JP2021078458A - Three-dimensional scaffold for cell culture, method for manufacturing the same, and cell dissemination method and cell culture method using the same - Google Patents

Abstract

To provide a three-dimensional scaffold for cell culture in which cells can easily enter a scaffold when disseminating cells, and falling of a cell from the scaffold can be suppressed, and a method for manufacturing the scaffold, and a cell culture method using the scaffold.SOLUTION: A three-dimensional scaffold for cell culture constituted of a laminate includes a gelatin nonwoven fabric and a gelatin film, in which gelatin fibers constituting the gelatin nonwoven fabric have an average fiber diameter after swelling of 2 μm or more and 400 μm or less, fiber intersections are at least partially welded, and the gelatin film is laminated on one surface of the gelatin nonwoven fabric, and is partially welded with the gelatin fibers constituting the gelatin nonwoven fabric. A ratio Tf/Tn between the thickness Tn of the gelatin nonwoven fabric and the thickness Tf of the gelatin film is 7.5×10-3 or less.SELECTED DRAWING: Figure 1

Description

The present invention relates to a three-dimensional scaffold for cell culture, which has high cell seeding efficiency and is suitable for cell culture, a method for producing the same, a cell seeding method using the same, and a cell culture method.

The fiber sheet is useful for medical use and as a scaffold for cell culture because of its high porosity and high specific surface area. In particular, fiber sheets using biocompatible polymers are suitably used as scaffolds for medical use and cell culture. For example, Patent Document 1 describes that nanofibers made of biopolymers such as gelatin, collagen and cellulose are formed on a support such as gauze or sponge and used as a culture medium. Patent Document 2 describes that a biocompatible long fiber non-woven fabric in which fiber intersections of biocompatible long fibers having an average fiber diameter of 1 to 70 μm are partially welded is used as a scaffold for cell culture.

WO2014 / 196549 WO2018 / 235745

However, in the case of a dense material using nanofibers such as the culture medium described in Patent Document 1, there is a problem that cells tend to stay on the surface of the culture medium. Further, in the scaffold described in Patent Document 2, if the fiber diameter is too large or the basis weight of the non-woven fabric is low, the material becomes sparse, and there is a problem that cells penetrate the inside of the scaffold and fall off from the scaffold.

In order to solve the above-mentioned conventional problems, the present invention provides a three-dimensional scaffold for cell culture, a method for producing the same, and a method for producing the same, which allows cells to easily invade the inside of the scaffold at the time of cell seeding and can suppress the detachment of cells from the scaffold. The cell culture method used is provided.

In the present invention, in a three-dimensional scaffold for cell culture composed of a gelatin non-woven fabric containing gelatin as a main component and a laminate containing a gelatin film containing gelatin as a main component, the gelatin fibers constituting the gelatin non-woven fabric are averaged after swelling. The fiber diameter is 2 μm or more and 400 μm or less, the fiber intersections are at least partially welded, and the gelatin film is laminated on one surface of the gelatin non-woven fabric and partially with the gelatin fibers constituting the gelatin non-woven fabric. The present invention relates to a three-dimensional scaffold for cell culture, which is welded and has a ratio Tf / Tn of the thickness Tn of the gelatin non-woven fabric to the thickness Tf of the gelatin film of 7.5 × 10 ^{-3 or less.}

The present invention is also a method for producing a three-dimensional scaffold for cell culture, in which a spinning fluid containing gelatin is extruded into the air from a nozzle discharge port, and is located behind the nozzle discharge port. A pressure fluid was injected forward from a non-contact fluid injection port, and the extruded spinning liquid was accompanied by the pressure fluid to form fibers, and the fibers were formed on a gelatin film containing gelatin as a main component. The present invention relates to a method for producing a three-dimensional scaffold for cell culture, which comprises obtaining a laminate of the gelatin film and the gelatin non-woven fabric by accumulating fibers to form a gelatin non-woven fabric.

The present invention is also a cell culture method using the above-mentioned three-dimensional cell culture scaffold, in which the three-dimensional scaffold for cell culture after swelling is arranged in the culture vessel so that the gelatin film is in contact with the inner bottom surface of the culture vessel. The present invention relates to a cell culture method comprising a step and a step of dropping a cell suspension onto a gelatin non-woven fabric of a three-dimensional scaffold for cell culture.

Further, in the above-mentioned cell seeding method, the present invention is a cell culture in which a cell suspension is dropped onto a gelatin non-woven fabric of a three-dimensional scaffold for cell culture, allowed to stand for a predetermined time, and then a liquid medium is added to perform cell culture. Regarding the method.

The present invention can provide a three-dimensional scaffold for cell culture in which cells can easily invade the inside of the scaffold at the time of cell seeding and can suppress the shedding of cells from the scaffold.
Further, according to the production method of the present invention, it is possible to obtain a three-dimensional scaffold for cell culture composed of a laminate in which a gelatin non-woven fabric and a gelatin film are integrated without using a binder component or thermocompression bonding means.
In addition, according to the cell seeding method of the present invention, cells can easily invade the inside of the scaffold and the shedding of cells from the scaffold can be suppressed.
Further, according to the cell culture method of the present invention, three-dimensional culture of cells can be performed.

FIG. 1 is a photograph of a scanning electron microscope (100 times) of a three-dimensional scaffold (laminate) for cell culture obtained in one embodiment of the present invention. FIG. 2 is a photograph of a three-dimensional scaffold (stacked body) for cell culture using a scanning electron microscope (500 times). FIG. 3 is a photograph of a scanning electron microscope (100 times) of a three-dimensional scaffold (laminate) for cell culture obtained in another embodiment of the present invention. FIG. 4 is a photograph of a scanning electron microscope (500 times) of the cell culture three-dimensional scaffold (laminate). FIG. 5 is a photograph of a scanning electron microscope (100 times) of a three-dimensional scaffold (laminate) for cell culture obtained in another embodiment of the present invention. FIG. 6 is a photograph of a scanning electron microscope (500 times) of the cell culture three-dimensional scaffold (laminate). FIG. 7 is a photograph of a scanning electron microscope (100 times) of a three-dimensional scaffold (laminate) for cell culture obtained in a comparative example of the present invention. FIG. 8 is a photograph of a three-dimensional scaffold (stacked body) for cell culture using a scanning electron microscope (500 times). FIG. 9 is a photograph of a scanning electron microscope (100 times) of a three-dimensional scaffold (nonwoven fabric) for cell culture obtained in another comparative example of the present invention. FIG. 10 is a schematic explanatory view of a manufacturing apparatus for manufacturing a three-dimensional scaffold (laminate) for cell culture used in one embodiment of the present invention. FIG. 11 is a photograph showing the results of hematoxylin eosin staining of scaffold sections after the cells were allowed to stand on the cell culture three-dimensional scaffold of Example 1 for 4 hours after cell seeding and the cells were adhered to the gelatin non-woven fabric. FIG. 12 is an enlarged photograph of the gelatin non-woven fabric portion. Arrowheads indicate cells. FIG. 13 is an enlarged photograph of the gelatin film portion. Arrowheads indicate cells.

