Complex containing hydrogel, dental pulp stem cells, and differentiated cells derived from dental pulp stem cells, method for producing same, and method for producing culture containing dental pulp stem cells and differentiated cells derived from dental pulp stem cells

Abstract

A complex according to the present disclosure contains: a hydrogel that has a compressive elastic modulus of 10-500 kPa and has a radiation-crosslinked structure of a hydrophilic polymer; and dental pulp stem cells and differentiated cells derived from dental pulp stem cells which adhere to the hydrogel.

Description

Composite comprising a hydrogel, dental pulp stem cells, and differentiated cells derived from dental pulp stem cells, method for producing the same, and method for producing a culture containing dental pulp stem cells and differentiated cells derived from dental pulp stem cells

[0001] The present disclosure relates to a composite comprising a hydrogel, dental pulp stem cells, and differentiated cells derived from dental pulp stem cells, a method for producing the same, and a method for producing a culture containing dental pulp stem cells and differentiated cells derived from dental pulp stem cells.

[0002] Many techniques related to tissue regeneration and organ formation using iPS cells have been reported so far. Although iPS cells can differentiate into almost all cells of the body and are considered to have the ability to proliferate infinitely, there is a possibility that transplanted cells may become tumorous or cancerous, and careful research has been continued (Non-Patent Document 1). On the other hand, dental pulp stem cells can be collected from deciduous teeth of infants or teeth of the elderly, and are considered to have an extremely low possibility of becoming tumorous or cancerous (Non-Patent Document 2). Therefore, there is a need for a culture method for utilizing dental pulp stem cells instead of iPS cells, particularly a culture method for three-dimensionally culturing dental pulp stem cells to induce differentiation into blood vessels, nerves, and calcification, and for constructing a three-dimensional form similar to living tissue.

[0003] For the three-dimensional culture of dental pulp stem cells or the medical application of the culture, a gel culture substrate composed of a biogenic component, having a hardness similar to that of living tissue, and having shape stability that can be used for several weeks of culture is required. So far, the inventors have found a quantum beam cross-linking technique for cross-linking and gelling various biopolymers containing collagen without using a drug and controlling its hardness (Patent Document 1 and Non-Patent Document 3).

[0004] International Publication No. 2020 / 004646

[0005] U. Ben-David & N. Benvenisty, “The tumorigenicity of human embryonic and induced pluripotent stem cells”, Nature Reviews Cancer, 11, 268-277 (2011)P. Hollands, D. Aboyeji & M. Orcharton, “Dental pulp stem cells in regenerative medicine”, British Dental Journal, 224, 747-750 (2018)TG Oyama et al., “Collagen hydrogels with controllable combined cues of elasticity and topography to regulate cellular processes”, Biomed Mater. 2021 Jun 11;16(4)

[0006] On conventional plastic culture substrates, dental pulp stem cells extend two-dimensionally and do not form three-dimensional structures. Furthermore, plastic culture substrates are more than 10,000 times harder than biological soft tissue, which is problematic because it subjects cells to physical stimuli that are completely different from the environment of dental pulp stem cells or differentiated tissue in vivo. Collagen physical gels, which are gelled by the self-assembly of type I collagen, are a possible substrate for three-dimensional culture, but they are fragile and shrink or decompose over time, making them unsuitable for three-dimensional cultures that require several weeks. Cross-linking is essential to increase the strength and adjust the hardness of the gel, but conventional cross-linking methods require highly toxic chemicals such as glutaraldehyde.

[0007] There is a need to develop a technology that differentiates dental pulp stem cells into desired cell types through long-term three-dimensional culture of dental pulp stem cells.

[0008] One aspect of this disclosure is the realization of a technology that enables the differentiation of dental pulp stem cells into desired cells by performing long-term three-dimensional culture of dental pulp stem cells.

[0009] As a result of diligent research, the inventors discovered that by using a specific hydrogel, long-term three-dimensional culture of dental pulp stem cells can be performed, enabling differentiation of dental pulp stem cells into desired cell types, thus completing the present invention.

[0010] To solve the aforementioned problems, a composite according to one aspect of the present disclosure is a composite comprising a hydrogel having a compressive modulus of 10 kPa or more and 500 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer, and dental pulp stem cells and differentiated cells derived from dental pulp stem cells that adhere to the hydrogel.

[0011] A method for producing a composite according to one aspect of the present disclosure is a method for producing a composite comprising a hydrogel having a compressive modulus of 10 kPa or more and 500 kPa or less, and a hydrogel having a radiation crosslinked structure of a hydrophilic polymer, and a step of differentiating the dental pulp stem cells, wherein the hydrogel is accompanied by dental pulp stem cells adhering to the hydrogel and differentiated cells derived from dental pulp stem cells.

[0012] A method for producing a culture according to one aspect of the present disclosure is a method for producing a culture that includes the steps of: contacting dental pulp stem cells with a hydrogel having a compressive modulus of 10 kPa or more and 500 kPa or less and a radiation crosslinked structure of a hydrophilic polymer; differentiating the dental pulp stem cells; and recovering a culture containing the dental pulp stem cells and differentiated cells derived from the dental pulp stem cells from the hydrogel.

