JP6450894B1 - Biocompatible long-fiber nonwoven fabric, production method thereof, solid scaffold for cell culture, and cell culture method using the same - Google Patents

Abstract

The present invention is a biocompatible long-fiber non-woven fabric mainly composed of a bio-compatible polymer, and the bio-compatible long fiber constituting the bio-compatible long-fiber non-woven fabric has a fiber diameter that changes in the length direction and a fiber intersection point. Relates to a biocompatible long-fiber non-woven fabric that is at least partially welded and does not sag when wet, but becomes transparent when wet. The biocompatible continuous fiber non-woven fabric pushes a spinning solution containing a biocompatible polymer into the air from the nozzle discharge port 3, is located behind the nozzle discharge port 3, and is in a non-contact state with the nozzle discharge port 3. The pressure fluid 7 is ejected from the front to the front, the extruded spinning solution is accompanied by the pressure fluid 7 to form fibers, and the long fibers 8 formed by the fibers are accumulated to form a long fiber nonwoven fabric 9. be able to. Thus, a biocompatible long-fiber nonwoven fabric having high moldability and molding stability useful for medical and cell culture scaffolds, a production method thereof, a solid scaffold for cell culture, and a cell culture method using the same.

Description

  The present invention relates to a biocompatible non-woven fabric suitable for medical use and a cell culture scaffold, a method for producing the same, a three-dimensional scaffold for cell culture, and a cell culture method using the same.

  The fiber sheet is useful for medical use and scaffolds for cell culture because of its high porosity and high specific surface area. In particular, gelatin is a material that has been widely used in medicine so far, and has precedents as a medical device and a pharmaceutical additive. Biocompatibility, cell affinity, and bioabsorbability have been demonstrated. Fibers made of gelatin are highly safe, and the bioabsorption period of several days to several months and the hardness can be controlled depending on the degree of chemical crosslinking of gelatin. For example, gelatin-like molded products of gelatin are actually put into practical use as a local hemostatic material in medical applications. Many three-dimensional molded bodies made of gelatin fibers have been reported. For example, Patent Document 1 proposes that gelatin is fiberized by an electrospinning method to form a nonwoven fabric. Patent Document 2 proposes that an aqueous solution containing a water-soluble linear polymer such as gelatin and polyethylene glycol is extruded into the air and spun. Patent Document 3 proposes that a gelatin solution is discharged into a coagulation bath to form a gel fiber, which is taken out and stretched to remove the remaining solution.

JP 2014-05519 A Japanese Patent Application Laid-Open No. 2012-167397 JP 2005-120527 A

  However, the three-dimensional structure (typified by cotton or non-woven fabric) made of gelatin fibers obtained by the prior art as described above has inferior mechanical properties, low strength when swollen in water, and when used for cell culture. As the cell grows, the molded body deforms, and there is a problem that the pore structure necessary for cell growth inside the molded body is lost. As described above, as a nonwoven fabric having high moldability and molding stability useful for medical and cell culture scaffolding, it is an indispensable material property that can maintain its internal structure during cell culture, and mechanical properties for that purpose. It is mandatory to have

  In order to solve the above-mentioned conventional problems, the present invention provides a biocompatible long-fiber nonwoven fabric having high moldability and high molding stability useful for medical and cell culture scaffolds, a production method thereof, a three-dimensional scaffold for cell culture, and The cell culture method used is provided.

  The biocompatible long fiber nonwoven fabric of the present invention is a biocompatible long fiber nonwoven fabric mainly composed of a biocompatible polymer, and the biocompatible long fiber constituting the biocompatible long fiber nonwoven fabric has a fiber diameter that varies in the length direction. The biocompatible long fibers constituting the biocompatible long-fiber nonwoven fabric are characterized in that the fiber intersections are at least partially welded and do not become dull even when wet, and become transparent when wet. And

  The method for producing a biocompatible long-fiber nonwoven fabric according to the present invention is a method for producing the biocompatible long-fiber nonwoven fabric described above, in which a spinning solution containing a biocompatible polymer is extruded from the nozzle outlet into the air, and the rear of the nozzle outlet. The body is obtained by ejecting a pressure fluid forward from a fluid ejection port that is not in contact with the nozzle ejection port and forming the fibers by causing the extruded spinning solution to accompany the pressure fluid. It is characterized by accumulating compatible long fibers into a non-woven fabric.

  The three-dimensional scaffold for cell culture of the present invention is characterized in that the biocompatible long fiber nonwoven fabric is a three-dimensional scaffold for cell culture.

  The cell culture method of the present invention is a cell culture method using the above-mentioned three-dimensional scaffold for cell culture, wherein cells are seeded on the scaffold, and then the seeded scaffold is placed in a liquid medium and cultured. It is characterized by.

  The three-dimensional scaffold for cell culture of the present invention is composed of a biocompatible long-fiber nonwoven fabric mainly composed of a biocompatible polymer, and the biocompatible long fiber constituting the non-woven fabric is composed of a single continuous fiber. The length is several tens of meters to several hundred meters, and the fiber diameter is changed in the length direction. The biocompatible long fibers constituting the non-woven fabric are medically bonded because the fiber intersections are partially welded. It is possible to provide moldability and molding stability useful for, for example, a three-dimensional scaffold for cell culture. That is, the biocompatible long fiber that constitutes the biocompatible long fiber non-woven fabric has uneven thickness and is partially welded, so that the biocompatible long fiber non-woven fabric has a bridge structure, is bulky and has a low density, and has a desired density. It is easy to mold into a shape and has high molding stability, and will not drip even when wet. This suppresses a change in the three-dimensional structure of the nonwoven fabric during cell culture and a change in the internal structure (void). For this reason, the cells are uniformly distributed and proliferated throughout the biocompatible long fiber nonwoven fabric, and the three-dimensional organization of the cells is achieved. Usually, when cells become three-dimensional, cells existing inside die due to lack of nutrient oxygen. However, the cells inside the three-dimensional tissue body cultured with a non-woven fabric composed of biocompatible long fibers such as the gelatin long fibers of the present invention do not die, and cells are produced by normal physiological production of proteins from the cells. Outer matrix formation was observed. This is probably because the nonwoven fabric material is a biocompatible hydrogel such as gelatin hydrogel, so that supply of nutrient oxygen from the outside through the hydrogel layer was obtained. Such an effect is not recognized in the nonwoven fabric which consists of fibers other than hydrogel reported previously. The biocompatible long-fiber non-woven fabric of the present invention is a non-woven fabric made of hydrogel fibers, and by improving the non-woven fabric preparation method, the mechanical strength is increased even when water is contained, and the deformation of the non-woven fabric during cell culture is suppressed. (Void) maintenance, biocompatible long fibers with high cell affinity, etc. are overlapped and the physiological environment of the cells is adjusted, so that cells can grow inside and outside the nonwoven fabric during cell culture It is considered that the three-dimensional organization of cells has become possible. The three-dimensional organization of cells is a technique necessary for cell research and drug discovery research. This is because it is known that the cell functions are enhanced by the three-dimensional interaction of the cells compared to the cells one by one and approach the cell functions in the body. The biocompatible long-fiber nonwoven fabric of the present invention is a bulky three-dimensional material made of hydrogel. When used as a scaffold by adding water or a liquid medium, the culture solution and oxygen are transferred to the hydrogel in the nonwoven fabric. It is easy to go around the entire scaffold through the gel fiber material, and nutrients and oxygen are efficiently supplied to the cells throughout the scaffold, enabling three-dimensional cell culture. Since the biocompatible long-fiber nonwoven fabric of the present invention is firm and firm even when wet with water, cell culture can be performed while flowing the liquid medium by at least one means selected from stirring, circulation and shaking. it can. In addition, it is transparent when wet and easy to observe with a microscope. This transparency is such that the inside of the scaffold can be observed with an inverted microscope in the culture solution. If it is transparent enough to be observed with an inverted microscope, the culture state can be confirmed, which is convenient. Furthermore, since the nonwoven fabric is composed of long fibers, there is a low risk of foreign matter contamination such as fiber dropping and the occurrence of dust. In addition, it is biocompatible. In addition, since an example of the biocompatible long fiber nonwoven fabric of the present invention is composed of a crosslinked hydrogel, when the hydrogel is swollen with a phosphate buffer or the like, by adding a drug such as bFGF, the hydrogel It can also be used as a carrier for taking a drug inside and releasing the drug slowly. Therefore, the biocompatible long fiber nonwoven fabric of the present invention is suitable as a scaffold for medical use or cell culture. In the production method of the present invention, the biocompatible long-fiber nonwoven fabric can be reasonably produced hygienically and efficiently without contamination.

