JP2006254722A - Cell anchorage material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a cell anchorage material containing a nanofiber sufficiently standing an actual use as an anchorage for cell culture in vitro, an anchorage for anagenesis or a molding for an embedded medical material and a cell anchorage material containing a nanofiber for simply adsorbing or releasing a protein, effective for cell culture, anagenesis, etc. <P>SOLUTION: The cell anchorage material comprises a nanofiber that comprises an organic polymer and has 1-200 nm number-average single fiber diameter. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell scaffold material containing nanofibers used for applications such as a scaffold material molded body for cell culture or tissue regeneration, and an implantable medical molded body.

  It is important to design various three-dimensional scaffold materials that serve as cell scaffolds as regenerative medicine and other medical materials. In particular, porous materials with high porosity and large surface area have attracted attention as effective scaffold materials in recent years. Has been. As a porous material, a foam or a fiber material is known. However, as a scaffold for cell culture or tissue regeneration, it is required to be highly compatible with living organisms or cells, or to have high adhesion to cells or tissues. It has been. In addition, it is important to imitate the environment of living tissues where cells grow such as bone marrow and connective tissue collagen fibers as scaffold materials, but these living tissues have a nano-level fibrous structure. It is common. For this reason, it is preferable that the scaffold material be a nano-level fibrous structure. Further, since the nano-level structure has a high specific surface area, it is considered to have a desirable shape as a cell scaffold material, and studies are being conducted. Yes.

For this reason, attempts have been made to use nanofibers, which are fibers having a nano-level diameter, for medical purposes such as scaffolds for cell culture outside the body, scaffolds for tissue regeneration, and implantable medical materials. One technique that has been in the limelight is electrospinning. In this method, the polymer is dissolved in the electrolyte solution and pushed out of the base, and at that time, a high voltage of several thousand to 30,000 volts is applied to the polymer solution, and the high-speed jet of the polymer solution and the subsequent bending of the jet are performed. It is a technology that makes it ultra fine by expansion. When this technology is used, the single yarn fineness may be equivalent to several tens of nanometers in terms of the single yarn diameter depending on conditions, but generally the fiber diameter is on the order of several hundreds of nanometers. The diameter of the nanofiber is generally non-uniform, and a necklace-like nanofiber in which a thread is passed through a bead having a diameter of a few micrometers in some parts (see Patent Document 1) may be formed. ). In addition, the shape of fibers and fiber products obtained by electrospinning is limited to non-woven fabrics, and the solvent evaporates during the fiberization process, so the resulting fiber assembly is often not oriented and crystallized, and the strength is also high. Since only a very weak thing is obtained compared with a normal textile product, it does not have the mechanical durability required as a scaffold material. In addition, cell culturing has been attempted on the nanofiber structure by this manufacturing method (see Patent Documents 1 and 2), but the fiber density is high, so that it is impossible for details to enter the fiber structure. There is no mechanical flexibility. For these reasons, nanofibers produced by electrospinning have significant restrictions on application development. Furthermore, electrospinning has a big problem as a manufacturing method, and the size of the obtained fiber product is at most about 100 cm ² , and the productivity is several g / hour at the maximum, which is much higher than that of ordinary melt spinning. There was a problem of being low. In addition, there is a problem that there is always a danger of electric shock, explosion, poisoning, because high voltage is required and harmful organic solvents and super fine yarns float in the air.

  On the other hand, in order to use nanofibers for research and medical materials such as scaffolding materials and tissue regeneration materials such as cell culture as described above, not only its physical properties but also cells etc. It is important to have effective protein effects on functions such as differentiation, and it is important to adsorb or fix these proteins on the scaffold material or to release the protein adsorbed on the scaffold material. There are many.

  We are studying protein adsorption performance and cell culture on polylactic acid nanofibers prepared by electrospinning, but electrospinning is not suitable for practical use due to the above-mentioned problems (Non Patents) Reference 1). In this study, only the adsorptivity of bovine serum proteins or adhesion molecules as proteins is examined, but proteins that are actually important for use in cell culture, tissue regeneration or medical applications in and outside the body are cytokines. It is a functional protein that acts on cell functions that are effective in cell culture and tissue regeneration, such as cell proliferation and differentiation, and studies on these proteins are important.

In order to use nanofibers as research and medical scaffold materials, the sustained release of the protein adsorbed on the scaffold material, that is, the carrier, to supply the protein to the cells or necessary locations in the living body. Sex is also important. If this sustained release property is utilized, there is an advantage that the protein concentration in the living body or in the culture solution can be kept constant, and the occasional addition of cytokine is unnecessary. However, as a carrier exhibiting sustained release, normally, a substance to be released is adsorbed by using a copolymer of degradable polymer such as lactic acid or enclosed in the inside of the carrier, and the sustained release is made by utilizing degradation of the polymer. (See Patent Document 3). However, the method of using a degradable polymer as a carrier in this way is very difficult to manufacture because it is limited in polymer and shape, and further requires copolymerization. Therefore, it is physically used as a scaffold material for cell culture and tissue regeneration. It was very difficult to obtain an effective nanofiber satisfying the mechanical and chemical properties as a molded body.
JP 2004-321484 A JP 2004-290133 A JP 2001-15893 A J. Biomed.Mat.Res 67A 531 (2003)

  The present invention eliminates the drawbacks of the prior art described above, and is sufficiently durable to be actually used as a molded body such as a scaffold for cell culture outside the body, a scaffold for tissue regeneration, or an implantable medical material. It is an object of the present invention to provide a cell scaffold material containing nanofibers, and a cell scaffold material containing nanofibers that can easily adsorb or slowly release proteins effective for cell culture, tissue regeneration, and the like.

(1) Cell scaffold material comprising nanofibers made of an organic polymer and having a number average single fiber diameter of 1 to 200 nm (2) Number average single fiber diameter is 1 to 100 nm (3) The cell scaffold material according to (1) or (2), wherein the nanofiber is produced from melt spinning (4) The organic polymer is a thermoplastic polymer, The cell scaffold material according to any one of (1) to (3) (5), wherein the thermoplastic polymer is selected from the group consisting of polyolefin, polyamide, polyurethane, polyester, fluorine-based polymer, polylactic acid, polyvinyl alcohol and derivatives thereof. The cell scaffold material according to (4), characterized in that the thermoplastic polymer is nylon or polybutylene tele The cell scaffold material according to (4) or (5), wherein the nanofiber is processed into the shape of paper, nonwoven fabric, or woven or knitted fabric (1) to (6) The cell scaffold material (8) according to any one of (1) to (7), characterized in that the nanofibers form a bundle structure. The cell scaffold material (10) according to any one of claims (1) to (8), wherein an aggregate is formed on another substrate, and the shape of the other substrate is for cell culture The cell scaffold material according to (9), wherein the material is in a shape used as an instrument (11) When 1 mg of nanofibers are immersed in 1 mL of a protein solution having a concentration of 100 μg / mL, the protein is adsorbed. Of protein in The cell scaffold material according to any one of (1) to (10), wherein 80% or more can be adsorbed (12) The protein is adsorbed to nanofibers (1) to (11) The cell scaffold material according to any one of (13), wherein the protein to be adsorbed is at least one selected from cytokines. (14) The cell scaffold material according to (12), wherein the protein once adsorbed is released slowly. (12) The cell scaffold material according to (12) or (13), wherein 1% or more of the protein once adsorbed is sustainedly released within 24 hours, and the sustained release is continued for 1 week or longer. The cell scaffold material (16) according to (14), characterized in that it is a molded body used for cell culture or tissue regeneration comprising the cell scaffold material according to any one of (1) to (15).

  The present invention provides a cell scaffold material including nanofibers having various shapes and uniform fiber diameters produced by sea-island melt spinning using a polymer alloy. Cells can be effectively cultured or tissue regenerated by the effect of high porosity or high specific surface area, or improved cell adhesion, imitation of the nano-level fibrous structure environment in vivo.

  In addition, by using sea-island melt spinning with a polymer alloy, the shape of the base material can be changed to a desired shape such as a nonwoven fabric or a woven fabric, and can be combined with another base material. In addition, the cell scaffold material containing the nanofiber of the present invention can adsorb proteins with high efficiency, and can further release the adsorbed proteins from the material. At this time, a protein effective for cell culture and tissue regeneration such as cytokine is used as a protein, and is adsorbed and sustained-released on the material, so that it can be used as a cell scaffold material for cell culture, tissue regeneration, and implantable medical use. Regardless of whether it is a molded body, medical use or research use such as inspection or measurement, it can be developed as a useful material.

