JP2009000100A - Scaffold material for cell culture, method for producing the same and module for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for readily and rapidly carrying out pharmacodynamic effect tests, toxicity tests and safety tests of substances such as medicines, chemical substances or cosmetics. <P>SOLUTION: A scaffold material for cell culture comprises hollow fiber membranes arranged in the mesh state and nanofibers having 1-1,000 nm diameter of single fibers arranged on the outer surface of the hollow fiber membranes and/or between the hollow fiber membranes. A method for producing the scaffold material for cell culture comprises arranging the hollow fiber membranes arranged in the mesh state between a nozzle and a target and carrying out electrospinning of the nanofibers. A module for cell culture is obtained by using the scaffold material for cell culture and has openings of hollow parts of the hollow fiber membranes at both ends of the hollow fiber membranes and port parts connecting to the openings. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a scaffold material for cell culture, a method for producing the same, and a scaffold material for cell culture for easily and quickly conducting a medicinal effect test, toxicity test, and safety test for substances such as pharmaceuticals, chemical substances, and cosmetics. The present invention relates to the cell culture module used.

  Traditionally, pharmaceuticals, chemicals, cosmetics, and other substances that may affect the human body have been required to be tested for efficacy, toxicity, and safety, and various animals are usually used for these tests. Animal experiments that were being conducted. However, animal experiments must take into account the differences between humans and animal species, and individual differences between animals, and it is necessary to repeat many experiments from the viewpoint of data reliability. At the same time, there were problems such as securing a large number of animals for the test and taking time and labor. In particular, it is practically impossible to co-test a large number of large animals such as pigs whose drug metabolic system is considered to be close to that of humans. In recent years, animal welfare is also increasing in animal experiments, and as a solution to the above problems, an evaluation test method using cultured animal cells, that is, an alternative method for animal experiments, has been developed.

  In order to efficiently culture cells in vitro, it is necessary to supplement the cultured cells with nutrients and air (oxygen). As an example, a method for supplying nutrients and air (oxygen) necessary for cell culture using a hollow fiber membrane has been proposed (Patent Document 1). According to this, it is disclosed that a culture solution and oxygen are supplied to a cell culture space by using a hollow fiber membrane having selective permeability and a hollow fiber membrane having gas permeability. However, although the efficiency of cell culture is increased, the cells are present in the culture liquid, which is suitable for the production of useful substances, etc., but it is difficult for the cultured cells to form tissues, and it is applicable to alternative animal experiments. It was difficult.

  As a method for culturing animal cells, particularly adherent animal cells, at high density, a cell culture device using a hollow fiber having a semipermeable membrane function and a fibrous cell support has been proposed (Patent Document 2). However, since the hollow fiber to be used is a semipermeable membrane that does not permeate proteinaceous substances produced by cells, it is not preferable for monitoring of cellular metabolites and the like. In addition, since the fibrous cell support is fixed to the cell culture vessel and disposed in the gap between the hollow fibers, the nutrient supply from the hollow fibers to the cultured cells and the recovery efficiency of metabolic waste products are inferior. It was.

  In order to promote the formation of cell tissue, the inner or outer cavity of the hollow fiber is filled with cells at high density using centrifugal force, and oxygen and nutrients are supplied and the metabolic waste products are removed using the hollow fiber. Has been proposed (Patent Document 3). However, in this method, since the filled cells and the hollow fiber are in direct contact with each other, the surface of the hollow fiber is blocked by cell adsorption, and there is a problem that supply of oxygen and nutrients and removal of metabolic waste products are insufficient.

  In recent years, attention has been focused on using nanofibers having nano-order fiber diameters as scaffolds for cell culture and tissue formation (Non-patent Documents 1 and 2). For example, nanofibers prepared by melt spinning are used. A method of using a fiber as a scaffold material is disclosed (Patent Document 4). In addition, a nanofiber pattern is formed using a mixed liquid in which organic nanofibers are dispersed in a solvent, and further, a functional drug effective for cell culture is adsorbed to this pattern, so that cell adhesion, proliferation, A method for controlling differentiation has been proposed (Patent Document 5). However, these methods have not taken into consideration the supply of nutrients and air (oxygen) necessary for cell growth.

  A cell culture module having a hollow fiber for supplying a culture solution and a porous carrier such as a nonwoven fabric for culturing cells has already been proposed (Patent Document 6). However, in this method, only a porous carrier is disposed in the hollow fiber module, and the hollow fiber supplying the culture solution and the porous carrier are not necessarily close to each other. The replenishment efficiency was poor, and the removal of metabolic waste was completely inadequate.

