Hydrogel, inkjet ink, method for preparing cell cultures, cell-containing gel particles, and method for producing the same.

Abstract

The purpose of the present invention is to provide a hydrogel that has both of gel shaping properties and cell adhesion properties and enables the long-term culture of cells.　Provided is a hydrogel 1 in which polyethylene glycol is contained as a backbone and a tetra-armed polymer 2 containing at least one electrophilic or nucleophilic functional group in a side chain and / or a terminal thereof and a linear polymer 3 containing at least two nucleophilic or electrophilic functional group in a side chain and / or a terminal thereof are crosslinked with each other, the hydrogel 1 being characterized in that the linear polymer 3 is a protein, a peptide or a polysaccharide, the electrophilic functional group is at least one group selected from the group consisting of a maleimidyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a phthalimidyl group, an imidazoyl group, an acryloyl group and a nitrophenyl group, and the nucleophilic functional group is at least one group selected from the group consisting of a thiol group, an amino group and -CO2PhNO2.

Description

[Technical Field]

[0001] The present invention belongs to the technical fields of hydrogels, inkjet inks, methods for preparing cell cultures using the inkjet inks, cell-containing gel particles and methods for producing the same, cell culture methods, and cell evaluation plates. [Background technology]

[0002] There is a need for a hydrogel composition that can pattern cells in three dimensions and is suitable for long-term culture. Conventional 3D models made from collagen materials exhibit cell adhesion, but their slow gelation time prevents rapid gel formation in the culture environment, resulting in random cell positions and making it difficult to obtain reproducible evaluation results. On the other hand, alginate gels are examples of materials that gel quickly and have good moldability, but gel materials with excellent moldability generally do not exhibit cell adhesion, making long-term culture of the model difficult and limiting their applications. In addition, guest materials are sometimes added to alginate gels and polymer materials to compensate for poor cell adhesion. For example, (Patent Document 1) discloses a hydrogel composition comprising a hydrogel made of a tetrabranched polymer with a polyethylene glycol backbone that is crosslinked with each other, and a guest substance, characterized in that the guest substance has cell adhesion factors such as collagen peptides. However, ensuring the amount and dispersibility of such guest materials while adding them often requires advanced technology, and there may be a trade-off with moldability, so there is still room for improvement.

[0003] Incidentally, in in vitro cell culture techniques, cells are seeded at the optimal seeding density and cultured under conditions suitable for the cells (adhesion / suspension conditions). When performing assays using cells that are difficult to obtain, such as patient-derived cells, it is necessary to culture them with a small number of cells. Generally, methods for culturing small numbers of cells include single-cell culture, spheroid culture, and liquid-phase droplet culture techniques. Among these, cell encapsulation technology, which cultures cells in a hydrogel, is already known as a method that offers high viability and allows for easy handling even with small numbers of cells.

[0004] However, conventional cell encapsulation techniques often use alginate for the hydrogel that encloses the cells, taking shapeability into consideration. However, depending on the cell type, it is difficult for cells to survive even for a short period of time in an alginate hydrogel. For example, (Patent Document 2) discloses a method for creating anisotropic collagen beads by enclosing cells in a hydrogel mixed with alginate and collagen, and maintaining the shapeability of the gel particles until the collagen gels through the rapid gelation of alginate. However, in this technique, the cells are brought into contact with a calcium solution during gelation, making it difficult to maintain cell viability.

[0005] Furthermore, methods using extracellular matrices such as collagen and gelatin, which take into account cell viability, are known. For example, (Patent Document 3) discloses a culture method in which animal cells are dispersed in a collagen solution, the collagen droplets containing the cells are gelled on the surface of a support to create a droplet-like collagen gel, and then a culture medium is added to culture the animal cells, with the aim of performing accurate and sufficient sensitivity tests even with a small amount of animal cells and clearly observing and measuring intercellular interactions during co-culture. In these methods using collagen, viability can be maintained, but the moldability is poor, and in order to maintain moldability, the particles containing cells and hydrogel are further coated with hydrogel or other materials. As a result, the particle size becomes large, making observation difficult, and for long-term culture, the size of the particles affects the permeability of nutrients and oxygen in the culture medium, resulting in a problem of low viability. In (Patent Document 2) mentioned above, the volume of the collagen gel particles is large at 3 μl, and the problem of reducing particle size has not been solved.

[0006] Furthermore, maintaining cell viability becomes even more difficult if the number of cells encapsulated in the hydrogel is small. In other words, using a hydrogel material that improves the encapsulation properties when the number of cells per particle is small makes it difficult to maintain cell viability. On the other hand, using a hydrogel material that can improve cell viability results in poor shaping, larger particle size, difficulty in observation, and ultimately, the inability to culture cells for extended periods. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2020-150846 [Patent Document 2] Patent No. 6817619 [Patent Document 3] International Publication No. 95 / 18216 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Therefore, the present invention aims to provide a hydrogel that achieves both gel-forming properties and cell adhesion properties, enabling long-term cell culture.

[0009] Furthermore, the present invention is not limited to cell types and aims to enable cell culture while maintaining the moldability of the hydrogel containing cells and the viability of the cells. [Means for solving the problem]

[0010] As a result of diligent research by the inventors, the above problem can be solved by forming a hydrogel by crosslinking a highly branched polymer with a specific structure based on polyethylene glycol, which has a fast gelling time, with a linear polymer such as a protein that has cell adhesion properties, and thus the invention has been completed.

[0011] In other words, the present invention is a hydrogel formed by crosslinking a highly branched polymer having polyethylene glycol as its backbone and containing one or more electrophilic or nucleophilic functional groups in its side chains and / or terminals with a linear polymer containing two or more nucleophilic or electrophilic functional groups in its side chains and / or terminals, wherein the linear polymer is a protein, peptide or polysaccharide, the electrophilic functional group is one or more selected from the group consisting of maleidyl group, N-hydroxy-succinimidyl (NHS) group, sulfosuccinimidyl group, phthalimidyl group, imidazoyl group, acryloyl group and nitrophenyl group, and the nucleophilic functional group is one or more selected from the group consisting of thiol group, amino group and -CO2PhNO2.

[0012] Furthermore, the inventors have discovered that the above problems can be solved by forming a hydrogel that encloses cells by crosslinking a highly branched polymer with a specific structure based on polyethylene glycol with a linear polymer such as a protein having specific functional groups in its side chains or terminals, and have completed the invention.

[0013] In other words, the present invention relates to cell-containing gel particles in which cells are enclosed by a hydrogel formed by crosslinking a highly branched polymer having polyethylene glycol as its backbone and containing one or more electrophilic or nucleophilic functional groups in its side chains and / or terminals with a linear polymer containing two or more nucleophilic or electrophilic functional groups in its side chains and / or terminals, wherein the linear polymer is a protein, peptide or polysaccharide, the electrophilic functional group is one or more selected from the group consisting of maleidyl group, N-hydroxy-succinimidyl (NHS) group, sulfosuccinimidyl group, phthalidyl group, imidazoyl group, acryloyl group and nitrophenyl group, and the nucleophilic functional group is one or more selected from the group consisting of thiol group, amino group and -CO2PhNO2.

[0014] This specification includes the disclosures of Japanese Patent Applications No. 2021-197665 and 2021-197639, which form the basis of the priority claim of this application. [Effects of the Invention]

[0015] According to the present invention, it is possible to provide a hydrogel that achieves both gel-forming properties and cell adhesion properties, enabling long-term cell culture.

[0016] Furthermore, according to the present invention, it is possible to culture even a single cell while maintaining cell viability, without being limited by cell type. [Brief explanation of the drawing]

[0017] [Figure 1] This figure schematically shows the hydrogel according to this embodiment. [Figure 2] This is a schematic diagram showing one embodiment of cell-containing gel particles according to the present invention. [Figure 3] This is a schematic diagram illustrating one embodiment of the cell culture method according to the present invention. [Figure 4] This is a schematic diagram showing another embodiment of the cell culture method according to the present invention. [Figure 5] This is a schematic diagram showing one embodiment of the cell evaluation plate according to the present invention. [Figure 6] This is a microscopic image of the cell culture according to Example 1. [Figure 7] This is a confocal microscope image of the cell culture according to Example 2. [Figure 8] This is a fluorescence microscope image of the cell culture according to Example 3. [Figure 9] (a) A plan view and (b) a side view of the laminate containing the cell culture prepared in Example 5. [Figure 10] (a) is a side view of the laminate containing the cell culture prepared in Example 6. (b) is an enlarged view of part A in (a). [Figure 11] This is a microscopic image of the cell culture according to Example 6. [Figure 12] This is a microscopic image of a cell culture according to Comparative Example 1. [Figure 13] This is a confocal microscope image of a cell culture according to Comparative Example 2. [Figure 14] This image shows the results of phase-contrast microscopy observation of cell-containing gel particles in Example 7. [Figure 15] This image shows the results of phase-contrast microscopy observation of cell-containing gel particles in Example 8. [Figure 16] This image shows the results of fluorescence microscopy observation of cell-containing gel particles in Example 10. [Figure 17] This image shows the results of phase-contrast microscopy observation of cell-containing gel particles in Example 11. [Figure 18] This image shows the results of phase-contrast microscopy observation of cell-containing gel particles in Example 12. [Figure 19] This image shows the results of phase-contrast microscopy observation of cell-containing gel particles in Example 13. [Figure 20] This graph shows the measurement results of IL-8 amount relative to LPS concentration in Example 14. [Modes for carrying out the invention]

[0018] The present invention will be described in detail below based on embodiments. The embodiments described below are preferred embodiments of the present invention and therefore have various technically preferable limitations. However, the scope of the present invention is not limited to these embodiments unless otherwise stated in the following description.

[0019] The hydrogel according to this embodiment is characterized by being formed by crosslinking a highly branched polymer having polyethylene glycol as its backbone and containing one or more electrophilic or nucleophilic functional groups in its side chains and / or terminals with a linear polymer containing two or more nucleophilic or electrophilic functional groups in its side chains and / or terminals.

[0020] A "hydrogel" is a gel that has a three-dimensional network structure and can hold a large amount of water within the spaces of that network structure. Good examples include polysaccharide gels such as agar, protein gels such as jelly, and superabsorbent polymer gels such as acrylic acid polymers. In general, hydrogels can incorporate various substances into the gel through the water they contain.

[0021] Figure 1 schematically shows a typical structure of the hydrogel of this embodiment. As shown in Figure 1, the hydrogel 1 of this embodiment is formed by crosslinking electrophilic functional groups 21, such as maleidyl groups, of the tetrabranched polymer 2 with nucleophilic functional groups 31, such as thiol groups, of the linear polymer 3, and has a structure in which the tetrabranched polymer and the linear polymer are dispersed. The example is not limited to Figure 1, and the tetrabranched polymer may have nucleophilic functional groups and the linear polymer may have electrophilic functional groups. The components constituting the hydrogel 1 will be described below.

[0022] <Multi-branched polymer> The multibranched polymer in this embodiment is a polymer having at least three polyethylene glycol branches, which crosslink with each other to form a network structure. In a preferred embodiment, the multibranched polymer is a polymer having four polyethylene glycol branches, and such a tetrabranched polymer with a polyethylene glycol backbone is known as Tetra-PEG. Tetra-PEG has one or more electrophilic or nucleophilic functional groups in its side chains and / or terminals. These functional groups crosslink with linear polymers to construct a network structure. Because this polymer is mainly composed of PEG, it also has excellent biocompatibility.

[0023] The electrophilic functional group in the highly branched polymer is one or more selected from the group consisting of maleimidyl group, N-hydroxysuccinimidyl (NHS) group, sulfosuccinimidyl group, phthalimidyl group, imidazoyl group, acryloyl group, and nitrophenyl group. Preferably, it is the maleimidyl group. The composition of electrophilic functional groups in the highly branched polymer may be the same or different, but it is preferable that they be the same. By having a uniform composition of functional groups, the reactivity with the nucleophilic functional groups of the linear polymer that forms the crosslinking bond becomes uniform, making it easier to obtain a gel with a uniform three-dimensional structure.

[0024] Examples of highly branched polymers having one or more maleidyl groups in the side chains and / or terminals and having polyethylene glycol as the backbone include compounds represented by the following formula (I) having maleidyl groups at the terminals.

