JP2013212088A - Hydrophilized base and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a hydrophilized base capable of easily imparting low adsorption properties at a low cost even for a large area product such as a flask.SOLUTION: A method produces a hydrophilized base having a base and in which a hydrophilized film having a film base material layer, a primer layer including a polysiloxane provided on the film base material layer, and a hydrophilic polymer layer including a polyalkylene glycol provided on the primer layer is fixed to at least one region of a surface of the base. The method includes the following steps: a cutting step of cutting a long-shaped hydrophilized film to acquire a leaf-like hydrophilized film; and a hydrophilized film fixing step of fixing the leaf-like hydrophilized film to the surface of the base.

Description

  The present invention relates to a hydrophilic substrate and a method for producing the same.

  Conventionally, for example, culture of blood cells, cancer cells, and the like is sometimes performed by suspension culture. In suspension culture, cells are seeded in a culture solution, and the cells are cultured in a suspended state while appropriately agitating the culture solution so that the cells do not adhere to the surface of the culture vessel. However, the cell may adhere to the bottom surface of the culture container or the like depending on the material of the culture container and the like, which may impair the properties of the cell. Therefore, it has been desired to develop a technique for making the surface of the culture vessel a low adsorptive surface in which cell adhesion is suppressed.

  In addition, an enzyme-linked immunosorbent assay (ELISA), which is a kind of immunoassay, is used in a wide range of fields including drug discovery, diagnosis, environmental measurement, and food. ELISA is known as a particularly sensitive method among many immunoassays, but in reality, the expected sensitivity cannot be obtained due to non-specific adsorption of biomolecules (proteins) to a solid support. There are also many. In particular, a complex such as an enzyme-labeled antibody is easily adsorbed on a solid support, which is a major cause of noise in ELISA. A blocking treatment with bovine serum albumin (BSA) to prevent such nonspecific adsorption is attempted, but the effect is limited. Therefore, it has been desired to develop a technique for making the surface of the solid support a low-adsorbing surface in which protein adsorption is suppressed.

On the other hand, (Patent Document 1) has a hydrophilic layer having a contact angle with water of 10 degrees or less on the inner surface of the container in contact with the culture solution, and the cell on the inner surface of the container is in contact with the portion. A cell culture vessel characterized by having a culture bed suitable for cell culture is disclosed. Further, as a material constituting the hydrophilic layer, polyhydroxyalkyl methacrylate, polyoxy-C ₂ -C ₄ -alkylene group-containing methacrylate polymer and the like are disclosed.

JP 2004-290111 A

  The cell culture container described in the above (Patent Document 1) obtains protein adsorption preventing performance by providing a coating made of a hydrophilic material such as polyhydroxyalkyl methacrylate on the surface of the container serving as a substrate. The performance was insufficient and there was room for further improvement. Accordingly, an object of the present invention is to provide a low-adsorbing hydrophilic substrate in which cell adhesion and protein adsorption are effectively suppressed, and a method for producing the same.

  In the above (Patent Document 1), when forming a coating of a hydrophilic material, it is necessary to prepare a solution in which the hydrophilic material is dissolved, and apply the solution directly to the surface of a substrate such as a flask and dry it. It was. Therefore, in particular, when the surface on which the coating is to be formed has a large area, a large amount of solution is required to apply the wet coating, which causes a problem that the production efficiency is lowered and the cost is increased. Accordingly, an object of the present invention is to provide a hydrophilic substrate and a method for producing the same, which can impart low adsorption performance easily and inexpensively even for a large-area product such as a flask.

  As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, an excellent low adsorption performance for cells and proteins can be obtained by providing a hydrophilic polymer layer containing polyalkylene glycol on the substrate surface via polysiloxane. The present invention has been completed.

  Further, the present invention has found that a film-like base material layer provided with a hydrophilic polymer layer via polysiloxane can be attached to the surface of a substrate such as a flask to provide low adsorption performance at low cost. Was completed. That is, the present invention includes the following inventions.