FIG. 1 is a photograph of a scanning electron microscope (SEM, Hitachi scanning microscope S-2600N, 100 times) of a gelatin long fiber nonwoven fabric obtained in one example of the present invention. FIG. 2 is a photograph of a scanning electron microscope (SEM, Hitachi scanning microscope S-2600N, 500 times) of the gelatin long-fiber nonwoven fabric. FIG. 3 is a photograph of a gelatin long fiber nonwoven fabric (length 35 mm, width 25 mm, thickness 0.95 mm) obtained in one example of the present invention. FIG. 4 is a schematic explanatory view of a nonwoven fabric manufacturing apparatus used in one embodiment of the present invention. FIG. 5 is a photograph showing the shape maintainability when the gelatin long fiber nonwoven fabric of Example 1 of the present invention is swollen in purified water and held with tweezers. FIG. 6 is a photograph showing the shape maintenance of the gelatin long-fiber nonwoven fabric of Example 2. FIG. 7 is a photograph showing the shape maintenance of the gelatin long-fiber nonwoven fabric of Example 3. FIG. 8 is a photograph showing the shape maintaining property of the gelatin long fiber nonwoven fabric of Example 4. FIG. 9 is a photograph showing the shape maintainability of the gelatin sponge swollen in the purified water of Comparative Example 1. FIG. 10 is an inverted microscopic photograph (20 ×) immediately after cell seeding when the cells were cultured using the scaffold made of the gelatin long-fiber nonwoven fabric of Example 4. FIG. 11 is an inverted microscope observation photograph (20 times) on the seventh day of culture. FIG. 12 is an inverted microscope observation photograph (10 times) immediately after cell seeding when the cells were cultured using the gelatin sponge of Comparative Example 1 as a scaffold. FIG. 13 is an inverted microscope observation photograph (10 times) immediately after cell seeding when the cells were cultured using the polypropylene nonwoven fabric of Comparative Example 2 as a scaffold. FIG. 14 is an inverted microscope observation photograph (10 times) immediately after cell seeding when cell culture is performed using the polylactic acid nonwoven fabric of Comparative Example 3 as a scaffold. FIG. 15 is an optical micrograph of a scaffold section stained with hematoxylin and eosin showing that cells have invaded the inside of the scaffold when cell culture was performed for 8 days using the scaffold made of the gelatin long-fiber nonwoven fabric of Example 4. It is. The photograph (10 times) observed with the optical microscope was connected on the software of the fluorescence microscope to obtain a whole image. FIG. 16 is a partial photograph (10 ×) observed with an optical microscope. FIG. 17 is an optical micrograph (4 ×) of a scaffold section stained with hematoxylin and eosin showing that the cells have penetrated only to the outside of the scaffold when cell culture was performed for 4 days using the polypropylene nonwoven fabric of Comparative Example 2 as a scaffold. ). FIG. 18 is an optical microscope observation photograph (20 ×) of the scaffold when cell culture was performed for 12 days using the gelatin long-fiber nonwoven fabric of Example 4 as the scaffold. FIG. 19 shows a fluorescence observation micrograph (high lightness portion (white portion) when the oxygen state in the scaffold was observed with a hypoxia marker when cell culture was performed for 12 days using the gelatin long-fiber nonwoven fabric of Example 4 as a scaffold. (Part close to): hypoxic site) (20 times). FIG. 20 is a graph showing cell proliferation. FIG. 21 is a schematic explanatory diagram of a cell culture device when performing agitation culture using a scaffold made of biocompatible long-fiber nonwoven fabric according to one embodiment. FIG. 22 is a photograph showing the change in the shape of the scaffold (observed from the lateral direction) during stirring culture. FIG. 23 is a photograph showing an appearance (observed from above) showing that the shape of the scaffold has changed after stirring culture. FIG. 24 is a photograph showing the appearance (observed from above) of the scaffold subjected to decellularization after stirring culture. FIG. 25 is a photograph showing the result (overall image) of hematoxylin and eosin staining of the cross section of the scaffold 14 days after stirring culture. FIG. 26 is a photograph showing the result of hematoxylin eosin staining (20 times) of the cross section of the scaffold 14 days after stirring culture. FIG. 27 is a photograph showing the result of hematoxylin and eosin staining (20 times) of a cross-section of a scaffold decellularized after 14 days of stirring culture. FIG. 28 is a photograph showing the result of hematoxylin and eosin staining of the scaffold cross section (overall image) after 25 days of stirring culture. FIG. 29 is a photograph showing the result of hematoxylin and eosin staining (20 times) of the cross section of the scaffold after 25 days of stirring culture. FIG. 30 is a photograph showing the result of hematoxylin and eosin staining (overall image) of the cross section of the scaffold subjected to decellularization after 25 days of stirring culture. FIG. 31 is a photograph showing the result of hematoxylin and eosin staining (20 ×) of the cross section of the scaffold subjected to decellularization after 25 days of stirring culture. FIG. 32 is a photograph showing a fluorescence microscope observation image (overall image) of a living cell having a cross section of the scaffold 14 days after stirring culture. FIG. 33 is a photograph showing a fluorescence microscope observation image (overall image) of dead cells on the cross section of the scaffold after 25 days of stirring culture. FIG. 34 is a photograph showing a fluorescence microscope observation image (overall image) of the living cells of the cross section of the scaffold after 25 days of stirring culture. FIG. 35 is a photograph showing a fluorescence microscope observation image (overall image) of dead cells on the cross section of the scaffold after 25 days of stirring culture. FIG. 36 is an explanatory diagram of a process for culturing cells by laminating a plurality of scaffolds made of gelatin long-fiber nonwoven fabric according to one embodiment of the present invention. FIG. 37 is a photograph showing the shape maintainability when the gelatin long-fiber nonwoven fabric of Example 6 was swollen in purified water and held with tweezers. FIG. 38 is a photograph showing an example in which the shape maintaining property is good when the scaffold is grasped with tweezers but is bent. FIG. 39 is an optical microscope (4 ×) photograph showing the appearance (observed from above) of the laminate of the scaffold and the cell sheet during the lamination culture using the gelatin long-fiber nonwoven fabric scaffold of Example 6; It is connected on the software to make a whole picture. FIG. 40 is a photograph (4 times) showing a cross section of the laminate cross section as a result of staining with hematoxylin and eosin. FIG. 41 is a photograph showing an example of good handling properties in the evaluation of handling properties by cantilever beams. FIG. 42 is a photograph showing an example of poor handling properties. FIG. 43 is a graph showing the relationship between handling property, thickness, and density when a scaffold made of gelatin long fiber nonwoven fabric is held with tweezers.

  The biocompatible long fiber nonwoven fabric of the present invention is a nonwoven fabric composed of long fibers. The length of the fiber is preferably several meters to several thousand meters. Long fibers can be produced by the melt blow method. In the melt blow method used in the present invention, a spinning solution containing a biocompatible polymer is extruded from a nozzle discharge port, is located behind the nozzle discharge port, and is pressured forward from a fluid discharge port in a non-contact state with the nozzle discharge port. Contamination (contamination) is generated by injecting the fluid, making the extruded spinning solution accompanying the pressure fluid directly into a dry fiber, and accumulating the resulting biocompatible long fibers into a non-woven fabric. Is prevented and can be manufactured hygienically. This long fiber is a non-divided fiber and is not subjected to a treatment such as division after spinning. In this nonwoven fabric, when fibers are accumulated (deposited) after spinning, the fibers are laminated in a state containing moisture, so that they are welded or entangled with each other. The nonwoven fabric density can be easily changed by changing the collection distance when the fibers are deposited.

  In contrast, the conventional melt-blowing method injects pressurized air from around the nozzle outlet of the spinning solution, so the spinning solution may leak into the pressure-air injection port, and the spinning solution that has accumulated for a long period of time is contained in the product. There was a phenomenon of contamination as a contaminant and product contamination.

  In the present invention, the biocompatible long fiber nonwoven fabric has a biocompatible polymer as a main component. Here, the main component means that 90% by mass or more of the biocompatible polymer is contained. The component of 10% by mass or less may be a crosslinking agent, a drug, another additive, and the like. The biocompatible polymer may be substantially 100% by mass. A commercially available biocompatible polymer can be used.

  The biocompatible long fibers constituting the biocompatible long fiber nonwoven fabric have a fiber diameter that changes in the length direction. The change in the fiber diameter in the length direction is caused by the turbulence (turbulent flow) of the pressure fluid because the spinning solution discharged from the nozzle is spun along with the pressure fluid (for example, pressurized air). Further, the change in the fiber diameter in the length direction also occurs when the discharge speed of each nozzle and / or the flow rate of the pressure fluid are different when a plurality of nozzles are used.

  In the biocompatible long fibers constituting the biocompatible long fiber nonwoven fabric, the fiber intersections are partially welded. This partial welding occurs when biocompatible long fibers such as gelatin long fibers blown away by the pressure fluid are deposited and are not completely solidified. By this partial welding, the biocompatible long-fiber non-woven fabric has a bridge structure, is bulky and has a low density, is easily molded into a desired shape, and has high molding stability. In the present invention, “the fiber intersection is at least partially welded” means that the biocompatible long fiber nonwoven fabric is welded to the extent that it does not sag even when wet. Some of the fiber intersections may be welded to such an extent that they do not sag even when wet with water, or all of the fiber intersections may be welded.