[0025] [ka]

[0026] In the above equation (I), n 21 ~n 24 These may be the same or different. 21 ~n 24The closer the value of 21 ~n 24 is, the more likely the gel can adopt a uniform three-dimensional structure and become highly strong, which is preferable, and it is particularly preferable that they are the same. When the value of 21 ~n 24 is too high, the strength of the gel becomes weak, and when the value of 21 ~n 24 is too low, it is difficult to form a gel due to the steric hindrance of the compound. Therefore, 3 ~n 4 is preferably a value of 5 or more and 300 or less, more preferably 20 or more and 250 or less, still more preferably 30 or more and 180 or less, even more preferably 45 or more and 115 or less, and particularly preferably 45 or more and 55 or less. The weight average molecular weight of the multi-branched polymer having an electrophilic functional group is 5×10 3 or more and 5×10 4 or less, more preferably 7.5×10 4 or more and 3×10 4 or less, and even more preferably 1×10

[0027] In the above formula (I), R 21 ~R 24 is a linker moiety connecting the functional group and the core moiety. R 21 ~R 24 may be the same or different, but in order to produce a high-strength gel having a uniform three-dimensional structure, it is preferably the same. In formula (I), R 21 ~R 24 are each the same or different and are a C1-C7 alkylene group, a C2-C7 alkenylene group, -NH-R 25 -, -CO-R 25 -, -R 26 -O-R 27 -, -R 26 -NH-R 27 -, -R 26 -CO2-R 27 -, -R 26 -CO2-NH-R 27 -, -R 26 -CO-R 27 -, -R 26 -NH-CO-R 27 -, or -R26 -CO-NH-R 27 -and so on. Here, R 25 R indicates a C1-C7 alkylene group. 26 R indicates a C1-C3 alkylene group. 27 This indicates a C1-C5 alkylene group.

[0028] Furthermore, the nucleophilic functional group in the highly branched polymer is one or more selected from the group consisting of thiol groups, amino groups, and -CO2PhNO2. Here, Ph represents an o-, m-, or p-phenylene group. In the highly branched polymer, the composition of nucleophilic functional groups may be the same or different, but it is preferable that they be the same. By having a uniform composition of functional groups, the reactivity with the electrophilic functional groups of the linear polymer that forms the crosslinking bond becomes uniform, making it easier to obtain a gel with a uniform three-dimensional structure.

[0029] Examples of highly branched polymers having one or more thiol groups in the side chains and / or terminals and having polyethylene glycol as the backbone include compounds represented by the following formula (II) having thiol groups at the terminals.

[0030] [ka]

[0031] In the above equation (II), n 11 ~n 14 These may be the same or different. 11 ~n 14 The closer the values ​​are, the more uniform the three-dimensional structure can be formed, resulting in higher strength, which is preferable, and it is particularly preferable that they are the same. 11 ~n 14 If the value of is too high, the strength of the gel will be weakened, 11 ~n 14 If the value of n is too low, gel formation will be difficult due to steric hindrance of the compound. 11 ~n 14The weight-average molecular weight of the highly branched polymer having a nucleophilic functional group is 5 × 10 3 The above 5 x 10 4 Preferably, it is 7.5 × 10 3 The above 3 x 10 4 More preferably, 1 × 10 4 The above 2 x 10 4 The following is even more preferable:

[0032] In the above equation (II), R 11 ~R 14 This is the linker region that connects the functional group to the core. 11 ~R 14 These may be the same or different, but it is preferable that they be the same in order to produce a high-strength gel with a uniform three-dimensional structure. In formula (II), R 11 ~R 14 These are either identical or different, and consist of a C1-C7 alkylene group, a C2-C7 alkenylene group, and -NH-R. 15 -, -CO-R 15 -, -R 16 -OR 17 -, -R 16 -NH-R 17 -, -R 16 -CO2-R 17 -, -R 16 -CO2-NH-R 17 -, -R 16 -CO-R 17 -, R 16 -NH-CO-R 17 -or-R 16 -CO-NH-R 17 -and so on. Here, R 15 R indicates a C1-C7 alkylene group. 16 R indicates a C1-C3 alkylene group. 17 This indicates a C1-C5 alkylene group.

[0033] Here, "C1-C7 alkylene group" refers to an alkylene group with 1 to 7 carbon atoms that may have branching, and means a straight-chain C1-C7 alkylene group or a C2-C7 alkylene group with one or more branches (with 1 to 7 carbon atoms including the branching). Examples include the methylene group, ethylene group, propylene group, and butylene group. More specifically, examples include -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)-, -(CH2)3-, -(CH(CH3))2-, -(CH2)2-CH(CH3)-, -(CH2)3-CH(CH3)-, -(CH2)2-CH(C2H5)-, -(CH2)6-, -(CH2)2-C(C2H5)2-, and -(CH2)3C(CH3)2CH2-.

[0034] A "C2-C7 alkenylene group" is a linear or branched alkenylene group having 2 to 7 carbon atoms and possessing one or more double bonds in the chain. For example, a divalent group having a double bond can be formed by removing 2 to 5 hydrogen atoms from adjacent carbon atoms from the aforementioned alkylene group.

[0035] In this specification, alkylene groups and alkenylene groups may have one or more substituents. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms (which may be fluorine, chlorine, bromine, or iodine atoms), amino groups, mono- or disubstituted amino groups, substituted silyl groups, acyl groups, or aryl groups. If there are two or more substituents, they may be the same or different.

[0036] Furthermore, in this specification, when a functional group is defined as "may have substituents," the type of substituent, the position of substitution, and the number of substituents are not particularly limited, and if there are two or more substituents, they may be the same or different. Examples of substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, oxo groups, etc. These substituents may have further substituents.

[0037] <Linear polymer> A linear polymer that forms a hydrogel through a crosslinking reaction with a highly branched polymer contains two or more nucleophilic or electrophilic functional groups in its side chains and / or terminals. For crosslinking purposes, if the highly branched polymer has electrophilic functional groups, the linear polymer has nucleophilic functional groups, and if the highly branched polymer has nucleophilic functional groups, the linear polymer has electrophilic functional groups. The content of the electrophilic and nucleophilic functional groups is the same as in the case of the highly branched polymer described above.

[0038] The molar ratio of nucleophilic functional groups to electrophilic functional groups in the branched polymer and linear polymer is preferably in the range of 0.5:1 to 1.5:1. Since these functional groups can crosslink by the reaction of each component in a 1:1 molar ratio, a mixed molar ratio closer to 1:1 is preferable, but in order to obtain a hydrogel of high strength, a nucleophilic functional group:electrophilic functional group ratio in the range of 0.8:1 to 1.2:1 is particularly preferable. It is even more preferable that these functional groups are present at the ends of the branched polymer and the linear polymer, respectively. In a preferred embodiment, two or more gel precursors with different mixing ratios can be formed first, and then these gel precursors can be further crosslinked to obtain the desired hydrogel.

[0039] Linear polymers are proteins, peptides, or polysaccharides. There are no particular restrictions on proteins, and they can be appropriately selected depending on the purpose. Examples include collagen, fibrin, albumin, fibronectin, laminin, tenascin, entactin, elastin, and compounds derived therefrom. Gelatin is particularly preferred as the linear polymer. Peptides are polymerized by two or more amino acid residues via peptide bonds (amide bonds). Applicable peptides include collagen peptides. Peptides may be dipeptides, tripeptides, oligopeptides (containing about 10 amino acids), or polypeptides (containing tens to hundreds of amino acids). Furthermore, there are no particular restrictions on polysaccharides, and they can be appropriately selected. Examples include cellulose, chitin, chitosan, hyaluronic acid, alginic acid, starch, and pectin. These linear polymers may be used individually or in combination of two or more. Protein-sugar complexes such as proteoglycans are also applicable. The molecular weight of the linear polymer is not particularly limited as long as it is within the range that can be present inside the hydrogel, but preferably it can be between 100 and 500,000. As a linear polymer containing two or more nucleophilic or electrophilic functional groups in the side chains and / or terminals, gelatin having thiol groups in the side chains is shown below.

[0040] [ka]

[0041] <cell> In this embodiment, the hydrogel can contain cells. The hydrogel of this embodiment has excellent gel-forming properties and cell adhesion due to the linear polymer, allowing for long-term cell culture within the hydrogel. There are no particular restrictions on the type of cells, and they can be appropriately selected according to the purpose. Taxonomically, for example, all cells can be used, regardless of whether they are eukaryotic cells, prokaryotic cells, multicellular organism cells, or unicellular organism cells.

[0042] Examples of eukaryotic cells include animal cells, insect cells, plant cells, and fungi. These may be used individually or in combination of two or more. Among these, animal cells are preferred, and if the cells form a cell aggregate, it is even more preferable that they are adherent cells that adhere to each other and have sufficient cell adhesion to not require physicochemical treatment to isolate them.

[0043] There are no particular restrictions on the type of adherent cells used; they can be appropriately selected depending on the purpose, and examples include differentiated cells and undifferentiated cells.

[0044] Differentiated cells include, for example, hepatocytes (parenchymal cells of the liver); stellate cells; Kupffer cells; vascular endothelial cells; endothelial cells such as endothelial cells and corneal endothelial cells; fibroblasts; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells such as keratinocytes; tracheal epithelial cells; gastrointestinal epithelial cells; cervical epithelial cells; epithelial cells such as corneal epithelial cells; mammary gland cells; pericytes; muscle cells such as smooth muscle cells and cardiomyocytes; renal cells; pancreatic islet cells; nerve cells such as peripheral nerve cells and optic nerve cells; chondrocytes; and osteocytes. In hydrogels containing nerve cells, the adhesive cells may be primary cells directly collected from tissues or organs, or they may be cells that have been passaged through several generations.

[0045] There are no particular restrictions on the undifferentiated cells used, and they can be appropriately selected depending on the purpose. Examples include undifferentiated embryonic stem cells, pluripotent stem cells such as mesenchymal stem cells with multipotency, unipotent stem cells such as vascular endothelial progenitor cells with monopotency, and iPS cells.

[0046] Examples of prokaryotic cells include bacteria and archaea.

[0047] Specific examples of cells include normal human dermal fibroblasts. Commercially available normal human dermal fibroblasts can be used, and examples of such commercial products include the product name: CC2507 (manufactured by Lonza).

[0048] Furthermore, the hydrogel may also contain cell aggregates (spheroids) that have undergone a prior culture process. Hydrogels containing cell aggregates can undergo superior maturation and differentiation compared to hydrogels in which cells are dispersed. The size of the cell aggregates is not particularly limited, but it is preferable that they have a diameter of 100 μm or less.

[0049] The hydrogel can contain at least one type of cell. In hydrogels containing two or more types of cells, it is possible to create cell cultures that promote cell function more effectively than single-cell cultures due to interactions between cell types.

[0050] Extracellular matrix may be added to the hydrogel enclosing the cells as needed. There are no particular restrictions on the extracellular matrix; it can be appropriately selected depending on the purpose. Examples include extracellular matrix proteins and glycoproteins such as collagen, laminin, fibronectin, elastin, fibrin, proteoglycans, hyaluronic acid, heparan sulfate proteoglycans, and chondroitin sulfate proteoglycans, as well as growth factors such as hepatocyte growth factor, fibroblast growth factor, and nerve growth factor, depending on the cell type. These may be used individually or in combination of two or more. These are known to promote cell adhesion, proliferation, and differentiation, and by including them in the hydrogel, they can function as triggers to change the morphology of cells within the formed hydrogel.

[0051] The hydrogel described above can be formed from an ink comprising a first solution containing a highly branched polymer with polyethylene glycol as its backbone and one or more electrophilic or nucleophilic functional groups in its side chains and / or terminals, and a second solution containing a linear polymer with two or more nucleophilic or electrophilic functional groups in its side chains and / or terminals. Specifically, a hydrogel can be obtained by mixing the first solution and the second solution and crosslinking them. Alternatively, the second solution may be mixed with the first solution before mixing the first and second solutions together, and the amount of the second solution to be mixed beforehand can be appropriately selected as long as it does not cause significant thickening or gelation. Similarly, the first solution may be mixed with the second solution before mixing the first and second solutions together.

[0052] The above ink is used as an inkjet ink. By ejecting either the first or second solution using the inkjet method and landing it on the other second or first solution, the first and second solutions can be mixed to form a hydrogel. The second or first solution, which is the target for landing, may be, for example, a liquid filling a container, or a liquid film pre-coated on a substrate. Alternatively, it may be a liquid droplet pre-dispenseed onto a substrate using the inkjet method, or a liquid film state in which numerous continuously dispensed droplets come into contact and combine to form various shapes such as lines. Here, "inkjet" refers to a means of ejecting a solution stored in a liquid chamber as a droplet from the ejection hole (nozzle) of an inkjet head and landing it on a target area. Because this ejection method can dispense minute solutions (droplets) from the ejection hole, it is possible to create highly accurate three-dimensional structures. By using the inkjet method, for example, the first and second solutions can be mixed with a resolution of 500 μm or less.

