JP2008029226A - Temperature responsive substrate material and cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material capable of finishing its function achieved by forming the structure of a structural material by collapsing the structure under a moderate condition after completing the function of the structural material, or the material capable of freely stopping the function of a functional substance, or recovering or releasing the functional substance after using the functional substance for various uses by keeping the functional substance on the surface or at the inside of the material. <P>SOLUTION: This temperature responsive substrate material is provided by containing a fibrous structural material consisting of an aggregate of short fibers having 1 to 50 μm number-average diameter of a single fiber and 0.2 to 30 mm fiber length of the single fiber and a temperature responsive polymer compound, and collapsing its structure by changing temperature from a temperature higher than a lower limit of critical dissolution temperature of the temperature responsive polymer compound in an aqueous solution to a lower temperature than the lower limit of the critical dissolution temperature. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a temperature-responsive base material that includes a fiber structure and a temperature-responsive polymer compound, and that collapses the structure in response to temperature, and a product that uses the temperature-responsive base material.

  Conventionally, as for various fibers such as natural fibers and synthetic fibers, fiber structures having functions suitable for various uses have been produced by processing the fibers by various methods. That is, the spatial arrangement, the physical structure, and the shape of the fibers forming the structure can express various functions and can be used for applications, and the fibers thus manufactured By holding a functional substance on the surface or inside of the structure, it has been possible to provide additional functions to the fiber structure and to develop applications.

  It is also important to collapse the produced fiber structure by external stimulation or the like. For example, if you want to stop the function that is manifested by the fiber structure forming its structure, or you want to take out and collect the functional substance held on the surface or inside of the fiber structure, or This is the case when you want to release it.

  When it is desired to collapse the fiber structure in this way, conventionally, for example, the fiber itself is deformed by heat treatment at a temperature at which the fiber melts or decomposes, or by treatment with an organic solvent in which the fiber dissolves. In general, the fiber structure is disrupted by modification or disappearance. However, such a method is generally an extreme condition and may adversely affect the surrounding environment, and is not considered to be a preferable method. For example, when a fiber structure is formed on a plastic substrate and only the fiber structure is desired to be collapsed, under the extreme processing conditions using heating or an organic solvent as described above, the plastic that is the substrate It will also affect the deformation and dissolution. Also, for example, when it is desired to recover or release the functional substance held inside the fiber structure, the functional substance held inside is weak against heat or dissolved in an organic solvent. In such a case, it becomes difficult to collect or release while maintaining the function of the functional substance.

  On the other hand, a material using a temperature-responsive polymer compound is known as a material whose structure changes under mild conditions. For example, it has been proposed to prepare a cell culture material using such a temperature-responsive polymer compound, and to recover the cells by changing its properties by changing the temperature (Patent Document 1 and Patent Document 2). reference.). However, since the material made of the temperature-responsive polymer compound itself becomes a gel-like material, the strength of the gel-like material is weak, and the structure collapses only by applying a slight external force including gravity. Therefore, it is impossible to hold an arbitrary shape, and there is a problem that it cannot be used under conditions where an external force such as shaking, centrifugation, and perfusion is applied.

In addition, it is impossible to fabricate a shape like a fiber by processing such a temperature-responsive polymer compound itself. In these Patent Documents 1 and 2, a cell culture material is produced by coating a surface of a support with a temperature-responsive polymer compound. This material depends on the properties of the temperature-responsive polymer compound. Only the surface properties of the substrate are changed, and the structure of the support itself is not destroyed.For example, it is possible to lose the function expressed by forming the support by disrupting the structure. For example, when the structure of the support itself has a complicated shape, it has been impossible to extract and collect functional substances such as cells that have entered the inside of the support body due to temperature changes. In addition, since the shape that can be produced is restricted, a free three-dimensional structure cannot be formed, and there is a drawback that the application is limited. That is, in the above conventional proposal, as a material whose structure changes (collapses) under a mild condition such as temperature change, it has an appropriate strength as a support, has flexibility in form and shape as a support, and is spatially Since there was no material that can form a support that can be used to hold a functional substance on the surface or inside because of its excellent function expression, it has an appropriate strength as a support and is free to shape and form There has been a demand for a material that has a degree and can collapse its structure under mild conditions such as temperature changes.
Japanese Patent Laid-Open No. 03-026690 JP 05-192138 A

  Accordingly, an object of the present invention is to eliminate the above-mentioned disadvantages of the prior art, and after the base material has fulfilled its function, the base material is structured by collapsing the base material structure under mild conditions. A material that can terminate the function that was manifested by forming the surface, or a functional substance that is retained on the surface or inside and used for various purposes, and then the functional substance can be freely As a material that can stop function, collect functional substances, and release to the outside, it has moderate strength as a base material, can take a free structure and shape, and is milder It is an object of the present invention to provide a temperature-responsive substrate as a material capable of collapsing the structure by temperature change.

  Another object of the present invention is to provide cells cultured on the surface and / or inside the above-mentioned temperature-responsive substrate.

  The present invention achieves the above-mentioned object, and the temperature-responsive base material of the present invention is a short fiber having a single fiber number average diameter of 1 nm to 50 μm and a single fiber length of 0.2 mm to 30 mm. The structure includes a fiber structure composed of an assembly of the above and a temperature-responsive polymer compound, and the structure is changed from a temperature higher than the lower critical solution temperature of the temperature-responsive polymer compound to a temperature lower than the lower critical solution temperature in an aqueous solution. It is a temperature-responsive substrate characterized by collapsing.

  According to a preferred embodiment of the temperature-responsive substrate of the present invention, the short fibers are nanofibers having a number average diameter of single fibers in the range of 1 nm to 500 nm, and the short fibers have a diameter greater than 500 nm. The composition ratio of is 3% by weight or less.

  According to a preferred embodiment of the temperature-responsive substrate of the present invention, the short fiber is made of an organic polymer.

  According to a preferred embodiment of the temperature-responsive base material of the present invention, the temperature-responsive polymer compound is insoluble in water at a temperature higher than the lower critical solution temperature and water at a temperature lower than the lower critical solution temperature. As a suitable temperature-responsive polymer compound, poly-N-substituted acrylamide can be mentioned. Moreover, the suitable content of this temperature-responsive high molecular compound is 1 to 20 weight% with respect to the weight of a fiber structure.

  According to a preferred embodiment of the temperature-responsive substrate of the present invention, the temperature-responsive substrate of the present invention is obtained by adding a temperature-responsive polymer compound to a dispersion obtained by dispersing an aggregate of short fibers in a solvent, It is formed by removing the solvent.

  According to a preferred embodiment of the temperature-responsive substrate of the present invention, the temperature-responsive substrate of the present invention is obtained by coating the surface of a short fiber constituting a fiber structure with a temperature-responsive polymer compound.

  According to a preferred embodiment of the temperature-responsive substrate of the present invention, the fiber structure is in a thin layer shape, a paper shape, a nonwoven fabric shape or a sponge shape.

  According to a preferred embodiment of the temperature-responsive substrate of the present invention, the temperature-responsive substrate of the present invention is formed on another substrate, and a predetermined pattern is formed on the other substrate. In addition, a functional substance is held on the surface and / or inside.

  The temperature-responsive substrate of the present invention is suitable for the field involving cell culture and / or tissue regeneration, and cells can be cultured on the surface and / or inside of the temperature-responsive substrate.

A fiber structure comprising a collection of short fibers having a number average diameter of single fibers of 1 nm to 50 μm and a fiber length of 0.2 mm to 30 mm provided by the present invention and a temperature-responsive polymer compound A temperature-responsive base material characterized in that the structure collapses by changing the temperature-responsive polymer compound in the aqueous solution from a temperature higher than the lower critical solution temperature to a temperature lower than the lower critical solution temperature. After the material has fulfilled its function, its structure is destroyed by a change in temperature so that the function that has been fulfilled by the base material forming the structure can be terminated, or the surface of the base material or After holding the functional substance inside, the function of the functional substance can be freely stopped, the functional substance can be recovered, Material that can be and out can be obtained. Moreover, the fiber structure which comprises a temperature-responsive base material has moderate intensity | strength as a three-dimensional structure, and can take a free structure and shape.
In the present invention, such a temperature-responsive base material can be provided, and by changing various functional substances to be retained on the surface or inside, clothing materials, interior products, living materials, environmental / industrial material products, It can be used for IT products, medical products and research products. In particular, if a drug is held as a functional substance on this temperature-responsive base material, the drug can be freely released by temperature change, so that it can be used for DDS (drug delivery system) applications. In addition, if this temperature-responsive substrate is used for cell culture, it is possible to disintegrate the substrate by changing the temperature after cell culture and take out the cultured cells from the surface or the inside. Makes it easy to collect cells cultured on substrates that have been difficult to culture, and is used in the fields of medicine, medicine, diagnosis, research and analysis related to cell medicine or tissue regeneration, especially in the medical field such as regenerative medicine and cell medicine. And can be applied.