The inventors of the present invention have repeated studies in order to solve the above-mentioned problems. As a result, the three-dimensional scaffold for cell culture is composed of a laminate containing a gelatin non-woven fabric containing gelatin as a main component and a gelatin film containing gelatin as a main component, and the average fiber diameter of the gelatin fibers constituting the gelatin non-woven fabric is 2 μm. The thickness is 400 μm or less, the fiber intersections are at least partially welded, the gelatin film is laminated on one surface of the gelatin non-woven fabric, and the gelatin fibers constituting the gelatin non-woven fabric are partially welded, and the gelatin non-woven fabric is partially welded. By setting the ratio Tf / Tn of the thickness Tn to the thickness Tf of the gelatin film to 7.5 × 10 ^-3 or less, cells can easily invade the inside of the scaffold at the time of cell seeding and the detachment of the cells from the scaffold is suppressed. Provide a three-dimensional scaffold for cell culture that can be used. In the present invention, "swelling" means swelling to saturation with water, buffer or liquid medium.

In the gelatin non-woven fabric, the average fiber diameter after swelling of the gelatin fibers is 2 μm or more and 400 μm or less, and the fiber intersections are partially welded, so that the gelatin non-woven fabric has a bulky bridge structure, and the cells are scaffolded at the time of cell seeding. Easy to invade. In addition, the mechanical strength of the non-woven fabric is not inferior even when it contains water, and by suppressing the deformation of the non-woven fabric during cell culture, maintaining the internal structure (voids), and using gelatin fibers with high cell affinity, the physiology of the cells It is considered that when the environment is prepared, cells can proliferate inside and outside the non-woven fabric during cell culture, and three-dimensional organization of cells becomes possible.

By partially welding the gelatin fibers constituting the gelatin non-woven fabric and the gelatin film, the gelatin non-woven fabric and the gelatin film are integrated, so that the cells are prevented from penetrating the scaffold and falling out from the cells. .. Further, the ratio Tf / Tn of the thickness Tn of the gelatin non-woven fabric to the thickness Tf of the gelatin film is 7.5 × 10 ^-3 or less, that is, the gelatin film has an appropriate thickness, so that the laminate is warped after swelling. Is suppressed and the cell suspension is less likely to flow out to the sides of the laminate, thus suppressing the shedding of cells from the scaffold. Furthermore, even when the laminate swells during cell culture, the gelatin film easily follows the gelatin non-woven fabric, and the gelatin film is less likely to peel off or break.

Both the gelatin non-woven fabric and the gelatin film contain gelatin as a main component. In the present invention, the main component means that gelatin is contained in an amount of 90% by mass or more. Other components of 10% by mass or less may be other biocompatible polymers, cross-linking agents, chemicals, plasticizers, other additives and the like, if necessary. It may be substantially 100% by mass gelatin. Since the three-dimensional scaffold for cell culture of the present invention contains gelatin as a main component, which is highly safe and has excellent bioabsorbability, the scaffold is necessary for regenerative therapy, cell research and drug discovery research by transplanting into a living body. It can be suitably used as a three-dimensional cultured tissue or the like.

The type and site of the animal from which collagen, which is the raw material of gelatin, is derived are not particularly limited. Collagen may be derived from, for example, vertebrates or fish. In addition, collagen derived from various organs and tissues such as dermis, ligaments, tendons, bones and cartilage can be appropriately used. Further, the method for preparing gelatin from collagen is not particularly limited, and examples thereof include acid treatment, alkali treatment, and enzyme treatment. The molecular weight of the gelatin is also not particularly limited, and those having various molecular weights can be appropriately selected and used. In addition, one type of gelatin may be used, or two or more types may be used in combination.

The gelatin is not particularly limited, but has appropriate flexibility and hardness, and from the viewpoint of enhancing the handleability of the scaffold, the jelly strength is preferably 100 g or more and 400 g or less, and more preferably 150 g or more and 360 g or less. is there. In the present invention, the jelly strength is measured according to JIS K 6503. The gelatin may be a commercially available product.

The other biocompatible polymer is not particularly limited, and for example, a natural polymer or a synthetic polymer can be used. Examples of natural polymers include proteins and polysaccharides. Examples of the protein include collagen, fibronectin, fibrinogen, laminin, fibrin and the like. Examples of polysaccharides include chitosan, calcium alginate, heparan sulfate, chondroitin sulfate, hyaluronic acid, heparin, starch, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, and dietangum. A molecule may be used, or a derivative of a natural polymer such as carboxymethyl cellulose may be used. Examples of the synthetic polymer include polyethylene glycol polyethylene glycol, polyethylene terephthalate, polyvinyl alcohol, thermoplastic elastomer, polypropylene, polyethylene, polystyrene, polymethylmethacrylate, polycarbonate, polydimethylsiloxane, cycloolefin polymer, and amorphous fluororesin. Examples thereof include non-absorbable synthetic polymers and bioabsorbable polymers such as polylactic acid, polyglucoric acid, polycaprolactone and polydioxanone. As the other biocompatible polymers described above, one type may be used, or two or more types may be used.

The gelatin fibers constituting the gelatin non-woven fabric have an average fiber diameter of 2 μm or more and 400 μm or less, preferably 5 μm or more and 300 μm or less, more preferably 10 μm or more and 200 μm or less, and further preferably 15 μm or more and 100 μm or less. Is. When the average fiber diameter of the gelatin fibers is within the above range, when the cells are seeded on the surface of the gelatin non-woven fabric of the three-dimensional scaffold for cell culture, the cells easily invade the three-dimensional scaffold for cell culture and the three-dimensional scaffold for cell culture. It is easy to distribute evenly inside the cell. In the present invention, the "average fiber diameter after swelling" means the average value of the diameters of 50 fibers arbitrarily selected from the gelatin non-woven fabric in the three-dimensional scaffold for cell culture after swelling.