[0013] According to one aspect of this disclosure, dental pulp stem cells can be differentiated into desired cells by performing long-term three-dimensional culture of dental pulp stem cells.

[0014] This figure shows phase-contrast microscope images of dental pulp stem cells cultured in standard medium using existing dishes and gels of various hardnesses. This figure shows the change in cell number of dental pulp stem cells cultured in standard medium using existing dishes and gels of various hardnesses. This figure shows electron microscope images of dental pulp stem cells cultured for 3 days in standard medium using existing dishes and gel hardness 3. This figure shows the expression level of the stem cell marker MCAM in dental pulp stem cells cultured for 14 days in standard medium using existing dishes and gels of various hardnesses. This figure shows the expression level of the bone / dentin differentiation marker RUNX2 in dental pulp stem cells cultured for 14 days in calcification differentiation induction medium using existing dishes and gels of various hardnesses. This figure shows the expression level of the stem cell marker MCAM in dental pulp stem cells cultured for 14 days in calcification differentiation induction medium using existing dishes and gels of various hardnesses. This figure shows phase-contrast microscope images of dental pulp stem cells cultured in vascular differentiation induction medium using gel. This figure shows the expression level of the vascular differentiation marker CD31 in dental pulp stem cells cultured for 10 weeks in vascular differentiation induction medium using gel. This figure shows the fluorescence staining image of MAP2 in dental pulp stem cells cultured on a gel for 31 days using a neuronal differentiation induction medium.

[0015] One aspect of this disclosure will be described in detail below. Unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B".

[0016] [A complex comprising a hydrogel, dental pulp stem cells, and differentiated cells derived from dental pulp stem cells] A complex according to one aspect of the present disclosure comprises dental pulp stem cells, differentiated cells derived from dental pulp stem cells, and a hydrogel.

[0017] [Dental Pulp Stem Cells] Dental pulp stem cells are mesenchymal stem cells isolated from dental pulp tissue. Dental pulp stem cells may be derived from the pulp tissue of deciduous or permanent teeth. Examples of dental pulp stem cells include those derived from mammals. Mammals include humans and non-human animals. Non-human animals include livestock such as cattle, horses, pigs, and sheep, as well as companion animals or laboratory animals such as dogs, cats, rats, mice, hamsters, monkeys, and rabbits.

[0018] Dental pulp stem cells may be isolated from the pulp tissue of removed teeth, such as deciduous or permanent teeth. Alternatively, commercially available dental pulp stem cells may be used.

[0019] [Differentiated cells derived from dental pulp stem cells] Examples of differentiated cells derived from dental pulp stem cells include osteoblasts, odontoblasts, vascular endothelial cells, nerve cells, adipocytes, myoblasts, chondrocytes, and mesenchymal cells such as stem cells.

[0020] The dental pulp stem cells and differentiated cells derived from dental pulp stem cells contained in the complex according to one aspect of this disclosure are adhered to a hydrogel described later. The complex may also contain other cells besides dental pulp stem cells and differentiated cells derived from dental pulp stem cells. Examples of other cells include umbilical cord blood-derived hematopoietic stem cells, hematopoietic stem cells, progenitor cells, etc.

[0021] Whether or not dental pulp stem cells are present in the complex can be confirmed, for example, by measuring the expression level of a gene (marker gene) specific to the dental pulp stem cells. An example of a marker gene for dental pulp stem cells is the MCAM (melanoma cell adhesion molecule) gene.

[0022] Whether or not differentiated cells derived from dental pulp stem cells are present in the complex can be confirmed, for example, by measuring the expression level of marker genes in those differentiated cells. Examples of marker genes for osteoblasts or odontoblasts (hereinafter sometimes referred to as bone / odontoblasts) include the RUNX2 (runt-related transcription factor 2) gene. Examples of marker genes for vascular endothelial cells include the CD31 (PECAM-1; platelet endothelial cell adhesion molecule-1) gene. Examples of marker genes for nerve cells include the MAP2 (microtubule-associated protein-2) gene.

[0023] The proportion of dental pulp stem cells to the total number of cells in the complex according to one aspect of this disclosure may be, for example, 5% or more, 10% or more, or 15% or more. Alternatively, the proportion of dental pulp stem cells to the total number of cells in the complex may be, for example, 60% or less, 50% or less, or 40% or less. An example of the range of the proportion of dental pulp stem cells to the total number of cells in the complex is 5 to 60% (for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 60%).

[0024] The proportion of differentiated cells derived from dental pulp stem cells among the total number of cells in the complex according to one aspect of this disclosure may be, for example, 5% or more, 10% or more, or 15% or more. Alternatively, the proportion of differentiated cells derived from dental pulp stem cells among the total number of cells in the complex may be, for example, 60% or less, 50% or less, or 40% or less. An example of the range of the proportion of differentiated cells derived from dental pulp stem cells among the total number of cells in the complex is 5 to 60% (for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 60%).

[0025] [Hydrogel] The hydrogel contained in the composite according to one aspect of the present disclosure has a radiation crosslinking structure of a hydrophilic polymer.

[0026] In this specification, a hydrogel is a gel that has hardened into a gel-like state by encapsulating water, and is composed of hydrophilic polymer molecules, such as proteins, that are cross-linked to each other.