  Hereinafter, the temperature-responsive base material and the manufacturing method thereof according to the present invention will be described in detail together with desirable embodiments.

  The temperature-responsive base material of the present invention includes a fiber structure composed of an assembly of short fibers having a single fiber number average diameter of 1 nm to 50 μm and a single fiber length of 0.2 mm to 30 mm, and a temperature response. Responsiveness in which the structure of the temperature-responsive substrate collapses when the temperature is changed from a temperature higher than the lower critical solution temperature of the temperature responsive polymer compound to a temperature lower than the lower critical solution temperature. It is a substrate. A base material here refers to the processed goods which can make various structures.

  In the present invention, “the structure collapses” means that the temperature-responsive base material assembled by the fibers becomes unstable as a whole or part of the structure, and maintains the original shape. This means that the entire structure collapses and breaks. For example, in an aqueous solution, the original shape cannot be maintained due to a weak external force such as a self-weight or water flow generated when shaking, centrifuging, perfusing, or swirling. It refers to such a state.

  The temperature-responsive polymer compound used in the present invention is a polymer compound having a lower critical solution temperature (LCST = Lower Critical Solution Temperature) (hereinafter sometimes referred to as LCST). When the temperature is lowered while the solvent is mixed, it refers to a polymer compound that transitions from an insoluble state to a soluble state with respect to the solvent with the LCST as a boundary. In particular, water is important as a solvent in various applications, and a temperature-responsive polymer compound having LCST with respect to water as a solvent changes from a dehydrated state to a hydrated state at the boundary of LCST by lowering the temperature. It is a polymer compound that undergoes transition, and is a polymer compound that is insoluble in water at a temperature higher than LCST and is soluble in water at a temperature lower than LCST. LCST is prepared by preparing a solution in which a polymer compound is mixed in a solvent, and changing the temperature from a high temperature to a low temperature while measuring the light transmittance using a turbidimeter, and the temperature at which the light transmittance rapidly decreases is the LCST. .

  In the temperature-responsive substrate of the present case, the fiber structure and the temperature-responsive polymer compound are included, and the temperature-responsive polymer compound covers the surface of the short fiber constituting the fiber structure, for example, The temperature-responsive polymer compound serves as a binder for binding the short fibers and the short fibers to the fiber structure, and the short fibers can maintain the structure formed as an aggregate. By using a temperature-responsive polymer compound as described above as a binder, the binder does not dissolve because the temperature-responsive polymer compound that is a binder is insoluble in water or other solvents at temperatures higher than the LCST. Even if the temperature-responsive base material is put in a solvent, it is possible to keep the state of the structure of the temperature-responsive base material as a whole. Conversely, since the temperature-responsive polymer compound as a binder is soluble in a solvent at a temperature lower than the LCST, the binder is easily dissolved in the solvent, and as a result, the temperature-responsive base material has a lower temperature than the LCST. When placed in a solvent in a state, the structure of the temperature-responsive substrate easily collapses due to a weak external force such as its own weight, shaking, or pipetting. That is, the temperature-responsive base material of the present invention can control the collapse of the structure as a whole under a mild condition such as a temperature change by such an action. From the above, in the present invention, it is preferable that the fiber surface of the fiber structure is coated with a temperature-responsive polymer compound.

As for the short fiber which comprises the fiber structure used by this invention, it is important that the number average diameter of a single fiber is 1 nm-50 micrometers. By making the number average diameter of the single fibers within such a range, the fiber is easily dispersed as a structure, and handling when producing the fiber structure is improved, and it is easy to obtain a fiber structure of an arbitrary shape. is there. On the other hand, when the number average diameter of the single fibers exceeds 50 μm, it becomes difficult to maintain the structure of the temperature-responsive base material due to the binder effect of the temperature-responsive polymer compound.
The number average diameter of the single fibers is preferably 1 nm to 10 μm, more preferably 1 to 500 nm, still more preferably 1 to 200 nm, and most preferably 1 to 100 nm. A fiber having a more free shape and structure by making the fiber diameter of a single fiber nanometer size (that is, in the present invention, a fiber having a fiber diameter smaller than 1 μm may be referred to as nanofiber). It becomes easier to fabricate the structure, and the specific surface area and space-forming ability of the fiber structure are increased compared to the fiber diameter being nano-sized, and the retention performance of the functional substance described later can be dramatically increased. In particular, when nanofibers having a single fiber diameter of 500 nm or less are used, not only the specific surface area dramatically increases, but also innumerable pores of several nanometers to several hundred nanometers can be formed between the fibers. It exhibits excellent adsorption, absorption, and release characteristics unique to nanofibers that were not seen with microfibers, and the number average diameter of single fibers is nano-level fiber diameter. And by, after collapsing the structure by temperature changes, it is easy to collect only functional material by removing the fibers.

  In the present invention, the number average diameter of single fibers can be determined as follows. That is, 150 cross-sections or vertical cross-sections of a temperature-responsive substrate or fiber structure are observed with a scanning electron microscope (SEM) or a transmission electron microscope (TEM), and 150 single samples randomly extracted in the same plane. The cross-sectional area of the fiber is analyzed by image processing software, the diameter in terms of a circle is further obtained, and the number average is calculated.

  Moreover, when using the short fiber whose number average diameter of a single fiber is 1-500 nm in this invention, it is preferable that the fiber constituent ratio of the short fiber larger than 500 nm is 3 weight% or less. The fiber constituent ratio is more preferably 1% by weight or less, and still more preferably 0.1% by weight or less. That is, this means that the presence of coarse short fibers exceeding 500 nm is close to zero.

Here, the fiber composition ratio of coarse short fibers means the ratio of the weight of coarse short fibers (larger than 500 nm) to the total weight of fibers larger than 1 nm in diameter, and is calculated as follows. To do. That is, each of the single fiber diameter of the short fibers of the fiber structure in a di, the square of the sum ^{(d1 2 + d2 2 + ··} + dn 2) = Σdi calculates ² (i = 1~n). Moreover, each fiber diameter of the short fiber larger than 500 nm in diameter is set to Di, and the sum total of the squares (D1 ² + D2 ² +... + Dm ² ) = ΣDi ² (i = 1 to m) is calculated. By calculating the ratio of the sum ΣDi ² to the sum Σdi ² , the area ratio of coarse fibers to the entire short fibers in the fiber structure, that is, the weight ratio can be obtained. By these, while being able to fully exhibit the function of the temperature-responsive base material of this invention and the fiber structure used by this invention, quality stability can also be made favorable.

  In addition, if the fiber length of the short fiber used in the present invention is too long, even if the temperature-responsive polymer compound is dissolved at a temperature lower than the LCST, the short fibers remain entangled, resulting in a structure. It may not collapse. On the other hand, if the fiber length is too short, the degree of entanglement of the short fibers is reduced when the fiber structure is formed, and as a result, the strength of the temperature-responsive substrate and the fiber structure used in the present invention is lowered. From these viewpoints, it is important that the fiber length of the short fibers constituting the temperature-responsive substrate and the fiber structure used in the present invention is 0.2 to 30 mm.

  Examples of fibers used in the present invention include cellulose fibers produced from wood pulp, natural fibers such as cotton, hemp, wool and silk, regenerated fibers such as rayon, semi-synthetic fibers such as acetate, nylon and polyester. In addition, synthetic fibers represented by acrylic and the like can be mentioned, but organic fibers are preferable because they can be used for various purposes. Among these, it is more preferable to be made of a thermoplastic polymer, whereby the fibers used can be produced by a melt spinning method, and thus the productivity can be made extremely high.

  Examples of the thermoplastic polymer referred to in the present invention include polyethylene terephthalate (hereinafter sometimes referred to as PET), polybutylene phthalate (hereinafter sometimes referred to as PBT), polylactic acid (hereinafter referred to as PET). Polyester such as PLA (sometimes referred to as PLA), polyamide such as nylon 6 (hereinafter sometimes referred to as N6) and nylon 66, polystyrene (hereinafter sometimes referred to as PS), polypropylene (hereinafter referred to as PP). And polyphenylene sulfide (hereinafter sometimes referred to as PPS). Among them, polycondensation polymers represented by polyesters and polyamides having a high melting point are preferably used.

  An organic polymer having a melting point of 165 ° C. or higher is preferably used because the fiber has good heat resistance. For example, PLA has a melting point of 170 ° C, PET has a melting point of 255 ° C, and N6 has a melting point of 220 ° C. The organic polymer may contain additives such as particles, a flame retardant, and an antistatic agent. Further, other components may be copolymerized as long as the properties of the organic polymer are not impaired. Furthermore, an organic polymer having a melting point of 300 ° C. or lower is preferably used because of easy melt spinning.

