JPWO2014034146A1 - Module for cell culture - Google Patents

Abstract

The present invention relates to a cell culturing module capable of taking a substance of any size into and out of an arbitrary position in the module, and a cell culturing module capable of being connected to a flow-feeding means such as a tube or a syringe with high liquid tightness or air tightness. The present invention also relates to a cell culturing module capable of efficiently culturing cells for a long period of time while maintaining the original function, and a cell culturing apparatus including the cell culturing module.

Description

  The present invention relates to a cell culture module. More specifically, a cell culture module that can put a substance of any size into and out of an arbitrary position in the module, and a cell culture module that can be connected to a flow feeding means such as a tube or a syringe with high liquid or air tightness. The present invention also relates to a cell culturing module capable of efficiently culturing cells for a long period of time while maintaining the original function, and a cell culturing apparatus including the cell culturing module.

  An evaluation test system for drug efficacy, toxicity, safety, etc. using cultured cells is important as an alternative test method to animal experiments. However, it is not always easy to culture cells, particularly primary cells, in vitro for a long period of time. In addition, in an evaluation test system using cultured cells, it is necessary to use cells having the same function as in vivo, but it is not easy to efficiently differentiate and proliferate cells while maintaining their original functions.

  In order to efficiently culture cells in vitro, it is necessary to supplement the cultured cells with nutrients and air (oxygen). As a technique for that, a method of supplying and discharging a culture solution using a hollow fiber membrane has been conventionally known (Patent Documents 1 to 3).

  On the other hand, in order to culture while maintaining the original function of the cell, it is preferable to culture the cell in an environment similar to that in the living body. As a technique for that purpose, a method of three-dimensionally culturing cells using a porous material such as hydroxyapatite or polylactic acid as a scaffold is known (Patent Document 4).

  In recent years, since a fibrous structure of tissue in a living body can be highly imitated, a technique using nanofibers having a nano-order fiber diameter as a scaffold material for cell culture or tissue formation has attracted attention (Non-Patent Document 1). Non-Patent Document 2). For example, a nanofiber pattern is formed using a method of using nanofibers produced by melt spinning as a scaffolding material (Patent Document 5), a mixed solution in which organic nanofibers are dispersed in a solvent, and further for cell culture. A method of controlling cell adhesion, proliferation, and differentiation by adsorbing an effective functional drug in this pattern has been shown (Patent Document 6).

  The inventors have developed a cell culturing module capable of efficiently culturing cells in vitro by using a hollow fiber membrane arranged in a mesh shape and a nanofiber layer as a scaffold material (Patent Literature). 7-10). In this module, nanofibers with nano-order fiber diameters create an environment similar to that in the living body, and the hollow fiber membrane replenishes the environment with culture medium and air (oxygen), and also provides metabolic waste products from cultured cells. Perform removal.

  In order to introduce fluid such as cell suspension, culture medium (culture solution), gas, etc. into the cell culture module, a tube is connected to a flow opening (opening) provided in a substrate constituting the module, or an injection needle It is being carried out. Under the present circumstances, in order to ensure the liquid-tightness or airtightness of the internal space of a module, the technique which seals a through-flow port with the sheet | seat which consists of silicon rubber is proposed (for example, refer patent document 10).

JP-A-4-341176 JP 2004-166717 A JP 2000-189189 A JP 2006-254722 A JP 2007-44149 A JP 2007-44149 A Japanese Unexamined Patent Publication No. 2009-100 JP 2010-148496 A JP 2010-148497 A JP 2011-239756 A

Polymer, 2006, Vol.55, No.3, pp.142 Tissue Engineering, 2005, Vol.11, No.1-2, pp.101

  In the cell culture module as described above, in order to introduce a substance from the outside into the inside or to take out a substance from the inside to the outside, an opening that can connect a tube, a syringe or the like to the substrate constituting the module ( Inlet or outlet) is provided. For example, introduction of a substance is performed by feeding a liquid containing the substance from a tube or the like connected to an inlet.

  Since these inlets or outlets usually have a hole diameter of about 0.5 to 5 mm, it is not possible to introduce a substance having a size larger than this into the module or discharge the substance to the outside. Further, even a substance smaller than this is configured to be introduced into the liquid flow, and therefore the substance cannot be disposed at an arbitrary position inside the module. Similarly, any object inside the module cannot be selected and discharged outside.

  By using the cell culture module as described above, the in vitro cell culture technology has achieved certain results. However, in order for a test system using cultured cells to be completed as an evaluation system that replaces animal experiments, cells are cultured while retaining the same functions as in vivo, and a system similar to biological tissue is reproduced. There must be. There has not yet been enough consideration for that.

  Accordingly, a first object of the present invention is to provide a cell culture module capable of taking a substance of any size into and out of an arbitrary position in the module.

  The present invention also provides a cell culture module that enhances the sealing properties of the cell suspension, culture medium, gas, etc., and prevents leakage of the cell suspension, etc., when cells are introduced. It is a second problem to provide a technique for this purpose.

  Furthermore, it is a third object of the present invention to provide a cultured cell evaluation system that can suppress dedifferentiation and the like and can cultivate cells for a long period of time while maintaining the same function as in vivo, and substitute for animal tests. .

  In order to solve the first problem, the present invention includes a culture field in which a hollow fiber membrane is disposed, a frame that forms an internal space including the culture field, and a removable lid, Provided is a module for cell culture in which an opening to which a lid is attached is provided with a size capable of exposing the entire culture site.

  In this cell culture module, by removing the lid and exposing the entire culture field, it is possible to introduce a substance into an arbitrary position of the culture field or to take out a substance at an arbitrary position to the outside. It is possible without size restrictions.

  In the cell culture module, the lid body and / or the frame body preferably has a flow port through which fluid flows from the outside to the internal space or from the internal space to the outside. It is preferable to have a female luer taper that decreases in diameter from the outside toward the internal space.

  One or a plurality of flow openings may be provided and can be provided at any position of the lid and / or the frame (two substrates or molds constituting the frame). As described above, it is particularly preferable that they are provided symmetrically with respect to the center of the fiber assembly.

  In order to solve the second problem, the present invention provides two substrates, a culture field in which a hollow fiber membrane is disposed in a culture region formed between the substrates, and an outside to the inside of the culture region. Or a plurality of flow ports for allowing fluid to flow from the inside to the outside, the flow ports being disposed at positions corresponding to the scaffold material of the substrate, and from the outside of the culture region Provided is a cell culture module having a female luer taper that is reduced in diameter toward the inside.

  A cell culture module having a female luer taper at the flow outlet can be connected to a flow feeding means such as a tube or a syringe having a male luer taper with high liquid tightness or air tightness.

  In the cell culture module, the plurality of flow holes may be provided on the same substrate or on different substrates, but are preferably provided on different substrates.

  In the cell culture module of the present invention, it is preferable that the flow opening is provided symmetrically with respect to the center of the culture field.

  In the cell culture module of the present invention, the fiber assembly is preferably arranged so as to be separable from the hollow fiber membrane. Moreover, the said fiber assembly shall consist of nanofibers.

  In order to solve the third problem, the present invention provides a cell culture module comprising a culture field in which a hollow fiber membrane and a fiber assembly made of nanofibers having an average diameter of 250 nm to 950 nm are arranged.

  Also in the cell culture module for solving the first and second problems described above, the fiber assembly can be made of nanofibers having an average diameter of 250 nm to 950 nm. The average diameter of the nanofiber is more preferably about 300 nm or about 600 nm, and particularly preferably about 600 nm.

  By culturing using the cell culture module of the present invention, cells having high activity can be obtained. The cytochrome 450 (CYP) activity of the obtained cells is preferably 2 pmol / 106 cells / min or more, more preferably 3 pmol / 106 cells / min or more, and particularly preferably 4 pmol / 106 cells / min or more.

  In the cell culture module of the present invention, the hollow fiber membrane is preferably composed of an oxygen supply hollow fiber membrane and a medium supply hollow fiber membrane. The hollow fiber membrane for oxygen supply supplies oxygen into the module to promote inspiration, and the hollow fiber membrane for medium supply supplies medium to the module to promote metabolism (nutrition addition and waste discharge). .

  The present invention also provides a cell culture apparatus including the cell culture module, oxygen supply means, and culture medium supply means.

  The cell culture device of the present invention may further include any one or more means selected from a monitoring means, an analysis means, and a sampling means.

  According to the present invention, there is provided a cell culture module capable of taking a substance of any size into and out of an arbitrary position in the module. Furthermore, by forming a tissue such as a cell sheet, it becomes possible to take out a cell mass that exhibits a behavior different from that of discrete cells in the state of culturing, and a highly accurate evaluation test can be performed.

  Further, according to the present invention, the cell suspension of the cell culture module, the culture medium, gas and the like can be sealed at the flow port, and the leakage of the cell suspension or the like can be prevented when cells are introduced. Technology is provided.

  Furthermore, according to the present invention, cells can be efficiently cultured for a long period of time while maintaining their original functions. The technology of the present invention can be applied to an existing scaffold material or a cell culture module using the same, thereby enabling better ex vivo evaluation of drug efficacy, toxicity, safety, and the like. .

