It is a conceptual diagram which shows the structure of the collagen Janus bead which concerns on 1st Embodiment. It is a conceptual diagram which shows the structural example of the microfluidic device for producing collagen Janus bead. It is a conceptual diagram for demonstrating the manufacturing method of a collagen Janus bead. (A)-(c) is a figure which shows the process of preparation. (D) is a diagram showing the interior A. FIG. (E) is a diagram showing the interior B. FIG. It is a conceptual diagram for demonstrating the culture method of the cell using a collagen Janus bead. (A) is a figure which shows differentiation induction. (B) is a diagram showing co-culture. It is a conceptual diagram which shows the structure of the scaffold using the anisotropic hydrogel which concerns on 2nd Embodiment. It is a conceptual diagram which shows the structure of the modification of a scaffold. (A) is the figure which equally divided the ratio of collagen. (B) is the figure which changed the ratio of collagen. It is a conceptual diagram which shows the structural example of the micro chamber array for producing a scaffold. It is a conceptual diagram for demonstrating the manufacturing method of a scaffold. (A)-(c) is a figure which shows the process of preparation. (D) is a diagram showing the interior C. FIG. (E) is a diagram showing the interior D. FIG. It is a conceptual diagram for demonstrating the shape of a collagen-alginate mixed bead. (A) is a figure which shows on an oblate ball. (B) is a figure which shows an oblong shape. It is a conceptual diagram for demonstrating the shape of the front-end | tip part of a theta pipe | tube. It is a conceptual diagram for demonstrating the manufacturing method of a scaffold. It is a conceptual diagram for demonstrating the culture | cultivation method of the cell using a scaffold. (A) is a figure which shows differentiation induction. (B) is a diagram showing co-culture. It is a conceptual diagram for demonstrating the observation method of the cultured cell. It is an image for demonstrating the experimental result of the culture | cultivation of the cell using a scaffold. (A) is a figure which shows before culture | cultivation. (B) is a figure which shows after culture | cultivation. It is a conceptual diagram for demonstrating the observation method using the conventional scaffold.

Next, first and second embodiments of the present invention will be described with reference to the drawings.
In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, it should be noted that the drawings are schematic and different from the actual ones. Therefore, specific components should be determined in consideration of the following description.
In addition, the first and second embodiments described below exemplify structures and methods for embodying the technical idea of the present invention, and the technical idea of the present invention is the shape of a component, The structure and arrangement are not specified as follows. The technical idea of the present invention can be variously modified within the technical scope defined by the claims described in the claims. Furthermore, in the following description, numerous specific details are set forth to provide a thorough understanding of the first and second embodiments of the present invention, but one or more implementations may be provided without such specific details. Obviously, embodiments can be implemented. In other instances, well-known structures and devices are schematically shown in order to simplify the drawing.

<First Embodiment>
As shown in FIG. 1, the anisotropic hydrogel according to the first embodiment has occupied areas (1a, 1b) as a three-dimensional shape surrounded by a boundary surface (outer surface), and there are a plurality of occupied areas. A plurality of regions 1a and 1b containing each of the types of components are partitioned from each other, and at least one of the types of components is a biological component and is an anisotropy hydrogel. As the microbeads using the anisotropic hydrogel according to the first embodiment, collagen Janus beads 10 having anisotropy using collagen as a bead material will be described as an example.

(Composition of collagen Janus beads)
As shown in FIG. 1, a collagen Janus bead 10 according to the first embodiment includes two collagens (hereinafter referred to as “first collagen 1a” and “second collagen” divided inside in a single particle. 1b ”) and the entire surface is covered with the thin film 2. In FIG. 1, for the sake of explanation, a part of the thin film 2 on the surface is broken and opened, and the first collagen 1a and the second collagen 1b inside are shown. In FIG. 1, the thin film 2 is shown as being thicker for the sake of explanation, but the thickness of the thin film 2 is actually extremely thin.

The first collagen 1a and the second collagen 1b are collagens having different properties from each other. As the properties of collagen, for example, at least one of molecular modification, concentration, and type (type) can be used. Examples of the molecular modification include fluorescent staining. Examples of the type include type I, type II, and type III representing the classification of collagen.
In addition, although the example which has two types of collagen inside is demonstrated as the collagen Janus bead 10 which concerns on 1st Embodiment, it is not limited to two types. It is technically possible to produce collagen Janus beads 10 having three or more types of collagen.

  Moreover, although the collagen is assumed as a kind of anisotropic hydrogel which concerns on 1st Embodiment, it is not limited to collagen. For example, in addition to collagen, a hydrogel based on chitosan gel, gelatin, hyaluronic acid gel, laminin gel, peptide gel, fibrin gel, or a mixture thereof can be used. For example, Matrigel (Nippon Becton Dickinson Co., Ltd.) may be used as a commercially available product. Moreover, you may use the hydrogel etc. which can be formed by irradiating ultraviolet-ray or a radiation to water-soluble polymers, such as polyvinyl alcohol, polyethylene oxide, and polyvinylpyrrolidone. Further, supramolecular hydrogel or the like may be used as the anisotropic hydrogel according to the first embodiment. Supramolecular hydrogel is a non-covalent hydrogel with self-assembled monomer molecules, Shinji Matsumoto et al., “Supramolecular hydrogel as smart biomaterial”, DojinNews, Vol. 118, p. .1-17, specifically explained in 2006.