  The fiber production method used in the present invention is not particularly limited, and can be obtained by a conventional melt spinning method, etc. In order to obtain nano-level fibers whose number average diameter of single fibers is smaller than 1 μm. The following method can be mentioned as an example of the manufacturing method.

  That is, two or more types of polymers having different solubility in a solvent are made into a polymer alloy melt, which is spun and then cooled and solidified to form a fiber. Then, if necessary, stretching and heat treatment are performed to obtain a polymer alloy fiber. And the fiber (nanofiber) used by this invention can be obtained by removing an easily soluble polymer with a solvent.

  Here, in the polymer alloy fiber that is the precursor of the nanofiber, the easily soluble polymer is the sea component (matrix), the hardly soluble polymer is the island component (domain), and the size of the island component is controlled. This is very important. Here, the island component size is a value obtained by observing a cross section of a polymer alloy fiber with a transmission electron microscope (TEM) and evaluating it in terms of diameter. Since the diameter of the nanofiber is substantially determined by the island component size in the nanofiber precursor, the distribution of the island component size is designed according to the diameter distribution of the nanofiber. For this reason, kneading of the polymer to be alloyed is very important, and it is preferable to knead with a kneading extruder, a static kneader or the like. In simple chip blends (for example, see JP-A-6-272114 and JP-A-10-53967), kneading is insufficient, and it is difficult to disperse island components with a size of several tens of nm.

  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. Further, 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 high kneading can be achieved by setting the length of the kneading part to 20% or more of the effective length of the screw. 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. In addition, by positioning the kneading part on the discharge side of the twin-screw extruder as much as possible, the residence time after kneading can be shortened and re-aggregation of the island component 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.

  Further, in order to disperse the island component with a size of several tens of nanometers, a combination of polymers is also important.

In order to make the island component domain (cross section of the nanofiber) close to a circular shape, the island component polymer and the sea component polymer are preferably incompatible. However, it is difficult for the island component 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. One of the indexes 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 Co., Ltd./Plastics Editorial Department, page 189. When the difference between the SP values of the two polymers is 1 to 9 (MJ / m ³ ) ^1/2, it is easy to achieve both circularization of the island component domain and ultrafine dispersion due to 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 ) is about ^½, which is an undesirable example.

  Further, when the melting point difference between polymers is 20 ° C. or less, particularly when kneading using an extrusion kneader, a difference in melting state in the extrusion kneader is unlikely to occur, so that highly efficient kneading is easy.

  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.

Further, the melt viscosity is also important, and the melt viscosity of the sea component polymer may greatly affect the spinnability. When a low viscosity polymer of 100 Pa · s or less is used as the sea component polymer, the island component 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 ultra-finely dispersed polymer alloy used in the present invention, the spinneret design is important, but the fiber cooling conditions are also important. As described above, since the polymer alloy is a very unstable molten fluid, it is preferable to cool and solidify immediately after being discharged from the die. Therefore, the distance from the die to the start of cooling is preferably 1 to 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.
By removing the easily soluble polymer of the polymer alloy fibers spun in this way (sea-island type fibers) with a solvent, nanofibers as fibers that are preferably used in the present invention can be obtained.
The nanofibers produced by the above production method have a crystallinity of 20% or more and have the same strength as ordinary clothing fibers, and therefore can be subjected to various processing.

  Moreover, the fiber used is surface partial hydrolysis treatment, chemical modification treatment, graft treatment, electron beam treatment, radiation treatment, ion implantation, heat treatment, steam treatment, pressurized steam treatment, gas treatment, acid treatment, alkali It is also possible to modify the surface properties by surface treatment, such as treatment, flame treatment, etching treatment, glow discharge treatment, plasma treatment, vacuum treatment, humidification treatment, alcohol treatment and polymer adsorption treatment. Affinity, adsorptivity and sustained release can be improved in accordance with the types of temperature-responsive polymer compounds and functional substances described later. These treatments may be performed at any time before, during or after the formation of the temperature-responsive substrate.

The fiber structure used in the present invention is composed of an assembly of the above-mentioned short fibers, and specifically, a sheet-like form such as a thin film, a paper-like material, and a nonwoven-like material or a sponge-like material. It can take a three-dimensional form. In the case of the sheet form, the basis weight is preferably 2000 g / m ² or less from the viewpoint of stability and durability. In the case of a three-dimensional form such as a sponge, the density is 0.0002 to 0.05 g / cm ³ and the porosity is 90% or more and 99.99 from the viewpoint of the inclusion and holding ability of the functional substance. % Or less is preferable. Moreover, it is preferable that the number average hole diameter of the pores which are relatively large communication holes surrounded by the wall structure in which the fibers are aggregated is 10 μm to 500 μm. By setting the number average pore diameter of the pores to 500 μm or less, it becomes possible to efficiently collect and retain when a functional substance such as a cell is added. Moreover, it is preferable that the minimum of the number average hole diameter of a void | hole is 10 micrometers or more. When the number average pore diameter of the pores is less than 10 μm, there is a problem that it becomes difficult for a functional substance such as a cell to enter the structure.

  The temperature-responsive polymer compound used in the present invention is preferably insoluble in water at a temperature higher than the LCST and preferably soluble in water at a temperature lower than the LCST. Examples of the polymer compound include poly N-substituted acrylamide, poly N-substituted methacrylamide, hydroxyalkyl cellulose, polyoxazolidone, polyvinyl methyl ether, polyalkylene oxide, polyvinyl alcohol, polymethacrylic acid, and copolymers and derivatives thereof. It is done.

  Among these, poly-N-substituted acrylamide is preferably used as the temperature-responsive polymer compound because it is easily available and LCST for water is present near room temperature. Examples of the poly N-substituted acrylamide include poly-Nn-propyl acrylamide, poly-N-isopropyl acrylamide, poly N, N-diethyl acrylamide, poly-N-cyclopropyl acrylamide, poly N, N-ethylmethyl acrylamide, Examples include poly-N-ethylacrylamide, polyacryloylpiperidine, and polyacryloylpyrrolidine. Among these, from the viewpoint of application development, poly-N-isopropylacrylamide having an LCST of around 30 to 32 ° C. is most preferably used.

  The molecular weight of the temperature-responsive polymer compound used is preferably 100 to 5000000, more preferably 1000 to 500000, and further preferably 3000 to 100000. When the molecular weight is less than 100, the responsiveness to temperature tends to decrease, and when the molecular weight exceeds 5000000, the solubility in water tends to decrease. The number of N-substituted acrylamide units in the temperature-responsive polymer compound is preferably 10 units or more. Further, the temperature-responsive polymer compound used preferably has a solubility in water of 1% by weight or more at a temperature of 25 ° C.

  In the present invention, these temperature-responsive high molecular weight compounds are contained in the fiber structure as a binder. If the amount to be contained is increased, for example, when nanofibers are used as short fibers, the nanofibers are formed. This causes a problem that the minute pore structure is buried, and the function expression due to the minute structure or spatial arrangement is hindered. Further, if the amount to be contained is small, the function as a binder cannot be achieved, and the short fibers cannot maintain the structure as the structure. Therefore, the content of the temperature-responsive polymer compound is preferably 1 to 20% by weight, more preferably 2 to 10% by weight, based on the weight of the fiber structure.

  The temperature-responsive substrate of the present invention is obtained by coating the surface of a short fiber constituting a fiber structure with a temperature-responsive polymer compound. The form of such a temperature-responsive base material is preferably formed by adding a temperature-responsive polymer compound to a dispersion in which a short fiber aggregate is dispersed in a solvent and removing the solvent. Can do.

  Therefore, next, the adjustment method of a short fiber dispersion liquid is demonstrated.

  For example, the fibers (nanofibers) obtained as described above are cut into a desired fiber length with scissors, a cutter, a guillotine cutter, a slicing machine, a cryostat, or the like to obtain short fibers. If the fiber length is too long, even if the temperature-responsive polymer compound is dissolved at a temperature lower than the LCST, the fibers remain entangled and the structure may not collapse. On the other hand, if the fiber length is too long, the dispersibility of the fibers in the solvent becomes poor, and the uniformity of the formed temperature-responsive substrate tends to be impaired. On the other hand, if the fiber length is too short, the degree of entanglement of the short fibers becomes small when the temperature responsive substrate is used, and it becomes difficult to maintain the shape as the temperature responsive substrate. From these viewpoints, it is important to cut the fiber length to 0.2 to 30 mm. The fiber length is more preferably 0.5 to 10 mm, and further preferably 0.8 to 5 mm.

  Next, the short fibers obtained by cutting are dispersed in a solvent. Solvents are not only water, but also hydrocarbons such as hexane and toluene, halogens and hydrocarbons such as chloroform and trichloroethylene, alcohols such as ethanol and isopropyl alcohol, ethyl ether and tetrahydrofuran. Ethers such as acetone, ketones such as methyl ethyl ketone, esters such as methyl acetate and ethyl acetate, polyhydric alcohols such as ethylene glycol and propylene glycol, amines such as triethylamine and N, N-dimethylformamide, and amide solvents However, it is preferable to use water in consideration of safety, environment, and the like.

