JP2005264364A - Method for producing floc - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a floc fiber structure suitable for cell culture. <P>SOLUTION: The floc having 0.1-20 μm average fiber diameter and 10-95 kg/m<SP>3</SP>average bulk density is obtained by a method comprising a step for producing a solution by dissolving an organic polymer in a solvent, a step for applying high voltage to the solution, a step for ejecting the solution, a step for forming fiber structures by evaporating the solvent from the ejected solution, a step for eliminating electric charge of the formed fiber structures, and a step for accumulating the fiber structures by the elimination of the electric charge. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing a cotton-like body. More specifically, the present invention relates to a method for producing a cotton-like body as a fiber structure useful as a substrate for culturing cells.

  In the field of regenerative medicine, a fiber structure may be used as a base material when cells are cultured. As the fiber structure, use of polyglycolic acid used for, for example, surgical sutures has been studied (for example, see Non-Patent Document 1). However, since the fiber structure obtained by these usual methods has a fiber diameter that is too large, the area to which cells can adhere is insufficient, and a fiber structure having a smaller fiber diameter has been desired to increase the surface area.

  On the other hand, an electrostatic spinning method is known as a method for producing a fiber structure having a small fiber diameter (see, for example, Patent Documents 1 and 2). The electrospinning method includes a step of introducing a liquid, for example, a solution containing a fiber-forming substance into an electric field, thereby causing the liquid to move toward an electrode and forming a fibrous substance. Usually, the fiber forming material is cured while it is squeezed out of solution. Curing is performed, for example, by cooling (for example, when the spinning liquid is solid at room temperature), chemical curing (for example, treatment with curing steam), or evaporation of the solvent. Moreover, the obtained fibrous substance is collected on a suitably arranged receptor, and can be peeled from there if necessary. In addition, since the electrospinning method can directly obtain a fibrous material in the form of a cotton-like material, it is not necessary to form a fiber structure once the fibers have been produced once, and the operation is simple.

  It is known to use a fiber structure obtained by an electrospinning method as a substrate for culturing cells. For example, the regeneration of blood vessels has been studied by forming a fiber structure made of polylactic acid by an electrospinning method and culturing smooth muscle cells thereon (see, for example, Non-Patent Document 2). However, the fiber structure obtained by using these electrospinning methods tends to take a dense structure in which the distance between the fibers is short, that is, a structure with a large apparent density. When this is used as a substrate (scaffold) for cell culture, the cultured cells are deposited on the surface of each individual fiber forming the fiber structure as the culture proceeds, and the surface of the fiber is thickly covered. End up. As a result, it is difficult for a solution containing nutrients or the like to sufficiently move into the fiber structure, and cell culture can be performed only near the surface of cells cultured and deposited on the fiber.

JP-A 63-145465 JP 2002-249966 A Noriya Ohno, Director of Masao Aizawa, “Regenerative Medicine”, NTS Corporation, January 31, 2002, page 258 “Journal of Biomaterials Applications” by Joel D. Stitzel, Kristin J. Pawlowski, Gary E. Wnek, David G. Simpson, Gary L. Bowlin. ), Sage Publications, 2001, 16, p. 22-33

  An object of the present invention is to provide a method for producing a cotton-like body having a large gap between fibers and having a sufficient thickness for cell culture so as to be suitable for long-time cell culture.

As a result of intensive studies in view of the above prior art, the present inventors have completed the present invention.
That is, the object of the present invention is to
A method for producing a cotton-like body comprising an organic polymer and having an average fiber diameter of 0.1 to 20 μm and an average apparent density of 10 to 95 kg / m ³ , wherein the organic polymer is dissolved in a solvent A step of applying a high voltage to the solution, a step of ejecting the solution, a step of evaporating a solvent from the ejected solution to form a fiber structure, and the formed fiber structure This is achieved by a method for producing a cotton-like body comprising the steps of losing the charge of the body and accumulating the fiber structure by the charge loss.

  The cotton-like body obtained by the production method of the present invention is effective as a substrate for cell culture because it has a small fiber diameter and is suitable for cell culture. Moreover, the obtained cotton-like body can also be used as it is, and can also be used in combination with another member according to handleability and other requirements.