  The present invention is a cell scaffold material comprising nanofibers made of an organic polymer and having a number average single fiber diameter of 1 to 200 nm.

  The cell scaffolding material as used in the present invention refers to all materials used for cells, tissues or organs in which cells have gathered inside or outside the body, and parts that come into contact with blood or body fluids that contain cells. This cell scaffold material expresses and promotes various cell functions such as cell adhesion, proliferation, differentiation, activation, migration, migration, and morphological change by contacting the cell with the material on or within the material. Refers to material. Specifically, as a cell culture scaffolding material such as a container or bag or column for culturing and forming cells, tissues and transplanted tissues, transplanted organs inside or outside the living body, artificial organs or artificial tissues such as artificial hearts or artificial corneas To heal diseases and wounds such as materials used, surgical materials such as sutures and fracture bonding templates, materials used for some or all of the tools and instruments used for treatment, syringes, catheters, wound protection materials, etc. Examples include materials used for some or all of the medical devices used in the above. In addition, it refers to a material that is effective for cell proliferation, activation, differentiation induction, colonization or tissue or organ repair, adhesion, engraftment, and shape formation when used as a medical material.

  In the cell scaffold material of the present invention, the nanofibers are mainly responsible for functions that affect adhesion, proliferation, differentiation, etc. to cells and tissues, or functions that adsorb and release proteins. These cell scaffold materials may be composed only of nanofibers, or may be materials in which the material or part of the molded body is nanofibers.

  Examples of the organic polymer referred to in the present invention include thermoplastic polymers, thermosetting polymers, biopolymers, and the like. From the viewpoint of moldability, thermoplastic polymers are preferable. The thermoplastic polymer is preferably one kind selected from the group consisting of polyolefin, polyamide, polyurethane, polyester, fluorine-based polymer, polylactic acid, polyvinyl alcohol, and derivatives thereof. Moreover, it is preferable that the melting point of the polymer is 165 ° C. or higher because the heat resistance of the nanofiber is good. For example, polylactic acid (PLA) is 170 ° C, polytrimethylene terephthalate (PTT) and polybutylene terephthalate (PBT) are 225 ° C, polyethylene terephthalate (PET) is 255 ° C, and nylon 6 (N6) is 220 ° C. It is particularly preferable to use nylon as the polyamide and polybutylene terephthalate as the polyester. The polymer may contain additives such as particles, a flame retardant, an antistatic agent, and an ionic substance. Further, other components may be copolymerized as long as the properties of the polymer are not impaired.

  The nanofiber referred to in the present invention refers to a single fiber having a single fiber diameter of 1 nm or more and less than 1000 nm. The nanofiber aggregate refers to an assembly of nanofibers in one, two, or three dimensions.

In the present invention, it is important to include nanofibers having a number average single fiber diameter of 1 to 200 nm in the cell scaffold material. Since the nanofiber aggregate of the present invention has a single yarn diameter of 1/10 to 1/100 or less that of a conventional microfiber fiber, the specific surface area is remarkably increased. For this reason, the property peculiar to nanofiber which was not seen with the usual fiber yarn grade is acquired. In particular, when the single fiber diameter is 200 nm or less, not only does the specific surface area dramatically increase, but also there are innumerable pores of several nanometers to several hundred nanometers between the fibers. Excellent adsorption or absorption characteristics. From this point of view, it is preferable that the single fiber diameter based on the number average of the nanofibers is smaller. Therefore, it is preferably 25 to 200 nm, more preferably 1 to 100 nm, still more preferably 30 to 100 nm, and still more preferably 30 to 80 nm. Here, the number average single fiber diameter can be determined as follows. That is, observe the cross section of a single fiber bundle with a transmission electron microscope (TEM), measure the diameter of 300 or more single fibers randomly extracted within the same cross section in a circle, and simply average these values. Can be obtained.

  By using such extremely thin fibers, nanofibers are smaller than cells with a size of several tens to several microns, so unlike conventional microfibers, cells are affected by multiple fibers. For example, cells can be directly affected by unevenness, cell adhesion, cell extension direction, nerve fiber extension, or adsorptivity of adhesive substances discharged from cells Can be affected. In addition, for example, if a woven or knitted fabric or a non-woven fabric is used, the nanofibers gather to form a bundle structure to form a nanofiber bundle, so that cells can enter between the bundles. On the other hand, a certain direction of physical direction can be given.

  In the present invention, it is preferable that the fiber ratio of single fibers having a nanofiber single fiber diameter exceeding 200 nm is 0 to 5% in the nanofiber aggregate. Here, the fiber ratio of a single fiber can be calculated | required as follows. First, as with the average diameter, the diameter in terms of a circle is obtained on the basis of the cross-sectional area of the fiber. And the ratio of the area of the whole single fiber of diameter 200nm with respect to the area of the whole of the nanofiber single fiber of 300 or more randomly extracted here, and the fiber ratio of the single fiber whose diameter of the nanofiber single fiber is 200nm in this invention To do. Here, by reducing the fiber ratio of coarse nanofibers having a diameter exceeding 200 nm, it is possible to effectively utilize excellent properties of nanofibers such as selective adsorption, sustained release, and cell recognition. Coarse nanofibers have a small surface area and are too thick for cell recognition, so that the properties of the nanofibers are difficult to be exhibited. It will be a negative effect. Furthermore, the ratio of coarse nanofibers with a diameter exceeding 200 nm is preferably 2% or less, more preferably 0% or less. Further, it is preferable to lower the lower limit of coarse nanofibers, and as coarse nanofibers, the fiber ratio of single fibers having a diameter exceeding 150 nm is preferably 0 to 5%, more preferably 2% or less, and even more preferably 0%. It is as follows. Furthermore, as a coarse nanofiber, it is preferable that the ratio of the single fiber exceeding a diameter of 100 nm is 0 to 5%, More preferably, it is 2% or less, More preferably, it is 0% or less.

  In the cell scaffold material of the present invention, the nanofibers preferably form a bundle structure.

  Although the manufacturing method of the nanofiber used by this invention is not specifically limited, It is preferable to employ | adopt the method of using the following polymer alloy melt spinning.

  That is, a polymer alloy melt obtained by alloying polymers having different solubility in two or more kinds of solvents is formed, and after spinning, it is solidified by cooling and fiberized. Then, stretching and heat treatment are performed as necessary to obtain polymer alloy fibers. Then, the nanofiber aggregate of the present invention can be obtained by removing the easily soluble polymer with a solvent.

  Here, in the polymer alloy fiber that is the precursor of the nanofiber assembly, the easily soluble polymer is the sea (matrix) and the hardly soluble polymer is the island (domain), and it is important to control the island size. . Here, the island size is obtained by observing the cross section of the polymer alloy fiber with a transmission electron microscope (TEM) and evaluating it in terms of diameter. Since the diameter of the nanofiber is almost determined by the island size in the precursor, the distribution of the island size is designed according to the diameter distribution of the nanofiber of the present invention. For this reason, kneading of the polymer to be alloyed is extremely important. In the present invention, it is preferable to highly knead by a kneading extruder, a stationary kneader or the like. Note that kneading is insufficient with a simple chip blend (for example, JP-A-6-272114), and thus it is difficult to disperse islands with a size of several tens of nm as in the present invention.

  Specifically, as a guide when kneading, depending on the polymer to be combined, when using a kneading extruder, it is preferable to use a biaxial extrusion kneader, and when using a static kneader, the number of divisions is It is preferable to set it to 1 million or more. Moreover, in order to avoid blend spots and fluctuations in the blend ratio over time, it is preferable to measure each polymer independently and supply the polymers to the kneading apparatus independently. At this time, the polymer may be supplied separately as pellets, or may be supplied separately in a molten state. Two or more kinds of polymers may be supplied to the root of the extrusion kneader, or may be a side feed that supplies one component from the middle of the extrusion kneader.