That is, the collagen Janus beads 10 according to the first embodiment can be microbeads having anisotropic hydrogel other than collagen. For example, at least one of the first collagen 1a and the second collagen 1b can be changed to an anisotropic hydrogel other than collagen.
In preparing the anisotropic hydrogel, an aqueous organic solvent having a property of mixing with water, ethanol, acetone, ethylene glycol, propylene glycol, glycerin, dimethylformamide, dimethyl sulfoxide, or the like may be added. In order to increase the strength of the anisotropic hydrogel, an appropriate component or solvent can be blended. From such a viewpoint, for example, dimethyl sulfoxide can be added as a solvent for preparing a polyvinyl alcohol anisotropic hydrogel.

  The thin film 2 is a poly-L-lysine (PLL) film. A film formed of a polycationic polymer may be used. Although details will be described later, in the microbead manufacturing method according to the first embodiment, the collagen-alginate mixed beads 6 are prepared using a mixed solution of a collagen solution and a sodium alginate solution as a pre-stage of the production of the collagen Janus beads 10. To do. At this time, a polymer (poly-L-lysine (PLL)) that is positively charged is bonded to alginic acid whose surface is negatively charged by electrostatic force, and the thin film 2 is formed.

(Configuration of microfluidic device)
In the microbead manufacturing method according to the first embodiment, the microfluidic device 20 using a centrifugal force as a drive source is used to produce the collagen Janus beads 10. As the microfluidic device 20, for example, a liquid gelation apparatus described in JP 2012-176374 A can be used.

As shown in FIG. 2, the microfluidic device 20 includes a microtube 21 and theta tubes 22a and 22b.
The microtube 21 is formed in a cylindrical body portion 21a, a liquid chamber 21b formed at one end of the body portion 21a, containing the gelling agent liquid 4, and the body portion 21a. A fixing portion 21c for fixing the theta tubes 22a and 22b to the gelling agent liquid 4 at a predetermined distance L is provided at the central axis position. As the gelling agent liquid 4, for example, a liquid capable of gelling the gelation target solutions 5a and 5b including a collagen solution (described later) can be used.

Theta tubes 22a and 22b are capillaries (capillaries) whose one ends (tips) on the liquid chamber 21b side are tapered. Then, the gelation target solutions 5a and 5b can be filled inside from the rear end, and the gelation target solutions 5a and 5b can be released into the gelling agent liquid 4 from the front end.
In the microfluidic device 20, a plurality of theta tubes 22 a and 22 b are fixed inside the microtube 21. For example, a multi-barrel tube in which a plurality of theta tubes 22a and 22b are bundled is fixed. Alternatively, a plurality of theta tubes 22a and 22b may be simulated by dividing a cavity inside one theta tube into a plurality of systems. Actually, the present invention is not limited to the theta tubes 22a and 22b, and at least a plurality of types of gelation target solutions 5a and 5b are simultaneously released from the tip, thereby releasing the gelation target solutions 5a and 5b. As long as the structures are in contact with each other. In the example of FIG. 2, two types (two systems) of theta tubes 22a and 22b are used so that two types of gelation target solutions 5a and 5b containing a collagen solution (collagen) can be released. That is, by increasing the number of theta tubes 22a and 22b (number of lines), the types of gelation target solutions 5a and 5b that come into contact with each other after release can be increased.

  Although not shown, the microtube 21 is attached to a small centrifuge, and centrifugal force is applied to the gelation target solutions 5a and 5b in the theta tubes 22a and 22b. Due to this centrifugal force, the gelation target solutions 5a and 5b are released from the tips of the theta tubes 22a and 22b. Beads are formed. In the microbead manufacturing method according to the first embodiment, the collagen-alginate mixed beads 6 are formed by using the mixed solution obtained by mixing the collagen solution and the sodium alginate solution as the gelation target solutions 5a and 5b. Details of the collagen-alginate mixed beads 6 will be described later.

  Further, since centrifugal force is applied by a small and high centrifugal force centrifuge and the gelation target solutions 5a and 5b are discharged from the tips of the theta tubes 22a and 22b, it is difficult to produce by the conventional method. Collagen-alginate mixed beads 6 having a diameter of 20 μm or more and 200 μm or less can be produced. In particular, a collagen-alginic acid mixed bead 6 having a diameter of 80 μm or more and 120 μm or less can be produced. Therefore, for example, extremely minute collagen-alginate mixed beads 6 (collagen Janus beads 10) having a diameter of about 100 μm can be produced. In the conventional method, it was not possible to produce a collagen-alginate mixed bead 6 having a minute size of about 100 μm in diameter. When cells or tissues are cultured inside the collagen Janus beads 10, it is very valuable to have a diameter of about 100 μm. For example, when the size of the collagen Janus bead 10 is large, no nutrient is contained in the inside due to passive diffusion, and thus cells and tissues tend to die at the center. However, if the diameter is about 100 μm, nutrients enter the inside by passive diffusion, so that the nutrients are sufficiently distributed to the center. It is also possible to produce collagen-alginic acid mixed beads 6 having a smaller size by increasing the centrifugal force or by reducing the tip diameters of the theta tubes 22a and 22b. Since the size of the single cell is about 10 μm in diameter, the size of the collagen-alginate mixed beads 6 is preferably 20 μm or more in diameter.