Hereinafter, the present invention will be described in detail. The cotton-like body obtained by the present invention is a three-dimensional structure formed by laminating the obtained fiber or fibers and fixing the fibers as necessary. The form of the cotton-like body has an average apparent density of 10 to 95 kg / m ³ and an average fiber diameter of 0.1 to 20 μm. Even if the shape of the cotton-like body is a square so that it can be easily subjected to secondary processing such as laminating the obtained cotton-like body with another sheet-like material or processing it into a tube shape or a mesh shape thereafter. Even if it is circular, its shape does not matter. The thickness of the cotton-like body is preferably 100 μm or more from the viewpoint of handling, and it is also possible to form a thick structure by overlapping the cotton-like bodies.

  The cotton-like body obtained by the present invention consists of an organic polymer that can be dissolved in a volatile solvent. Examples of organic polymers that can be dissolved in a volatile solvent include polyvinyl chloride, polyacrylonitrile, polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, polybutylene succinate, and polyethylene succinate. , Polystyrene, polycarbonate, polyhexamethylene carbonate, polyarylate, polyvinyl isocyanate, polybutyl isocyanate, polymethyl methacrylate, polyethyl methacrylate, polynormal propyl methacrylate, polynormal butyl methacrylate, polymethyl acrylate, polyethyl acrylate, polybutyl acrylate, Polyethylene terephthalate, polytrimethylene terephthalate, polyethylene naphthalate, polyparaphenylene terephthalate , Polyparaphenylene terephthalamide-3,4'-oxydiphenylene terephthalamide copolymer, polymetaphenylene isophthalamide, cellulose diacetate, cellulose triacetate, methylcellulose, propylcellulose, benzylcellulose, fibroin, natural rubber, Polyvinyl acetate, polyvinyl methyl ether, polyvinyl ethyl ether, polyvinyl normal propyl ether, polyvinyl isopropyl ether, polyvinyl normal butyl ether, polyvinyl isobutyl ether, polyvinyl tertiary butyl ether, polyvinylidene chloride, poly (N-vinylpyrrolidone), poly (N-vinyl) Carbazole), poly (4-vinylpyridine), polyvinyl methyl ketone, polymethyl isopropenyl ketone Polyethylene oxide, polypropylene oxide, polycyclopentene oxide, polystyrene sulfone, nylon 6, nylon 66, nylon 11, nylon 12, nylon 610, nylon 612, as well as their copolymers.

  Of these, preferred are polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, polybutylene succinate, and polyethylene succinate and aliphatic polyesters such as these copolymers, and Preferably, polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, and polycaprolactone are used. Of these, polylactic acid is particularly preferred.

  In the present invention, other polymers and other compounds may be used in combination (for example, polymer copolymerization, polymer blending, compound mixing) as long as the purpose is not impaired.

  The cotton-like body obtained by the present invention is formed from fibers having an average fiber diameter of 0.1 to 20 μm. If the average fiber diameter is smaller than 0.1 μm, it is not preferable for use as a cell culture substrate for regenerative medicine because the biodegradability is too early. On the other hand, if the average fiber diameter is larger than 20 μm, the area to which cells can adhere becomes small, which is not preferable. A more preferable average fiber diameter is 0.1 to 10 μm. The fiber diameter represents the diameter of the fiber cross section. However, sometimes the cross-sectional shape of the fiber can be elliptical. With respect to the fiber diameter in this case, the average of the length in the major axis direction and the length in the minor axis direction of the ellipse is calculated as the fiber diameter. When the fiber cross section is neither circular nor elliptical, the fiber diameter is calculated by approximating a circle or an ellipse.

The cotton-like body obtained by the present invention is a cotton-like body having an average apparent density of 10 to 95 kg / m ³ . Here, the average apparent density means the density calculated from the area, average thickness, and mass of the produced cotton-like body. A preferable average apparent density is 50 to 90 kg / m ³ . If the average apparent density is greater than 95 kg / m ³ , it is not preferable because a solution containing nutrients does not sufficiently penetrate into the substrate during cell culture. If the average apparent density is less than 10 kg / m ³ , the mechanical strength required during cell culture cannot be maintained, which is not preferable.

  Next, the step of producing a solution by dissolving an organic polymer in a solvent will be described. In the production method of the present invention, the concentration of the organic polymer with respect to the solvent in the solution is preferably 1 to 30% by weight. If the concentration of the organic polymer is less than 1% by weight, it is not preferable because the concentration is too low, making it difficult to form a fiber structure. On the other hand, if it is greater than 30% by weight, the fiber diameter of the resulting fiber structure is undesirably large. A more preferable concentration of the organic polymer with respect to the solvent in the solution is 2 to 20% by weight.