(1) provided with a substrate;
A hydrophilic polymer containing a film base layer, a primer layer containing polysiloxane provided on the film base layer, and a polyalkylene glycol provided on the primer layer in at least one region of the surface of the substrate A method for producing a hydrophilic substrate on which a hydrophilic film having a layer is fixed,
The following steps:
A cutting process for obtaining a sheet-like hydrophilized film by cutting a long hydrophilized film;
The said method including the hydrophilization film fixing process which fixes a sheet-like hydrophilization film on the surface of a base | substrate.
(2) further comprising a hydrophilized film production process for producing an elongated hydrophilized film,
The hydrophilized film manufacturing process unwinds and conveys a long film base layer wound in a roll shape, and sequentially forms a primer layer and a hydrophilic polymer layer on the unrolled film base layer. The method for producing a hydrophilic substrate according to the above (1), comprising a step of winding the subsequent hydrophilic film into a roll.
(3) The method for producing a hydrophilic substrate according to (1) or (2), wherein the hydrophilic film fixing step includes a step of sticking a sheet-like hydrophilic film to the surface of the substrate via an adhesive.
(4) The above-mentioned (1), wherein the hydrophilized film fixing step includes a step of obtaining a hydrophilized substrate by filling a resin in an injection mold in which a sheet-like hydrophilized film is previously disposed and molding the substrate. Or the manufacturing method of the hydrophilization base | substrate as described in (2).
(5) provided with a container-shaped substrate;
A hydrophilized substrate in which a primer layer containing polysiloxane and a hydrophilic polymer layer containing polyalkylene glycol are provided on at least one region of the inner surface of the substrate.
(6) A container-shaped base is provided,
At least one region of the inner surface of the substrate has a film base layer, a primer layer containing polysiloxane provided on the film base layer, and a hydrophilic containing polyalkylene glycol provided on the primer layer A hydrophilic substrate on which a hydrophilic film having a polymer layer is disposed.
(7) The hydrophilized substrate according to (5) or (6), which is for cell culture.
(8) A long shape having a film base layer, a primer layer containing polysiloxane provided on the film base layer, and a hydrophilic polymer layer containing polyalkylene glycol provided on the primer layer A hydrophilic film of
An elongated release film disposed on the side of the hydrophilic film on which the primer layer and the hydrophilic polymer layer are not provided,
The hydrophilic film is formed with a plurality of regions surrounded by a cut line that can be separated as sheet pieces, and the cut line extends from the surface of the hydrophilic film in the thickness direction of the film. The half-cut line is formed up to a position in the thickness direction that cuts the hydrophilic film but does not completely cut the release film.
Half-cut hydrophilized film with long release film.

  The hydrophilic substrate of the present invention can effectively suppress undesirable cell adhesion and protein adsorption. Moreover, even if it is a large-area product such as a flask, low adsorption performance can be imparted efficiently and inexpensively.

FIG. 1A is a perspective view showing the structure of a flask-type hydrophilic substrate produced according to the present invention. FIG. 1B shows a cross-sectional view taken along the line I-I ′ of the substrate 100, and FIG. 1C shows a cross-sectional view taken along the line II-II ′. FIG. 2A is a perspective view showing the structure of a flask-type hydrophilic substrate produced according to the present invention. 2B shows a cross-sectional view taken along the line III-III ′ of the substrate 200, and FIG. 1C shows a cross-sectional view taken along the line IV-IV ′. FIG. 3A is a perspective view showing the structure of a bottle-type hydrophilic substrate produced according to the present invention. FIG. 3B shows the structure of a member for producing a bottle-type hydrophilic substrate produced according to the present invention. It is a figure explaining the process of joining a 1st member and one other member, and manufacturing a flask type hydrophilic base | substrate. It is a figure explaining the process of joining a 1st member and two other members, and manufacturing a flask type hydrophilic base | substrate. It is a schematic diagram which shows one Embodiment of the cross section of a hydrophilization film. It is a figure which shows typically a mode that the hydrophilic film is joined to the surface of the 1st member through the adhesive agent. It is a schematic diagram which shows one Embodiment of the cross section of a hydrophilization film. It is a schematic diagram which shows one Embodiment of the cross section of a hydrophilization film. It is a figure which shows the outline of the method of manufacturing a hydrophilic film. It is a figure which shows typically one Embodiment of a cutting process. FIG. 12A is a schematic cross-sectional view showing that the laminate is half-cut in the cutting step. FIG. 12B is a schematic view showing that a long laminate is half-cut in a cutting step to obtain a long-half-cut hydrophilized film with a half-cut release film. It is a schematic diagram which shows one Embodiment of a hydrophilization film fixing process which fixes a hydrophilization film via an adhesive, and a member joining process. It is a schematic diagram which shows one Embodiment and the member joining process of a hydrophilic film fixing process which fixes a hydrophilic film by heat seal. It is a schematic diagram which shows the outline | summary of embodiment which fixes a hydrophilization film to a 1st member by ultrasonic welding. It is a schematic diagram which shows one Embodiment and a member joining process of the hydrophilic film fixing process which fixes a hydrophilic film by ultrasonic welding. It is a schematic diagram which shows one Embodiment and the member joining process of the hydrophilic film fixing process which fixes a hydrophilic film by laser welding. It is a schematic diagram which shows one Embodiment and member joining process of a hydrophilic film fixing process which fixes a hydrophilic film by integration by in-mold molding. It is a schematic diagram which shows other one Embodiment and the member joining process of the hydrophilic film fixing process which fixes a hydrophilic film by integration by in-mold shaping | molding. It is a flowchart which shows the outline of the manufacturing method of the hydrophilization base | substrate of this invention. It is a graph which shows the measurement result of the protein adsorption amount about Example 1 and Comparative Example 1. It is a figure which shows the observation result of the cell adhesiveness about Example 1 and Comparative Example 1. FIG. It is a graph which shows the measurement result of the protein adsorption amount about Example 2 and Comparative Example 2. It is a figure which shows the observation result of the cell adhesiveness about Example 2 and Comparative Example 2. FIG.