  When a twin screw extrusion kneader is used as the kneading apparatus, it is preferable to achieve both high kneading and suppression of the polymer residence time. The screw is composed of a feeding part and a kneading part, but it is preferable that the kneading part has a length of 20% or more of the effective length of the screw so that high kneading can be achieved. In addition, by setting the length of the kneading part to 40% or less of the effective screw length, excessive shear stress can be avoided and the residence time can be shortened. Can be suppressed. Further, the kneading part is positioned as close as possible to the discharge side of the twin-screw extruder, so that the residence time after kneading can be shortened and re-aggregation of the island polymer can be suppressed. In addition, when strengthening kneading, it is possible to provide a screw having a backflow function for sending the polymer in the reverse direction in an extrusion kneader.

  In addition, a combination of polymers is also important for ultra-fine dispersion of islands with a size of several tens of nanometers.

In order to make the island domain (cross section of the nanofiber) close to a circular shape, the island polymer and the sea polymer are preferably incompatible. However, it is difficult for the island polymer to be sufficiently finely dispersed by a simple combination of incompatible polymers. For this reason, it is preferable to optimize the compatibility of the polymer to be combined, but one index for this purpose is the solubility parameter (SP value). The SP value is a parameter reflecting the cohesive strength of a substance defined by (evaporation energy / molar volume) ^1/2 , and a polymer alloy having good compatibility may be obtained between materials having close SP values. The SP value is known for various polymers, and is described, for example, in “Plastic Data Book”, edited by Asahi Kasei Amidus Corporation / Plastics Editorial Department, page 189. It is preferable that the difference between the SP values of the two types of polymers is 1 to 9 (MJ / m ³ ) ^{1/2 because} it is easy to achieve both the island domain circularization and ultrafine dispersion by incompatibility. For example, nylon 6 (N6) and PET have a SP value difference of about 6 (MJ / m ³ ) ^1/2, which is a preferable example. N6 and polyethylene (PE) have a SP value difference of 11 (MJ / m ³ ) About ^1/2, which is an undesirable example.

  Further, it is preferable that the difference in melting point between polymers is 20 ° C. or less because kneading with an extrusion kneader is easy to knead with high efficiency because a difference in melting state in the extrusion kneader hardly occurs.

  In addition, when a polymer which is easily decomposed or thermally deteriorated is used as one component, it is necessary to keep the kneading and spinning temperature low, which is also advantageous. Here, in the case of an amorphous polymer, since there is no melting point, it is replaced by the glass transition temperature, Vicat softening temperature or heat distortion temperature.

Furthermore, melt viscosity is also important, and if the polymer forming the island is set lower, the island polymer is likely to be deformed by shearing force. However, if the island polymer is excessively low in viscosity, it tends to be seamed and the blend ratio with respect to the whole fiber cannot be increased. Therefore, the island polymer viscosity is preferably 1/10 or more of the sea polymer viscosity. In addition, the melt viscosity of the sea polymer may greatly affect the spinnability, and it is preferable to use a low viscosity polymer of 100 Pa · s or less as the sea polymer because the island polymer is easily dispersed. This can also significantly improve the spinnability. At this time, the melt viscosity is a value at a shear rate of 1216 sec ⁻¹ at the die surface temperature during spinning.

  When spinning the ultrafinely dispersed polymer alloy used in the present invention, the spinneret design is important, but the cooling condition of the yarn is also important. As described above, since the polymer alloy is a very unstable molten fluid, it is preferable to quickly cool and solidify after discharging from the die. For this reason, it is preferable that the distance from a nozzle | cap | die to the cooling start shall be 1-15 cm. Here, the start of cooling means a position where positive cooling of the yarn is started, but in the actual melt spinning apparatus, it is replaced with this at the upper end of the chimney.

The spun polymer alloy fiber is preferably subjected to stretching and heat treatment. However, the preheating temperature during stretching is higher than the glass transition temperature (T _g ) of the island polymer, so that the yarn unevenness can be reduced. It is possible and preferable.

  The polymer alloy fibers obtained in this way can be made into woven or knitted fabrics according to known methods, or into pile fabrics or nonwoven fabrics. Therefore, structures and assemblies having shapes that cannot be produced by conventional electrospinning techniques. Can be molded as For example, when using a non-woven fabric, a known method such as a needle punch method or a hydroentanglement method can be used.

  By eluting the readily soluble polymer, which is a sea polymer, from the polymer alloy fibers and fabrics thus obtained with a solvent, nanofibers and a fabric comprising the same are obtained. In this case, the solvent is an aqueous solution. Is preferable from the viewpoint of reducing the environmental load. Specifically, it is preferable to use an alkaline aqueous solution or hot water. For this reason, as an easily soluble polymer, hot water soluble polymers, such as a polymer hydrolyzed alkali, such as polyester and polycarbonate (PC), polyalkylene glycol, polyvinyl alcohol, and derivatives thereof are preferable.

  The nanofibers produced by the above production method have a crystallinity of 20% or more, and have the same strength as ordinary clothing fibers.

  In addition, in the above production method, particularly when a stationary kneader is positioned directly above the die, a nanofiber aggregate having a long fiber shape in which nanofibers are theoretically extended infinitely may be obtained.

  Nanofibers produced by the above manufacturing method are completely different from conventional nanofibers. For the first time, nanofibers can be drawn and heat-treated by drawing and heat-treating polymer alloy fibers, which are precursors. The tensile strength and shrinkage rate can be freely controlled. Furthermore, it is possible to crimp the polymer alloy fiber as a precursor.

  In order to disperse nanofibers into single fibers one by one, for example, the following wet papermaking method can be used. That is, the polymer alloy fiber of the present invention is scraped or combined to form a tow, and then the readily soluble polymer is eluted. Next, this nanofiber aggregate fiber is cut into a fiber length of 0.1 to 10 mm with a guillotine cutter or the like to obtain nanofiber short fibers. By defeating this, single fibers are broken apart. At the production level, the beating is processed with a Niagara beater or refiner. Experimentally, there are household mixers and cutters, laboratory grinders and mixers and cutters, biomixers, roll mills, mortars, and PFI beaters for papermaking. And this is injected | thrown-in to a liquid and a nanofiber liquid dispersion is obtained using a dispersing agent as needed. Furthermore, when a dispersion having a high affinity with the polymer constituting the nanofiber is used, the diameter of the nanofiber aggregate can be dispersed to 300 nm or less, and further to a single fiber level. Then, a nanofiber paper in which single fibers are dispersed can be obtained by papermaking these dispersions. It is also possible to form an aggregate of nanofibers on another substrate by casting the dispersion on another substrate and evaporating the dispersion.

  The nanofibers used in the present invention can be processed into any shape such as yarn, cotton, fabric, etc., depending on the material and the convenience of the molded product. Here, the yarn refers to a one-dimensional structure including nanofibers, and more specifically refers to long fibers, short fibers, and spun yarn. Moreover, cotton means the thing which crimped the short fiber and opened it. As the fabric, various forms such as a woven / knitted fabric, a pile fabric, a non-woven fabric, and paper can be adopted. As the cell scaffold material, a nanofiber structure is preferably formed on a non-woven fabric, paper shape, woven or knitted shape or other molding substrate. For example, it is preferable to use a woven or knitted fabric in order to give shape followability to tissues or bones by transplanting into a living body, and in order to improve the stability of the shape as a scaffold for culture, a woven fabric or nonwoven fabric, It is preferable to use paper. In order to increase the thickness of these fabrics and improve the operability during cell culture or tissue transplantation, it is preferable to use a nonwoven fabric. In order to disperse nanofibers as much as possible and improve cell dispersibility during culture or tissue regeneration, it is preferable to use paper by wet papermaking. Also, in order to maintain the stability of the shape as a scaffold for culture and tissue regeneration, and to improve the moldability of the shape, the nanofiber dispersion is cast on another substrate or the substrate is dispersed in the dispersion. After impregnation, it is preferable to form a nanofiber aggregate or structure on another substrate by evaporating the dispersion.

  Moreover, although the thread | yarn, cotton, and fabric used for the cell scaffold material of this invention may be comprised only from nanofiber, in order to ensure form stability, bulkiness, and moldability, a single fiber diameter is 1-50 micrometers. Fibers may be mixed, polystyrene, polypropylene, polycarbonate, polyvinyl chloride, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), cellulose, polysulfone, etc., film shape, dish shape, well shape Alternatively, it may be combined with a plastic material that can be molded into a certain shape such as a hollow fiber shape or a porous shape. The existence form of nanofibers in such mixed use and composite products can take various forms such as mixed fiber, covering, mixed twist, mixed cotton, kneading, lamination, melt mixing, kneading, coating and the like. In particular, in the case of mixed cotton, kneading, and coating, it is preferable from the viewpoint of familiarity with nanofiber that the fiber to be mixed is a microfiber having a single fiber diameter of 1 to 5 μm.