  Moreover, a solvent may be used individually by 1 type and may combine several solvent. The solvent is not particularly limited as long as it can dissolve the organic polymer and evaporates at the spinning stage to form a fiber. For example, acetone, chloroform, ethanol, isopropanol, methanol, toluene, tetrahydrofuran, Water, benzene, benzyl alcohol, 1,4-dioxane, propanol, dichloromethane, carbon tetrachloride, cyclohexane, cyclohexanone, phenol, pyridine, trichloroethane, acetic acid, formic acid, hexafluoroisopropanol, hexafluoroacetone, N, N-dimethylformamide, N, N-dimethylacetamide, acetonitrile, N-methyl-2-pyrrolidinone, N-methylmorpholine-N-oxide, 1,3-dioxolane, methyl ethyl ketone, mixed solvents of the above solvents, etc. It is. Of these, dichloromethane is preferably used in view of handling properties and physical properties.

  Next, a step of applying a high voltage to the solution, a step of ejecting the solution, and a step of evaporating the solvent from the ejected solution to form a fiber structure will be described.

  In the present invention, it is necessary to apply a high voltage to the solution in order to eject a solution in which an organic polymer is dissolved to form a fiber structure. The method of applying a voltage is not particularly limited as long as a solution in which an organic polymer is dissolved is ejected and a fiber structure is formed, but a method of inserting an electrode into a solution and applying a voltage, or a solution ejection nozzle There is a method of applying a voltage.

  In addition, an auxiliary electrode can be provided separately from the electrode applied to the solution. Further, the value of the applied voltage is not particularly limited as long as the fiber structure is formed, but it is usually in the range of 5 to 50 kV, and when the applied voltage is smaller than 5 kV, the fiber structure is not ejected. Since the body is not formed, it is not preferable. When the applied voltage is higher than 50 kV, discharge is generated from the electrode toward the ground electrode, which is not preferable. More preferably, it is the range of 10-30 kV. The desired potential may be generated by any appropriate method known in the art.

  Further, immediately after the solution in which the organic polymer is dissolved is ejected, the solvent in which the organic polymer is dissolved volatilizes and a fiber structure is formed. Ordinary spinning is performed at room temperature in the atmosphere, but when volatilization is insufficient, it can be performed under negative pressure or in a high-temperature atmosphere. The spinning temperature depends on the evaporation behavior of the solvent and the viscosity of the spinning solution, but is usually in the range of 0 to 50 ° C.

  Next, the step of eliminating the charge of the formed fiber structure will be described. The method for eliminating the charge of the fiber structure is not particularly limited as long as it is a method for eliminating the charge of the fiber structure, and a preferable method is a method of eliminating the charge with an ionizer. An ionizer is an apparatus that can generate ions by a built-in ion generator and discharge the charged substances to discharge the ions, thereby eliminating the charge of the charged objects. As a preferable ion generator constituting the ionizer used in the present invention, there is an apparatus that generates ions by applying a high voltage to a built-in discharge needle.

  Next, the step of accumulating the fiber structure due to the charge disappearance will be described. The method of accumulating the fiber structure due to the charge loss is not particularly limited as long as the fiber structure is accumulated, but as a normal method, the electrostatic force of the fiber structure is lost due to the charge loss, There is a method of dropping and accumulating by its own weight. Moreover, you may perform the method of attracting | sucking the fiber structure which lost the electrostatic force and accumulating it on a mesh as needed, the method of making the air in an apparatus convect, and accumulating on a mesh, etc.

  Moreover, although the cotton-like body obtained by this invention may be used independently, according to a handleability and other requirements, you may use it in combination with another member.

  Moreover, the use of the cotton-like body obtained by this invention is not limited to the cell culture base material for regenerative medicine, It can be used for various uses, such as various filters and a catalyst support base material.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. The evaluation items in the following examples and comparative examples were carried out by the following methods.
Moreover, each value in an Example was calculated | required with the following method.
1. Average fiber diameter:
The surface of the obtained fiber structure was photographed with a scanning electron microscope (S-2400, manufactured by Hitachi, Ltd.) (magnification 2000 times), and randomly selected 20 points from the photograph, and the fiber diameter was measured. The average value of all the fiber diameters (n = 20) was determined and used as the average fiber diameter.
2. Area measurement:
The calculation was performed by placing a cotton-like body on a 0.1 mm square graph paper, copying the outline of the cotton-like body, and counting the number of wrinkles in the outline.
3. Average thickness:
Using a high-precision digital length measuring machine (Mitutoyo Co., Ltd .: trade name “Lightmatic VL-50”), an average value obtained by measuring the film thickness of the cotton-like body at a length measuring force of 0.01 N and n = 10 was calculated. . In this measurement, the measurement was performed with the minimum measurement force that can be used by the measuring instrument.
4). Average apparent density:
The mass of the cotton-like body was measured, and the average apparent density was calculated based on the area and average thickness determined by the above method.