  Furthermore, a test method and apparatus for filling cells in the lumen of the porous hollow fiber membrane, flowing a test substance such as a chemical substance or a drug outside the hollow fiber together with nutrients, and monitoring the metabolism and rate of metabolism of the test substance by the cells. Known (Patent Document 7). However, in this method, since the filled cells and the hollow fiber are in direct contact with each other, the surface of the hollow fiber is also blocked by cell adsorption, and there is a problem that supply of oxygen and nutrients and removal of metabolic waste products are insufficient.

Patent No. 3026516 JP 2002-112763 A Japanese Patent No. 3725147 JP 2006-254722 A JP 2007-44149 A JP 2003-180334 A JP 2000-189189 A Polymer, 55, 3, 142 (2006) Tissue Eng. , 11, 101 (2005)

  The present invention is intended to solve the above-mentioned problems, and in cell culture, a sufficient amount of nutrients and air (oxygen) is supplied, metabolic waste products are removed, and a cell culture scaffolding material having the ability to promote cell growth. An object of the present invention is to provide a manufacturing method thereof, and a cell culture module using the scaffold material.

The present invention provides a cell culture scaffold in which hollow fiber membranes are arranged in a mesh shape, and nanofibers having a single fiber diameter of 1 to 1000 nm are arranged on the outer surface of the hollow fiber membrane and / or between the hollow fiber membranes. Regarding materials.
Furthermore, the present invention relates to a method for producing the above-mentioned cell culture scaffold material, in which a hollow fiber membrane arranged in a mesh shape is arranged between a nozzle and a target, and nanofibers are electrospun.
The present invention also relates to a cell culture module using the cell culture scaffold material described above.

  According to the present invention, in addition to being able to cultivate cells efficiently, it is also possible to monitor the metabolic waste products on-line, and to perform tests using animal substitute experimental methods such as chemical substances and drugs. Can be performed efficiently.

Hereinafter, preferred embodiments of the present invention will be described.
The scaffold for cell culture of the present invention is a nanofiber having a single fiber diameter of 1-1000 nm disposed on a hollow fiber membrane disposed on a mesh or in a void of a mesh formed of a hollow fiber membrane. is there.

  As the membrane base polymer of the hollow fiber membrane, any polymer can be used as long as it can be formed into a hollow fiber membrane, for example, polyolefins such as polyethylene and polypropylene, polysulfone, polyallyl sulfone, polyether sulfone, polyacrylonitrile, cellulose acetate, Examples thereof include polyvinylidene fluoride, a mixture of two or more kinds of polymers, and the like.

  The outer diameter of the hollow fiber membrane is preferably 50 μm or more and 3000 μm or less, and more preferably 100 μm or more and 2000 μm or less. When such a range is exceeded, when the outer diameter is less than 50 μm, the hollow portion of the hollow fiber membrane becomes small, so that, for example, the differential pressure when the culture solution is fed increases, thereby removing metabolic waste products. If the outer diameter is 3000 μm or more, the surface area of the hollow fiber membrane occupies a unit volume becomes small, and for example, nutrient supply and gas supply to cultured cells are insufficient. There is a risk.

The film thickness of the hollow fiber membrane is preferably 5 μm or more and 500 μm or less. When the film thickness is 5 μm or less, the hollow fiber membrane may be destroyed by pressurization during nutrient supply or gas supply. When the film thickness is 500 μm or more, the filtration resistance increases, Gas supply and removal of metabolic waste products may be insufficient. The film thickness when the hollow fiber membrane is a multilayer membrane is the sum of the thicknesses of the respective layers (the thickness of all layers).
The inner diameter of the hollow fiber membrane is not generally known because it depends on the outer diameter and film thickness of the hollow fiber membrane, but is usually 10 μm or more.

  As the hollow fiber membrane, a microfiltration membrane, an ultrafiltration membrane, a gas separation membrane, or the like can be used. These can be appropriately selected as necessary. For example, nutrients can be supplied to the cultured cells by using a microfiltration membrane, and oxygen-containing gas is supplied to the cultured cells by using a gas separation membrane. be able to.