  As a method for dispersing the short fibers in the solvent, a stirrer such as a mixer or a homogenizer can be used. In the case of a form in which thin short fibers are strongly agglomerated like nanofibers, it is preferable to beat in a solvent as a pretreatment step for dispersion by stirring. Niagara beater, refiner, sonicator, cutter, laboratory A shearing force is applied by a pulverizer, a biomixer, a home mixer, a roll mill, a mortar, a PFI beating machine, etc., and the short fibers can be dispersed one by one before being administered into a solvent.

  Also, in order to make the dispersibility of the short fibers uniform in the fiber dispersion or to improve the mechanical strength of the temperature responsive substrate when used as a temperature responsive substrate, the short fibers in the fiber dispersion The fiber concentration is preferably in the range of 0.001 to 30% by weight with respect to the total weight of the dispersion. In particular, since the mechanical strength of the temperature-responsive substrate greatly depends on the presence state of short fibers in the dispersion, that is, the interfiber distance, it is preferable to control the concentration of short fibers in the fiber dispersion within the above range. The short fiber concentration in the fiber dispersion is more preferably in the range of 0.01 to 10% by weight, and still more preferably in the range of 0.05 to 5% by weight.

  Next, the preferable manufacturing method of the temperature-responsive base material of this invention is demonstrated.

  A temperature-responsive polymer compound is added to the fiber dispersion prepared as described above in the above-described concentration range to prepare a dispersion. Then, by removing the solvent from the dispersion, various forms of temperature-responsive base materials in which the temperature-responsive polymer compound is coated on the short fiber surface as a binder are formed.

  For example, in order to remove the solvent from the dispersion containing the short fiber, the solvent, and the temperature-responsive polymer compound, the non-woven fabric in which the short fibers are entangled by removing the solvent by a conventional heat treatment or a reduced pressure treatment. It is possible to form a temperature-responsive substrate. Further, for example, a paper-like form in which short fibers are dispersed in a single fiber state can be obtained by papermaking the dispersion. It is also possible to form a temperature-responsive substrate in the form of a coating (film) on another substrate by casting the dispersion on another substrate and removing the solvent.

  In addition, the short fibers in the dispersion obtained above are fixed in a dispersed state and the solvent is removed to form a sponge-like form containing a large number of pores. Examples of the method include the following methods. That is, the dispersion liquid to which the temperature-responsive polymer compound is added is placed in a suitable container or mold. Thereafter, the solvent is removed from the dispersion liquid placed in the container or the mold. The method for removing the solvent is not particularly limited, but it is preferable to remove the solvent by drying. Examples of the drying method include natural drying, hot air drying, vacuum drying, freeze drying and the like. In order to obtain a particularly uniform structure, freeze drying is preferable. In lyophilization, first, the dispersion is instantly frozen with liquid nitrogen or an ultra-low temperature freezer. Thereby, it is possible to fix the state in which the dispersion is frozen, that is, the dispersion state of the short fibers in the solid. Thereafter, the dispersion medium is sublimated by evacuation, but only the dispersion medium is removed in a state where the temperature-responsive polymer compound becomes a binder while the dispersion state of the short fibers is fixed.

  In addition, when producing the above-mentioned sponge-shaped temperature-responsive substrate, it is possible to mold the sponge-like structure into a desired three-dimensional shape by arbitrarily changing the shape of the container and the formwork. It is.

  Moreover, the temperature-responsive base material of the present invention may be formed on another base. Here, the other substrate is a base, a foundation, and a base on which a short fiber, a fiber dispersion in which short fibers are dispersed in a solvent, or a dispersion to which a temperature-responsive polymer compound is added can be placed. It refers to materials that have a solid structure that can serve as a foundation and that can serve as a scaffold for various functions. Further, the shape of the other substrate may be flat or have a three-dimensional structure, for example, a plate shape, a film shape, a sheet shape, a fiber shape, a rod shape, a cylindrical shape, a spiral shape, a sponge shape, a particle shape, a flake shape, and the like. Any shape can be used, such as flasks, petri dishes, wells, plates, multi-wells, multi-well plates, slides, films, bags, columns, tanks, bottles, hollow fibers, non-woven fabrics, and microbeads. Shape.

  Further, when forming a temperature-responsive substrate in the form of a thin film on the surface of another substrate, by using a method of removing the solvent after forming a dispersion pattern on the surface of the other substrate, A temperature-responsive substrate pattern can be formed on the surface of another substrate. Here, when forming a pattern on a substrate, if a dispersion containing a temperature-responsive polymer compound is spotted on the substrate using a pipette, a spotter, an arrayer, etc., a circular temperature-responsive substrate The pattern can be formed. Furthermore, when a stamp method, a stencil method, a spray method, or the like is used using a dispersion liquid, a precise pattern can be formed, but the method is not limited to these methods. The pattern referred to in the present invention refers to an object in which a two-dimensional expansive shape can be recognized as a pattern, a pattern, a style, and the like, and is generally a space such as a figure, a picture, an image, a pattern, a character, and a pattern It refers to what is recognized as the shape of a natural object. The pattern may be any shape, and the pattern itself may or may not have regularity.

  Other base materials include plastics with excellent moldability such as polystyrene, polypropylene, polyolefin, polyethylene, polyvinyl chloride, polylactic acid, polyester, polyvinyl alcohol, cellulose, polyacrylate, polysulfone, polycarbonate, polyurethane and polyimide. Alternatively, inorganic substances such as glass, gold, platinum, copper, titanium, tantalum, zirconia, silica, alumina, hydroxyapatite, calcium phosphate, and magnesium phosphate can also be used.

  The temperature-responsive base material of the present invention can hold a functional substance inside or on the surface of the base material. In particular, if nanofibers are used as the fibers, the specific surface area of the fiber surface is remarkably improved, and a nano-level space is formed between the fibers. In addition, it is possible to develop unique holding performance such as closeness and high density by spatial arrangement at the nano level. The method for retaining the functional substance is not particularly limited, and after the temperature-responsive substrate is formed, the temperature-responsive substrate is post-processed by adsorption treatment, impregnation treatment, coating, etc. on the surface of the temperature-responsive substrate. Or may be previously contained in the dispersion before forming the temperature-responsive base material, and may be held simultaneously with the formation of the temperature-responsive base material.

  The functional substance used in the present invention refers to all substances that can improve the function of the temperature-responsive substrate as a material. Functions include, for example, color development, water repellency, waterproofing, windproofing, moisture retention, flame retardancy, heat resistance, heat absorption, silencer, heat insulation, color, gloss, touch, biocompatibility and cell adhesion. However, it is not limited to these. In addition, as functional substances, dyes, antistatic agents, ionic substances, hygroscopic agents, moisturizers, water repellents, heat retention agents, heat-resistant agents, flame retardants, catalysts, photocatalysts, enzymes, ligands, receptors, light emitting elements, light absorption Examples include, but are not limited to, an element, a fluorescent element, a phosphorescent element, a reflective agent, a heat generating agent, an endothermic agent, a cooling agent, a reducing agent, and an oxidizing agent. The functional substance may be a high molecular compound or a low molecular compound, and the shape thereof is not limited.

  By holding these functional substances on the surface or inside of the temperature-responsive substrate, the temperature-responsive substrate can express various functions, such as clothing materials, interior products, living materials, and the environment.・ Can be used in industrial material products, IT products, medical products, and research products, and the temperature-responsive substrate collapses by deactivating the temperature in an aqueous solution to a temperature lower than LCST, thereby deactivating its function. It is also possible. For example, it is possible to release the dye in response to the temperature by holding the dye as a functional substance in the temperature-responsive base material of the present invention and lowering the temperature to collapse the structure. For example, the light-emitting element and the excitation element can be brought close to each other as a functional substance in the temperature-responsive base material so that the light-emitting element can be extinguished by collapsing the structure due to a temperature change.

In addition, if a drug such as a protein such as a cytokine or an antibody or a low molecular weight compound such as a steroid, alkaloid, or aminoglycoside is used as a functional substance, a drug delivery system or diagnosis that can release the drug in response to temperature This means that it can also be applied to other uses.
Further, for example, if proteins, peptides, amino acids and vitamins are used as functional substances to be retained, the temperature-responsive substrate can be suitably used for cell culture for cell culture or tissue regeneration. . Culture means expressing, promoting and suppressing various cell functions such as cell attachment, adhesion, proliferation, differentiation, activation, migration, migration, morphological change and colonization. As a functional substance to be retained for such cell culture use, it is preferable to adsorb proteins. However, in order to control various functions of cells, the adsorbed proteins can be used for cell adhesion, proliferation or growth. More preferably, the protein is related to differentiation function control. Examples of such proteins include, but are not limited to, cytokines, extracellular matrix, and adhesion factors. It is also possible to adsorb these proteins from serum components in the cell culture medium.