  The biocompatible continuous fiber preferably has an average fiber diameter (D) in the range of 1 to 70 μm, and the fiber diameter is changed in the range of D ± 0.5D. A more preferable range of the average fiber diameter (D) is 5 to 60 μm. When the diameter of the biocompatible long fiber and the degree of change thereof are as described above, it is further useful for medical use and cell culture scaffolds. In the present invention, “average fiber diameter” and “degree of change” are respectively the average value of the diameters of 50 fibers arbitrarily selected from scanning electron micrographs (500 times) of biocompatible long-fiber nonwoven fabrics, and It means the degree of change.

  The biocompatible continuous fiber is preferably substantially unstretched. In the above, the substantially unstretched state refers to a state in which mechanical stretch has not been applied. When the spinning solution discharged from the nozzle is blown off by accompanying the compressed air, the fibers become thin but are substantially unstretched.

The biocompatible long-fiber nonwoven fabric does not drip even when wet. In the present invention, “does not drip even when wet with water” means a compressive deformation rate at a compressive stress of 1.0 kPa after swelling to a saturated state of a biocompatible non-woven fabric with water (hereinafter simply referred to as “compression”). It is also referred to as “deformation rate”.) Means 40% or less. The saturated state is a state in which water is contained to the maximum extent, and means a state in which the water content remains at a certain limit and does not increase further. The compressive deformation rate of the biocompatible long fiber nonwoven fabric is preferably 40% or less, more preferably 35% or less, and further preferably 30% or less. In addition, the lower limit of the compressive deformation rate of the biocompatible continuous fiber nonwoven fabric is not particularly limited, but is preferably 1% or more, and more preferably 5% or more, for example. In addition, the biocompatible long-fiber nonwoven fabric has an initial elastic modulus after swelling to a saturated state with water (hereinafter, also simply referred to as “initial elastic modulus”) from the viewpoint of not sagging even when wet. The pressure is preferably 30 kPa or more, more preferably 0.35 kPa or more. The upper limit of the initial elastic modulus of the biocompatible long fiber nonwoven fabric is not particularly limited, but is preferably 20 kPa or less, and more preferably 10 kPa or less, for example. In the present invention, the compressive deformation rate is defined as the thickness when no load is applied to the biocompatible long-fiber nonwoven fabric after swelling to a saturated state with water (H1), and the thickness at a compressive stress of 1.0 kPa ( In the case of H2), it is calculated by the following formula. When the compressive deformation rate after swelling with water is in the above-described range, it does not drip even when wet. In the present invention, the initial elastic modulus means a compressive elastic modulus with a strain of 1 to 5%. The compression test is performed as described below.
Compression deformation rate (%) = 100 − {(H2 / H1) × 100}

  The biocompatible long-fiber nonwoven fabric becomes transparent when wet with water. This transparency is such that the inside of the scaffold can be observed with an inverted microscope in the culture solution. If it is transparent enough to be observed with an inverted microscope, the culture state can be confirmed, which is convenient. Specifically, in the present invention, “transparent when wet with water” means in a wavelength range of 400 to 800 nm of a biocompatible long-fiber nonwoven fabric (thickness 1.5 mm) after swelling to a saturated state with water. It means that the average transmittance is 10% or more. The average transmittance in the wavelength range of 400 to 800 nm of the biocompatible long-fiber nonwoven fabric (thickness 1.5 mm) after swelling to saturation with water is preferably 15% or more, more preferably 20% or more. .

  The biocompatible long fiber nonwoven fabric is preferably formed in a sheet shape. If it is a sheet, it can be formed into various shapes.

  The biocompatible long fiber nonwoven fabric is preferably crosslinked. Thereby, shape stability and heat resistance can be maintained. Crosslinking is preferably heat dehydration crosslinking for biological safety. In addition, the biocompatible long fiber or the biocompatible polymer before crosslinking is preferably water-soluble. When the biocompatible polymer is water-soluble, it can be spun in the state of an aqueous solution as a spinning solution, and the safety to the living body can be increased.

  The biocompatible polymer is at least one selected from the group consisting of water-soluble polymers such as gelatin, collagen, chitosan, alginic acid, hyaluronic acid, mucopolysaccharide, dextran, carboxymethylcellulose, polyvinyl alcohol, polyethylene glycol, and modified products thereof. A polymer is preferred. Here, the “modified product” means that the molecular ends, side chains and the like are modified to such an extent that the water solubility is not impaired. These are preferable because they are easily dissolved in water (including heated water), can be solution-spun, can be gelled and cross-linked after fiber formation, and can be formed into a nonwoven fabric with high form stability. Of these, gelatin is more preferable.

  In the method for producing a biocompatible long-fiber nonwoven fabric of the present invention, a spinning solution containing a biocompatible polymer is extruded from the nozzle discharge port into the air, is located behind the nozzle discharge port, and is in a non-contact state with the nozzle discharge port. A pressure fluid is ejected forward from the fluid ejection port, the extruded spinning solution is caused to form a fiber along with the pressure fluid, and the obtained biocompatible long fibers are accumulated to form a nonwoven fabric. In the present invention, a manufacturing apparatus used for manufacturing a biocompatible long-fiber nonwoven fabric includes: a means for extruding a spinning solution containing a biocompatible polymer into air from a nozzle discharge port; and the nozzle discharge port located behind the nozzle discharge port. Includes means for ejecting pressure fluid forward from a fluid ejection port in a non-contact state, and means for accumulating biocompatible long fibers formed by the extruded spinning solution accompanying the pressure fluid. Since the pressure fluid injection port is disposed rearwardly in a non-contact state independently of the nozzle discharge port, the spinning solution is not mixed. For this reason, contamination can be prevented from entering the product.

  The temperature of the spinning solution is preferably not less than the temperature at which the spinning solution flows and less than the decomposition temperature of the biocompatible polymer. If the temperature is not higher than the temperature at which the spinning solution flows, spinning cannot be performed. If the temperature is higher than the decomposition temperature of the biocompatible polymer, the decomposition product may be mixed into the product.

  The pressure pressure of the pressure fluid is preferably 0.05 to 0.5 MPa. If it is the said range, it can fiberize by blowing off the spinning liquid extruded in the air from the nozzle discharge port. The temperature of the pressure fluid is preferably around the temperature of the spinning solution, more preferably the temperature of the pressure fluid is the temperature of the spinning solution ± 50 ° C., and more preferably the temperature of the spinning solution ± 30 ° C. In this state, the spinning solution extruded from the nozzle discharge port into the air is not rapidly cooled, but is fiberized in a fluidized state, and then cooled in the air to form solid fibers.

  The spinning solution preferably has a viscosity of 500 to 3000 mPa · s at a temperature of 60 ° C. If the viscosity is within the above range, it is convenient for fiberization.

  The biocompatible long fiber nonwoven fabric is preferably freeze-dried in vacuum and then crosslinked. The reason for vacuum freeze-drying is to prevent the deterioration of the biocompatible long fiber (biocompatible polymer) and to dry it rapidly. Then, it is preferable to crosslink because of shape stability. The cross-linking may be cross-linking using a chemical reagent, heat dehydration cross-linking, photo cross-linking, energy ray cross-linking such as ultraviolet ray, electron beam, radiation, or the like. The temperature of heat dehydration crosslinking varies depending on the type of biopolymer, but is preferably, for example, from the glass transition point to the softening point. The heat dehydration crosslinking temperature of the gelatin long fiber is preferably 100 to 160 ° C.

  Hereinafter, the gelatin long fiber nonwoven fabric will be described as an example. The gelatin long fiber nonwoven fabric is suitable for a medical nonwoven fabric or a scaffold for cell culture. Gelatin itself is biocompatible and biodegradable, and it is easy to use if it is a non-woven fabric.

The method for producing a gelatin long fiber nonwoven fabric includes the following steps.
1. Preparation step (1) Gelatin is dissolved in heated water. The dissolution temperature (heated water temperature) is preferably 20 to 90 ° C. After dissolution, it may be filtered to remove foreign matter or dust.
(2) Thereafter, the dissolved air may be removed by decompression or vacuum degassing.
2. Step (1) A heated gelatin aqueous solution (spinning solution) is discharged from a nozzle of a spinning machine.
(2) A pressure fluid is supplied from the periphery of the nozzle, and the discharged gelatin aqueous solution is accompanied with the pressure fluid to form fibers.
(3) The obtained gelatin long fibers are accumulated to obtain a gelatin long fiber nonwoven fabric.
3. Post-process (1) The gelatin long fiber nonwoven fabric is vacuum freeze-dried.
(2) The dried gelatin long fiber nonwoven fabric may be cut into a predetermined size and formed into a predetermined shape. For the molding, press molding or the like can be used.
(3) Crosslinking the gelatin long fiber nonwoven fabric. For crosslinking, heat dehydration crosslinking, thermal crosslinking, electron beam crosslinking, radiation crosslinking such as γ rays, ultraviolet crosslinking, or the like can be employed.
(4) The gelatin long fiber nonwoven fabric or sheet having a predetermined shape is sterilized. Sterilization can be performed using ethylene oxide gas sterilization, water vapor, electron beam irradiation, irradiation with γ rays, or the like. In the case of radiation such as electron beam irradiation and γ-ray irradiation, crosslinking can be carried out simultaneously with sterilization.
4). Sterilization at the time of use As a preparatory step for use in medical or cell culture scaffolding, ethylene oxide sterilization or steam sterilization may be performed. The cross-linked gelatin long fiber nonwoven fabric can be steam sterilized.