  In the cell scaffold material of the present invention, the nanofibers preferably form an aggregate on another substrate.

  In addition, the nanofibers used in the present invention can be subjected to various pretreatments depending on the intended use. Pretreatment includes heat treatment, hydrolysis treatment with acid and alkali, hot water treatment, glow discharge treatment, electron beam treatment, autoclave treatment, gas treatment, steam treatment, flame treatment, coating treatment, graft polymerization treatment, stretching Although a process etc. are mention | raise | lifted, it is not limited to these. In particular, coating treatment or graft polymerization treatment using a polymer is important for variously changing the properties of nanofibers, and polymers used for such treatment include polyvinylpyrrolidone (PVP), polyethyleneimine, polylysine, poly Cationic polymers such as allylamine, ionic polymers such as anionic polymers such as polyacrylic acid and polymethacrylic acid, hydrophilic polymers such as polyvinyl alcohol, polyethylene glycol, and cellulose, and hydrophobic properties such as polystyrene, polyethylene terephthalate, and polybutylene terephthalate Polymers such as polymers, collagen, fibronectin, extracellular matrix, biopolymers such as chitin, chitosan, etc. are mentioned, but not limited thereto.

  In the nanofiber of the present invention, in particular, when the single fiber diameter is 200 nm or less, not only does the specific surface area dramatically increase but also there are innumerable pores of several nanometers to several hundred nanometers between the fibers. In order to show the excellent adsorption / absorption characteristics unique to nanofibers that were not seen, functional drugs can be adsorbed and supported on the nanofibers. The functional drug here refers to all substances that can improve the function of the fiber as a material. For example, a hygroscopic agent, a moisturizing agent, a water repellent, a heat retaining agent or a smoothing agent can be used as a target. The properties are not limited to fine particles, and polymers such as functional polymers, amino acids, proteins, vitamins, polyamines, photocatalyst nanoparticles, low molecular weight substances, drugs, and the like can be used. In particular, it is useful as a cell scaffold material to carry various proteins effective for cell culture and tissue regeneration on the surface.

  In addition, the adsorption / absorption characteristics of the nanofibers not only mean that functional drugs can be carried, but also their sustained release properties are excellent. The sustained release here refers to the property that a substance once adsorbed to the nanofiber surface is gradually released to the surrounding environment over a long period of time. For this reason, it means that it can be applied to a drug delivery system and the like as an excellent sustained-release substrate for functional molecules, functional drugs, and proteins.

  Because of these characteristics, nanofibers can easily absorb and release functional drugs, especially proteins, or absorb and release culture fluid and culture fluid components, and can be used effectively as scaffolds for cell culture and tissue regeneration. .

  In order to make further use of adsorption / absorption characteristics, it has an aromatic functional group such as polyethylene terephthalate and polybutylene terephthalate as a nanofiber with excellent absorbability of functional drugs such as proteins and excellent moldability as a nanofiber. It is preferable to use nanofibers made from hydrophobic thermoplastic polymers such as polyesters, polypropylenes, polyolefins such as polyethylene. Also, nanofibers made from polyamides such as nylon and hydrophilic polymers such as polyurethane, polylactic acid, and polyvinyl alcohol as nanofibers with excellent sustained release of functional drugs such as proteins and excellent moldability as nanofibers. It is preferable to use fibers.

  Moreover, if an ionic polymer or a polymer having an ionic functional group on the surface is used as the nanofiber, it is possible to adsorb the functional drug by electrostatic interaction. Similarly, after the nanofiber surface is coated with an ionic polymer or a polymer having an ionic functional group on the surface, the functional drug may be adsorbed by electrostatic interaction. Conversely, if you want to prevent adsorption due to electrostatic interaction, use a substance or polymer that has a counter ion to block it to remove ionicity from the surface of the nanofiber, so that adsorption due to electrostatic interaction is avoided. It can also be prevented.

  It is also possible to bind a functional agent directly to a functional group on the nanofiber surface by a covalent bond. Such functional groups include carboxyl groups, amino groups, mercapto groups, pyridyl disulfide groups, isocyanate groups, phenyl azide groups, diazocarbene groups, hydrazine groups, N-hydroxysuccinimide groups, imide ester groups, nitroaryl halide groups, imidazolylcarbamic acids. Group, maleimide group, thiophthalimide group, activated halogen group, in order to introduce such functional groups to the nanofiber surface, use polymers and molecules with such functional groups as raw materials for nanofiber production. Alternatively, a functional group may be introduced on the surface of the nanofiber using a commercially available crosslinking agent having an active group that reacts with two different types of functional groups. Moreover, you may introduce | transduce the functional group which has binding properties, such as a biotin group, an avidin group, streptavidin, and polyhistidine.

  In the present invention, it is more preferable to use nanofibers made of polymers such as nylon, polyurethane, polylactic acid, etc., particularly from the standpoint of protein adsorption, sustained release performance balance, biocompatibility, and moldability as a nanofiber.

  In addition, when used as a scaffold for cell culture or tissue regeneration, or as an implantable medical material, cell culture fluid, body fluid, or blood is carried between single fibers, and its retention performance has been greatly improved compared to conventional microfibers. In addition to being able to carry a large amount of liquid, it has a favorable property for operability during culture and transplantation, such as being less likely to spill from a nanofiber bundle. Thus, in order to impregnate culture fluid, body fluid, and blood, it is preferable to include nanofibers made of a hydrophilic polymer such as polyamide such as nylon.

  There is no particular limitation on the loading method of the functional drug, and it may be supported on the nanofiber by post-processing by adsorption treatment or coating in a solution, or it may be contained in the polymer alloy fiber that is the precursor of the nanofiber. You can leave it. In addition, the functional drug itself may be directly supported on the nanofiber assembly, or after the functional drug precursor substance is supported on the nanofiber, the precursor substance is converted into a desired functional drug. You can also More specific examples of the latter method include impregnating the nanofiber aggregate with an organic monomer and then polymerizing it, or impregnating the nanofiber aggregate with an easily soluble substance by treatment in a bath, There are a method of making it hardly soluble by oxidation-reduction reaction, ligand substitution, counter ion exchange reaction, enzyme reaction, photochemical reaction, hydrolysis reaction and the like, and a method of converting the structure into an activated form. In addition, when a functional drug precursor is supported in the spinning process, it is also possible to adopt a method in which a molecular structure with high heat resistance is maintained in the spinning process and the molecular structure is returned to a functional structure by post-processing. .

  The nanofiber assembly fiber surface described above may not be a natural substrate for cell attachment, cell growth, proliferation, differentiation induction, and activation. For this reason, in order to use nanofiber aggregates as cell scaffold materials such as cell culture scaffolds or tissue regeneration scaffolds, they can be coated with another biodegradable polymer or hydrophilic polymer as a binder, or partially alkaline hydrolyzed. Cell adhesion and inoculation density can also be improved by modifying the adsorption of serum proteins to the fibers that have been treated with and hydrolyzed on the surface. In particular, it is effective to adsorb functional proteins necessary for cell function expression such as cell adhesion, cell proliferation and differentiation, and activation to the fiber surface.

  In the cell scaffold material of the present invention, it is preferable that the protein is adsorbed to the nanofiber. Furthermore, in the cell scaffold material of the present invention, the protein to be adsorbed is preferably one or more types selected from cytokines.

