Scaffold, method for producing scaffold, cell culture, and cell culture method

Abstract

A scaffold (10) for culturing cells has a hydrogel (12) and a plasma-derived or platelet-derived component or a fibrin-containing substance (14) attached to the hydrogel (12).

Description

[Technical Field]

[0001] The present invention relates to a scaffold for culturing cells, a method for producing a scaffold, a cell culture comprising the scaffold, and a cell culture method using the scaffold. [Background technology]

[0002] Currently, attempts are being made to culture a variety of cells in various fields, including developmental biology, drug discovery, and regenerative medicine. These cells are typically cultured two-dimensionally on the surface of plastic tissue culture vessels (two-dimensional culture). In recent years, attention has been focused on techniques for culturing cells in a three-dimensional culture environment using scaffolds such as porous membranes and hydrogels (three-dimensional culture). The following Patent Documents 1 to 6 disclose the cultivation of cells inside tubular hydrogels.

[0003] It is known that whether cells can be cultured efficiently depends greatly on the type of cell and the culture conditions. Cultured cells that grow in a suspended state in the medium are called "suspension cells." On the other hand, cells that grow while attached to a scaffold are called "adherent cells." [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2011 / 046105 [Patent Document 2] Patent Publication No. 2017-99303 [Patent Document 3] International Publication No. 2017 / 091662 [Patent Document 4] International Publication No. 2018 / 098295 [Patent Document 5] International Publication No. 2019 / 178549 [Patent Document 6] International Publication No. 2020 / 032221 Summary of the Invention

[0005] It is known that whether cells can be cultured efficiently depends greatly on the type of cell and the culture conditions. In particular, the culture efficiency of adherent cells can vary greatly depending on the scaffold used for culture. The inventors of the present application have found that when culturing adherent cells using a hydrogel as a scaffold, it is difficult to culture the adherent cells under certain conditions, and there is still room for improvement.

[0006] Therefore, there is a demand for a scaffold suitable for culturing adherent cells, a cell culture containing the scaffold, and a method for culturing adherent cells using the scaffold.

[0007] A scaffold for culturing cells according to one embodiment comprises a hydrogel and a plasma-derived or platelet-derived component or a fibrin-containing substance attached to the hydrogel.

[0008] In a preferred embodiment, the hydrogel is in the form of a string, a tube, a sphere, or a spherical shell.

[0009] In a preferred embodiment, the plasma-derived or platelet-derived component or the fibrin-containing substance is provided inside or outside the hydrogel.

[0010] In a preferred embodiment, the plasma-derived or platelet-derived component or the fibrin-containing material comprises a component derived from human platelet lysate.

[0011] In a preferred embodiment, the hydrogel comprises an alginate gel.

[0012] In a preferred embodiment, the hydrogel comprises an alginate gel and gelatin mixed with the alginate gel.

[0013] A cell culture according to one embodiment comprises the scaffold described above and cells adhered to the scaffold.

[0014] In a preferred embodiment, the cells are adherent cells.

[0015] In a preferred embodiment, the cells are mesenchymal stem cells.

[0016] A cell culture method according to one embodiment includes forming a scaffold having a hydrogel and a plasma-derived or platelet-derived component or a fibrin-containing substance attached to the hydrogel, adhering cells to the scaffold, and culturing the cells.

[0017] In one embodiment, a method for producing a scaffold for culturing cells includes contacting a hydrogel with a suspension containing plasma-derived or platelet-derived components, or fibrinogen, fibrin, or a mixture thereof.

[0018] In a preferred embodiment, the method for producing the scaffold includes gelling a hydrogel precursor to form the hydrogel, and immersing the hydrogel in the suspension.

[0019] In a preferred embodiment, the method for producing the scaffold includes flowing the suspension while flowing a hydrogel precursor around the suspension, and gelling the hydrogel precursor to form a tubular hydrogel that surrounds the suspension.

[0020] In a preferred embodiment, the suspension comprises human platelet lysate.

[0021] In a preferred embodiment, the hydrogel precursor comprises an alginate solution.

[0022] According to the above aspects, it is possible to provide a scaffold suitable for culturing adherent cells, a method for manufacturing the scaffold, a cell culture containing the scaffold, and a method for culturing adherent cells using the scaffold. [Brief explanation of the drawings]

[0023] [Figure 1]1 is a photograph showing the structure of a scaffold for cell culture according to the first embodiment. [Figure 2] FIG. 1 is a schematic diagram showing a cross section of a scaffold for cell culture according to a first embodiment. [Figure 3] 1 is a photograph showing an example of the structure of a cell culture having a scaffold for cell culture according to the first embodiment and cells adhered to the scaffold. [Figure 4] FIG. 10 is a schematic diagram showing the structure of a scaffold for cell culture according to a second embodiment. [Figure 5] FIG. 10 is a schematic diagram showing a cross section of a scaffold for cell culture according to a second embodiment. [Figure 6] 10 is a photograph showing an example of the structure of a cell culture having a scaffold for cell culture according to the second embodiment and cells adhered to the scaffold. [Figure 7] FIG. 10 is a schematic diagram showing an example of an apparatus for producing a scaffold for cell culture according to a second embodiment. [Figure 8] 1 is a graph showing the proliferation rates of cells cultured in Examples 13 to 16 and Reference Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0024] As a result of extensive research, the present inventors have discovered a scaffold suitable for culturing adherent cells and a cell culture method using the scaffold.

[0025] The scaffold for culturing cells comprises a hydrogel and a component attached to the hydrogel. The component attached to the hydrogel is preferably a component having cell adhesive properties. The component attached to the hydrogel may be a plasma-derived or platelet-derived component, or a fibrin-containing material.

[0026] The hydrogel may be in the form of a string, a tube, a sphere, or a spherical shell. From the viewpoints of ensuring the surface area of ​​the scaffold and ease of handling of the scaffold, the hydrogel is more preferably in the form of a string or a tube.

[0027] FIG. 1 is a photograph showing the structure of a cell culture scaffold according to the first embodiment. FIG. 2 is a schematic diagram showing a cross section of the cell culture scaffold according to the first embodiment. The string-like structure in the container in the photograph shown in FIG. 1 is a scaffold 10. In the embodiment shown in FIGS. 1 and 2, the scaffold has a continuously extending string-like hydrogel 12. A plasma-derived or platelet-derived component or a fibrin-containing substance 14 is provided on the outer surface of this hydrogel 12 (see FIG. 2). In this case, adhesive cells are cultured while adhering to the component 14 on the outer surface of the hydrogel 12 (see FIG. 3). FIG. 3 shows an enlarged portion of a cell culture in which mesenchymal stem cells (MSCs) have been adhered and cultured on the cell culture scaffold. It is possible to culture a larger number of cells than that shown in FIG. 3 by continuing the cell culture.