Hereinafter, the present invention will be described in detail.
The hydrophilized substrate of the present invention comprises a substrate, and a primer layer containing polysiloxane and a hydrophilic polymer layer containing polyalkylene glycol provided on the primer layer in at least one region of the inner surface of the substrate. It is provided.

  As another embodiment, the hydrophilized substrate of the present invention comprises a substrate, and a film substrate layer and a polysiloxane provided on the film substrate layer are provided in at least one region of the inner surface of the substrate. A hydrophilic film having a primer layer containing and a hydrophilic polymer layer containing polyalkylene glycol provided on the primer layer is disposed. In this case, since a hydrophilic film provided with a primer layer and a hydrophilic polymer layer is disposed as a separate member on a general substrate such as a flask, low adsorption performance can be imparted easily and inexpensively.

  As the substrate, any substrate that is desired to suppress cell adhesion or protein adsorption can be used. Examples include petri dishes, flasks, multi-well plates, cell factory type culture containers, roller bottle type culture containers used for suspension culture.

  An example of a preferred embodiment of the hydrophilic substrate according to the present invention is shown in FIG. The embodiment of FIG. 1 is an example in which a hydrophilic film is disposed inside a container-shaped substrate. A base body 100 shown in FIG. 1A includes at least a bottom portion 101, a side wall portion 102 erected on the periphery of the bottom portion 101, and a top surface portion 103 that is joined to the upper end portion of the side wall portion 102 and is disposed to face the bottom portion 101. Prepare. A through-hole 104 is formed in a part of the side wall 102, and the base has a shape called “flask type” including a neck 105 extending from the periphery of the through-hole 104 to the outside of the base. A locking portion 106 for locking the lid 110 is formed on the neck portion 105 of the base body 100, and the lid 110 is detachably mounted via the locking portion. A flask-type cell culture vessel 120 is formed by combining the substrate 100 and the lid 110.

  FIG. 1B shows a cross-sectional view taken along the line I-I ′ of the substrate 100, and FIG. 1C shows a cross-sectional view taken along the line II-II ′. In the internal space surrounded by the bottom portion 101, the side wall portion 102, and the top surface portion 103 of the base body 100, an inner chamber 130 for accommodating cells and a culture medium is formed. A hydrophilized film 140 is fixed to a region of the inner surface facing the inner chamber 130 (the bottom 101 in the embodiment shown in FIG. 1).

  Another preferred embodiment of a flask-type hydrophilized substrate is shown in FIG. The base 200 shown in FIG. 2A has a shape in which a part of the bottom 201 is bent in the direction of the top surface 103. The side wall portion 202 is erected on the periphery of the bottom portion 201 and connects the bottom portion 201 and the top surface portion 103 disposed to face the bottom portion 201. The substrate 200 having such a shape is also classified as a “flask type”. The base body 200 is combined with the lid 110 to constitute a flask-type cell culture container 220.

  As another embodiment of the hydrophilized substrate, as shown in FIG. 3A, a cylindrical body 301, a bottom 302 joined to one end of the body 301 and closing the one end, A top portion 303 that is joined to the end and closing the other end, a through hole 304 is formed in a part of the top portion 303, and a neck portion 305 that extends from the periphery of the through hole 304 to the outside of the base body, and has a bottle shape. Examples include a substrate in a container form. The neck portion 305 is formed with a locking portion 306 for locking the lid 310, and the lid 310 is detachably mounted via the locking portion 306. Such a substrate 300 is called a “bottle type”. An inner chamber 340 for accommodating cells and a medium is formed in the internal space of the substrate 300. A hydrophilized film 350 to be described later is fixed on the inner wall surface facing the inner chamber 340 of the trunk portion 301. The base body 300 is combined with a lid 310 to form a bottle-type cell culture container 330. FIG. 3A shows an example in which the body 301 has a cylindrical shape with a circular cross section, but the present invention is not limited to this, and as long as the body is cylindrical, a triangle, a quadrangle, a pentagon, a hexagon, etc. It can be a cylindrical shape having various cross sections such as a polygon.