[0053] In this case, either or both of the first and second solutions of the inkjet ink may contain the aforementioned cells. For example, the first solution containing cells can be ejected by an inkjet method onto the second solution, which does not contain cells, on a substrate to form a hydrogel (cell culture) containing cells.

[0054] When preparing cell cultures from inkjet ink containing cells, the first solution and the second solution can be mixed on a substrate having a patterned structure on its surface. Examples of patterned structures include uneven surfaces formed in a periodic or arbitrary pattern. The patterned structure may be formed so that the surface of the substrate itself has an uneven pattern, or, for example, the aforementioned hydrogel without cells may be arranged in a predetermined pattern on the surface of a flat substrate. By forming cell cultures in a patterned structure, experiments can be conducted to investigate, for example, how the migration and spread of cells within the hydrogel are affected by the patterned structure.

[0055] Furthermore, cell cultures formed from inkjet ink containing cells can be combined with other cell cultures by heating them as needed and bringing them into contact. The heating temperature varies depending on the hydrogel composition and cell type, but as long as it does not cause excessive damage to the cells contained in the cell culture, there is no particular problem; for example, a temperature between 0°C and 40°C is preferable. Raising the temperature above 40°C is acceptable for partial and very short periods of time.

[0056] The present invention also relates to a method for producing a cell culture, comprising a holding step of holding a first solution containing a highly branched polymer having a polyethylene glycol backbone and having one or more nucleophilic or electrophilic functional groups at its side chains and / or terminals, and a gel forming step of forming one or more hydrogels by depositing droplets of a second solution containing a linear polymer having one or more nucleophilic or electrophilic functional groups at its side chains and / or terminals into contact with the held first solution using a droplet dispensing device. Here, the highly branched polymer and linear polymer are as described above for hydrogels. Furthermore, at least one of the first or second solution may contain cells. The cells to be included are as described above.

[0057] The retention step is the step of retaining the first solution on a support or hydrogel. For example, the first solution may be impregnated into the support by immersing it in the first solution, thereby retaining the first solution on its entire surface and / or inside. Alternatively, the first solution may be ejected using a droplet ejection method such as the inkjet method described in detail in the next step, thereby fixing and retaining the first solution at specific positions on the surface of the support or hydrogel. A cell layer may also be used as a support to retain the first solution. Here, "support" refers to a component that retains the first solution. There are no particular restrictions on the material of the support, as long as it does not inhibit cell activity or proliferation, and it can be appropriately selected according to the purpose. Preferably, it is a material to which cells can adhere, or a material to which cell adhesion materials are easily adsorbed.

[0058] Supports can be broadly classified into organic and inorganic materials. Examples of organic materials include synthetic resins, silicone-based materials, natural resins, cellulose structures (wood, paper, etc.), chitinous structures, and natural fibers (silk, wool, cotton, spongy fibers, etc.). Cellular layers fixed to substrates can also serve as organic material supports. Examples of synthetic resins include polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetylcellulose), polyimide (PI), nylon (Ny), low-density polyethylene (LDPE), medium-density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, and acrylic materials such as urethane acrylate. Examples of silicone-based materials include polydimethylsiloxane (PDMS). Examples of inorganic materials include glass (including fiberglass), pottery (ceramics, enamel, etc.), metals, carbon fibers, and calcium phosphate structures (bones, teeth, seashells, etc.).

[0059] The above materials may be used individually, or two or more organic materials, inorganic materials, or a combination of organic and inorganic materials may be used in combination. For example, fiber-reinforced plastics (FRP) made by combining carbon fibers or glass fibers with synthetic resins are examples.

[0060] There are no particular restrictions on the size of the support; it can be selected appropriately depending on the purpose. There are no particular restrictions on the shape of the support; it can be selected appropriately depending on the purpose. For example, it may be a three-dimensional shape such as a dish, multiplate, flask, membrane, or cell insert, or it may be a flat or flat film shape such as a glass plate, slide glass, or coverslip.

[0061] There are no particular restrictions on the structure of the support, and it can be appropriately selected depending on the purpose. Examples include porous structures, mesh structures, uneven structures, honeycomb structures, etc. Porous structures and mesh structures are particularly preferred as supports because they can hold a large amount of solution.

[0062] The gel formation process involves dispensing a second solution using a droplet dispenser so that it comes into contact with the first solution held during the holding process. This mixing of the first and second solutions causes the nucleophilic functional groups and electrophilic reactive groups of the branched polymers and linear polymers contained in each solution to react and bond, forming a hydrogel. "In contact" means that the first and second solutions are mixed through contact.

[0063] A "droplet ejection device" is a means of ejecting a solution stored in a liquid chamber as droplets and landing them on a target area. Dispensing methods for ejection devices include inkjet methods and gel extrusion dispensers. In the inkjet method, the solution is ejected from an ejection port (nozzle). Because this ejection method allows for the ejection of minute solutions (sometimes called droplets) from the ejection port, it is possible to create highly accurate three-dimensional structures. In this case, the droplets ejected from the droplet ejection device may or may not contain cells.

[0064] The amount of liquid droplets discharged can be any amount. Preferably, it is 9 pL or more, 15 pL or more, 20 pL or more, 30 pL or more, 40 pL or more, 50 pL or more, 60 pL or more, 70 pL or more, 80 pL or more, 90 pL or more, or 100 pL or more, and 900 pL or less, 800 pL or less, 700 pL or less, 600 pL or less, 500 pL or less, 400 pL or less, or 300 pL or less.

[0065] "Impact" refers to bringing a solution into contact with a target area. This is achieved by dispensing droplets onto the target area using a specific dispensing method.

[0066] If the second solution contains cells, the cell content is 5 × 10 5 cells / mL ~ 1 × 10 8 cells / mL is preferred, 1 × 10 6 cells / mL ~ 5 × 10 7 A cell / mL ratio is more preferable. If the cell density is lower than this, it becomes difficult to form a hydrogel containing the appropriate number of cells, and conversely, if it is higher, it becomes difficult to dispense the solution using dispensing methods such as inkjet printing.

[0067] There are no particular restrictions on the position from which the solution is dispensed. The solution should be dispensed so that it lands at the desired target location. Furthermore, the impact points may be separate, or they may partially touch or overlap.

[0068] Gel formation occurs through the reaction of polymers having nucleophilic and electrophilic functional groups; therefore, a hydrogel is formed when the second solution lands on the first solution. When the second solution is dispensed using a droplet dispenser, a single application can typically form a dot-shaped hydrogel, although this is not limited to dot-shaped hydrogels. In this specification, "dot-shaped" refers to a point-like shape. Therefore, it is not limited to circular or hemispherical shapes, but can be approximately circular, approximately hemispherical, polygonal, irregular, or a combination thereof. Furthermore, the dot-shaped hydrogel only needs to have a predetermined length and thickness in three dimensions. If the second solution is dispensed multiple times, a variety of hydrogel shapes will be formed, with dot-shaped hydrogels as the smallest unit.

[0069] The volume of the hydrogel formed by the crosslinking reaction caused by a single impact, i.e., the dot-shaped hydrogel, depends on the number of times the second solution is dispensed to the same location. Furthermore, the larger the dispensing pore diameter and the more times the solution is dispensed to the same location, the larger the volume of the dot-shaped hydrogel. Therefore, the volume of the dot-shaped hydrogel can be adjusted by changing the number of times the solution is dispensed to the same location and the dispensing pore diameter. Although not limited, the preferred volumes of the dot-shaped hydrogel in this specification are 9 pL or more, 15 pL or more, 20 pL or more, 30 pL or more, 40 pL or more, 50 pL or more, 60 pL or more, 70 pL or more, 80 pL or more, 90 pL or more, or 100 pL or more, and 900 pL or less, 800 pL or less, 700 pL or less, 600 pL or less, 500 pL or less, 400 pL or less, and 300 pL or less. The diameter of the dot-shaped hydrogel is not limited, but it should be between 10 μm and 300 μm, and the thickness should be between 5 μm and 150 μm.

[0070] In this process, the second solution is dispensed an arbitrary number of times in a single step, which may result in the formation of multiple dot-shaped hydrogels. The dot-shaped hydrogels may be in contact with each other, either entirely or partially. By arranging multiple dot-shaped hydrogels in a series, hydrogels of any shape, not just dots, can be formed. The shape can be appropriately selected according to the purpose. For example, a linear hydrogel can be formed by arranging dots in a single axis direction. Furthermore, a membrane-like (planar) hydrogel can be formed by arranging linear hydrogels without gaps on the same plane. By controlling the fusion and isolation of the dot-shaped hydrogels, the movement and spread of cells contained within each dot-shaped hydrogel can be controlled and suppressed when linear or membrane-like hydrogels are formed. In addition, by forming membrane-like hydrogels, the gel can be easily stacked.

[0071] Furthermore, when cells are included in a solution, the number of cells contained within the dot-shaped hydrogel depends on the concentration of the solution. Therefore, the cell density can be controlled by the number of times the dot-shaped hydrogel is formed.

[0072] <culture medium> The prepared cell culture can be placed in a suitable culture medium to allow for long-term cell culture within the hydrogel. The culture medium is a solution that contains components necessary for maintaining the shape of the cell culture and the survival of the cells, prevents drying, and regulates the external environment, such as osmotic pressure. There are no particular restrictions on the culture medium; it can be appropriately selected from known culture media depending on the application.

[0073] Examples of the culture media include those classified by composition, such as natural media, semi-synthetic media, and synthetic media; and those classified by form, such as semi-solid media, liquid media, and powdered media (hereinafter sometimes referred to as "powdered media"). These may be used individually or in combination of two or more. If the cells are of animal origin, any culture medium used for animal cell culture can be used.

[0074] There are no particular restrictions on the culture medium used for animal cell culture, and it can be appropriately selected according to the purpose. For example, Dulbecco's Modified Eagles's Medium (D-MEM), Ham's Nutrient Mixture F12, D-MEM / F12 medium, McCoy's 5A medium, Eagles's Minimum Essential Medium (EMEM), αMEM medium (alpha Modified Eagles's Minimum Essential Medium (αMEM)), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemPro34 (Invitrogen), X-VIVO 10 (Kembrex), X-VIVO 15 (Kembrex), HPGM (Kembrex), StemSpan H3000 (Stem Cell Technologies), StemSpanSFEM (Stem Cell Technologies), StemlineII (Sigma-Aldrich), QBSF-60 (Quality Biological), StemProhESCSFM (Invitrogen), Essential8® medium (Gibco), Stemfit AK02N (Ajinomoto Co., Inc.), Stemfit Basic 02 (Ajinomoto Co., Inc.), mTeSR1 or 2 medium (Stem Cell Technologies), ReproFF or ReproFF2 (ReproCELL), PSGro hESC / iPSC medium (System Biosciences), NutriStem® medium (Biological Industries, Inc.), CSTI-7 medium (Cell Science Institute), MesenPRO Examples include RS medium (Gibco), MF-Medium® mesenchymal stem cell proliferation medium (Toyobo Co., Ltd.), Sf-900II (Invitrogen), and Opti-Pro (Invitrogen).These can be used individually or in combination of two or more types.

[0075] There are no particular restrictions on the carbon dioxide concentration in the culture medium, and it can be appropriately selected depending on the purpose, but a concentration of 2% to 5% is preferred. When the carbon dioxide concentration is 2% to 5%, cells can be cultured effectively.

[0076] Next, an embodiment of the cell-containing gel particles according to the present invention will be described with reference to Figure 2. As shown in Figure 2, the cell-containing gel particles 5 of this embodiment have cells 51 encased in a hydrogel 50 formed by crosslinking a highly branched polymer with polyethylene glycol as its backbone and containing one or more electrophilic or nucleophilic functional groups at its side chains and / or terminals with a linear polymer containing two or more nucleophilic or electrophilic functional groups at its side chains and / or terminals. The individual components constituting the cell-containing gel particles 1 will be described below.

[0077] <Multi-branched polymer> The branched polymer in this embodiment is a polymer having multiple polyethylene glycol branches that crosslink with each other to form a network structure. In a preferred embodiment, the branched polymer has four polyethylene glycol branches, and such a tetrabranched polymer with a polyethylene glycol backbone is known as Tetra-PEG. Tetra-PEG has one or more electrophilic or nucleophilic functional groups in its side chains and / or terminals. These functional groups crosslink with linear polymers to construct a network structure.