  The temperature of the heated gelatin aqueous solution (spinning solution) is preferably 20 to 90 ° C. If it is the said range, gelatin can maintain the stable sol state. The gelatin concentration of the heated gelatin aqueous solution is preferably 30 to 55% by mass when the gelatin aqueous solution is 100% by mass. A more preferable concentration is 35 to 50% by mass. If it is the said density | concentration, the stable sol state can be maintained. The viscosity of the heated gelatin aqueous solution (spinning solution) is preferably 500 to 3000 mPa · s. With the above viscosity, stable spinning can be performed.

  The temperature of the pressure fluid is preferably 80 to 120 ° C. in the case of an aqueous gelatin solution (spinning solution). Although depending on the flow rate of the pressure fluid and the temperature of the surrounding atmosphere, stable spinning can be performed within the above temperature range. The pressure fluid is preferably air, and the pressure is preferably 0.1 to 1 MPa.

The three-dimensional scaffold for cell culture of the present invention is produced using the biocompatible long-fiber nonwoven fabric. As an example, the non-woven fabric (sheet) after cross-linking is molded by punching it into a predetermined shape to obtain a cell culture scaffold. Alternatively, after swelling with a predetermined liquid medium, a target scaffold for cell culture is obtained. The scaffold thickness before swelling (dry state) in the liquid medium is preferably 0.2 mm or more, more preferably 0.3 mm or more and 10 mm or less. When the thickness is in the above-mentioned range, when it is swollen with water or a liquid medium and then gripped with tweezers, the shape maintaining property is high, and it is difficult to bend and the handling property is good. Further, when used for layered culture, the density of the scaffold before swelling (dry state) in the liquid medium is preferably 0.8 g / cm ³ or more, more preferably 1.0 g / cm ³ or more and 1.3 g / cm ^3. It is as follows. When the density is within the above range, when used for layered culture, when it is swelled with water or a liquid medium and then gripped with tweezers, the shape maintaining property is high and it is difficult to bend and the handling property is good.

Cell culture using the scaffold is not particularly limited, and can be performed, for example, as follows. First, a target cell is suspended in a liquid medium at a predetermined concentration to prepare a cell suspension, and a scaffold made of biocompatible long fiber nonwoven fabric such as a gelatin long fiber nonwoven fabric scaffold is prepared in the same liquid medium. And let stand for a predetermined time (for example, 30 minutes) to swell sufficiently. Next, a swollen scaffold made of biocompatible long fiber nonwoven fabric is placed in a tube or a culture dish, to which a cell suspension is added, shaken on a shaker in an incubator, and seeded with cells. The cell suspension may be a suspension of one type of cell or a suspension of two or more different types of cells. That is, cell seeding may be performed by seeding a single cell or simultaneously seeding different cell types. After cell seeding, the scaffold made of biocompatible long fiber nonwoven fabric is washed with a phosphate buffer solution to remove unadhered cells, and a cell seeded scaffold made of biocompatible long fiber nonwoven fabric is obtained. The cell-seeded scaffold made of biocompatible long-fiber nonwoven fabric may be transferred to a plate or a culture dish, and a liquid medium may be added, followed by stationary culture in an incubator under predetermined conditions (for example, temperature 37 ° C., 5% CO ₂ ). The liquid medium may be changed every 2-3 days. Alternatively, a cell-seeded biocompatible long-fiber nonwoven fabric scaffold is placed in a culture vessel, and a liquid medium is added until the cell-seeded biocompatible long-fiber nonwoven fabric scaffold is immersed, in a 37 ° C., 5% CO ₂ incubator. The agitation culture may be performed while the liquid medium is agitated and circulated on a magnetic stirrer. The medium may be changed every 3 to 4 days by removing half of the liquid medium and adding an equal amount of new liquid medium. Alternatively, a cell-seeded biocompatible long-fiber nonwoven fabric scaffold is placed in a culture vessel, and a liquid medium is added until the cell-seeded biocompatible long-fiber nonwoven fabric scaffold is immersed, in a 37 ° C., 5% CO ₂ incubator. You may culture | cultivate, making it shake. The medium may be changed every 3 to 4 days by removing half of the liquid medium and adding an equal amount of new liquid medium. The biocompatible long fiber nonwoven fabric and the solid scaffold for cell culture using the biocompatible long fiber nonwoven fabric of the present invention become transparent when wet. This transparency is such that the inside of the scaffold can be observed with an inverted microscope in the culture solution. If it is transparent enough to be observed with an inverted microscope, the culture state can be confirmed, which is convenient. As described above, when cells are seeded on a biocompatible long-fiber nonwoven fabric scaffold and cell culture is performed, a three-dimensional cell aggregate is obtained as described later. The obtained three-dimensional cell aggregate can be used in combination with a cell sheet of a single cell or a cell sheet of a plurality of types of cells.

Further, cell culture may be performed by laminating a plurality of scaffolds made of biocompatible long-fiber nonwoven fabric. For example, cell culture can be performed by alternately laminating a plurality of cell sheets obtained by cell culture and scaffolds made of biocompatible long-fiber nonwoven fabric swollen with a liquid medium. From the viewpoint of improving the handling property when performing the layered culture, as described above, when used for the layered culture, the thickness of the scaffold before swelling (dry state) in the liquid medium is 0.2 mm or more, and the density is 0. It is preferable that it is 0.8 g / cm ³ or more. In the method of culturing a plurality of cells, the cell sheet is not limited to the same cell type, and a cell sheet composed of a plurality of types of cells and a cell sheet of a plurality of types of different cells may be laminated and co-cultured.

  Next, it demonstrates using drawing. FIG. 1 is a photograph of a scanning electron microscope (SEM, Hitachi scanning microscope S-2600N, 100 ×) of a gelatin long fiber nonwoven fabric obtained in one example of the present invention. FIG. 2 is a photograph of a scanning electron microscope (SEM, Hitachi scanning microscope S-2600N, 500 times) of the gelatin long-fiber nonwoven fabric. It turns out that the fiber diameter of the gelatin long fiber which comprises a nonwoven fabric is changing in the length direction. Moreover, it can also confirm that the fiber intersection is partially welded to the gelatin long fiber which comprises a nonwoven fabric.

  FIG. 3 is a photograph of the long fiber nonwoven fabric (length 35 mm, width 25 mm, thickness 0.95 mm) obtained in one example of the present invention. According to the method for producing a biocompatible long-fiber nonwoven fabric of the present invention, a long sheet-shaped nonwoven fabric can be obtained. In view of handling in the case of a three-dimensional scaffold for cell culture, the size is set as an example. be able to.

FIG. 4 is a schematic explanatory view of a nonwoven fabric manufacturing apparatus used in one embodiment of the present invention. In the nonwoven fabric manufacturing apparatus 10, the spinning solution 2 containing the biocompatible polymer placed 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 a compressor 4. Reference numeral 12 denotes a heat retaining container.
Further, the pressure fluid 7 is ejected forward from a fluid ejection port 5 that is located behind the nozzle ejection port 3 and is not in contact with the nozzle ejection port 3. A pressure fluid (for example, compressed air) is supplied to the fluid ejection port 5 from the compressor 6. The distance between the fluid ejection port 5 and the nozzle ejection port 3 is preferably 5 to 30 mm.
The extruded spinning solution is accompanied by the pressure fluid 7 to become long fibers 8 and is deposited on the take-up roll 11 as long fiber nonwoven fabrics 9. At this time, since the accumulated long fibers contain moisture or are not completely solidified, the fibers that are in contact at least at a part of the fiber intersections are welded to each other. In addition to the take-up roll, long fibers may be collected and deposited by a net or the like to form a nonwoven fabric.

  FIG. 21 is a schematic explanatory diagram of a cell culture device when performing agitation culture using a scaffold made of a biocompatible long-fiber nonwoven fabric according to one embodiment. The stirring culture test apparatus 20 includes a stirring bar 22 and a stirring bar 23 in a spinner flask 21, and the liquid medium 24 is stirred by rotating the stirring bar 22. For example, a scaffold 26 made of a biocompatible non-woven fabric passed through a 22-gauge needle 25 is placed in the liquid medium 24, and cell culture is performed. A predetermined atmospheric gas may be fed into the flask. In addition to stirring, the culture solution may be shaken or circulated. When cells are cultured in this way, large three-dimensional cell aggregates are obtained in a short period of time, and the cells have a high survival rate within the aggregates. In addition, the obtained three-dimensional cell aggregate may lose a scaffold made of a biocompatible long fiber nonwoven fabric such as a gelatin long fiber nonwoven fabric, and may be replaced with cells and an extracellular matrix produced by the cells. Furthermore, the obtained three-dimensional cell aggregate exhibits high strength. The three-dimensional cell aggregates thus obtained can be used in appropriate combination with other single cell cell sheets or cell sheets of plural types of cells.