(Method for producing collagen Janus beads)
First, as gelation target solutions 5a and 5b, a collagen solution (5 [mg / mL]) and a 5% ([w / w]) sodium alginate solution were mixed at 3: 2 ([v / v]). A mixed solution is produced. Alginic acid is very easy to gel as compared with collagen, and instantly hardens when it comes into contact with the gelling agent solution 4. Therefore, before the gelation target solutions 5a and 5b are mixed with each other, each collagen in the gelation target solutions 5a and 5b can be hardened with alginic acid. Therefore, the collagen can be temporarily solidified without waiting for the completion of collagen gelation.

  Moreover, when producing the collagen Janus beads 10 having a plurality of collagens with different fluorescent stains, a plurality of types of mixed solutions subjected to different fluorescent stains are used. For example, two types of mixed solutions, a mixed solution that has been subjected to blue fluorescent staining and a mixed solution that has been subjected to red fluorescent staining, are used as the gelation target solutions 5a and 5b, thereby forming collagen that has been fluorescently stained red. Collagen Janus beads 10 having a hemisphere (collagen hemisphere) and a hemisphere (collagen hemisphere) made of collagen fluorescently stained in blue and having pseudo anisotropy can be produced.

  Further, when producing the collagen Janus beads 10 having a plurality of collagens having different concentrations and types, the concentration or type of collagen contained in the collagen solution, the ratio of the collagen solution contained in the mixed solution, etc. were changed. Multiple types of mixed solutions are used. For example, the mechanical strength of the collagen after gelation can be changed by changing the collagen concentration of each of the gelation target solutions 5a and 5b. Thereby, the collagen Janus bead 10 which has a hard collagen hemisphere and a soft collagen hemisphere, and has anisotropy is producible.

  Next, the mixed solution (the gelation target solutions 5a and 5b) is filled into the theta tubes 22a and 22b, and the theta tubes 22a and 22b are fixed in the microtube 21 containing the gelling agent solution 4, Centrifugal force is applied to the microtube 21 using a centrifuge. By this centrifugal force, the mixed solution (gelation target solutions 5a and 5b) in the theta tubes 22a and 22b is released toward the gelling agent solution 4 in the microtube 21. As the centrifugal force, 2000 [G] is applied for 3 minutes. As the gelling agent solution 4, a calcium chloride solution (500 [mM]) is used at 80 [μL]. By this calcium chloride solution, sodium alginate contained in the mixed solution (gelation target solutions 5a and 5b) becomes a calcium alginate hydrogel and hardens, and collagen-alginate mixed beads 6 are formed as shown in FIG. 3 (a). Is done.

  The details of the interior A of the collagen-alginate mixed beads 6 shown in FIG. 3A are shown in FIG. As shown in FIG. 3D, in the inside A of the collagen-alginate mixed bead 6, the first collagen 1a and the second collagen among the gelation target solutions 5a and 5b constituting the collagen-alginate mixed bead 6 are used. Collagen 1b is not yet gelled, but alginic acid 7 is gelled to form a calcium alginate hydrogel network and retains the non-gelled first collagen 1a and second collagen 1b. .

Next, the formed collagen-alginic acid mixed beads 6 are washed with a sodium chloride solution (150 [mM]) and then immersed in a poly-L-lysine (PLL) solution. By dipping in this poly-L-lysine (PLL) solution, a poly-L-lysine (PLL) film is formed as a thin film 2 on the surface of the collagen-alginic acid mixed beads 6 as shown in FIG. The
Next, the collagen-alginate mixed beads 6 on which the thin film 2 is formed are washed with physiological saline, and then an alginate lyase solution is added to the physiological saline to a final concentration of 200 [μg / mL]. ] For 10 minutes, and the collagen-alginate mixed beads 6 are treated with arginase. As the physiological saline, for example, Dulbecco's phosphate buffered physiological saline is used. By this arginase treatment, the dissolution (melting) of the hydrogel of calcium alginate in the collagen-alginate mixed beads 6 and the gelation of collagen are performed. Then, the hydrogel of calcium alginate is removed from the inside of the collagen-alginic acid mixed beads 6 having the thin film 2 formed on the surface, and the gelled first collagen 1a and second collagen 1b are left inside, as shown in FIG. As shown in (c), collagen Janus beads 10 are produced (manufactured). In FIG. 3C, for the sake of explanation, a part of the thin film 2 on the surface of the collagen Janus bead 10 is broken and opened to show the first collagen 1a inside.

  Details of the interior B of the collagen Janus beads 10 shown in FIG. 3C are shown in FIG. As shown in FIG. 3 (e), alginic acid 7 (calcium alginate hydrogel) is completely removed in the interior B of the collagen Janus beads 10, and the gelation of the first collagen 1a and the second collagen 1b is completed. A fiber network of the first collagen 1a and the second collagen 1b is constructed inside the collagen Janus beads 10.

By following each of the above procedures, a single particle has a first collagen 1a and a second collagen 1b partitioned inside, and the surface is a thin film 2 (poly-L-lysine (PLL) film) The collagen Janus beads 10 in a state of being coated with () can be produced.
It should be noted that the beads (collagen-alginate beads 6 in the above-described procedures of injection formation of collagen-alginate mixed beads 6 by microfluidic device 20, coating of poly-L-lysine (PLL) membrane, and dissolution of calcium alginate hydrogel by alginate lyase The diameters of the alginate mixed beads 6 and the collagen Janus beads 10) are approximately uniform.