[0028] FIG. 4 is a schematic diagram showing the structure of a cell culture scaffold according to a second embodiment. FIG. 5 is a schematic diagram showing a cross section of a cell culture scaffold according to a second embodiment. In the embodiment shown in FIGS. 4 and 5, a scaffold 20 has a continuously extending tubular hydrogel 22. A plasma-derived or platelet-derived component or a fibrin-containing substance 24 is provided on the inner surface of the hydrogel 22. In this case, adhesive cells 28 are cultured while adhering to the component 24 on the inner surface of the hydrogel 22. That is, the adhesive cells 28 are cultured inside the tubular scaffold (see FIG. 6). FIG. 6 shows an enlarged portion of a cell culture in which mesenchymal stem cells (MSCs) are adhered and cultured inside the cell culture scaffold. It is also possible to continue culturing the cells until the interior of the tubular scaffold becomes dense by continuing the cell culture.

[0029] The type of adhesive cells is not particularly limited. Adherent cells may be various types of pluripotent stem cells, human ES cells or human iPS cells, or unipotent stem cells. Examples of various types of pluripotent stem cells include iPS cells, ES cells, mesenchymal stem cells, and neural stem cells. Examples of unipotent stem cells include hepatic stem cells, germline stem cells, respiratory progenitor cells, and digestive progenitor cells. Adherent cells may also be various differentiated cells, such as muscle cells such as skeletal muscle cells and cardiac muscle cells, nerve cells such as cerebral cortical cells, fibroblasts, epithelial cells, endothelial cells, adipocytes, osteoblasts, macrophages, dendritic cells, hepatocytes, pancreatic beta cells, keratinocytes, kidney cells, and renal tubule cells.

[0030] The hydrogel can be obtained by gelling a liquid hydrogel precursor. The hydrogel is sufficient as long as it has sufficient strength to function as a scaffold for adherent cells, and preferably has sufficient permeability to cell culture medium components. In a specific example, the hydrogel may be a gel whose main component is alginate gel. In this case, the hydrogel precursor may be a solution whose main component is alginate solution.

[0031] The hydrogel may contain other materials mixed into the alginate gel. For example, gelatin or collagen may be mixed into the alginate gel. The gelatin or collagen may be cross-linked with a cross-linking agent. The cross-linking agent may be, but is not limited to, genipin.

[0032] Alginate gel can be formed by crosslinking an alginate solution with divalent metal ions. The alginate solution may be, for example, sodium alginate, potassium alginate, ammonium alginate, or a combination thereof. At room temperature or near room temperature, the alginate solution is easily crosslinked by divalent metal ions in a short time to form an alginate gel. Furthermore, alginate gel is not cytotoxic. Therefore, it is preferable that the hydrogel constituting the scaffold for cell culture contains alginate gel as its main component.

[0033] Alginic acid may be a natural extract or chemically modified. Examples of chemically modified alginic acid include methacrylate-modified alginic acid. The hydrogel may also be a mixture of the aforementioned alginate with agar, agarose, polyethylene glycol (PEG), polylactic acid (PLA), nanocellulose, or the like. The weight of the alginate relative to the weight of the solvent in the alginate solution is, for example, 0.1 to 10.0 wt %, preferably 0.25 to 7.0 wt %, and more preferably 0.5 to 5.0 wt %.

[0034] Examples of divalent metal ions used to obtain an alginate gel include calcium ions, magnesium ions, barium ions, strontium ions, zinc ions, iron ions, etc. Preferably, the divalent metal ions are calcium ions or barium ions.

[0035] The divalent metal ions are preferably provided to the alginic acid in the form of a solution. Examples of solutions containing divalent metal ions include solutions containing calcium ions. Examples of such solutions include aqueous solutions of calcium chloride, calcium carbonate, and calcium gluconate. Such solutions may preferably be aqueous solutions of calcium chloride or barium chloride.

[0036] The concentration of the divalent metal ions in the solution containing the divalent metal ions is, for example, 1 mM to 1 M, preferably 20 to 500 mM, and more preferably 100 mM.

[0037] The raw material for the alginate gel may preferably be sodium alginate. In this case, the M / G ratio of sodium alginate is preferably 0.4 to 1.8, more preferably 0.1 to 0.4, from the viewpoints of the strength as a scaffold and the permeability of medium components. The M / G ratio is determined by the composition ratio of D-mannuronic acid and L-guluronic acid in the alginic acid group.

[0038] The plasma- or platelet-derived components may include components derived from human platelet lysate (hPL). Such components may be, for example, insoluble components in human platelet lysate or fibrin-containing components. Here, the fibrin-containing components may include insoluble components derived from plasma, platelets, or human platelet lysate.

[0039] The "fibrin-containing material" may be fibrin itself, a material containing fibrin as a major component, or a material containing components other than fibrin. Examples of components other than fibrin include proteins such as albumin and fibronectin. For example, the fibrin-containing material may contain both fibrin and albumin. Furthermore, a liquid medium containing human platelet lysate (hPL) may contain, for example, plasma or hPL-derived components, fibrin, fibrinogen, or a mixture thereof. Fibrinogen is converted to fibrin by the action of thrombin. Fibrin is an insoluble component and can be attached to the inner or outer surface of a hydrogel. It is also expected that proteins such as albumin and fibronectin can facilitate the attachment of fibrin to the inner or outer surface of a hydrogel.

[0040] The plasma- or platelet-derived component or fibrin-containing substance may be attached to the hydrogel by any method, for example, by contacting the hydrogel with a suspension containing the plasma- or platelet-derived component or fibrinogen.

[0041] Plasma- or platelet-derived components, fibrin, or fibrinogen, are contained in a liquid medium containing, for example, platelet lysate. Therefore, a scaffold can be formed by contacting a hydrogel with a suspension containing a liquid medium containing platelet lysate, such as human platelet lysate (hPL). Here, this suspension may be a cell suspension containing cells to be cultured and attached to the scaffold. Alternatively, fibrinogen-containing solution can be brought into contact with the hydrogel, and fibrin can be attached to the hydrogel by the action of thrombin. Note that the use of thrombin is not essential as long as fibrin can be precipitated.

[0042] Preferably, the interior or exterior surface of the hydrogel is coated with a plasma-derived or platelet-derived component, or a fibrin-containing material.

[0043] When the scaffold is string-like or tubular, the length of the hydrogel may be, for example, preferably 5 cm or more, more preferably 10 cm or more, and even more preferably 20 cm or more. The longer the length of the hydrogel, the more cells can be cultured.

[0044] When the scaffold has a continuously extending string shape as shown in Figures 1 and 2, the outer diameter of the scaffold (reference symbol R1 in Figure 2) is not particularly limited. From the viewpoint of adhesiveness of adherent cells, the outer diameter of the scaffold may be, for example, in the range of 10 µm to 5000 µm, preferably in the range of 40 µm to 2000 µm, and more preferably in the range of 80 µm to 1000 µm. Here, the outer diameter may be defined as the average value of outer diameters measured at multiple positions, for example, 10 positions.

[0045] When the scaffold has a continuously extending tubular shape as shown in Figures 4 and 5, the outer diameter of the scaffold (reference symbol R2 in Figure 5) is not particularly limited. The outer diameter of the scaffold may be, for example, in the range of 10 µm to 4000 µm, preferably in the range of 40 µm to 1000 µm, and more preferably in the range of 80 µm to 500 µm. Here, the outer diameter may be defined as the average value of outer diameters measured at multiple positions, for example, 10 positions.