In order to supply a specific substance to a cell or to remove a specific metabolic waste product, a hollow fiber membrane having a substance selective permeability can be used. Examples of the hollow fiber membrane having the material selective permeability include the above-mentioned microfiltration membrane and ultrafiltration membrane, and further, the hollow fiber membrane having the material selective permeability to the membrane material itself and the hollow portion are selected. Examples thereof include a porous hollow fiber membrane filled with a permeable substance.
By using such a hollow fiber membrane having selective permeability, it becomes possible to efficiently supply a test substance such as a specific component in a culture solution, a chemical substance, or a drug to a cultured cell. It is also possible to monitor things by discharging them out of the system.

  In order to selectively supply air, oxygen, and other gases to the cultured cells efficiently, a gas separation membrane can be used as described above. The gas can be supplied using a porous hollow fiber membrane. For example, a hollow fiber membrane in which a non-porous substrate having a gas separation function is supported by a porous substrate is suitable. By using such a hollow fiber membrane having a gas separation function, gas can be supplied into the culture solution without bubble, and damage to the cultured cells can be prevented. Further, it is possible to prevent a liquid such as a culture solution from entering the hollow portion of the hollow fiber and closing the hollow portion.

  In the present invention, a hydrophilic fiber-treated hollow fiber membrane can be used. By hydrophilizing the hollow fiber membrane, it is possible to facilitate the supply of liquid components such as a culture solution, and to control cell adhesion to the surface of the hollow fiber membrane. Examples of the method for hydrophilizing the hollow fiber membrane include treating the hollow fiber membrane with a hydrophilic polymer such as an ethylene-vinyl alcohol copolymer, glycerin, and ethanol.

  Two or more types of hollow fiber membranes can also be used in the cell culture scaffold material of the present invention. For example, microfiltration membranes for supplying nutrients, gas separation membranes for supplying gas, substance-selective permeable hollow fibers for supplying specific components to cultured cells or discharging metabolic waste products of specific components Membranes can be arbitrarily combined.

  The hollow fiber membrane used in the present invention is arranged in a mesh shape. For example, a hollow fiber membrane arranged in a sheet shape at a predetermined interval and a hollow fiber membrane arranged in a sheet shape at a predetermined interval in a direction substantially perpendicular thereto are arranged, and the hollow fiber membrane is arranged in a mesh shape can do. The number of hollow fiber membrane sheets arranged in a mesh shape is not particularly limited, but is preferably 2 or more and 10 or less. When forming a mesh with two or more types of hollow fiber membranes, different types of hollow fiber membranes may be mixed in the same hollow fiber membrane sheet, or after the same hollow fiber membranes are arranged in a sheet shape, It is also possible to form a mesh by stacking different types of arranged hollow fiber membranes. What kind of hollow fiber membrane mesh is used to supply a desired liquid or gas through the hollow fiber membrane or to remove metabolic waste products is a port when the cell culture scaffold material of the present invention is modularized. Depends on the design of the part.

  In the present invention, nanofibers are used. The diameter of the nanofiber is 1 nm to 1000 nm, preferably 10 nm to 500 nm, more preferably 20 nm to 300 nm. When the diameter of the single fiber is out of such a range, the strength of the single fiber may be insufficient if it is less than 1 nm, and the effect of the specific surface area may be insufficient if it is 1000 nm or more. One of the characteristics of nanofibers is the specific surface area. The smaller the single fiber diameter, the larger the specific surface area and the greater the adsorption properties, making it suitable as a scaffold for cultured cells. . The nanofiber substrate is not particularly limited, but a solvent-soluble polymer is preferable from the viewpoint of nanofiber spinning. However, the spinning method of the nanofiber used in the present invention is not particularly limited, and methods such as wet spinning, melt spinning, electrospinning, etc. can be appropriately selected.

The diameter of the nanofiber single fiber in the present invention can be measured as follows. First, the surface of the nanofiber or its aggregate is observed using an electron microscope. As the electron microscope, a scanning electron microscope (SEM) is preferable. The observation magnification is about 5000 to 50,000 times. When the observation magnification is less than 5000 times, it is difficult to determine the fiber diameter of the nanofiber. Subsequently, the diameter of the single fiber of a nanofiber is calculated | required from the obtained SEM photograph. As for the diameter of the single fiber, the width of the surface of any 20 nanofibers in the SEM photograph is measured, and the average is defined as the diameter of the single nanofiber.
When obtaining the diameter of a single fiber, it is preferable to use image analysis software. The diameter of the single fiber obtained by the image analysis software varies slightly depending on the image quality adjustment for image analysis, the type of the image analysis software, etc., but the difference is within the range of normal experimental error.