[0027] Radiation crosslinking, while not specifically limited, refers to the formation of a three-dimensional network structure through radiation irradiation without the use of crosslinking agents. Radiation irradiation creates active sites on polymer chains, and these active sites serve as the starting point for cross-linking of polymer chains into either an X-shape (cross-linking) or a T-shape (linking of the ends of polymer chains to other polymer chains), thereby forming a three-dimensional network structure. In other words, radiation crosslinked structures are crosslinked structures formed by intramolecular or intermolecular crosslinking of polymer chains through radiation irradiation without the use of additives such as crosslinking agents. A characteristic of radiation crosslinking is that it proceeds even at room temperature or below without the use of additives such as crosslinking agents, and it is applied to inducing gelation of materials, improving heat resistance, and imparting shape memory properties.

[0028] Hydrogels with a radiation-crosslinked structure of hydrophilic polymers constitute a radiation-crosslinked structure that does not contain any crosslinking agent. Therefore, they can maintain their hydrogel state even when left unattended under cell culture conditions.

[0029] (Hydrophilic Polymer) The hydrophilic polymer refers to a polymer having hydrophilic groups in its molecule. Examples of hydrophilic groups include hydroxyl groups, amino groups, carboxyl groups, ether groups, acyl groups, and sulfo groups. That is, the hydrophilic polymer is a polymer having at least one, preferably two or more, such hydrophilic groups in its molecule.

[0030] Furthermore, the molecular weight of the hydrophilic polymer is not particularly limited; for example, a polymer in the range of 150 to 2,000,000 may be appropriately selected and used. Typically, a polymer in the range of 1,000 to 1,000,000 may be appropriately selected and used. For polymers with the same composition, the higher the molecular weight, the greater the compressive modulus of the hydrogel tends to be. In this specification, unless otherwise specified, "molecular weight" refers to the weight-average molecular weight, and such weight-average molecular weight may be measured by conventionally known size exclusion chromatography.

[0031] Examples of the hydrophilic polymers include hydrophilic polymers derived from natural products or their derivatives, such as proteins, peptides, polysaccharides, and nucleic acids. "Derived from natural products" means that they can be obtained by extraction or purification from natural products (earth resources, typically living organisms, such as animals, plants, and fungi), and is not limited to being natural products themselves. For example, synthetic proteins artificially synthesized using extracts or purified products from natural products are included in the category of hydrophilic polymers derived from natural products (hereinafter also referred to as natural polymers). Synthetic proteins include both proteins synthesized in cell-based protein synthesis systems and proteins synthesized in cell-free protein synthesis systems.

[0032] The term "protein" refers to a macromolecule formed by the linkage of multiple amino acids via peptide bonds, and is not limited by the number of amino acids constituting the protein. For example, it includes peptides consisting of two or three or more amino acids. In this specification, "peptide" specifically refers to a macromolecule consisting of two to 2,000 amino acids.

[0033] Examples of hydrophilic polymers derived from natural products include, for example, polysaccharides such as dextrin, dextran, chitin, chitosan, agar, agarose, gellan gum, xanthan gum, karaya gum, carrageenan, cellulose, and starch; proteins such as collagen, gelatin, fibrin, albumin, laminin, fibronectin, keratin, ovalbumin, myosin, globulin, and peptides; and nucleic acids such as DNA or RNA.

[0034] The hydrophilic polymer derived from natural products may be used as a raw material for hydrogels using only a single subtype, or a combination of multiple different subtypes. For example, collagen is known to have subtypes such as type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen. Therefore, one or more of these subtypes of collagen can be used in combination. Type I collagen is preferred because it is the most abundant type in the body and can be obtained relatively inexpensively. Type IV collagen is also preferred because it is the collagen found in the basement membrane of the skin and can be obtained relatively easily.

[0035] Furthermore, the derivatives of the hydrophilic polymer (natural polymer) derived from the natural product are not particularly limited, but examples include derivatives obtained by substituting the natural product-derived polymer with a lower alkyl group, a lower alkoxyalkyl group, or a hydroxy lower alkyl group. Specifically, examples include natural polymer derivatives selected from the group consisting of lower alkyl group-substituted cellulose derivatives, lower alkoxyalkyl group-substituted cellulose derivatives, hydroxy lower alkyl group-substituted cellulose derivatives, lower alkoxyalkyl group-substituted chitosan derivatives, lower alkoxyalkyl group-substituted chitin derivatives, lower alkoxyalkyl group-substituted starch derivatives, and lower alkoxyalkyl group-substituted carrageenan derivatives.

[0036] The hydrophilic polymer may be an artificially synthesized synthetic polymer (synthetic resin). Conventionally known hydrophilic polymers can be used without particular limitation, but examples include synthetic polymers selected from the group consisting of polyethylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, polymethacrylamide, and polyethylene glycol. Furthermore, polymers having artificially designed sequences are also included in the category of synthetic polymers, and examples include artificial proteins (including peptides), nucleic acids, and polysaccharides having artificially designed sequences.

[0037] The hydrogel may be formed by cross-linking (bonding) only one of the hydrophilic polymers, or by cross-linking (bonding) two or more hydrophilic polymers.