  Cytokines are examples of functional proteins important for cell adhesion, cell proliferation and cell function to be adsorbed as described above. Cytokines refer to proteins that are biologically active through specific receptors on the cell surface in minute amounts, and are responsible for the regulation of immunity and the inflammatory response, the damage and death of virus-infected cells and tumor cells, and the proliferation and differentiation of cells. A generic term for proteins. Cytokines include interleukins, growth factors, chemokines, tumor necrosis factors, interferons and the like. Specifically, insulin, IGF (insulin-like growth factor) -I, IGF-II, EGF (epidermal growth factor), TGF (transforming growth factor) -α, TGF-β1, TGF-β2, FGF (fibroblasts) Growth factor) -1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, VEGF (vascular endothelial growth factor) -A, VEGF-B, VEGF-C, VEGF-D , NGF (nerve growth factor), IL (interleukin) -1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, GM-CSF, G-CSF, M-CSF, SCF ( Stem cell factor), FL (flt-3 ligand), EPO (erythropoietin), TPO (thrombopoietin), OSM, LIF, activin, inhibin, BMP (bone morphogenetic protein), PDGF, HGF, TNF (tumor necrosis factor) -α, Examples include, but are not limited to, TNF-β, Fas-L (Fas ligand), CD40 ligand, MIP, MCP, IFN (interferon) α, IFNβ, IFNγ, GDNF, and the like. In addition to cytokines, proteins that affect cells include Notch ligands (Delta 1-3, jaggide / selate 1, 2), stimulating antibodies such as anti-CD3 antibody and anti-CD28 antibody, T cell receptor (TCR), Wnt Secreted proteins, Tie receptors and the like can be used.

  In addition to these cytokines, there are proteins related to cell adhesion, called extracellular matrix, and adhesion factors, which are effective for cell culture and tissue regeneration. It is effective in terms of culture and tissue regeneration. The extracellular matrix refers to complex aggregates of biopolymers synthesized by cells and secreted and accumulated outside the cells. That is, it corresponds to the structural support of the tissue deposited around the cell and regulates cell adhesion, cytoskeletal orientation, cell shape, cell migration, cell proliferation, intracellular metabolism, and cell differentiation from the cell. Examples of such substances include fibronectin, laminin, collagen, glycosaminoglycan (such as heparan sulfate and hyaluronic acid), heparin, chitin, and chitosan. The adhesion factor refers to a factor that exists on the cell surface and is related to cell-cell and cell-extracellular matrix adhesion. As factors related to cell-cell adhesion, cadherin family, Ig superfamily, selectin family, sialomucin The integrin family is a factor involved in the binding between the cell and the extracellular matrix. In addition, artificially synthesized peptides, extracellular matrices, and genetically engineered proteins include “Pronectin F”, “Pronectin L” manufactured by Sanyo Chemical Industries, and “RetroNectin” manufactured by Takara Shuzo.

  As described above, when protein is adsorbed to nanofibers, it is preferable that a large amount of protein is efficiently adsorbed. For this reason, 1 mg of nanofiber is immersed in 1 mL of protein solution having a concentration of 100 μg / mL as a cell scaffold material. When protein is adsorbed, it is preferable that 80% or more of the protein in the solution can be adsorbed.

Moreover, it is preferable that the protein once adsorbed as a cell scaffold material is released gradually.
Proteins and functional drugs adsorbed on the nanofiber surface by the method as described above are gradually released over a long period after adsorption. The amount released and the period of sustained release depend on the nature of the nanofiber surface, the amount of adsorbed protein, the nature of the protein itself, and the environment in which it is released slowly, such as pH, temperature, and salt concentration. For example, a protein adsorbed by hydrophobic interaction is more likely to be released as the surface of the nanofiber becomes more hydrophilic. Therefore, it is preferable to use a hydrophilic polymer as a material for the nanofiber. In addition, in order to make the nanofiber surface hydrophilic, for example, the nanofiber surface can be hydrolyzed with hydrochloric acid or sodium hydroxide, or coated with a hydrophilic polymer. In addition, when the protein adsorbed on the nanofiber surface is slowly released into the solution, the amount of protein released is increased by adding a highly adsorbent substance such as a high molecular weight protein to the sustained release environment, that is, the solution. Can be made. Albumins, serum proteins, milk proteins, skim milk, lipids, etc. are examples of highly adsorbable substances added to the solution for such purposes, and the amount of protein released and the sustained release period are adjusted by adjusting the amount added. It becomes possible. In addition, by using a degradable polymer such as polylactic acid as a material for the nanofiber, it is possible to release the protein adsorbed along with the degradation of the polymer itself. Alternatively, a degradable substance such as gelatin is supported on the surface of the nanofiber, and then the protein can be adsorbed and released slowly.

  As the cell scaffold material, it is preferable that 1% or more of the protein once adsorbed is sustainedly released within 24 hours, and the sustained release is continued for 1 week or more.

  Moreover, it is preferable that the cell scaffold material containing the nanofiber of the present invention is used as a molded body used for cell culture or tissue regeneration.

  Cell scaffold materials containing the nanofibers of the present invention include, for example, hematopoietic stem cells, neural stem cells, mesenchymal hepatocytes, mesodermal stem cells, ES cells, immune cells, blood cells, nerve cells, blood vessels in vivo and in vitro Endothelial cells, fibroblasts, epithelial cells, hepatocytes, pancreatic β cells, cardiomyocytes, bone marrow cells, amniotic cells, umbilical cord blood cells or NIH3T3 cells, Hela cells, COS cells, HEK cells, L929 cells, Daudi cells, Jurkat cells, Part or all of a molded body for scaffold material used for cell culture, such as cell lines such as KG-1a cells and CTLL-2 cells, or various hybridoma cell lines that are antibody-producing cells, and cell culture bags Partial or complete regeneration of tissues such as nerve, heart, blood vessel, cartilage, skin, cornea, kidney, liver, hair, heart muscle, muscle, tendon, and formation of tissue for transplantation Part or all of the molded body for the scaffold material used, or aneurysm coil, embolic material, artificial nerve, artificial mucosa, artificial esophagus, artificial trachea, artificial blood vessel, artificial valve, artificial chest wall, artificial pericardium, artificial myocardium, Artificial diaphragm, artificial peritoneum, artificial ligament, artificial tendon, artificial cornea, artificial skin, artificial joint, artificial joint, artificial cartilage, dental material, part or all of a medical molded body for implantation in vivo such as an intraocular lens, surgical use A part of a molded body used for medical activities such as sutures, surgical filling materials, surgical reinforcement materials, wound protection materials, fracture joint materials, catheters, syringes, infusion / blood bags, blood filters, extracorporeal circulation materials, etc. All of them can be used as a part of or all of other materials such as contact lenses and intraocular lenses, and moldings. As polymers used as nanofibers, polylactic acid and polyglycolic acid are used as scaffolds for tissue regeneration / restoration of skin, periodontal tissue, jawbone, etc., and as cell scaffold materials as templates. Nylon is used as a material for a portion that comes into contact with cells, tissues, body fluids, and blood as a surgical suture or the like. PMMA is used as a material for artificial kidneys and contact lenses. For this reason, it can be said that these polymers are suitable as materials for nanofibers used as cell scaffold materials.

  Most of the above-mentioned uses are fields where the nanofiber nonwoven fabric produced by electrospinning cannot be developed due to lack of strength and shape stability, lack of size (width) itself, etc. Nanofiber aggregates using a new polymer alloy melt spinning method can be used for the first time in terms of strength and form stability. For example, since a cell scaffold material for implantation in a living body requires product strength, it can be achieved with excellent yarn strength like the nanofiber of the present invention. In general, the nonwoven fabric structure may lack the controllability of the scaffold microstructure, and it is impossible to obtain a uniform porosity that allows cells to enter. Furthermore, a scaffold using a nonwoven fabric generally has a weak mechanical structure. Specific bonding materials or backing materials may be required to ensure structural stability, but nanofibers made with the polymer alloy melt spinning of this patent are not compatible with textile technology or other materials. In vivo bone marrow that can provide a method to realize a well-structured and ordered structure scaffold using composite technology, and is formed into a three-dimensional scaffold with various design patterns, and is optimal for various porosity or cell culture and tissue regeneration Fine distribution of functional ligands such as cytokines can be realized by using microstructures and surface-modified fibers that mimic the environment such as the inner or interstitial layer.

When the cell scaffold material containing the nanofiber of the present invention is used as a molded body and cell culture and tissue culture are performed as a culture scaffold, MEM medium, DMEM medium, αMEM medium, WE medium, F12 medium, RPMI1640 medium, L-15 are used as the medium. Medium, MCDB153 medium, BME medium, IMEM medium, ES medium, DM-160 medium, Fisher medium, StemSpan medium, StemPro medium, Hybridoma SFM medium, and mixtures thereof can be mentioned as the culture medium for cell culture. It is not limited. In addition, serum such as bovine serum, fetal bovine serum, horse serum, human serum, plasma components, or cytokines and additives such as interleukin, interferon, insulin, transferrin, selenium, and mercaptoethanol may be added to these culture solutions. good. When culturing, culturing in a 5% CO ₂ incubator, culturing in a gas permeable bag, using a column incorporating a cell scaffold material containing the nanofiber of the present invention, or cells Cells can also be cultured using a perfusion culture system that incorporates a reservoir containing a suspension, an oxygen load device using a commercially available artificial lung, a dialysis column for exchanging the medium, etc. .