  By adsorbing or fixing a physiologically active substance to the nanofiber of the present invention, for example, adhesion, proliferation, differentiation, and function expression of cultured cells can be controlled. Examples of the physiologically active substance include functional polymers, amino acids, proteins, sugar chains, vitamins and the like. In particular, it is effective to use a physiologically active substance effective for cell culture, for example, a cytokine which is a general term for a physiologically active substance having an important role in cell proliferation, differentiation and functional expression. Cytokines include interleukins, blood boosting factors, growth factors and the like. Alternatively, it is also effective to use a cell adhesion substrate such as a cell-extracellular matrix, an integrin involved in cell-cell adhesion, or cadherin. It is also possible to conduct a cell culture test by adsorbing or fixing a test substance such as a drug or a chemical substance on the nanofiber.

As a method of obtaining the cell culture scaffold material of the present invention by arranging nanofibers on the outer surface of the hollow fiber membrane arranged in a mesh and / or between the membranes, for example, on the hollow fiber membrane sheet or the hollow fiber membrane Examples include a method of arranging nanofibers on a mesh and laminating another hollow fiber membrane sheet or a hollow fiber membrane mesh thereon, and a method of forming a hollow fiber membrane previously covered with nanofibers into a mesh shape. .
In addition, the cell culture scaffold material of the present invention may be prepared by dropping a mixed solution in which nanofibers are dispersed in a solvent onto a hollow fiber membrane mesh, disposing a hollow fiber membrane mesh in the mixed solution, or hollow fiber membrane. Can also be obtained by dispersing nanofibers in the solvent in which the nanofibers are immersed and removing the solvent, or by arranging the nanofibers on the hollow fiber membrane mesh or in the voids of the hollow fiber membrane mesh. Can do.

Moreover, the cell culture scaffold material of this invention can be obtained by arrange | positioning on the hollow fiber membrane which arrange | positioned the sheet-like thing which consists of a some nanofiber on the mesh shape, and laminating | stacking this. This sheet-like material can be obtained by paper-making nanofibers or applying an electrospinning method which is one of nanofiber spinning methods.
The method for producing the sheet-like material can be selected as appropriate. However, when the electrospinning method is adopted, the sheet-like material can be formed simultaneously with the spinning of the nanofibers.

Furthermore, as will be described below, nanofibers can be spun in the presence of a hollow fiber membrane arranged in a mesh shape.
Therefore, in the present invention, it is particularly preferable to employ the electrospinning method as a method for producing the cell culture scaffold material because this production can be efficiently performed.

  Here, a method for producing the cell culture scaffold material of the present invention using the electrosping method will be briefly described. The basic apparatus configuration of the electrospinning method includes a nozzle, a target, and a DC high-voltage power supply. When a high voltage is applied while supplying the spinning dope to the nozzle, an electric repulsive force is generated in the spinning dope, and the spinning dope is injected toward the target, and when the spinning conditions are appropriate, nano-order fibers on the target, that is, Nanofibers can be obtained.

  The spinning conditions for obtaining the nanofiber are the molecular weight of the polymer, the solvent, the concentration of the solution, the applied voltage, the distance between the nozzle and the metal collector, etc., and these are adjusted as appropriate. The polymer concentration of the solution varies depending on the type, molecular weight, and solvent of the polymer to be used, so it cannot be generally stated. However, if the polymer concentration is too low, the productivity may be lowered or the fibrous material may be difficult to obtain. In addition, if the polymer concentration is too high, the viscosity increases and it is difficult to inject the polymer. Therefore, it is generally preferable to adjust the concentration to about 0.1 to 40 wt%.

  The nanofiber substrate used in the electrospinning method is preferably a polymer soluble in a solvent. The polymers that can be electrospun include nylon 4, 6, nylon 6, nylon 6, 6, nylon 12, polyacrylic acid, polyacrylonitrile, polyamide, polycarbonate, polyetherimide, polyethylene oxide, polyethylene terephthalate, polystyrene, styrene-butadiene. Examples thereof include a copolymer, polysulfone, polyurethane, polyvinyl alcohol, polyvinyl chloride, polyvinyl pyrrolidone, polyvinylidene fluoride, polycaprolactone, polylactic acid, silk, cellulose acetate, chitosan, and collagen. However, it is not limited to these, It is also possible to contain these polymer blends, an inorganic substance, and a carbon material. Moreover, even if it is a polymer insoluble in a solvent, as long as it is a thermoplastic polymer, the laser electrospinning method can also be applied.