The gelatin fibers constituting the gelatin non-woven fabric have fiber intersections partially welded. As will be described later, this partial welding occurs when gelatin fibers blown off by a pressure fluid are deposited during the production of a three-dimensional scaffold for cell culture and are not completely solidified. By this partial welding, the gelatin non-woven fabric has a bridge structure, is bulky and has a low density, is easy to be molded into a desired shape, and has high molding stability. In the present invention, since the fiber intersections are partially welded, the gelatin non-woven fabric does not become flat even when wet with water. Further, in the gelatin non-woven fabric, some of the fiber intersections may be welded, or all of the fiber intersections may be welded.

The gelatin non-woven fabric is not particularly limited, but for example, from the viewpoint of enhancing handleability and cell invasion, the thickness is preferably 0.1 mm or more, more preferably 0.2 mm or more, and more preferably 0.3 mm. The above is more preferable, and 0.4 mm or more is particularly preferable. The gelatin non-woven fabric is not particularly limited, but for example, from the viewpoint of increasing the viability of cells in three-dimensional culture, the thickness is preferably 2 mm or less, more preferably 1.5 mm or less, and 1 mm or less. Is more preferable, and 0.7 mm or less is particularly preferable.

The gelatin nonwoven fabric is not particularly limited, for example, from the viewpoint of suppressing detachment of cells, it is preferable that the basis weight is 10 g / ^{m 2} or more, more preferably 25 g / ^{m 2} or more, 50 g / ^{m 2} The above is more preferable, and 100 g / m ² or more is particularly preferable. The gelatin non-woven fabric is not particularly limited, but for example, from the viewpoint of enhancing cell invasion and cell viability in 3D culture, the basis weight ^{is preferably 600 g / m 2} or less, and 500 g / m ² or less. Is more preferable, and 400 g / m ² or less is further preferable.

The gelatin non-woven fabric is not particularly limited, but for example, from the viewpoint of enhancing cell invasion and cell viability in 3D culture, the pore diameter is preferably 20 μm or more, more preferably 30 μm or more. It is more preferably 40 μm or more. The gelatin non-woven fabric is not particularly limited, but for example, from the viewpoint of suppressing cell shedding, the pore diameter is preferably 400 μm or less, more preferably 300 μm or less, and further preferably 200 μm or less. preferable. In the present invention, the pore size of the gelatin nonwoven fabric can be calculated by the following formula (1) based on the assumption of Wrotnowski.

The gelatin film is arranged on one surface of the gelatin nonwoven fabric and is partially welded to the gelatin fibers constituting the gelatin nonwoven fabric. This partial welding occurs when the gelatin fibers blown off by the pressure fluid during the production of the cell culture scaffold are not completely solidified when they are deposited on the gelatin film, as will be described later. By this partial welding, the gelatin non-woven fabric and the gelatin film are integrated, and the cells are prevented from penetrating the scaffold and falling out from the cells.

The ratio Tf / Tn of the thickness Tn of the gelatin non-woven fabric to the thickness Tf of the gelatin film is 7.5 × 10 ^-3 or less. As a result, the warp of the laminate after swelling is suppressed, the cell suspension is less likely to flow out to the side surface of the laminate, and therefore the shedding of cells from the scaffold is suppressed. Further, even when the laminate is swollen, the gelatin film easily follows the gelatin non-woven fabric, and the gelatin film is less likely to be peeled off or broken. The Tf / Tn is ^{preferably 7.0 × 10 -3} or less, and ^{more preferably 6.0 × 10 -3} or less. ^{Further, the Tf / Tn is preferably 1.0 × 10 -3} or more, and more preferably 1.5 × 10 ^-3 or more, from the viewpoint of easily suppressing peeling or breakage of the gelatin film.

As an example, from the viewpoint of convenience and handleability, the gelatin film preferably has a thickness of 0.5 μm or more, more preferably 0.6 μm or more, and further preferably 0.7 μm or more. , 0.8 μm or more is particularly preferable. Further, as an example, the gelatin film preferably has a thickness of 10 μm or less, more preferably 8 μm or less, and 6 μm or less, from the viewpoint of enhancing the integrity with the gelatin non-woven fabric and the efficiency of three-dimensional culture of cells. It is more preferably 4 μm or less.

The gelatin film is preferably a non-porous film, but may have micropores having a size such that cells do not penetrate, for example, a pore diameter of about 10 μm or less or about 5 μm or less.

The laminate is not particularly limited, but for example, from the viewpoint of maintaining strength during cell culture and increasing cell viability in three-dimensional culture, compression under compressive stress of 1.0 kPa after swelling to a saturated state with water. The deformation rate (hereinafter, also simply referred to as “compression deformation rate”) is preferably 40% or less, more preferably 35% or less, still more preferably 30% or less. The saturated state means a state in which water is contained to a maximum, and a state in which the water content stays at a certain limit and does not increase any more. In the present specification, the compressive deformation rate is defined as (H1) the thickness under no load and (H2) the thickness under compressive stress of 1.0 kPa in the laminated body after being swollen to a saturated state with water. If, it is calculated by the following formula. The compression test is performed as described below.
Compression deformation rate (%) = 100-{(H2 / H1) x 100}

The gelatin non-woven fabric and the gelatin film are preferably crosslinked from the viewpoint of increasing water resistance, easily maintaining the morphology during cell culture, and effectively performing three-dimensional culture of cells. The cross-linking may be a chemical cross-linking using a compound such as a cross-linking agent, but from the viewpoint of biosafety, a cross-linking using a cross-linking agent having biosafety or a cross-linking without using a cross-linking agent is preferable. .. Examples of the cross-linking without using a cross-linking agent include thermal cross-linking, electron beam cross-linking, radiation cross-linking such as γ-ray, ultraviolet cross-linking, and the like. Preferably, it is a thermal dehydration crosslink.

In one or more embodiments of the present invention, the laminate of gelatin non-woven fabric and gelatin film suppresses or enhances cell adhesion factors, cell inducing factors, cell growth factors, substances that nourish or energize cells, and cell functions. It may be coated with a substance or the like. The cell adhesion factor is not particularly limited, and examples thereof include fibronectin and the like. The substance that gives nutrients and energy to cells is not particularly limited, and examples thereof include ATP, pyruvic acid, and glutamine. Further, in one or more embodiments of the present invention, the laminate of the gelatin non-woven fabric and the gelatin film may be immersed in a solution containing a physiologically active substance such as a cell-inducing factor and a cell growth factor to contain these components. .. In the cell culture process, these physiologically active substances are gradually released from the laminate, so that cell culture can be promoted.