[Example 1]
1 part by weight of polylactic acid (manufactured by Shimadzu Corporation: trade name “Lacty 9031”) and 9 parts by weight of dichloromethane (Wako Pure Chemical Industries, Ltd., special grade) were mixed at room temperature (25 ° C.) to prepare a solution. Spinning was performed using the apparatus shown in FIG. 1 to obtain a cotton-like body. The inner diameter of the ejection nozzle 1 was 0.8 mm, the voltage was 15 kV, the distance from the ejection nozzle 1 to the ionizer 7 was 15 cm, and the distance from the ejection nozzle 1 to the electrode 4 was 25 cm. The average diameter of the fiber structure constituting the obtained cotton-like body was 3.4 μm, and the average apparent density was 70 kg / m ³ . Electron micrographs of the flocculent surface are shown in FIGS.

[Reference example]
1 part by weight of polylactic acid (Shimadzu Corporation: trade name “Lacty 9031”) and 9 parts by weight of dichloromethane (made by Wako Pure Chemical Industries, Ltd., special grade) were mixed at room temperature (25 ° C.) to prepare a solution. Spinning was performed with the ionizer 7 of the apparatus shown in FIG. 1 turned off, and a fiber structure was obtained on the electrode 4. The inner diameter of the ejection nozzle 1 was 0.8 mm, the voltage was 15 kV, and the distance from the ejection nozzle 1 to the electrode 4 was 25 cm. The fiber structure constituting the obtained fiber assembly had an average diameter of 3.5 μm and an average apparent density of 180 kg / m ³ . Electron micrographs of the fiber assembly are shown in FIGS.

  As a result of electron microscope observation, no significant difference was observed in the surface structure between the surface structure of the cotton-like body having a low apparent density obtained in Example 1 and the surface structure of the fiber structure obtained in Reference Example. It was confirmed that the product obtained in Example 1 can be suitably used as a substrate for culturing cells.

  In addition, the fiber structure obtained in the present Example has a lower apparent density and is more suitable for cell culture.

It is the figure which showed typically the manufacturing apparatus for performing the manufacturing method of this invention. It is the photograph figure obtained by image | photographing (2000 times) the surface of the cotton-like body obtained by operation of Example 1 with a scanning electron microscope. It is the photograph figure obtained by imaging | photography (20000 times) the surface of the cotton-like body obtained by operation of Example 1 with a scanning electron microscope. 6 is a photograph obtained by photographing the surface of a fiber assembly obtained by the operation of Comparative Example 1 with a scanning electron microscope (2000 times). FIG. 6 is a photograph obtained by photographing the surface of a fiber assembly obtained by the operation of Comparative Example 1 with a scanning electron microscope (magnification 20000 times). FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Solution ejection nozzle 2 Solution 3 Solution holding tank 4 Electrode 5 High voltage generator 6 Cotton body deposit stand 7 Ionizer

Claims (7)

A method for producing a cotton-like body comprising an organic polymer and having an average fiber diameter of 0.1 to 20 μm and an average apparent density of 10 to 95 kg / m ³ , wherein the organic polymer is dissolved in a solvent A step of applying a high voltage to the solution, a step of ejecting the solution, a step of evaporating a solvent from the ejected solution to form a fiber structure, and the formed fiber structure A method for producing a cotton-like body, comprising: a step of losing the charge of the body; and a step of accumulating the fiber structure by the charge loss.   The manufacturing method according to claim 1, wherein the step of erasing the electric charge of the jetted solution is erasing the electric charge due to the charged air.   The manufacturing method of Claim 1 whose solvent used for the said melt | dissolution is a volatile organic solvent.   The production method according to claim 3, wherein the volatile organic solvent is an organic solvent containing a halogenated hydrocarbon.   The production method according to claim 4, wherein the halogenated hydrocarbon is dichloromethane.   The manufacturing method of Claim 1 whose said organic polymer is aliphatic polyester.   The production method according to claim 6, wherein the aliphatic polyester is polylactic acid, polyglycolic acid, or polylactic acid-polyglycolic acid copolymer.

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