JP2011239756A - Cell culture module - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture module which can: perform three-dimensional cell culture; supply nutrients and air (oxygen) to the cultured cells and remove metabolic waste matters from the cultured cells with high efficiency, while easily controlling the supply and the removal with more than three conditions independently without using a special device; and downsize the module.SOLUTION: The cell culture module 1 includes: a cell culture scaffold material 10 having hollow fiber membrane meshes and a nanofiber layer disposed fluid-tightly between a lower bottom 20 and an upper cover 30; and cell injection ports 21a, 22a, 31a and 32a and openings 21b, 22b, 31b and 32b covered by sheets at the position where the lower bottom 20 and the upper cover 30 correspond to the nanofiber layer.

Description

  The present invention relates to a cell culture module.

  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 differences between humans and animal species, and individual differences between animals, and many experiments have to be repeated from the viewpoint of data reliability. For this reason, there are problems such as requiring a lot of expenses and test time, and it takes time and effort to secure a large number of animals to be tested and a place to breed them. 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. More recently, animal welfare has been increasing in animal experiments. Therefore, as a solution to such a problem, 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). In this method, a culture solution and oxygen are supplied to a cell culture space using a hollow fiber membrane having selective permeability and a hollow fiber membrane having gas permeability. Thereby, the efficiency of cell culture can be increased, which is suitable for production of useful substances. However, in this method, since cells exist in the culture liquid, it is difficult for the cultured cells to form a tissue, and it is difficult to apply the method to alternative animal experiments.

  As a technique for culturing animal cells, particularly adherent animal cells at a high density, a cell culture vessel using a hollow fiber having a semipermeable membrane function and a fibrous cell support is shown (Patent Document 2). However, this cell culture device is not preferable for monitoring cell metabolites and the like because the hollow fiber used is a semipermeable membrane that does not permeate the proteinaceous substance produced by the cells. In addition, since the fibrous cell support is fixed to the cell culture container and disposed in the gap between the hollow fibers, nutrients can be supplied from the hollow fibers to the cultured cells, and metabolic waste products can be recovered from the cultured cells. The efficiency was low.

  In addition, in order to promote the formation of cell tissue bodies, the inner or outer cavity of the hollow fiber is filled with cells at high density using centrifugal force, and the supply of oxygen and nutrients, metabolic waste products using the hollow fiber A method for removing the film is shown (Patent Document 3). However, in this method, since the filled cells and the hollow fiber are in direct contact with each other, the hollow fiber surface may be blocked by cell adsorption, which may result in insufficient supply of oxygen and nutrients and removal of metabolic waste products. It was.

  In addition, there is 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 together with nutrients outside the hollow fiber, and monitoring the metabolism and rate of metabolism of the test substance by the cell. (Patent Document 4). However, in this method, since the filled cells and the hollow fiber are in direct contact with each other, the hollow fiber surface may be blocked by cell adsorption, which may result in insufficient supply of oxygen and nutrients and removal of metabolic waste products. It was.

  In addition, 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 is disclosed (Patent Document 5). However, in this cell culture module, the porous carrier is only 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. For this reason, cell secretions are easy to retain, but nutritional supplementation efficiency is poor, and removal of metabolic waste products is quite insufficient.

  In recent years, nanofibers having nano-order fiber diameters are attracting attention as a scaffold for cell culture and tissue formation because they can highly mimic the fibrous structure of tissue in vivo (Non-patent Document 1). Non-Patent Document 2). For example, a method of using nanofibers produced by melt spinning as a scaffold material is shown (Patent Document 6). In addition, a nanofiber pattern is formed using a mixture of organic nanofibers dispersed in a solvent, and a functional drug effective for cell culture is adsorbed to the pattern, thereby allowing cell adhesion, proliferation, and differentiation. A method for controlling the above is disclosed (Patent Document 7).

Japanese Patent No. 3026516 JP 2002-112763 A Japanese Patent No. 3725147 JP 2000-189189 A JP 2003-180334 A JP 2006-254722 A JP 2007-44149 A

Polymer, 55, 3, 142 (2006) Tissue Eng. , 11, 101 (2005)

  If the nanofiber-based scaffold material can supply nutrients and air (oxygen) to cultured cells and recover metabolic waste products from cultured cells with high efficiency, it is expected to be an excellent alternative to animal experiments. Is done. However, in the methods of Patent Documents 6 and 7, supplementation of nutrients and air (oxygen) necessary for cell growth has not been considered, and it has been difficult to efficiently culture cells.

In addition, in the scaffold material using nanofibers, even when a hollow fiber membrane is used, when the three or more types of components are to be supplied uniformly to the cultured cells via the hollow fiber membrane, the arrangement of the hollow fiber membrane is It will be complicated. Therefore, it is considered difficult to supply three or more kinds of components and control each condition sequentially.
Furthermore, since the conventionally used cell culture module is used in a state where the cell culture scaffold material to be used is immersed in a culture solution in a container, a large amount of culture space is required especially in the case of simultaneous culturing of multiple samples and other conditions. Was necessary. For this reason, it has been desired to make the module compact.

  Therefore, in the present invention, three-dimensional cell culture is possible, and nutrition, air (oxygen) supply to the cultured cells, and removal of metabolic waste products from the cultured cells can be performed with high efficiency. It is an object of the present invention to provide a module for cell culture that can be easily supplied and removed under three or more conditions and independently controlling those conditions without making use of the module, and the module can be made compact.

  The cell culture module of the present invention is a cell culture scaffold material having a hollow fiber membrane mesh in which hollow fiber membranes are arranged in a mesh shape, and a nanofiber layer made of an assembly of nanofibers in contact with the hollow fiber membrane mesh. Is arranged in a liquid-tight manner between two covers, and a cell inlet for injecting cultured cells and a opening other than the cell inlet are formed at a position corresponding to the nanofiber layer in the cover, In the module, the cell inlet and the opening are closed with a sheet.

  The cell culturing module of the present invention can perform three-dimensional cell culture, and can efficiently supply nutrients and air (oxygen) to the cultured cells and remove metabolic waste products from the cultured cells. Moreover, even if it does not use a special apparatus, the said supply and removal can be performed easily by controlling these conditions independently by 3 or more types of conditions. Further, the module can be made compact.