[0038] From the viewpoint of achieving culture in an environment similar to the survival environment of dental pulp stem cells, it is preferable to select an animal-derived hydrophilic polymer or a derivative thereof as the hydrophilic polymer, and more preferably an animal-derived hydrophilic polymer (hereinafter also referred to as a bio-derived polymer) or a derivative thereof obtained by extraction or purification from a living organism (animal living organism). Examples of bio-derived polymers include gelatin, collagen, or collagen peptides.

[0039] Furthermore, when a composite according to one aspect of this disclosure is used as a transplantable component, the hydrophilic polymer is preferably a biocompatible and biodegradable hydrophilic polymer, and more preferably a bio-derived polymer, in that it eliminates the need to remove the hydrogel from the composite. The biodegradability refers to the ability to be degraded by enzymes such as collagenase or protease of the target to be transplanted.

[0040] Since the complex according to one aspect of this disclosure includes the hydrogel, it enables long-term three-dimensional culture of dental pulp stem cells adhering to the hydrogel and induction of differentiation into desired cells.

[0041] (Compressive modulus of hydrogel) The compressive modulus of the hydrogel is 10 kPa or more and 500 kPa or less (for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kPa). In this specification, "compressive modulus" is defined by a known method of measuring a stress-strain curve in accordance with JIS K 6272. For example, the compressive modulus may be calculated based on a stress-strain curve from the hydrogel surface to 10% of its thickness. The measurement conditions for the compressive modulus may be, for example, a compression rate of 50 μm / s and a maximum test load of 2 N. The compressive modulus may be measured immediately after removing the hydrogel from a 37°C incubator to a room temperature environment (15°C or more and 25°C or less).

[0042] When the compressive elastic modulus is within the above range, differentiation of dental pulp stem cells into desired cells is possible.

[0043] When the desired cells are osteoblast / odontoblast cells, in order to further promote differentiation into osteoblast / odontoblast cells, the compressive elastic modulus is preferably 300 kPa or more, more preferably 350 kPa or more, and even more preferably 400 kPa or more. Also, the compressive elastic modulus is preferably 500 kPa or less, more preferably 480 kPa or less, and even more preferably 450 kPa or less. As an example of the range of the compressive elastic modulus, 300 to 500 kPa (for example, 300, 325, 350, 375, 400, 425, 450, 475 or 500 kPa) can be mentioned.

[0044] When the desired cells are vascular endothelial cells or nerve cells, in order to further promote differentiation into vascular endothelial cells or nerve cells, the compressive elastic modulus is preferably 10 kPa or more, more preferably 20 kPa or more, and even more preferably 30 kPa or more. Also, the compressive elastic modulus is preferably 120 kPa or less, more preferably 100 kPa or less, and even more preferably 80 kPa or less. As an example of the range of the compressive elastic modulus, 10 to 120 kPa (for example, 10, 25, 50, 75, 100 or 120 kPa) can be mentioned.

[0045] Also, the compressive elastic modulus of the hydrogel may be non-uniform. For example, it can be set to be non-uniform in the thickness direction. For example, it may be set to be composed of a plurality of hydrogel layers with different compressive elastic moduli, or it may be a gradient gel in which the elastic force of the cell culture surface is set low and the elastic force increases in the depth direction (thickness direction). By setting the elastic modulus to be non-uniform (typically with a low elastic modulus on the cell culture surface) in the thickness direction like this, for example, cells can easily penetrate into the hydrogel and three-dimensional culture can be realized. Also, the compressive elastic modulus may be set to be non-uniform in the horizontal direction. For example, a site with a different compressive elastic modulus from the surroundings may be provided, or it may be a gradient gel in which the compressive elastic modulus gradually decreases in a specific direction.

[0046] (Water content of the hydrogel) The water content of the hydrogel is, for example, 10% by mass or more, preferably 30% by mass or more. The upper limit of the water content of the hydrogel is not particularly limited and may be appropriately set, for example, at 99% by mass or less.

[0047] In the hydrogel, the content of the hydrophilic polymer with respect to the hydrogel is, for example, 1% by mass or more, preferably 3% by mass or more. The upper limit of the content of the hydrophilic polymer with respect to the hydrogel is not particularly limited and is, for example, 50% by mass or less, preferably 40% by mass or less, and more preferably 30% by mass or less.

[0048] The hydrogel may have at least one of concavo-convex portions and parallel grooves on its surface. The grooves constituting the parallel grooves may be straight lines or dotted lines. The interval between the parallel grooves may be 1 μm or more, 2 μm or more, or 3 μm or more. The interval between the parallel grooves may be 200 μm or less, 50 μm or less, or 30 μm or less.

[0049] The depth of the groove recess of the parallel groove may be 0.5 μm or more or 1 μm or more. Also, the depth of the groove recess of the parallel groove may be 5 μm or less, 4 μm or less, or 3 μm or less.

[0050] By having the concavo-convex portions on the surface of the hydrogel, cells can be seeded and cultured in the recesses. In this specification, having concavo-convex portions on the surface of the hydrogel means having at least one recess and at least one protrusion on the surface of the hydrogel. The shape of the concavo-convex portions may be any shape, for example, dot-shaped or substantially circular. Also, the hydrogel having concavo-convex portions on its surface can be suitably used, for example, for the purpose of isolating, holding, and culturing about 1 to 10 cells per recess.