  In addition, the use of nanofibers as short fibers means that the fibrous extracellular matrix typified by collagen surrounding cells in the bone marrow, basement membrane, and amniotic membrane is a nano-level fibrous substance. Fibers can structurally resemble these extracellular matrices, and the temperature-responsive substrate of the present invention that can use nanofibers can also be used for culturing cells that were difficult to cultivate conventionally. It becomes possible.

  Examples of cells that can be cultured in or on the temperature-responsive substrate of the present invention include, for example, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, mesodermal stem cells, ES cells (embryonic stem cells), many Potent stem cells, CD34 positive cells, immune cells, blood cells, neurons, vascular endothelial cells, fibroblasts, epithelial cells, hepatocytes, pancreatic β cells, cardiomyocytes, osteoblasts, chondrocytes, myoblasts Cells derived from living bodies such as bone marrow cells, amniotic cells and umbilical cord blood cells, NIH3T3 cells, 3T3-L1 cells, 3T3-E1 cells, Hela ( HeLa) cells, PC-12 (PC Zelb) cells, P19 (Pineteen) cells, CHO (Chinese hamster oocytes) cells, C S (SEOS) cells, HEK (HEC) cells, Hep-G2 (Hepsey two) cells, CaCo2 (Kako two) cells, L929 (El Nine to Nine) cells, C2C12 (C2 C12) cells, Daudi ( Daudi cells, Jurkat cells, KG-1a cells, CTLL-2 cells, NS-1 cells, MOLT-4 cells , Cell lines such as HUT78 (HQ78) cells and MT-4 (MT4) cells, various hybridoma cell lines that are antibody-producing cells, and cells obtained by genetically modifying these cells. However, it is not limited to these.

  In the case of adherent cells, adhesion is promoted without any special treatment, but in the case of floating cells, proteins such as extracellular matrix, adhesion factors and antibodies are retained on a temperature-responsive substrate. It becomes possible to promote adhesion.

  In addition, by using tissue-derived cells as cells, for example, muscle tissue when using myoblasts, myocardium when using myocardial cells, and blood vessels when using vascular endothelial cells are temperature-responsive groups. A structure can be formed in the shape of the material. That is, when cultivating cells in a two-dimensional state, the temperature-responsive substrate is formed in a thin-film shape or paper form, and when culturing cells in a three-dimensional state, the temperature-responsive substrate is formed in a sponge state or non-woven fabric form. Can be kept. That is, the temperature-responsive substrate of the present invention is a scaffold molded body used for tissue regeneration such as nerve, heart, blood vessel, cartilage, skin, cornea, kidney, liver, hair, myocardium, muscle, and tendon, or tissue formation for transplantation. Can be used for some or all of

  By culturing cells using the temperature-responsive substrate of the present invention as a culture material for cell culture or tissue regeneration as described above, the temperature-responsive substrate is disrupted by changing the temperature. And tissue can be recovered from the temperature-responsive substrate. Such an effect makes it possible to collect cells without using a cell peeling agent such as a proteolytic enzyme such as trypsin, which has been conventionally used for peeling cells from the support structure. Cell detachment agents are unfavorable techniques for culturing cells because they damage proteins such as receptors and ligands on the cell surface. Short fibers that have been disintegrated by disintegrating the temperature-responsive substrate by changing the temperature can be separated by a sieving structure such as a filter, so the cultured cells can be collected under mild conditions of temperature changes. It becomes possible to do.

In addition, when cell culture is performed inside or on the temperature-responsive substrate of the present invention, the cells are cultured in a 5% CO ₂ incubator, cultured in a gas-permeable bag, It seems that a column with a built-in structure is used, or a reservoir containing a cell suspension, an oxygen load device using a commercially available artificial lung, a dialysis column for exchanging the medium, etc. It is also possible to cultivate cells using a simple perfusion culture system.

  In addition, the temperature-responsive base material of the present invention is a part or all of a medical molded body for implantation in vivo such as an aneurysm coil, an embolic material, a dental material and an intraocular lens, a surgical suture, a surgical filling material, a surgical Can be used as a part or all of medical materials such as stiffeners, wound protection materials, wound treatment materials, fracture joint materials, prosthetic materials and catheters, and after achieving the purpose as medical molded bodies and medical materials, temperature It is also possible to collapse the structure by change.

  In addition, by forming a structure pattern on the surface of the temperature-responsive substrate and adsorbing or slow-releasing proteins to the structure, cell adhesion, proliferation, and mobility on the surface of the temperature-responsive substrate In addition, functions such as differentiation ability can be controlled two-dimensionally or three-dimensionally according to a pattern. In particular, by attaching cells to a specific shape according to the pattern, for example, after forming a tissue or cell aggregate having a specific shape, it is possible to change the temperature without using a cell release agent that adversely affects the cells. It is also possible to recover only cells and tissues having a specific shape by collapsing the structure. By applying such technology, it becomes possible to develop devices in the medical, research and analytical fields using regenerative medicine, tissue engineering, cell culture, biosensors, biochips and drug screening.

  Hereinafter, the temperature-responsive base material of the present invention will be described in detail using examples. The following methods were used for the measurement methods in the examples.

A. Polymer melt viscosity The polymer melt viscosity was measured with a Capillograph 1B manufactured by Toyo Seiki Seisakusho. The polymer storage time from sample introduction to measurement start was 10 minutes.

B. Polymer Melting Point Using DSC-7 manufactured by Perkin Elmaer, the peak top temperature showing the melting of the polymer at 2nd run was defined as the melting point of the polymer. The rate of temperature increase at this time was 16 ° C./min, and the sample amount was 10 mg.

C. Shear stress at the base discharge hole The shear stress between the base hole wall and the polymer is calculated from the Hagen-Poiseuille equation (shear stress (dyne / cm ² ) = R × P / 2L). Here, R is a radius (cm) of the die discharge hole, P is a pressure loss (dyne / cm ² ) at the die discharge hole, and L is a length of the die discharge hole (cm). P = (8LηQ / πR ⁴ ), η is the polymer viscosity (poise), Q is the discharge rate (cm ³ / sec), and π is the circumference. Further, 1 dyne / cm ² of the CGS unit system is 0.1 Pa in the SI unit system.

D. Worcester spots of polymer alloy fibers (U%)
Measurement was performed in a normal mode at a yarn feeding speed of 200 m / min, using a USTER TESTER 4 manufactured by Zerbegger Worcester.

E. SEM observation Platinum was vapor-deposited on the sample and observed with an ultra-high resolution electrolytic emission scanning electron microscope. UHR-FE-SEM manufactured by Hitachi, Ltd. was used as the SEM apparatus.

F. Observation of cross section of nanofiber by TEM Using a nanofiber bundle before dispersion, an ultrathin section was cut out in the direction of the cross section of the nanofiber, and the cross section of the nanofiber was observed with a TEM apparatus. Moreover, metal dyeing | staining was given as needed. As the TEM apparatus, H-7100FA type manufactured by Hitachi, Ltd. was used.

G. Number average diameter of single fibers The number average diameter of single fibers (nanofibers) is determined as follows. That is, the single fiber diameter of the fiber was calculated in terms of a circle using image processing software (“WinROOF” (registered trademark)) from the photograph obtained by observing the SEM apparatus or TEM apparatus, and a simple average value thereof was obtained. At that time, the diameters of 150 fibers randomly extracted in the same cross section were analyzed and used for calculation.

H. Fiber component ratio The fiber component ratio of the short fibers (nanofibers) is evaluated as follows. That is, using the data used in determining the number average diameter, a respective single fiber diameter and di, the square of the sum ^{(d1 2 + d2 2 + ··} + dn 2) = Σdi 2 (i = 1~n ) Is calculated. Moreover, each fiber diameter of the single fiber larger than 500 nm in diameter is set to Di, and the sum total of the squares (D1 ² + D2 ² +... + Dm ² ) = ΣDi ² (i = 1 to m) is calculated. By calculating the ratio of ΣDi ² to Σdi ² , the area ratio of coarse fibers to the total fibers, that is, the fiber composition ratio was obtained.