[0078] As the electrophilic functional group in the multi-branched polymer, it is one or more selected from the group consisting of maleimidyl group, N-hydroxy-succinimidyl (NHS) group, sulfosuccinimidyl group, phthalimidyl group, imidazolyl group, acryloyl group and nitrophenyl group. Among the multi-branched polymers, the composition of the electrophilic functional groups may be the same or different, but the same is preferred. When the composition of the functional groups is the same, the reactivity with the nucleophilic functional groups of the linear polymer forming the cross-linkage becomes uniform, and it becomes easier to obtain a gel having a uniform three-dimensional structure.

[0079] Examples of the multi-branched polymer having one or more maleimidyl groups at the side chain and / or the terminal and having polyethylene glycol as the backbone include the compound represented by the following formula (I) having a maleimidyl group at the terminal.

[0080] [Chemical formula]

[0081] In the above formula (I), n 21 ~n 24 may be the same or different from each other. The closer the values of n 21 ~n 24 are to each other, the more likely the gel can take on a uniform three-dimensional structure and become high-strength, which is preferred, and being the same is particularly preferred. If the values of n 21 ~n 24 are too high, the strength of the gel becomes weak, and if the values of n 21 ~n 24 are too low, it is difficult to form a gel due to the steric hindrance of the compound. Therefore, it is appropriate that n 21 ~n 24 is a value of 5 or more and 300 or less, preferably 20 or more and 250 or less, more preferably 30 or more and 180 or less, still more preferably 45 or more and 115 or less, and particularly preferably 45 or more and 55 or less. The weight average molecular weight of the multi-branched polymer having an electrophilic functional group is preferably 5×10 3 or more and 5×10 4 or less, preferably 7.5×10 3 or more and 3×104 More preferably, 1 × 10 4 The above 2 x 10 4 The following is even more preferable:

[0082] In the above equation (I), R 21 ~R 24 This is the linker region that connects the functional group to the core. 21 ~R 24 These may be the same or different, but it is preferable that they be the same in order to produce a high-strength gel with a uniform three-dimensional structure. In formula (I), R 21 ~R 24 These are either identical or different, and consist of a C1-C7 alkylene group, a C2-C7 alkenylene group, and -NH-R. 25 -, -CO-R 25 -, -R 26 -OR 27 -, -R 26 -NH-R 27 -, -R 26 -CO2-R 27 -, -R 26 -CO2-NH-R 27 -, -R 26 -CO-R 27 -, -R 26 -NH-CO-R 27 -, or -R 26 -CO-NH-R 27 -and so on. Here, R 25 R indicates a C1-C7 alkylene group. 26 R indicates a C1-C3 alkylene group. 27 This indicates a C1-C5 alkylene group.

[0083] Furthermore, the nucleophilic functional group in the highly branched polymer is one or more selected from the group consisting of thiol groups, amino groups, and -CO2PhNO2. The composition of nucleophilic functional groups in the highly branched polymer may be the same or different, but it is preferable that they be the same. By having a uniform composition of functional groups, the reactivity with the electrophilic functional groups of the linear polymer that forms the crosslinking bond becomes uniform, making it easier to obtain a gel with a uniform three-dimensional structure.

[0084] Examples of highly branched polymers having one or more thiol groups in the side chains and / or terminals and having polyethylene glycol as the backbone include compounds represented by the following formula (II) having thiol groups at the terminals.

[0085] [ka]

[0086] In the above equation (II), n 11 ~n 14 These may be the same or different. 11 ~n 14 The closer the values ​​are, the more uniform the three-dimensional structure can be formed, resulting in higher strength, which is preferable, and it is particularly preferable that they are the same. 11 ~n 14 If the value of is too high, the strength of the gel will be weakened, 11 ~n 14 If the value of n is too low, gel formation will be difficult due to steric hindrance of the compound. 11 ~n 14 The weight-average molecular weight of the highly branched polymer having a nucleophilic functional group is 5 × 10 3 The above 5 x 10 4 Preferably, it is 7.5 × 10 3 The above 3 x 10 4 More preferably, 1 × 10 4 The above 2 x 10 4 The following is even more preferable:

[0087] In the above equation (II), R 11 ~R 14 This is the linker region that connects the functional group to the core. 11 ~R 14 These may be the same or different, but it is preferable that they be the same in order to produce a high-strength gel with a uniform three-dimensional structure. In formula (II), R 11 ~R 14These are either identical or different, and consist of a C1-C7 alkylene group, a C2-C7 alkenylene group, and -NH-R. 15 -, -CO-R 15 -, -R 16 -OR 17 -, -R 16 -NH-R 17 -, -R 16 -CO2-R 17 -, -R 16 -CO2-NH-R 17 -, -R 16 -CO-R 17 -, R 16 -NH-CO-R 17 -or-R 16 -CO-NH-R 17 -and so on. Here, R 15 R indicates a C1-C7 alkylene group. 16 R indicates a C1-C3 alkylene group. 17 This indicates a C1-C5 alkylene group.

[0088] Here, "C1-C7 alkylene group" refers to an alkylene group with 1 to 7 carbon atoms that may have branching, and means a straight-chain C1-C7 alkylene group or a C2-C7 alkylene group with one or more branches (with 1 to 7 carbon atoms including the branching). Examples include the methylene group, ethylene group, propylene group, and butylene group. More specifically, examples include -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)-, -(CH2)3-, -(CH(CH3))2-, -(CH2)2-CH(CH3)-, -(CH2)3-CH(CH3)-, -(CH2)2-CH(C2H5)-, -(CH2)6-, -(CH2)2-C(C2H5)2-, and -(CH2)3C(CH3)2CH2-.

[0089] A "C2-C7 alkenylene group" is a linear or branched alkenylene group having 2 to 7 carbon atoms and possessing one or more double bonds in the chain. For example, a divalent group having a double bond can be formed by removing 2 to 5 hydrogen atoms from adjacent carbon atoms from the aforementioned alkylene group.

[0090] In this specification, alkylene groups and alkenylene groups may have one or more substituents. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms (which may be fluorine, chlorine, bromine, or iodine atoms), amino groups, mono- or disubstituted amino groups, substituted silyl groups, acyl groups, or aryl groups. If there are two or more substituents, they may be the same or different.

[0091] Furthermore, in this specification, when a functional group is defined as "may have substituents," the type of substituent, the position of substitution, and the number of substituents are not particularly limited, and if there are two or more substituents, they may be the same or different. Examples of substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, oxo groups, etc. These substituents may have further substituents.

[0092] <Linear polymer> A linear polymer that forms a hydrogel through a crosslinking reaction with a highly branched polymer contains two or more nucleophilic or electrophilic functional groups in its side chains and / or terminals. For crosslinking purposes, if the highly branched polymer has electrophilic functional groups, the linear polymer has nucleophilic functional groups, and if the highly branched polymer has nucleophilic functional groups, the linear polymer has electrophilic functional groups. The content of the electrophilic and nucleophilic functional groups is the same as in the case of the highly branched polymer described above.

[0093] Linear polymers are proteins, peptides, or polysaccharides. As proteins, there are no particular restrictions as long as they do not adversely affect cells, and they can be appropriately selected depending on the purpose. Examples include collagen, fibrin, and albumin. Gelatin is particularly preferred as the linear polymer. Peptides are polymerized by two or more amino acid residues via peptide bonds (amide bonds). Peptides may be dipeptides, tripeptides, oligopeptides (containing about 10 amino acids), or polypeptides (containing tens to hundreds of amino acids), but it is preferable that they do not gel at room temperature and pressure. Furthermore, there are no particular restrictions on polysaccharides, and they can be appropriately selected. Examples include cellulose, chitin, chitosan, hyaluronic acid, alginic acid, starch, and pectin. These linear polymers may be used individually or in combination of two or more. As a linear polymer containing two or more nucleophilic or electrophilic functional groups in its side chains and / or terminals, gelatin having thiol groups in its side chains is shown below.

[0094] [ka]

[0095] <cell> There are no particular restrictions on the type of cell used; cells can be selected as appropriate according to the purpose. Taxonomically, all cells can be used, regardless of whether they are eukaryotic, prokaryotic, multicellular, or unicellular organisms.

[0096] Examples of eukaryotic cells include animal cells, insect cells, plant cells, and fungi. These may be used individually or in combination of two or more. Among these, animal cells are preferred, and if the cells form a cell aggregate, it is even more preferable that they are adherent cells that adhere to each other and have sufficient cell adhesion to not require physicochemical treatment to isolate them.

[0097] There are no particular restrictions on the type of adherent cells used; they can be appropriately selected depending on the purpose, and examples include differentiated cells and undifferentiated cells.

[0098] Differentiated cells include, for example, hepatocytes (parenchymal cells of the liver); stellate cells; Kupffer cells; vascular endothelial cells; endothelial cells such as endothelial cells and corneal endothelial cells; fibroblasts; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells such as keratinocytes; tracheal epithelial cells; gastrointestinal epithelial cells; cervical epithelial cells; epithelial cells such as corneal epithelial cells; mammary gland cells; pericytes; muscle cells such as smooth muscle cells and cardiomyocytes; renal cells; pancreatic islet cells; nerve cells such as peripheral nerve cells and optic nerve cells; chondrocytes; and osteocytes. The aforementioned adherent cells may be primary cells directly collected from tissues or organs, or they may be cells that have been passaged through several generations.

[0099] There are no particular restrictions on the undifferentiated cells used, and they can be appropriately selected depending on the purpose. Examples include undifferentiated embryonic stem cells, pluripotent stem cells such as mesenchymal stem cells with multipotency, unipotent stem cells such as vascular endothelial progenitor cells with monopotency, and iPS cells.

[0100] Examples of prokaryotic cells include bacteria and archaea.

[0101] Specific examples of cells include normal human dermal fibroblasts. Commercially available normal human dermal fibroblasts can be used, and examples of such commercial products include the product name: CC2507 (manufactured by Lonza).

[0102] <Cell-containing gel particles> Cell-containing microgel particles are formed in vitro, and for this purpose, culture vessels, culture media, and other known materials necessary for functional expression and cell survival are used as appropriate depending on the application. The cell-containing gel particles contain one or more cells and are spherical or ellipsoidal in shape. Multiple types of cells may be included, and they may be in contact with a substrate such as a culture vessel. There are no particular restrictions on the cell density, and the cells may be directly bound to each other. The hydrogel surrounding the cells is a gel obtained by a cross-end coupling reaction between a highly branched polymer and a linear polymer. The gel concentration (mass % concentration (w / w) of gel molecules in the hydrogel) is preferably 0.6% to 8%.

[0103] The diameter of the cell-containing gel particles can be appropriately set depending on the type of cell and the conditions under which the cells are cultured, but a range of 60 μm to 500 μm is preferable because it makes it easier to observe the cells contained in the hydrogel. Here, diameter refers to the average of the long axis and short axis of the gel particle in the microscopic image obtained by observing the cell-containing gel particle under a microscope.

[0104] Extracellular matrix may be added to the hydrogel 50 enclosing the cells 51 as needed. The extracellular matrix is ​​not particularly limited and can be appropriately selected depending on the purpose. Examples include extracellular matrix proteins and glycoproteins such as collagen, laminin, fibronectin, elastin, fibrin, proteoglycans, hyaluronic acid, heparan sulfate proteoglycans, and chondroitin sulfate proteoglycans, as well as growth factors such as hepatocyte growth factor, fibroblast growth factor, and nerve growth factor, depending on the cell. These may be used individually or in combination of two or more. These are known to promote cell adhesion, proliferation, and differentiation, and by including them in the hydrogel 50, they can function as triggers to change the morphology of cells within the formed gel particles.

[0105] <Method for producing cell-containing gel particles> The method for producing the cell-containing gel particles described above will be explained with reference to Figure 3. The cell-containing gel particles 62 of this embodiment can be produced by mixing droplets of a first solution 60 containing a multi-branched polymer and cells with a second solution 61 containing a linear polymer but no cells, which is placed in a culture vessel 63. Alternatively, the cells may be contained in the second solution instead of the first solution, and droplets of the second solution may be mixed with the first solution which does not contain cells.

[0106] <Culture container> The culture vessel 63 consists of a substrate that serves as a base or support necessary for the formation and maintenance of cell-containing gel particles, and examples include containers made of resin, glass, or metal. The substrate can be not only flat, but also perforated, mesh-like, uneven, or honeycomb-like, and can be selected as appropriate depending on the application. It is preferable to use a multi-well type culture plate or insert that has high light transmittance and does not self-fluoresce or emit light.