It is a figure explaining the structure of 101 A of cell culture modules which concern on 1st embodiment of this invention. A is a sectional view when the cap 150 is removed, and B is a top view. It is a figure explaining the structure of the through-flow port. It is a figure explaining the structure of the culture place 110. FIG. A is a top view, B is a cross-sectional view taken along line P-P in A, and C is a cross-sectional view taken along line Q-Q in A. It is a figure explaining the structure of the modification of 101 A of cell culture modules which concern on 1st embodiment, and sectional drawing at the time of the removal of the cap 150 is shown. It is a figure explaining the structure of the modification of the culture field. A is a top view and B is a cross-sectional view taken along the line P-P in A. FIG. It is a figure explaining the structure of the module 101B for cell cultures concerning 2nd embodiment of this invention. A is a top view when the cap 150 is removed, and B is a cross-sectional view. It is a figure explaining the structure of 101 C of cell culture modules which concern on 3rd embodiment of this invention. A is a top view when the cap 150 is removed, and B is a side view. It is a figure explaining the structure of module for cell culture 101D which concerns on 4th embodiment of this invention. A is a top view when the cap 150 is removed, and B is a cross-sectional view. It is a figure explaining the structure of module 201A for cell culture which concerns on 5th embodiment of this invention. A is a plan view, and B is a cross-sectional view taken along the line P-P in A. FIG. It is a figure explaining the connection aspect of the port and syringe by a Luer taper. It is a figure explaining the sealing aspect of a port. It is a figure explaining the structure of the scaffold material 210. FIG. A is a plan view, B is a cross-sectional view taken along line P-P in A, and C is a cross-sectional view taken along line Q-Q in A. It is a figure explaining the procedure of the cell culture method using the module 201A for cell cultures. It is a figure explaining the structure of the module 201B for cell cultures concerning 6th embodiment of this invention. A is a plan view, and B is a cross-sectional view taken along the line P-P in A. FIG. It is a figure explaining the procedure of the cell culture method using the module for cell culture 201B. An example of the structure of the cell culture apparatus of this invention is shown. The cytochrome 3A4 (CYP) enzyme activity is shown when Hc cells (upper) and hepatoblasts derived from Hc cells are statically cultured using each nanofiber nonwoven fabric (No. 1 to No. 6) as a scaffold. The result of having observed the cell growth state when stationary culture | cultivation of Hc cell origin hepatoblast using each nanofiber nonwoven fabric as a scaffold is shown with a position scanning microscope (from the top, No.1, No.2, No.3). The results of observing the cell growth state with stationary scanning microscope when Hc cell-derived hepatoblasts were statically cultured using each nanofiber nonwoven fabric as a scaffold are shown (No. 4, No. 5, No. 6 from above). It shows cytochrome 3A4 (CYP3A4) enzyme activity when human frozen hepatocytes are statically cultured using a nanofiber nonwoven fabric or petri dish as a scaffold (No. 2, No. 4, No. 6, Petri dish from the left). It shows changes over time in the number of cells (× 106 cells / plate) when Hc cell-derived hepatoblasts were statically cultured using each nanofiber nonwoven fabric (-□-: No. 2, -Δ-: No. 3). ,-■-: No. 4,-▲-: No. 5,-◇-: No. 6). FIG. 6 shows changes over time in cell density (× 107 cells / cm 3) when Hc cells are induced into hepatoblasts using the cell culture apparatus of the present invention and further cultured under reflux. 2 shows changes over time in cell density (× 107 cells / cm 3) when circulating human frozen hepatocytes using the cell culture apparatus of the present invention. The change with time of dissolved oxygen concentration when circulating human frozen hepatocytes using the cell culture apparatus of the present invention is shown.

  This specification includes the contents described in the specifications of Japanese Patent Application Nos. 2012-192980, 2012-265436, 2013-051179, and 2013-101805, which are the basis of the priority of the present application. Include.

1. Cell Culture Module According to First Embodiment (1) Configuration of Frame and Lid FIG. 1 shows the configuration of a cell culture module 101A according to the first embodiment of the present invention. The cell culture module 101A includes a frame including the substrates 120 and 130 and the mold 140, and a lid 150 (hereinafter referred to as “cap 150”). The substrate 130 is provided with an opening 131 to which the cap 150 is attached.

  A screw groove 132 is provided around the circumferential surface (inner circumferential surface) of the opening 131. The cap 150 has an outer peripheral surface that is provided with a thread 151 and has the same diameter as the opening 131. The cap 150 is attachable to or detachable from the opening 131 (screw cap) by the engagement between the screw thread 151 and the screw groove 132. 1A shows a cross-sectional view of the cell culture module 101A when the cap 150 is removed, and FIG. 1B shows a top view of the substrates 120 and 130 and the mold 140 when the cap 150 is removed.

  When the cap 150 is attached to the opening 131, it forms a liquid-tight (or air-tight) internal space 102 (see FIG. 2B described later) together with the substrates 120 and 130 and the mold 140. Reference numeral 152 in FIG. 1 indicates a sealing member (for example, an O-ring) for increasing the liquid tightness (or air tightness) of the internal space 102. The sealing member 152 is disposed at a contact portion between the cap 150 and the substrate 130, and seals the contact portion when the cap 150 is attached to the opening 131.

  In the internal space 102, a culture place 110 configured by arranging a hollow fiber membrane 111a (113a) and a hollow fiber membrane 111b (113b) in a mesh shape is located. The hollow fiber membrane 111a (113a) passes through two opposite sides of the mold 140 and is inserted into the mold 140, and is fixed to the mold 140 with potting resin. Similarly, the hollow fiber membrane 111b (113b) passes through the other two opposite sides of the mold 140 and is inserted into the mold 140 to be fixed to the mold 140. The configurations of the hollow fiber membranes 111a (113a) and 111b (113b) and the culture site 110 will be described later in detail with reference to FIG.

  The substrates 120 and 130 only need to have strength suitable for firmly fixing the hollow fiber membranes 111a (113a) and 111b (113b) and the mold 140, and are, for example, acrylic plates. Each of the substrates 120 and 130 may be composed of one acrylic plate or the like, or may be composed of two or more acrylic plates or the like. The cap 150 may also be an acrylic plate, for example, and may be composed of one or two or more acrylic plates.

  The material of the mold 140 may be any material as long as sufficient adhesion with the potting resin can be obtained, for example, silicon rubber. Moreover, the potting resin should just have sufficient adhesiveness, for example, various thermosetting resins, such as an epoxy resin, a urethane resin, an unsaturated polyester resin, a silicon resin, a thermoplastic resin, etc. are used.

  The opening 131 of the substrate 130 is provided with a size capable of exposing the entire culture field 110 when the cap 150 is removed. In other words, when viewed from above (see FIG. 1B), the opening 131 is sized so that the entire culture field 110 can be located inside thereof.

  The diameter D of the opening 131 is preferably equal to or larger than the diameter d of the culture field 110. More specifically, it is preferable that the opening 131 and the culture field 110 are arranged concentrically and that the diameter D of the opening 131 is greater than or equal to the diameter d of the culture field 110. In other words, when viewed from above (see FIG. 1B), the opening area of the opening 131 is preferably equal to or larger than the area of the culture field 110. More specifically, when viewed from the top (see FIG. 1B), the opening 131 and the culture field 110 are preferably arranged so that the entire culture field 110 is located inside the opening 131.

  Thereby, in the cell culture module 101A, when the cap 150 is removed, the entire culture site 110 can be exposed so that it can be visually recognized and operated from the outside. Therefore, in the cell culture module 101A, by removing the cap 150 and exposing the entire culture field 110, a substance can be introduced into an arbitrary position of the culture field 110 or a substance at an arbitrary position can be taken out. However, it is possible without restriction of the size of the substance.

(2) Flow outlet Next, FIG. 2 is referred. The cap 150 may have a flow opening 153 for allowing fluid such as a cell suspension to flow from the outside of the module to the internal space 102 or from the internal space 102 to the outside (see FIG. A). The flow opening 153 is provided through the cap 150. The through-flow port 153 communicates between the outside and the internal space 102 when the cap 150 is attached. In the figure, an example is shown in which the flow port 153 is configured by screwing and attaching a port (connection member) 154 to a screw groove formed in the cap 150.

  Although the figure shows an example in which two flow ports 153 are provided in the cap 150, one or three or more flow ports 153 can be provided. Further, the flow port 153 can also be provided in the substrate 120 as shown in FIG. 2B. The flow port 153 has a cap 150, substrates 120, 130, and a mold as long as a fluid such as a cell suspension, a culture medium, and a gas can flow from the outside to the inside of the internal space 102 or from the inside to the outside. Any number of frames 140 can be provided at any position.

  The material of the port 154 is not particularly limited, but is usually polycarbonate, polymethyl methacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, polydimethylsiloxane (PDMS), or the like. As the port 154, a so-called syringe port widely used in the medical field can be suitably used.

  The port 154 preferably has a female luer taper, and its inner wall is preferably gradually reduced in diameter in the direction from the outside to the inside of the internal space 102. As a result, the port 154 is connected to a flow feeding means such as a tube or a syringe having a male luer taper with good liquid tightness. Conventionally known standards can be applied to the taper angle and size of the female and male luer tapers.

  Although the magnitude | size and shape of the through-flow port 153 are not specifically limited, For example, it is circular and a diameter shall be about 500 micrometers-about 5 mm. The female luer taper of the port 154 only needs to be provided on at least a part of the inner wall of the port 154 so that a flow feeding means having a male luer taper can be inserted.

(3) Culture Field Next, the configuration of the culture field 110 will be described with reference to FIG. The culture field 110 includes a hollow fiber membrane mesh 112 in which hollow fiber membranes 111a and 111b (the hollow fiber membranes 111a and 111b are collectively referred to as a hollow fiber membrane 111) are arranged in a mesh shape, and a scaffold material 115 made of a fiber assembly. And hollow fiber membrane mesh 114 in which hollow fiber membranes 113a and 113b (hollow fiber membranes 113a and 113b are collectively referred to as hollow fiber membrane 113) are arranged in a mesh shape are laminated in this order.

(3-1) Hollow fiber membrane The membrane base polymer of the hollow fiber membrane 111 that forms the hollow fiber membrane mesh 112 may be any material that can be molded on the hollow fiber membrane. For example, polyolefins such as polyethylene and polypropylene, poly Examples include sulfone, polyallyl sulfone, polyether sulfone, polyacrylonitrile, cellulose acetate, polyvinylidene fluoride, and a mixture of two or more of these.

  The outer diameter of the hollow fiber membrane 111 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 111, 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 111 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 111 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. The film thickness when the hollow fiber membrane 111 is a multilayer film is the sum of the film thicknesses of the respective layers (film thickness of all layers). The inner diameter of the hollow fiber membrane 111 is preferably 10 μm or more and 2995 μm or less.

  As the hollow fiber membrane 111, 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

  Further, 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.