  The molecular weight of the polymer to be used is not particularly limited, but if the molecular weight is too low, the spinnability may be lowered, and if the molecular weight is too high, the viscosity becomes high and spinning may be difficult. Even in these cases, electrospinning can be enabled by adjusting the polymer concentration. It is also possible to adjust the surface tension and conductivity of the spinning dope by adding additives.

  The solvent to be used is not particularly limited as long as the polymer can be dissolved at the above concentration. For example, solvents such as acetone, triacetin, dimethylformamide, and dimethylacetamide can be used. However, if a highly volatile solvent is used, the solvent may volatilize, which may cause problems such as polymer clogging at the nozzle tip. is there. In order to improve solubility, several kinds of solvents may be blended and used.

  Although the preferable range of the voltage to be applied differs depending on the type and physical properties of the polymer used, it cannot be generally stated, but is generally in the range of 0.1 to 50 kV, more preferably 1 to 40 kV. The distance between the nozzle and the target depends on the type of polymer used, the physical properties, and the applied voltage, so it cannot be said unconditionally. However, if the distance is too short, discharge will occur, and if it is too long, the jettability will deteriorate. The range is preferably 30 mm to 500 mm. The target is not particularly limited, such as a flat plate shape, a roll shape, an edge shape, or a grid shape, and by placing a hollow fiber membrane mesh between the nozzle and the target, on the hollow fiber membrane mesh or on the hollow fiber membrane. Nanofibers can be formed in the voids of the mesh.

  For example, by placing a hollow fiber membrane mesh on a target and performing electrospinning, by arranging nanofibers on the mesh of the hollow fiber membrane or by increasing the mesh interval of the hollow fiber membrane, Nanofibers can also be placed inside. Alternatively, a plurality of hollow fiber membrane meshes having nanofibers disposed on the surface or voids may be laminated. Furthermore, a hollow fiber membrane whose surface is covered with nanofibers is produced by placing the hollow fiber membrane before meshing between the nozzle and the target and performing electrospinning, and laminating this on the mesh It is also possible to do.

  Using the cell culture scaffold material of the present invention, for example, a cell culture module can be obtained by enclosing it in a liquid-tight manner in a container. Furthermore, by providing the cell culture module with openings in the hollow portions of the hollow fiber membranes at both ends of the hollow fiber membranes and ports connected to the openings, waste products and metabolites discharged from the hollow fiber membranes can be removed from the outside. It can be installed in equipment and can be analyzed online.

  Moreover, in the scaffold for cell culture of the present invention, a hollow fiber membrane that is a gas separation membrane and a hollow fiber membrane that has substance-selective permeability are used as the two types of hollow fiber membranes. While supplying oxygen-containing gas from one end hollow portion, the gas is discharged from the other end hollow portion, and the nutrient of cultured cells is supplied from the one end hollow portion of the hollow fiber membrane having selective permeability, The state of cell growth continuously while culturing cells by discharging waste and metabolites of the cultured cells from the other end hollow part or analyzing these discharged components online Can also be evaluated.

Furthermore, by administering a test substance to the cells cultured in the cell culture module of the present invention, discharging a solution containing waste products and metabolites from one end hollow portion of the hollow fiber membrane, and analyzing and monitoring this solution The stress received by the cells when the test substance is administered can be evaluated over time.
In addition, a test substance is supplied to one end hollow part of the hollow fiber membrane in the cell culture module of the present invention in which cells are cultured, and a solution containing waste products and metabolites is discharged from the other end part. By analyzing and monitoring, the state of the cells can be evaluated.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, it is not limited to these. In addition, the evaluation method and manufacturing apparatus used in the Example are as follows.
<Manufacturing equipment>
For the arrangement of the nanofibers on the mesh-shaped hollow fiber membrane, a NEU nanofiber electrosping unit manufactured by Kato Tech was used. The spinning conditions were such that the peripheral speed of the metal drum as the target was 1 m / min, the applied voltage was 12 kV, and the distance between the nozzle tip and the target was 120 mm.

<Observation of nanofiber>
For observation of the nanofiber, a scanning electron microscope JSM-6060A manufactured by JEOL Ltd. was used.