[0046] The thickness of the hydrogel constituting the scaffold (the difference between R2 and R3 in Figure 5) is preferably substantially uniform. Here, "substantially uniform" means that the difference (thickness) between the outer diameter and inner diameter measured at multiple locations, for example, 10 locations, is within a range of ±10% from the average value. The inner diameter, outer diameter, and thickness can be measured, for example, using a phase-contrast optical microscope.

[0047] When the hydrogel constituting the scaffold is tubular, both ends of the hydrogel in the extending direction are preferably closed by the hydrogel, which can prevent cells inside the hydrogel from leaking out of the hydrogel.

[0048] From the viewpoint of protecting cells, it is preferable that the tubular hydrogel has a mechanical strength higher than that of the substrate provided inside the hydrogel. The mechanical strength of the hydrogel can be measured by a method well known to those skilled in the art, such as tensile strength or load strength, using a tensile tester in water.

[0049] The interior of the tubular hydrogel may contain a cell suspension. The cell suspension may include the cells to be cultured, a plasma- or platelet-derived component or a component that will serve as a source of a fibrin-containing substance to be attached to the hydrogel, and a substrate. The cell suspension may be the one used to attach the plasma- or platelet-derived component or the fibrin-containing substance. The substrate may contain, for example, an extracellular matrix, chitosan gel, collagen solution, Matrigel, collagen gel, gelatin, alginate solution, alginate gel, peptide gel, laminin, agarose, nanocellulose, methylcellulose, hyaluronic acid, proteoglycan, elastin, pullulan, dextran, pectin, gellan gum, xanthan gum, guar gum, carrageenan, glucomannan, or a mixture thereof. Furthermore, the cell suspension may contain a culture medium, a culture supernatant, a buffer solution, human platelet lysate, platelet-rich plasma (PRP), serum, or a mixture thereof. Preferably, the cell suspension contains human platelet lysate.

[0050] The substrate may contain various growth factors suitable for cell culture, cell maintenance and proliferation, or cell function expression, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), etc.

[0051] (Scaffolding manufacturing method 1) When a scaffold for culturing cells has a continuously extending string shape as shown in Figures 1 and 2, the scaffold can be manufactured, for example, as follows. First, a hydrogel precursor is gelled to form a hydrogel. The hydrogel precursor may be any precursor that can be gelled. Such a precursor may be, for example, a solution containing an alginic acid solution as a main component. The alginic acid solution may be, for example, an aqueous solution of sodium alginate. If necessary, a solution such as a gelatin solution or a collagen solution may be mixed into the alginic acid solution.

[0052] Next, the hydrogel precursor is loaded into a syringe and ejected at a predetermined speed from the syringe outlet into a solution containing a gelling agent. When the hydrogel precursor is an alginate solution, the solution containing a gelling agent may be a solution containing the aforementioned divalent cations. The solution containing a divalent cation may be, for example, a solution in which calcium chloride or barium chloride is dissolved in a desired liquid medium. When the hydrogel precursor contains a gelatin solution or a collagen solution, the solution containing a gelling agent may contain a crosslinker such as genipin.

[0053] The hydrogel precursor is discharged into a solution containing a gelling agent, and then gels to form a hydrogel. By continuously discharging the hydrogel precursor from the syringe outlet, a continuously elongated string-like hydrogel is formed.

[0054] Next, the string-like hydrogel obtained as described above is immersed in a suspension containing plasma- or platelet-derived components, fibrin, fibrinogen, or a mixture thereof. The components of this suspension are as described above. This suspension may be, for example, a liquid medium containing insoluble components derived from plasma or human platelets, more preferably a medium containing human platelet lysate.

[0055] Through the above process, plasma-derived or platelet-derived components and / or fibrin-containing substances converted from fibrinogen are attached to the outer surface of the string-like hydrogel. This results in the formation of the scaffold described above. Furthermore, by intermittently ejecting the hydrogel precursor from the syringe outlet described above, numerous spherical hydrogels are formed. By attaching fibrin-containing substances to these numerous spherical hydrogels as described above, spherical scaffolds are formed.

[0056] When culturing adherent cells using such a scaffold, the scaffold is placed in a desired liquid medium and the adherent cells are seeded in. The adherent cells are cultured while adhering to the plasma- or platelet-derived components or fibrin-containing material on the outer surface of the hydrogel.

[0057] (Scaffolding manufacturing method 2) When the scaffold for culturing cells has a continuously extending tubular shape as shown in Figures 4 and 5, the scaffold can be manufactured, for example, as follows: Figure 7 is a schematic diagram of an apparatus used to form a tubular scaffold.

[0058] First, a suspension 1 containing a plasma- or platelet-derived component, fibrin, fibrinogen, or a mixture thereof is flowed, while a hydrogel precursor 3 is flowed around the suspension 1. Here, the suspension 1 is a cell suspension containing cells to be cultured and a substrate containing fibrin and / or fibrinogen. The material of the substrate is as described above. Preferably, the substrate contains an extracellular matrix and / or human platelet lysate. In this case, the cell suspension contains a plasma- or hPL-derived component, fibrin, fibrinogen, or a mixture thereof. The material of the hydrogel precursor 3 is as described above.

[0059] Preferably, the suspension 1 is caused to flow as a laminar flow. This laminar flow is formed within the first inlet pipe 2. The hydrogel precursor 3 is caused to flow so as to surround the outer periphery of the flow of suspension 1. In other words, the hydrogel precursor 3 flows coaxially with and in the same direction as the suspension 1. Preferably, the hydrogel precursor 3 is caused to flow as a laminar flow. As a result, a flow of hydrogel precursor 3 is formed at the second inlet pipe 4 that surrounds the flow of cell suspension 1.

[0060] The hydrogel precursor is gelled to form a tubular hydrogel that covers the suspension 1. This can be achieved by contacting the outer periphery of the flow of hydrogel precursor 3 with a solution 5 containing a gelling agent that gels the hydrogel precursor. In the embodiment shown in Figure 7, the solution 5 surrounds the hydrogel precursor (second laminar flow) 3 at the third inlet tube 6. That is, the solution 5 flows coaxially with the suspension 1 and hydrogel precursor 3 and in the same direction.

[0061] The flow of the cell suspension 1, the hydrogel precursor 3, and the gelling agent-containing solution 5 may be started in any order at the start of production, and may be stopped in any order at the end of production. However, from the viewpoint of confining cells without leakage, it is preferable that the flow of the cell suspension 1 be started last at the start of production, and that it be stopped first at the end of production. The flow rates of the cell suspension 1, the hydrogel precursor 3, and the gelling agent-containing solution 5 are not particularly limited as long as a scaffold can be formed.

[0062] The cell suspension 1, hydrogel precursor 3, and solution 5 containing a gelling agent flow out of a third inlet tube 6 and are immersed in a liquid or suspension, such as physiological saline or a liquid medium. Here, the hydrogel precursor 3 flows out of the third inlet tube 6 while being gelled by the addition of the gelling agent. This results in the formation of a tubular hydrogel that covers the suspension. The insoluble components formed inside the tubular hydrogel, i.e., plasma- or platelet-derived components and / or fibrin-containing substances, adhere to the inner surface of the tubular hydrogel over time.

[0063] Through the above process, plasma-derived or platelet-derived components and / or fibrin-containing substances are attached to the inner surface of the tubular hydrogel, thereby forming the scaffold described above.