  The finally produced collagen Janus beads 10 have two different collagen hemispheres, and each collagen hemisphere has an anisotropic collagen gel of collagen (first collagen 1a, second collagen 1b). A fiber network has been established. That is, the collagen Janus bead 10 has an anisotropic fiber network in which a plurality of collagens are partitioned within a single particle.

  Each of the plurality of compartmentalized collagens (first collagen 1a, second collagen 1b) is a gel of uniform size. Therefore, it is possible to imitate the cell surface state in the living body including the cell layer. It is also possible to encapsulate and culture at least one type of cell inside the gel. In addition, it is possible to configure a plurality of different cell layers inside the collagen Janus beads 10 and imitate an anisotropic tissue formed by the plurality of cell layers. Furthermore, it is also possible to mimic a plurality of different anisotropic cell boundary environments on the boundaries of multiple collagens.

(Culture of cells using collagen Janus beads)
As described above, the collagen Janus beads 10 can be cultured by adding the cells 8a and 8b to the first collagen 1a and the second collagen 1b, respectively, as shown in FIGS. 4 (a) and 4 (b). . 4A and 4B, illustration and description of the thin film 2 are omitted.

  In the example of FIG. 4A, cell differentiation is induced by adding cells 8a and 8b to each of the first collagen 1a and the second collagen 1b and culturing them. In this case, the cells 8a cultured by adding to the first collagen 1a become the first type cells, and the cells 8b cultured by adding to the second collagen 1b become the second type cells, which are different types. Changes to cells. Further, in the example of FIG. 4B, co-culture between the heterogeneous cells 8a and 8b is performed by performing layered culture on the boundary between the first collagen 1a and the second collagen 1b. In this case, the cell 8a cultured after being added to the first collagen 1a and the cell 8b cultured after being added to the second collagen 1b are combined to form a network of cells 8a, 8b.

  Thus, by using the collagen Janus beads 10 according to the first embodiment, differentiation induction of individual cells 8a and 8b (stem cells), co-culture between different cells 8a and 8b in a single particle, and the like are conventional. It becomes possible to carry out a cell culture method using an anisotropic collagen gel fiber network, which was difficult with this method.

  Further, when the collagen Janus beads 10 are produced, as a molecular modification, when the collagen Janus beads 10 are produced by mixing the cells 8a subjected to red fluorescent staining and the cells 8b subjected to blue fluorescent staining as a molecular modification, the color is blue. The fluorescently stained collagen hemisphere is encapsulated in red fluorescently stained cells 8a, and the red fluorescently stained collagen hemisphere is encapsulated in blue fluorescently stained cells 8b. For example, NIH / 3T3 cells subjected to fluorescent staining are used as the cells 8a and 8b. Collagen hemisphere and cells 8a and 8b to be encapsulated can be visually discriminated by fluorescent staining to control the encapsulation of cells 8a and 8b and selectively encapsulate cells 8a and 8b in a microenvironment. Is possible. When the encapsulated cells 8a and 8b are determined to be alive, the survival rate of the cells 8a and 8b is about 86%, and it becomes possible to encapsulate with a very high survival rate.

  In the above description, the case where the cells 8a and 8b are encapsulated in the collagen Janus beads 10 has been described. However, in the collagen Janus beads 10, biological components such as cells, proteins, lipids, saccharides, nucleic acids, and antibodies are contained. One or more kinds can be added. The types of cells 8a and 8b are not particularly limited. For example, ES cells and iPS cells having pluripotency, various stem cells having pluripotency (hematopoietic stem cells, neural stem cells, mesenchymal stem cells, etc.), differentiation In addition to stem cells having a unity (hepatic stem cells, reproductive stem cells, etc.), various types of differentiated cells such as muscle cells such as skeletal muscle cells and cardiomyocytes, nerve cells, fibroblasts, epithelial cells, hepatocytes, Examples thereof include pancreatic β cells and skin cells. Of course, the cells and biological components are not limited to those exemplified above. Moreover, you may add a non-biological component to the collagen Janus bead 10 simultaneously. For example, fibers such as carbon nanofibers, inorganic substances such as a catalyst substance, beads coated with an antibody or the like, or an artificial object such as a microchip can be added.

(Effect of 1st Embodiment)
According to the microbead manufacturing method according to the first embodiment, by using the microfluidic device 20 using a centrifugal force as a driving source, collagen having anisotropy, which has been difficult to produce by a conventional method, is used. Janus beads 10 can be produced. In addition, a very small amount of collagen Janus beads 10 having a diameter of about 100 μm can be produced in large quantities without using reagents such as oils and organic solvents that are toxic to the cells 8a and 8b.

Further, the produced collagen Janus beads 10 have a thin film 2 of a poly-L-lysine (PLL) film on the surface and two types of collagen (first collagen 1a, second collagen 1b) hemisphere inside. In each collagen hemisphere, a fiber network of collagen (first collagen 1a, second collagen 1b) can be confirmed.
In addition, by making it possible to visually distinguish two different types of collagen hemispheres inside the collagen Janus beads 10, encapsulation is performed using a plurality of cells 8a and 8b (for example, NIH / 3T3 cells) that have undergone different fluorescent staining. Each target cell 8a, 8b can be selectively encapsulated in a plurality of different collagen hemispheres inside the collagen Janus beads 10. At that time, the survival rate of the cells 8a and 8b was 86%, and the cells could be encapsulated with a very high survival rate.
Further, by using the collagen Janus beads 10 according to the first embodiment, further understanding of the behavior of the cells 8a and 8b in an anisotropic microenvironment, and engineering such as cell culture and tissue engineering based on the understanding. Application is expected.