  Further, the hollow fiber membrane 111 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 also be used for the hollow fiber membrane mesh 112. 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 112 is formed by arranging a plurality of hollow fiber membranes in a mesh shape so that a mesh is formed. As a form of the hollow fiber membrane mesh 112, for example, a plurality of hollow fiber membranes 111a are arranged in parallel at a predetermined interval to form a sheet, and a plurality of hollow fibers are formed along a direction orthogonal to the hollow fiber membranes 111a. A configuration in which the film 111b is arranged in parallel at a predetermined interval to form a sheet can be used (FIG. 3A). The number of layers of the sheet made of the hollow fiber membrane 111 in the hollow fiber membrane mesh 112 is not particularly limited, and preferably 2 to 10 layers. In this example, the sheet has two layers (a layer made of the hollow fiber membrane 111a and a layer made of the hollow fiber membrane 111b).

  The hollow fiber membrane mesh 114 and the hollow fiber membrane 113 can be the same as the hollow fiber membrane mesh 112 and the hollow fiber membrane 111, and the preferred forms are also the same.

  The form of the hollow fiber membranes 112 and 114 may be selected according to the supply of nutrients and gas through the hollow fiber membranes 111 and 113 and the removal of metabolic waste products. When the hollow fiber membranes 111 and 113 are modularized Depends on port design. In the culture place 110, the hollow fiber membrane 111a of the hollow fiber membrane mesh 112 and the hollow fiber membrane 113a of the hollow fiber membrane mesh 114 are preferably the same type of hollow fiber membrane. Similarly, the hollow fiber membrane 111b of the hollow fiber membrane mesh 112 and the hollow fiber membrane 113b of the hollow fiber membrane mesh 114 are preferably the same type of hollow fiber membrane. By adopting such a configuration, when the hollow fiber membranes 111 and 113 are modularized, for example, the hollow fiber membranes 111a and 113a are gathered and connected to one port portion to form a gas supply hollow fiber membrane. The hollow fiber membrane 111b and the hollow fiber membrane 113b can be gathered and connected to another port portion to be used as a medium supply hollow fiber membrane.

(3-2) Scaffold material As the fiber assembly constituting the scaffold material 115, an assembly of nanofibers is preferably used. The aggregate of nanofibers is preferably formed by electrospinning (electrospinning) on a substrate. The substrate is not particularly limited as long as it can form an assembly of nanofibers 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.

  Further, 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 becomes high 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 average diameter (thread diameter) of the nanofiber (single fiber) is preferably 250 nm to 950 nm, more preferably about 300 or about 600 nm, and further preferably about 600 nm. If nanofibers having a thread diameter in this range are used, cells can be cultured for a long time with high activity. The obtained cell has a cytochrome P450 (CYP) activity of preferably 2 or more, more preferably 3 or more, and particularly preferably 4 or more.

  In addition, the diameter of the single fiber of a nanofiber can be measured by the method of the said patent document 10. For example, the average diameter of nanofiber single fibers can be measured, for example, as follows. The surface of the nanofiber or its aggregate (nanofiber layer) is observed with an electron microscope, the width of the surface of any 20 nanofibers in the obtained electron micrograph is measured, and the average is calculated as the single nanofiber. The diameter of the fiber. 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 fiber assembly 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 fiber assembly 115 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 scaffold material 15, so that the efficiency of cell culture is improved.

  Adsorption or growth of cultured cells can be controlled by adsorbing or fixing a physiologically active substance to the scaffold material 115. 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. In addition, a cell culture test can be performed by adsorbing or fixing a test substance such as a drug or a chemical substance to the scaffold material 115.

  The culture field 110 has a laminate in which the scaffold material 115 is disposed between the hollow fiber membrane mesh 112 and the hollow fiber membrane mesh 114 described above. Thus, by arranging the nanofiber layer between the hollow fiber membrane meshes, the scaffold material 115 can be firmly fixed to the hollow fiber membrane meshes 112 and 114, and supply of nutrients and gas to the cultured cells, It is possible to more stably remove metabolic waste products from the cell culture. In addition, since the scaffold material 115 is disposed between the hollow fiber membrane meshes 112 and 114, the distance between the cultured cells adhered to the scaffold material 115 and the hollow fiber membranes 111 and 113 is reduced. Gas supply and removal of metabolic waste products from the cell culture can be performed with higher efficiency, and necrosis of the cultured cells is reduced and the efficiency of the cell culture is improved.

  The size of the culture field 110 may be any size as long as the number of cells necessary for the drug test can be secured, preferably 1 m 2 to 0.1 cm 2, more preferably 600 cm 2 to 0.2 cm 2, and particularly preferably 59 cm 2 to 0.35 cm 2. . Within this range, the uniformity of the scaffold is increased.

  In the cell culture module according to the present invention, the number of laminated layers of the hollow fiber membrane meshes 112 and 114 and the scaffold material 115 in the culture site 10 is not particularly limited, and the hollow fiber membrane mesh 114, the scaffold material 115, and the hollow fiber membrane mesh. 112, the scaffold material 115, and the hollow fiber membrane mesh 112 may be laminated in this order, or a larger number of the hollow fiber membrane meshes 112 and 114 and the scaffold material 115 may be laminated.

  In the cell culture module 101A, for example, a tube or the like is connected to the above-described port 154, so that the hollow fiber membranes 112 and 114 of the hollow fiber membrane meshes 112 and 114 can be used as a route for supplying nutrients and gases and removing metabolic waste products. A third path other than 111 and 113 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.

(4) Modification (4-1) Modification of Lid In the above, the screw groove 132 is provided on the peripheral surface (inner peripheral surface) of the opening 131 of the substrate 130, and the screw thread 151 is provided on the outer peripheral surface of the cap 150. An example in which the cap 150 is attached to and detached from the opening 131 has been described. This form is simple in structure and easy to process, and the cap 150 can be easily attached and detached. In addition, the size of the module can be reduced.

  As shown in FIG. 4, the cap 150 may be attached to and detached from the opening 131 by providing a screw groove 132 on the outer peripheral surface of the opening 131 of the substrate 130 and providing a screw thread 151 on the inner peripheral surface of the cap 150. . FIG. 4A shows a cross-sectional view of the cell culture module 101A when the cap 150 is removed.

  In this modification, the screw thread 151 and the screw groove 132 do not come into contact with the medium or the like introduced into the internal space 102. For this reason, it is possible to prevent the screw thread 151 and the screw groove 132 from adhering to each other due to a dried medium or the like, and to improve the detachability operability of the cap 150.

(4-2) Modification Example of Culture Field In the above, the example in which the culture field 110 is configured by arranging the scaffold material 115 between the hollow fiber membrane mesh 112 and the hollow fiber membrane mesh 114 has been described. As shown in FIG. 5, the culture field 110 may be configured by laminating a hollow fiber membrane mesh 114, a hollow fiber membrane mesh 112, and a scaffold material 115 in this order. Thereby, the scaffold material 115 can be arrange | positioned on the hollow fiber membrane mesh 112 so that isolation | separation is possible. Therefore, if the cap 50 is removed to expose the entire culture field 110, the scaffold material 115 on the hollow fiber membrane mesh 112 can be taken out from the culture field 110 as it is.

2. Cell Culture Method in Cell Culture Module According to First Embodiment A cell culture method using the above-described cell culture module 101A will be described.

(1) Cell introduction procedure First, cells are introduced into the culture site 110. The introduction of cells can be performed with the cap 150 removed (see FIG. 1). When the cap 150 is removed, the entire culture field 110 is exposed through the opening 131. For this reason, it is possible to introduce a cell into an arbitrary position on the culture field 110 while visually checking the entire culture field 110. The introduction of cells is performed, for example, by inserting a pipette or the like into the internal space 102 from the opening 231 and dropping the cell suspension at an arbitrary position on the culture field 110. A substance such as a test substance may be introduced together with the cell suspension. For example, by inserting tweezers or the like into the culture space 102 and placing the solid substance on the culture place 110, the substance can be introduced at an arbitrary position without restriction on the size of the substance.

  Introducing the cells may be performed by connecting a cell suspension liquid feeding means to the port 154 when the cap 150 is provided with the flow port 153 (see FIG. 2). By sending the cell suspension from the through-flow port 153, a flow of the cell suspension is formed in the internal space 102, and the cells are transported by the flow and dispersed on the culture site 110. However, in this case, it is difficult to introduce the cells only at arbitrary positions on the culture place 110. In addition, a substance such as a test substance may be introduced together with the cell suspension. In this case, the substance needs to be introduced as a solution or a dispersion, and the substance can be introduced only at an arbitrary position on the culture place 110. It is difficult to introduce to.

(2) Culturing procedure After the introduction of the cells, for example, the hollow fiber membrane 111a and the hollow fiber membrane 113a are gathered and connected to one port portion to supply gas, and the hollow fiber membrane 111b and the hollow fiber membrane 113b are gathered. Cell culture is performed by supplying a medium connected to another port.

  Specifically, the cell culture uses, for example, a hollow fiber membrane that is a gas separation membrane as the hollow fiber membranes 111a and 113a, and a hollow fiber membrane having substance-selective permeability as the hollow fiber membranes 111b and 113b, and the hollow fiber membrane 111a, While supplying a gas containing oxygen from one end of 113a, the gas is discharged from the other end. Further, nutrients of the cultured cells are supplied from one end of the hollow fiber membranes 111b and 113b, and waste products and metabolites of the cultured cells are discharged from the other end. Thereby, it is also possible to analyze the discharged components online and to evaluate the growth status of the cultured cells continuously while culturing the cultured cells.

  In addition, a test substance is administered to cultured cells from one end of the hollow fiber membranes 111b and 113b, and a solution containing waste products and metabolites is discharged from the other end of the hollow fiber membranes 111b and 113b, and this solution is analyzed. By monitoring, it is possible to evaluate the stress applied to the cultured cells and the state of the cultured cells over time when the test substance is administered.