It is the figure which showed one example of embodiment of the module for cell cultures of this invention. (A) Top view, (B) Side view, (C) Exploded view of upper lid and lower bottom. It is a top view which shows a mode (B) which provided the bottom frame (A) used for preparation of the cell culture scaffold material of this embodiment, and the square frame in this bottom frame. It is the figure which showed the cell culture scaffold material of this embodiment. 4A is a plan view, FIG. 3B is a cross-sectional view taken along line A-A ′ in FIG. 4A, and FIG. 4C is a cross-sectional view taken along line B-B ′ in FIG. It is the figure which expanded the cell culture scaffold material of this embodiment. (A) The enlarged view to which a part of cell culture scaffold material of FIG. 3 (A) was expanded. (B) The enlarged view which expanded a part of cell culture scaffold material of FIG.3 (C). It is process drawing which showed a mode that the cell culture scaffold material of FIG. 3 was produced. (A) It is a top view which shows a mode that the upper frame was installed in the scaffold material 10 of FIG.5 (C). (B), (C) It is process drawing which bundles and fixes the outer side part of the formwork 40 of a hollow fiber membrane. 2 is a SEM photograph of a nanofiber layer 15A in Example 1. (A) Layer cross section (observation magnification 5000 times), (B) Layer surface (observation magnification 10000 times). 2 is a photograph of cell culture scaffold material 10A in Example 1. FIG. 4 is a SEM photograph of a nanofiber layer 15B in Example 2. (A) Layer cross section (observation magnification 2000 times), (B) Layer surface (observation magnification 10000 times). 4 is a graph showing changes in cell density in the culture of Example 3. It is the microscope picture which observed the cell after the culture | cultivation of Example 3 with the phase-contrast microscope (observation magnification of 32 times). It is the microscope picture which observed the cell after the culture | cultivation of Example 3 with the phase-contrast microscope (observation magnification 56 times). 6 is a graph showing changes in cell density in the culture of Comparative Example 1. It is the microscope picture which observed the cell after the culture | cultivation of the comparative example 1 with the phase-contrast microscope (observation magnification 56 times). It is the microscope picture which observed the cell after the culture | cultivation of the comparative example 1 with the confocal laser scanning microscope (observation magnification 200 times).

[Cell culture module]
The cell culture module of the present invention is a cell culture scaffold material having a hollow fiber membrane mesh in which hollow fiber membranes are arranged in a mesh shape, and a nanofiber layer made of an assembly of nanofibers in contact with the hollow fiber membrane mesh. Are disposed in a liquid-tight manner between the two covers. In the cell culture module of the present invention, a cell inlet for injecting cultured cells and an opening other than the cell inlet are formed at a position corresponding to the nanofiber layer in the cover, and the cell inlet And the opening is closed with a sheet. Hereinafter, an exemplary embodiment of the module for cell culture of the present invention will be described in detail.

  As shown in FIGS. 1 (A) and 1 (B), the cell culture module 1 (hereinafter referred to as “module 1”) of the present embodiment has a cell culture scaffold material 10 (hereinafter “scaffold material 10”). Is fixed to the mold 40 with potting resin, and is sandwiched between the lower base 20 and the upper lid 30 which are two covers, and the scaffold material 10 is sealed in a liquid-tight manner.

As shown in FIG. 1C, the lower bottom 20 has a sheet 23 disposed between the plates 21 and 22. In the flat plates 21 and 22, cell injection ports 21a and 22a and openings 21b and 22b are formed. The shape of the flat plates 21 and 22 may be a shape that matches the shape of the scaffold material 10.
The flat plates 21 and 22 may have any strength suitable for firmly fixing the scaffold material 10 therebetween, and examples thereof include acrylic plates.
The sheet 23 may be any sheet as long as it can seal the cell injection ports 21a and 22a and the openings 21b and 22b and can connect a microscope, a tube, etc., and insert a needle as necessary. A sheet made of silicon rubber is exemplified.

The cell injection ports 21a and 22a are openings for injecting cultured cells. For example, the cultured cells can be injected into the scaffold material 10 by inserting an injection needle into the sheet 23 at the cell injection ports 21a and 22a.
The shapes of the cell injection ports 21a and 22a are not particularly limited, and a circular shape is preferable from the viewpoint of ease of drilling.
The size of the cell injection ports 21a and 22a is not particularly limited as long as each is formed independently of the openings 21b and 22b and can inject cultured cells. For example, when the shapes of the cell injection ports 21a and 22a are circular, the diameter is preferably 500 μm or more and 5 mm or less.

The openings 21b and 22b are openings formed independently of the cell injection ports 21a and 22a. The openings 21b and 22b can be used, for example, for feeding nutrients or air by connecting tubes, removing metabolic waste products, or observing the culture state by connecting a microscope.
The shape of the openings 21b and 22b is not particularly limited, and a circular shape is preferable from the viewpoint of ease of drilling.
Moreover, the magnitude | size of opening 21b, 22b is not specifically limited, What is necessary is just the range in which each is formed independently of cell injection hole 21a, 22a. For example, when the shapes of the openings 21b and 22b are circular, the diameter thereof depends on the size of the module 1, but is preferably 1 mm or more and the short side of the nanofiber layer as the upper limit.

As shown in FIG. 1C, the upper lid 30 has a sheet 33 disposed between the flat plates 31 and 32. In the flat plates 31 and 32, cell injection ports 31a and 32a and openings 31b and 32b are formed.
The flat plates 31 and 32 and the sheet 33 can be the same as the flat plates 21 and 22 and the sheet 23.
The shapes and sizes of the cell injection ports 21a and 22a and the cell injection ports 31a and 32a may be the same or different, but are preferably the same. The shapes and sizes of the openings 21b and 22b and the openings 31b and 32b may be the same or different, but are preferably the same.

  In this embodiment, the scaffold material 10 is fixed to the mold 40 by potting resin. Since the scaffold material 10 is fixed to the mold 40 with the potting resin, the side portions of the scaffold material 10 in the module 1 are sealed by the potting resin and the mold 40, and the scaffold material 10 is liquid in the module 1. It is tightly enclosed. Further, the use of the mold 40 facilitates the production of the scaffold material 10.

As shown in FIGS. 1B and 2A and 2B, the mold frame 40 includes a bottom frame 41 having an opening 41a, square frames 42 provided at four corners of the bottom frame 41, and an upper frame. 44.
The material of the bottom frame 41 and the upper frame 44 may be any material as long as sufficient adhesion to the potting resin is obtained, and examples thereof include silicon rubber.
The material of the square frame 42 is preferably silicon rubber. If the material of the square frame 42 is silicon rubber, even if a high pressure is applied when the scaffold material 10 fixed to the mold frame 40 is sandwiched between the lower base 20 and the upper lid 30 and then fixed with clips or the like. It is easy to suppress the module 1 from being damaged.