Second Embodiment
As shown in FIG. 5, the anisotropic hydrogel according to the second embodiment has occupied regions (3a, 3b) as a three-dimensional shape surrounded by a boundary surface (outer surface), and a plurality of the occupied regions. A plurality of regions 3a and 3b containing each of the types of components are partitioned from each other, and at least one of the types of components is a biological component and is a hydrogel having anisotropy. In the scaffold 30 using the anisotropic hydrogel according to the second embodiment and the manufacturing method thereof, the microchamber 42 is used instead of the thin film 2 of the microbeads (collagen Janus beads 10) according to the first embodiment. The microbeads according to the first embodiment and the method for manufacturing the microbeads are different from each other in that the anisotropic hydrogel (for example, collagen) mass (occupied region) is held.

(Structure of the scaffold)
As shown in FIG. 5, the scaffold 30 using the anisotropic hydrogel according to the second embodiment includes two collagens (hereinafter referred to as “first collagen 3 a”) inside the microchamber 42. And a normal line of the boundary surface 3c between the first collagen 3a and the second collagen 3b (also referred to as “second collagen 3b”). The direction intersects the normal direction of the bottom surface 42 b of the microchamber 42. In the example of FIG. 5, the normal direction of the boundary surface 3c is orthogonal to the normal direction of the bottom surface 42b. The first collagen 3a and the second collagen 3b are anisotropic collagens having different properties.

  In addition, although the anisotropic and composite example which has two types of collagen inside is demonstrated as the scaffold 30 using the anisotropic hydrogel which concerns on 2nd Embodiment, it is not limited to two types. As shown in FIGS. 6A and 6B, it is technically possible to produce a scaffold 30 having three or more types of collagen. It is also possible to change the ratio of each collagen. In the example of FIG. 6A, there are three types of collagen (first collagen 3a, second collagen 3b, and third collagen 3d), and the ratios of the collagens are equal to each other. Further, in the example of FIG. 6B, similarly, there are three types of collagen, but the ratio of one collagen (third collagen 3d) is larger than the ratio of the other two collagens.

  Moreover, although the collagen is assumed as a kind of anisotropic hydrogel which concerns on 2nd Embodiment, it is not limited to collagen. For example, in addition to collagen, a hydrogel based on chitosan gel, gelatin, hyaluronic acid gel, laminin gel, peptide gel, fibrin gel, matrigel, supramolecular hydrogel, or a mixture thereof can be used. Moreover, you may use the hydrogel which can be formed by irradiating water-soluble polymers, such as polyvinyl alcohol, polyethylene oxide, and polyvinylpyrrolidone, with an ultraviolet-ray or a radiation.

(Configuration of micro chamber array)
In the method for manufacturing the scaffold 30 using the anisotropic hydrogel according to the second embodiment, the microchamber array 40 is used to manufacture the anisotropic / complex scaffold 30. As shown in FIG. 7, the microchamber array 40 includes a plate-shaped main body plate 41 and a plurality of microchambers 42 arranged in an array on one surface of the main body plate 41.

  The microchamber 42 is a recess having a circular cross section that can accommodate only one collagen-alginate mixed bead 6. The diameter of the microchamber 42 is slightly larger than the diameter of the collagen-alginate mixed beads 6. For example, when the diameter of the collagen-alginate mixed beads 6 is about 100 [μm], the diameter of the microchamber 42 is about 150 [μm]. As a material of the microchamber array 40, silicon rubber is preferable in consideration of easy adhesion of the first collagen 3a and the second collagen 3b.

(Manufacturing method of scaffold)
First, in the same procedure as the method for manufacturing the anisotropic hydrogel according to the first embodiment, the microfluidic device 20 is used and, as shown in FIG. Collagen-alginate mixed beads 6 are formed as a mass (occupied region) of isotropic hydrogel.
Details of the interior C of the collagen-alginate mixed beads 6 shown in FIG. 8A are shown in FIG. As shown in FIG. 8D, in the inside C of the collagen-alginic acid mixed bead 6, the first collagen 3a and the second of the gelation target solutions 5a and 5b constituting the collagen-alginic acid mixed bead 6 are used. Collagen 3b is not yet gelled, but alginic acid 7 is gelled to form a calcium alginate hydrogel network and retains the non-gelled first collagen 3a and second collagen 3b. .

  In addition, as shown in FIG. 9A, the collagen-alginate mixed beads 6 are oblate (spheroids are flattened) crushed in a direction intersecting the normal direction of the boundary surface 5c between the gelation target solutions 5a and 5b. 9 (b), or a small lump of an oval shape (a shape in which a sphere is elongated) extending in the normal direction of the boundary surface 5c, as shown in FIG. 9B. . In the example shown in FIG. 9A, the shape is a small oblate sphere that is crushed in a direction perpendicular to the normal direction of the boundary surface 5c. Moreover, in the example of FIG. 9B, it is a fine oval lump extending in a direction parallel to the normal direction of the boundary surface 5c. Note that “oblong shape” and “ellipsoid” include not only a perfect oblate shape or an oblong shape, but also an approximate shape such as a combination of the oblate shape and the oblong shape.