  FIG. 36 is an explanatory diagram of a process for culturing cells by laminating a plurality of scaffolds made of biocompatible long-fiber nonwoven fabric according to an embodiment of the present invention. As shown in (a), a cell suspension 32 having a predetermined concentration is seeded in a culture dish 31 containing a predetermined liquid medium 30, and statically cultured for a predetermined time (for example, 10 days), As shown in (b), the cell sheet 33 is produced. Next, as shown in (c), the cell sheet 33 is peeled off from the culture dish by a pipette flow. After removing the liquid medium 30 from the culture dish 31, a scaffold 34 made of a biocompatible non-woven fabric that has been swollen in a predetermined liquid medium for a predetermined time (for example, 30 minutes) is held by tweezers, and (d) As shown in Fig. 4, the cell sheet 33 is placed on the peeled cell sheet 33. Next, as shown in (e), the cell sheet 33 and the scaffold 34 can be integrated by pushing them from above with a predetermined pressure 35 (for example, 1 kPa) for several seconds. A cell sheet 33 and a scaffold 34 integrated with each other are held with tweezers, and a plurality of sheets are laminated as shown in FIG. In this way, a large three-dimensional cell mass is produced. In the past, the scaffolds were sucked up with a pipette and laminated, which required time and labor, a low success rate, and skill. Compared with this, since the scaffold made of biocompatible long fiber nonwoven fabric such as the gelatin long fiber nonwoven fabric scaffold of the present invention can be grasped with tweezers, the handleability can be improved dramatically. The operation of gripping with tweezers is performed as shown in FIGS. 6-9, for example. FIG. 36 illustrates a method of culturing a plurality of cells by laminating cell sheets and scaffolds, but the cell suspension is seeded on a scaffold made of gelatin long-fiber nonwoven fabric that has been swollen for a predetermined time in a predetermined liquid medium. A plurality of scaffolds after cell culture obtained by time cell culture may be laminated to produce a large three-dimensional cell mass. The scaffold after cell culture obtained by culturing cells for a predetermined time may infiltrate cells inside the scaffold. Alternatively, the cells may be cultured so as to remain on the scaffold surface to have a cell sheet-like structure. In the method of culturing a plurality of cells, the cell sheet is not limited to the same cell type, and a cell sheet composed of a plurality of types of cells and a cell sheet of a plurality of types of different cells may be laminated and co-cultured.

  Hereinafter, more specific description will be made using examples. In addition, this invention is not limited to the following Example.

The measuring method is as follows.
<Fiber diameter>
Using 50 fibers arbitrarily selected from a scanning electron microscope (SEM, Hitachi scanning microscope S-2600N, 500 times) photograph of a biocompatible long-fiber nonwoven fabric, the average fiber diameter and the degree of change were measured. .
<Others>
As described later, the measurement was performed according to a measurement method defined by JIS or industry.

Example 1
As the gelatin, Nitta Gelatin Co., Ltd. (jelly strength: 262 g, raw material: alkali-treated beef bone) was used, and the mass ratio of gelatin: water = 3: 5 (gelatin concentration: 37.5% by mass) was dissolved at 60 ° C. The viscosity at 60 ° C. was 960 to 970 mPa · s. Using this gelatin aqueous solution as a spinning solution, a nonwoven fabric nonwoven fabric was produced using the nonwoven fabric production apparatus shown in FIG. The temperature of the spinning solution is 60 ° C., the nozzle diameter (inner diameter) is 250 μm, the discharge pressure is 0.3 MPa, the nozzle height is 5 mm, the air pressure is 0.3 MPa, the air temperature is 100 ° C., and the distance between the fluid injection port 5 and the nozzle discharge port 3 is The long fiber nonwoven fabric 9 was wound up by a winding roller 11 with a collection distance of 20 mm and a collection distance of 100 cm.
Subsequently, the long fiber nonwoven fabric was freeze-dried in vacuum (−80 ° C., 13 Pa, 72 hours), and then heat dehydrated and crosslinked. The crosslinking conditions were a temperature of 140 ° C. and 48 hours.

  The obtained nonwoven fabric of Example 1 was observed with a scanning electron microscope (SEM, Hitachi scanning microscope S-2600N), and the results are shown in FIG. 1 (100 times) and FIG. 2 (500 times). When the diameters of 50 fibers arbitrarily selected from the scanning electron micrograph (500 times) of the biocompatible long fiber nonwoven fabric of FIG. 2 were measured, the average fiber diameter of the long fibers constituting the nonwoven fabric was 51 μm, 51 The fiber diameter changed in the range of ± 20 μm. Moreover, as shown in FIGS. 1-2, the component fiber was welded partially in the fiber intersection.

  The obtained nonwoven fabric of Example 1 was punched into a cylindrical shape having a diameter of 4 mm. The punched nonwoven fabric sample was allowed to stand in purified water at 37 ° C. overnight, fully swollen until saturated, and confirmed for shape maintenance when held with tweezers. The results are shown in FIG. As shown in FIG. 5, the nonwoven fabric of Example 1 had good shape maintenance after swelling, and even when held with tweezers after swelling, the shape was maintained and handling properties were excellent. Further, after measuring the diameter of the swollen nonwoven fabric sample, the thickness (H1) was measured with a creep meter physical property test system RE2-30005C manufactured by Yamaden Co., Ltd., and a 0.05 mm diameter plunger was used. Compressed at / sec. The compression elastic modulus (kPa) with a strain of 1 to 5% and the intermediate compressive stress (kPa) with a strain of 10%, 20% and 30% were measured. Further, the thickness change rate (strain rate) at the time of compressive stress of 1 kPa was calculated based on the thickness at the time of no load (strain 0%) as the compressive deformation rate. A compression elastic modulus (kPa) with a strain of 1 to 5% corresponds to the initial elastic modulus. Moreover, the transmittance | permeability in the wavelength range 400-800nm of the swollen nonwoven fabric sample (thickness 1.5mm) was measured at 10nm intervals using the plate reader company make, model number "Spectra Max i3", and the range of 400-800nm. The average transmittance was calculated.

(Example 2)
A nonwoven fabric was produced in the same manner as in Example 1 except that the discharge pressure was 0.15 MPa, and the physical properties were measured in the same manner as in Example 1.

Example 3
A nonwoven fabric was produced in the same manner as in Example 1 except that the discharge pressure was 0.2 MPa, and the physical properties were measured in the same manner as in Example 1.

(Example 4)
A non-woven fabric was prepared in the same manner as in Example 1 except that the discharge pressure was 0.1 MPa, the air pressure was 0.2 MPa, and the collection distance was 50 cm, and the physical properties were measured in the same manner as in Example 1.

(Example 5)
The nonwoven fabric was produced in the same manner as in Example 4 except that the production time of the nonwoven fabric was increased, and after swelling, the thickness at no load was 3.03 mm, and the physical properties were measured in the same manner as in Example 1. did.

(Example 6)
The nonwoven fabric was prepared in the same manner as in Example 4 except that the nonwoven fabric production time was reduced and the thickness at the time of no load after swelling was 0.69 mm. The physical properties were measured in the same manner as in Example 1 except that.

(Comparative Example 1)
As Comparative Example 1, gelatin sponge after thermal crosslinking (Pfizer Co., Ltd., trade name “Zelfoam”) was sliced to an arbitrary thickness, punched out to a diameter of 4 mm, and left in purified water at 37 ° C. overnight. The sample was sufficiently swollen until saturated, and the physical properties were measured in the same manner as in Example 1.

  The above conditions and results are summarized in Table 1. In addition, the results of confirming the shape maintainability when the gelatin long fiber nonwoven fabrics of Examples 2 to 4, the gelatin sponge of Comparative Example 1 and the gelatin long fiber nonwoven fabric of Example 6 were swollen in purified water and held with tweezers were obtained. These are shown in FIGS. As can be seen from FIGS. 6 to 9 and 37, the gelatin long-fiber nonwoven fabrics of Examples 2 to 4 also have good shape retention after swelling, similar to the gelatin long-fiber nonwoven fabric of Example 1, and have tweezers after swelling. However, the shape was maintained and the handling was excellent. On the other hand, the gelatin sponge of Comparative Example 1 had poor shape maintenance after swelling, and its shape collapsed when held with tweezers after swelling.

  As is clear from Table 1, the gelatin sponge of Comparative Example 1 was thinner after swelling when compared with the thickness before and after swelling. Further, the gelatin sponge of Comparative Example 1 has a low compressive elasticity modulus in compression characteristics and a small change in compressive stress due to strain. Therefore, the gelatin sponge is very easily deformed and has no stiffness. On the other hand, the gelatin long fiber nonwoven fabric of each Example of the present invention was thicker after swelling when the thickness before and after swelling was compared. Compared to gelatin sponge, the compression modulus was about 1.2 to 3.0 times higher. Furthermore, as the fiber diameter increased, the compressive stress increased with strain and was more resistant to deformation. In addition, the compression deformation rate (strain rate) at a compressive stress of 1.0 kPa is greatly deformed to 56.8% in the gelatin sponge, but the gelatin long-fiber nonwoven fabric of the example is 35% or less and has a strong waist. It was.