[0107] In the example shown in Figure 3, cell-containing gel particles 62, formed by crosslinking between the first solution 60 and the second solution 61, are present in the culture medium 64 filled in the culture vessel 63, allowing for cell culture in this state. As shown in Figure 3, in cell culture performed with the cell-containing gel particles 62 surrounded by the culture medium 64, even a single cell can be cultured, and cells surviving and extending within the cell-containing gel particles 62 can be observed. Furthermore, when nerve cells are used, the neurites remain within the cell-containing gel particles 62. In the example shown in Figure 3, the top of the culture vessel 63 is open for the addition and removal of the culture medium 64 and drugs, but a closed system capable of perfusion culture or a tip-type vessel with microchannels may also be used.

[0108] In another embodiment, as shown in Figure 4, droplets of the first solution 70 containing cells are dropped onto the second solution 71 on a substrate to form cell-containing gel particles 72 by a crosslinking reaction. These cell-containing gel particles 72 are then attached to and held in a culture vessel 73, and cell culture can be performed with the culture vessel 73 filled with culture medium 74. Performing cell culture with the cell-containing gel particles 72 in contact with the culture vessel 73 has the advantage of allowing suspension cells to be cultured under contact conditions, and also facilitating observation with a microscope.

[0109] <culture medium> The culture medium filling the culture vessel is a solution that contains components necessary for maintaining the shape of cell-containing gel particles and cell survival, prevents drying, and regulates the external environment such as osmotic pressure. There are no particular restrictions on the culture medium; it can be appropriately selected from known culture media depending on the application.

[0110] Examples of the culture media include those classified by composition, such as natural media, semi-synthetic media, and synthetic media; and those classified by form, such as semi-solid media, liquid media, and powdered media (hereinafter sometimes referred to as "powdered media"). These may be used individually or in combination of two or more. If the cells are of animal origin, any culture medium used for animal cell culture can be used.

[0111] There are no particular restrictions on the culture medium used for animal cell culture, and it can be appropriately selected according to the purpose. For example, Dulbecco's Modified Eagles's Medium (D-MEM), Ham's Nutrient Mixture F12, D-MEM / F12 medium, McCoy's 5A medium, Eagles's Minimum Essential Medium (EMEM), αMEM medium (alpha Modified Eagles's Minimum Essential Medium (αMEM)), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemPro34 (Invitrogen), X-VIVO 10 (Kembrex), X-VIVO 15 (Kembrex), HPGM (Kembrex), StemSpan H3000 (Stem Cell Technologies), StemSpanSFEM (Stem Cell Technologies), StemlineII (Sigma-Aldrich), QBSF-60 (Quality Biological), StemProhESCSFM (Invitrogen), Essential8® medium (Gibco), Stemfit AK02N (Ajinomoto Co., Inc.), Stemfit Basic 02 (Ajinomoto Co., Inc.), mTeSR1 or 2 medium (Stem Cell Technologies), ReproFF or ReproFF2 (ReproCELL), PSGro hESC / iPSC medium (System Biosciences), NutriStem® medium (Biological Industries, Inc.), CSTI-7 medium (Cell Science Institute), MesenPRO Examples include RS medium (Gibco), MF-Medium® mesenchymal stem cell proliferation medium (Toyobo Co., Ltd.), Sf-900II (Invitrogen), and Opti-Pro (Invitrogen).These can be used individually or in combination of two or more types.

[0112] There are no particular restrictions on the carbon dioxide concentration in the culture medium, and it can be appropriately selected depending on the purpose, but a concentration of 2% to 5% is preferred. When the carbon dioxide concentration is 2% to 5%, cells can be cultured effectively.

[0113] Figure 5 shows one embodiment of the cell evaluation plate according to the present invention. The cell evaluation plate 8 in Figure 5 has a substrate with a number of wells 80, and each well 80 contains cell-containing gel particles 81. The number of wells is not particularly limited, and the individual cell-containing gel particles 81 may have the same composition with respect to the type of cell and hydrogel, or they may have different compositions. Such a cell evaluation plate 8 can be suitably used for high-throughput screening. [Examples]

[0114] The present invention will be described in more detail below using examples and comparative examples. However, the technical scope of the present invention is not limited to these examples.

[0115] (Example 1) (1) Preparation of the first solution 0.02 g of Tetra-PEG-maleimidil (SUNBRIGHT PTE-100MA, manufactured by Yuka Sangyo Co., Ltd.) was dissolved in 1 ml of phosphate-buffered saline (manufactured by Life Technologies, hereafter also referred to as PBS(-)) to prepare a first solution containing 2% Tetra-PEG-maleimidil.

[0116] (2) Preparation of the second solution 0.05 g of gelatin-SH (Thiol functionalized gelatin, manufactured by Sigma-Aldrich) was dissolved in 1 ml of PBS(-) at 37°C overnight to prepare a second solution containing 5% gelatin-SH.

[0117] (3) Preparation of hydrogels (2) After dropping 10 μL of the second solution prepared in (2) onto a 96-well plate, 10 μL of the first solution prepared in (1) was stirred and mixed with the dropped second solution to prepare a hydrogel.

[0118] (4) Seeding of nerve cells After thawing vials containing cryopreserved glutamatergic neurons in a 37°C bath, the cells were transferred to a 15 mL centrifuge tube and suspended in 5 mL of DMEM. 100 μL of the cell suspension was taken into an Eppendorf tube, the sample was aspirated into a cassette, and the cell count was measured using a cell counter to determine the number of cells in the suspension. The cell suspension in the 15 mL centrifuge tube was centrifuged (200 × g, 3 min), the supernatant was removed using an aspirator, and the cell concentration was reduced to 1 × 10⁶ in PBS(-). 6 The cells were prepared as a cell suspension at a concentration of cells / mL. This cell suspension was then placed on the hydrogel prepared in (3) with a cell count of 1 × 10⁶. 4 Cell cultures were prepared by seeding the cells individually.

[0119] (5) Observation of cells The morphology of cells on the hydrogel surface of the prepared cell cultures was evaluated after 7 days of culture based on whether the cells were spreading. The morphology of cells within the hydrogel was observed using a microscope (CKX41, Olympus Corporation). The results are shown in Figure 6. As shown in Figure 6, it was confirmed that nerve cells were spreading on the hydrogel surface.

[0120] (Example 2) (1) Preparation of the first solution 0.02 g of Tetra-PEG-maleimidil (SUNBRIGHT PTE-100MA, manufactured by Yuka Sangyo Co., Ltd.) was dissolved in 1 ml of phosphate-buffered saline (manufactured by Life Technologies, hereafter also referred to as PBS(-)) to prepare a first solution containing 2% Tetra-PEG-maleimidil.

[0121] (2) Preparation of the second solution 0.05 g of gelatin-SH (Thiol functionalized gelatin, manufactured by Sigma-Aldrich) was dissolved in 1 ml of PBS(-) at 37°C overnight to prepare a second solution containing 5% gelatin-SH.

[0122] (3) Culture of human umbilical vein endothelial cells (hereinafter referred to as "HUVEC" or "HUVEC cells") HUVEC cells were cultured in 100 mm dishes using endothelial cell basal medium (Culture system containing EBMTM-2 Basal Medium (CC-3156), EBMTM-2 SingleQuots™ Supplements (CC-4176), manufactured by Lonza) in an incubator (KM-CC17RU2, manufactured by Panasonic Corporation, 37°C, 5% CO2 environment).

[0123] (4) Preparation of the first solution containing HUVEC cells 5 mL of PBS(-) was added to a dish, and the PBS(-) was removed by aspirator, washing the surface. After washing with PBS(-), 2 mL of 0.05% trypsin-0.05% EDTA solution (life technologies) was added to the dish, and it was heated in an incubator for 5 minutes to detach the cells from the dish. After confirming cell detachment with a phase-contrast microscope (instrument name: CKX41, Olympus Corporation), 2 mL of DMEM containing FBS was added to the dish to inactivate the trypsin. The cell suspension from the dish was transferred to one 15 mL centrifuge tube, and centrifugation (H-19FM, KOKUSAN, 200 × g, 3 min) was performed, and the supernatant was removed using an aspirator. After removal, 2 mL of DMEM containing FBS was added to the centrifuge tube, and the cells were gently pipetted to disperse and obtain a cell suspension. 100 μL of the cell suspension was taken into an Eppendorf tube, the sample was aspirated into a cassette (Via1-Cassette, chemometec), and the number of cells was measured using a cell counter (NucleoCounter NC-3000, chemometec) to determine the number of cells in the solution. A portion of the cell suspension was transferred to a 15 ml centrifuge tube and centrifuged (200 × g, 3 minutes), and the supernatant was removed using a pipette. The first solution prepared in (1) was added, and a cell concentration of 5 × 10 was obtained, containing 2% Tetra-PEG-maleimidyl. 6 A cell-containing first solution was obtained, consisting of a cell suspension with a concentration of cells / mL.

[0124] (5) Preparation of cell cultures (2) After dropping 3 μL of the second solution prepared in (2) onto a 35 mm dish (model number: 3000-035, manufactured by AGC Techno Glass), 3 μL of the cell-containing first solution prepared in (4) was stirred and mixed with the dropped second solution to prepare a cell culture in which cells were encapsulated in a hydrogel.

[0125] (6) Observation of cells The morphology of the cells in the hydrogel after 7 days of culture was evaluated to see if the cells were spreading. Calcein AM (-Cellstain®-Calcein-AM solution, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the culture medium in a 35 mm dish, and the cells were cultured again for 1 hour in an incubator (as described above) at 37°C in a 5 volume % CO2 environment. After 1 hour of culture, the morphology of the cells in the hydrogel was observed using a confocal microscope. The results are shown in Figure 7. As shown in Figure 7, it was confirmed that the HUVEC cells were spreading within the hydrogel.

[0126] (Example 3) The basic procedure was the same as in Example 2. However, this example differs from Example 2 in that two or more gel precursors with different mixing ratios were formed in the first solution, nerve cells were used instead of HUVEC cells, and cell cultures were prepared using inkjet printing.

[0127] (1) Preparation of the first solution 0.02 g of Tetra-PEG-maleimidil was dissolved in 1 ml of PBS(-). Furthermore, a first solution containing 2% Tetra-PEG-maleimidil and 0.1% Thiol Gelatin was prepared using the second solution prepared in Example 2.

[0128] (2) Preparation of the first solution containing nerve cells After thawing the cryopreserved glutamatergic nerve cells in a 37°C bath, the cells were transferred to a 15 mL centrifuge tube and suspended in 5 mL of DMEM. 100 μL of the cell suspension was taken out into an Eppendorf tube, the sample was aspirated into a cassette, and the number of cells was counted using a cell counter to determine the number of cells in the solution. The cell suspension in the 15 mL centrifuge tube was centrifuged (200 × g, 3 min), and the supernatant was removed using an aspirator. The first solution prepared in (1) was added, and the cell concentration was 1 × 10⁶, containing 1% Tetra-PEG-maleimidyl and 0.1% gelatin-SH. 7 A cell-containing first solution was obtained, consisting of a cell suspension with a concentration of cells / mL.

[0129] (3) Preparation of cell cultures The second solution from Example 2 was applied to an 18mm square cover glass (No. 1, Thickness 0.13~0.17mm, manufactured by Matsunami Glass Co., Ltd.). After filling the liquid chamber of the inkjet head with the cell-containing first solution prepared in (2), it was dropped drop by drop onto the cover glass to deposit the cell-containing first solution into the second solution, and a cell culture was prepared by the reaction of the two solutions. After gel formation, the cover glass was transferred to a 35mm dish, filled with culture medium, and the formation of the gel was confirmed under a microscope. The dish was then incubated in an incubator at 37°C and 5% CO2 for 14 days.

[0130] (4) Immunofluorescence staining After removing the supernatant, the cells were washed with PBS(-) (hereinafter referred to as "PBS washing"). The supernatant was removed, and the cells were fixed by adding 4% paraformaldehyde (hereinafter referred to as "4% PFA") and letting them stand for 20 minutes. After removing the supernatant, the PBS washing was repeated three times. The supernatant was removed, and the cells were permeabilized with 0.1% Triton for 10 minutes. After repeating the PBS washing three times, the cells were blocked with 1% BSA for 1 hour. β3-tubulin antibody (β3-Tubulin(D71G9)XP Rabbit mAb#5568, Cell Signaling Technology, hereinafter referred to as "TUBB3") and PEG antibody (Anti-PEG antibody-BSA and Azidefree by Rat monoclonal, abcam) were diluted with 1% BSA. After blocking, the supernatant was removed, and the above primary antibody dilution was added and incubated overnight at 4°C. The following day, the cells were washed three times with PBS, and a secondary antibody solution diluted with 1% BSA solution was added and incubated for 1 hour. From this point onward, the work was carried out while shielding from light with aluminum foil or similar material. After removing the supernatant, Hoechst solution diluted with PBS was added and nuclear staining was performed for 5 minutes. The cells were washed twice with PBS and mounting medium was added dropwise. Observation using a fluorescence microscope (Zeiss) confirmed that the cells in the hydrogel on day 14 of culture expressed TUBB3, a marker for nerve cells (Figure 8). In addition, the neurites were observed to bend along the contour of the gel, indicating that the neurites remained within the gel.