In one or more embodiments of the present invention, the three-dimensional scaffold for cell culture is not particularly limited, but it suppresses the generation of contaminants, prevents product contamination, and uses a gelatin non-woven fabric without using a binder component or heat-bonding means. From the viewpoint of integrating the gelatin film, the spinning liquid containing gelatin is extruded into the air from the nozzle discharge port, located behind the nozzle discharge port, and forward from the fluid injection port in a non-contact state with the nozzle discharge port. A pressure fluid is jetted toward the surface, the extruded spinning liquid is associated with the pressure fluid to form fibers, and the fiber-formed fibers are accumulated on a gelatin film containing gelatin as a main component to form a gelatin non-woven fabric. Therefore, it is preferable to obtain a laminate of the gelatin film and the gelatin non-woven fabric.

The gelatin film is not particularly limited, and can be produced by a known method for producing a film. For example, it can be produced by applying a gelatin solution to the surface of a substrate and then drying it. As the base material, for example, a polyethylene terephthalate film (PET film), a glass plate, a polystyrene sheet, a fluororesin sheet, or the like can be used. The PET film, glass plate, polystyrene sheet, fluororesin sheet and the like may be treated with water repellency.

The gelatin solution can be obtained by dissolving gelatin alone or, if necessary, gelatin and other biocompatible polymers that can be used as the above-mentioned other components in a solvent. Examples of the solvent include water, ethanol, alcohols such as 1-propanol, 2-propanol and glycerin, and amides such as dimethylformamide and dimethylacetamide. One of these solvents may be used alone, or two or more of these solvents may be mixed and used. Above all, from the viewpoint of handleability, it is preferable to use water such as distilled water, pure water, ultrapure water, and ion-exchanged water. Since gelatin is water-soluble, it can be used for film formation in the state of an aqueous solution, and the safety to the living body is enhanced.

The concentration of gelatin is not particularly limited, but for example, from the viewpoint of film forming property and ductility, when the gelatin solution is 100% by mass, it is preferably 0.1% by mass or more and 35% by mass or less, and 1% by mass. It is more preferably% or more and 30% by mass or less, and further preferably 3% by mass or more and 20% by mass or less. The dissolution temperature (solvent temperature) is preferably 10 ° C. or higher and 90 ° C. or lower, more preferably 20 ° C. or higher and 80 ° C. or lower, and further preferably 30 ° C. or higher and 70 ° C. or lower. If necessary, gelatin may be dissolved in a solvent and then filtered to remove foreign substances, dust and the like. Further, if necessary, the dissolved air may be removed by depressurizing or vacuum defoaming thereafter. From the viewpoint of efficiently removing gas (air bubbles), the degree of vacuum at the time of defoaming under reduced pressure is preferably 5 kPa or more and 30 kPa or less.

The drying is not particularly limited, and can be performed by, for example, natural drying, heat drying, vacuum drying (vacuum drying), forced exhaust drying, forced circulation convection, or the like. Specifically, the drying temperature may be, for example, −40 ° C. or higher and 90 ° C. or lower, 0 ° C. or higher and 60 ° C. or lower, or 10 ° C. or higher and 40 ° C. or lower. The drying time may be, for example, in the range of 1 to 200 hours, preferably in the range of 3 to 100 hours, and more preferably in the range of 5 to 48 hours.

In the melt blow method used in the present invention, the spinning liquid containing gelatin is extruded from the nozzle discharge port, and the pressure fluid is directed forward from the fluid injection port which is located behind the nozzle discharge port and is not in contact with the nozzle discharge port. The extruded spinning liquid is sprayed and accompanied by the pressure fluid to be directly fiberized by a dry method, and the obtained gelatin fibers are accumulated on a gelatin film to form a non-woven fabric, so that contamination (contamination) is generated. Is prevented and can be manufactured hygienically. When the fibers are accumulated (deposited) after spinning, the fibers are laminated in a state of containing water, so that the fibers are welded or entangled with each other and integrated, and the gelatin fibers constituting the non-woven fabric are gelatin films. The gelatin non-woven fabric and the gelatin film are integrated by welding. The density of the non-woven fabric can be easily changed by changing the collection distance when the fibers are deposited.

FIG. 10 is a schematic explanatory view of an apparatus for manufacturing a three-dimensional scaffold for cell culture used in one embodiment of the present invention. In the cell culture three-dimensional scaffold manufacturing apparatus 20, the gelatin-containing spinning liquid 2 contained in the heating tank 1 is pushed out into the air from the nozzle discharge port 3. A predetermined pressure is applied to the heating tank 1 by the compressor 4. Reference numeral 12 is a heat insulating container.
Further, the pressure fluid 7 is injected forward from the fluid injection port 5 which is located behind the nozzle discharge port 3 and is in a non-contact state with the nozzle discharge port 3. A pressure fluid (for example, compressed air) is supplied to the fluid injection port 5 from the compressor 6. The distance between the fluid injection port 5 and the nozzle discharge port 3 is preferably 5 to 30 mm.
The extruded spinning liquid is accompanied by the pressure fluid 7 to become gelatin fibers 8, and is deposited as gelatin non-woven fabric 9 on the gelatin film 10 arranged on the take-up roll 11. At this time, since the deposited fibers contain water or are not completely solidified, the fibers in contact with each other at at least a part of the fiber intersections are welded to each other, and the gelatin fibers constituting the non-woven fabric are formed with the gelatin film. By welding, the gelatin non-woven fabric and the gelatin film are integrated. In addition, other collecting means such as a net may be used instead of a take-up roll.

First, gelatin alone or, if necessary, gelatin and other biocompatible polymers that can be used as the above-mentioned other components are dissolved in a solvent, preferably water, to prepare a spinning solution. The dissolution temperature (temperature of a solvent such as water) is preferably 20 ° C. or higher and 90 ° C. or lower, and more preferably 40 ° C. or higher and 90 ° C. or lower. If necessary, gelatin may be dissolved in a solvent such as water and then filtered to remove foreign substances and dust. Further, if necessary, the dissolved air may be removed by depressurizing or vacuum defoaming thereafter. From the viewpoint of efficiently removing gas (air bubbles), the degree of vacuum at the time of defoaming under reduced pressure is preferably 5 kPa or more and 30 kPa or less. Since gelatin is water-soluble, it can be spun in an aqueous solution as a spinning solution, and the safety to the living body is improved. As the water, for example, pure water, distilled water, ultrapure water or the like can be appropriately used. When another biocompatible water-soluble polymer is used as another component, a spinning solution can be prepared by dissolving it in water at the same time as gelatin.