  The potting resin in the present invention is not particularly limited as long as it has sufficient adhesiveness, and examples thereof include various thermosetting resins and thermoplastic resins such as epoxy resins, urethane resins, unsaturated polyester resins, and silicon resins. .

The cell culture scaffold material in the present invention is preferably the scaffold material 10 illustrated in FIG. However, it is not limited to this.
As shown in FIGS. 3 (A) to 3 (C), the scaffold material 10 includes a hollow fiber membrane mesh 12 in which hollow fiber membranes 11a and 11b (11) are arranged in a mesh shape, and a nano-fiber assembly. The fiber layer 15 and the hollow fiber membrane mesh 14 in which the hollow fiber membranes 13a and 13b (13) are arranged in a mesh shape are laminated in this order.

  The membrane base polymer of the hollow fiber membrane 11 that forms the hollow fiber membrane mesh 12 may be any polymer that can be molded on the hollow fiber membrane, for example, polyolefins such as polyethylene and polypropylene, polysulfone, polyallylsulfone, and polyether. Examples thereof include sulfone, polyacrylonitrile, cellulose acetate, polyvinylidene fluoride, and a mixture of two or more of these.

  The outer diameter of the hollow fiber membrane 11 is preferably 50 to 3000 μm, and more preferably 100 to 2000 μm. If the outer diameter is 50 μm or more, it is easy to secure a sufficiently large hollow portion in the hollow fiber membrane 11, so that the differential pressure when feeding the culture solution becomes large, and removal of metabolic waste products is insufficient. It is easy to suppress becoming. In addition, when the outer diameter is 3000 μm or less, the surface area of the hollow fiber membrane 11 per unit volume can be increased, so that it becomes easy to supply sufficient nutrients and gas to the cultured cells.

The film thickness of the hollow fiber membrane 11 is preferably 5 to 500 μm. If the film thickness is 5 μm or more, it is easy to prevent the hollow fiber membrane from being destroyed by pressurization during nutrient supply or gas supply. In addition, when the film thickness is 500 μm or less, the filtration resistance is further reduced, so that the efficiency of nutrient supply and gas supply to the cultured cells and removal of metabolic waste products from the cultured cells is improved.
In addition, the film thickness in case the hollow fiber membrane 11 is a multilayer film is the sum total of the film thickness of each layer (film thickness of all layers).
The inner diameter of the hollow fiber membrane 11 is preferably 10 μm or more and 2995 μm or less.

As the hollow fiber membrane 11, a microfiltration membrane, an ultrafiltration membrane, a gas separation membrane, or the like can be used. These can be appropriately selected as necessary.
By using a hollow fiber membrane having a substance-selective permeability, a specific substance can be supplied to cultured cells, or a specific metabolic waste product can be removed from the cultured cells. In addition, it becomes possible to efficiently supply test substances such as specific components, chemical substances, drugs, etc. in the culture medium to the cultured cells, and to discharge waste products and metabolites out of the system and monitor them. .
Examples of the hollow fiber membrane having selective substance permeability include the above-mentioned microfiltration membrane and ultrafiltration membrane, and further, the hollow fiber membrane having substance selective permeability in the membrane material itself and the selective permeability in the hollow part. A porous hollow fiber membrane filled with a substance having

  Moreover, by using a gas separation membrane, air, oxygen, and other gases can be efficiently and selectively supplied to the cultured cells. The gas supply may be performed using a porous hollow fiber membrane. In this case, a hollow fiber membrane in which a nonporous membrane substrate having a gas separation function is supported by a porous membrane substrate is preferable. By using such a hollow fiber membrane having a gas separation function, gas can be supplied into the culture solution without bubble, and thus it is easy to prevent damage to the cultured cells. In addition, it is easy to prevent a liquid such as a culture solution from entering the hollow portion of the hollow fiber membrane and closing the hollow portion.

The hollow fiber membrane 11 is preferably subjected to a hydrophilic treatment. By hydrophilizing the hollow fiber membrane, it becomes easy to supply a liquid component such as a culture solution to the cultured cells, and it is also easy to suppress cell adhesion to the surface of the hollow fiber membrane. Examples of the method for hydrophilizing the hollow fiber membrane include a method of 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 be used for the hollow fiber membrane mesh 12. For example, microfiltration membranes that supply nutrients, gas separation membranes that supply gases, hollow fibers with selective permeability to substances that supply specific components to cultured cells or discharge specific metabolic waste products from cultured cells Membranes can be used in any combination.

The hollow fiber membrane mesh 12 is a plurality of hollow fiber membranes arranged in a mesh shape so that a mesh is formed. As a form of the hollow fiber membrane mesh 12, for example, a plurality of hollow fiber membranes 11a (hollow fiber membranes 11) are arranged in parallel at a predetermined interval to form a sheet, and in a direction orthogonal to the hollow fiber membranes 11a. A plurality of hollow fiber membranes 11b (hollow fiber membranes 11) are arranged in parallel at a predetermined interval to form a sheet shape, thereby forming a mesh shape (FIG. 3A).
The distance d ₁ (FIG. 4A) between the hollow fiber membranes 11a is preferably 0.05 to 5 mm, and more preferably 0.05 to 1 mm. The distance d ₁ is the equal to or greater than the outer diameter of the hollow fiber membrane 11a, as long as 5mm or less, the removal of the supply and metabolic waste of nutrients and gas can be performed uniformly throughout the cells adhered to the nanofiber layer 15, the cell The efficiency of culture is improved. For the same reason, the distance d ₂ (FIG. 4A) between the hollow fiber membranes 11b is preferably 0.05 to 5 mm, and more preferably 0.05 to 1 mm.
The number of sheets of the hollow fiber membrane 11 in the hollow fiber membrane mesh 12 is not particularly limited, and 2 to 10 layers are preferable. In this example, the sheet has two layers (a layer made of the hollow fiber membrane 11a and a layer made of the hollow fiber membrane 11b).