[0051] The hydrogel contained in the composite according to one aspect of this disclosure can be obtained, for example, by irradiating solutions of each hydrophilic polymer with appropriately adjusted concentrations. For example, the hydrogel contained in the composite according to one aspect of this disclosure can be produced in accordance with the hydrogel production method described in Patent Document 1. It is preferable to lower the oxygen concentration in the solutions of each hydrophilic polymer or their physical gels before irradiation in order to further increase the radiation crosslinking efficiency. As a method for lowering the oxygen concentration in the solutions of each hydrophilic polymer or their physical gels, for example, a method of preparing a physical gel by sealing and standing the solutions of each hydrophilic polymer in the presence of an oxygen scavenger can be used.

[0052] Furthermore, a hydrogel having at least one of the uneven surface and parallel grooves on its surface can also be manufactured in accordance with the hydrogel manufacturing method described in Patent Document 1.

[0053] [Uses of the composite] The composite according to one aspect of this disclosure can be used as a medical component such as an implantable component, a component for pharmacological testing (for example, a component for drug screening), and an experimental component, etc.

[0054] A complex according to one aspect of this disclosure includes dental pulp stem cells and differentiated cells derived from dental pulp stem cells, with their stem cell properties maintained. The cells included in the complex exhibit diversity. Furthermore, the hydrogel included in the complex is composed of biologically derived components. Therefore, the complex is similar to biological tissue and can be used for the regeneration of target tissue.

[0055] [Method for producing the complex] A method for producing the complex according to one aspect of the present disclosure includes the steps of contacting dental pulp stem cells with the hydrogel and differentiating the dental pulp stem cells. The dental pulp stem cells and hydrogel have already been described and will not be repeated here.

[0056] (Process of bringing dental pulp stem cells into contact with hydrogel) Dental pulp stem cells can be brought into contact with hydrogel by seeding them onto the hydrogel.

[0057] A culture medium may be added to the hydrogel before or after contact with dental pulp stem cells. Any known culture medium used for culturing dental pulp stem cells can be used. Examples of such culture media include minimum essential alpha medium.

[0058] To promote the proliferation of dental pulp stem cells, the culture medium may contain physiologically active factors (e.g., cell growth factors, differentiation-inducing factors, adhesion factors, chemotactic factors, extracellular matrix, etc.).

[0059] The number of dental pulp stem cells to be in contact with the hydrogel can be appropriately selected depending on the intended use of the complex. For example, 5 × 10⁶ cells. ４ pieces / cm ２ The dental pulp stem cells may be brought into contact with the hydrogel as described below.

[0060] After contacting the dental pulp stem cells with the hydrogel, it is preferable to culture and proliferate the dental pulp stem cells so that the area percentage (cell occupancy area percentage, confluence) of the dental pulp stem cells occupying the hydrogel is 70% or more, before the step of differentiating the dental pulp stem cells into the desired cells.

[0061] (Process for differentiating dental pulp stem cells) After contacting dental pulp stem cells with hydrogel, the culture medium is replaced with a differentiation medium, thereby differentiating the dental pulp stem cells into the desired cells.

[0062] The differentiation medium may be a known medium used for differentiation into the desired cell type. Examples of differentiation media for bone / odontoblast cells include a medium supplemented with β-glycerophosphates such as β-glycerophosphate sodium n-hydrate, dexamethasone, and ascorbic acid such as L-ascorbic acid magnesium phosphate n-hydrate. Examples of differentiation media for nerve cells include a medium supplemented with FGF (fibroblast growth factor), such as FGF-2, and EGF (epidermal growth factor). Examples of differentiation media for vascular endothelial cells include a medium supplemented with VEGF (vascular endothelial growth factor). Examples of commercially available differentiation media for vascular cells include EBM®-2 medium, which consists of EGM®-2MV singleQuots® from Lonza.

[0063] By replacing the culture medium with one for differentiation into bone / odontoblasts and culturing dental pulp stem cells for more than seven days, they can be differentiated into bone / odontoblasts. Furthermore, by replacing the culture medium with one for differentiation into bone / odontoblasts and culturing dental pulp stem cells for more than seven days, they can be differentiated into bone-like structures that extend in three dimensions.

[0064] By replacing the culture medium with one for differentiation into vascular endothelial cells and culturing dental pulp stem cells for 14 days or more, they can be differentiated into vascular endothelial cells. Furthermore, by replacing the culture medium with one for differentiation into vascular endothelial cells and culturing dental pulp stem cells for 14 days or more, they can be differentiated into three-dimensionally extending vascular-like structures.

[0065] By replacing the culture medium with a medium for differentiation into nerve cells and culturing dental pulp stem cells for 14 days or more, they can be differentiated into nerve cells. Furthermore, by replacing the culture medium with a medium for differentiation into nerve cells and culturing dental pulp stem cells for 14 days or more, they can be differentiated into nerve-like structures that extend in three dimensions.