(3) Cell recovery procedure The cultured cells can be recovered by removing the cap 150 again. When the cap 150 is removed, the entire culture field 110 is exposed through the opening 131. For this reason, it is possible to selectively collect cells at an arbitrary position on the culture field 110 while visually checking the entire culture field 110. For example, the cells are collected by inserting a pipette into the culture space 102 through the opening 131 and sucking the cells at an arbitrary position on the culture field 110. Alternatively, for example, a spatula can be inserted into the culture space 102, and cells at an arbitrary position on the culture field 110 can be scraped and collected.

  Furthermore, when the scaffold material 115 is detachably disposed on the hollow fiber membrane mesh 112, the cells may be collected by taking out the scaffold material 115 to which the cells are attached as it is.

  In addition, when a substance such as a test substance is introduced into the culture place 110, the substance can be recovered together with the cells. For example, by selectively taking out a solid substance at an arbitrary position on the culture site 110 with tweezers or the like, it can be recovered from an arbitrary position without any restriction on the size of the substance.

3. Cell Culture Module According to Second Embodiment FIG. 6 shows the configuration of a cell culture module 101B according to the second embodiment of the present invention. In the cell culture module 101A according to the first embodiment described above, the configuration in which the cap 150 is attached to and detached from the opening 131 by engaging the screw groove 132 of the opening 131 and the thread 151 of the cap 150 has been described. The cell culture module 101B according to this embodiment is different from the cell culture module 101A in that the cap 150 is attached to and detached from the opening 131 by screws 161 and screw holes 162.

  The screw hole 162 is provided through the cap 150, the substrate 130, the mold 140, and the substrate 120. The screw holes 162 are provided at positions that do not interfere with the opening 131 and the internal space 102, the hollow fiber membrane 111a (113a), and the hollow fiber membrane 111b (113b) (preferably at the four corners of the cell culture module 1B as shown in the figure). It has been. The cap 150 is attached to the opening 131 by superimposing the cap 150, the substrate 130, the mold 140, and the substrate 120 so that the positions of the screw holes 162 formed in each of the caps 150, and the screws 161 are inserted into the screw holes 162. Do by screwing.

  The cell culturing module 101B is easy to process, and there is no problem of fixation between the thread 51 and the thread groove 132 due to drying of the medium that can occur in the cell culturing module 101A. Note that the screw hole 162 needs to penetrate through the cap 150 to at least a part of the substrate 130 in order to attach the cap 150 to the opening 131. However, it is not essential that the screw holes 162 are formed in the entire board 130, and further up to the mold 140 and the board 120. Further, the number and positions of the screw holes 162 are not limited to the form shown in the drawing, and can be appropriately changed.

4). Cell Culture Module According to Third Embodiment FIG. 7 shows a configuration of a cell culture module 101C according to the third embodiment of the present invention. The cell culture module 101 </ b> C is configured such that the cap 150 is attached to and detached from the opening 131 by engagement between an engagement hole 133 provided in the substrate 130 and an engagement claw 155 provided in the cap 150. This is different from the cell culture module 101A according to the first embodiment described above.

  The engagement hole 133 is provided in the periphery of the opening 131 of the substrate 130. Further, the engagement claw 155 is provided at a position that can correspond to the position of the engagement hole 133 when the cap 150 is closed in the opening 131. The engagement hole 133 and the engagement claw 155 need only have shapes that can be engaged. For example, as shown in the figure, after the engagement claw 155 is inserted into the engagement hole 133, the cap 150 is rotated. In general, a structure in which the engagement claw 155 is engaged with the engagement hole 133 can be adopted.

5. Cell Culture Module According to Fourth Embodiment FIG. 8 shows a configuration of a cell culture module 101D according to the fourth embodiment of the present invention. The cell culture module 101D is different from the cell culture module 101A according to the first embodiment described above in that the cap 150 is attached to and detached from the opening 131 by a fastener 163.

  As the fastener 163, for example, one that holds the cap 150 and the substrate 120 with the opening 131 closed therebetween can be adopted. In this case, a recess (see reference numeral 164 in the figure) for fitting the fastener 163 may be provided on the upper surface of the cap 150 and the lower surface of the substrate 120.

  The cell culture modules 101C and 101D are excellent in the detachability operability of the cap 150. Further, in the cell culture modules 101C and 101D, there is no problem of fixation between the screw thread 151 and the screw groove 132 due to drying of the medium that can occur in the cell culture module 101A.

  As described above, in the cell culture module according to the present invention, when the cap 150 is removed, the entire culture field 110 can be exposed so that it can be visually recognized and operated from the outside. For this reason, in the cell culture module according to the present invention, the cap 150 is removed to expose the entire culture field 110, whereby a substance can be introduced into an arbitrary position of the culture field 110, or a substance at an arbitrary position can be externally provided. It can be taken out without any restriction on the size of the substance. It is also possible to perform measurement by inserting a probe for pH measurement, dissolved oxygen concentration measurement and the like in the internal space 102.

6). Cell Culture Module (1) Schematic Configuration According to Fifth Embodiment FIG. 8 shows a configuration of a cell culture module 201A according to the fifth embodiment of the present invention. In the cell culture module 201A, a substrate 220, 230 and a mold 240 constitute a liquid-tight (or air-tight) culture space (culture region) 202. In the culture space 202, a scaffold material 210 made of a hollow fiber membrane and a fiber assembly is disposed. The scaffold material 210 is fixed to the mold 240 with a potting resin, and the substrates 220 and 230 form a culture space 202 by sandwiching them.

  The substrates 220 and 230 may have any strength suitable for being firmly fixed with the scaffold material 210 and the mold 240 interposed therebetween, and are, for example, acrylic plates. In the cell culture module 201A, each of the substrates 220 and 230 may be composed of one acrylic plate or the like, or may be composed of two or more acrylic plates or the like. Further, when a plurality of culture spaces 202 are provided in the cell culture module 201A, there may be two or more substrates 220 and 230 forming these.

  The material of the mold 240 may be any material as long as sufficient adhesion with the potting resin can be obtained, for example, silicon rubber. Moreover, the potting resin should just have sufficient adhesiveness, for example, various thermosetting resins, such as an epoxy resin, a urethane resin, an unsaturated polyester resin, a silicon resin, a thermoplastic resin, etc. are used.

(2) Flow port A screw hole 221 is formed in the substrate 220. The screw hole 221 penetrates the substrate layer 220 and communicates the outside and the inside culture space 202 of the cell culture module 201A. A part of the screw hole 221 is threaded, and a port (connecting member) 222 is screwed in and attached.

  The screw hole 221 and the port 222 constitute a flow opening for allowing a fluid such as a cell suspension, a culture medium, and a gas to flow from the outside to the inside of the culture space 202 or from the inside to the outside. The screw holes 221 and the ports 222, and the flow holes formed from these are arranged on the substrate 220 at positions corresponding to the scaffold material 210 (specifically, a fiber assembly 215 to be described later). In addition, the two flow openings are disposed at positions that are symmetrical (point-symmetric) with respect to the center of the scaffold material 210 (specifically, a fiber assembly 215 described later).

  The material of the port 222 is not particularly limited, but is usually polycarbonate, polymethyl methacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, polydimethylsiloxane (PDMS), or the like. As the port 222, a so-called syringe port widely used in the medical field can be suitably used.

  A screw hole 231 is formed in the substrate 230, and a port 232 is attached to the screw hole 231. The configuration of the screw hole 231 and the port 232 is the same as that of the screw hole 221 and the port 222.

  The port 222 and the port 232 have a female luer taper, and the inner wall is gradually reduced in diameter in the direction from the outside to the inside of the culture space 202. As a result, the port 222 and the port 232 are connected to a flow feeding means such as a tube or a syringe having a male luer taper with good liquid tightness. Conventionally known standards can be applied to the taper angle and size of the female and male luer tapers.

  The sizes and shapes of the screw holes 221 and 231 and the ports 222 and 232 are not particularly limited, but are, for example, circular and have a diameter of about 500 μm to 5 mm. The female-type luer taper of the port 222 and the port 232 is only required to be provided so that a flow-feeding means having a male-type luer taper can be inserted into at least a part of their inner walls. The screw holes 221 and 231 may be perforations as long as they communicate between the outside of the cell culture module 201A and the culture space 202 and can be attached to the ports 222 and 232.

(3) Connection by Luer Taper FIG. 9 shows a connection mode with a port using a syringe as an example of liquid feeding means. In the syringe denoted by reference numeral 205 in the figure, the fluid held in the syringe body 252 (here, cell suspension is taken as an example) is pushed into the syringe body 252 by pushing the plunger 253 into the syringe body 252. Can be discharged to the outside. A disposable syringe generally used in the medical field can be suitably used as the syringe 205.

  The distal end portion 251 of the syringe 205 includes a male luer taper that decreases in diameter from the connection portion with the port 222 and / or the syringe main body 252 of the cell culture module 201A. The male luer taper of the distal end portion 251 of the syringe 205 and the female luer taper of the port 222 and / or the port 232 of the cell culture module 201A have substantially the same shape and can be fitted. Therefore, the syringe 205 and the cell culture module 201 </ b> A can be easily and fluid-tightly connected by inserting the distal end portion 251 into the port 222 and / or the port 232. Thereby, in the cell culture module 201A, the cell suspension in the syringe 205 can be introduced into the culture space 202 without causing leakage.

  Furthermore, the port 222 and the port 232 can be easily sealed by a luer plug (sealing member) (see FIG. 10). FIG. 13A shows luer plugs 223a and 233a that can be detachably attached to the port 222 and the port 232. FIG. 10B shows luer plugs 223b and 233b having male luer tapers that can be fitted to the female luer tapers of the port 222 and the port 232.

  According to these luer plugs, after introducing the cell suspension in the syringe 205 into the culture space 202, the port 222 and / or the port 232 from which the syringe 205 has been removed can be easily sealed. Note that the luer plug can be attached to the port 222 and the port 232 of the unused cell culture module 201A. In addition, the cell culture module 201A in use can be attached to the port 222 and the port 232 when it is not necessary to attach the syringe 205 or the like.