When forming a hollow fiber membrane mesh with two or more types of hollow fiber membranes, different types of hollow fiber membranes may be mixed in the same sheet, and different types of hollow fiber membranes should be used for each sheet. Also good. That is, in the case of the present embodiment, as the aspect (1) in which two or more types of hollow fiber membranes having different types are used as the hollow fiber membrane 11a and two or more types of hollow fiber membranes having different types are used as the hollow fiber membrane 11b. It is also possible to adopt an embodiment (2) in which the same type of hollow fiber membrane is used as the hollow fiber membrane 11a, and the same type of hollow fiber membrane different from the hollow fiber membrane 11a is used as the hollow fiber membrane 11b. Alternatively, an embodiment (3) may be employed in which two or more different types of hollow fiber membranes are used for one of the hollow fiber membranes 11b and the same type of hollow fiber membrane is used for the other. When two or more types of hollow fiber membranes 11 are used in the hollow fiber membrane mesh 12, the same type of hollow fiber membranes are gathered to provide a port portion, and the port portion is connected to an external device via the hollow fiber membrane. The aspect (2) is preferable because it is easy to analyze the discharged waste products and the like online.
Specifically, for example, by using a microfiltration membrane as the hollow fiber membrane 11a and a gas separation membrane as the hollow fiber membrane 11b, nutrients are supplied to the cultured cells by the hollow fiber membrane 11a, and to the cultured cells by the hollow fiber membrane 11b. Gas can be supplied (aspect (2)).
The positions of the hollow fiber membranes 11a and 11b in the hollow fiber membrane mesh 12 can be fixed by fixing a portion other than the mesh-shaped portion with a potting resin. The aforementioned potting resin can be used as the potting resin.

The hollow fiber membrane mesh 14 and the hollow fiber membrane 13 in the present invention can be the same as the hollow fiber membrane mesh 12 and the hollow fiber membrane 11, and the preferred forms are also the same.
In the scaffold material 10, the form of the hollow fiber membrane meshes 12, 14 may be selected according to the supply of nutrients and gas through the hollow fiber membranes and the removal of metabolic waste, and when the scaffold material 10 is modularized. Depends on port design.
In the scaffold material 10, the hollow fiber membrane 11a of the hollow fiber membrane mesh 12 and the hollow fiber membrane 13a of the hollow fiber membrane mesh 14 are preferably the same type of hollow fiber membrane. Similarly, the hollow fiber membrane 11b of the hollow fiber membrane mesh 12 and the hollow fiber membrane 13b of the hollow fiber membrane mesh 14 are preferably the same type of hollow fiber membrane. By adopting such a form, when the scaffold material 10 is modularized, the hollow fiber membrane 11a and the hollow fiber membrane 13a, and the hollow fiber membrane 11b and the hollow fiber membrane 13b are gathered together in the same port portion. By linking, it becomes easy to perform cell culture while analyzing on-line wastes discharged through the hollow fiber membrane.

In the scaffold material 10 of the present embodiment, as shown in FIGS. 3B and 3C, the hollow fiber membranes 11 and 13 parallel to each other in the hollow fiber membrane mesh 12 and the hollow fiber membrane mesh 14 are each ½. It is preferable that the pitches are shifted from each other. Thereby, since the hollow fiber membrane can be uniformly arranged at a higher density with respect to the nanofiber layer 15 in the scaffold material 10, the efficiency of cell culture is improved.
The distance d ₃ (FIG. 4B) between the hollow fiber membrane 11b and the hollow fiber membrane 13b in the hollow fiber membrane mesh 12 and the hollow fiber membrane mesh 14 is preferably 0.5 to 1000 μm, more preferably 1.0 to 50 μm. . If the distance d ₃ is at 0.5μm or more 1000μm or less, the removal of the supply and metabolic waste products nutrition and gas for performed uniformly throughout the cells adhered to the nanofiber layer 15, thereby improving the efficiency of the cell culture.

The nanofiber layer 15 of the present embodiment is a layer made of an assembly of nanofibers.
The nanofiber layer 15 is preferably formed by electrospinning (electrospinning) on a substrate. The base material is not particularly limited as long as the nanofiber layer 15 can be formed on the surface by electrospinning, and examples thereof include aluminum foil.

  The nanofiber material is preferably a solvent-soluble polymer from the viewpoint of easy spinning. Particularly, solvent-soluble polymers that can be electrospun include, for example, nylon 4, 6, nylon 6, nylon 6, 6, nylon 12, polyacrylic acid, polyacrylonitrile, polyamide, polycarbonate, polyetherimide, polyethylene oxide, polyethylene Examples include terephthalate, polystyrene, styrene-butadiene copolymer, polysulfone, polyurethane, polyvinyl alcohol, polyvinyl chloride, polyvinyl pyrrolidone, polyvinylidene fluoride, polycaprolactone, polylactic acid, silk, cellulose acetate, chitosan, and collagen. However, the present invention is not limited to these, and a mixture of these polymers, an inorganic material, or a carbon material may be included.

The molecular weight of the polymer 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 may increase and spinning may be difficult.
Even if the polymer is insoluble in a solvent, a thermoplastic polymer can be used when the laser electrospinning method is applied. Examples of the thermoplastic polymer applicable to the laser electrospinning method include polyethylene terephthalate.

The diameter of the nanofiber (single fiber) is preferably 1 nm to 1000 nm, more preferably 10 nm to 500 nm, and still more preferably 20 nm to 300 nm. If the said diameter is 1 nm or more, it will become a nanofiber which has sufficient intensity | strength easily. Moreover, if the said diameter is 1000 nm or less, it will become a nanofiber which has sufficient specific surface area easily. One of the characteristics of the nanofiber is the size of the specific surface area, and the specific surface area increases exponentially as the diameter of the single fiber decreases, and the adsorption characteristics increase. Therefore, it is suitable as a scaffold material for cell culture.
The diameter of the nanofiber single fiber can be measured as follows. The surface of the nanofiber or its aggregate (nanofiber layer 15) is observed with an electron microscope, and the width of the surface of any 20 nanofibers in the obtained electron micrograph (FIG. 9B) is measured. The average is defined as the diameter of a single nanofiber. As the electron microscope, a scanning electron microscope (SEM) is preferable. The observation magnification is preferably 5000 to 50,000 times. If the observation magnification is 5000 times or more, the diameter of the nanofiber can be easily determined. Moreover, when calculating | requiring the diameter of a single fiber, it is preferable to use image analysis software. The diameter of the nanofiber 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.

  The thickness of the nanofiber layer 15 is preferably 0.5 to 100 μm, and more preferably 1 to 50 μm. If thickness is 0.5 micrometer or more, it will become easy to arrange | position the nanofiber layer 15 between hollow fiber membrane meshes. If the thickness is 100 μm or less, the supply of nutrients and gas and the removal of metabolic waste products can be performed uniformly on the entire cells adhered to the nanofiber layer 15, thereby improving the efficiency of cell culture.

In the present embodiment, adhesion, proliferation, differentiation, and function expression of cultured cells can be controlled by adsorbing or fixing a physiologically active substance to the nanofiber layer 15.
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 physiologically active substances having an important role in the growth, differentiation and functional expression of cultured cells. Cytokines include interleukins, blood boosting factors, growth factors and the like. It is also effective to use cell adhesion substrates such as integrins and cadherins involved in cell-extracellular matrix, cell-cell adhesion. 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 layer 15.