  The material of the substrate is not particularly limited. Specifically, polystyrene (PS), polyethylene terephthalate (PET), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), medium density polyethylene (MDPE) ), Resin materials such as polyvinyl chloride, polyvinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, and acrylic resin, and inorganic materials such as glass and quartz. Among them, polystyrene is particularly preferable for cell culture applications because of its low toxicity.

  In the container-shaped substrate, the inner surface on which the hydrophilized film is disposed is usually not released to the outside, and in the direction in which it faces the other region (in the embodiment of FIG. 1, There is a top surface portion 103) located in a direction facing the bottom portion 101 to which the hydrophilic film 140 is fixed. For this reason, it is not easy to introduce and fix the hydrophilic film on the inner surface of the inner chamber after the substrate is completed. Therefore, in order to make it easy to fix the hydrophilic film, the base is a portion having an inner surface to which the hydrophilic film is fixed, and the first surface having a shape in which the side on which the inner surface exists is opened. Prepare the member as one or more other members, fix the hydrophilic film to the first member, and then join the first member to which the hydrophilic film is fixed and one or more other members. A hydrophilized substrate is formed.

  The point which forms a hydrophilization base | substrate by combining a some member is demonstrated along FIG. As shown in FIG. 4, in one embodiment for forming the base body 100, the first member 401 includes a bottom portion 101 and a side wall portion 102, and the bottom portion 101 of the hydrophilic film 140 fixing surface side is opened. The base body 100 is formed by joining the second member 402 corresponding to the top surface portion 103. At this time, the hydrophilic film 140 is fixed to the inner surface of the bottom 101 of the first member 401 before joining with the second member 402. In this embodiment, the first member to which the hydrophilic film is fixed is particularly referred to as “film-fixed first member”. The first member 401 and the second member 402 are joined in a liquid-tight manner so that the culture solution does not leak out according to the purpose of cell culture.

  In the embodiment shown in FIG. 5, the first member 501 corresponding to the bottom portion 101, the second member 502 corresponding to the side wall portion 102 including the neck portion 105, and the third member 503 corresponding to the top surface portion 103 are joined. As a result, the substrate 100 is formed. The hydrophilized film 140 is fixed to a portion of the first member 501 corresponding to the inner surface of the bottom 101.

  The combination of a plurality of members is not limited to the form shown in FIGS.

  Note that the number of first members for manufacturing one substrate is not limited to one, and a plurality of members may be provided. For example, when fixing a hydrophilized film on a plurality of mutually facing inner surfaces of an inner chamber of a substrate, a first member corresponding to each of the plurality of inner surfaces is prepared, and the plurality of first members A hydrophilic film is fixed to each to form a plurality of film-fixed first members. Thereafter, the plurality of film-fixed first members are joined together with other members as necessary to complete the hydrophilic substrate. The case where only a plurality of film-fixed first members are joined to complete a hydrophilic substrate is not excluded. For example, in the case of the bottle-shaped base 300 in which the hydrophilized film 350 is fixed to the inner surface of the body 301 shown in FIG. 3A, two firsts having substantially the same shape obtained by dividing the base 300 along the axis of the body 301. Preparing a member, fixing a hydrophilized film to each of the members to form a film-fixed first member, and joining two film-fixed first members (360 and 361 in FIG. 3B) to produce a hydrophilized substrate. it can.

  Commercially available products may be purchased and used as each member constituting the cell culture container, the first member for manufacturing the substrate, and one or more other members, and the method of the present invention may be carried out. A person may make and use it himself. A first member for manufacturing a base made of a resin material and one or more other members are manufactured by means such as injection molding, and a scale for measuring the volume of contents is printed if necessary. It can manufacture by providing on a member surface by means, such as. As will be described later, the first member and the film-fixed first member are formed at the same time by filling a resin in an injection mold in which a sheet-like hydrophilic film is placed in advance. Also good.

<Structure of hydrophilic film>
The structure of the hydrophilic film in the present invention will be described with reference to FIGS.
The hydrophilized film 610 includes a film base layer 601, a primer layer 602 including polysiloxane provided on the film base layer 601, and a hydrophilic polymer layer 603 including polyalkylene glycol provided on the primer layer 602. And at least.

  It is preferable that the hydrophilic film 610 has flexibility that can be wound up in a long form.

  The hydrophilized film 610 can further include a necessary layer depending on a bonding method to a member constituting the base material.