The temperature of the spinning liquid is preferably 20 ° C. or higher and 90 ° C. or lower, and more preferably 40 ° C. or higher and 90 ° C. or lower. Within the above range, gelatin can maintain a stable sol state. The gelatin concentration of the gelatin aqueous solution is preferably 30% by mass or more and 55% by mass or less when the gelatin aqueous solution is 100% by mass. A more preferable concentration is 35% by mass or more and 50% by mass or less. At the above concentration, a stable sol state can be maintained. The viscosity of the gelatin aqueous solution (spinning solution) is preferably 500 mPa · s or more and 3000 mPa · s or less. Stable spinning can be achieved if the viscosity of the gelatin aqueous solution is within the above range.

The spinning liquid is discharged from a nozzle of a spinning machine, a pressure fluid is supplied from around the nozzle, the discharged gelatin aqueous solution is associated with the pressure fluid to form fibers, and the obtained gelatin fibers are accumulated on a gelatin film. To make a gelatin non-woven fabric. The discharge pressure of the nozzle is not particularly limited, but may be, for example, 0.1 MPa or more and 1 MPa or less.

The temperature of the pressure fluid is preferably 20 ° C. or higher and 120 ° C. or lower, and more preferably 80 ° C. or higher and 120 ° C. or lower. Although it depends on the flow velocity of the pressure fluid and the temperature of the ambient atmosphere, stable spinning can be achieved within the above temperature range. Air is preferably used as the pressure fluid, and the pressure is preferably 0.1 MPa or more and 1 MPa or less. Within the above range, the spinning liquid extruded into the air from the nozzle discharge port can be blown off to form fibers.

In the gelatin non-woven fabric, the average fiber diameter after swelling of gelatin fibers is 2 μm or more and 400 μm or less, preferably 5 μm or more and 300 μm or less, more preferably 10 μm or more and 200 μm or less, and further preferably 15 μm or more and 100 μm or less. .. The gelatin non-woven fabric having a desired average fiber diameter can be obtained by appropriately adjusting the nozzle diameter (inner diameter) and the like.

The laminate of the gelatin non-woven fabric and the gelatin film is preferably crosslinked. Thereby, morphological stability and water resistance can be improved. The cross-linking may be a chemical cross-linking using a compound such as a cross-linking agent, but from the viewpoint of biosafety, a cross-linking using a cross-linking agent having biosafety or a cross-linking without using a cross-linking agent is preferable. Examples of cross-linking without using a cross-linking agent include thermal cross-linking, electron beam cross-linking, radiation cross-linking such as γ-rays, and ultraviolet cross-linking. In the case of electron beam irradiation or radiation irradiation such as γ-rays, sterilization and cross-linking can be performed at the same time. From the viewpoint of easily obtaining the desired crosslinking effect, thermal crosslinking is preferable, and thermal dehydration crosslinking is more preferable. The thermal dehydration crosslinking may be performed, for example, at 100 ° C. or higher and 160 ° C. or lower for 24 hours or longer and 96 hours or shorter. Further, the thermal dehydration crosslinking may be performed under a vacuum of 1 kPa or less, for example. The laminate may be dried before cross-linking. The drying may be air drying at room temperature or vacuum freeze drying.

A laminate in which a gelatin non-woven fabric and a gelatin film are integrated can be cut into a predetermined shape and size as needed and used as a three-dimensional scaffold for cell culture. Since gelatin has biocompatibility and biodegradability, a laminate in which a gelatin non-woven fabric and a gelatin film are integrated is suitable for medical use or a scaffold for cell culture. At the time of use, the three-dimensional scaffold for cell culture can be sterilized by ethylene oxide gas sterilization, steam (autoclave), electron beam irradiation, irradiation with γ-rays or the like, or by ethanol treatment or the like. In the case of electron beam irradiation and radiation irradiation such as γ-rays, cross-linking can be performed at the same time as sterilization.

The manufacturing process of the gelatin film or the laminate is preferably performed aseptically in, for example, a clean bench or a clean room. It is possible to prevent the gelatin film and the laminate from being contaminated by the propagation of various germs during the work. As the manufacturing instrument to be used, for example, it is preferable to use one that has been sterilized by autoclave, electron beam irradiation, irradiation with γ-rays or the like. Further, it is preferable that the gelatin solution is also subjected to, for example, conventionally known filter filtration sterilization and then subjected to the film manufacturing process.

In the present invention, as an example, the crosslinked laminate is formed by punching into a predetermined shape to obtain a scaffold for cell culture. Alternatively, after swelling with water, a buffer solution or a predetermined liquid medium, the scaffold for target cell culture is used.

When cell seeding is performed using the cell culture scaffold, the shedding of cells from the scaffold is suppressed and the seeding efficiency is increased. Specifically, the three-dimensional scaffold for cell culture after swelling is placed in the culture vessel so that the gelatin film is in contact with the inner bottom surface of the culture vessel, and the cell suspension is dropped onto the gelatin non-woven fabric of the three-dimensional scaffold for cell culture. This allows the cells to be seeded. Specifically, the scaffold can be arranged by gripping the end of the scaffold with tweezers.

The culture vessel is not particularly limited, and for example, a dish, a plate, a flask, or the like can be used. A culture vessel whose inner surface has been hydrophilized in order to promote cell adhesion may be used, or an untreated culture vessel which has not been subjected to such treatment may be used.

When a culture vessel in which the inner surface such as the inner bottom surface is hydrophilized in order to promote cell adhesion is used as the culture vessel, the size (for example, diameter) Ls of the three-dimensional scaffold for cell culture after swelling and the cell culture The ratio Ls / Lb of the size (for example, diameter) Lb of the inner bottom surface of the culture vessel in which the three-dimensional scaffold is arranged and the cells are cultured is preferably 1.01 times or more and 1.30 times or less, preferably 1.05 times or more. It is more preferably 1.25 times or less, and further preferably 1.10 times or more and 1.20 times or less. As a result, the adhesion between the three-dimensional scaffold for cell culture and the side surface of the culture vessel is improved, and it is suppressed that the cells wrap around the scaffold and fall off from the scaffold at the time of cell seeding. Here, for Ls and Lb, the size of the inner bottom surface of the culture vessel and the size of the inner bottom surface of the culture vessel measured at a predetermined arrangement location after arranging the three-dimensional scaffold for cell culture after swelling so that the gelatin film is in contact with the inner bottom surface of the culture vessel, respectively. It means the size of the three-dimensional scaffold for cell culture after swelling at the place of arrangement. For example, when the inner bottom surface of the culture vessel is circular, the size Ls after swelling of the three-dimensional scaffold for cell culture corresponds to the diameter after swelling of the three-dimensional scaffold for cell culture, and the size Lb of the inner bottom surface of the culture vessel is the culture vessel. Corresponds to the diameter of the inner bottom surface of.