The scaffold material 10 of this embodiment has a laminate in which the nanofiber layer 15 is disposed between the hollow fiber membrane mesh 12 and the hollow fiber membrane mesh 14 described above. As described above, by arranging the nanofiber layer between the hollow fiber membrane meshes, the nanofibers can be firmly fixed to the hollow fiber membrane meshes, supplying nutrients and gases to the cultured cells, Removal of metabolic waste products can be performed more stably.
Compared to the case where a nanofiber layer is formed on a hollow fiber membrane mesh and cultured cells are adhered on the nanofiber layer, the nanofiber layer is disposed between the hollow fiber membrane meshes to adhere to the nanofiber layer. Since the distance between the cultured cells and the hollow fiber membranes is reduced, it is possible to supply nutrients and gas to the cultured cells and remove metabolic waste products from the cell culture with higher efficiency. Decreases and the efficiency of cell culture improves.

In the cell culture scaffold material in the present invention, the number of layers of the hollow fiber membrane mesh and the nanofiber layer is not particularly limited, and the hollow fiber membrane mesh, the nanofiber layer, the hollow fiber membrane mesh, the nanofiber layer, and the hollow fiber membrane mesh are not limited thereto. The cell culture scaffold material laminated | stacked in order may be sufficient, and the cell culture scaffold material laminated | stacked more than this may be sufficient.
Further, the form of the mesh portion of the hollow fiber membrane mesh is not limited to the form in which the hollow fiber membrane 11a (13a) and the hollow fiber membrane 11b (13b) in the scaffold material 10 are orthogonal to each other. For example, the hollow fiber membrane 11a (13a) and the hollow fiber membrane 11b (13b) can be used as long as the efficiency of supplying nutrients and gases and discharging metabolic waste products to the cultured cells adhered to the nanofiber layer is not too low. ) May not be orthogonal to each other. Also, the diagonal direction between the vertical direction and the vertical direction is α (0 ° <α <90 °) and β (−90 ° <β <0 °) (for example, ± 60 ° with respect to the vertical direction). The hollow fiber membrane mesh may be meshed by arranging the hollow fiber membranes in parallel in three directions (two directions). In this case, it is preferable that the nanofiber layer has a hexagonal shape corresponding to the shape. Moreover, it is preferable to use a hexagonal form for fixing the scaffold material.

Hereinafter, the form of the scaffold material 10 fixed to the mold 40 in the present embodiment will be described.
The hollow fiber membranes 13a are arranged at regular intervals in the frame portion 43 of the bottom frame 41, and further the hollow fiber membranes 13b are arranged along the direction orthogonal to the hollow fiber membranes 13a to form the hollow fiber membrane mesh 14. (FIG. 5A). Further, the nanofiber layer 15 is arranged on the hollow fiber membrane mesh 14, and the four corners of the nanofiber layer 15 are fixed to the square frame 42 by a potting resin (FIG. 5B).

  Moreover, the hollow fiber membrane 11b is arrange | positioned on the nanofiber layer 15, and also the hollow fiber membrane 11a is arrange | positioned along the direction orthogonal to the hollow fiber membrane 11b, and the hollow fiber membrane mesh 12 is formed (FIG. 5 ( C)), these hollow fiber membranes 11b and 11a and the previously disposed hollow fiber membranes 13a and 13b are fixed to the bottom frame 41 with a potting resin. On the hollow fiber membrane mesh 12, an upper frame 44 is fixed with a potting resin (FIG. 6A).

  Since it becomes easy to provide the port part of the scaffold material 10, it is preferable that the outer part of the mold 40 between the hollow fiber membranes connected to the same port part is bundled and fixed. For example, a portion of the hollow fiber membrane outside the mold 40 is bundled and tied through the silicon tube 50, and potting resin is filled and fixed in the silicon tube 50 (FIG. 6B). Thereafter, the hollow fiber membrane portion from the mold 40 to the silicon tube 50 is covered and fixed with a potting resin, and the portion ahead of the silicon tube 50 is cut and removed (FIG. 6C).

  The module 1 of the present invention is configured to supply nutrients and gas and remove metabolic waste products by connecting tubes, for example, to the openings 21b and 22b of the lower base 20 and the openings 31b and 32b of the upper lid 30. As a third path other than the hollow fiber membranes 11 and 13 of the hollow fiber membrane meshes 12 and 14 can be provided. Therefore, compared with the case of supplying nutrients and gas using only the hollow fiber membrane or removing metabolic waste products, cell culture under more conditions and independently controlling those conditions, It can be performed more simply. In addition, the nanofiber layer 20 of the scaffold material 10 can be easily formed even when a tube, a microscope, or the like is connected to the opening by the cell inlets 21a and 22a in the lower base 20 and the cell inlets 31a and 32a in the upper lid 30. The cultured cells can be injected into and adhered. In addition, since the module 1 uses the lower base 20 and the upper lid 30 (cover) to enclose the scaffold material 1 in a liquid-tight manner, the module 1 is compared with a mode in which the scaffold material 10 is immersed in the culture solution in the container. It can be made compact.

Examples of the usage method of the module 1 include the following usage methods.
In module 1, the hollow fiber membrane mesh with both ends of the hollow fiber membrane is connected to the port, and the waste and metabolites discharged from the hollow fiber membrane are introduced to external equipment. Analyzing with is possible.
For example, in the module 1, when the hollow fiber membranes 11a and 13a are hollow fiber membranes which are gas separation membranes, and the hollow fiber membranes 11b and 13b are hollow fiber membranes having a material selective permeability, the hollow fiber membranes 11a and 13a While supplying oxygen-containing gas from one end, the gas is discharged from the other end, and nutrients of the cultured cells are supplied from one end of the hollow fiber membranes 11b and 13b, and the cultured cells are aged from the other end. Products and metabolites can be excreted. Thus, by analyzing the discharged components online, it is possible to continuously evaluate the growth state of the cultured cells while culturing the cultured cells. Further, a tube or the like is connected to the openings 21b and 22b of the lower base 20 and the openings 31b and 31b of the upper lid 30, and components and conditions other than the hollow fiber membranes 11a and 13a are connected to the cultured cells from the tubes. Nutrition can also be supplied.

  In addition, a test substance is administered to the cultured cells cultured in the module 1 from one end of the hollow fiber membranes 11b and 13b, and a solution containing waste products and metabolites is discharged from the other end of the hollow fiber membranes 11b and 13b. By analyzing and monitoring this solution, it is possible to evaluate the stress received by the cultured cells when the test substance is administered and the state of the cultured cells over time. Also in this case, administration of the test substance and discharge of the solution containing waste products and metabolites may be performed by tubes connected to the openings 21b and 22b of the lower base 20 and the openings 31b and 32b of the upper lid 30.