  As a method for adjusting the flatness of the collagen-alginate mixed beads 6, for example, a method for adjusting the distance L (see FIG. 2) between the tips of the theta tubes 22 a and 22 b and the liquid level of the gelling agent solution 4. Can be adopted. In this method, as the distance L becomes longer, the speed at which the gelation target solutions 5a and 5b collide with the gelling agent solution 4 increases, so that the collagen-alginate mixed beads 6 can be flattened. Further, for example, as shown in FIG. 10, the collagen-alginate mixture is obtained by extending the cavity at the tip of the theta tubes 22a and 22b in the normal direction of the boundary portion 22c that partitions the theta tubes 22a and 22b. The beads 6 can be extended.

Next, as shown in FIG. 11, a plurality of microchamber arrays 40 are placed in the petri dish 50 with the surface on which the microchambers 42 are formed facing upward, and a buffer solution 51 is injected into the petri dish 50. Submerge all the microchamber arrays 40 under the buffer 51. As the buffer solution 51, for example, Dulbecco's phosphate buffered saline (DPBS) is used.
Next, a plurality of collagen-alginate mixed beads 6 are spread on each microchamber array 40 submerged in buffer 51, and collagen-alginate mixed beads 6 are placed in each microchamber 42 as shown in FIG. 8 (b). It is accommodated as a fine mass of anisotropic hydrogel. At that time, the collagen-alginic acid mixed beads 6 extend in the normal direction of the boundary surface 5c or the oblate shape collapsed in the direction intersecting the normal direction of the boundary surface 5c between the gelation target solutions 5a and 5b. Since the shape is oblong, the normal direction of the boundary surface 5c between the first collagen 3a and the second collagen 3b intersects the normal direction of the bottom surface 42b of the microchamber 42. In the example of FIG. 8B, the normal direction of the boundary surface 5c is orthogonal to the normal direction of the bottom surface 42b.

  Next, an alginate lyase solution is added to the petri dish 50 into which the buffer solution 51 is injected so that the final concentration becomes 200 [μg / mL], and the mixture is warmed at 37 [° C.] for 10 minutes, and the collagen-alginate mixed beads 6 are treated with arginase. I do. By this arginase treatment, the dissolution of the calcium alginate hydrogel network in the collagen-alginate mixed beads 6 and the gelation of the collagen are performed. Then, the hydrogel of calcium alginate is removed from the inside of the collagen-alginate mixed beads 6 accommodated in the microchamber 42, and the first collagen 3a and the second collagen 3b gelled inside the collagen-alginate mixed beads 6 are formed. Left behind. Then, as shown in FIG. 8C, the first collagen 3a and the second collagen 3b spread into the microchamber 42 in a state where the compartmentation is maintained, and the microchambers of the microchamber array 40 in the petri dish 50 are spread. An anisotropic / composite scaffold 30 is produced for each 42. By using such a scaffold 30 for culturing and observing a culture target such as a cell, the culture target can be observed more efficiently.

Details of the interior D of the scaffold 30 shown in FIG. 8C are shown in FIG. As shown in FIG. 8 (e), in the inside D of the scaffold 30, the alginate 7 (calcium alginate) is completely removed, the first collagen 3 a and the second collagen 3 b gel, and the first inside the scaffold 30. A fiber network of one collagen 3a and second collagen 3b is constructed.
By following each of the above procedures, in the microchamber 42, a minute mass of anisotropic / complex anisotropic hydrogel composed of the first collagen 3a and the second collagen 3b partitioned inside. The scaffold 30 in a state where the normal direction of the boundary surface 3c between the first collagen 3a and the second collagen 3b intersects the normal direction of the bottom surface 42b of the microchamber 42 can be produced. . Each of the plurality of compartmentalized collagens consists of an anisotropic hydrogel of uniform size. Therefore, it is possible to imitate the cell surface state in the living body including the cell layer. It is also possible to encapsulate and culture at least one type of cell inside the hydrogel. It is also possible to configure a plurality of different cell layers inside the scaffold 30 and imitate an anisotropic tissue formed by the plurality of cell layers. Furthermore, it is also possible to mimic a plurality of different anisotropic cell boundary environments on the boundaries of multiple collagens.

(Cell culture using anisotropic hydrogel)
As described above, the anisotropic hydrogel according to the second embodiment, as shown in FIGS. 12 (a) and 12 (b), has the cells 8a and 8a in each of the first collagen 3a and the second collagen 3b. 8b can be added and used as a scaffold 30 for culturing in an anisotropic microenvironment. As a method for adding the cells 8a and 8b, for example, a method of mixing the cells 8a and 8b with the gelation target solutions 5a and 5b, or a method of spreading the cells 8a and 8b on the scaffold 30 after the formation of the scaffold 30 can be used. .

  In the example of FIG. 12A, cell differentiation is induced by adding cells 8a and 8b to each of the first collagen 3a and the second collagen 3b and culturing them. In this case, the cells 8a cultured by adding to the first collagen 3a become the first type cells, and the cells 8b cultured by adding to the second collagen 3b become the second type cells, which are different types. Changes to cells. In the example of FIG. 12B, co-culture between the heterogeneous cells 8a and 8b is performed by layered culture on the boundary surface 3c between the first collagen 3a and the second collagen 3b. In this case, the cell 8a cultured by adding to the first collagen 3a and the cell 8b cultured by adding to the second collagen 3b are combined to form a cell network.