<Diameter of nanofiber single fiber>
The diameter of the single fiber of the nanofiber was determined by image analysis of SEM photographs using image analysis software (Image-Pro Plus ver. 4.5.0.24, manufactured by Planetron Co., Ltd.). After adjusting the scale of the image analysis to match the scale of the SEM photograph, the width of the surface of any 20 nanofibers in the SEM photograph is obtained, and the average value is the diameter of the nanofiber single fiber. did.

<Observation of cell shape>
The cell shape was observed using a confocal laser scanning microscope LSM5PASCL manufactured by Carl Zeiss.

(Example 1: Production of cell culture scaffold material)
As a hollow fiber membrane, a hydrophilic porous hollow fiber membrane (EX270FS manufactured by Mitsubishi Rayon Engineering) (hollow fiber membrane A) and a three-layer composite hollow fiber membrane having a gas separation function (MHF200TL manufactured by Mitsubishi Rayon Engineering) (hollow fiber) Membrane B) was used.
The hollow fiber membranes A were arranged at intervals of 2 mm, and then the hollow fiber membranes B were arranged at intervals of 2 mm so as to be substantially orthogonal thereto. Next, the hollow fiber membranes A are arranged between the hollow fiber membranes A arranged in advance, and the hollow fiber membranes are arranged also between the hollow fiber membranes B arranged in the same manner. A hollow fiber membrane was placed to prepare a hollow fiber membrane mesh.
At this time, the mesh portion constituted by the hollow fiber membrane A and the hollow fiber membrane B was set to about 20 mm × 20 mm. The produced hollow fiber membrane mesh was fixed on an aluminum foil, and the portion other than the mesh portion was covered with tape so that nanofibers were formed only on the mesh portion, and then fixed to a drum-type target together with the aluminum foil.
As the nanofiber substrate, a polyacrylonitrile polymer having a weight average molecular weight (Mw) of about 350,000 and an acrylonitrile content of about 97% was used. This polyacrylonitrile-based polymer was dissolved in dimethylacetamide so as to be 10 wt% to obtain a spinning dope. This spinning stock solution was filled in a syringe equipped with an 18G nozzle (needle), and electrospinning was performed on the produced hollow fiber membrane mesh to produce a scaffold material for cell culture.
After electrospinning, in order to fix the electrospun hollow fiber membrane mesh, a position about 15 mm away from the periphery of the mesh portion was potted. Polyurethane resin was used for potting. The polyurethane resin used is a mixture of Coronate 4403 and Nippon Run 4223 of Nippon Polyurethane Industry Co., Ltd. at 1/1 (wt ratio), and a mold is provided at a position about 15 mm away from the periphery of the mesh part. The outer hollow fiber membrane part was hardened with potting resin.
1 and 2 show optical micrographs of the obtained cell culture scaffold material. FIG. 1 is an overall view of the fabricated scaffold material, and FIG. 2 is a cross-sectional photograph of the mesh portion.
From these, it is understood that nanofibers are formed on the hollow fiber membrane mesh by electrospinning on the hollow fiber membrane mesh.
FIG. 3 and FIG. 4 are photographs of the obtained cell culture scaffold material observed with a scanning electron microscope, and are a surface and a cross section of the nanofiber, respectively. As a result of image analysis from the photograph of FIG. 3, it can be seen that the diameter of the nanofiber monofilament is about 150 nm and the thickness of the nanofiber layer is 5 to 50 μm from FIG.

(Example 2: Production of cell culture module)
The same hollow fiber membrane A and hollow fiber membrane B as in Example 1 were used. Further, the hollow fiber membranes A were arranged at intervals of 4 mm, and then the hollow fiber membranes B were arranged at intervals of 4 mm so as to be substantially orthogonal thereto. Next, the hollow fiber membranes A are arranged between the hollow fiber membranes A arranged in advance, and the hollow fiber membranes are arranged also between the hollow fiber membranes B arranged in the same manner. A scaffold material for cell culture was prepared in the same manner as in Example 1 except that the hollow fiber membrane was disposed and the mesh portion constituted by the hollow fiber membrane A and the hollow fiber membrane B was about 10 mm × 10 mm.
Next, a mold was made using the same potting resin as in Example 1 with a width of about 5 mm outward from a position of about 1 mm from the periphery of the hollow fiber membrane mesh portion. Further, the tip of the hollow fiber membrane was bundled using a silicon tube, and the outside of the hollow fiber membrane bundled with the inside of the silicon tube was hardened with the same potting resin as described above to produce a port portion (FIG. 5).