  In the cell culture module of the present invention described above, a cell culture scaffold material is placed between two covers (lower base 20 and upper lid 30) and sealed in a liquid-tight manner so that the culture area is made constant. Since it is fractionated, it is easy to control the culture conditions. In addition, the module can be made more compact than a module in which the cell culture scaffold material is immersed in the culture solution in the container. Therefore, even when multiple samples are arranged side by side, multiple samples and multiple conditions can be cultured simultaneously. The culture space can be further reduced.

  The cover of the module for cell culture of the present invention has a cell inlet and an opening other than the cell inlet, and the cell inlet and the opening are closed with a sheet. Therefore, a new path other than the hollow fiber membrane of the hollow fiber membrane mesh can be provided by connecting a tube or the like to the opening of the cover. This increases the number of pathways to cultured cells, so it is possible to increase the conditions for supplying nutrients and gases and removing metabolic waste products with a simpler method, and to perform cell culture with independent control of those conditions. it can. In addition, since the cell injection port for injecting cultured cells is provided independently, even when a tube or a microscope is connected to the opening after the module is manufactured, the cultured cells can be easily added to the cell culture scaffold material in the module. Can be injected and glued.

  In addition, in normal culture using a cell culture dish or plate, only planar cell culture on the cell culture dish or plate can be performed, whereas the cell culture module of the present invention is a cell having a nanofiber layer. Since the culture scaffold material is provided, the cultured cells can enter the nanofiber layer and adhere and proliferate three-dimensionally. Therefore, the cultured cell form is close to that in the living body, and a cultured cell with higher reliability of evaluation can be obtained in drug efficacy tests, toxicity tests, safety tests, etc. of substances such as pharmaceuticals, chemical substances and cosmetics.

The cell culture module of the present invention is not limited to the module 1 illustrated in FIG. For example, a module may be formed without using the above-described mold as long as the cell culture scaffold material can be sealed in a liquid-tight manner.
Further, the cell culture scaffold material in the cell culture module is not limited to the scaffold material 10 described above, and may be a cell culture scaffold material in which a nanofiber layer is formed on a hollow fiber membrane mesh. It may be a cell culture scaffold material having nanofibers between the hollow fiber membranes. Moreover, the cell culture scaffold material which formed the nanofiber layer using methods, such as wet spinning other than electrospinning, and melt spinning, may be sufficient.

[Production method]
Hereinafter, the manufacturing method of the module 1 which has the above-mentioned scaffold material 10 as an example of the manufacturing method of the module for cell cultures of this invention is demonstrated.
The nanofiber layer 15 is preferably formed by electrospinning (electrospinning) on a substrate having no irregularities on the aforementioned surface from the viewpoint that a uniform and uneven nanofiber layer is easily obtained and the production efficiency is high. . However, the method is not limited to electrospinning, and methods such as wet spinning and melt spinning may be used.

  Hereinafter, the production of the nanofiber layer 15 using the electrospinning 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. In this apparatus, when a high voltage is applied while supplying the spinning dope to the nozzle, an electric repulsive force is generated in the spinning dope. At this time, by setting the base material between the nozzle and the target, the spinning stock solution is jetted toward the target, and when the spinning conditions are appropriate, a layer composed of nano-order fibers on the base material, that is, nano A fiber layer can be obtained.

The spinning conditions for obtaining the nanofiber layer 15 are the molecular weight of the polymer forming the nanofiber, the solvent, the concentration of the solution, the applied voltage, the distance between the nozzle and the target, etc. A fiber layer can be obtained.
As the polymer, it is preferable to use the above-described solvent-soluble polymer from the viewpoint of easy electrospinning. In addition, when a thermoplastic polymer is used, a laser electrospinning method can 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 when the solubility of the polymer is low, electrospinning can be enabled by adjusting the polymer concentration to be low. It is also possible to adjust the surface tension and conductivity of the spinning dope by adding additives.

  The polymer concentration of the spinning dope varies depending on the type of polymer used, the molecular weight, and the solvent, and cannot be generally stated, but it is preferably adjusted to 0.1 to 40 wt%. When the polymer concentration is 0.1 wt% or more, the productivity is excellent and the nanofiber layer 15 is easily obtained. Moreover, if the polymer concentration is 40 wt% or less, it is easy to prevent the viscosity of the spinning dope from becoming too high and difficult to eject from the nozzle.

The solvent to be used is not particularly limited as long as the polymer can be dissolved at the above concentration, and for example, a solvent such as acetone, triacetin, dimethylformamide, dimethylacetamide or the like can be used. As the solvent, it is preferable to use a highly volatile solvent because it is easy to suppress the solvent from volatilizing and clogging the polymer at the tip of the nozzle. A solvent may be used by blending a plurality of solvents in order to improve the solubility of the polymer.

Although the voltage to be applied varies depending on the type and physical properties of the polymer used, it cannot be generally stated, but is preferably 0.1 to 50 kV, and more preferably 1 to 40 kV. When the voltage is 0.1 kV or more, the production efficiency of the nanofiber layer is improved. Moreover, if a voltage is 40 kV or less, it will become difficult to short-circuit between a nozzle and a target.
Although the distance between the nozzle and the target varies depending on the type and physical properties of the polymer used and the applied voltage, it cannot be generally stated, but is preferably 10 to 1000 mm, more preferably 30 to 500 mm. If the distance is 10 mm or more, it is difficult for discharge to occur. Moreover, if the said distance is 1000 mm or less, the injection property of the spinning dope from a nozzle will improve.
Further, the shape of the target is preferably a flat plate shape or a roll shape from the viewpoint that a uniform nanofiber layer 15 can be easily obtained.
The nanofiber layer 15 is obtained by the method described above.

Next, the corner frames 42 are fixed with potting resin at the four corners of the bottom frame 41 (FIG. 2A) having the opening 41a in the center (FIG. 2B). Thereafter, the hollow fiber membrane 13a is arranged on the bottom frame 41, and further the hollow fiber membrane 13b is arranged along the direction orthogonal to the hollow fiber membrane 13a, and the end of the hollow fiber membrane outside the bottom frame 41 is temporarily fixed. Thus, the hollow fiber membrane mesh 14 is formed (FIG. 5A).
Next, the nanofiber layer 15 is placed on the formed hollow fiber membrane mesh 14, and the four corners of the nanofiber layer 15 are fixed to the square frame 42 with a potting resin (FIG. 5B).