Thus, by using the anisotropic scaffold 30 using the anisotropic hydrogel according to the second embodiment, differentiation induction of individual cells 8a and 8b (stem cells) and heterogeneous cells in a single particle are performed. It becomes possible to carry out a cell culturing method that has been difficult with conventional techniques such as co-culture between (cells 8a, 8b).
As shown in FIG. 13, the cultured cells 8a and 8b are observed on the side opposite to the formation surface of the microchamber 42, and the cells 8a of the first and second collagens 3a and 3b in the microchamber 42 are observed. , 8b can be enlarged with a microscope 60.

  As described above, in the scaffold 30, the normal direction of the boundary surface 3 c intersects (for example, orthogonal) with the normal direction of the bottom surface 42 b of the microchamber 42. Therefore, in the microscope 60, the cells 8a and 8b near the boundary surface 3c can be enlarged and observed from the direction intersecting the normal direction of the boundary surface 3c. As a result, the state of the cells 8a and 8b in the vicinity of the boundary surface 3c can be observed in more detail. Therefore, it is particularly suitable for cancer cell migration tests, stem cell differentiation induction tests in heterogeneous microenvironment, and neuronal cell protrusion development tests.

  Incidentally, as shown in FIG. 15, the scaffold 30 in which the normal direction of the boundary surface 3c between the first collagen 3a and the second collagen 3b and the normal direction of the bottom surface 42b of the microchamber 42 do not intersect, that is, In the conventional scaffold 30 in which the boundary surface 3c and the bottom surface 42b are parallel, the microscope 60 displays the cells 8a and 8b from the normal direction of the boundary surface 3c. Therefore, it becomes difficult to observe in detail the state of the cells 8a and 8b in the vicinity of the boundary surface 3c.

  Further, when the scaffold 30 is prepared by mixing the cells 8a having been subjected to red fluorescent staining and the cells 8b having been subjected to blue fluorescent staining as a molecular modification when the scaffold 30 is prepared, the second fluorescently stained first Cells 8a stained with red fluorescence are encapsulated in one collagen 3a, and cells 8b stained blue are encapsulated in second collagen 3b stained red. As the cells 8a and 8b, for example, NIH / 3T3 cells subjected to fluorescent staining are used. By making the first and second collagens 3a, 3b and the cells 8a, 8b visually distinguishable by fluorescent staining, the encapsulation of the cells 8a, 8b is controlled, and the cells 8a, 8b are selectively placed in the microenvironment. It becomes possible to encapsulate.

  In the above description, the case where the cells 8a and 8b are enclosed in the scaffold 30 has been described. However, one or more biological components such as cells, proteins, lipids, saccharides, nucleic acids, and antibodies are added to the scaffold 30. can do. The types of cells 8a and 8b are not particularly limited. For example, ES cells and iPS cells having pluripotency, various stem cells having pluripotency, stem cells having differentiation unity, and other various differentiated cells. Examples thereof include muscle cells such as skeletal muscle cells and cardiomyocytes, nerve cells, fibroblasts, epithelial cells, hepatocytes, pancreatic β cells, skin cells and the like. Of course, the cells and biological components are not limited to those exemplified above. Moreover, you may add a non-biological component to the scaffold 30 simultaneously. For example, fibers such as carbon nanofibers, inorganic substances such as a catalyst substance, beads coated with an antibody or the like, or an artificial object such as a microchip can be added.

(Effect of 2nd Embodiment)
According to the scaffold using the anisotropic hydrogel and the manufacturing method thereof according to the second embodiment, the microchamber 42 has the first collagen 3a and the second collagen 3b partitioned inside. An anisotropic and complex microenvironment is realized, and the normal direction of the boundary surface 3c between the first collagen 3a and the second collagen 3b intersects the normal direction of the bottom surface 42b of the microchamber 42. A scaffold 30 having an anisotropic / complex structure can be produced. In addition, a very small scaffold 30 having a diameter of about 150 μm can be produced in a large amount in an array without using a reagent such as an oil or an organic solvent that is toxic to the cells 8a and 8b.

The prepared scaffold 30 has two types of collagen (first collagen 3a and second collagen 3b), and in each collagen, the collagen (first collagen 3a and second collagen 3b). ) Fiber network can be confirmed.
In addition, by making two different types of collagen inside the scaffold 30 visually identifiable, a plurality of cells 8a and 8b that have undergone different fluorescence staining are used to scaffold each cell 8a and 8b to be encapsulated. 30 can be selectively encapsulated in a plurality of different collagens.

  Further, as shown in FIG. 14A, an experiment was performed in which NIH / 3T3 cells were seeded on the scaffold 30 as cells 8a and 8b and then heated at 37 [° C.] for 24 hours in an environment filled with carbon dioxide. This experiment confirmed that NIH / 3T3 cells can be cultured as shown in FIG. 14 (b). In FIGS. 14A and 14B, the collagen (the first collagen 3a and the second collagen 3b) is not fluorescently stained, so that both have the same color.

  Further, by using the anisotropic scaffold 30 using the anisotropic hydrogel according to the second embodiment, the normal direction of the boundary surface 3c of two different types of collagen is normal to the bottom surface 42b of the microchamber 42. Since it intersects the direction, the cultured cells 8a and 8b can be observed from the direction intersecting the normal direction of the boundary surface 3c. Therefore, the state of the cells 8a and 8b in the vicinity of the boundary surface 3c can be observed in more detail as an anisotropic microenvironment.