[0064] In the embodiment shown in FIG. 7 , a scaffold was formed by forming a flow of cell suspension 1, a flow of hydrogel precursor 3, and a flow of gelling agent-containing solution 5, which were then discharged from the third inlet tube 6. Alternatively, a scaffold with a similar structure can be formed by forming a flow of cell suspension 1, forming a flow of hydrogel precursor 3 that surrounds the outer periphery of the first laminar flow, and discharging these flows into a container containing gelling agent-containing solution 5. In this case, by intermittently discharging the flow of cell suspension 1 and the flow of hydrogel precursor 3, numerous spherical hydrogel shells are formed. As described above, plasma- or platelet-derived components and / or fibrin-containing substances adhere to the inner surfaces of these spherical hydrogel shells. In this case, a spherical scaffold encasing cells can be formed.

[0065] When culturing adherent cells using such a scaffold, the adherent cells to be cultured can be contained inside the hydrogel, and the cells are cultured while adhering to plasma- or platelet-derived components and / or fibrin-containing materials on the inner surface of the hydrogel.

[0066] The hydrogel constituting the scaffold for cell culture is preferably alginate gel. Alginate gel has the advantage that it can be easily and instantly gelled from an alginate solution by a solution containing divalent cations at or near room temperature. Alginate gel also has the advantage of low cytotoxicity.

[0067] However, alginate gel is not cell-adhesive and is therefore not necessarily suitable as a scaffold for adhesive cells. The inventors of the present application discovered a scaffold that is easy to adhere and culture adhesive cells to by attaching plasma- or platelet-derived components and / or fibrin-containing substances to the outer or inner surface of a hydrogel, particularly an alginate gel.

[0068] Furthermore, the inventors of the present application have found that a liquid medium (suspension) containing human platelet lysate can be suitably used to effectively attach or coat plasma- or platelet-derived components and / or fibrin-containing substances to the aforementioned string-, tubular-, spherical-, or spherical-shell-shaped hydrogels, particularly alginate gels. However, as long as it is possible to attach plasma- or platelet-derived components and / or fibrin-containing substances to the outer or inner surface of the hydrogel, the attachment or coating method is not limited to the methods described above.

[0069] Furthermore, to facilitate the attachment of plasma- or platelet-derived components and / or fibrinogen-containing substances to the hydrogel, a suspension may be prepared by mixing a solution containing plasma- or platelet-derived components or fibrinogen with a medium, and then allowing the suspension to stand at room temperature (e.g., 22-37°C) for 10-120 minutes. After allowing the suspension to stand, the suspension may be brought into contact with the hydrogel. Furthermore, when the scaffold for cell culture is a continuously extending tubular structure as shown in Figures 4 and 5, it is preferable to prepare a suspension by mixing a solution containing plasma- or platelet-derived components or fibrinogen with a medium, then allowing the suspension to stand at room temperature (e.g., 22-37°C) for 10-120 minutes before introducing the cells to be cultured into the suspension 1. The cell suspension 1 thus obtained can be used to manufacture a tubular scaffold using the apparatus shown in Figure 7, as described above.

[0070] Furthermore, the inventors of the present application have discovered that, if necessary, mixing collagen gel or gelatin gel into alginate gel can enhance adhesion of adhesive cells and facilitate their cultivation. Collagen gel or gelatin gel has higher cell adhesiveness than alginate gel, making it possible to compensate for the disadvantage of alginate gel, namely, its low cell adhesiveness. In this case, the ratio of collagen gel or gelatin gel to alginate gel may be, for example, 0.1 to 20 mass percent, preferably 1.0 to 20 mass percent.

[0071] [Example 1] (Scaffolding manufacturing) Next, the examples will be described in detail. First, the following reagents were prepared. Sodium alginate (Kimika Co., Ltd. "I-3G") Gelatin ("beMatrix gelatin LS-H" manufactured by Nitta Gelatin Co., Ltd.) Dulbecco's modified Eagle's medium: DMEM (Sigma-Aldrich "D6429") Genipin (Fujifilm Wako Pure Chemical Industries, Ltd., product code: 078-03021) MSC medium (Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use): C-28009) ·hPL liquid (AventaCell, UltraGRO-PURE:HPCHXCRL50))

[0072] First, the gelatin solution was prepared by adding the gelatin to physiological saline and autoclaving it. The gelatin concentration relative to the physiological saline was equivalent to 20 volume percent at 37°C.

[0073] The sodium alginate was added to saline and stirred to prepare an alginic acid solution. The concentration of sodium alginate relative to the saline was 1.96 mass percent.

[0074] The gelatin solution and the alginate solution were mixed at a volume ratio of 1:1 at 37°C to prepare a mixed solution.

[0075] Calcium chloride and the above-mentioned genipin were dissolved in Dulbecco's modified Eagle's medium (DMEM). The genipin concentration in the medium (DMEM) was 1 mM at 37°C. The calcium chloride concentration in the medium (DMEM) was 100 mM at 37°C. Here, "M" means molar concentration (mol / L) (the same applies below). This produced a liquid medium containing calcium chloride.

[0076] Next, a mixture of the gelatin solution and the alginate solution was filled into a glass syringe, and the mixture was ejected from an injection needle attached to the syringe into the liquid medium containing calcium chloride.

[0077] Here, the alginate solution in the mixture is cross-linked by calcium ions in the liquid medium, forming a gel. Meanwhile, the gelatin in the mixture is cross-linked by genipin in the liquid medium. This results in the formation of a string-like hydrogel (scaffold) in the liquid medium (see also Figure 1).

[0078] After the mixed solution was discharged from the syringe, the liquid medium containing the string-like hydrogel was left standing at 37°C for a desired period of time.

[0079] After standing, the string-shaped hydrogel was washed and then transferred to a mixture of the above hPL solution and the above MSC medium. The medium containing the hydrogel was left standing for a desired period of time while maintained at 37°C. The concentration of the hPL solution to the MSC medium was such that the volume ratio was 1:1 at 37°C. The above hPL solution contained plasma- or platelet-derived components, fibrinogen, fibrin, or a mixture thereof.

[0080] This procedure resulted in the formation of a scaffold with plasma- or platelet-derived components and / or fibrin-containing material attached to the outer surface of the string-like hydrogel. The plasma- or platelet-derived components and / or fibrin-containing material were derived from components in the hPL solution. The diameter of the string-like scaffold was approximately 420 μm.

[0081] (Cell culture) Next, we will explain the method for culturing cells using the scaffold produced in Example 1. First, the following cells, materials, and reagents were prepared. -String-like scaffolds manufactured as described above Human Mesenchymal Stem Cells from Bone Marrow (hMSC-BM) (Promo Cell "C-12974") Culture medium (Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use): C-28009)

[0082] The aforementioned string-like scaffold was transferred to the above-mentioned MSC medium. Human bone marrow-derived mesenchymal stem cells dispersed in this medium were seeded. The number of seeded cells was 4.0 × 10 5 The container containing the medium is non-cell-adhesive.

[0083] The cells were cultured under these conditions for the number of days shown in Table 1. The entire medium was replaced every two days. The medium used after replacement was the same as the medium used before replacement. If any cells were not attached to the scaffold, they were collected by centrifuging the medium supernatant. The collected cells were reseeded in the medium after the medium replacement.