It is also possible to impregnate a cell scaffold material containing nanofibers in advance with these culture solutions and additives, and add the cells therein to culture the cells. By adopting such a method, the operability of the cultured cells is improved.
When nanofibers form an aggregate on another substrate, the shape of the other substrate is preferably a shape used as a cell culture instrument.
The cell scaffold material containing the nanofiber of the present invention can be used for purposes other than actual medical practice for research that leads to medical treatment. For example, cell culture to examine detailed culture and tissue culture, flask for tissue culture, petri dish, well, plate, multi-well, multi-well plate, slide, film, back, column, tank, bottle, hollow fiber, non-woven fabric It is also possible to form a nanofiber aggregate obtained in the present invention on a substrate having a shape used as a cell culture instrument.

  The cell scaffold material containing the nanofiber of the present invention can also be used in a method for producing cells for transplantation used for stem cell transplantation and the like, for example, in recent years, supplying umbilical cord blood instead of bone marrow transplantation for severe blood diseases such as leukemia. Hematopoietic stem cell transplantation used as a source has been performed. Umbilical cord blood transplantation is mainly used for the treatment of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), aplastic anemia, congenital immune deficiency, congenital metabolic disorders, etc. Compared to bone marrow transplantation and peripheral blood stem cell transplantation, post-transplant graft-versus-host disease (GVHD) is lighter and proliferative capacity is about one tenth of the number of cells used during bone marrow transplantation. But transplantation is possible. The absolute total number of stem cells contained in umbilical cord blood is small, and the number of cells necessary for engraftment of hematopoietic stem cells in adults cannot be secured, and so far transplantation has been performed mainly for children. For example, using the material of the present invention, by expanding while maintaining undifferentiated hematopoietic stem cells and progenitor cells in umbilical cord blood, avoiding expansion of adaptation such as stem cell transplantation to adults, engraftment failure, promoting hematopoietic recovery, Reduction of blood transfusion volume, transplantation to multiple patients, shortening of hospitalization period of patients and safer transplantation can be realized. In addition, it can also be used for culturing transplantation cells such as helper T cells, killer T cells, and dendritic cells used in cancer immunotherapy. Thus, in order to culture stem cells or immune cells, it is preferable to adsorb and slowly release cytokines that induce functions such as cell proliferation or cell division on the cell scaffold material of the present invention.

  In addition, by using the cell scaffold material containing the nanofiber of the present invention as a scaffold material molding for cell culture, it is also possible to produce cell preparations by culturing cells effective against various diseases and diseases It is. Cell preparations refer to drugs and medical devices processed from tissues and cells, and cell preparation manufacturing methods refer to cell separation, cell proliferation, cell stimulation, cell differentiation induction, cell apoptosis induction, etc. As a cell preparation, it includes all processes for processing into a form effective for diseases and diseases. In order to produce a cell preparation, first, a tissue, a body fluid, or the like that is a supply source of a cell group is collected. The source of these cell groups is preferably derived from humans, but is not limited thereto. Sources of such cell groups include, but are not limited to, peripheral blood, umbilical cord blood, bone marrow fluid, amniotic tissue, placental tissue, gonad, peripheral blood of G-CSF mobilization, fetal tissue, and the like. When using bodily fluids as a supply source, it is common to obtain a uniform cell group that excludes extra components for cell culture by centrifugation, unit gravity sedimentation, centrifugation, etc. before culturing. . In addition, it is preferable that the cell group with high purity of cells to be transplanted is made into a cell group using a cell separation method such as flow cytometry, magnetic bead method, affinity column method, etc. before culturing. After performing such various processes, cell culture and tissue regeneration are performed by the above-described method using the cell scaffold material and the scaffold material molded body containing the nanofiber of the present invention, thereby obtaining cells necessary as a cell preparation. Can be obtained with high purity. In addition, it is preferable as a method for producing a cell preparation to perform cell separation again after culturing cells using a cell scaffold material containing the nanofiber of the present invention. By this production method, the intended useful cells can be obtained in large quantities with high purity, and a cell preparation with excellent effects can be produced.

  Examples are shown below, but the present invention is not limited thereto. The method for producing nanofibers used in the examples is shown in the following reference examples.

(Reference Example 1) [Method for producing N6 nanofiber aqueous dispersion]
Melt viscosity 53 Pa · s (262 ° C., shear rate 121.6 sec ⁻¹ ), melting point 220 ° C. N6 (20 wt%), melt viscosity 310 Pa · s (262 ° C., shear rate 121.6 sec ⁻¹ ), melting point 225 ° C. Copolymerized PET (80 wt%) having a melting point of 225 ° C. copolymerized with 8 mol% of isophthalic acid and 4 mol% of bisphenol A was kneaded at 260 ° C. with a biaxial extrusion kneader to obtain a polymer alloy chip with b ^* value = 4. Obtained. The copolymerized PET had a melt viscosity of 180 Pa · s at 262 ° C. and 1216 sec ⁻¹ . The kneading conditions at this time were as follows.

Screw type Same direction complete meshing type Double thread screw Screw diameter 37mm, effective length 1670mm, L / D = 45.1
The kneading part length is 28% of the effective screw length
The kneading part was located on the discharge side from 1/3 of the effective screw length.

There are three backflow sections along the way. Polymer supply N6 and copolymerized PET were weighed separately and supplied separately to the kneader.

260 ° C
Two vents This polymer alloy was melted at 275 ° C. and led to a spin block with a spinning temperature of 280 ° C. The polymer alloy melt was filtered with a metal nonwoven fabric having a limit filtration diameter of 15 μm, and then melt-spun from a die having a die surface temperature of 262 ° C. At this time, a base having a measuring portion having a diameter of 0.3 mm above the discharge hole, having a discharge hole diameter of 0.7 mm and a discharge hole length of 1.75 mm was used. And the discharge amount per single hole at this time was 2.9 g / min. Furthermore, the distance from the base lower surface to the cooling start point was 9 cm. The discharged yarn is cooled and solidified with a cooling air of 20 ° C. over 1 m, and is supplied by an oil supply guide installed 1.8 m below the base 5, and then passed through unheated first and second take-up rollers. Was wound up at 900 m / min. The spinnability at this time was good, and there was no yarn breakage during continuous spinning for 24 hours. This was subjected to a stretching heat treatment with the first hot roller at 98 ° C. and the second hot roller at 130 ° C. At this time, the draw ratio between the first hot roller and the second hot roller was 3.2 times.

  The obtained polymer alloy fiber showed excellent properties as a raw material for cell scaffolding material of 120 dtex, 12 filaments, strength 4.0 cN / dtex, elongation 35%, U% = 1.7%. Further, when a cross section of the obtained polymer alloy fiber was observed with a TEM, N6 was an island component and copolymerized PET was a sea component, and the island component N6 had a diameter of 53 nm, and N6 was very finely dispersed. A polymer alloy fiber was obtained. The “polymer alloy fiber” was scraped to about 10,000 dtex. And it processed with 98 degreeC and 10-% sodium hydroxide for 1 hour, the polyester component of a sea component was removed, and the casse which consists of N6 nanofiber aggregates was obtained. And this was cut into 2 mm fiber length with a guillotine cutter, and N6 nanofiber short fiber was obtained. From this, N6 nanofibers were sampled and the cross section of the fibers was observed by TEM. As a result, the number average single fiber diameter of N6 nanofibers was 60 nm. The fiber ratio of single fibers having a single fiber diameter of 100 nm or more was 0%. And this N6 nanofiber short fiber was first beaten with a Niagara beater. Water was removed from the fiber with a centrifugal separator to obtain a primary beaten fiber having a fiber concentration of 10%. Further, the primary beating fiber was secondarily beaten with a PFI beating apparatus, and then dehydrated to obtain N6 nanofiber pulp having a concentration of 10% of nanofibers.