[Production Example 1 of Dispersion]
Melt viscosity 57 Pa · s (temperature 240 ° C., shear rate 2432 sec ⁻¹ ), melting point 220 ° C. N6 (nylon 6) (20 wt%), weight average molecular weight 120,000, melt viscosity 30 Pa · s (temperature 240 ° C., shear speed 2432Sec ^-1), poly L-lactic acid with a melting point of 170 ° C. (optical purity of 99.5% or more) (80 wt%) was then melt-kneaded at a temperature of 220 ° C. in the biaxial extrusion kneader to obtain a polymer alloy chip . Here, the weight average molecular weight of poly L lactic acid was determined as follows. That is, THF (tetrahydrofuran) was mixed with a chloroform solution of a sample (poly L lactic acid) to obtain a measurement solution. The chromatograph was measured at 25 ° C. using a gel permeation chromatography (GPC) Waters 2690 manufactured by Waters, and the weight average molecular weight was calculated in terms of polystyrene. The melt viscosity of N6 at a temperature of 262 ° C. and a shear rate of 121.6 sec ⁻¹ was 53 Pa · s. The poly L-lactic acid had a melt viscosity of 86 Pa · s at a temperature of 215 ° C. and a shear rate of 1216 sec ⁻¹ . The kneading conditions at this time were as follows.
-Polymer supply: N6 and poly L lactic acid were weighed separately and supplied separately to the kneader.
・ Screw type: Fully meshed in the same direction, double threaded screw ・ Screw: Diameter (D) 37 mm, Effective length (L) 1670 mm,
(L) / (D) = 45.1
(The kneading part length was positioned on the discharge side from 1/3 of the effective screw length.)
-Temperature: 220 ° C
Vent: 2 places This polymer alloy chip was melted at a melting portion at a temperature of 230 ° C. and led to a spin block at a spinning temperature of 230 ° C. Next, 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 215 ° C. At this time, a base having a base hole diameter of 0.3 mm and a hole length of 0.55 mm was used, but almost no ballast phenomenon was observed. Moreover, the discharge amount per single hole at this time was 0.94 g / min. Furthermore, the distance from the base lower surface to the cooling start point (the upper end of the chimney) was 9 cm. The discharged yarn is cooled and solidified for 1 m with cooling air at a temperature of 20 ° C., and is supplied with an oil supply guide installed 1.8 m below the base, and then the unheated first take-up roller and second take-up roller are used. Winded up through. Next, this was stretched and heat-treated at a temperature of the first hot roller of 90 ° C. and a temperature of the second hot roller of 130 ° C. At this time, the draw ratio between the first hot roller and the second hot roller was 1.5 times. The obtained polymer alloy fiber exhibited excellent properties of a total fineness of 62 dtex, a filament count of 36 filaments, a strength of 3.4 cN / dtex, an elongation of 38%, and U% = 0.7%. Moreover, when the cross section of the obtained polymer alloy fiber was observed with a TEM, poly L lactic acid was a sea component, N6 showed an island-island sea-island structure, and the number average diameter of the island component N6 was 55 nm, A polymer alloy fiber which is a precursor of N6 nanofibers in which N6 is ultrafinely dispersed was obtained.

  By immersing the obtained polymer alloy fiber in a 5% aqueous sodium hydroxide solution at a temperature of 95 ° C. for 1 hour, 99% by weight or more of the poly-L lactic acid component in the polymer alloy fiber is removed by hydrolysis, and acetic acid is used. After neutralization, it was washed with water and dried to obtain a fiber bundle of N6 nanofibers. As a result of analyzing the fiber bundle of N6 nanofibers from a TEM photograph, the number average diameter of single fibers of N6 nanofibers is 60 nm, which is an unprecedented thinness, and the fiber composition ratio of N6 nanofibers having a diameter of more than 100 nm is 0 weight. %Met.

  The obtained fiber bundles of N6 nanofibers were cut into 0.01 mm, 0.2 mm, 2 mm, 30 mm and 50 mm lengths to obtain N6 nanofiber short fibers. Tappy Standard Niagara Test Beata (manufactured by Toyo Seiki Seisakusho Co., Ltd.) is charged with 23 liters (L) of water and 30 g of the short fiber of each fiber length obtained above, pre-beaten for 5 minutes, and then excess water Cut and recovered the short fibers. The weight of this short fiber was 250 g, and its water content was 88% by weight. 250 g of water-containing short fibers were charged as they were into an automatic PFI mill (manufactured by Kumagaya Riki Kogyo Co., Ltd.) and beaten for 6 minutes at a rotation speed of 1500 rpm and a clearance of 0.2 mm. 42 g of short fibers beaten into an oster blender (manufactured by oster) and 500 g of water are stirred and stirred for 30 minutes at a rotational speed of 13900 rpm. Further, the fiber dispersion thus obtained is mixed with poly as a temperature-responsive polymer compound. 0.25 g of N-isopropylacrylamide (manufactured by Aldrich, number average molecular weight 20000-250,000, LCST: 30-32 ° C.) was added and stirred for 30 minutes at 30 rpm on a rotary shaker, and 1.0 weight of N6 nanofibers % And N6 nanofiber-containing dispersion liquid 1 (fiber length 0.2 mm), dispersion liquid 2 (fiber length 2 mm), dispersion liquid 3 (fiber length 30 mm), and dispersion Liquid 4 (fiber length 50 mm) and dispersion 5 (fiber length 0.01 mm) were obtained. In the dispersion liquid 4, the nanofibers were not uniformly dispersed, and a large aggregate (floc) in which the nanofibers were associated was formed. In addition, for the short fiber having a fiber length of 2 mm, a dispersion 6 to which the temperature-responsive polymer compound is not added in the same production method as the dispersion 2 is used as the dispersion 6, and a production method similar to the dispersion 2 is used as the dispersion 7. In the same preparation method as dispersion 2, poly-N-isopropylacrylamide was added in an amount of 0.5 g and the temperature-responsive polymer compound concentration was 0.1% by weight. A dispersion liquid in which 0.05 g of N-isopropylacrylamide was added and the concentration of the temperature-responsive polymer compound was 0.01% by weight was used as dispersion liquid 9 in the same production method as dispersion liquid 2, and the temperature-responsive polymer was prepared. Polyvinyl methyl ether (Scientific Polymer Co., Ltd., weight average molecular weight 90000, LCST: 37-38 ° C.) was used as the compound, and 0.25 g of this was added. The thermoresponsive polymer compound 0.05 wt% containing dispersed liquid was, respectively prepared.

[Production Example 2 of Dispersion]
Polystyrene (PS) copolymerized with PBT (polybutylene terephthalate) (20 wt%) having a melt viscosity of 120 Pa · s (temperature: 262 ° C., shear rate: 121.6 sec ⁻¹ ) and melting point of 225 ° C. and 22 mol% of 2-ethylhexyl acrylate. (80 wt%) was used, and the kneading temperature was 240 ° C., and melt kneading was performed in the same manner as in Production Example 1 of the dispersion to obtain a polymer alloy chip. At this time, the melt viscosity of copolymerized PS at a temperature of 262 ° C. and a shear rate of 121.6 sec ⁻¹ was 140 Pa · s, and the melt viscosity at a temperature of 245 ° C. and a shear rate of 1216 sec ⁻¹ was 60 Pa · s.

  The obtained polymer alloy chip was melt-spun in the same manner as in Production Example 1 of the dispersion 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, a die having a discharge hole diameter of 0.7 mm and a discharge hole length of 1.85 mm, having a measuring portion having a diameter of 0.3 mm above the discharge hole, was used. The spinnability was good and the yarn breakage was 1 in 1 t spinning. The discharge rate per single hole at this time was 1.0 g / min. The obtained undrawn yarn was drawn and heat treated in the same manner as in Production Example 1 of the dispersion at a drawing temperature of 100 ° C., a draw ratio of 2.49 times, and a heat setting temperature of 115 ° C. The obtained drawn yarn had a total fineness of 161 dtex, a filament number of 36, a strength of 1.4 cN / dtex, an elongation of 33%, and U% = 2.0%. When the cross section of the obtained polymer alloy fiber was observed with a TEM, the copolymer PS was a sea component, the PBT was an island component, and the number average diameter of the PBT was 70 nm, and the PBT was nano-sized. Uniformly dispersed polymer alloy fibers were obtained.

  By immersing the obtained polymer alloy fiber in trichlorethylene, 99% by weight or more of copolymer PS, which is a sea component, was eluted and then dried to obtain a fiber bundle of PBT nanofibers. As a result of analyzing the fiber bundle of the PBT nanofiber from the TEM photograph, the number average diameter of the single fiber of the PBT nanofiber is 85 nm, which is an unprecedented thinness, and the fiber composition ratio of the PBT nanofiber having a diameter larger than 200 nm is 0. The fiber ratio of PBT nanofibers with a diameter of greater than 100 nm was 1% by weight.

  The obtained fiber bundle of PBT nanofibers was cut into a length of 2 mm to obtain short fibers of PBT nanofibers. The obtained short fibers of PBT nanofibers were preliminarily beaten in the same manner as in Production Example 1 of the dispersion to obtain PBT nanofibers having a water content of 80% by weight, and then beaten in the same manner as in Production Example 1 of the dispersion. Then, stirring and addition of a temperature-responsive polymer compound were performed to obtain a dispersion 10 containing PBT nanofibers containing 1.0% by weight of PBT nanofibers and 0.05% by weight of a temperature-responsive polymer compound.