[0084] (Cell recovery) We will now explain the method for recovering cells cultured using the scaffold described above. First, the string-like hydrogel (scaffold) to which the cells have attached is removed from the culture medium and placed in a recovery solution made from a mixture of EDTA / PBS solution and a cell dispersion enzyme solution (Tryple). The scaffold is then incubated in the recovery solution at 37°C for 5 minutes to dissolve the scaffold and disperse the cells.

[0085] Next, the cells were further incubated at 37°C for 3 minutes, and when the scaffold shape was no longer visible, the collected solution was centrifuged for 5 minutes. The supernatant was then discarded, the cells were suspended in medium, and the collected cells were stained with trypan blue and the number of viable cells was counted.

[0086] [Example 2] The scaffold and its manufacturing method according to Example 2 are the same as those of Example 1, except for the following points. In Example 2, the above-mentioned hPL solution (AventaCell, UltraGRO-PURE:HPCHXCRL50) was introduced into the MSC medium during cell culture. The concentration of the hPL solution in the MSC medium was equivalent to 10 volume percent at a temperature of 37°C. Other details were the same as those of Example 1.

[0087] [Reference example 1] The scaffold of Reference Example 1 is almost the same as that of Example 1, except that plasma-derived or platelet-derived components and / or fibrin-containing substances are not attached to the outer surface of the string-shaped hydrogel. Therefore, in Reference Example 1, after creating the string-shaped hydrogel as in Example 1, the string-shaped hydrogel is not immersed in an hPL solution. Other details are the same as those of Example 1.

[0088] [Reference example 2] The scaffold and its manufacturing method according to Reference Example 2 are the same as those of Reference Example 1, except for the following points. In Reference Example 2, the above-mentioned hPL solution (AventaCell, UltraGRO-PURE:HPCHXCRL50) was introduced into the MSC medium during cell culture. The concentration of the hPL solution in the MSC medium was equivalent to 10 volume percent at a temperature of 37°C. Other details were the same as those of Reference Example 1.

[0089] The results of cell culture for Examples 1, 2, Reference Examples 1 and 2 are shown in Table 1 below. [Table 1]

[0090] The cell proliferation rates in Example 1, Example 2, and Reference Example 1 were 9 times or more higher than that in Reference Example 1. Furthermore, the cell proliferation rates in Example 1 and Example 2 were slightly higher than that in Reference Example 2. Similarly, the doubling times of cell culture in Example 1 and Example 2 were improved compared to those in Reference Examples 1 and 2. This indicates that a scaffold in which plasma-derived or platelet-derived components and / or fibrin-containing substances are attached to the outer surface of a string-like hydrogel is more suitable for cell culture.

[0091] Furthermore, a comparison between Example 1 and Example 2, or a comparison between Example 1 and Reference Example 2, shows that the cell proliferation rate can be sufficiently increased by using a scaffold in which plasma- or platelet-derived components and / or fibrin-containing materials are attached to the outer surface of a string-shaped hydrogel, even without introducing human platelet lysate (hPL) into the medium during cell culture. In other words, the use of a scaffold in which plasma- or platelet-derived components and / or fibrin-containing materials are attached to the outer surface of a string-shaped hydrogel eliminates the need to introduce human platelet lysate into the medium during cell culture. This makes it possible to reduce the amount of relatively expensive human platelet lysate used.

[0092] [Example 3] Example 3 corresponds to the subculture of the cells collected in Example 1. Specifically, except for the following explanation, a scaffold is produced in the same manner as in Example 1, and cells are cultured in the same manner as in Example 1. However, the cells seeded in Example 3 are the cells collected in Example 1. The number of cells seeded in Example 3 is 4.0 × 10 5 cells.

[0093] [Example 4] Example 4 corresponds to the subculture of the cells collected in Example 2. Specifically, except for the following explanation, a scaffold is produced in the same manner as in Example 2, and cells are cultured in the same manner as in Example 2. That is, in Example 4, human platelet lysate is introduced into the MSC medium during cell culture. However, the cells seeded in Example 4 are the cells collected in Example 2. The number of cells seeded in Example 4 is 4.0 × 10 5 cells.

[0094] [Reference example 3] Reference Example 3 corresponds to the subculture of the cells collected in Reference Example 1. Specifically, except for the following explanation, a scaffold is produced in the same manner as in Reference Example 1, and cells are cultured in the same manner as in Reference Example 1. However, the cells seeded in Reference Example 3 are the cells collected in Reference Example 1. The number of cells seeded in Reference Example 3 is 4.0 × 10 5 cells.

[0095] [Reference example 4] Reference Example 4 corresponds to the subculture of the cells collected in Reference Example 2. Specifically, except for the following explanation, a scaffold is produced in the same manner as in Reference Example 2, and cells are cultured in the same manner as in Reference Example 2. That is, in Reference Example 4, human platelet lysate is introduced into the MSC medium during cell culture. However, the cells seeded in Reference Example 4 are the cells collected in Reference Example 2. The number of cells seeded in Reference Example 4 is 4.0 × 10 5 cells.

[0096] The results of cell culture for Examples 3, 4, Reference Examples 3 and 4 are shown in Table 2 below. [Table 2]

[0097] When using hydrogel scaffolds with plasma-derived or platelet-derived components and / or fibrin-containing substances attached, as in Examples 3 and 4, a proliferation rate of more than three times higher was maintained even in cell subculture. On the other hand, in Comparative Examples 3 and 4, the number of recovered cells decreased. This indicates that the scaffolds of Examples 3 and 4 are more suitable for cell culture.

[0098] [Example 5] Example 5 corresponds to the re-subculture of the cells recovered in Example 3. Specifically, except for the following explanation, a scaffold is produced in the same manner as in Example 1, and cells are cultured in the same manner as in Example 1. However, the cells seeded in Example 5 are the cells recovered in Example 3. The number of cells seeded in Example 5 is 4.0 × 10 5 cells.

[0099] In Example 5, after culturing the cells for 5 days, the number of recovered cells was 1.6 x 10 6 The cells were cultured in a manner similar to that of the conventional scaffolds, and a proliferation rate of approximately four times higher was maintained. Thus, when using hydrogel scaffolds to which plasma- or platelet-derived components and / or fibrin-containing substances were attached, a high proliferation rate was maintained even in the re-subculture of the cells.

[0100] It was found that mesenchymal stem cells are adhesive cells and easily adhere to the scaffolds of Examples 1 to 5. Therefore, the effectiveness of culturing adhesive cells can be improved by using a scaffold in which plasma-derived or platelet-derived components and / or fibrin-containing substances are attached to the outer surface of the hydrogel.

[0101] Furthermore, as mentioned above, forming the scaffold into a string shape makes it easier to handle the scaffold during culture and facilitates three-dimensional culture in a liquid medium. However, from the viewpoint of the effect of culturing adherent cells, the scaffold is not limited to a string shape and may be, for example, spherical.