[0066] [Method for producing a culture containing dental pulp stem cells and differentiated cells derived from dental pulp stem cells] A method for producing a culture containing dental pulp stem cells and differentiated cells derived from dental pulp stem cells according to one aspect of the present disclosure includes the steps of contacting dental pulp stem cells with the hydrogel, differentiating the dental pulp stem cells, and recovering the culture from the hydrogel. The steps of contacting dental pulp stem cells with the hydrogel and differentiating the dental pulp stem cells have already been described and will not be repeated here.

[0067] (Step for recovering cultures containing dental pulp stem cells and differentiated cells derived from dental pulp stem cells from hydrogel) The cultures containing dental pulp stem cells and differentiated cells derived from dental pulp stem cells can be detached from the hydrogel and recovered from the hydrogel by enzymatic treatment with a proteolytic enzyme (e.g., trypsin) or mechanical treatment such as pipetting.

[0068] The culture containing the dental pulp stem cells and differentiated cells derived from dental pulp stem cells can be used, for example, in therapeutic applications (e.g., transplantation, regenerative medicine), pharmacological testing methods (e.g., drug screening methods), and the like.

[0069] A kit for producing the aforementioned complex or a culture containing the dental pulp stem cells and differentiated cells derived from dental pulp stem cells is also included in one aspect of the present disclosure. The kit is used in a method for producing the aforementioned complex or a method for producing a culture containing the dental pulp stem cells and differentiated cells derived from dental pulp stem cells, and includes the hydrogel.

[0070] The kit may further include at least one of the following: a bioactive factor different from the hydrogel, a culture medium (e.g., a growth medium, a differentiation medium), and dental pulp stem cells. The kit may also include instructions describing the manufacturing procedure for the complex. These instructions may be written or printed on paper or other media, or attached to electronic media such as magnetic tape, a computer-readable disk, or a CD-ROM.

[0071] [Summary] The composite according to Embodiment 1 of the present disclosure comprises a hydrogel having a compressive modulus of 10 kPa or more and 500 kPa or less, and a radiation crosslinked structure of a hydrophilic polymer, and dental pulp stem cells and differentiated cells derived from dental pulp stem cells that adhere to the hydrogel.

[0072] The composite according to aspect 2 of the present disclosure is, in aspect 1, wherein the compressive modulus is 300 kPa or more and 500 kPa or less, and the differentiated cells derived from dental pulp stem cells may be osteoblasts or odontoblasts.

[0073] The composite according to aspect 3 of the present disclosure is, in aspect 1, wherein the compressive modulus is 10 kPa or more and 120 kPa or less, and the differentiated cells derived from dental pulp stem cells may be vascular endothelial cells or nerve cells.

[0074] A method for producing a composite according to aspect 4 of the present disclosure is a method for producing a composite comprising a hydrogel having a compressive modulus of 10 kPa or more and 500 kPa or less, and a hydrogel having a radiation crosslinked structure of a hydrophilic polymer, and a step of differentiating the dental pulp stem cells, wherein the hydrogel is accompanied by dental pulp stem cells adhering to the hydrogel and differentiated cells derived from dental pulp stem cells.

[0075] The method for producing the composite according to aspect 5 of the present disclosure is, in aspect 4, wherein the compressive modulus is 300 kPa or more and 500 kPa or less, and the differentiated cells derived from dental pulp stem cells may be osteoblasts or odontoblasts.

[0076] The method for producing the composite according to aspect 6 of the present disclosure is, in aspect 4, wherein the compressive modulus is 10 kPa or more and 120 kPa or less, and the differentiated cells derived from dental pulp stem cells may be vascular endothelial cells or nerve cells.

[0077] A method for producing a culture according to aspect 7 of the present disclosure is a method for producing a culture that includes the steps of: contacting dental pulp stem cells with a hydrogel having a compressive modulus of 10 kPa or more and 500 kPa or less and a radiation crosslinked structure of a hydrophilic polymer; differentiating the dental pulp stem cells; and recovering a culture containing the dental pulp stem cells and differentiated cells derived from the dental pulp stem cells from the hydrogel.

[0078] The method for producing a culture according to aspect 8 of the present disclosure is, in aspect 7, wherein the compressive modulus is 300 kPa or more and 500 kPa or less, and the differentiated cells derived from dental pulp stem cells may be osteoblasts or odontoblasts.

[0079] The method for producing a culture according to aspect 9 of this disclosure is, in aspect 7, wherein the compressive modulus is 10 kPa or more and 120 kPa or less, and the differentiated cells derived from dental pulp stem cells may be vascular endothelial cells or nerve cells.

[0080] In any one of embodiments 1 to 3, the composite according to embodiment 10 of this disclosure may be one or more hydrophilic polymers selected from the group consisting of gelatin, collagen, and collagen peptides.

[0081] [Example 1] Preparation of gel culture substrate Gelatin, a highly purified type I collagen which is the main component of the environment surrounding dental pulp stem cells (extracellular matrix) in vivo, was selected as the component of the gel. First, type A gelatin derived from pigskin (Nitta Gelatin) was dissolved in ultrapure water in a 50°C water bath to obtain a 10 wt% or 15 wt% aqueous solution. The gelatin aqueous solution was poured into a polystyrene dish or microplate, sealed in a gas barrier bag with an oxygen absorber, and left to stand overnight at 20°C to form a physical gel. Then, it was irradiated with cobalt-60 gamma rays at a dose rate of 5 kGy / h at a 15-20°C environment for 15-40 kGy. Irradiation introduces crosslinking into the gelatin and forms a three-dimensional network structure, so that it does not dissolve even at a 37°C environment and forms a hydrogel containing water.