[0131] (Example 4) The basic procedure was the same as in Example 3. However, this example differs from Example 3 in that it uses HUVEC cells and HepG2 cells (human liver cancer-derived cell lines) and the cell cultures are layered using inkjet printing.

[0132] (1) Culture of HepG2 cells HepG2 cells were cultured for 72 hours in a 100 mm dish in Dulbecco's Modified Eagle Medium (trade name: DMEM(1X), manufactured by Thermo Fisher Scientific, hereinafter referred to as "DMEM") containing 10% by mass fetal bovine serum (hereinafter referred to as "FBS") and 1% by mass antibiotic (Antibiotic-Antimycotic Mixed Stock Solution (100x), manufactured by Nacalai Tesque Co., Ltd.) in an incubator.

[0133] (2) Preparation of the first solution containing HepG2 cells HepG2 cells were dispersed in the same manner as HUVEC cells to obtain a cell suspension. The prepared first solution was added, and the cell concentration reached 1 × 10⁶, containing 2% Tetra-PEG-maleimidil. 7 A first solution containing HepG2 cells was obtained, consisting of a cell suspension of cells / mL.

[0134] (3) Preparation of the first solution containing HUVEC cells In the same manner as in Example 2, the cell concentration was 1 × 10⁻⁶. 7 A first solution containing HUVEC cells was obtained, consisting of a cell suspension of cells / mL.

[0135] (4) Preparation of cell cultures A first solution containing HepG2 cells was loaded onto an 18mm square coverslip and dropped onto a 10mm x 20mm area at a 100μm pitch, one drop at a time, using an inkjet head. This was then immersed in a dish containing the second solution, impregnating the droplet-like HepG2 cell-containing first solution with the second solution to form a cell culture. Next, using an inkjet head filled with the first solution containing HUVEC cells, droplets were dropped one by one onto a 5 mm x 10 mm area on the cell culture at a 100 μm pitch. Subsequently, the second solution was impregnated into the droplet-like first solution containing HUVEC cells to form a second layer of cell culture. By repeating the same process with an inkjet head, the third layer (HepG2 cells) and the fourth layer (HUVEC cells) of cell cultures were formed, and finally a three-dimensional cell culture consisting of four layers of cell-containing hydrogel was obtained. Finally, the three-dimensional cell cultures were transferred to a 35mm dish containing 1 mL of DMEM containing 10% FBS and 1% antibiotic by mass, and 1 mL of endothelial cell basic medium, and placed in an incubator (as described above) at 37°C with a 5% CO2 environment.

[0136] (5) Immunofluorescence staining After removing the supernatant, the cells were washed with PBS(-). The supernatant was removed, and the cells were fixed by adding 4% PFA and letting them stand for 20 minutes. After removing the supernatant, the PBS wash was repeated three times. The supernatant was removed, and the cells were permeabilized with 0.1% Triton for 10 minutes. After repeating the PBS wash three times, the cells were blocked with 1% BSA for 1 hour. Albumin antibody (Anti-Albumin antibody, abcam) and CD31 antibody (Anti-CD31 antibody, abcam) were diluted with 1% BSA. After blocking, the supernatant was removed, and the above primary antibody dilution was added and incubated overnight at 4°C. The next day, the cells were washed with PBS three times, and the secondary antibody solution diluted with 1% BSA was added and incubated for 1 hour. From this point onward, the work was carried out while shielding from light with aluminum foil, etc. After removing the supernatant, nuclear staining was performed with Hoechst solution diluted with PBS for 5 minutes. The PBS wash was repeated twice, and mounting medium was added dropwise. Observations using a confocal microscope (FV10, Olympus Corporation) confirmed that cells in the hydrogel on day 7 of culture were expressing albumin, a marker for HepG2 cells, and CD31, a marker for HUVEC cells, and were spreading. Furthermore, a layered structure was observed, with HepG2 cells in the first and third layers and HUVEC cells in the second and fourth layers.

[0137] (Example 5) The basic procedure was the same as in Example 3. However, this example differs from Example 3 in that it uses HUVEC cells and stacks cell cultures using inkjet printing.

[0138] (1) Preparation of cell-free first solution 0.02 g of Tetra-PEG-maleimidil (SUNBRIGHT PTE-100MA, manufactured by Yuka Sangyo Co., Ltd.) was dissolved in 1 ml of phosphate-buffered saline (manufactured by Life Technologies, hereafter also referred to as PBS(-)) to prepare a cell-free first solution containing 2% Tetra-PEG-maleimidil.

[0139] (2) Preparation of the first solution containing HUVEC cells A first solution containing 1% Tetra-PEG-maleimidil was prepared, and the cell concentration was 1 × 10⁻⁶. 7 A first solution containing HUVEC cells was obtained, consisting of a cell suspension of cells / mL.

[0140] (3) Preparation of cell cultures Using an inkjet head filled with the cell-free first solution, droplets were dropped one by one onto a 5 mm x 10 mm area on an 18 mm square coverslip at a 100 μm pitch. Then, the coverslip was placed on a dish containing the second solution prepared in Example 2, and the droplet-like cell-free first solution was impregnated with the second solution to form a hydrogel. Next, as a gel layer formation step, one drop at a time was dropped across the hydrogel at a 100 μm pitch using an inkjet head filled with the first solution containing HUVEC cells. As shown in Figure 9, the hydrogel 20 was placed on the coverslip 30, and a layer was created in which a line-shaped cell culture 10 with a length of 15 mm was formed crossing the hydrogel 20.

[0141] (4) Immunofluorescence staining After removing the supernatant, the cells were washed with PBS(-). The supernatant was removed, and the cells were fixed by adding 4% PFA and letting them stand for 20 minutes. After removing the supernatant, the PBS wash was repeated three times. The supernatant was removed, and the cells were permeabilized with 0.1% Triton for 10 minutes. After repeating the PBS wash three times, the cells were blocked with 1% BSA for 1 hour. The CD31 antibody (Anti-CD31 antibody, abcam) was diluted with 1% BSA. After blocking, the supernatant was removed, and the above primary antibody dilution was added and incubated overnight at 4°C. The next day, the cells were washed with PBS three times, and the secondary antibody solution diluted with 1% BSA solution was added and incubated for 1 hour. From this point onward, the work was carried out while shielding from light with aluminum foil, etc. After removing the supernatant, nuclear staining was performed with Hoechst solution diluted with PBS for 5 minutes. The PBS wash was repeated twice, and the mounting medium was added dropwise. Observation using a confocal microscope (FV10, Olympus Corporation) confirmed that cells in the hydrogel on day 7 of culture were expressing CD31, a marker for HUVEC cells, and were spreading.

[0142] (Example 6) The basic procedure was the same as in Example 5. However, this example differs from Example 5 in that it uses the nerve cells used in Example 3. By dropping ink containing cells using an inkjet printer, a hydrogel 20 was placed on a coverslip 30, as shown in Figure 10(a), and a laminate was created in which a line-shaped cell culture 10 was formed traversing the hydrogel 20. In this laminate, cells C spread within the cell culture 10, as shown in Figure 10(b), which is a schematic magnified view of portion A in Figure 10(a). Figure 11 is an image of the line-shaped cell culture 10 observed under a microscope. From the results in Figure 11, it was confirmed that when a line-shaped cell culture was created using an inkjet printer, the cells spread along the entire line, regardless of the shape of the ejected droplets.

[0143] (Comparative Example 1) When a hydrogel was prepared using Tetra-PEG-maleimidil as the first solution and Tetra-PEG-SH as the second solution, and observed in the same manner as in Example 1, as shown in Figure 12, the nerve cells remained round and did not extend, and most of the cells were not extended.

[0144] (Comparative Example 2) Cell cultures were prepared in the same manner as in Example 2, except that a hydrogel was used to coat the cells, which consisted of phosphate-buffered saline mixed with Tetra-PEG-maleimidyl and Tetra-PEG-SH (SUNBRIGHT PTE-100SH, manufactured by Yuka Sangyo Co., Ltd.) at a mass ratio of 1%, thrombin (Thrombin from bovine Plasma, manufactured by Sigma-Aldrich) at 20 U / mL, and fibrinogen (Fibrinogen from bovine plasma, manufactured by Sigma-Aldrich) at a mass ratio of 1%. Cell expansion was then observed. The results are shown in Figure 13. From Figure 13, it can be seen that after incubation, some HUVEC cells remained round while others were expanded, suggesting that it is difficult to expand almost all cells within the hydrogel and culture them long-term.

[0145] (Comparative Example 3) As Comparative Example 3, a cell culture was prepared in the same manner as in Example 2, except that a calcium alginate gel was formed by mixing a sodium alginate solution, prepared by dissolving sodium alginate (SKAT ONE, manufactured by Kimika) at a mass ratio of 1% and gelatin-SH at a mass ratio of 2% in phosphate-buffered saline (manufactured by Life Technologies, hereafter also referred to as PBS(-)), with a calcium chloride solution, prepared by dissolving calcium chloride dihydrate (manufactured by Wako Pure Chemical Industries, Ltd.) at a mass ratio of 100 mmol / L in distilled water, in a mass ratio of 1:1 to form a calcium alginate gel, and cell expansion was observed. As a result, the morphology of the HUVEC cells after incubation did not expand and remained round, and it was also observed that the calcium alginate gel gradually disintegrated when the dish was shaken, confirming that it lacked stability as a scaffold material during culture.

[0146] (Comparative Example 4) When cell cultures were prepared and observed in the same manner as in Example 3, using Tetra-PEG-maleimidil and Matrigel as the first solution and Tetra-PEG-SH as the second solution, the nerve cells remained round and did not extend, and most cells were not extended.

[0147] The cell cultures formed by the methods of each example and each comparative example were evaluated using the methods described below, and the results are summarized in Table 1.

[0148] (Evaluation of cell morphology after long-term culture in gel) Based on the morphology of the cells after 7 days of culture, a "○" was given if more than 50% of the cells had extended, and a "×" if they had not. Cell extension was determined if the pseudopods of the cells were visible.

[0149] [Table 1]

[0150] Table 1 shows that cells can spread on the hydrogel surface of Example 1, while cells cannot spread on the normal Tetra-PEG gel surface of Comparative Example 1. The cell culture medium of Example 2 was found to be more efficient at spreading HUVEC cells compared to a normal Tetra-PEG gel with guest material added (Comparative Example 2) and an alginate gel (Comparative Example 3). Furthermore, Examples 3 and Comparative Example 4 confirmed that similar results can be obtained even with cell culture mediums produced by inkjet printing. In other words, by crosslinking a polymer material with a fast gelling time with a water-soluble protein having cell adhesion properties to form a hydrogel as described in the present invention, it was possible to obtain a cell culture medium that achieves both gel-forming properties and cell adhesion properties, and it was confirmed that long-term cell culture is possible.

[0151] (Example 7) Preparation of cell-containing gel particles using 3T3 cells (adherent cells) (1) Preparation of the first solution 0.02 g of Tetra-PEG-maleimidil (product name: SUNBRIGHT PTE-100MA, manufactured by Yuka Sangyo Co., Ltd.) was dissolved in 1 ml of phosphate-buffered saline (manufactured by Life Technologies, hereafter also referred to as PBS(-)) to prepare a first solution containing 2% Tetra-PEG-maleimidil.

[0152] (2) Preparation of the second solution 0.05 g of Thiol Gelatin (trade name: Thiol functionalized gelatin, manufactured by Sigma-Aldrich) was dissolved in 1 ml of PBS(-) and incubated overnight at 37°C to prepare a second solution containing 5% Thiol Gelatin.