[0102] [Example 6] (Scaffolding manufacturing) Next, Example 6 will be described in detail. The scaffold produced in Example 6 is a tubular scaffold as shown in Figures 4 and 5. First, the following reagents were prepared. Sodium alginate (Kimika Co., Ltd. "I-3G") Human Mesenchymal Stem Cells from Bone Marrow (hMSC-BM) (Promo Cell "C-12974") Dulbecco's modified Eagle's medium: DMEM (Sigma-Aldrich "D6046") Human Platelet Lysate (STEMCELL TECHNOLOGIES, 06960)

[0103] First, a cell suspension, a sodium alginate solution, and a calcium chloride solution were prepared. The sodium alginate solution was prepared by adding the above sodium alginate to physiological saline and stirring. The concentration of sodium alginate relative to the physiological saline was 1.44 mass percent.

[0104] The cell suspension contained the human bone marrow-derived mesenchymal stem cells, the Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS), and the human platelet lysate. Specifically, DMEM and FBS were mixed at a volume ratio of 9:1 at 37°C. Then, the human platelet lysate was mixed with the DMEM and FBS mixture at a volume ratio of 7:3 at 37°C. The mixture was then left to stand at room temperature for 10 to 120 minutes. Next, a cell suspension was generated by introducing human bone marrow-derived mesenchymal stem cells into the mixture of DMEM, FBS, and human platelet lysate. The density of the human bone marrow-derived mesenchymal stem cells in the suspension was 2 x 10 5 cells / mL. FBS is rich in albumin.

[0105] Using these materials, a tubular hydrogel scaffold encasing the cell suspension was created according to the scaffold creation method shown in Figure 7 and the accompanying explanation. That is, a flow of cell suspension, a flow of sodium alginate solution around the flow of cell suspension, and a flow of calcium chloride aqueous solution around the flow of sodium alginate solution were formed, and these flows were then ejected into physiological saline (see also Figure 7). The sodium alginate solution was cross-linked by contact with the calcium chloride aqueous solution to form an alginate gel. This resulted in the creation of a long, thin tubular hydrogel encasing the cell suspension in physiological saline. The number of cells in the tubular hydrogel was approximately 2 × 10 4 The cross-sectional diameter of the formed tubular hydrogels was 400-500 μm.

[0106] The tubular hydrogel containing the cell suspension was left to stand in saline for the desired time. The cylindrical hydrogel containing the cell suspension was then transferred to a liquid medium, and cells were cultured within the tubular hydrogel. During this process, insoluble components derived from the human platelet lysate, specifically plasma- or platelet-derived components and / or fibrin-containing components, are believed to have adhered to the inside of the hydrogel. In this way, a scaffold for cell culture was produced.

[0107] Human bone marrow-derived mesenchymal stem cells were cultured in the scaffolds at 37°C in a liquid medium for the number of days shown in Table 3. This liquid medium was the aforementioned Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich, "D6046"). This liquid medium was replaced every two days. The medium used after replacement was the same as the medium used before replacement.

[0108] (Cell recovery) The method for recovering cells cultured using the scaffold described above is explained below. First, the tubular hydrogel (scaffold) containing the cells was removed using an EDTA / PBS solution, and the cells were recovered by centrifugation. The recovered cells were then stained with trypan blue and counted for viability. The cells were recovered using this method on the fourth day after the start of cell culture, and the recovered cells were subcultured. For subculture, the recovered cells were cultured in the tubular scaffold in the same manner as described above.

[0109] [Example 7] The scaffold manufacturing method and cell culture method of Example 7 were the same as those of Example 6, except for the following: In Example 7, when preparing the cell suspension, human platelet lysate was mixed with a mixture of DMEM and FBS at a volume ratio of 4:6 at 37°C. The other steps were the same as those of Example 6.

[0110] [Example 8] The scaffold manufacturing method and cell culture method of Example 8 were the same as those of Example 6, except for the following: In Example 8, human platelet lysate was used instead of a mixture of DMEM and FBS when preparing the cell suspension; the other steps were the same as those of Example 6.

[0111] [Reference example 5] The scaffold manufacturing method and cell culture method of Reference Example 5 were the same as those of Example 6, except for the following explanation. However, in Reference Example 5, human platelet lysate was not mixed into the mixture of DMEM and FBS when preparing the cell suspension. That is, the cell suspension was formed by introducing human bone marrow-derived mesenchymal stem cells into the mixture of DMEM and FBS. The other steps were the same as those of Example 6.

[0112] The results of cell culture for Examples 6, 7, 8 and Reference Example 5 are shown in Table 3 below. [Table 3]

[0113] The cell proliferation rates in Examples 6 to 8 were higher than that in Reference Example 5. Therefore, it was found that the cell proliferation rate was higher when human platelet lysate was mixed into the cell suspension. In other words, it was found that a scaffold in which plasma-derived or platelet-derived components or a fibrin-containing substance was attached to the inside of a tubular hydrogel was more suitable for culturing adherent cells.

[0114] Furthermore, referring to Examples 6 to 8, it can be seen that the higher the concentration of human platelet lysate in the cell suspension when producing a scaffold, the higher the cell proliferation rate and the shorter the culture time.

[0115] [Example 9] The scaffold manufacturing method and cell culture method of Example 9 were the same as those of Example 6, except for the following explanation. However, in Example 9, when preparing the cell suspension, human platelet lysate was mixed with a mixture of DMEM and FBS at a volume ratio of 9:1 at 37°C. Furthermore, the liquid medium used for cell culture was prepared by mixing the aforementioned mixture of DMEM and FBS with hPL solution (AventaCell, UltraGRO-PURE: HPCHXCRL50). Here, the hPL solution was mixed with the mixture of DMEM and FBS at a volume ratio of 9:1 at 37°C. The remaining steps were the same as those of Example 6. The hPL solution was a solution containing fibrinogen, although it had been subjected to a fibrinogen-reducing treatment.

[0116] [Example 10] The scaffold manufacturing method and cell culture method of Example 10 were the same as those of Example 9, except for the following: In Example 10, when preparing the cell suspension, human platelet lysate was mixed with a mixture of DMEM and FBS at a volume ratio of 7:3 at 37°C. The other steps were the same as those of Example 9.

[0117] [Example 11] The scaffold manufacturing method and cell culture method of Example 11 were the same as those of Example 9, except for the following: In Example 11, when preparing the cell suspension, human platelet lysate was mixed with a mixture of DMEM and FBS at a volume ratio of 4:6 at 37°C. The other steps were the same as those of Example 9.

[0118] [Example 12] The scaffold manufacturing method and cell culture method of Example 12 were the same as those of Example 9, except for the following: In Example 12, human platelet lysate was used instead of DMEM and FBS when preparing the cell suspension. The other steps were the same as those of Example 9.

[0119] [Reference example 6] The scaffold manufacturing method and cell culture method of Reference Example 6 were the same as those of Example 9, except for the following explanation. However, in Reference Example 6, human platelet lysate was not mixed with the mixture of DMEM and FBS when preparing the cell suspension. That is, the cell suspension was formed by introducing human bone marrow-derived mesenchymal stem cells into the mixture of DMEM and FBS. The other steps were the same as those of Example 9.