[0082] The hardness (compressive modulus) of the gel was adjusted by controlling the concentration of the gelatin aqueous solution and the amount of gamma radiation, as shown in Table 1, to mimic the hardness of the dental pulp tissue environment. The compressive modulus of the gel was determined by pouring PBS into the resulting gel and incubating it overnight at 37°C. Then, the gel was compressed using a rheometer with a load of 2N and a cylindrical rod with a diameter of 3 mm, and the slope of the straight section was calculated from the resulting stress-strain curve.

[0083]

[0084] From the above results, it can be seen that the gel in this disclosure is formed using only gelatin and water without the use of crosslinking agents or other drugs, and that its compressive modulus can be adjusted to a desired value. The obtained gel is sterilized by gamma irradiation during preparation, and after pouring in the culture medium and incubating at 37°C to fill the gel with the culture medium components, it can be used for culturing dental pulp stem cells.

[0085] In the following examples, the culture and differentiation induction of dental pulp stem cells were performed under incubation conditions of 37°C and 5% CO2.

[0086] [Example 2] Culture of dental pulp stem cells using gel. Human dental pulp stem cells were obtained from extracted teeth at Nippon Dental University Hospital with approval from the Ethics Review Committee of the Faculty of Life Dentistry, Nippon Dental University. As a standard culture medium, minimum essential alpha medium (Gibco) was used, supplemented with 20% fetal bovine serum (FBS), 100 μM magnesium L-ascorbic acid phosphate n-hydrate (Wako), 2 mM L-glutamine (Gibco), 100 units / ml penicillin, and 100 μg / ml streptomycin (Gibco). Dental pulp stem cells were seeded on existing polystyrene dishes and on gels of hardness 1 to 3 formed inside the dishes. Figure 1 shows phase-contrast microscope images on day 1, day 3, and day 7. Regardless of the gel hardness used, the dental pulp stem cells showed adhesion equivalent to that when polystyrene dishes were used.

[0087] Furthermore, dental pulp stem cells were seeded in existing dishes and in gels of hardness 1 to 3 formed within the dishes, and cultured using the standard culture medium described above. Figure 2 shows the results of counting the number of cells on days 1, 2, 3, and 4. Regardless of the gel hardness used, the dental pulp stem cells showed a proliferation rate equivalent to that when polystyrene dishes were used.

[0088] Based on these results, it was found that the gel of this disclosure exhibits cell adhesion and proliferation rates comparable to existing dishes, regardless of its hardness, and can be used without problems for culturing dental pulp stem cells.

[0089] On the other hand, differences were observed in cell morphology between the existing dish and the gel disclosed herein. Figure 3 shows electron microscope images of cells fixed and dried after 3 days of culture (scale bar: 20 μm). While cells cultured on the dish spread out flat, cells cultured using the gel with stiffness 1 showed three-dimensional proliferation, with cells overlapping each other. Cells cultured using gels with stiffness 2 and 3 also proliferated three-dimensionally, similar to the gel with stiffness 1.

[0090] [Example 3] Maintaining Stem Cell Properties Dental pulp stem cells were seeded on existing dishes and gels of various hardness levels and cultured for 14 days using the standard culture medium described in Example 2. To perform gene expression analysis by digital PCR (dPCR), total RNA was extracted from the cells using the RNeasy® Mini Kit (QIAGEN), and cDNA was synthesized using the ReverTra Ace® qPCR RT Master Mix (Toyobo). dPCR analysis was performed using the Clarity® Plus Consumables Kit (JN Medsys), Luna® Universal Probe qPCR Master Mix (New England Biolabs), and PrimeTime® qPCR Assays (IDT) on a C1000 Touch Thermal Cycler (Bio-Rad). Figure 4 shows the expression level of the stem cell marker MCAM in dental pulp stem cells. The gel with hardness level 3 expressed MCAM to the same extent as the gel on the dish, indicating that it maintained stem cell properties to a similar degree. It can be seen that gels with hardness levels 1 and 2 maintain stem cell properties while changing to a state that is more conducive to differentiation compared to the dish and the gel with hardness level 3.

[0091] [Example 4] Calcification Dental pulp stem cells were seeded in existing dishes and gels of various hardnesses and cultured in standard medium until 80-100% confluence was achieved. Next, calcification was induced using differentiation induction medium, and dPCR analysis was performed after 14 days of culture. The differentiation medium used was MEMα supplemented with 10% FBS, 100 μM L-ascorbic acid magnesium phosphate n-hydrate, 2 mM L-glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin, 10 mM β-glycerophosphate sodium n-hydrate (Wako), and 10 nM dexamethasone (Wako).

[0092] Figure 5A shows the data for the calcification (bone / dentin differentiation) marker RUNX2 analyzed by dPCR, and Figure 5B shows the data for the stem cell marker MCAM. In both cases, differentiation into bone was promoted compared to the dish, and it was found that differentiation into bone was most easily achieved when using the gel with hardness 3. Furthermore, the stem cell marker MCAM was expressed even after calcification induction, and it was found that the gel with hardness 3 contained more cells that maintained stem cell properties compared to the dish.