(Example A1)
This example is an example of a cell culture test by stationary culture. The cells were statically cultured using the scaffold made of the gelatin long-fiber nonwoven fabric obtained in Example 4 (dry thickness 1.2 mm, density 0.44 g / cm ³ , diameter 4 mm).
1. Details of test (1) Cell culture (a) Human bone marrow-derived mesenchymal stem cells (hMSC) were placed in a liquid medium (αMEM medium containing 10% by mass of fetal bovine serum and 1% by mass of penicillin streptomycin) at 2.0 ×. A cell suspension was prepared by suspending to 10 ⁶ cells / mL.
(B) The gelatin long fiber non-woven fabric scaffold after sterilization with ethylene oxide gas was allowed to stand in a liquid medium for 30 minutes to be sufficiently swollen.
(C) Place the swollen gelatin long fiber non-woven scaffold into a 15 mL PP tube, add 200 μL of the cell suspension, and shake in an incubator at 37 ° C. on an orbital shaker at 300 rpm for 6 hours. Cell seeding.
(D) After cell seeding, the gelatin long fiber nonwoven fabric scaffold was washed with phosphate buffer (pH 7.4), unadhered cells were removed, transferred to a 12-well plate with tweezers, 3 mL of liquid medium was added, The culture was stationary in an incubator at a temperature of 37 ° C. and 5% CO ₂ . The liquid medium was changed every 2-3 days.
(2) Observation during culture The scaffold during cell culture was observed and photographed with a routine inverted microscope (“Primo Vert” manufactured by Carl Zeiss Microscopy).
(3) Number of cells The cultured scaffold was washed with a phosphate buffer (pH 7.4), and a 30 mM sodium chloride-citrate solution (SSC solution) containing 0.2 mg / mL sodium dodecyl sulfate at 37 ° C. The cells were collected by stirring at 300 rpm in 300 μL for 24 hours. To 100 μL of the collected cell dispersion, 400 μL of 30 mM sodium chloride-citric acid solution (SSC solution) was added, and Hoech33258 (Nacalai Tesque), which is a nuclear stain, was added to the resulting solution to 1 μL / mL. . The obtained solution was subjected to fluorescence intensity measurement at Ex 355 nm and Em 460 nm, and the cell number was determined from a standard line obtained from a solution with a known cell number.
(4) Cell distribution in the scaffold The cultured scaffold was washed with a phosphate buffer, fixed with 4% paraformaldehyde, and further washed three times with a phosphate buffer (pH 7.4). After washing, after embedding with an OCT compound (Sakura Finetech Japan Co., Ltd.), a slice was prepared in a frozen state in a direction perpendicular to the diameter of the columnar scaffold. The frozen section was stained with hematoxylin and eosin, and the manner of cell distribution in the scaffold was observed with an optical microscope (manufactured by Keyence Corporation, model number “BZ-X710”).
(5) Evaluation of hypoxic condition in the scaffold The liquid medium in the cell culture was removed, and a predetermined amount of Hypoxia Probe LOX-1 (Organogenix) was added to the newly added αMEM medium and incubated overnight. The scaffold in culture was observed with a fluorescence microscope ("BZ-X710" manufactured by Keyence Corporation) at Ex510-560 nm and Em580 nm. The contrast was adjusted with Image J.

(Example A2)
Cell culture was performed as described in Example A1, except that the gelatin long-fiber nonwoven fabric scaffold obtained in Example 2 was used instead of the gelatin long-fiber nonwoven fabric scaffold obtained in Example 4. It was.

(Comparative Examples A1 to A3)
Instead of the gelatin long-fiber nonwoven fabric scaffold obtained in Example 4, the gelatin sponge scaffold of Comparative Example 1 (Comparative Example A1), and Comparative Example 2 made of a polypropylene long-fiber nonwoven fabric scaffold (Comparative Example 2) Cell culture was performed as described in Example A1 except that as a comparative example 3, a scaffold made of a polylactic acid long fiber nonwoven fabric having a fiber diameter of 2 μm (comparative example A3) was used.

2. Results (1) Cell culture and observability during culture FIG. 10 shows an inverted microscope observation photograph (20 times) immediately after cell seeding when the cells were cultured using the gelatin long-fiber nonwoven fabric scaffold of Example 4. 11 shows an inverted microscope observation photograph (20 times) on the seventh day of the same culture. As can be seen from FIGS. 10 and 11, the gelatin long-fiber nonwoven fabric scaffold of Example 4 was transparent in the culture medium, and the state of cell culture could be observed. Further, after cell seeding, cell adhesion was observed at the intersection of the gelatin long fibers, and the cells covered the fiber gaps on the 7th day of culture. On the other hand, Comparative Example A1 (FIG. 12) using the gelatin sponge scaffold of Comparative Example 1, Comparative Example A2 (FIG. 13) using the polypropylene nonwoven scaffold of Comparative Example 2, and Polylactic acid nonwoven fabric of Comparative Example 3 In Comparative Example A3 using a scaffold (FIG. 14), light did not pass through the scaffold during cell culture, and the state of the cells could not be observed. It was confirmed that the scaffolds using the gelatin long-fiber nonwoven fabric obtained in the Examples can be easily used for research purposes because the cell morphology can be observed while culturing cells.
(2) Number of cells Table 2 and FIG. 20 show data on cell proliferation when using the gelatin long-fiber non-woven scaffolds obtained in Example 2 and the gelatin long-fiber non-woven scaffolds obtained in Example 4. Indicates. When the scaffold made of the gelatin long-fiber nonwoven fabric of Example 4 was used, it was about 3.5 times after cell seeding on day 7 of culture, about 8.2 times after cell seeding on day 14 of culture, culture 21 On the day, the cells proliferated approximately 19.5 times after cell seeding.

(3) Cell distribution in scaffold FIG. 15 shows the whole of the scaffold section stained with hematoxylin and eosin after cell culture for 8 days using the gelatin long-fiber nonwoven fabric scaffold obtained in Example 4 and observed with an optical microscope. The photograph of the image (the photograph observed at 10 times is connected on the software) is shown, and the partially enlarged photograph (10 times) is shown in FIG. As is clear from FIGS. 15 and 16, when cells were cultured using the scaffold made of non-woven fabric made of gelatin long fiber nonwoven fabric obtained in Example 4 of the present invention, the cells had penetrated into the inside of the scaffold. It was. On the other hand, in Comparative Example A2 using the scaffold made of polypropylene nonwoven fabric of Comparative Example 2, cell invasion was up to the outside of the scaffold (FIG. 17: photograph at a magnification of 4). 15 to 17, the portion that appears dark is the cell nucleus.
(4) Oxygen partial pressure in the scaffold FIG. 18 shows an optical microscope observation photograph (20 times) of the scaffold when the cell culture was performed for 12 days using the gelatin long fiber nonwoven fabric scaffold obtained in Example 4. Indicated. FIG. 19 is a fluorescence observation micrograph (high lightness) in which the oxygen state in the scaffold was observed with a hypoxic marker after cell culture was performed for 12 days using the gelatin long-fiber nonwoven fabric scaffold obtained in Example 4. Part (part close to white): hypoxic part) (20 times) was shown. Since the scaffold made of the gelatin long-fiber nonwoven fabric obtained in Example 4 can be identified as a fiber in the optical microscope observation photograph of FIG. 18, the fluorescence intensity by the low oxygen marker is weak (FIG. 19). It was confirmed that oxygen was diffusing. In the scaffold made of the gelatin long fiber nonwoven fabric obtained in the examples of the present invention, since the constituent fibers are partially welded and do not sag even when wet with water, oxygen goes to the entire scaffold made of the gelatin long fiber nonwoven fabric. It was useful as a three-dimensional scaffold.