[Production Example 3 of Dispersion]
Dispersion except that N6 in Production Example 1 of dispersion was changed to PP (polypropylene) (23 wt%) having a melt viscosity of 350 Pa · s (temperature 220 ° C., shear rate 121.6 sec ⁻¹ ) and melting point 162 ° C. In the same manner as in Production Example 1, the polymer alloy chip was obtained by melt-kneading. The melt viscosity of poly L lactic acid at a temperature of 220 ° C. and a shear rate of 121.6 sec ⁻¹ was 107 Pa · s. Using this polymer alloy chip, melting was performed in the same manner as in Production Example 1 of the dispersion at a melting temperature of 230 ° C., a spinning temperature of 230 ° C. (die surface temperature of 215 ° C.), a single hole discharge rate of 1.5 g / min, and a spinning speed of 900 m / min. Spinning was performed. The obtained undrawn yarn was drawn and heat treated in the same manner as in Production Example 1 of the dispersion at a drawing temperature of 90 ° C., a draw ratio of 2.7 times, and a heat setting temperature of 130 ° C.

  By immersing the obtained polymer alloy fiber in a 5% aqueous sodium hydroxide solution at a temperature of 98 ° C. for 1 hour, 99% by weight or more of the poly-L lactic acid component in the polymer alloy fiber is hydrolyzed and removed with acetic acid. After summing, washing with water and drying were performed to obtain a fiber bundle of PP nanofibers. As a result of analyzing the fiber bundle of the PP nanofiber from the TEM photograph, the number average diameter of the single fiber of the PP nanofiber was 240 nm, and the fiber ratio of the PP nanofiber having a diameter larger than 500 nm was 0% by weight.

  The obtained PP nanofiber bundle was cut into 2 mm lengths to obtain PP nanofiber short fibers. The obtained short fibers of PP nanofibers were preliminarily beaten in the same manner as in Production Example 1 of the dispersion to obtain PP nanofibers having a water content of 75% by weight, and then beaten in the same manner as in Production Example 1 of the dispersion. , Stirring, addition of temperature-responsive polymer compound, stirring for 30 minutes at 30 rpm on a rotary shaker, PP containing 1.0% by weight of PP nanofibers and 0.05% by weight of temperature-responsive polymer compound A dispersion 11 containing nanofibers was obtained.

[Production Example 4 of Dispersion]
A 60% by weight alkali-soluble copolymer polyester resin is used for the sea component, 40% by weight of the N6 resin is used for the island component, the island component is 100 islands by melt spinning, and a polymer array composite fiber having a single fiber fineness of 5.3 dtex ( (Hereinafter referred to as composite fiber), and then stretched 2.5 times to obtain a composite fiber having a single fiber fineness of 2.1 dtex. The composite fiber had a strength of 2.6 cN / dtex and an elongation of 35%. Thereafter, the composite fiber was treated with a 3% aqueous sodium hydroxide solution at a temperature of 98 ° C. for 1 hour to hydrolyze and remove 99% by weight or more of the polyester component in the composite fiber, neutralized with acetic acid, After washing with water and drying, N6 ultrafine fibers were obtained. When the average single fiber fineness of the obtained ultrafine fibers of N6 was analyzed from a TEM photograph, the single fiber fineness was equivalent to 0.02 dtex (average fiber diameter 2 μm). The obtained N6 ultrafine fiber was cut into 2 mm lengths to make short fibers, and then 5 g of these short fibers and 495 g of water were charged into an oster blender (manufactured by oster Co., Ltd.) and stirred at a rotational speed of 13900 rpm for 30 minutes. 0.25 g of the same temperature-responsive polymer compound as in the above was added and stirred for 30 minutes at 30 rpm on a rotary shaker, containing 1.0% by weight of N6 ultrafine fiber and 0.05% by weight of temperature-responsive polymer compound. A dispersion 12 was obtained.

[Production Example 5 of Dispersion]
After obtaining PET (polyethylene terephthalate) fiber having a single fiber fineness of 10 dtex (average fiber diameter of 30 μm) by a conventionally known melt spinning method, this was cut into a length of 2 mm to obtain short fibers. After charging 5 g of these short fibers and 495 g of water into an Oster blender (manufactured by Oster) and stirring for 1 minute at a rotational speed of 10,000 rpm, 0.25 g of the same temperature-responsive polymer compound as in Production Example 1 was added, and The mixture was stirred at 30 rpm for 30 minutes to obtain a dispersion 13 containing 1.0% by weight of PET fibers and 0.05% by weight of a temperature-responsive polymer compound.

[Production Example 6 of Dispersion]
After obtaining a PET fiber having a single fiber fineness of 33 dtex (average fiber diameter 55 μm) by a conventionally known melt spinning method, this was cut into a length of 2 mm to obtain a short fiber. After charging 5 g of this short fiber and 495 g of water into an Oster blender (manufactured by Oster Co., Ltd.) and stirring for 1 minute at a rotational speed of 10,000 rpm, 0.25 g of the same temperature-responsive polymer compound as in Production Example 1 was added and rotary shaking was performed. The mixture was stirred at 30 rpm for 30 minutes to obtain a dispersion 14 containing 1.0% by weight of PET fibers and 0.05% by weight of a temperature-responsive polymer compound.

(Examples 1-3)
A sponge-like temperature-responsive substrate was prepared using the dispersions 1 to 3 obtained in Production Example 1 of the dispersion. In the production method, 1 mL of each of dispersions 1 to 3 was placed in a well (diameter: 16 mm) of a 24-well plate, and left in an ultra-low temperature freezer at a temperature of -80 ° C for 12 hours. The frozen sample was put into a chamber of a freeze dryer (FD-5N) manufactured by EYELA, and freeze-dried at a vacuum degree of 0.1 kPa or less to obtain a temperature-responsive base material having a sponge-like three-dimensional structure. (See FIG. 1).

  Next, the structure of the temperature-responsive substrate remains even if the obtained temperature-responsive substrate is removed from the well, and hot water at 37 ° C. higher than the LCST of the used temperature-responsive polymer compound is added and pipetting is performed. Although it did not collapse (see FIG. 2), the structure of the temperature-responsive substrate collapsed when pipetting was performed by adding 4 ° C. cold water lower than LCST (see FIG. 3). It was confirmed that it had a temperature responsiveness of.

(Comparative Example 1)
The dispersion 4 obtained in Production Example 1 of the dispersion was lyophilized in the same manner as in Example 1 to try to form a sponge-like temperature-responsive substrate, but the fiber length was as large as 50 mm. However, since a non-uniform aggregate was formed, a sponge-shaped temperature-responsive substrate could not be formed.

(Comparative Example 2)
Using the dispersion 5 obtained in Production Example 1 of the dispersion, freeze-drying was performed in the same manner as in Example 1 to try to form a sponge-like temperature-responsive substrate. However, the obtained structure has a flat sheet-like structure. Further, when 37 ° C. warm water was added to the resulting structure and pipetting was performed, the fiber length was as small as 0.01 mm, and the structure collapsed.

(Comparative Example 3)
Using the dispersion 6 obtained in Production Example 1 of the dispersion, lyophilization was performed in the same manner as in Example 1 to try to form a sponge-like temperature-responsive substrate. As a result, a clean sponge-like temperature-responsive substrate was obtained. Formed. However, it was found that there was no structure controllability due to temperature because the structure collapsed when pipetting was performed by adding warm water at 37 ° C. higher than the LCST of the temperature-responsive polymer compound used.

(Examples 4 to 6)
Using the dispersions 7 to 9 obtained in Production Example 1 of the dispersion, lyophilization was performed in the same manner as in Example 1 to try to form a sponge-like temperature-responsive substrate. A conductive substrate was formed. About each obtained temperature-responsive base material, like Example 1, warm water and cold water were added separately, the collapse of the structure was observed, and the temperature-responsiveness of the temperature-responsive base material was confirmed. It was confirmed that the temperature responsive substrate also has temperature responsiveness.

(Examples 7 to 10)
Using the dispersions 10 to 13 obtained in Production Examples 2 to 5 of the dispersion, lyophilization was performed in the same manner as in Example 1 to try to form a sponge-like temperature-responsive substrate. A temperature responsive substrate was formed. About each obtained temperature-responsive base material, like Example 1, warm water and cold water were added separately, the collapse of the structure was observed, and the temperature-responsiveness of the temperature-responsive base material was confirmed. It was confirmed that the temperature responsive substrate also has temperature responsiveness.

(Comparative Example 4)
Using the dispersion 14 obtained in Production Example 6 of the dispersion, freeze-drying was performed in the same manner as in Example 1 to try to form a sponge-like temperature-responsive substrate. However, the obtained structure has a flat sheet-like structure. Further, when pipetting was performed by adding 37 ° C. warm water, the average fiber diameter was as large as 55 μm, and the structure collapsed. Thus, it was found that there was no structure controllability due to temperature.