  In FIG. 9, the syringe 205 is described as an example of the flow feeding means connected to the port 222 and the port 232, but a similar connection using a Luer taper feeds various fluids including a liquid such as a medium and a gas such as oxygen. It can carry out between various flow-feeding means, such as a tube, a pump, and a stopcock. When connecting a tube or a pump, it is connected to the port 222 or the port 232 through a luer fitting having a male luer taper that can be fitted to the female luer taper of the port 222 or the port 232. Moreover, what is necessary is just to use what was provided with the male type luer taper which can be fitted to the female type luer taper of the port 222 or the port 232 for a stopcock.

  By connecting with the flow feeding means by the Luer taper, the cell culture module 201A can easily introduce a fluid such as a cell suspension, a culture medium, or gas without sealing the connection portion using a curable resin or the like (or Discharge).

(4) Scaffold Material Next, the structure of the scaffold material 210 will be described with reference to FIG. The scaffold material 210 includes a hollow fiber membrane mesh 212 in which hollow fiber membranes 211a and 211b (211) are arranged in a mesh shape, a fiber assembly 215, and a hollow fiber membrane 213a and 213b (213) in a mesh shape. The hollow fiber membrane mesh 214 is laminated in this order.

(4-1) Hollow fiber membrane The membrane base polymer, outer diameter, film thickness, etc. of the hollow fiber membrane 111 forming the hollow fiber membrane mesh 212 are as described in the first embodiment.

  The hollow fiber membrane mesh 212 is formed by arranging a plurality of hollow fiber membranes in a mesh shape so that a mesh is formed. As a form of the hollow fiber membrane mesh 212, for example, a plurality of hollow fiber membranes 211a (hollow fiber membranes 211) are arranged in parallel at a predetermined interval to form a sheet, and in a direction perpendicular to the hollow fiber membranes 211a. A plurality of hollow fiber membranes 211b (hollow fiber membranes 211) are arranged in parallel at a predetermined interval to form a sheet shape, thereby forming a mesh shape (FIG. 11A). The number of layers of the sheet made of the hollow fiber membrane 211 in the hollow fiber membrane mesh 212 is not particularly limited, and preferably 2 to 10 layers. In this example, the sheet has two layers (a layer made of the hollow fiber membrane 211a and a layer made of the hollow fiber membrane 211b).

  The hollow fiber membrane mesh 214 and the hollow fiber membrane 213 can use the same thing as the hollow fiber membrane mesh 212 and the hollow fiber membrane 211, and their preferable forms are also the same.

  The form of the hollow fiber membrane meshes 212 and 214 may be selected according to the supply of nutrients and gas through the hollow fiber membranes and the removal of metabolic waste products, and also for the design of the port part when modularizing the scaffold material 210 Dependent. In the scaffold material 210, the hollow fiber membrane 211a of the hollow fiber membrane mesh 212 and the hollow fiber membrane 213a of the hollow fiber membrane mesh 214 are preferably the same type of hollow fiber membrane. Similarly, the hollow fiber membrane 211b of the hollow fiber membrane mesh 212 and the hollow fiber membrane 213b of the hollow fiber membrane mesh 214 are preferably the same type of hollow fiber membrane. By adopting such a form, when the scaffold material 210 is modularized, for example, the hollow fiber membrane 211a and the hollow fiber membrane 213a are gathered and connected to one port portion to form a gas supply hollow fiber membrane. The membrane 211b and the hollow fiber membrane 213b can be gathered and connected to another port portion to be used as a medium supply hollow fiber membrane.

(4-2) Fiber Assembly As the fiber assembly 215, an assembly of nanofibers is preferably used. The material, molecular weight, diameter, and thickness of the nanofiber assembly will be described later.

  The scaffold material 210 has a laminate in which the fiber assembly 215 is disposed between the hollow fiber membrane mesh 212 and the hollow fiber membrane mesh 214 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.

  In the cell culture module 201A, for example, a tube or the like is connected to the ports 222 and 232 described above, so that the hollow fiber membrane meshes 212 and 214 can be used as a route for supplying nutrients and gases and removing metabolic waste products. A third path other than the thread films 211 and 213 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.

(5) Modified Example In this embodiment, an example in which one flow port (screw hole 221 and port 222) is provided on the substrate 220 and one flow port (screw hole 231 and port 232) is provided on the substrate 230. explained. In the cell culture module according to the present invention, the number of flow ports may be one, and in this case, it may be disposed on either the substrate 220 or the substrate 230. In the module for cell culture according to the present invention, three or more flow ports may be provided. In this case, one or more may be provided on the substrate 220 and the substrate 230, respectively.

  In the present embodiment, the example in which the flow hole is configured by the screw holes 221 and 231 and the ports 222 and 232 has been described. In the module for cell culture according to the present invention, the flow port may be configured to have a female luer taper and be capable of being liquid-tightly connected to a flow feeding means having a corresponding male luer taper. As another form of the flow opening, for example, a form in which the holes perforated in the substrates 220 and 230 have a female-type luer taper can be cited.

  Furthermore, in the cell culture module according to the present invention, the number of layers of the hollow fiber membrane mesh and the fiber assembly of the scaffold material 210 is not particularly limited, and the hollow fiber membrane mesh, nanofiber layer, hollow fiber membrane mesh, nanofiber layer The cell culture scaffold material in which the hollow fiber membrane meshes are laminated in this order may be used, or a cell culture scaffold material in which more than this is laminated may be used.

7). Cell Culture Method in Cell Culture Module According to Fifth Embodiment A cell culture method using the above-described cell culture module 201A will be described with reference to FIG.

(1) Cell introduction procedure [Procedure 1]
First, a cell suspension feeding means is connected to the ports 222 and 232 of the cell culture module 201A. Although not shown in FIG. 12, it is assumed that the syringe (syringe 205) described in FIG. 9 is used as the liquid feeding means. However, the liquid feeding means may be a tube or a pump.

  In this procedure, two syringes are prepared. One syringe is connected to the port 222 with the plunger pushed into the syringe body. The other syringe is connected to the port 232 while the syringe body is filled with the cell suspension.

  The connection between the ports 222 and 232 and the syringe can be made fluid-tight by inserting the male luer taper at the tip of the syringe into the female luer taper of the ports 222 and 232.

[Procedure 2]
Next, the plunger of the syringe filled with the cell suspension connected to the port 232 is pushed in to introduce the cell suspension into the culture space 202 (see FIG. 12A). At the same time, the plunger of the syringe connected to the port 222 is pulled to collect at least a part of the cell suspension introduced into the culture space 202 from the culture space 202 into the syringe body.

  Thereby, in the culture space 202, the flow of the cell suspension is formed from the port 232 side to the port 222 side (see arrow in FIG. 12A). As described above, the ports 222 and 232 are disposed on the substrates 220 and 230 at positions corresponding to the scaffold material 210 (specifically, the fiber aggregate 215). For this reason, the cells introduced into the culture space 202 are transported in the flow and dispersed on the fiber assembly 215.

  Furthermore, in the cell culture module 201A, the ports 222 and 232 are disposed at positions that are symmetrical (point-symmetric) with respect to the center of the scaffold material 210 (specifically, the fiber assembly 215). For this reason, the flow of the cell suspension from the port 132 side to the port 222 side can be effectively generated in the entire culture space 202, and the cells are efficiently dispersed on the fiber assembly 215 by the flow. It is possible.

[Procedure 3]
When all or most of the cell suspension in the syringe connected to the port 232 disappears, this time, the plunger of the syringe connected to the port 222 is pushed in, and the cell suspension recovered in the syringe body is re-introduced into the culture space. It introduces into 202 (refer FIG. 12B). At the same time, the plunger of the syringe connected to the port 232 is pulled to collect at least a part of the cell suspension reintroduced into the culture space 202 from the culture space 202 into the syringe body.

  As a result, a flow of cell suspension is formed in the culture space 202 from the port 222 side to the port 232 side (see the arrow in FIG. 12B), introduced into the culture space 202, and dispersed on the fiber assembly 215. The cells are further uniformly dispersed on the fiber assembly 215 by being conveyed in the flow.

[Procedure 4]
When all or most of the cell suspension in the syringe connected to the port 222 has disappeared, repeat steps 2 and 3 as desired. In the procedure 2 and the procedure 3, by alternately generating the flow from the port 232 side to the port 222 side and the flow in the opposite direction, the cells can be uniformly dispersed on the fiber assembly 215.

  The number of repetitions of the procedure 2 and the procedure 3 can be appropriately set depending on conditions such as the type of cells, the amount (concentration) in the suspension, and the target culture density. However, the procedure 4 is not essential, and depending on the above conditions, the cells may be sufficiently uniformly dispersed on the fiber assembly 215 even in the procedures 2 and 3 (without the procedure 4).

[Procedure 5]
Finally, the plungers of both the syringe connected to the port 222 and the syringe connected to the port 232 are pushed in to introduce all the cell suspension into the culture space 202. At this time, the liquid exceeding the volume of the culture space 202 is discharged through the hollow fiber membranes 211 and 213 (see FIG. 11).

  According to the above procedures 1 to 5, it is possible to introduce cells with a highly uniform concentration distribution without unevenly distributing cells in the cell culture module 201A, specifically, on the fiber assembly 215. .

(2) Culture procedure After the introduction of the cells, for example, the hollow fiber membrane 211a and the hollow fiber membrane 213a are gathered and connected to one port portion to supply gas, and the hollow fiber membrane 211b and the hollow fiber membrane 213b are gathered. Cell culture is performed by supplying a medium connected to another port. At this time, since the cells are uniformly introduced into the cell culturing module 201A, the growth rate decreases due to lack of nutrients and oxygen in the high density portion, cell degeneration and necrosis, etc. that occur when the cells are unevenly distributed. Can be prevented.

  Specifically, cell culture uses, for example, a hollow fiber membrane that is a gas separation membrane as the hollow fiber membranes 211a and 213a, and a hollow fiber membrane having substance-selective permeability as the hollow fiber membranes 211b and 213b. While supplying a gas containing oxygen from one end of 213a, the gas is discharged from the other end. Further, nutrients of the cultured cells are supplied from one end of the hollow fiber membranes 211b and 213b, and waste products and metabolites of the cultured cells are discharged from the other end. Thereby, it is also possible to analyze the discharged components online and to evaluate the growth status of the cultured cells continuously while culturing the cultured cells.