  For example, as shown in FIG. 7, the hydrophilized film 610 constitutes a substrate via an adhesive 703 on the side of the film base layer 601 that is different from the side on which the primer layer 602 and the hydrophilic polymer layer 603 are formed. The first member 720 to be attached can be fixed. In this case, as shown in FIG. 8, a hydrophilized film 810 further including an adhesive layer 803 can be used. The surface of the pressure-sensitive adhesive layer 803 of the hydrophilized film 810 can be further protected by a release film 804 as necessary at the stage before sticking.

  Moreover, the hydrophilic film 610 can fix the base and the side of the film base layer 601 different from the side on which the primer layer 602 and the hydrophilic polymer layer 603 are formed, if necessary. In this case, as shown in FIG. 9, a hydrophilic film 910 further including a heat-sealable resin layer 905 can be used.

  A primer layer and a hydrophilic polymer layer are each fixed to the surface of the film base layer so as to have a predetermined thickness, thereby producing a hydrophilic film. There is no restriction | limiting in particular in the film thickness of a primer layer. Although there is no restriction | limiting in particular in the film thickness of a hydrophilic polymer layer, For example, it is good to set it in the range of 5 nm-10 nm.

<Film base material layer>
The material of the film base layer is polystyrene (PS), polyethylene terephthalate (PET), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), medium Examples thereof include resin materials such as density polyethylene (MDPE), polyvinyl chloride, polyvinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, and acrylic resin. In particular, polystyrene is preferably used because of its low toxicity and excellent flexibility.

  The surface of the film base material layer on which the primer layer and the hydrophilic polymer layer are formed can be a surface subjected to an easy adhesion treatment. “Easy adhesion treatment” refers to treatment with an easy adhesive such as polyester, acrylic ester, polyurethane, polyethyleneimine, silane coupling agent, perfluorooctane sulfonic acid (PFOS), and the like. In addition, a commercial item may be used for the film base material layer by which the surface was easily adhesive-treated if necessary.

  The thickness of the film base layer (in the case where the film base layer further includes an easy-adhesion layer, refers to the entire thickness of the film base layer including the easy-adhesion layer) is not particularly limited, but in a roll shape The thickness is preferably such that it can be wound up, and is, for example, 5 to 500 μm, preferably 20 to 400 μm, more preferably 50 to 250 μm.

<Primer layer>
The primer layer provided on the film substrate layer contains at least polysiloxane. Here, polysiloxane is a polymer composed of repeating units containing a siloxane bond (Si—O—Si), and can be obtained by condensation polymerization of a silanol compound. The condensation of the silanol compound is a reaction that occurs between the molecules of the silanol compound. When the molecule on the surface of the film base layer does not have a reactive functional group, no reaction occurs between the silanol compound and the surface of the film base layer. That is, the silanol compound and the formed polysiloxane are bonded to the surface of the film substrate layer by physical adsorption. Such a physical adsorption force of the silanol compound to the surface of the film substrate layer is extremely weak in the monomer, but becomes stronger if the condensation proceeds to some extent and becomes a polymer (polysiloxane) state. A primer layer containing polysiloxane can be formed on the film substrate layer by appropriately condensing the silanol compound. The polysiloxane in the primer layer may be linear, or may have a branched or network structure, but preferably has a branched or network structure.

The silanol compound used for synthesizing the polysiloxane preferably has an organic group containing a carbon atom directly connected to a silicon atom and a functional group in addition to a silanol group (Si—OH). This organic group becomes a side chain of the polysiloxane. Silanol compounds typically have a structure represented by Formula 1:
(R ¹ ) _p (R ² ) _4-pq Si (OH) _q ... (Formula 1)
(P is 1 or 2, q is 2 or 3, p + q is 3 or 4, and R ¹ is an organic group containing a carbon atom directly connected to a silicon atom and containing a functional group. R ² is an organic group containing a carbon atom directly connected to a silicon atom). It is preferable that p = 1 and q = 2 or 3, and it is more preferable that p = 1 and q = 3. When p + q = 4, R ² is not present.

R ¹ is preferably substituted with a functional group having one or more (preferably one) hydrogen atom through an appropriate linker structure as necessary, and has 1 to 20 carbon atoms, preferably 1 to 15, more preferably 1 to 10, and particularly preferably 1 to 6 hydrocarbon groups (provided that the hydrocarbon group itself is a vinyl group as in the case where all or a part of the hydrocarbon groups are vinyl groups). If it is a functional group, it need not be substituted by a functional group). The hydrocarbon group is a saturated or unsaturated aliphatic hydrocarbon group (an alkyl group, an alkenyl group having 2 or more carbon atoms, or an alkynyl group having 2 or more carbon atoms) having a linear or branched chain or ring structure. The aromatic hydrocarbon group may be a monocyclic or polycyclic aromatic hydrocarbon group having 6 or more carbon atoms, or the aromatic hydrocarbon group substituted by one or more aliphatic hydrocarbon groups. Alternatively, the aliphatic hydrocarbon group substituted by one or more aromatic hydrocarbon groups may be used. In the hydrocarbon group, the carbon-carbon bond may be interrupted by one or two identical or different atoms selected from oxygen, nitrogen and sulfur. Preferred examples of the hydrocarbon group include a propyl group and an ethyl group.