(Example 3: Culture of bovine vascular endothelial cells)
A scaffold material for cell culture was produced in the same manner as in Example 2 except that the hollow fiber membrane mesh composed of the hollow fiber membrane A and the hollow fiber membrane B was 100 mm × 100 mm.
The cell culture scaffold material mesh portion cut into a circle with a diameter of 21 mm is fixed to a circular cover glass (Matsunami Glass Industry Co., Ltd., φ21 mm) using the potting urethane resin used in Example 1. Then, this was placed on the bottom of the well of a 6-well cell culture plate to perform cell culture. At that time, as a comparison, cell culture was carried out using only the hollow fiber membrane mesh by removing the nanofibers from the cell culture scaffold material prepared above.
Dulbecco's modified Eagle medium (DMEM) used for cell culture was prepared using a DMEM reagent manufactured by Nippon Pharmaceutical.
Bovine vascular endothelial cells (obtained from RIKEN Cell Bank) were seeded at 0.5 × 10 ⁶ cells / well in each well of the cell culture plate on which the cover glass was placed, and were incubated at 37 ° C., 5% CO _{2 in} DMEM medium. Cultured under conditions.
After culturing for one week, the cover glass was removed from the cell culture plate and observed with a confocal laser scanning microscope.
6 and 7 are photographs of cells adhered to the cell culture scaffold material observed with a confocal laser scanning microscope, FIG. 6 is a cell culture scaffold material with nanofibers, and FIG. 7 is a cell with nanofibers removed. It is a photograph with a culture scaffold material.
FIG. 6 shows that bovine vascular endothelial cells spread over the entire surface of the cell culture scaffold material in the cell culture scaffold material with nanofibers. This shows that the cell culture scaffold material is a material suitable for cell culture. In addition, it is advantageous that cells adhere to a part of the hollow fiber when nutrients and oxygen are supplied from the hollow fiber membrane.
It can be seen from FIG. 7 that the bovine vascular endothelial cells are only adhered to a part of the hollow fiber membrane in the cell culture scaffold material from which the nanofibers are removed. This indicates that it is effective to have nanofibers to adhere many cells to the cell culture scaffold material.

  According to the present invention, a medicinal effect test, a toxicity test, and a safety test of substances such as pharmaceuticals, chemical substances, and cosmetics can be easily and rapidly performed.

Optical micrograph of the cell culture scaffold material obtained in the example Optical micrograph of the cell culture scaffold material obtained in the example Scanning electron micrograph of the scaffold material for cell culture obtained in the examples Scanning electron micrograph of the scaffold material for cell culture obtained in the examples Photograph of module for cell culture having port part obtained in Example 2 Confocal micrograph of the cells adhered to the cell culture scaffold obtained in Example 3 Confocal micrograph of the cells adhered to the cell culture scaffold obtained in Example 3

Claims (10)

A cell culture scaffold material in which hollow fiber membranes are arranged in a mesh shape, and nanofibers having a single fiber diameter of 1 to 1000 nm are arranged on the outer surface of the hollow fiber membrane and / or between the hollow fiber membranes. The cell culture scaffold material according to claim 1, wherein two or more types of hollow fiber membranes are disposed. The cell culture scaffold material according to claim 1 or 2, wherein a hollow fiber membrane having substance-selective permeability is disposed. The cell culture scaffold material according to any one of claims 1 to 3, wherein a hollow fiber membrane that is a gas separation membrane is disposed. The cell culture scaffold material according to any one of claims 1 to 4, wherein a hollow fiber membrane subjected to hydrophilic treatment is disposed. The cell culture scaffold material according to claim 3, wherein the hollow fiber membrane having selective substance permeability has a function of feeding cultured cells and a function of removing waste products and metabolites from the cultured cells. The cell culture scaffold material according to claim 4, wherein the gas separation membrane has a function of supplying oxygen-containing gas to the cultured cells. The manufacturing method of the cell culture scaffold material of Claim 1 which arrange | positions the hollow fiber membrane arrange | positioned in mesh shape between a nozzle and a target, and electrospins a nanofiber. A module for cell culture using the cell culture scaffold material according to claim 1. The module for cell culture of Claim 9 which has the opening part of the hollow part of hollow fiber membrane of a hollow fiber membrane both ends, and the port part connected with this opening part.