[0153] (3) Cell culture 3T3 / NIH cells (JCRB Cell Bank, hereinafter referred to as "3T3") were cultured for 72 hours in a 100 mm dish using Dulbecco's Modified Eagle Medium (product name: DMEM(1X), Life Technologies, hereinafter referred to as "DMEM") containing 10% newborn calf serum (product name: Newborn Calf Serum, manufactured by Sigma-Aldrich, hereinafter referred to as "NCS") in an incubator (product name: KM-CC17RU2, manufactured by Panasonic Corporation, 37°C, 5% CO2 environment).

[0154] (4) Preparation of cell suspension 5 mL of PBS(-) was added to a dish, and the PBS(-) was removed by aspirator, washing the surface. After washing with PBS(-), 2 mL of 0.05% trypsin-0.05% EDTA solution (life technologies) was added to the dish, and it was heated in an incubator for 5 minutes to detach the cells from the dish. After confirming cell detachment with a phase-contrast microscope (instrument name: CKX41, Olympus Corporation), 2 mL of DMEM containing NCS was added to the dish to inactivate the trypsin. The cell suspension from the dish was transferred to one 15 mL centrifuge tube, and centrifugation (product name: H-19FM, KOKUSAN, 200 × g, 3 min) was performed, and the supernatant was removed using an aspirator. After removal, 2 mL of DMEM containing NCS was added to the centrifuge tube, and the cells were gently pipetted to disperse and obtain a cell suspension. 100 μL of the cell suspension was taken into an Eppendorf tube, the sample was aspirated into a cassette (product name: Via1-Cassette, manufactured by chemometec), and the number of cells was measured using a cell counter (product name: NucleoCounter NC-3000, manufactured by chemometec) to determine the number of cells in the solution. A portion of the cell suspension was transferred to a 15 ml centrifuge tube and centrifuged (200 × g, 3 minutes), and the supernatant was removed using a pipette. The first solution prepared in (1) was added, and a cell concentration of 1 × 10⁶ was obtained, containing 2% Tetra-PEG-maleimidyl. 7 A cell-containing first solution was obtained, consisting of a cell suspension with a concentration of cells / mL.

[0155] (5) Preparation of cell-containing gel particles A 13mm diameter porous polyester culture membrane (product name: ipCELLCULTURE Track Etched Membrane, pore size: 0.45μm, pore density: 4E6cm-2, thickness: 12μm, manufactured by it4ip) was placed in a 35mm dish, and 2mL of the second solution was added to impregnate the membrane with the second solution. After filling the liquid chamber of the inkjet head with the cell-containing first solution prepared in (4), it was dropped drop by drop onto the membrane, causing the cell-containing first solution to deposit into the second solution, and cell-containing gel particles were produced by the reaction of the two solutions. After gel formation, the membrane was transferred to a 35mm dish, and the gel particles were suspended using DMEM containing NCS, and then dispensed into a 96-well plate. The presence of gel particles was confirmed under a microscope, and the plate was heated at 37°C and 5% CO2. 2 The samples were placed in an environmental incubator and cultured for 7 days.

[0156] (6) Observation of cell-containing gel particles The cell-containing gel particles were observed using a phase-contrast microscope. As a result, as shown in Figure 14, it was confirmed that 3T3 cells, which are adherent cells, could be cultured within the gel. Even without direct adhesion to the substrate, it was observed that the cells were spreading within the gel.

[0157] (Example 8) Preparation of cell-containing gel particles using CHO cells (suspension cells) (1) Preparation of the first solution The procedure was carried out in the same manner as in Example 7.

[0158] (2) Preparation of the second solution The procedure was carried out in the same manner as in Example 7.

[0159] (3) Cell culture In an incubator, CHO (pMAM-luc) cells (JCRB Cell Bank, hereinafter referred to as "CHO") were cultured for 72 hours in a 100 mm dish using Ham's F12 (product name: F-12 Ham, manufactured by Sigma-Aldrich, hereinafter referred to as "Ham's F12") containing 10% fetal bovine serum (product name: Fetal Bovine Serum, manufactured by GE Healthcare, hereinafter referred to as "FBS").

[0160] (4) Preparation of cell suspension 5 mL of PBS(-) was added to a dish, and the PBS(-) was removed by aspirator to wash the surface. After washing with PBS(-), 2 mL of 0.05% trypsin-0.05% EDTA solution was added to the dish, and it was heated in an incubator for 5 minutes to detach the cells from the dish. 2 mL of Ham's F12 with FBS was added to the dish to inactivate the trypsin. The cell suspension from the dish was transferred to a 15 mL centrifuge tube and centrifuged (200 × g, 3 min), and the supernatant was removed using an aspirator. After removal, 2 mL of Ham's F12 with FBS was added to the centrifuge tube, and the cells were dispersed by gentle pipetting to obtain a cell suspension. 100 μL of the cell suspension was taken out into an Eppendorf tube, the sample was aspirated into a cassette, and the number of cells was counted using a cell counter to determine the number of cells in the solution. A portion of the cell suspension was transferred to a 15 ml centrifuge tube and centrifuged (200 × g, 3 min), and the supernatant was removed using a pipette. The first solution prepared in (1) was added, and a cell concentration of 1 × 10 was obtained, containing 2% Tetra-PEG-maleimidil. 7 A cell-containing first solution was obtained, consisting of a cell suspension with a concentration of cells / mL.

[0161] (5) Preparation of cell-containing gel particles The procedure was the same as in Example 7, and the samples were incubated for 5 days in an incubator at 37°C and 5% CO2.

[0162] (6) Observation of cell-containing gel particles The cell-containing gel particles were observed using a phase-contrast microscope. As a result, as shown in Figure 15, it was confirmed that the suspension-type CHO cells could be cultured within the gel.

[0163] (Example 9) Evaluation of cell viability (1) Cell staining The cell-containing gel particles prepared in Example 7 were used for evaluation. Propidium (Sigma Aldrich, hereinafter referred to as "PI") and Hoechst 33342 (H3570, Thermo Fisher Scientific, hereinafter referred to as "Hoechst") were diluted in culture medium to 0.5 ng / ml and incubated at 37°C. After incubation for 1 hour, the cell-containing gel particles were observed using a fluorescence microscope (Olympus Corporation) and images were obtained.

[0164] (2) Calculation of cell viability Based on the images obtained in (1), the number of cells stained with PI was taken as the number of dead cells, and the total number of cells stained with Hoechst was taken as the total number of cells. The survival rate (%) was calculated as (number of dead cells / total number of cells) × 100.

[0165] (3) Judgment criteria Cell viability was marked as 75% or higher after 4 and 7 days of culturing cell-containing gel particles, and this is summarized in Table 2. As shown in Table 2, it was confirmed that cell viability could be maintained even after culturing in the gel particles for one week.

[0166] [Table 2]

[0167] (Example 10) Preparation of cell-containing gel particles using nerve cells (1) Preparation of the first solution The procedure was carried out in the same manner as in Example 7.

[0168] (2) Preparation of the second solution The procedure was carried out in the same manner as in Example 7.

[0169] (3) Preparation of cell suspension After thawing the cryopreserved glutamatergic nerve cells in a 37°C bath, the cells were transferred to a 15 mL centrifuge tube and suspended in 5 mL of DMEM. 100 μL of the cell suspension was taken out into an Eppendorf tube, the sample was aspirated into a cassette, and the number of cells was counted using a cell counter to determine the number of cells in the solution. The cell suspension in the 15 mL centrifuge tube was centrifuged (200 × g, 3 min), and the supernatant was removed using an aspirator. Using the first and second solutions prepared in (1) and (2), a solution containing 1% Tetra-PEG-maleimidyl and 0.1% Thiol Gelatin with a cell concentration of 1 × 10⁶ was prepared. 7 A cell-containing first solution was obtained, consisting of a cell suspension with a concentration of cells / mL.

[0170] (4) Preparation of cell-containing gel particles The procedure was the same as in Example 7, and the samples were incubated in an incubator at 37°C and 5% CO2 for 7 days.

[0171] (5) Immunofluorescence staining of cell-containing gel particles After removing the supernatant, the cells were washed with PBS(-) (hereinafter referred to as "PBS washing"). The supernatant was removed, and 4% paraformaldehyde (product name: 4% paraformaldehyde-phosphate buffer, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "4% PFA") was added and the cells were allowed to stand for 20 minutes to fix them. After removing the supernatant, PBS washing was repeated three times. The supernatant was removed, and 0.1% Triton was added and the cells were permeabilized for 10 minutes. After repeating PBS washing three times, the cells were blocked with 1% BSA for 1 hour. β3-tubulin antibody (product name: β3-Tubulin(D71G9)XP Rabbit mAb#5568, manufactured by Cell Signaling Technology, hereinafter referred to as "TUBB3") and PEG antibody (product name: Anti-Polyethylene glycol antibody [26A04]BSA and Azide free (ab94764), abcam) were diluted with 1% BSA. After blocking, the supernatant was removed, and the primary antibody dilution described above was added and incubated overnight at 4°C. The next day, PBS washing was performed three times, and the secondary antibody solution diluted with 1% BSA solution was added and incubated for 1 hour. From this point onward, the work was carried out while shielding from light with aluminum foil or similar material. After removing the supernatant, Hoechst solution diluted with PBS was added and nuclear staining was performed for 5 minutes. PBS washing was repeated twice, and mounting medium (product name: Fluoromount, DBS) was added dropwise. Observation using a fluorescence microscope (Zeiss) confirmed that the cells in the gel particles on day 7 of culture expressed TUBB3, a marker for nerve cells (Figure 16). In addition, the neurites were observed to bend along the contour of the gel, indicating that the neurites remained within the gel.

[0172] (Example 11) Size of cell-containing gel particles (1) Preparation of the first solution The procedure was carried out in the same manner as in Example 7.

[0173] (2) Preparation of the second solution The procedure was carried out in the same manner as in Example 7.

[0174] (3) Preparation of cell suspension After thawing the vial containing cryopreserved cardiomyocytes in a 37°C water bath, it was transferred to a single 15 mL centrifuge tube, and 10 mL of DMEM was added and suspended. 100 μL was taken out from the cell suspension into an Eppendorf tube, the sample was aspirated into a cassette, and the cell count was measured using a cell counter to determine the number of cells in the solution. A part of the cell suspension was dispensed from the 15 mL centrifuge tube and centrifuged (200×g, 3 minutes), and the supernatant was removed using an aspirator. Using the first solution and the second solution prepared in (1) and (2), a cell-containing first solution containing 1% Tetra-PEG-maleimidyl and 0.1% Thiol Gelatin and having a cell concentration of 1×10 7 cells / mL was obtained.

[0175] (4) Preparation of gel particles The preparation of gel particles with a diameter of 200 μm was carried out in the same manner as in Example 7. For gel particles with a diameter of 2 mm, 1 μL of the second solution was dropped into a 35 mm dish, and 1 μL of the cell-containing first solution prepared in (3) was injected into this droplet. After gel formation, DMEM was gently added to the 35 mm dish, and it was placed in an incubator at 37°C and 5% CO2 for 7 days of culture.

[0176] (5) Observation of cell-containing gel particles The cell-containing gel particles were observed with a phase-contrast microscope. As a result, as shown in Fig. 17, it was confirmed that the cardiomyocytes were pulsating inside the gel particles. Also, it was confirmed that individual cells could be observed in the cell-containing gel particles with a diameter of 200 μm, but individual cells could not be observed in detail in the gel particles with a diameter of 2 mm.

[0177] (Example 12) Cell-containing gel particles containing one cell (1) Preparation of the first solution Performed in the same manner as in Example 7.

[0178] (2) Preparation of the second solution Performed in the same manner as in Example 7.

[0179] (3) Cell culture Performed in the same manner as in Example 7.

[0180] (4) Preparation of cell suspension The procedure was carried out in the same manner as in Example 7, and the cell concentration was 1 × 10⁻⁶. 6 A cell-containing first solution was obtained, consisting of a cell suspension with a concentration of cells / mL.

[0181] (5) Preparation of cell-containing gel particles The procedure was carried out in the same manner as in Example 7.

[0182] (6) Observation of cell-containing gel particles The cell-containing gel particles were observed using a phase-contrast microscope after 1, 3, and 7 days of culture. As shown in Figure 18, cell proliferation was observed in the cell-containing gel particle that initially contained only one cell after 7 days. This confirmed that it is possible to culture even a single cell within a cell-containing gel particle.

[0183] (Comparative Example 5) Cell-containing gel particles using alginate The basic procedure was carried out in the same manner as in Example 7. However, the first and second solutions were prepared using the following materials.

[0184] (1) Preparation of the first solution 0.584 g of calcium chloride (model number: 192-13925, manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as "CaCl2") was dissolved in 100 mL of ultrapure water, and a first solution consisting of a 100 mmol / L CaCl2 aqueous solution was prepared.