A functional group for substituting one or more hydrogens of the hydrocarbon group in R ¹ through an appropriate linker structure as necessary reacts with a hydroxyl group of polyalkylene glycol to form a covalent bond. The functional group is not particularly limited as long as the functional group can be converted to a functional group capable of reacting with a hydroxyl group of a polyalkylene glycol to form a covalent bond, but typically (1H-imidazole -1-yl) carbonyl group, succinimidyloxycarbonyl group, glycidyl group, epoxy group, aldehyde group, amino group, thiol group, carboxyl group, azide group, cyano group, active ester group (1H-benzotriazole-1 -Yloxycarbonyl group, pentafluorophenyloxycarbonyl group, paranitrophenyloxycal Alkylsulfonyl group), a halogenated carbonyl group, an isocyanate group, a maleimide group, and the like, in particular, glycidyl group or an epoxy group is preferable. The glycidyl group or epoxy group can itself react with the hydroxyl group of polyalkylene glycol to form a covalent bond, but the glycidyl group or epoxy group is converted according to the method described in JP-A-2009-156864. It may be converted to an aldehyde group, and the formed aldehyde group may be reacted with the hydroxyl group of polyalkylene glycol. These functional groups may be substituted directly on the hydrogen atom of the hydrocarbon group or may be substituted via an appropriate linker structure. Examples of the linker structure include a divalent group having 0 to 3 carbon atoms and 0 to 3 identical or different heteroatoms selected from nitrogen, oxygen and sulfur. When the group is bonded to the left side and the functional group is bonded to the right side, -O-, -S-, -NH-,-(C = O) O-, -O (C = O)-, -NH (C = O)-,-(C = O) NH-,-(C = O) S-, -S (C = O)-, -NH (C = S)-,-(C = S) NH-, Structures represented by-(N = C = N)-, -CH = N-, -N = CH-, -O-O-, -S-S-,-(O = S = O)-are mentioned. It is done.

Particularly preferred embodiments of R ¹ include a 3-glycidoxypropyl group, a 2- (3,4-epoxycyclohexyl) ethyl group, and the like.

R ² is preferably a hydrocarbon group similar to that described above for R ¹ except that it is not substituted by a substituent (but selected independently of R ¹ ), of which A linear or branched alkyl group having 1 to 6 carbon atoms is preferable, and a methyl group or an ethyl group is particularly preferable.

The silanol compound can be obtained from a silicon compound having a group capable of generating a silanol group by hydrolysis. Such silicon compounds have a structure represented by Formula 2:
(R ¹ ) _p (R ² ) _4-pq Si (Y) _q ... (Formula 2)
(Y is a group that can independently generate a silanol group by hydrolysis, and p, q, R ¹ , and R ² are as defined for the silanol compound).

  Y is preferably an alkoxy group, a halogen atom, an aryloxy group, an alkoxy group substituted by an alkoxy group or an aryloxy group, an aryloxy group substituted by an alkoxy group or an aryloxy group, an alkylcarbonyloxy group, or the like. Y is particularly an alkoxy group having 1 to 6 carbon atoms (particularly a methoxy group, ethoxy group, isopropoxy group, tert-butoxy group), an alkoxy group having 1 to 6 carbon atoms substituted by an alkoxy group (for example, methoxyethoxy group). Group), an alkylcarbonyloxy group having 1 to 6 carbon atoms (for example, an acetoxy group), and a chlorine atom are preferable.

  As the silicon compound of Formula 2, a commercially available compound can be suitably used as the silane coupling agent, and 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropyltriethoxysilane, and the like are particularly preferable. .

  The primer layer containing polysiloxane can be formed by a method including a step of polymerizing the silanol compound of Formula 1 on the film substrate layer (primer layer forming step). Preferably, the step comprises hydrolyzing the silicon compound of formula 2 as follows to produce a silanol compound of formula 1 and a solution in which the produced silanol compound and base are dissolved in an alcohol. Contacting on the layer. Hydrolysis conditions are not particularly limited, but for example, the following method is possible. First, dilute hydrochloric acid is added to the silicon compound of formula 2 to hydrolyze the group Y. The pH of dilute hydrochloric acid is desirably adjusted to 2.0 to 3.0. The molar ratio of water molecules to silicon compound is 2-4. By this operation, the group Y is converted to a silanol group, and a silanol compound of the formula 1 is formed.