Next, the hollow fiber membrane 11b is arranged on the nanofiber layer 15, and the hollow fiber membrane 11a is arranged along the direction orthogonal to the hollow fiber membrane 11b to form the hollow fiber membrane mesh 12 (FIG. 5 ( C)) These hollow fiber membranes 11b, 11a and the previously disposed hollow fiber membranes 13a, 13b are fixed by potting resin at a frame portion 43 between the square frames 42.
On the upper side of the hollow fiber membrane mesh 12, the upper frame 44 is fixed with a potting resin so that the scaffold material 10 is fixed to the mold 40 (FIG. 6A).

  Next, the portion outside the mold 40 of the hollow fiber membrane is bundled and tied through the silicon tube 50, and the tube is filled with potting resin and fixed (FIG. 6B). Thereafter, the hollow fiber membrane portion outside the mold 40 is covered and fixed with a potting resin, and the knot is cut (FIG. 6C).

Subsequently, the scaffold material 10 fixed to the mold 40 is sandwiched between the lower base 20 and the upper lid 30 and fixed with clips or the like. However, the method for fixing the scaffold material 10 sandwiched between the lower base 20 and the upper lid 30 is not limited to the method using the clip or the like, and is not particularly limited as long as these can be firmly fixed.
In addition, the cultured cells are adhered to the nanofiber layer 15 by fixing the scaffold material 10 fixed by the mold 40 between the lower base 20 and the upper lid 30 and then inserting the needle of the syringe into the sheet of the cell injection port. It can be carried out by injection or the like. If cultured cells are injected from the cell inlet, the cultured cells can be adhered regardless of the shape of the openings of the lower base 20 and the upper lid 30 and the connection status of tubes, microscopes, etc. to the openings. In addition, when injecting cultured cells from one cell inlet of the lower base 20 or the upper lid 30, it is possible to adjust the pressure by cell injection using the other cell inlet or opening. The adhesion of the cultured cells to the nanofiber layer 15 is not limited to the method of injecting the cultured cells from the cell injection port, and may be performed before being fixed by being sandwiched between the lower base 20 and the upper lid 30, such as a tube. It may be performed by a syringe or the like through the opening of the lower bottom 20 or the upper lid 30 before connecting the.
By the above method, the module 1 in which the scaffold material 10 is sealed in a liquid-tight manner is obtained.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited by the following description.
The method for measuring the diameter of the nanofiber in this example is as follows.
[Observation of nanofiber layer]
For observation of the nanofiber layer, 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 obtained by image analysis of the obtained SEM photograph 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.

[Example 1]
The nanofiber layer was formed using a NEU nanofiber electrospinning unit manufactured by Kato Tech. Aluminum foil was fixed to the drum-type target of the electrospinning unit. As a polymer for forming the nanofiber, a polyacrylonitrile polymer having a mass 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 to produce a nanofiber layer 15A on the aluminum foil. The spinning conditions were such that the peripheral speed of the metal drum as a target was 1 m / min, the applied voltage was 12 kV, the distance between the nozzle tip and the target was 100 mm, and the spinning time was 60 minutes.
SEM photographs of the nanofiber layer 15A are shown in FIGS. 7 (A) and 7 (B). The thickness of the nanofiber layer 15 was about 5 μm. The diameter of the nanofiber was about 200 nm.

As a hollow fiber membrane, a hydrophilic porous hollow fiber membrane (manufactured by Mitsubishi Rayon Engineering, EX270FS) (hollow fiber membranes 11a and 13a) and a three-layer composite hollow fiber membrane having a gas separation function (manufactured by Mitsubishi Rayon Engineering, MHF200TL) ) (Hollow fiber membranes 11b and 13b).
The hollow fiber membranes 13a are arranged at intervals of 2 mm on the bottom frame 41 (length 35 mm × width 35 mm, width of the frame portion 10 mm), and then the hollow fiber membranes 13 b are arranged 2 mm along the direction orthogonal to the hollow fiber membranes 13 a. Arranged at intervals. At this time, the mesh portion composed of the hollow fiber membrane 13a and the hollow fiber membrane 13b is arranged so as to be about 15 mm × 15 mm, and the end of the hollow fiber membrane outside the bottom frame is temporarily fixed to fix the hollow fiber membrane mesh 14. Formed (FIG. 5A).

  Next, the nanofiber layer 15A peeled from the aluminum foil was placed on the hollow fiber membrane mesh 14, and a potting resin (Nihon Polyurethane Industry Co., Ltd. Coronate (registered trademark) 4403 and Nippon Runan (registered trademark) 4223 1/1 The four corners of the nanofiber layer 15A were fixed to the square frame 42 (prepared with (wt ratio)) (FIG. 5B). Further, on the nanofiber layer 15A, the hollow fiber membranes 11b were arranged at intervals of 2 mm, and then the hollow fiber membranes 11a were arranged at intervals of 2 mm along a direction orthogonal to the hollow fiber membranes 11b. The hollow fiber membrane 11a and the hollow fiber membrane 11b were respectively installed so as to be positioned between the adjacent hollow fiber membranes 13a and 13b, that is, shifted by ½ pitch to form the hollow fiber membrane mesh 12 ( FIG. 5C). The hollow fiber membrane 11b, the hollow fiber membrane 11a, and the previously disposed hollow fiber membrane 13a and hollow fiber membrane 13b were fixed at the frame portion 43 by potting resin. Thereafter, the upper frame 44 was placed on the hollow fiber membrane mesh 10 and fixed with potting resin to obtain a cell culture scaffold material 10A fixed to the mold 40 (FIG. 6 (A)).

Next, the hollow fiber membranes 11a and 13a and the hollow fiber membranes 11b and 13b in the hollow fiber membrane meshes 12 and 14 are respectively bundled and tied through a silicon tube 50 (length 10 mm), and potted in the silicon tube 50 After filling and fixing the resin, the hollow fiber membrane part outside the mold 40 was covered and fixed with potting resin, and the part ahead of the silicon tube 50 was cut and removed.
A photograph of the cell culture scaffold material 10A fixed to the mold 40 is shown in FIG.

  Next, the cell culture scaffold material 10A fixed to the mold 40 is obtained by arranging a cell made of silicon rubber between two acrylic plates having a cell inlet and an opening, and a cell similar to the lower base 20. A cell culture module 1A was obtained by sandwiching and fixing between a top cover 30 in which a sheet made of silicon rubber was placed between two acrylic plates having an inlet and an opening.