  As mentioned above, although this invention was demonstrated with reference to 1st and 2nd embodiment, it is not intending limiting invention by these description. From the description of the invention, other embodiments of the invention will be apparent to persons skilled in the art, along with various variations of the disclosed embodiments. Therefore, it is to be understood that the claims encompass these modifications and embodiments that fall within the scope and spirit of the present invention.

  DESCRIPTION OF SYMBOLS 1a ... 1st collagen, 1b ... 2nd collagen, 2 ... Thin film, 3a ... 1st collagen, 3b ... 2nd collagen, 3c ... Interface, 3d ... 3rd collagen 3d, 4 ... Gelling agent Solution, 5a ... Gelation target solution, 5b ... Gelation target solution, 5c ... Interface, 6 ... Alginic acid mixed beads, 7 ... Alginic acid, 8a, 8b ... Cells, 10 ... Collagen Janus beads, 20 ... Microfluidic device, 21 ... Microtube, 21a ... Body part, 21b ... Liquid chamber, 21c ... Fixing part, 22a ... Theta tube, 22b ... Theta tube, 22c ... Boundary part, 30 ... Scaffolding, 40 ... Microchamber array, 41 ... Main body plate, 42 ... Microchamber, 42a ... Inner peripheral surface, 42b ... Bottom, 50 ... Petri dish, 51 ... Buffer, 60 ... Microscope

Claims (18)

  A microbead having anisotropy by containing a plurality of types of components partitioned in a single particle, wherein at least one of the plurality of types of components is a biological component.   The microbead according to claim 1, comprising a plurality of collagens having different properties as the plurality of types of components.   Each of the plurality of collagens is a gel having a uniform size, imitates a cell surface state in a living body, and encapsulates and cultures at least one type of cells in the gel to further increase the tissue surface state in the living body. The microbead according to claim 2, which mimics   The microbead according to claim 3, wherein a plurality of different cell layers are formed inside the bead to mimic a tissue formed by the plurality of cell layers.   The microbead according to claim 3 or 4, which mimics a plurality of different cell boundary environments on the boundary of the plurality of collagens.   The microbead according to any one of claims 1 to 5, which has a diameter of 20 µm to 200 µm. Produce a mixed solution of collagen and alginic acid,
By the gelation of the alginic acid contained in the mixed solution, a mixed bead containing the non-gelled collagen is produced,
Forming a thin film on the surface of the mixed beads;
A method for producing microbeads, comprising dissolving the gel of alginic acid and gelling the collagen in the mixed beads. Producing a plurality of types of the mixed solution,
Capillary tubes are individually filled with each of a plurality of types of the mixed solution,
Each of a plurality of types of the mixed solution is released from the tip of the capillary tube toward the gelling agent solution by centrifugal force,
The microbead manufacturing method according to claim 7, wherein the mixed beads are produced by gelation in the gelling agent solution in a state where each of the plurality of types of the mixed solutions released from the tip of the capillary is in contact with each other. Method.   The method for producing microbeads according to claim 7 or 8, wherein the plurality of types of the mixed solutions contain the collagen having different properties.   The microchamber contains a plurality of types of components partitioned, at least one of the plurality of types of components is a biological component, and a normal direction of a boundary surface between the plurality of types of components has a normal direction of the microchamber. A scaffold characterized by intersecting the normal direction of the bottom.   The scaffold according to claim 10, comprising a plurality of collagens having different properties as the plurality of types of components. Produce a mixed solution of collagen and alginic acid,
By the gelation of the alginic acid contained in the mixed solution, a mixed bead containing the non-gelled collagen is produced,
Storing the mixed beads in a microchamber;
A method for producing a scaffold, comprising dissolving the gel of alginic acid and gelling the collagen inside the microchamber. Producing a plurality of types of the mixed solution,
Capillary tubes are individually filled with each of a plurality of types of the mixed solution,
Each of a plurality of types of the mixed solution is released from the tip of the capillary tube toward the gelling agent solution by centrifugal force,
A method of a boundary surface between a plurality of types of the mixed solution that has been gelled in the gelling agent solution in a state where each of the plurality of types of the mixed solution released from the tip of the capillary is in contact with each other. The manufacturing method of the scaffold of Claim 12 which produces the oblate spherical shape which is crushed in the direction which cross | intersects a linear direction, or the oval shape extended in the normal line direction of the said interface.   The manufacturing method of the scaffold of Claim 13 which adjusts the flatness degree of the said mixing bead by adjusting the distance of the front-end | tip of the said capillary, and the said gelatinizer liquid.   The method for manufacturing a scaffold according to any one of claims 12 to 14, wherein a plurality of types of the mixed solutions contain the collagen having different properties. It has an occupied area as a three-dimensional shape surrounded by a boundary surface,
The occupied area is partitioned into a plurality of areas each containing a plurality of types of components;
An anisotropic hydrogel, wherein at least one of the plurality of types of components is a biological component.   The anisotropic hydrogel according to claim 16, wherein the entire occupied region is covered with a thin film.   The occupied region is accommodated in a micro chamber, and a normal direction of a boundary surface between the plurality of types of components intersects a normal direction of a bottom surface of the micro chamber. Anisotropic hydrogel.

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