  A silanol compound is then applied onto the film substrate layer to form polysiloxane by condensation polymerization. The silanol compound of Formula 1 is soluble in alcohol along with the base. It is desirable to adjust the final concentration of the silanol compound in the range of 0.1 to 10% (v / v). As the base, triethylamine, N, N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine and the like can be used, but the base is not limited thereto. It is desirable to adjust the final concentration of the base in the range of 0.1 to 10% (v / v). As the alcohol, ethanol, 2-propanol, tert-butyl alcohol, and the like can be used, but the alcohol is not limited thereto. This silanol solution is brought into contact with the surface of the film substrate layer and left for 10 minutes to 24 hours. Although the reaction temperature can be set in the range of 4 ° C to 80 ° C, room temperature (20 ° C to 25 ° C) is particularly preferable. By the above operation, a primer layer containing polysiloxane can be formed on the film substrate layer. The coating density of the primer layer can be controlled by the concentration of silanol or base, or the time during which the silanol solution is brought into contact with the film substrate layer. The higher the coating density of the primer layer, the higher the bond density of the polyalkylene glycol chain subjected to the addition reaction in the next step.

  When the functional group derived from the silanol compound on the side chain of the formed polysiloxane is derivatized and converted to another functional group, the functional group is converted to the hydroxyl group of the polyalkylene glycol after the formation of the primer layer. A derivatization step is performed that converts to a functional group that can react to form a covalent bond.

<Hydrophilic polymer layer>
After forming the primer layer, a hydrophilic polymer layer is further provided thereon to obtain a hydrophilic film. The hydrophilic polymer layer contains at least polyalkylene glycol. Here, polyalkylene glycol means the following formula:
− {(CH ₂ ) _n —O} _m −
(M is an integer indicating the degree of polymerization). n is preferably 2 or more. That is, examples of applicable polyalkylene glycols include polyethylene glycol, polypropylene glycol, and polybutylene glycol. Among them, polyethylene glycol (PEG) is preferably used because of its lower adsorptivity for cells, proteins and the like.

  Moreover, the number average molecular weight of the polyalkylene glycol chain in the hydrophilic polymer layer affects the adsorptivity of cells and proteins. In order to obtain sufficiently low adsorptivity, the number average molecular weight of the polyalkylene glycol is preferably 176 or more. For example, the number average molecular weight of PEG is preferably 176 or more (m is 4 or more), more preferably 362 or more. The upper limit of the number average molecular weight of the polyalkylene glycol is not particularly limited, but it is difficult to handle because the viscosity increases as the number average molecular weight increases, and it is difficult to arrange the polyalkylene glycol chain at high density. The number average molecular weight is preferably 25000 or less, more preferably 10,000 or less.

  The number average molecular weight of the polyalkylene glycol can be determined by a vapor pressure osmotic pressure method or a membrane osmotic pressure method. The vapor pressure osmotic pressure method can be used when the number average molecular weight of the polyalkylene glycol is less than 100,000. The membrane osmotic pressure method can be used when the number average molecular weight of the polyalkylene glycol is 10,000 to 1,000,000.

  The hydrophilic polymer layer can be formed by subjecting a polyalkylene glycol to an addition reaction to a primer layer chemically or physically fixed on the film substrate layer. Specifically, for example, a polyalkylene glycol containing a catalytic amount of concentrated sulfuric acid is brought into contact with the surface of the primer layer containing polysiloxane. Here, the polyalkylene glycol having a number average molecular weight exceeding 1000 is previously heated and melted. If necessary, polyalkylene glycol may be diluted with tert-butyl alcohol or the like. This polyalkylene glycol solution is brought into contact with the surface of the primer layer and heated. In the case of PEG, for example, the heating temperature can be set in the range of 60 ° C. to 90 ° C., but it is preferably about 80 ° C. (75 ° C. to 85 ° C.) in consideration of the heat resistance of the substrate. The heating time can be set in the range of 10 minutes to 24 hours, but when the heating temperature is around 80 ° C., it is preferably 30 minutes to 60 minutes. By the above operation, the polyalkylene glycol chain is covalently bonded to the primer layer. At this time, the bond density of the polyalkylene glycol chain depends on the coating density of the primer layer.

  The hydrophilic polymer layer thus formed may be further washed as necessary. It is considered that free polymer molecules that are not immobilized are present on the surface of the hydrophilic polymer layer after the addition reaction, and these free polymers can be removed by washing, which is preferable. Here, the cleaning method is not particularly limited, but typical examples include immersion cleaning, swing cleaning, shower cleaning, spray cleaning, and ultrasonic cleaning. The cleaning liquid typically includes various water-based, alcohol-based, hydrocarbon-based, chlorine-based, and acid / alkali cleaning liquids.