(Example A3)
This example is an example of a cell culture test by stirring culture.
1. Details of test (1) Production of gelatin long fiber nonwoven fabric Scaffold made of gelatin long fiber nonwoven fabric obtained in Example 5 (dry thickness 1.6 mm, density 0.44 g / cm ³ , diameter 4 mm) was used. Cell culture was performed by stirring culture.
(2) Cell culture Cells were seeded by the same cell seeding method as in the stationary culture described above, using the gelatin long-fiber nonwoven fabric scaffold obtained above. Using the apparatus shown in FIG. 21, a plurality of gelatin-spun fiber non-woven scaffolds 26 seeded with cells were stabbed into a 22-gauge sterilization needle 25, and the distance between the scaffolds 26 was adjusted to 2 mm. A 22G needle 25 pierced with a gelatin long fiber non-woven fabric scaffold 26 was stabbed into a rubber stopper at the top of the spinner flask 21 and fixed. The liquid medium was added until the scaffold made of the gelatin long-fiber nonwoven fabric was immersed, and the culture was stirred on a magnetic stirrer placed in a 37 ° C., 5% CO ₂ incubator at 100 rpm. Every 3 to 4 days, half of the liquid medium was removed and an equal amount of new liquid medium was added to change the medium.
(3) Confirmation of cell viability The scaffold in culture was washed with a phosphate buffer. The solution was adjusted so that it might become Calcein AM (Life Technologies) 0.75 microliters / mL and Ethium homodimer-1 (Life Technologies) 2 microliters / mL to a phosphate buffer solution. 1 mL of the above solution was added to a scaffold placed in a 24-well plate and incubated at 37 ° C. for 30 minutes. After the incubation, the solution was removed, and 1 mL of phosphate buffer was added, followed by washing at 37 ° C. for 5 minutes for washing. After washing, the scaffold was embedded with OCT compound (Sakura Finetech Japan Co., Ltd.), and then frozen and sliced in the direction perpendicular to the diameter of the cylindrical scaffold. Using a fluorescence microscope (manufactured by Keyence Corporation, model number “BZ-X710”), live cells were observed for fluorescence at Ex: 470 nm, Em: 525 nm, and dead cells were observed at Ex: 545 nm, Em: 605 nm.
(4) Decellularization treatment The cultured scaffolds were washed with a phosphate buffer solution, and the cells were washed in a 30 mM sodium chloride-citrate (SSC) solution (300 μL) containing 0.2 mg / mL sodium dodecyl sulfate at 37 ° C. The cells were agitated at 300 rpm for a period of time to extract the cells and perform decellularization treatment.
(5) Cell distribution The scaffold after stirring culture and the scaffold subjected to the decellularization treatment were washed with a phosphate buffer, fixed with 4% paraformaldehyde, and further washed three times with a phosphate buffer. After washing, after embedding with an OCT compound (Sakura Finetech Japan Co., Ltd.), a slice was prepared in a frozen state so as to be perpendicular to the diameter of the cylindrical scaffold. The frozen section was stained with hematoxylin and eosin, and the manner of cell distribution in the scaffold was observed with an optical microscope (manufactured by Keyence Corporation, model number “BZ-X710”).
(6) Strength measurement of scaffold The diameter of the scaffold after cell culture and the scaffold after decellularization treatment was measured, and the thickness was measured with a creep meter physical property test system RE2-30005C manufactured by Yamaden Co., Ltd. Were compressed at a speed of 0.05 mm / sec. The compression elastic modulus (kPa) of 1-5% strain and the intermediate compressive stress (kPa) of 10%, 20%, 30% strain were measured. The strain at the time of compressive stress of 1 kPa was defined as the dimensional change rate with no load (strain 0%) as a reference.

2. Results (1) Change in scaffold shape by stirring culture The shape of the scaffold made of gelatin long-fiber nonwoven fabric changed with the passage of the culture date. FIG. 22 shows a photograph of the scaffold in culture taken with a digital camera from the lateral direction. FIG. 23 shows a photograph of the appearance of a cell scaffold obtained by cutting out one scaffold after culturing. FIG. 24 shows a photograph of the cell scaffold that has been decellularized. By the stirring culture, the scaffold made of the gelatin long-fiber non-woven fabric had the corner of the gelatin long-fiber non-woven fabric scaffold taken on the seventh day of culture, and the diameter became small and clouded white after the 14th day. On the 25th day of the culture, the scaffolds arranged on the needles were connected at an interval of 2 mm and integrated. The scaffold after cell culture maintained its shape even after decellularization.
(2) Cell distribution In FIG. 25, in Example A3, a section (scaffold cross section) of the scaffold after 14 days of stirring culture was stained with hematoxylin-eosin, and a photograph of the whole image observed with an optical microscope (observed at 10 times). , Linked on software). FIG. 26 shows a partially enlarged photograph (20 ×) at the end. As is clear from FIGS. 25 and 26, the cells have penetrated into the scaffold, and the cells proliferated more densely, particularly at the end of the scaffold. In FIGS. 25 and 26, the dark-colored portion is the cell nucleus.
In FIG. 27, in Example A3, the section of the scaffold after 14-day stirring culture and decellularization treatment was stained with hematoxylin eosin, and the end portion was observed with an optical microscope (20 times). At the end of the scaffold where the cells grew at a high density, the tissue remained between the gelatin fibers even after the scaffold was removed, indicating that the cells were producing extracellular matrix.
FIG. 28 shows a photograph of the whole image (observed at 10 times and linked on software) of the scaffold section after stirring culture for 25 days in Example A3, stained with hematoxylin-eosin and observed with an optical microscope. FIG. 29 shows a partially enlarged photograph (20 ×) at the end. As is clear from FIGS. 28 and 29, the cells proliferated at high density to the center of the tissue. In FIGS. 28 and 29, the dark-colored portion is the cell nucleus.
In FIG. 30, in Example A3, a section of the scaffold after 25 days of agitation culture and decellularization was stained with hematoxylin-eosin, and a photograph of the whole image observed with an optical microscope (observed at 10 times and connected on software) )showed that. FIG. 31 shows a partially enlarged photograph (20 ×) at the end and the center. As apparent from FIGS. 30 and 31, on the 25th day of culturing, gelatin fibers are not seen but only extracellular matrix is seen, so that the gelatin long fiber nonwoven fabric is decomposed while the cells are densely aggregated. It was found that all of the extracellular matrix produced by the cells was replaced.
(3) Cell Life and Death FIG. 32 is a photograph of the whole image of the living cells of the section of the scaffold after stirring and culturing for 14 days in Example A3 (observed at 10 times and connected on the software). Indicated. Since the fluorescence intensity is strong at the live cell site, the site with high brightness (site close to white) is the live cell.
FIG. 33 shows a photograph of the whole image of the dead cells of the section of the scaffold after 14 days of stirring culture in Example A3 (observed at 10 times and connected on the software). Since the fluorescence intensity is strong at the dead cell site, a site with high brightness (a site close to white) is the dead cell site.
As is clear from FIGS. 32 to 33, many living cells are observed inside the gelatin long fiber nonwoven fabric, and almost no dead cells are observed. It was found that by using a gelatin long fiber nonwoven fabric as a cell culture scaffold, even in a three-dimensional cell aggregate, cells showed a high survival rate at the center of the tissue.
FIG. 34 shows, in Example A3, a living cell of a section of a scaffold after stirring and culturing for 25 days, as a whole image (10 times) observed with a fluorescence microscope, and linked on software). Since the fluorescence intensity is strong at the live cell site, the site with high brightness (site close to white) is the live cell.
FIG. 35 shows a photograph of a whole image (observed at 10 × and connected on software) of dead cells in a section of the scaffold after stirring and culturing for 25 days in Example A3. Since the fluorescence intensity is strong at the dead cell site, a site with high brightness (a site close to white) is the dead cell site.
As apparent from FIGS. 34 and 35, even after the gelatin long-fiber nonwoven fabric disappears and is replaced with a tissue composed of cells and extracellular matrix, many living cells are observed inside the cell tissue, and dead cells are observed. It was hardly seen. When cell culture was performed using a gelatin long-fiber nonwoven fabric as a scaffold, it was found that the gelatin long-fiber nonwoven fabric showed a high survival rate even after being replaced with a high-density three-dimensional cell aggregate having a large diameter of 2 mm or more.
(4) Strength measurement of scaffold Table 3 shows the mechanical properties of the scaffold after stirring culture and the decellularized scaffold in Example A3. Both the 1-5% elastic modulus and the intermediate stress increased by stirring culture using gelatin long fiber nonwoven fabric. The strength of the gelatin long-fiber non-woven fabric was improved by densely growing cells in the voids. Furthermore, these mechanical behaviors are the same in the decellularized scaffolds, and the strength was improved by filling the voids of the gelatin long-fiber nonwoven fabric with the extracellular matrix produced by the cells. In particular, on the 25th day of culturing, the gelatin long-fiber nonwoven fabric disappeared, and although it became a cell aggregate composed only of cells and extracellular matrix, the strength was high and the extracellular produced by the cells The matrix contains elastic fibers such as elastin and can be expected to be a cell aggregate closer to the human ecological environment.

From the results of cell culture under stirring using the gelatin long-fiber nonwoven fabric scaffold of this example, the following can be understood.
(1) Three-dimensional cell aggregates having a size of 2 mm or more were obtained in a short period of 25 days, and the cells had a high survival rate within the aggregates.
(2) In the obtained three-dimensional cell aggregate, the gelatin long-fiber nonwoven fabric disappeared and replaced with cells and an extracellular matrix produced by the cells.
(3) The obtained three-dimensional cell aggregate showed high strength.

(Example A4)
This example is an example of a cell culture test using stacked culture. Using the gelatin long-fiber nonwoven fabric scaffold obtained in Example 6 (dry thickness 0.2 mm, density 0.8 g / cm ³ , diameter 6 mm), cells were statically cultured.