  Further, 5.5 g of this 10% concentration N6 nanofiber pulp and 0.55 g of an anionic dispersant (“Charol AN-103P” manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) were placed in a disaggregator with water and dispersed for 5 minutes. After further adding water to the dispersion in the disaggregator, 500 ml was taken in a lab mixer, (1) dispersed at 6000 rpm for 30 minutes in a lab mixer, and (2) a solution filtered through a 50 mesh stainless steel wire mesh was obtained. (3) The nanofibers on the stainless steel wire net were returned to water, and the operations (1) and (2) were repeated three times to obtain a nanofiber aqueous dispersion having a concentration of about 0.01 wt%.

Reference Example 2 [PBT nanofiber aqueous dispersion]
Polystyrene (PS) copolymerized with 22% PBT and 2-ethylhexyl acrylate having a melt viscosity of 120 Pa · s (262 ° C., 121.6 sec ⁻¹ ) and a melting point of 225 ° C., the PBT content is 25% by weight, and the kneading temperature Was 240 ° C. and melt-kneaded in the same manner as in Reference Example 1 to obtain a polymer alloy chip. At this time, the melt viscosity at 262 ° C. and 121.6 sec ⁻¹ of the copolymerized PS was 140 Pa · s, and the melt viscosity at 245 ° C. and 1216 sec ⁻¹ was 60 Pa · s.

  This was melt-spun in the same manner as in Reference Example 1 at a melting temperature of 260 ° C., a spinning temperature of 260 ° C. (die surface temperature of 245 ° C.), and a spinning speed of 1200 m / min. At this time, the same spinneret as that used in Reference Example 1 was used as the base. The spinnability was good and the yarn breakage was 1 in 1 t spinning. The single-hole discharge rate at this time was 1.0 g / min. The obtained undrawn yarn was subjected to drawing heat treatment in the same manner as in Reference Example 1. The obtained drawn yarn was 161 dtex, 36 filaments, had a strength of 1.4 cN / dtex, an elongation of 33%, and U% = 2.0%, and exhibited excellent characteristics as a raw material of the cell scaffold material.

  When the cross section of the obtained polymer alloy fiber was observed with TEM, the copolymer PS showed the sea (thin part), the PBT was the island (dense part) sea-island structure, and the PBT number average diameter was 70 nm. A polymer alloy fiber in which PBT was nano-sized and uniformly dispersed was obtained. A 0.01 wt% PBT nanofiber aqueous dispersion is obtained using a nonionic dispersant (“Neugen EA-87” manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) in the same manner as in Reference Example 1 using the polymer alloy fiber. It was. In the sea removal treatment, 99% or more of the sea polymer was eluted by treating with trichlorethylene. The number average single fiber diameter of the PBT nanofibers was 85 nm.

Reference Example 3 [N6 nanofiber nonwoven fabric]
Using N6 of Reference Example 1 and a weight average molecular weight of 120,000, a melt viscosity of 30 Pa · s (240 ° C., 2432 sec ⁻¹ ), a poly-L lactic acid (optical purity of 99.5% or more) having a melting point of 170 ° C., the content of N6 is Polymer alloy chips were obtained by melting and kneading in the same manner as in Reference Example 1 at 20% by weight and a kneading temperature of 220 ° C. A highly oriented undrawn yarn which was melt-spun in the same manner as in Reference Example 1 at a melting temperature of 230 ° C., a spinning temperature of 230 ° C. (die surface temperature of 215 ° C.) and a spinning speed of 3500 m / min was drawn at a drawing temperature of 90 ° C. and a draw ratio of 1 The film was stretched and heat-treated at a heat setting temperature of 130 ° C. 39 times. The obtained drawn yarn is 67 dtex, 36 filaments, has a strength of 3.6 cN / dtex, an elongation of 40%, a boiling water shrinkage of 9%, and U% = 0.7%, and is an excellent property as a raw material for cell scaffold materials. showed that.

  When the cross section of the obtained polymer alloy fiber was observed with a TEM, the sea-island structure where PLA was the sea and N6 was the island, the diameter by the number average of the island N6 was 55 nm, and N6 was nano-sized and uniformly dispersed. A polymer alloy fiber was obtained.

This polymer alloy fiber was combined into a tow of 100,000 dtex, and then subjected to mechanical crimping to obtain a crimped yarn having a number of crimps of 15 threads / 25 mm. This was cut into a fiber length of 51 mm, defibrated with a card, and then made into a web with a cross wrap weber. Next, 3000 needle / cm ² needle punches were applied to obtain a 750 g / m ² fiber-entangled nonwoven fabric. After applying polyvinyl alcohol to this nonwoven fabric, it was subjected to an alkali treatment with a 3% aqueous sodium hydroxide solution (60 ° C., bath ratio 1: 100) for 2 hours, and 99% or more of PLA was hydrolyzed and removed. As a result of extracting and analyzing the nanofiber aggregate from the nanofiber structure, the single fiber diameter based on the number average of the nanofibers is 55 nm, which is an unprecedented fineness. The fiber fineness ratio was 70%, and the single fiber fineness variation was very small.

Reference Example 4 [N6 microfiber nonwoven fabric]
Nylon 6 (N6: 60% by weight) island component with a melting point of 220 ° C. Polystyrene (PS) is used as the sea component, and sea island component composite yarn is drawn as described in JP-A-53-106872, and stretched. The drawn island component composite yarn was obtained. This polymer alloy fiber was combined in the same manner as in Reference Example 2 to obtain a 100,000 dtex tow, and then subjected to mechanical crimping to obtain a crimped yarn having 15 crimps / 25 mm. This was cut into a fiber length of 51 mm, defibrated with a card, and then made into a web with a cross wrap weber. Next, 3000 needle / cm ² needle punches were applied to obtain a 750 g / m ² fiber-entangled nonwoven fabric. After applying polyvinyl alcohol to this nonwoven fabric, 99% or more of PS was removed by trichlorethylene treatment to obtain a nonwoven fabric of N6 ultrafine fibers having a single fiber diameter of about 2 μm. When the cross section of the fiber was observed with a TEM, the single fiber diameter of the ultrafine fiber was 2.2 μm.

Reference Example 5 [N6 nanofiber knitted fabric]
A circular knitting was produced using the polymer alloy fiber produced in Reference Example 1, and this was immersed in a 3% aqueous sodium hydroxide solution (95 ° C., bath ratio 1: 100) for 2 hours, thereby co-polymerizing the polymer alloy fiber. More than 99% of the polymerized PET was hydrolyzed and removed. As a result, the circular knitting made of N6 single yarn maintained the circular knitting shape even though the copolymerized PET, which is a sea polymer, was removed. Moreover, the basis weight of this circular knitting was 60 g / m ² .

  When the yarn was pulled out from the circular knitting made of this N6 single yarn and the side of the fiber was observed by SEM, this yarn was not a single yarn but an infinite number of nanofibers gathered together, and as a whole it was a nanofiber bundle. I found out. Moreover, the space | interval of the nanofibers of this N6 nanofiber bundle is about several nanometers-several hundred nanometers, and the very fine space | gap existed. Furthermore, as a result of observing the cross section of the fiber by TEM, it was found that this N6 nanofiber had a single fiber diameter of about several tens of nanometers. And the single fiber diameter by the number average of a nanofiber was 60 nm and the thinness which was not in the past. The fiber ratio of single fibers having a single fiber diameter of 100 nm or more was 0%.

Example 1 [Cell Adhesion on Cell Culture Well Formed with N6 Nanofibers]
100 μL of the N6 nanofiber aqueous solution of Reference Example 1 was added to a 96-well plate for cell culture, and then dried in a dryer at 60 ° C. to form a structure of N6 nanofiber as a cell scaffold material on the well. Wells were made. C ₂ C ₁₂ myoblasts are suspended in DMEM medium containing 10% FBS (fetal bovine serum), seeded at 2 × 10 ⁵ cells per well, incubated for 1 hour in a 5% CO ₂ incubator, and then attached cells. When the number was confirmed, it was confirmed that about 1.5 × 10 ⁵ cells (about 75% of the seeded cells) were adhered.

Comparative Example 1 [Cell adhesion on a cell culture well that does not form N6 nanofibers]
In Example 1, when adhesion of C ₂ C ₁₂ myoblasts was confirmed without forming nanofibers on the wells, the number of adherent cells was about 0.2 × 10 ⁵ (about 10% of the seeded cells). The cells were only attached.