In the present invention, the cell may be an animal cell, and its origin is not particularly limited. The animal may be a human or a non-human animal. Examples of animals other than humans include primates such as monkeys and chimpanzees, rodents such as mice, rats and hamsters, and ungulates such as cows, sheep, goats and pigs. Further, in the present invention, the cells include individual cells, cell lines, cells obtained by culturing such as primary culture, and the like. The cells are not particularly limited, and examples thereof include somatic cells, stem cells, progenitor cells, germ cells, immune cells, and the like.

Somatic cells include somatic cells that make up a living body and cancer cells derived from somatic cells. The somatic cells constituting the living body are not particularly limited, and are, for example, fibroblasts, muscle cells, endothelial cells, osteoblasts, endothelial cells, bladder cells, lung cells, osteoblasts, nerve cells, hepatocytes, and chondrocytes. , Epithelial cells, mesenteric cells and the like. The cancer cells are not particularly limited, and examples thereof include breast cancer cells, renal cancer cells, prostate cancer cells, lung cancer cells, liver cancer cells, cervical cancer cells, esophageal epithelial cancer, pancreatic cancer, colon cancer, and bladder cancer.

Stem cells are cells that can differentiate into a variety of specialized cell types. The stem cells are not particularly limited, and are, for example, embryonic stem cells (ES cells), embryonic cancer tumor cells (EC), embryonic germ stem cells (EG), artificial pluripotent stem cells (iPS cells), adult stem cells, and scutellum. Spore-derived stem cells, reproductive ridge-derived stem cells, malformation-derived stem cells, oncostatin-independent stem cells (OISC), bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, sheep water-derived mesenchymal stem cells, skin-derived mesenchymal stem cells Examples thereof include stem cells and bone membrane-derived mesenchymal stem cells.

Progenitor cells are cells that can develop from the stem cells and differentiate into terminally differentiated cells that constitute a living body.

Examples of germ cells include sperm, sperm cells, eggs, and egg cells.

The immune cell is not particularly limited, and examples thereof include macrophages, lymphocytes, and dendritic cells.

As the above-mentioned cells, one type may be used alone, or two or more types may be used in combination depending on the purpose and the like.

The seeding amount of cells per unit surface area of the three-dimensional scaffold for cell culture is not particularly limited and can be appropriately determined based on the cell type, the thickness of the scaffold, the texture, etc. 200 cells / mm ² or more and 20000 cells / cm ² or less, 2000 cells / mm ² or more and 15000 cells / mm ² or less, 4000 cells / mm ² or more and 12000 cells / mm ² or less It is more preferable to have.

The liquid medium is not particularly limited, and a medium containing components necessary for the survival and proliferation of cells can be appropriately used depending on the type of cells. The medium may contain serum, antibiotics, growth factors and the like. As the serum, for example, bovine serum, fetal bovine serum, horse serum, human serum and the like can be appropriately used. As the antibiotic, penicillin, streptomycin, gentamicin, amphotericin, ampicillin, minocycline, kanamycin and the like can be appropriately used. As the growth factor, a cell proliferation factor, a differentiation inducing factor, a cell adhesion factor and the like can be appropriately used.

In one or more embodiments of the present invention, after dropping the cell suspension on the gelatin non-woven fabric of the three-dimensional scaffold for cell culture, the cells are allowed to stand for a predetermined time, for example, 3 to 4 hours, and precultured to adhere the cells. After that, a liquid medium can be added to carry out cell culture.

The culture may be carried out at, for example, 27 ° C. or higher and 40 ° C. or lower, or 31 ° C. or higher and 37 ° C. or lower. Carbon dioxide may be in the range of 2% or more and 10% or less.

The culturing time may be appropriately determined according to the cell type, the number of cells, etc., but for example, the culturing may be continued for 2 to 8 days, continuously for 3 to 7 days, or 4 to 6 days. You may continue to do so. The medium may be changed every 2-3 days.

As an example, after confirming that the cells adhered to the three-dimensional scaffold for cell culture 4 hours after cell seeding, a liquid medium was added to the culture vessel, and the cells were placed in an incubator under _{predetermined conditions (for example, temperature 37 ° C., 5% CO 2).} May be statically cultured in. The liquid medium may be changed every 2-3 days. Alternatively, after cell seeding, a liquid medium may be added to a culture vessel, and the liquid medium may be stirred and cultured on a magnetic stirrer placed in an incubator at _{37 ° C. and 5% CO 2 while stirring and culturing.} Medium may be replaced every 3-4 days by removing half the amount of liquid medium and adding an equal amount of fresh liquid medium. Alternatively, after cell seeding, a liquid medium may be added to the culture vessel, and the cells may be cultured while shaking in an incubator at _{37 ° C. and 5% CO 2.} Medium may be replaced every 3-4 days by removing half the amount of liquid medium and adding an equal amount of fresh liquid medium. Since the three-dimensional scaffold for cell culture of the present invention becomes transparent when it gets wet with water, the inside of the scaffold can be observed with an inverted microscope in the culture solution.

When cells are seeded on a three-dimensional scaffold for cell culture as described above and cell culture is performed, the cells easily invade the inside of the scaffold and the cells are suppressed from falling off from the scaffold. Three-dimensional cells can be made.

Hereinafter, a more specific description will be given with reference to Examples. The present invention is not limited to the following examples.

The measurement / evaluation method is as follows.
<Average fiber diameter>
The scaffold after swelling was observed with an optical microscope (manufactured by KEYENCE CORPORATION, model number BZ-X700), and the average fiber diameter after swelling was measured using 50 fibers arbitrarily selected.
<Thickness>
The cross section of the laminate was observed with a scanning electron microscope (FlexSEM1000 manufactured by Hitachi High-Technologies Corporation, 100 times and 500 times), and 10 gelatin film layer thicknesses and gelatin non-woven fabrics were arbitrarily selected from the obtained scanning electron micrographs. And the thickness of the laminated body were measured, and the average value was calculated.
<Metsuke (mass per unit area)>
The basis weight of the gelatin non-woven fabric was measured according to JIS L 1913.
<Apparent density>
The density of the gelatin non-woven fabric was calculated based on the thickness and basis weight of the non-woven fabric.
<Pore diameter>
The pore diameter of the gelatin non-woven fabric can be calculated by the following formula (1) based on the assumption of Wrotnowski.