  In addition, a test substance is administered to cultured cells from one end of the hollow fiber membranes 211b and 213b, and a solution containing waste products and metabolites is discharged from the other end of the hollow fiber membranes 211b and 213b, and this solution is analyzed. By monitoring, it is possible to evaluate the stress applied to the cultured cells and the state of the cultured cells over time when the test substance is administered.

(3) Modified Example Here, the cell culture method using the cell culture module 201 </ b> A in which one flow port is provided in the substrate 220 and one flow port is provided in the substrate 230 has been described. In the cell culture module according to the present invention, when three or more flow ports are disposed, any two flow ports are selected, and a cell suspension is introduced from one flow port, What is necessary is just to produce the flow of a cell suspension as demonstrated in FIG. 12 by discharging | emitting from the other through-flow port. In this case, it is preferable to select two flow openings that are symmetric with respect to the center of the scaffold material 210 (point symmetry). Alternatively, one or two or more flow ports are made into one group, the flow ports are divided into two groups, a cell suspension is introduced from one flow port group A, and discharged from the other flow port group B. To generate a flow of the cell suspension as described in FIG. Even in this case, it is preferable that the flow port group A and the flow port group B are symmetrical (point-symmetric) with respect to the center of the scaffold material 210 (specifically, the fiber assembly 215). .

8). Cell Culture Module (1) Schematic Configuration According to Sixth Embodiment FIG. 13 shows the configuration of a cell culture module 201B according to the sixth embodiment of the present invention. The cell culture module 201B is different from the cell culture module 201A according to the fifth embodiment in that a screw hole 221 and a port 222, and a through-flow port constituted by these are arranged in the substrate 230. . That is, in the cell culture module 201A, two flow ports are provided on different substrates 220 and 220 (see FIG. 8), whereas in the cell culture module 201B, the two flow ports are the same. It is provided on the substrate 230. Also in the cell culture module 201B, the two flow openings are arranged at positions that are symmetric (in this embodiment, line symmetric) with respect to the center of the scaffold material 210 (specifically, a fiber assembly 215 described later). It is installed.

  The configuration of the cell culture module 201B other than the configuration of the above-described flow port is the same as that of the cell culture module 201A.

(2) Modification In this embodiment, two flow holes (screw hole 221 and port 222, screw hole 231 and port 232) are provided on the substrate 230. In the module for cell culture according to the present invention, three or more through holes may be provided. In this case, the through holes may be provided on either the substrate 220 or the substrate 230.

9. Cell Culture Method in Cell Culture Module According to Sixth Embodiment (1) Cell Introduction Procedure FIG. 12 shows the cell introduction procedure of the cell culture method using the cell culture module 201B. Also in the cell culture module 201B, the flow from the port 232 side to the port 222 side and the flow in the opposite direction are alternately generated according to the above-described procedures 1 to 5, so that the cells can be uniformly distributed on the fiber assembly 215. It can be dispersed and introduced with a highly uniform concentration distribution. As a result, it is possible to perform culture without causing a decrease in cell growth rate or causing degeneration / necrosis.

(2) Modification In the cell culture module according to the present invention, when three or more flow ports are provided, any two flow ports are selected, and the cell suspension is flowed through one of the flow channels. The flow of the cell suspension as described in FIG. 12 may be generated by introducing from the mouth and discharging from the other flow port. Alternatively, one or two or more flow ports are made into one group, the flow ports are divided into two groups, a cell suspension is introduced from one flow port group A, and discharged from the other flow port group B. To generate a flow of the cell suspension as described in FIG. This point is as described in the section of the cell culture method in the cell culture module according to the fifth embodiment.

10. Module for Cell Culture According to Seventh Embodiment The present invention provides a module for cell culture comprising a culture field in which a hollow fiber membrane and a fiber assembly made of nanofibers having an average diameter of 250 nm to 950 nm are arranged. The average diameter of the nanofiber is preferably about 300 nm or about 600 nm, more preferably about 600 nm.

  The hollow fiber membranes are preferably arranged in a mesh shape, and a nanofiber layer composed of an assembly of nanofibers is in contact with the two on the outer surface of the hollow fiber membrane and / or between the hollow fiber membranes (flat plate). ) Are arranged in a liquid-tight manner. A cell inlet for injecting cultured cells and an opening other than the cell inlet are formed at positions corresponding to the nanofiber layer of the cover. The opening is used for supplying nutrients and air by connecting a tube, removing metabolic waste, or observing the culture state by connecting a microscope. The cell inlet and the opening are sealed by a sheet that can be connected to a microscope, a tube, etc., and can be inserted with an injection needle, if necessary.

  In the cell culture module of the present invention, a tube or the like can be connected to the opening as a route for supplying nutrients and gas and removing metabolic waste products. Thereby, cell culture can be performed more easily under more conditions and independently controlling those conditions.

  The module has a hollow fiber membrane mesh with both ends of the hollow fiber membrane gathered, the opening is connected to the port part, and waste products and metabolites etc. discharged from the hollow fiber membrane are introduced to an external device. Analyzing with is possible.

  In addition, a test substance is administered to cultured cells cultured in the module from one end of the hollow fiber membrane, and a solution containing waste products and metabolites is discharged from the other end of the hollow fiber membrane, and this solution is analyzed and monitored. By doing so, the stress received by the cultured cells when the test substance is administered and the state of the cultured cells can be evaluated over time. In this case, administration of the test substance and discharge of the solution containing waste products and metabolites may be performed by a tube connected to the opening.

  The cell culturing module of the present invention described above has a cell culture scaffolding material placed between two covers and sealed in a liquid-tight manner, thereby dividing the culture region into a fixed region. Condition control is easy. 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.

  Preferred configurations of the cell culture module according to the seventh embodiment of the present invention include, for example, JP 2009-100, JP 2010-14896, JP 2010-148497, and JP 2011-239756. It is described in the publication.

11. Cell Culture Device The present invention also provides a (circulating) cell culture device including the cell culture module of the present invention. The apparatus for cell culture of the present invention includes a cell culture module, oxygen supply means, and medium supply means. Moreover, a monitoring means, an analysis means, a sampling means, etc. may be included as needed. An example of the apparatus for cell culture of the present invention is shown in FIG. 16 (hereinafter, the numbers in parentheses correspond to the numbers in FIG. 16).

(1) Oxygen supply means The oxygen supply means is means for supplying oxygen to the cells through the hollow fiber membrane, and includes, for example, an air pump 302 for sending out oxygen, a tube 303 constituting an oxygen supply circuit to the module 301, A filter 304 for preventing fine dust (for example, a diameter of 0.22 μm) from entering the hollow fiber membrane, a moisturizing water storage tank 305, and the like can be included.

(2) Medium supply means The medium supply means includes, for example, a storage tank (medium bottle 306) for the medium to be supplied, a medium supply for supplying the medium to the cells through the hollow fiber membrane and circulating while removing metabolic waste products. A (circulation) pump 307 and a tube 308 as a medium supply (circulation) circuit are included.

(3) Sampling means The sampling means is means for appropriately collecting a medium from the cell culture module 301 and includes a sampling port 309 connected to the cell culture module 301, a storage tank for the collected medium, and the like 310.

(4) Others In addition to the above-mentioned means, the cell culture module of the present invention is a means for monitoring the sampled medium and circulating medium components, test substances, metabolite concentrations, etc. over time (monitoring means) Means for analyzing (analyzing means) may be included.

  The cell culture module of the present invention can cultivate cells for a long period of time while preventing their dedifferentiation and maintaining their functions.

  The cells used in the cell culture module of the present invention are not particularly limited, but include adherent animal cells such as human fetal foreskin fibroblasts, Chinese hamster lung fibroblasts, chicken fetal fibroblasts, and Syrian hamster neonatal kidney cells. Examples include fibroblasts, human cervical cancer cells, Chinese hamster ovary cells, mouse breast cancer cells, epithelial cells such as African green monkey kidney cells, and vascular endothelial cells. In particular, culture of primary cells such as hepatocytes, coronary artery endothelial cells, Golden Hamter Langerhans cell group, rat thymic epithelial cells, rat renal epithelial cells, etc., which are difficult to culture while maintaining the function of the cells It is suitable for.

  The cell culture module of the present invention is also applicable to frozen cells. Frozen cells generally have a slow growth rate and are difficult to maintain for a long time. In particular, it is known that the function of hepatocytes is greatly reduced within a few days. According to the cell culture method of the present invention, it is possible to perform culture for a long time while maintaining a high function.

  The medium used for the culture is determined according to the type of the cultured cell, and may be any medium that is usually used as a medium for the cell. The culture method of the present invention can also be used for serum-free culture.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited by the following description.

Example 1 Cell Culture Using Cell Culture Scaffold Material Consisting of Nanofiber Layer Fabrication and analysis of nonwoven fabric 1.1 Fabrication of nanofiber layer
Add 10% to 15% polyacrylonitrile powder by weight to dimethylacetamide (Wako Pure Chemical Industries) heated in a constant temperature water bath at 80 ° C, stir well to dissolve completely, and leave it at room temperature for stable poly An acrylonitrile dope was prepared. Furthermore, the dope was filled in a disposable syringe (Terumo 20 ml, SS-20ESz), a nozzle (18G × 11/2) was mounted, and the dope was installed in an electrocross pinning apparatus (Katek).

  In the apparatus, silver paper is stuck around the target (roll). The voltage is 12kV, the plunger push-out speed of the syringe is 0.03mm / min, the distance to the target (roll) is 200mm, the target rotation speed is 1m / min, the left and right reciprocating movement (traverse) speed is 10cm / min, and the spinning time is 120min. To prepare a nanofiber layer (nonwoven fabric). Finally, the non-woven fabric for each silver paper was peeled off, and at the time of experiment use, it was collected from the silver paper with a necessary sheet with tweezers and used for a cell culture experiment described later.