[0120] The results of cell culture for Examples 9 to 12 and Reference Example 6 are shown in Table 4 below. [Table 4]

[0121] The cell proliferation rates in Examples 9 to 12 were higher than that in Reference Example 6. Therefore, it was found that the cell proliferation rate was higher when human platelet lysate was mixed into the cell suspension. In other words, it was found that a scaffold in which plasma-derived or platelet-derived components or a fibrin-containing substance was attached to the inside of a tubular hydrogel was more suitable for culturing adherent cells.

[0122] Furthermore, referring to Examples 9 to 12, it can be seen that the higher the concentration of human platelet lysate in the cell suspension when producing a scaffold, the higher the cell proliferation rate and the shorter the incubation time.

[0123] Furthermore, a comparison of Examples 6 to 8 with Examples 10 to 12 reveals that increasing the concentration of fibrinogen in the liquid medium used for cell culture increases the cell proliferation rate and shortens the culture time.

[0124] Furthermore, when a scaffold having a tubular hydrogel was manufactured and when mesenchymal stem cells were cultured using the scaffold, if the scaffold was treated with nattokinase, which specifically degrades fibrin, no adhesion or proliferation of mesenchymal stem cells to the inner surface of the tubular scaffold was observed. In other words, in the above-mentioned examples, it can be seen that at least fibrin contributes to cell adhesion and proliferation.

[0125] [Example 13] (Scaffolding manufacturing) Next, Example 13 will be described in detail. The scaffold produced in Example 13 is a tubular scaffold as shown in Figures 4 and 5. First, the following reagents were prepared. Sodium alginate (Kimika Co., Ltd. "I-1G") Human Mesenchymal Stem Cells from Bone Marrow (hMSC-BM) (Promo Cell "C-12974") MSC medium (Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use): C-28009) Fibrinogen: derived from human plasma (Fujifilm Wako Pure Chemical Industries, Ltd.: JAN 4987481365186) Phosphate buffered saline: PBS (Fujifilm Wako Pure Chemical Industries, Ltd.: JAN 4987481628489)

[0126] First, a cell suspension, a sodium alginate solution, and a calcium chloride aqueous solution were prepared. The sodium alginate solution was prepared by adding the above sodium alginate to physiological saline and stirring. The concentration of sodium alginate relative to the physiological saline was 0.99 mass percent.

[0127] The cell suspension was prepared as follows. First, the above-mentioned phosphate buffered saline (PBS) in which the above-mentioned fibrinogen was dissolved was prepared. Here, the concentration of fibrinogen in the phosphate buffered saline was 5 mg / mL at a temperature of 37°C. Next, 10% of the fibrinogen-containing phosphate buffered saline was added. 5 ~10 6 A cell suspension was produced by introducing human bone marrow-derived mesenchymal stem cells at a density of 1000 cells / mL.

[0128] Next, a tubular hydrogel scaffold encasing the cell suspension was created according to the scaffold creation method shown in Figure 7 and the accompanying description. Specifically, a flow of the cell suspension described above, a flow of sodium alginate solution around the flow of the cell suspension, and a flow of calcium chloride solution around the flow of the sodium alginate solution were created, and these flows were then ejected into saline (see also Figure 7). The sodium alginate solution was crosslinked by contact with the calcium chloride solution to form an alginate gel. This resulted in the creation of a long, thin tubular hydrogel encasing the cell suspension produced as described above in saline.

[0129] Next, the resulting tubular hydrogel was transferred into physiological saline containing thrombin and allowed to stand for 30 minutes at 37°C. The concentration of thrombin relative to the physiological saline was selected to be sufficient to convert the fibrinogen inside the tubular hydrogel into fibrin.

[0130] The above process resulted in the formation of a tubular hydrogel scaffold containing fibrin inside, whose concentration roughly corresponds to the concentration of fibrinogen introduced inside the tubular hydrogel.

[0131] Next, the scaffold with fibrin inside the tubular hydrogel was immersed in the above-mentioned MSC medium, and the above-mentioned human bone marrow-derived mesenchymal stem cells encapsulated inside the scaffold were cultured for 7 days. During the culture, the MSC medium was changed every 2 to 3 days.

[0132] (Cell recovery) We will now explain the method for recovering cells cultured using the scaffold described above. First, fibrin was dissolved along with the tubular hydrogel using an EDTA / PBS solution containing nattokinase. After confirming that the scaffold had sufficiently collapsed, the cells were recovered by centrifugation. The number of recovered cells was then counted using a hemocytometer.

[0133] [Example 14] The scaffold manufacturing method and cell culture method of Example 14 are the same as those of Example 13, except for the following: In Example 14, when preparing the cell suspension, the concentration of fibrinogen in phosphate buffered saline was set to 10 mg / mL at a temperature of 37°C. All other points are the same as those of Example 13. In Example 14, the concentration of fibrin inside the tubular hydrogel is considered to roughly correspond to the concentration of fibrinogen introduced inside the tubular hydrogel.

[0134] [Example 15] The scaffold manufacturing method and cell culture method of Example 15 are the same as those of Example 13, except for the following: In Example 15, when preparing the cell suspension, the concentration of fibrinogen in phosphate buffered saline was set to 25 mg / mL at a temperature of 37°C. All other points are the same as those of Example 13. In Example 15, the concentration of fibrin inside the tubular hydrogel is considered to roughly correspond to the concentration of fibrinogen introduced inside the tubular hydrogel.

[0135] [Example 16] The scaffold manufacturing method and cell culture method of Example 16 are the same as those of Example 13, except for the following: In Example 16, when preparing the cell suspension, the concentration of fibrinogen in phosphate buffered saline was set to 50 mg / mL at a temperature of 37°C. All other points are the same as those of Example 13. In Example 16, the concentration of fibrin inside the tubular hydrogel is considered to roughly correspond to the concentration of fibrinogen introduced inside the tubular hydrogel.

[0136] [Reference example 7] The scaffold manufacturing method and cell culture method of Reference Example 7 were the same as those of Example 13, except for the following: In Reference Example 7, fibrinogen was not added to the phosphate buffered saline when preparing the cell suspension. The other points were the same as those of Example 13. Therefore, in Reference Example 7, fibrin was not precipitated inside the tubular hydrogel.

[0137] Fig. 8 is a graph showing the proliferation rates of the cells cultured in Examples 13 to 16 and Reference Example 7. In the graph of Fig. 8, the case where the fibrin concentration is 0 corresponds to Reference Example 7. The proliferation rates shown in Fig. 8 are values ​​calculated by dividing the number of cells recovered after 7 days by the number of cells at the time the scaffold was produced.

[0138] In Reference Example 7, the cells in the tubular hydrogel aggregated into a round shape, and the cell number decreased. On the other hand, in Examples 13 to 16, the cells proliferated compared to Reference Example 7. Therefore, it was found that a scaffold with fibrin attached to the inside of a tubular hydrogel is more suitable for cell culture. Furthermore, it was found that when the fibrin concentration inside the tubular hydrogel, in other words, the fibrinogen concentration when preparing the cell suspension, was at least 5 to 50 mg / mL, it was significantly superior to a tubular hydrogel that did not contain fibrinogen.