  The results of Examples 1 to 10 and Comparative Examples 1 to 4 are summarized in Table 1.

(Example 11)
100 μL of dispersion 2 prepared in Production Example 1 of dispersion was added to a well (diameter: 6.4 mm) of a 96-well plate for cell culture, and the plate was allowed to stand in an ultra-low temperature freezer at a temperature of −80 ° C. for 12 hours. . The frozen sample was put in a chamber of a freeze dryer (FD-5N) manufactured by EYELA, and freeze-dried at a vacuum degree of 0.1 kPa or less to prepare a sponge-like temperature-responsive substrate on the well. . 100 μL of mouse osteoblastic 3T3-E1 cell suspension (cell concentration 5 × 10 ⁵ / mL, 10% fetal bovine serum-added αMEM (alpha minimum basic) medium) was added to the obtained temperature-responsive substrate. The cells were allowed to stand for 1 hour at a temperature of 37 ° C. to retain the cells in the temperature-responsive substrate, and then 100 μL of 10% fetal bovine serum-added αMEM medium was added thereto, and the temperature was 37 ° C., 5%. Culturing was carried out for 48 hours in a CO ₂ atmosphere. After 48 hours, the culture solution was removed, 200 μL of PBS (phosphate buffer solution) cooled to a temperature of 4 ° C. was added, and pipetting was performed. As a result, the temperature-responsive substrate collapsed as shown in FIG. did.
The cells and the disintegrated temperature-responsive substrate were collected, and the cultured cells were collected by repeatedly passing through a nylon mesh having a mesh interval of 40 μm to remove the fibers (N6 nanofibers). When the number of cells was measured with a hemocytometer, the number of cells increased 4.6 times.

Example 12
Using the fiber prepared in Dispersion Production Example 1, 0.1% by weight of N6 nanofibers having a fiber length of 2 mm and a temperature-responsive polymer compound of 0.005% by weight were produced in the same manner as in Dispersion Production Example 1. A dispersion 15 containing was prepared. The dispersion 15 was spotted on a polystyrene petri dish with a micropipette at 0.5 μL / spot and then dried at a temperature of 65 ° C. As a result, a film having a circular pattern with a diameter of about 1 mm as shown in FIG. A temperature-responsive substrate was formed. The pattern of the temperature-responsive substrate remained retained even when sprayed with PBS warmed to 37 ° C. on the obtained temperature-responsive substrate (see FIG. 5), but when PBS cooled to 4 ° C. was sprayed Since the temperature-responsive substrate collapsed and the pattern disappeared (see FIG. 6), it was confirmed that the pattern of the temperature-responsive substrate had temperature responsiveness.

(Example 13)
A circular film-like temperature-responsive substrate was formed on an untreated polystyrene petri dish by the same method as in Example 12. PBS containing 1 mg / mL fibronectin (manufactured by Sigma) was added onto the temperature-responsive substrate, and incubated at 37 ° C. for 30 minutes to adsorb and retain fibronectin on the temperature-responsive substrate. Prepare a mouse myoblast C2C12 cell suspension (cell concentration 1 × 10 ⁴ / mL, DMEM (Dulbecco's Modified Eagle) medium supplemented with 10% fetal bovine serum), and place this cell suspension on a temperature-responsive substrate. After adding 100 μL and incubating for 1 hour at a temperature of 37 ° C. and 5% CO ₂ , the non-adherent cells were washed with PBS warmed to a temperature of 37 ° C., the non-adherent cells were removed, and 10% fetal bovine serum was added again. When DMEM medium was added and cultured at 37 ° C. in a 5% CO ₂ atmosphere for 1 week, the cells proliferated and associated cells according to the temperature-responsive substrate pattern (circular shape) as shown in FIG. Tissue formation was observed. When a PBS solution cooled to 4 ° C. was sprayed on a temperature-responsive substrate containing cells in which such a tissue was formed, the temperature-responsive substrate collapsed, and the tissue associated with the cells was circular. It was confirmed that it was peeled from the temperature-responsive substrate while maintaining the shape (see FIG. 8).

  A fiber structure comprising a collection of short fibers having a number average diameter of 1 nm to 50 μm and a single fiber length of 0.2 to 30 mm and a temperature-responsive polymer compound of the present invention in an aqueous solution The temperature-responsive base material is characterized in that its structure collapses when the temperature is changed from a temperature higher than the LCST of the temperature-responsive polymer compound to a temperature lower than the LCST, such as clothing materials, interior products, living materials, environmental and industrial materials. It can be applied to products, IT products, medical products, and research products, and is particularly suitable as a material used for cell culture, tissue regeneration, DDS and diagnosis.

FIG. 1 is a drawing (perspective view) substituted photograph (same size) showing a temperature-responsive base material having a sponge-like three-dimensional structure obtained in Example 2. FIG. 2 is a substitute for a drawing (perspective view) showing a state after pipetting by adding 37 ° C. warm water higher than the LCST of the temperature-responsive polymer compound used to the temperature-responsive base material of FIG. It is a photograph (same size). FIG. 3 is a drawing (perspective view) showing a state after pipetting by adding 4 ° C. cold water lower than the LCST of the temperature-responsive polymer compound used to the temperature-responsive base material of FIG. It is a photograph (same size). FIG. 4 is a drawing (plan view) substitute photograph (25 times) showing a film-like temperature-responsive substrate having a circular pattern obtained in Example 7. FIG. 5 is a substitute photograph (plan view) showing a state after spraying PBS heated to 37 ° C., which is higher in temperature than the LCST of the temperature-responsive polymer compound used, on the temperature-responsive base material of FIG. Yes (25 times). 6 is a drawing (plan view) showing a state after spraying PBS cooled to 4 ° C. at a temperature lower than the LCST of the temperature-responsive polymer compound used on the temperature-responsive substrate of FIG. (25 times). FIG. 7 is a drawing (perspective view) substitute photograph showing a state in which cells are proliferated and cells are associated with each other according to the pattern (circular shape) of the temperature-responsive substrate obtained in Example 8. (25 times). FIG. 8 shows that the temperature-responsive base material collapsed by spraying a PBS solution cooled to 4 ° C. onto the temperature-responsive base material containing the tissue associated with the cells in FIG. It is drawing (perspective view) substitute photograph which shows the state peeled from the temperature-responsive base material, keeping the shape of (25 times).

Claims (15)

  It includes a fiber structure composed of an assembly of short fibers having a single fiber number average diameter of 1 nm to 50 μm and a single fiber length of 0.2 mm to 30 mm, and a temperature responsive polymer compound, and is temperature responsive in an aqueous solution. A temperature-responsive substrate characterized in that its structure collapses by changing the temperature from a temperature higher than the lower critical solution temperature of the polymer compound to a temperature lower than the lower critical solution temperature.   The temperature-responsive substrate according to claim 1, wherein the short fibers are nanofibers having a single fiber number average diameter of 1 nm to 500 nm.   The temperature-responsive substrate according to claim 1 or 2, wherein the fiber constituting ratio of single fibers having a diameter larger than 500 nm is 3% by weight or less.   The temperature-responsive substrate according to any one of claims 1 to 3, wherein the short fibers are made of an organic polymer.   The temperature-responsive polymer compound is insoluble in water at a temperature higher than the lower critical solution temperature, and is soluble in water at a temperature lower than the lower critical solution temperature. The temperature-responsive base material in any one of.   6. The temperature-responsive base material according to claim 5, wherein the temperature-responsive polymer compound is poly N-substituted acrylamide.   The temperature-responsive base material according to any one of claims 1 to 6, wherein the content of the temperature-responsive polymer compound is 1 to 20% by weight based on the weight of the fiber structure.   The temperature-responsive polymer compound is added to a dispersion obtained by dispersing an aggregate of short fibers in a solvent, and the solvent is removed to form the dispersion. The temperature-responsive substrate as described.   The temperature-responsive base material according to any one of claims 1 to 8, wherein a surface of the short fiber constituting the fiber structure is coated with a temperature-responsive polymer compound.   The temperature-responsive substrate according to any one of claims 1 to 9, wherein the fiber structure is in a thin layer shape, a paper shape, a nonwoven fabric shape, or a sponge shape.   The temperature-responsive substrate according to claim 1, wherein the temperature-responsive substrate is formed on another substrate.   The temperature-responsive substrate according to claim 11, wherein a predetermined pattern is formed on another substrate.   The temperature-responsive substrate according to any one of claims 1 to 12, wherein a functional substance is held on the surface and / or inside thereof.   The temperature-responsive substrate according to any one of claims 1 to 13, which is used in a field involving cell culture and / or tissue regeneration.   The cell cultured on the surface and / or inside of the temperature-responsive substrate according to claim 14.