1.2 Analysis of non-woven fabric (analysis of average diameter of single fiber of nanofiber)
The nonwoven fabric was observed using a scanning electron microscope JSM-6060A manufactured by JEOL. 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 surface width of any 20 nanofibers in the SEM photograph is obtained, and the average value is calculated as the diameter of the nanofiber single fiber ( Thread diameter). Hereinafter, the nonwoven fabric produced with the nanofiber of each thread diameter is described using the numbers (No. 1 to 6) shown in Table 1.

2. Cell culture experiment 2.1 Materials and equipment used [equipment used]
(1) Cell culture module The cell culture module was produced according to the previous report (Unexamined-Japanese-Patent No. 2011-239756). A hollow fiber membrane for oxygen supply and a hollow fiber membrane for nutrient supply of the medium are arranged inside the module, and a nanofiber nonwoven fabric sheet for cell attachment is charged and sealed between the hollow fiber membranes. Cells adhering to the non-woven fabric can be proliferated by always being supplied with abundant nutrients and oxygen from a large amount of medium in the medium bottle and the air pump in a three-dimensional culture state.

(2) Cell culture device A circulating cell culture device (FIG. 16) having the following configuration was used.
・ One tube pump (perista pump) for medium circulation Flow rate 8mL / hr, 60mL / hr
・ 1 pump for air supply Flow rate 1.2L / hr, 3L / hr (Pump is filter (ADVANTEC, pore size 0.2μm), so that suction gas of 0.2μm or more will not enter the hollow fiber membrane. Yes.)
・ 3 bottles (medium bottle, air disposal empty bottle, intake air moisturizing water bottle)
・ Air line and culture medium circulation line (both are silicon tubes)
・ Sampling port (port for collecting medium)
・ Tube joint (resin)
・ Plate for placing the device

(3) Others ・ CO2 incubator: Direct incubator (Thermo)
・ Air pump: CHIKARA Nα1500 (NISSO)
・ Medium pump: Low flow rate SJ-1211L type (TOTO)
・ SEM: KEYENCE

[cell]
-Hc cells: normal human fetal liver cells (purchased from DS Pharma Biomedical)
・ Frozen human hepatocytes: Adult hepatocytes sold by BIOPREDIC International, France (Purchased Human Cryopreserved Hepatocytes, Bach: HEP187082 from domestic CA Corporation)

[Culture medium]
・ Hc cell culture medium: GIBCO D-MEM / F-12 ・ 11330032 Add 500% Fetal Bovine Serum, Hyclone ・ 10437028/10% of the required medium volume, and Nacalai Tesque ・ Sodium bicarbonate and injection Penicillin G potassium (manufactured by Meiji Seika Co., Ltd.), streptomycin sulfate (Meiji Seika Co., Ltd.), and Human-acidic FGF (Cosmo Bio) were used.
-Cryopreservation medium: Cryopreservation solution for normal cells-R-055-50 (Kurabo)
・ Adult hepatocyte culture medium: Incubation medium (BIOPREDIC)
Long-term culture medium (BIOPREDIC)
Thawing medium (BIOPREDIC)
Celling medium (Seeding medium) (BIOPREDIC)

[Reagent / Equipment]
・ PBS buffer (Invitrogen)
・ Trypan Blue: Trypan Blue solution ・ T8154 (20mL) (SIGMA)
・ Trypsin: Trypsin-EDTA (Cat: 27250-018, Lot: 1219490) (GIBCO BRL)
・ Sodium n-butyrate (for biochemical experiments) (SIGMA)
・ Dicumarol (Wako Pure Chemical Industries)
・ Β-glucuronidase / arylsulfatase (ROCHE)
・ Benzyloxyresorufin (Molecular Probes)
・ Resorufin (Molecular Probes)
・ Rifampicin for biochemical experiments (Wako Pure Chemical Industries)
・ Glucose measurement kit: Glucose CII-Test Wako ・ 439-90901 (WAKO)
・ Hematocytometer (including cover glass): Birkelturk hemocytometer ・ A112 (Fuji Science Industry Co., Ltd.)

2.2 Cell culture (1) Functional evaluation by stationary culture of Hc cell-derived hepatoblasts Nanofiber nonwoven fabrics having various thread diameters (average diameters) were prepared according to the method of 1.1. The thawed cells were seeded on each non-woven fabric, and the functional expression of the cells was analyzed by the following method.

  The petri dish (35 mm in diameter) was laid with a non-woven fabric for scaffolding on the bottom and fixed to the bottom with a 2 mm diameter silicon tube. Next, 9 mL of a medium preliminarily warmed to 37 ° C. was dispensed into a 15 mL centrifuge tube, the cell-containing tube was taken out of the freezer, and warmed in a constant temperature water bath. It was removed after warming for about 0.5 minutes, and thawed thoroughly with a pipette. Further, after centrifugation with a swing centrifuge (1000 G), the supernatant was removed, and the cells precipitated with a pipette were suspended in the medium. The cells were stained with trypan blue, the number of cells was counted with a hemocytometer under a phase contrast microscope, and the cell density in the suspension medium was estimated.

  The cells were diluted with a medium to a density suitable for culture (104 cells to 105 cells / 2 mL), and seeded with 2 mL of the medium suspension added to a petri dish with a nonwoven fabric fixed to the bottom. The lid was covered, and the medium was gently shaken several times back and forth and left and right to shake the medium on the non-woven fabric on the bottom of the petri dish so that the cells were evenly dispersed. Then, the culture was started after standing in a CO2 incubator. After 5 to 12 hours, the medium was changed, cells that did not adhere to the bottom surface were removed together with the medium, and left in a CO2 incubator.

  The medium was changed between 2 and 3 days. On the 7th day of culture, the medium was replaced with a medium supplemented with 1 mM sodium butyrate. On the 8th day, 1 mM dairy sodium and cytochrome 3A4 (CYP3A4) enzyme inducer The medium was replaced with rifampsin 10 μM, and on the 14th day, the substrate benzyloxylysorphine was added and reacted at 37 ° C. for 30 minutes. The reaction solution was treated with β-glucuronidase and arylsulfatase acetate buffer and precipitated. The function of the cultured cells was measured using the value obtained by quantifying the product resorufin dissolved in ethanol by fluorescence measurement (excitation 550 nm fluorescence 600 nm) as the enzyme activity. For the calculation of the concentration, a calibration curve prepared with ethanol in which resorufin was dissolved was used.

(2) Functional evaluation by stationary culture of human frozen hepatocytes As a method of cell thawing, a cell thawing medium was used (30 ml) to dilute DMSO until the ampoule was thawed, as in Hc. Centrifugation was performed at × g for 1 minute, and only the cell thawing medium was removed. Then, the cells were suspended in a seeding medium (1 ml), seeded in a petri dish and used.

  As in (1), No. 2, no. 4, no. 6 non-woven fabrics (control: petri dish only) were spread, and thawed adult hepatocytes were replaced with a seeding medium in a thawing medium, and then seeded and cultured in 105 cells / Petri dish. At this time, add 2 mL of stationary culture medium, replace the medium with rifampicin 10 μM medium in 24 hr immediately after seeding to remove the non-adherent floating cells, and replace the medium with rifampicin 10 μM daily after seeding. did. Cell functions were compared by activity analysis of the CYP3A4 enzyme described above.

(3) Evaluation of proliferation rate by stationary culture of Hc cells The thawed cells were seeded on a nonwoven fabric prepared according to 1.1, and the proliferation rate of the cells was analyzed.
Nonwoven fabrics No. 1 to No. 1 listed in Table 1. Hc cells (105 cells) were seeded on a petri dish with 6 fixed on the bottom, and a nonwoven fabric was spread on the bottom of a 35 mm diameter petri dish, and Hc cells were seeded on 105 cells / pet. At this time, 2 mL of medium for Hc cells was added, the medium was changed for 5 hr immediately after seeding to remove cells that did not adhere and floated, and the medium was changed every day after 3 days, 5 days, and 7 days of culture. The number of cells was analyzed and compared to the concentration at which glucose in the supplemented medium decreased around the elapsed days.

(4) Functional evaluation by reflux culture of Hc cell-derived hepatoblasts Hc cells were proliferated and cultured in reflux cultures, and were subjected to long-term maintenance culture as liver parenchymal cells by induction and maturation, and the cell functions were measured. .

1) Cell preparation Hc cells (fetal human hepatocytes) were thawed and seeded in a petri dish, and it was confirmed that the cells were proliferating in the petri dish.

2) Preparation of culture The line to which the joint was connected, the bottle, and the filter were packaged with silver paper, and sterilized by autoclaving as it was. In a clean bench, the packaging was removed and connected to the developed cell culture module to assemble a reflux culture apparatus (FIG. 16).

  Ethanol water was circulated in the medium line to sterilize the inside of the module, the ethanol water was discarded, and water was circulated in the medium line to completely remove the ethanol water. Water was discarded and the medium was circulated in the medium line to confirm stable operation.

3) Circulating cell culture The apparatus was installed in a CO2 incubator and circulated with a medium to confirm the stable operation of the apparatus. The medium volume in the medium bottle was started at 40 mL.

  With 1 mL of disposable syringe, 26 needles were stabbed into a silicone rubber sheet, and a medium in which predetermined cells were dispersed was injected into the module. In the module, cells were attached to a nanofiber nonwoven fabric, and the cells were grown in three-dimensional culture. Thereafter, reflux culture was started in a CO2 incubator.

  The culture medium was sampled from the sampling port, the glucose concentration was analyzed, the number of cells in the module was estimated from the glucose consumption rate, and the change over time was traced. Similarly, the increase in ammonia, which is an inhibitor of cell growth, was traced in the same manner, and the medium was changed when it did not exceed 2 mM. The first medium change was performed for the first 9 days, and when no consumption of 50 mg / dL or more of glucose was observed, the medium was changed regardless of the glucose concentration. The medium was replaced with fresh medium when the glucose concentration of the medium fell below 250 mg / dL and when the consumption rate of glucose was 50 mg / dL or more per day. When the cells grew and the glucose consumption rate improved, the amount of medium in the medium bottle was increased as necessary in accordance with the frequency of medium exchange.