[0093] [Example 5] Differentiation into blood vessels After seeding dental pulp stem cells in existing dishes and gels of various hardnesses, they were cultured in normal medium and differentiation induction medium for 10 weeks and then analyzed in various ways. EBM®-2 medium (Lonza), composed of EBM®-2MV singleQuots®, was used as the differentiation induction medium. Figure 6 shows phase-contrast microscope images of dental pulp stem cells differentiated using gel. It can be seen that blood vessel-like structures were formed in all cases where gel was used. On the other hand, no blood vessel-like structures were formed on the existing dishes where the cells extended two-dimensionally.

[0094] Next, the vascular differentiation marker CD31 was analyzed by dPCR. As shown in Figure 7, it can be seen that vascular differentiation was most promoted when using a gel with a stiffness of 1.

[0095] [Example 6] Differentiation into nerves Dental pulp stem cells were seeded in existing dishes and gels of various hardnesses, cultured in normal medium for 1 day, and then differentiation into nerves was induced using differentiation induction medium. After 31 days of culture (32 days total), various analyses were performed. Neurobasal medium A (Gibco) was used as the differentiation induction medium, supplemented with 40 ng / ml recombinant human fibroblast growth factor basic FGF-2 human (Prospect), 20 ng / ml recombinant human EGF (Peprotech), B27 supplement (Gibco), 100 units / ml penicillin, and 100 μg / ml streptomycin.

[0096] Figure 8 shows a fluorescence staining image of dental pulp stem cells that have undergone neurogenic differentiation, stained for the neural differentiation marker MAP2. After fixing the cells with 4% paraformaldehyde (Wako), they were stained with a MAP2 antibody (ab32454, 1:1000; Abcam). Dental pulp stem cells differentiated using gel showed a fibrous distribution of MAP2, indicating that differentiation into nerves was promoted. In particular, hardness level 1 showed the highest density of MAP2 distribution, suggesting that this level is suitable for differentiation into nerves.

[0097] As demonstrated in the above examples, the prepared gel enabled long-term three-dimensional culture of dental pulp stem cells and induction of differentiation into nerve, blood vessel, and bone / dentin cells. Furthermore, the hardness of the gel was optimal for differentiation into bone / dentin, while a soft gel was optimal for differentiation into blood vessels and nerve cells, suggesting the possibility that differentiation into specific cells can be promoted depending on the gel's hardness. The gel and cells form a three-dimensional structure (cell-gel complex). This complex is similar to living tissue in that the cells forming it include both dental pulp stem cells that maintain stem cell properties and differentiated cells, and is expected to lead to the elucidation of the tissue regeneration mechanisms of dental pulp stem cells in vivo and in vitro, and their application to the regeneration of target tissues (regenerative medicine).

[0098] The composites of this disclosure can be used, for example, as medical components such as implantable components, pharmacological testing components such as drug screening components, and experimental components.

Claims

1. A complex comprising a hydrogel having a compressive modulus of 10 kPa or more and 500 kPa or less, and a radiation-crosslinked structure of a hydrophilic polymer, and dental pulp stem cells and differentiated cells derived from dental pulp stem cells that adhere to the hydrogel.

2. The complex according to claim 1, wherein the compressive modulus is 300 kPa or more and 500 kPa or less, and the differentiated cells derived from dental pulp stem cells are osteoblasts or odontoblasts.

3. The composite according to claim 1, wherein the compressive modulus is 10 kPa or more and 120 kPa or less, and the differentiated cells derived from dental pulp stem cells are vascular endothelial cells or nerve cells.

4. A method for producing a complex comprising a hydrogel having a compressive modulus of 10 kPa or more and 500 kPa or less, and having a radiation crosslinked structure of a hydrophilic polymer, and differentiating the dental pulp stem cells, the method comprising the steps of contacting the hydrogel with dental pulp stem cells and differentiated cells derived from dental pulp stem cells that adhere to the hydrogel.

5. The method for producing the composite according to claim 4, wherein the compressive modulus is 300 kPa or more and 500 kPa or less, and the differentiated cells derived from dental pulp stem cells are osteoblasts or odontoblasts.

6. The method for producing the composite according to claim 4, wherein the compressive modulus is 10 kPa or more and 120 kPa or less, and the differentiated cells derived from dental pulp stem cells are vascular endothelial cells or nerve cells.

7. A method for producing a culture, comprising the steps of: contacting dental pulp stem cells with a hydrogel having a compressive modulus of 10 kPa or more and 500 kPa or less, and a radiation crosslinking structure of a hydrophilic polymer; differentiating the dental pulp stem cells; and recovering a culture containing the dental pulp stem cells and differentiated cells derived from the dental pulp stem cells from the hydrogel.

8. The method for producing a culture according to claim 7, wherein the compressive modulus is 300 kPa or more and 500 kPa or less, and the differentiated cells derived from dental pulp stem cells are osteoblasts or odontoblasts.

9. The method for producing a culture according to claim 7, wherein the compressive modulus is 10 kPa or more and 120 kPa or less, and the differentiated cells derived from dental pulp stem cells are vascular endothelial cells or nerve cells.

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