[Example 2]
A nanofiber layer 15B was formed in the same manner as in Example 1 except that the spinning time was changed to 120 minutes in the production of the nanofiber layer, and the cell culture scaffold material 1B was formed.
9A and 9B show SEM photographs of the nanofiber layer 15B. The thickness of the nanofiber layer 15B was about 10 μm. The diameter of the nanofiber was about 200 nm.

  The module for cell culture of the present invention was compact. Therefore, the culture space can be further reduced in multi-sample, multi-condition simultaneous culture. In addition, since nutrients and gas are supplied and metabolic waste products are removed through the hollow fiber membrane, cell culture can be performed with high efficiency. In addition, by connecting a tube, etc., to the opening of the lower base and upper lid (cover), it is possible to supply nutrients and gas and remove waste products under three or more conditions without using a special device. It is easy to control the conditions independently. Furthermore, by injecting cultured cells from the cell injection port, it is possible to easily adhere the cultured cells to the nanofiber layer even when a tube or a microscope is connected to the opening.

<Cell culture>
Hereinafter, cell culture using the cell culture module of the present invention will be described.
[Example 3]
Human fetal liver cells (Hc cells, manufactured by Dainippon Sumitomo Pharma Co., Ltd.) were cultured for 32 days using the cell culture module 1A produced in Example 1. In the culture, a medium circulation circuit including the hollow fiber membranes 11a and 31a is formed, and a CS-C medium (manufactured by Cell Systems) is continuously added at 6.8 mL / hr in the cell culture module 100A using the circuit. While feeding. The CS-C medium to be supplied is replaced at a time when the pH of the medium (pH indicator) and the glucose concentration (measuring reagent: glucose CII-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.) are measured. , 25, 27, 29, and 31 days were replaced with fresh medium. Further, from the 19th day after the start of the culture, the culture was performed while supplying air (about 1 L / min) from the hollow fiber membranes 11b and 31b to the cell culture module 100A by an air pump.
FIG. 10 shows changes in cell density during 32 days of culture. The cell density was calculated from the degree of decrease in glucose concentration in the medium. The broken line in FIG. 10 shows the start time of air supply. Further, micrographs obtained by observing the cell morphology of the cell scaffold material after culturing with a phase-contrast inverted microscope (ECLIPSE-TE300, manufactured by Nikon) are shown in FIG. 11 (observation magnification: 32 times) and FIG. 12 (observation magnification: 56 times). Show. In addition, (A) and (B) in FIG. 11 and FIG. 12 are photographs in which different parts of the same cell culture scaffold material are observed.

[Comparative Example 1]
As a comparison target with the culture of Example 3, stationary culture using the cell culture scaffold material 1A was performed.
The cell culture scaffold 1A produced in Example 1 is placed in a CS-C medium (manufactured by Cell Systems) in a 35 mm culture dish, and human fetal liver cells (Hc cells, manufactured by Dainippon Sumitomo Pharma Co., Ltd.) are used for 22 days. The culture was stationary. The culture was performed while shaking the dish in order to improve the mass transfer into the cell culture scaffold material 1A. The CS-C medium exchange time is determined from the measurement results of the medium pH (pH indicator) and glucose concentration (measuring reagent: glucose CII-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.). The interval was changed every day, from 7 to 10 days at 1 day intervals, from 11 to 14 days at 18 hour intervals, and from 15 to 23 days at 12 hour intervals.
FIG. 13 shows changes in cell density in the static culture for 31 days. The cell density was calculated from the degree of decrease in glucose concentration in the medium. Moreover, the microscope picture which observed the cell form in the scaffold material for cells after 22-day culture | cultivation with the phase-contrast inverted microscope (ECLIPSE-TE300, Nikon company) is shown in FIG. 14 (observation magnification 56 times). Moreover, after fixing the cell on the scaffold material for cells 22 days later with formalin, the cytoskeleton and the nucleus were stained, and a microscope photograph observed with a confocal laser microscope is shown in FIG. 15 (observation magnification 200 times). 14 and 15, (A) and (B) are photographs in which different parts of the same cell scaffold material are observed. In FIG. 15, the upper left is a photograph of the cytoskeleton, the upper right is a photograph of the nucleus, the lower left is a photograph of the cell scaffold material, and the lower right is a photograph of the cytoskeleton and nucleus.

As shown in FIG. 10, in the culture of Example 3 using the cell culture module of the present invention, the maximum value of the cell density was 1.2 × 10 6 by continuously supplying the CS-C medium and air. ^The density became ⁸ cells / cm ³ (reached on the 32nd day after the start of culture). On the other hand, as shown in FIG. 13, in the static culture using the culture dish of Comparative Example 1, the maximum value of the cell density was 1.9 × 10 ⁷ cells / cm ³ (reached on the 20th day after the start of the culture). . As described above, the culture performed while continuously supplying the culture medium and air using the cell culture module of the present invention has a cell density about 6 times that of stationary culture, and promotes excellent cell growth. Showed the ability.

  Moreover, in the photograph (FIG. 15, upper right) which observed the nucleus of the cell in the cell culture scaffold material stationary culture | cultivated with the confocal laser microscope, the nucleus overlapped and was observed. From this result, it was found that the cells that adhered and proliferated to the cell culture scaffold material in the stationary culture entered between the fibers in the nanofiber layer and proliferated three-dimensionally. On the other hand, as shown in FIGS. 11, 13 and 14, in both cultures of Example 3 and static culture of Comparative Example 1, cells adhered and proliferated similarly to the nanofiber layer of the cell culture scaffold material. It was confirmed that The cell culture scaffold material in the culture of Example 3 and the stationary culture of Comparative Example 1 is the same. In the culture of Example 3, the cells are considered to be three-dimensionally proliferating in 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.

  DESCRIPTION OF SYMBOLS 1 Cell culture module 10 Cell culture scaffold material 11, 11a, 11b Hollow fiber membrane 12 Hollow fiber membrane mesh 13, 13a, 13b Hollow fiber membrane 14 Hollow fiber membrane mesh 15 Nanofiber layer 20 Lower bottom (cover) 21a, 22a Cell Inlet 21b, 22b Opening 30 Upper lid (cover) 31a, 32a Cell inlet 31b, 32b Opening

Claims (1)

A cell culture scaffold material having a hollow fiber membrane mesh in which hollow fiber membranes are arranged in a mesh shape and a nanofiber layer made of an assembly of nanofibers in contact with the hollow fiber membrane mesh is provided between two covers. Liquid-tightly arranged,
A cell injection port for injecting cultured cells and an opening other than the cell injection port are formed at a position corresponding to the nanofiber layer in the cover, and the cell injection port and the opening are closed with a sheet Culture module.