(Example 1)
<Preparation of laminate (three-dimensional scaffold for cell culture)>
Nitta Gelatin Co., Ltd. (jelly strength 262 g, raw material: alkali-treated beef bone) was used as gelatin, and the mass ratio of gelatin: water = 92.5: 7.5 (gelatin concentration 7.5% by mass) was used, and the temperature was 60. Dissolved at ° C. This gelatin aqueous solution was placed on a polytetrafluoroethylene film (thickness: 50 μm) on a bar coater No. 1 manufactured by TP Giken Co., Ltd. A gelatin film was obtained by applying at 20 and air-drying at room temperature overnight.
Next, using Nitta Gelatin Co., Ltd. (jelly strength 262 g, raw material: alkali-treated beef bone) as gelatin, the mass ratio of gelatin: water = 3: 5 (gelatin concentration 37.5 mass%) was set, and the temperature was 60 ° C. Dissolved. The viscosity at 60 ° C. was 960-970 mPa · s. A laminate was produced by using this aqueous gelatin solution as a spinning solution and using the production apparatus shown in FIG. 10 to accumulate gelatin fibers on a gelatin film arranged on a take-up roll to form a non-woven fabric. The temperature of the spinning liquid is 60 ° C, the nozzle diameter (inner diameter) is 250 μm, the discharge pressure is 0.2 MPa, the nozzle height is 5 mm, the air pressure is 0.375 MPa, the air temperature is 100 ° C, and the distance between the fluid injection port and the nozzle discharge port is 5 mm. The collection distance was 50 cm. The laminate was air dried overnight at room temperature and then heat dehydrated and crosslinked. The cross-linking conditions were a temperature of 140 ° C. and 48 hours.
The obtained laminate was swollen with purified water and then punched into a cylinder having a diameter of 7 mm to prepare a three-dimensional scaffold for cell culture.
In the laminated body, the weight of the gelatin non-woven fabric was 150 g / m ² , the average fiber diameter after swelling of the gelatin fibers was 47 μm, and the pore diameter was 119.6 to 157.1 μm.
<Cell seeding>
(1) The laminate (three-dimensional scaffold for cell culture) obtained above was allowed to stand in a liquid medium (MEM Alpha basic manufactured by Gibco) for 30 minutes and swollen in the liquid medium.
(2) The swelled laminate was placed in a 96-well plate having a diameter of 6.3 mm at the bottom of the well so that the gelatin film was in contact with the bottom of the well. The laminate was sterilized by washing with 70% ethanol and washing with PBS.
(3) The laminate was pecked to ensure that it was in close contact with the bottom surface and wall surface of the well to prevent bubbles.
(4) A laminate obtained by suspending mouse-derived fibroblast-like MC3T3-E1 cells in a liquid medium (MEM Alpha basic manufactured by Gibco) so as to have a concentration ^{of 1 × 10 6 cells / mL.} 100 μL was added dropwise on the surface of the gelatin non-woven fabric. After allowing the cells to adhere to the laminate by static culture for 4 hours in an incubator at a temperature of 37 ° C. and 5% CO _{2, 100 μL of liquid medium was further added.}
(5) Focusing on the bottom surface of the well, bright field observation was performed with an optical microscope (manufactured by Olympus Corporation, model number CKX53), and the warp of the laminate and the adhesion to the bottom surface and the wall surface of the well were observed.
(6) The laminate was carefully removed, and the cells that fell on the bottom surface of the well were observed in a bright field with an optical microscope (manufactured by KEYENCE CORPORATION, model number BZ-X700). A 1/4 area (that is, 90 °) of the well bottom surface is extracted in a bright field observation photograph, the cell adhesion area is measured by Image J, and the ratio of the cell adhesion area to the 1/4 area of the well is calculated. did.
(7) The laminate was fixed with 4% paraformaldehyde (PFA), dried in a blower dryer at 50 ° C. for 2 to 3 hours, and then the gelatin film portion was subjected to a scanning electron microscope (FlexSEM1000 manufactured by Hitachi High-Technologies Corporation, 500 times). By observing, it was confirmed whether or not the gelatin film was torn.
(8) The laminate was fixed with 4% PFA and then further washed with a phosphate buffer solution (pH 7.4). After cleaning, it is embedded in OCT compound (Sakura Finetech Japan Co., Ltd.), frozen in liquid nitrogen, and in the frozen state, the diameter of the columnar laminate and the vertical direction (maximum split plane of the laminate) become 10 μm thick. Sections were prepared as described above. A cryofilm (manufactured by Leica Microsystems, model number "2C (9)") was used to prepare the sections. Frozen sections were stained with hematoxylin eosin, and the cell distribution in the scaffold was observed with an optical microscope (manufactured by KEYENCE CORPORATION, model number "BZ-X700").

(Example 2)
A three-dimensional scaffold for cell culture was prepared and cell seeding was carried out in the same manner as in Example 1 except that the concentration of the gelatin aqueous solution at the time of preparing the gelatin film was 5% by mass.

(Example 3)
When making gelatin film, TP Giken Co., Ltd. Bar Coater No. A three-dimensional scaffold for cell culture was prepared and cell seeding was carried out in the same manner as in Example 2 except that No. 10 was used.

(Comparative Example 1)
When making gelatin film, TP Giken Co., Ltd. Bar Coater No. A three-dimensional scaffold for cell culture was prepared and cell seeding was carried out in the same manner as in Example 2 except that 40 was used.

(Comparative Example 2)
As gelatin, a product manufactured by Nitta Gelatin Co., Ltd. (jelly strength 262 g, raw material: alkali-treated beef bone) was used, the mass ratio was gelatin: water = 3: 5 (gelatin concentration 37.5 mass%), and the mixture was dissolved at a temperature of 60 ° C. The viscosity at 60 ° C. was 960-970 mPa · s. Using this aqueous gelatin solution as a spinning solution, gelatin fibers were directly accumulated on a take-up roll using the production apparatus shown in FIG. 10 to produce a long-fiber non-woven fabric. The temperature of the spinning liquid is 60 ° C, the nozzle diameter (inner diameter) is 250 μm, the discharge pressure is 0.2 MPa, the nozzle height is 5 mm, the air pressure is 0.375 MPa, the air temperature is 100 ° C, and the distance between the fluid injection port and the nozzle discharge port is 5 mm. The collection distance was 50 cm. The long fiber non-woven fabric was air dried overnight at room temperature and then heat dehydrated and crosslinked. The cross-linking conditions were a temperature of 140 ° C. and 48 hours.
The weight of the obtained gelatin non-woven fabric was 150 g / m ² , the average fiber diameter after swelling of the gelatin fibers was 47 μm, and the pore diameter was 119.6 to 157.1 μm.
The obtained long-fiber non-woven fabric was swollen with purified water and then punched into a cylinder having a diameter of φ7 mm to prepare a three-dimensional scaffold for cell culture.
Cell seeding was carried out in the same manner as in Example 1 except that the three-dimensional scaffold for cell culture obtained above was used.