  When the cell density reached 1 × 10 8 cells / mL, sodium butyrate was added to induce Hc cells into hepatoblasts and matured into hepatocytes.

  The cell function was quantitatively evaluated from the relationship between the given substrate and the reaction rate by inducing cytochrome 3A4 enzyme in the cell. Specifically, in addition to sodium butyrate in the medium, the inducer rifampsin was further added to induce cytochrome 3A4 enzyme in the cells, and the substrate benzyloxylysorphine was added and reacted at 37 ° C. for 30 minutes. . The reaction solution was treated with β-glucuronidase and arylsulfatase acetate buffer, the precipitate was removed, and the product resorufin dissolved in ethanol (special grade, Wako Pure Chemical Industries) was measured by fluorescence measurement (excitation 550nm fluorescence 600nm) Quantified. This value was defined as the enzyme activity, and the function of the cultured cells was measured. For the calculation of the concentration, a calibration curve prepared with ethanol in which resorufin was dissolved was used.

  Sodium butyrate was further added to the medium to maintain and culture hepatoblasts, and the function of the cells was analyzed by the method described above.

3. Results (1) Functional evaluation by stationary culture of Hc cell-derived hepatoblasts 1-No. FIG. 17 (upper) shows cytochrome 3A4 enzyme activity in cells when Hc cells were cultured using 6 as a scaffold. In addition, hepatoblasts derived from Hc cells were treated with functional nonwoven fabric No. 1-No. FIG. 4 (bottom) shows cytochrome 3A4 enzyme activity when 6 is maintained as a scaffold.

  Nonwoven fabric No. 2 and No. When cultured with 4 hepatoblasts expressed high function (FIG. 17). On the other hand, no. In case of 5, the function of hepatoblasts was low (bottom of FIG. 17). The cell density (arrival density) was not different before and after maturation from Hc cells to hepatoblasts.

Moreover, the result of having observed the cell growth state in each nonwoven fabric with a position scanning microscope in FIG. 18 is shown.
No. In 1 (average diameter of about 170 nm), the entire surface of the nonwoven fabric was covered with cells and multilayered on the surface, and the cell shape was broken. This result suggests that cell function (cytochrome 3A4 enzyme activity) may be lowered when the cell density is too high.

  No. In 2 (average diameter of about 300 nm), in the loose layer cross section of the nonwoven fabric, cells grew in a spherical shape inside the nonwoven fabric, and high development of microvilli (small balls) was observed. These suggest high cell functions.

  No. In 3 (average diameter of about 500 nm), the voids were large, the cells grew along the fibers, and no microvilli were seen.

  No. In 4 (average diameter of about 600 nm), cells grew on the surface of the nonwoven fabric, and agglomeration of several cells was confirmed. Although there were not many cells inside the nonwoven fabric, the cells inside the nonwoven were surrounded by fibers and microvilli developed. These suggest high cell functions.

  No. In 5 (average diameter of about 1000 nm), the cells grew in a large space and not in a dense fiber. It is thought that it was growing on the nonwoven fabric under the fluff on the surface of the nonwoven fabric to have submerged a little. In addition, the microvilli were not seen in the cell. These suggest a low function of the cell.

  No. In 6 (average diameter of about 1300 nm), the cells were densely proliferated by entwining the fibers in the inner multilayer of the nonwoven fabric. No microvilli were seen in the cells. This result suggests that cell function (cytochrome 3A4 enzyme activity) may be lowered when the cell density is too high.

(2) Functional evaluation by stationary culture of human frozen hepatocytes 2, No. 4, no. FIG. 19 shows the cytochrome 3A4 enzyme activity in the cells when cultured using the nonwoven fabric of No. 6 (control: petri dish only).
In the case of using human frozen hepatocytes, as in the case of Hc cell-derived hepatoblasts, non-woven fabric No. 2 and No. 4, the cell expresses a high function, and has a nonwoven fabric No. In the case of 6 and petri dish, only a low function was expressed (FIG. 19).

(3) Evaluation of proliferation rate by stationary culture of Hc cells 2-No. FIG. 20 shows the daily change in cell density when cultured using the nonwoven fabric of No. 6. No. 2-No. No. 5 compared with the nonwoven fabric of No. 5. A good growth rate was observed in the nonwoven fabric No. 6, and it was confirmed that the growth rate was suppressed in the nonwoven fabric having a yarn diameter of φ1000 nm or less. In addition, no. Only the nonwoven fabric of 6 was confirmed to increase the growth rate (results are not shown).

(4) Function maintenance by reflux culture of Hc cell-derived hepatoblasts FIG. 21 shows changes in cell density over time when perfusion culture is performed using the apparatus shown in FIG. It was confirmed that by using the cell culture module of the present invention, Hc cells were grown to a high density of 2.5 × 10 8 cells / mL by circulating culture.

(5) Function maintenance by reflux culture of frozen human hepatocytes Adult hepatocytes were thawed in the same manner as stationary culture, seeded in a module from a seeding port, and refluxed cell culture was performed. Using the medium for long-term maintenance, the medium replacement frequency was 80 ml of medium daily until the third day of culture, and 100 mL daily after the fourth day of culture.

  Seed human frozen hepatocytes at 4.8 × 10 6 cells / module. FIG. 22 shows the daily change in cell density when the perfusion culture is performed using the apparatus shown in FIG. It was confirmed that by using the cell culture module of the present invention, long-term maintenance culture for 33 days could be performed at the cell density seeded by circulating culture.

  Further, when the dissolved oxygen concentration of the line was analyzed during the long-term culture, the cells were maintained while being stably maintained at 5.8 ppm by consuming the initial dissolved oxygen concentration of 6.6 ppm as shown in FIG. It was confirmed that it was made.

4). Discussion With the cell culture scaffold material (nanofiber nonwoven fabric) of the present invention, highly functional hepatocytes could be obtained from Hc cells. Moreover, when the yarn diameter (average diameter) of the nonwoven fabric used was 250-950 nm, high functional expression could be realized.

  By circulating culture with the apparatus using the module for cell culture of the present invention, Hc cells were induced to differentiate into hepatoblasts showing high function (CYP3A4 enzyme activity), and this could be cultured and maintained for a long time. Also, human frozen hepatocytes could be cultured and maintained for a long period of time while maintaining high function by carrying out perfusion culture using an apparatus using the module for cell culture of the present invention.

  According to the present invention, cells can be efficiently cultured for a long period of time while maintaining their original functions. Therefore, the present invention is useful for the construction of a cell culture evaluation system that substitutes for animal experiments for evaluating the efficacy, toxicity, and safety of substances such as pharmaceuticals, chemical substances, and cosmetics.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

101A, 101B, 101C, 101D: Module for cell culture, 110: Culture place, 111, 111a, 111b, 113, 113a, 113b: Hollow fiber membrane, 112, 114: Hollow fiber membrane mesh, 115: Scaffold material, 102: Internal space, 120, 130: substrate, 131: opening, 132: screw groove, 133: engagement hole, 140: mold, 150: lid (cap), 151: screw thread, 152: sealing member, 153: Flow port, 154: port, 155: engagement claw, 161: screw, 162: screw hole, 163: fastener, 164: recess 201A, 201B: module for cell culture, 210: scaffold material, 211, 211a, 211b 213, 213a, 213b: hollow fiber membrane, 212, 214: hollow fiber membrane mesh, 215: fiber assembly, 202: culture space (culture Area), 220, 230: substrate, 221, 231: screw hole, 222, 232: port, 223a, 223b, 233a, 233b: luer plug, 240: mold, 250: syringe, 251: tip, 252: syringe Main body, 253: Plunger 301: Module for cell culture, 302: Air pump, 303: Tube constituting oxygen supply circuit, 304: Filter, 305: Reservoir for moisturizing water, 306: Reservoir for medium, 307: Supply of medium (Circulation) pump, 308: tube as medium supply (circulation) circuit, 309: sampling port, 310: storage tank of collected medium, etc.

Claims (14)

A culture site where a hollow fiber membrane is disposed;
A frame constituting an internal space including the culture site;
A removable lid,
With
A cell culture module in which an opening to which the lid is attached is provided with a size capable of exposing the entire culture site.   The module for cell culture according to claim 1, wherein the lid and / or the frame has a flow passage through which a fluid flows from the outside to the internal space or from the internal space to the outside.   The cell culture module according to claim 1 or 2, wherein the flow-through port has a female luer taper whose diameter decreases from the outside toward the internal space.   The cell culture module according to claim 3, wherein a plurality of flow openings are provided. Two substrates,
A culture site in which a hollow fiber membrane is disposed in a culture region formed between the substrates;
A plurality of flow ports for allowing fluid to flow from the outside to the inside of the culture region or from the inside to the outside, and the flow ports are disposed at positions corresponding to the culture field of the substrate, And the module for cell culture provided with the female type lure taper diameter-reducing toward the inside from the exterior of the said culture | cultivation area | region.   The cell culture module according to claim 5, wherein the plurality of flow openings are provided on different substrates.   The cell culture module according to claim 4 or 6, wherein the through-flow port is provided symmetrically with respect to the center of the culture field.   The module for cell culture according to any one of claims 1 to 7, wherein the fiber assembly is disposed so as to be separable from the hollow fiber membrane.   The cell culture module according to claim 8, wherein the fiber assembly is made of nanofibers.   The module for cell culture according to claim 9, wherein the fiber assembly is composed of nanofibers having an average diameter of 250 nm to 950 nm. Hollow fiber membranes,
A module for cell culture comprising a culture field in which fiber assemblies composed of nanofibers having an average diameter of 250 nm to 950 nm are arranged.   The cell culture module according to any one of claims 1 to 11, wherein the hollow fiber membrane includes a gas supply hollow fiber membrane and a medium supply hollow fiber membrane.   An apparatus for cell culture, comprising the module for cell culture according to any one of claims 1 to 12, an oxygen supply means, and a medium supply means.   Furthermore, the apparatus for cell culture of Claim 13 containing any 1 or 2 or more means chosen from a monitoring means, an analysis means, and a sampling means.

Images