[0139] As explained in Examples 9 to 12, when fibrin was precipitated on the inside of a tubular hydrogel by treating fibrinogen-containing phosphate buffered saline with thrombin, it was sometimes difficult to obtain a uniform coating of fibrin depending on the conditions. In contrast, as explained in Examples 6 to 12, when plasma- or platelet-derived components or fibrin-containing substances were attached to the inside of a tubular hydrogel using human platelet lysate (hPL), coating was easy and more suitable for cell culture.

[0140] Therefore, it is more preferable to attach plasma- or platelet-derived components or fibrin-containing materials to the inside of a tubular hydrogel by using human platelet lysate (hPL) containing plasma- or platelet-derived components, fibrinogen, fibrin, or a mixture thereof. However, it should be noted that the scope of the present invention also includes embodiments in which fibrin is precipitated on the inside of a tubular hydrogel by treating a fibrinogen-containing solution with thrombin, as described in Examples 9 to 12.

[0141] From the above-described embodiments and / or examples and the additional explanations below, it can be understood that at least the following inventions are explicitly stated in this specification.

[0142] "Appendix 1" A scaffold for culturing cells, comprising: A hydrogel, and a plasma-derived or platelet-derived component or a fibrin-containing material attached to the hydrogel. Here, the plasma-derived or platelet-derived components are as described above. As described above, the fibrin-containing substance may be fibrin itself, for example, a fibrin polymer, or a mixture of fibrin and other polymers.

[0143] "Appendix 2" 2. The scaffold of claim 1, wherein the hydrogel is string-shaped, tubular, spherical, or shell-shaped.

[0144] "Appendix 3" 3. The scaffold of claim 2, wherein the plasma-derived or platelet-derived component or the fibrin-containing material is provided inside or outside the hydrogel.

[0145] "Appendix 4" 4. The scaffold of any one of claims 1 to 3, wherein the plasma-derived or platelet-derived component or the fibrin-containing material comprises a component derived from human platelet lysate.

[0146] "Appendix 5" 5. The scaffold of any one of claims 1 to 4, wherein the hydrogel comprises an alginate gel.

[0147] "Appendix 6" 6. The scaffold of any one of claims 1 to 5, wherein the hydrogel comprises an alginate gel and gelatin mixed with the alginate gel.

[0148] "Appendix 7" A scaffold for culturing cells, comprising: A hydrogel, a fibrin polymer disposed on the hydrogel. Here, the hydrogel may be, for example, in the form of a string, a tube, a sphere, or a spherical shell. In this case, the fibrin polymer may be provided on the outer surface of the string-shaped or spherical hydrogel, or on the inside of the tubular or spherical hydrogel.

[0149] "Appendix 8" 8. The scaffold of claim 7, comprising a fibrin polymer and albumin disposed on the hydrogel. Here, the hydrogel may be, for example, in the form of a string, a tube, a sphere, or a spherical shell. In this case, the fibrin polymer and albumin may be provided on the outer surface of the string-shaped or spherical hydrogel, or on the inside of the tubular or spherical hydrogel.

[0150] "Appendix 9" A scaffold according to any one of appendices 1 to 8; and cells attached to the scaffold. Here, the types of cells adhered to the scaffold are as already listed.

[0151] "Appendix 10" 10. The cell culture of claim 9, wherein the cells are adherent cells.

[0152] "Appendix 11" 11. The cell culture of claim 10, wherein the cells are mesenchymal stem cells.

[0153] "Appendix 12" A cell culture method comprising adhering cells to the scaffold described in any one of appendices 1 to 8 and culturing the cells.

[0154] "Appendix 13" A method for producing a scaffold for culturing cells, comprising: A method for producing a scaffold, comprising contacting a hydrogel with a suspension comprising a plasma-derived or platelet-derived component, fibrinogen, fibrin, or a mixture thereof. Preferably, the suspension contains fibrinogen. In this case, the amount of fibrinogen introduced into the suspension, for example, the amount of fibrinogen introduced relative to phosphate buffered saline, may be, for example, 1 mg / mL or more, preferably 3 mg / mL or more, and more preferably 5 mg / mL or more. The upper limit of the amount of fibrinogen introduced is not particularly limited, but may be, for example, 300 mg / mL. Fibrin polymers may also be formed by allowing thrombin to act on the fibrinogen in the suspension.

[0155] "Appendix 14" 14. The method for producing a scaffold described in Appendix 13, wherein the suspension comprises fibrinogen and albumin.

[0156] "Appendix 15" gelling a hydrogel precursor to form the hydrogel; 15. A method for producing a scaffold according to claim 13 or 14, comprising soaking the hydrogel in the suspension.

[0157] "Appendix 16" flowing a hydrogel precursor around the suspension while flowing the suspension; and gelling the hydrogel precursor to form a tubular hydrogel that covers the suspension.

[0158] "Appendix 17" 17. A method for producing a scaffold according to any one of claims 13 to 16, wherein the suspension comprises human platelet lysate.

[0159] "Appendix 18" 18. A method for producing a scaffold according to any one of claims 13 to 17, wherein the hydrogel precursor comprises an alginate solution.

[0160] "Appendix 19" 12. A cell population obtained by removing the scaffold from the cell culture of any one of claims 9 to 11.

[0161] "Appendix 20" 12. A biological material produced by at least one of the cells attached to the scaffold in the cell culture of any one of Supplementary Notes 9 to 11, the cells recovered from the scaffold in the cell culture of any one of Supplementary Notes 9 to 11, and the cell population of Supplementary Notes 19. Here, the biological material may be any material produced by cells. The biological material may be, for example, a polymer such as a nucleic acid, a protein, or a polysaccharide. Such biological material may be produced during the cultivation of cells or cell populations in the cell culture described above.

[0162] Furthermore, cells adhered to a scaffold include not only cells attached to the outer surface of the scaffold but also cells encapsulated in the hydrogel that forms the scaffold. When the hydrogel is, for example, tubular or spherical, the produced substance includes a substance produced by cells encapsulated in the tubular or spherical hydrogel.

[0163] As described above, the contents of the present invention have been disclosed through embodiments and examples, but the descriptions and drawings that form part of this disclosure should not be understood to limit the present invention. Various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art from this disclosure. Therefore, the technical scope of the present invention is defined only by the inventive features of the claims that can be reasonably understood from the above description.

Claims

1. A method for producing a scaffold for culturing adherent cells, comprising: flowing a suspension containing human platelet lysate while flowing an alginate solution around the suspension; forming a hydrogel by gelling the alginate solution, and depositing plasma-derived or platelet-derived components or fibrin-containing substances on the inner surface of the hydrogel; A method for producing a scaffold, wherein the proportion of human platelet lysate in the suspension is 10% by volume or more at a temperature of 37°C.

2. 2. A method for producing the scaffold of claim 1, comprising utilizing the human platelet lysate to deposit fibrin polymer on the inner surface of the hydrogel.

3. The method for producing a scaffold according to claim 1 or 2, wherein the proportion of the human platelet lysate in the suspension is 30% by volume or more at a temperature of 37°C.

4. The method for producing a scaffold according to claim 1 , wherein the suspension contains adherent cells.

5. The method for producing a scaffold according to claim 4 , wherein the adhesive cells are mesenchymal stem cells.

6. A culture method comprising culturing adherent cells inside the hydrogel using a scaffold manufactured by the scaffold manufacturing method described in any one of claims 1 to 5.

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