Example 2 [Formation of nerve fibers on wells for cell culture formed with PBT nanofibers]
After adding 500 μL of the PBT nanofiber aqueous solution of Reference Example 2 to a 24-well plate for cell culture, it was dried in a dryer at 60 ° C. to form a PBT nanofiber structure as a cell scaffold material on the well, and cell culture Wells were prepared. After adding 0.01% collagen aqueous solution to this well and coating with collagen, PC12 cells are suspended in RPMI1640 medium containing 5% FBS, 10% HS (bovine serum) and seeded at 1 × 10 ⁵ cells per well. After that, nerve growth factor (NGF) was added at a concentration of 50 ng / mL and cultured for 5 days. When observed under a microscope after 5 days, nerve fibers were extended from 80% of the cells, and the length of the nerve fibers averaged about 200 μm.

Comparative Example 2 [Formation of nerve fibers on cell culture wells not forming PBT nanofibers]
After adding a 0.01% collagen aqueous solution directly to a 24-well plate for cell culture to coat collagen, PC12 cells were suspended in RPMI 1640 medium containing 5% FBS, 10% HS (bovine serum) in the same manner as in Example 2. After seeding 1 × 10 ⁵ cells per well, NGF was added at a concentration of 50 ng / mL and cultured for 5 days. Observation with a microscope 5 days later revealed that nerve fibers were extended from 50% of the cells, and the average length of the nerve fibers was about 20 μm.

Example 3 [Adsorption of protein on N6 nanofiber nonwoven fabric]
1 mg of the N6 nanofiber nonwoven fabric prepared in Reference Example 3 was immersed in 1 mL of PBS solution containing serum albumin, fibronectin, insulin, and EGF at a concentration of 100 μg / mL, respectively, and protein was adsorbed at 37 ° C. all day and night. The remaining protein concentration was detected by the Bradford method, and the amount of protein adsorbed on the nonwoven fabric was detected. As a result, 95% of serum albumin, 85% of fibronectin, 90% of insulin and 90% of EGF were adsorbed on the N6 nanofiber nonwoven fabric on average.

Comparative Example 3 [Protein adsorption to N6 microfiber nonwoven fabric]
1 mg of the N6 microfiber nonwoven fabric prepared in Reference Example 4 was immersed in 1 mL of PBS solution containing serum albumin, fibronectin, insulin, and EGF at a concentration of 100 μg / mL, respectively, and protein was adsorbed at 37 ° C. all day and night. The remaining protein concentration was detected by the Bradford method, and the amount of protein adsorbed on the nonwoven fabric was detected. As a result, on average, 75% of serum albumin, 40% of fibronectin, 2% of insulin, and 5% of EGF were adsorbed to the N6 microfiber nonwoven fabric.

Example 4 [Slow release of protein from N6 nanofiber nonwoven fabric]
0.2 mg of N6 nanofiber nonwoven fabric prepared in Reference Example 3 was immersed in 1 mL of PBS solution containing insulin at a concentration of 1 μg / mL, and the insulin was adsorbed overnight at 37 ° C., and then washed three times with PBS solution. Then, it was immersed in 1 mL of PBS solution containing 5% BSA in an atmosphere of 37 ° C., and the solution was exchanged every day for one week. The insulin concentration in the solution was measured by ELISA. As a result, the released insulin concentration was 120 ng / mL on the first day, 100 ng / mL on the second day, 60 ng / mL on the third day, 50 ng / mL on the fourth day, 40 ng / mL on the fifth day, 6 The day was 40 ng / mL and the 7th day was 30 ng / mL.

Comparative Example 4 [Prolonged Release of Protein from N6 Microfiber Nonwoven Fabric]
0.2 mg of the N6 microfiber nonwoven fabric produced in Reference Example 4 was immersed in 1 mL of PBS solution containing insulin at a concentration of 1 μg / mL, and the insulin was adsorbed overnight at 37 ° C., and then washed three times with the PBS solution. Then, it was immersed in 1 mL of PBS solution containing 5% BSA in an atmosphere of 37 ° C., and the solution was changed every day. The insulin concentration in the solution was measured by ELISA. As a result, the released insulin concentration was 100 ng / mL on the first day and 10 ng / mL on the second day, and was not detectable after the third day.

Example 4 [Culture using protein-adsorbed nanofibers]
After immersing 0.1 mg of N6 nanofiber nonwoven fabric prepared in Reference Example 3 in 0.5 mL of PBS solution containing NGF at a concentration of 1 μg / mL, NGF was adsorbed overnight at 37 ° C., and then washed three times with PBS solution. The NGF adsorption N6 nanofiber nonwoven fabric was produced.

Next, 0.01% collagen aqueous solution was added onto a 24-well plate for cell culture to coat collagen, and then PC12 cells were suspended in RPMI 1640 medium containing 5% FBS, 10% HS, and 1 × 10 per well. ^After seeding ⁵ cells and attaching the cells, the NGF-adsorbed N6 nanofiber nonwoven fabric prepared above was added to the culture solution and cultured for 5 days. When observed under a microscope after 5 days, nerve fibers were extended from 60% of the cells.

Comparative Example 4 [Culture using nanofiber not adsorbing protein]
In Example 4, a non-woven fabric was prepared using a PBS solution not containing NGF, and this non-woven fabric was added to the culture solution in the same manner as in Example 4 and observed with a microscope after 5 days of culture. Fiber extension could not be confirmed.

Example 5 [Implant material using N6 nanofiber fabric]
The effectiveness of tissue regeneration was confirmed by conducting transplantation experiments on model animals using the N6 nanofiber fabric produced in Reference Example 3. The N6 nanofiber fabric 1 cm × 1 cm produced in Reference Example 3 was immersed in 1 mL of a phosphate buffer containing 100 μg of bFGF at 37 ° C. for a whole day and night to produce a tissue regeneration material in which bFGF was adsorbed on the nanofiber surface. . This biological tissue regeneration fiber material was implanted subcutaneously in the back of a ddY mouse (7 weeks old, female), and the mouse was sacrificed one week later. When the tissue section was stained with hematoxylin and eosin and microscopic observation was performed, it was confirmed that new blood vessels were uniformly induced in the bFGF-containing nanofiber fabric.

Claims (16)

  A cell scaffold material comprising nanofibers made of an organic polymer and having a number average single fiber diameter of 1 to 200 nm.   The cell scaffold material according to claim 1, wherein the diameter of the single fiber diameter by number average is 1 to 100 nm.   The cell scaffold material according to claim 1 or 2, wherein the nanofiber is produced from melt spinning.   The cell scaffold material according to any one of claims 1 to 3, wherein the organic polymer is a thermoplastic polymer.   The cell scaffold material according to claim 4, wherein the thermoplastic polymer is one kind selected from the group consisting of polyolefin, polyamide, polyurethane, polyester, fluoropolymer, polylactic acid, polyvinyl alcohol, and derivatives thereof.   The cell scaffold material according to claim 4 or 5, wherein the thermoplastic polymer is nylon or polybutylene terephthalate.   The cell scaffold material according to any one of claims 1 to 6, wherein the nanofiber is processed into a shape of paper, nonwoven fabric, or woven or knitted fabric.   The cell scaffold material according to any one of claims 1 to 7, wherein the nanofibers form a bundle structure.   The cell scaffold material according to any one of claims 1 to 8, wherein the nanofiber forms an aggregate on another substrate.   The cell scaffold material according to claim 9, wherein the shape of the other substrate is a shape used as a cell culture instrument.   11. When 1 mg of nanofibers are immersed in 1 mL of a protein solution having a concentration of 100 μg / mL to adsorb proteins, 80% or more of the proteins in the solution can be adsorbed. A cell scaffold material according to 1.   The cell scaffold material according to any one of claims 1 to 11, wherein a protein is adsorbed to the nanofiber.   The cell scaffold material according to claim 12, wherein the protein to be adsorbed is at least one selected from cytokines.   The cell scaffold material according to claim 12 or 13, wherein the protein once adsorbed is gradually released.   The cell scaffold material according to claim 14, wherein 1% or more of the protein once adsorbed is sustainedly released within 24 hours, and the sustained release continues for 1 week or more.   A molded article used for cell culture or tissue regeneration comprising the cell scaffold material according to any one of claims 1 to 15.