[0185] (2) Preparation of the second solution 20 mg of sodium alginate (product name: Kimika Algin SKAT-ONE, manufactured by Kimika Co., Ltd.) was dissolved in 2 mL of ultrapure water to prepare a 1.0% sodium alginate aqueous solution.

[0186] (3) Criteria for determining particle size Table 3 below summarizes the results, with a circle (○) indicating a diameter of 500 μm or less for cell-containing gel particles and a cross (×) indicating a diameter exceeding 500 μm. As shown in Table 3, it was confirmed that microgel particles of 500 μm or less can be produced using sodium alginate.

[0187] (4) Evaluation of cell viability The procedure was carried out in the same manner as in Example 9, and cells with a viability of 75% or higher after 7 days of culture were marked with ○, and those with a viability of less than 75% were marked with ×, and the results are summarized in Table 3 below. As shown in Table 3, it was confirmed that cell viability could not be maintained for up to one week within the alginate microgel particles.

[0188] [Table 3]

[0189] (Comparative Example 6) Cell-containing gel particles using collagen (1) Preparation of collagen solution The collagen gel was prepared using a collagen gel culture kit (Nitta Gelatin Co., Ltd.) and stored at 4°C.

[0190] (2) Preparation of cell suspension Using the collagen solution prepared in (1), the cell concentration was 1 × 10 6 A cell suspension was prepared at a concentration of cells / mL.

[0191] (3) Preparation of cell-containing gel particles (2) The cell suspension prepared in (2) was dropped into 35 mm dishes in 1 μL increments, gelled at 37°C for 10 minutes, and then culture medium was added. The gel particles were placed in an incubator at 37°C in a 5% CO2 environment and cultured for 7 days.

[0192] (4) Criteria for determining particle size Table 4 below summarizes the results, with a circle (○) indicating a diameter of 500 μm or less for cell-containing gel particles and a cross (×) indicating a diameter exceeding 500 μm. As shown in Table 4, it was confirmed that it is not possible to produce microgel particles of 500 μm or less when using collagen.

[0193] (5) Evaluation of cell viability Performed in the same manner as in Example 9, when the cell viability after 7 days of culture was 75% or more, it was marked as ○, and when it was less than 75%, it was marked as ×, and summarized in Table 4 below. As shown in Table 4, it was confirmed that the cell viability could be maintained until 1 week in the collagen gel particles.

[0194] [Table 4]

[0195] (Comparative Example 7) Cell-containing gel particles using gelatin (1) Preparation of gelatin solution 1 ml of PBS(-) was added to 0.05 g of Thiol Gelatin and dissolved overnight at 37°C to prepare a 5% Thiol Gelatin solution.

[0196] (2) Preparation of cell suspension Using the gelatin solution prepared in (1), a cell suspension with a cell concentration of 1×10 6 cells / mL was prepared.

[0197] (3) Preparation of cell-containing gel particles 1 μL of the cell suspension prepared in (2) was dropped into a 35 mm dish and gelled at 4°C, and then a medium was added. The gel particles were placed in an incubator at 37°C and 5% CO2 environment and cultured for 7 days.

[0198] (4) Criteria for determining particle size When the diameter of the prepared cell-containing gel particles was 500 μm or less, it was marked as 〇, and when it was more than 500 μm, it was marked as ×, and summarized in Table 5 below. As shown in Table 5, it was confirmed that when using gelatin, microgel particles of 500 μm or less could not be prepared.

[0199] (5) Evaluation of cell viability As a result of culturing the gel particles gelled at 4°C in an incubator at 37°C, the gel melted, so the viability of the cells cultured in the gel could not be evaluated.

[0200] [Table 5]

[0201] (Comparative Example 8) Cell-containing gel particles using PEG-SH The basic procedure was carried out in the same manner as in Example 7. However, the second solution was prepared using the following materials. (1) Preparation of the second solution Tetra-PEG-SH (product name: SUNBRIGHT PTE-100SH, manufactured by Yuka Sangyo Co., Ltd.) was dissolved in PBS(-) to prepare a first solution containing 2% Tetra-PEG-SH.

[0202] (2) Criteria for determining particle size Table 6 below summarizes the results, with a circle (○) indicating a diameter of 500 μm or less for cell-containing gel particles and a cross (×) indicating a diameter exceeding 500 μm. As shown in Table 6, it was confirmed that it is not possible to produce microgel particles of 500 μm or less when using collagen.

[0203] (3) Evaluation of cell viability The procedure was carried out in the same manner as in Example 9, and cells with a viability of 75% or higher after 7 days of culture were marked with ○, and those with a viability of less than 75% were marked with ×, and the results are summarized in Table 6 below. As shown in Table 6, it was confirmed that cell viability could not be maintained for up to one week within the gel particles using PEG-SH.

[0204] [Table 6]

[0205] (Example 13) Cell-containing gel particles, including suspended cells, are cultured under adhesive conditions. (1) Preparation of the first solution The procedure was carried out in the same manner as in Example 7.

[0206] (2) Preparation of the second solution The procedure was carried out in the same manner as in Example 7.

[0207] (3) Preparation of cell suspension The procedure was carried out in the same manner as in Example 10, and the cell concentration of antibody-producing hybridomas (JCRB Cell Bank, hereinafter referred to as "HyB") containing 1% Tetra-PEG-maleimidyl and 0.1% Thiol Gelatin was 1 × 10⁻¹⁶. 7 A cell-containing first solution was obtained, consisting of a cell suspension with a concentration of cells / mL.

[0208] (4) Preparation of cell-containing gel particles An 18mm square coverslip (product name: No.1 Thickness 0.13~0.17mm, manufactured by Matsunami Glass Co., Ltd.) was placed in a 35mm dish, and 1 mL of the second solution was added to coat the surface of the coverslip with the second solution. After filling the liquid chamber of the inkjet head with the cell-containing first solution prepared in (3), it was dropped drop by drop onto the coverslip, causing the cell-containing first solution to deposit into the second solution, and cell-containing gel particles were produced by the reaction of the two solutions. After gel formation, the coverslip was transferred to a 35mm dish, 10% FBS-containing DMEM was gently added, and the mixture was cultured for 7 days in an incubator at 37°C and 5% CO2.

[0209] (5) Observation of cell-containing gel particles The cell-containing gel particles were observed using a phase-contrast microscope 1, 4, and 7 days after culturing. As shown in Figure 19, it was confirmed that by adhering the cell-containing gel particles to the culture medium, it was possible to track and observe the cells without losing them.

[0210] (6) Calculation of cell viability PI / Hoechst staining was performed in the same manner as in Example 9, and stained images were obtained. The number of cells stained with PI was taken as the number of dead cells, and the total number of cells stained with Hoechst was taken as the total number of cells. The survival rate (%) was calculated as (number of dead cells / total number of cells) × 100.

[0211] (7) Judgment criteria Table 7 summarizes the results, indicating that cells with a viability of 75% or higher after 4 and 7 days of culture of the cell-containing gel particles were summarized. As shown in Table 7, it was confirmed that cell viability could be maintained even when HyB, a suspension cell, was cultured in cell-containing gel particles attached to a substrate.

[0212] [Table 7]

[0213] (Example 14) Measurement of IL-8 secretion by LPS stimulation (1) Preparation of the first solution The procedure was carried out in the same manner as in Example 7.

[0214] (2) Preparation of the second solution The procedure was carried out in the same manner as in Example 7.

[0215] (3) Preparation of cell suspension The procedure was carried out in the same manner as in Example 7, and a solution containing 2% Tetra-PEG-maleimidil and 0.1% Thiol Gelatin was used, resulting in a cell concentration of 1 × 10⁻¹⁶ HUVEC Umbilical Vein Endo Cells (LONZA, hereinafter referred to as "HUVEC"). 7 A cell-containing first solution was obtained, consisting of a cell suspension with a concentration of cells / mL.

[0216] (4) Preparation of cell-containing gel particles The second solution was added to each well of a 96-well plate. After filling the liquid chamber of the inkjet head with the cell-containing first solution prepared in (3), nine drops were added to each of the 96-well plates, causing the cell-containing first solution to deposit into the second solution, and cell-containing gel particles were produced by the reaction of the two solutions. After gel formation, Endothelial Cell Growth Basal Medium-2 (LONZA, hereinafter referred to as EBM-2) was gently added, and the gels were cultured for two days in an incubator at 37°C and 5% CO2.

[0217] (5) LPS processing After culturing the cell-containing gel particles for two days, lipopolysaccharide (hereinafter referred to as LPS) was added to the culture medium at concentrations of 10 pg / mL, 100 pg / mL, and 10,000 pg / mL, and the cells were incubated for 24 hours. As a control, wells without LPS treatment were also prepared.

[0218] (6) ELISA An IL-8 ELISA was performed using the culture supernatant of cell-containing gel particles treated with the aforementioned LPS for 24 hours. The IL-8 Human Uncoated ELISA Kit (Invitrogen) was used, and the experiment was conducted according to the specified protocol. The absorbance at 450 nm was measured with a plate reader, and the IL-8 concentration of each sample was estimated from the standard. As shown in Figure 20, the amount of IL-8 in the supernatant increased in a concentration-dependent manner with LPS. [Explanation of symbols]

[0219] 1 Hydrogel 2. Quadrilateral polymer 3 Linear polymers 10 cell culture 20 Hydrogels 21 Electrophilic functional group 30 Cover glass 31 Nucleophilic functional groups 5 Cell-containing gel particles 50 Hydrogels 51 cells 60 Solution 1 61 Second Solution 62 Cell-containing gel particles 63 Culture vessel 64 culture medium 70 Solution 1 71 Second Solution 72 Cell-containing gel particles 73 Culture vessel 74 Culture medium 8 Cell evaluation plate 80 wells 81 Cell-containing gel particles C cell All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A method for preparing cell cultures, A holding step of holding a first solution containing a highly branched polymer having polyethylene glycol as its backbone and having one or more nucleophilic or electrophilic functional groups at its side chains and / or terminals, and Gel formation process: A second solution containing a linear polymer having one or more nucleophilic functional groups or the other functional group of an electrophilic functional group at its side chains and / or terminals is ejected by an inkjet method using a droplet ejection device so as to come into contact with the held first solution, thereby forming one or more hydrogels. Includes, The linear polymer is a protein, peptide, or polysaccharide. The electrophilic functional group is one or more selected from the group consisting of maleimidyl group, N-hydroxysuccinimidyl (NHS) group, sulfosuccinimidyl group, phthalimidyl group, imidazoyl group, acryloyl group, and nitrophenyl group. The nucleophilic functional group is a thiol group, an amino group, and -CO ２ PhNO ２ One or more species selected from a larger group, At least one of the first solution or the second solution contains cells, The first solution further comprises gelatin having thiol groups in its side chains and / or terminals. A method for preparing the aforementioned cell culture.

2. A method for preparing a cell culture according to claim 1, wherein the first solution and the second solution are brought into contact with each other at a resolution of 500 μm or less.

3. A first solution comprising a polybranched polymer having polyethylene glycol as its backbone and containing one or more electrophilic or nucleophilic functional groups in its side chains and / or terminals, A second solution comprising a linear polymer having two or more nucleophilic or electrophilic functional groups in its side chains and / or terminals, A process of mixing, The linear polymer is a protein, peptide, or polysaccharide. The electrophilic functional group is one or more selected from the group consisting of maleimidyl group, N-hydroxysuccinimidyl (NHS) group, sulfosuccinimidyl group, phthalimidyl group, imidazoyl group, acryloyl group, and nitrophenyl group. The nucleophilic functional group is a thiol group, an amino group, and -CO ２ PhNO ２ One or more species selected from a larger group, The first solution or the second solution contains cells, The first solution further comprises gelatin having thiol groups in its side chains and / or terminals, The process includes mixing droplets of the first solution or the second solution containing the cells into the second solution or the first solution that does not contain the cells by an inkjet method. Method for producing cell-containing gel particles.

4. The method for producing cell-containing gel particles according to claim 3, wherein the linear polymer is gelatin.

5. A method for culturing cells, comprising placing cell-containing gel particles prepared by the manufacturing method described in claim 3 or 4 into a culture vessel, and culturing cells in a manner that prevents the cell-containing gel particles from contacting the culture vessel.

6. A method for culturing cells, comprising adhering cell-containing gel particles prepared by the manufacturing method described in claim 3 or 4 to a culture vessel, and culturing cells while the cell-containing gel particles are held in the culture vessel.

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