The results of various measurements and evaluations of the three-dimensional scaffolds for cell culture of Example 1 and Comparative Example 2 are shown in Table 1 below. In Table 1 below, the thickness is measured for the cell culture three-dimensional scaffold before swelling, and Ls is the diameter of the cell culture three-dimensional scaffold after swelling.

As can be seen from the data in Tables 1 and 2, the ratio Tf / Tn of the thickness Tf of the gelatin film to the thickness Tn of the gelatin non-woven fabric is 7 in the three-dimensional scaffold for cell culture in which the gelatin film and the gelatin non-woven fabric are laminated and integrated. In the examples having a size of .5 × 10 ^-3 or less, the shedding of cells from the scaffold was suppressed at the time of cell seeding, and the seeding efficiency was improved.

On the other hand, although the gelatin film and the gelatin non-woven fabric are laminated and integrated, the scaffold of Comparative Example 1 in which the ratio Tf / Tn of the thickness Tf of the gelatin film and the thickness Tn of the gelatin non-woven fabric ^{exceeds 7.5 × 10 -3.} In this case, the laminate was warped due to the difference in the degree of swelling between the gelatin film and the gelatin non-woven fabric, and the inclination caused the cell suspension to flow out to the side surface of the laminate, resulting in significant loss of cells from the scaffold. .. Also, in the case of the scaffold of Comparative Example 2 containing no gelatin film, cells were significantly shed from the scaffold after cell seeding.

(2) Cell distribution In FIG. 11, in Example 1, cells were seeded on a scaffold, left to stand for 4 hours to adhere the cells to a gelatin non-woven fabric, and then a section of the scaffold (scaffold cross section) was stained with hematoxylin eosin. , A photograph of the whole image observed with an optical microscope (observed at 10 times and connected on software) was shown. In FIG. 11, the scale is 1 mm. FIG. 12 shows an enlarged photograph of the gelatin non-woven fabric portion. In FIG. 12, the scale is 100 μm. FIG. 13 shows an enlarged photograph of the gelatin film portion. In FIG. 13, the scale is 100 μm. As is clear from FIGS. 11 to 13, the cells seeded on the surface of the laminated gelatin non-woven fabric have invaded the inside of the scaffold.

The three-dimensional scaffold for cell culture of the present invention can be applied not only to cell culture but also to various medical uses.

1 Heating tank 2 Spinning liquid 3 Nozzle discharge port 4, 6 Compressor 5 Fluid injection port 7 Pressure fluid 8 Gelatin fiber 9 Gelatin non-woven fabric 10 Gelatin film 11 Winding roll 12 Heat insulation container 20 Manufacturing equipment

Claims (9)

In a three-dimensional scaffold for cell culture composed of a laminate containing a gelatin non-woven fabric containing gelatin as a main component and a gelatin film containing gelatin as a main component.
The gelatin fibers constituting the gelatin non-woven fabric have an average fiber diameter of 2 μm or more and 400 μm or less after swelling, and the fiber intersections are at least partially welded.
The gelatin film is laminated on one surface of the gelatin nonwoven fabric and partially welded to the gelatin fibers constituting the gelatin nonwoven fabric.
A three-dimensional scaffold for cell culture, wherein the ratio Tf / Tn of the thickness Tn of the gelatin non-woven fabric to the thickness Tf of the gelatin film is 7.5 × 10 ^{-3 or less.} The three-dimensional scaffold for cell culture according to claim 1, wherein the gelatin non-woven fabric and the gelatin film are thermally dehydrated and crosslinked. The three-dimensional scaffold for cell culture according to claim 1 or 2, wherein the gelatin film has a thickness of 0.80 μm or more and 3.20 μm or less. The three-dimensional scaffold for cell culture according to any one of claims 1 to 3, wherein the gelatin non-woven fabric has a thickness of 0.1 mm or more and 2.0 mm or less and a grain size of 10 g / m ² or more and 600 g / m ^{2 or less.} .. The method for producing a three-dimensional scaffold for cell culture according to any one of claims 1 to 4.
The spinning liquid containing gelatin is extruded into the air from the nozzle discharge port,
A pressure fluid is injected forward from a fluid injection port located behind the nozzle discharge port and in a non-contact state with the nozzle discharge port.
The extruded spinning liquid is associated with the pressure fluid to form fibers, and the fiber-formed fibers are accumulated on a gelatin film containing gelatin as a main component to form a gelatin non-woven fabric, whereby the gelatin film and the gelatin are formed. A method for producing a three-dimensional scaffold for cell culture, which comprises obtaining a laminate of non-woven fabric. The method for producing a three-dimensional scaffold for cell culture according to claim 5, wherein the laminate is thermally dehydrated and crosslinked. The cell seeding method using the three-dimensional scaffold for cell culture according to any one of claims 1 to 4.
The step of arranging the three-dimensional scaffold for cell culture after swelling in the culture vessel so that the gelatin film is in contact with the inner bottom surface of the culture vessel, and the step of dropping the cell suspension onto the gelatin non-woven fabric of the three-dimensional scaffold for cell culture are included. A cell seeding method characterized by this. The inner surface of the culture vessel is hydrophilized, and the ratio of the size Ls after swelling of the three-dimensional scaffold for cell culture to the size Lb of the inner bottom surface of the culture vessel in which the three-dimensional scaffold for cell culture is arranged and cell culture is performed. The cell seeding method according to claim 7, wherein Ls / Lb is 1.05 times or more and 1.3 times or less. In the cell seeding method according to claim 7 or 8, cell culture is performed by dropping a cell suspension onto a gelatin non-woven fabric of a three-dimensional scaffold for cell culture, allowing it to stand for a predetermined time, and then adding a liquid medium to perform cell culture. Method.

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