1. Cell culture (1) Alternating lamination of gelatin long fiber non-woven scaffold and cell sheet (a) Human bone marrow-derived mesenchymal stem cells (hMSC) in liquid medium (fetal bovine serum 10% by mass, penicillin streptomycin 1% by mass) Cell suspension) to 2.0 × 10 ⁶ cells / mL to prepare a cell suspension.
(B) 1 mL of a liquid medium was added to a 12-well well plate, and 50 μL of the cell suspension was seeded there, followed by stationary culture for 10 days to prepare a cell sheet.
(C) Using a 1000 mL micropipette, the cell sheet was peeled from the well plate with 12 holes by a liquid flow.
(D) The gelatin long fiber non-woven fabric scaffold after sterilization with ethylene oxide gas was allowed to stand in a liquid medium for 30 minutes to be sufficiently swollen.
(D) The medium in the well plate was removed, and a swollen gelatin long fiber non-woven fabric scaffold was placed on the cell sheet. A pressure of 1.3 kPa was applied from above the gelatin long fiber nonwoven fabric scaffold to adhere the cell sheet and the gelatin nonwoven fabric.
(E) Using tweezers, three layers of the same cell sheet / gelatin long fiber nonwoven fabric scaffold made in the same manner were laminated (FIG. 36-f), under conditions of a temperature of 37 ° C. and 5% CO ₂ . Incubate for 60 minutes to bond the layers together.
(2) Observation of the upper surface of a laminated body The laminated body of the scaffold and cell sheet after lamination | stacking was observed and image | photographed with the optical microscope (the Keyence Corporation make, model number "BZ-X710").
(3) Observation of cross section of laminate The laminate was washed with a phosphate buffer, fixed with 4% paraformaldehyde, and further washed three times with a phosphate buffer (pH 7.4). After washing, after embedding with an OCT compound (Sakura Finetech Japan Co., Ltd.), a slice was prepared in a frozen state in a direction perpendicular to the diameter of the columnar scaffold. The frozen section was stained with hematoxylin and eosin, and the cell distribution in the scaffold was observed with an optical microscope (manufactured by Keyence Corporation, model number “BZ-X710”).

2. Result (1) Observation of Laminate In FIG. 39, a micrograph (4 times) of the laminate observed from the top is connected on the software of a fluorescence microscope (manufactured by Keyence Co., Ltd., model number “BZ-X710”). I showed that. It was confirmed that the cell sheet overlapped the upper surface of the gelatin nonwoven fabric without wrinkles.
FIG. 40 shows a micrograph of a cross-sectional view of a laminate in which three layers of gelatin long fiber nonwoven fabric and cell sheets are alternately laminated. The cell nucleus and cytoplasm stained with hematoxylin and eosin were seen in a linear form, confirming the presence of the cell sheet. In addition, the cell sheet was seen in three layers at regular intervals. The interval was equivalent to the thickness (about 0.7 mm) of the swollen gelatin long fiber nonwoven fabric, and it was confirmed that the gelatin long fiber nonwoven fabric and the cell sheets were alternately laminated.

(Example B1)
Using gelatin long fiber nonwoven fabrics of different thickness (dry state) and density (dry state), gelatin long fiber nonwoven fabrics preferred for a scaffold for lamination were studied. Good hand rigging means that when the scaffold after swelling in the liquid medium is grasped with tweezers, the shape maintaining property is good and it is difficult to bend. For example, FIGS. 5 to 8 and FIG. 37 are examples in which, when the scaffold after swelling with water or a liquid medium is gripped with tweezers, the shape maintaining property is good and not bent. On the other hand, FIG. 38 shows an example in which the shape maintainability is good when gripped with tweezers but is bent. The handling property of the scaffold made of gelatin long fiber nonwoven fabric was evaluated with a cantilever beam. A rectangular parallelepiped base was used as a test base. The test piece was placed on the end of the pedestal so that half of the area of the test piece came out of the pedestal so that it could be a cantilever. When the included angle of the test piece was measured within 45 degrees, it was judged that the handleability was good (A), and when it was greater than 45 degrees, the handleability was poor (B). In the cantilever test, an example of good handling properties is shown in FIG. 41, and an example of poor handling properties is shown in FIG. The results of handling properties are shown in FIG. From FIG. 43, when a gelatin long fiber nonwoven fabric scaffold is swollen with a liquid medium and grasped with tweezers, the dry thickness before swelling is 0.2 mm or more, and the density is 0.8 g / cm ³ or more. It turned out to be preferable.

  The biocompatible long-fiber nonwoven fabric of the present invention is suitable for a three-dimensional scaffold for cell culture and can also be applied to various medical uses.

DESCRIPTION OF SYMBOLS 1 Heating tank 2 Spinning liquid 3 Nozzle discharge port 4,6 Compressor 5 Fluid injection port 7 Pressure fluid 8 Long fiber 9 Long fiber nonwoven fabric 10 Nonwoven fabric manufacturing apparatus 11 Winding roll 12 Heat insulation container 20 Stirring culture test apparatus 21 Spinner flask 22 Stirring Bar 23 Stirrer 24, 30 Liquid medium 25 22 gauge needle 26, 34 Scaffold 31 Culture dish 32 Cell suspension 33 Cell sheet 35 Pressure

Claims (18)

A biocompatible long-fiber nonwoven fabric mainly composed of a biocompatible polymer,
The biocompatible long fiber constituting the biocompatible long fiber nonwoven fabric has a fiber diameter that changes in the length direction,
The biocompatible long fiber constituting the biocompatible long-fiber nonwoven fabric has a fiber intersection at least partially welded,
The biocompatible long fiber nonwoven fabric is heat dehydrated and crosslinked,
A biocompatible non-woven fabric that does not sag when wet and becomes transparent when wet.   The biocompatible long fiber nonwoven fabric according to claim 1, wherein the biocompatible long fiber has an average fiber diameter (D) in the range of 1 to 70 µm, and the fiber diameter varies in a range of D ± 0.5D.   The biocompatible long fiber nonwoven fabric according to claim 1 or 2, wherein the biocompatible long fiber is substantially in an unstretched state.   The biocompatible long-fiber nonwoven fabric according to any one of claims 1 to 3, wherein the biocompatible long-fiber nonwoven fabric has a compressive deformation rate of 40% or less at a compressive stress of 1.0 kPa. The biocompatible long fiber nonwoven fabric according to any one of claims 1 to 4 , wherein the biocompatible long fiber constituting the biocompatible long fiber nonwoven fabric is water-soluble before crosslinking. The biocompatible polymer is gelatin, collagen Contact and biocompatible filament nonwoven fabric according to any one of claims 1 to 5, at least one water soluble polymer selected from the group consisting of modifications. It is a manufacturing method of the biocompatible continuous fiber nonwoven fabric of any one of Claims 1-6 ,
Push the spinning solution containing biocompatible polymer into the air from the nozzle outlet,
The pressure fluid is ejected forward from the fluid ejection port which is located behind the nozzle ejection port and is not in contact with the nozzle ejection port,
The extruded spinning solution is accompanied with the pressure fluid to form a fiber, and the fiber-formed fiber is accumulated to form a nonwoven fabric .
A method for producing a biocompatible long-fiber non-woven fabric, wherein the bio-compatible long-fiber non-woven fabric is vacuum freeze-dried and then subjected to heat dehydration crosslinking . The method for producing a biocompatible non-woven fabric according to claim 7 , wherein the temperature of the spinning solution is not less than the temperature at which the spinning solution flows and not more than the decomposition temperature of the biocompatible polymer. The method for producing a biocompatible continuous fiber non-woven fabric according to claim 7 or 8 , wherein an injection pressure of the pressure fluid is 0.05 to 0.5 MPa, and a temperature of the pressure fluid is a temperature of the spinning solution ± 30 ° C. The method for producing a biocompatible nonwoven fabric according to any one of claims 7 to 9 , wherein the spinning solution has a viscosity of 500 to 3000 mPa · s at a temperature of 60 ° C. A three-dimensional scaffold for cell culture, wherein the biocompatible long-fiber nonwoven fabric according to any one of claims 1 to 6 is used as a three-dimensional scaffold for cell culture. The three-dimensional scaffold for cell culture according to claim 11 , wherein the scaffold has a thickness of 0.2 mm or more, and cells survive inside the scaffold. A cell culture method using the three-dimensional scaffold for cell culture according to claim 11 or 12 ,
A cell culture method comprising seeding cells on the scaffold, then placing the seeded scaffold in a liquid medium and culturing cells. The cell culture method according to claim 13 , wherein the scaffold has a thickness of 0.2 mm or more, and the cells survive inside the scaffold. The cell culture method according to claim 13 or 14 , wherein the cell culture is performed while flowing the liquid medium by at least one means selected from stirring, circulation, and shaking. The cell culture method according to any one of claims 13 to 15 , wherein during the cell culture, the scaffold disappears and is replaced with cultured cells and an extracellular matrix produced by the cells. The cell culture method according to any one of claims 13 to 16 , comprising a step of grasping the scaffold with tweezers and laminating the scaffold with a cell sheet. The cell culture method according to claim 17 , wherein the scaffold has a thickness of 0.2 mm or more and a density of 0.8 g / cm ³ or more.