<Adhesive layer>
Examples of the pressure-sensitive adhesive constituting the pressure-sensitive adhesive layer include polyester resins, acrylic ester resins, polyurethane resins, polyethyleneimine resins, silane coupling agents, perfluorooctane sulfonic acid (PFOS), and particularly acrylic ester resins, A polyurethane resin or the like can be preferably used.

  Although the thickness of an adhesive layer is not specifically limited, It is preferable that they are 10 micrometers-300 micrometers, and it is more preferable that they are 20 micrometers-200 micrometers.

  As shown in FIG. 8, a release film 804 for protecting the pressure-sensitive adhesive layer 803 is bonded to the outside of the pressure-sensitive adhesive layer 803 of the hydrophilized film 810 before being attached to the substrate. preferable. The release film 804 is made of a release member having peelability, and is peeled and removed at the time of sticking. The release member is not particularly limited as long as it has the necessary strength and flexibility. For example, a film made of a resin such as polyethylene terephthalate, polypropylene, or polyethylene, or a foamed film thereof containing a silicone-based, fluorine-based, long-chain alkyl group Examples thereof include those subjected to release treatment with a release agent such as carbamate. The thickness of the release film 804 is not particularly limited, but is preferably 10 μm to 100 μm.

  The pressure-sensitive adhesive layer 803 is obtained by applying a pressure-sensitive adhesive layer and a coating solution for forming a pressure-sensitive adhesive layer containing a solvent as necessary to form a coating film on the surface of the film substrate layer or the release film, and drying as necessary. Can be formed. As the coating method, for example, a comma coating method, a blade coating method, a gravure coating method, a rod coating method, a knife coding method, a reverse roll coating method, an offset gravure coating method and the like can be used.

<Heat sealable resin layer>
A heat-sealable resin layer is a heat-sensitive adhesive (melts and adheres when heated). A heat-seal layer is applied in advance, and heat or pressure is applied during use to target the film. Used to adhere to. The heat-sealable resin layer can be formed by using a printing method such as gravure coating, die coating, roll coating, spray coating or the like.

<Method for producing hydrophilic substrate>
The outline of the method for producing a hydrophilic substrate of the present invention is as shown in FIG. 20, in which a long hydrophilized film is prepared, and the hydrophilized film is formed by a cutting step, a hydrophilized film fixing step, and as necessary. A step of joining the fixed base (first member) and another member and a sterilization step performed as necessary are sequentially performed. Hereinafter, each process is explained in full detail. However, the present invention is not limited to the following specific modes.

<Preparation of hydrophilic film>
In the method of the present invention, a step of preparing a long hydrophilized film may be referred to as a “hydrophilic film preparatory step”.

  The hydrophilic film preparation step is a step of preparing a long hydrophilic film. Here, “preparing a long hydrophilized film” preferably refers to producing a long hydrophilized film, but in the step of preparing a preliminarily produced long hydrophilized film. There may be. In the following description, the former process for producing a long hydrophilic film is referred to as a “hydrophilized film production process”.

  In the hydrophilic film manufacturing process, it is preferable to manufacture a long hydrophilic film by a roll-to-roll method. The roll-to-roll method is a method in which a flexible long base material wound in a roll shape is fed out, subjected to a predetermined treatment on the base material while being intermittently or continuously conveyed, and again It is a production system that winds up on a roll. The roll-to-roll method can efficiently produce a hydrophilic film with high uniformity as compared with a batch method in which a hydrophilic film is produced for each sheet.

  One embodiment of the hydrophilized film manufacturing process is to feed and convey a long film base layer wound in a roll shape, and sequentially apply a primer layer and a hydrophilic polymer layer on the fed film base layer. Forming, and winding the formed hydrophilic film into a roll.

  As an example, the manufacturing method by the roll-to-roll system of the hydrophilic film 610 provided with the film base material layer 601, the primer layer 602, and the hydrophilic polymer layer 603 is demonstrated along FIG. First, a long film base material layer (raw roll) wound in a roll shape is prepared (S1). As the film base material layer, a film whose surface has been subjected to easy adhesion treatment in advance may be used. If necessary, easy adhesion treatment may be performed by a roll-to-roll method. A film base layer wound in a roll shape is fed out, conveyed, and a coating film is formed on the surface of the fed film base layer by applying the above-described composition for forming polysiloxane, followed by a polymerization reaction. To form a primer layer (S2). Subsequently, the film base material layer on which the primer layer is formed is transported, and a coating film is formed by applying a coating composition containing polyalkylene glycol on the surface of the drawn primer layer, and is hydrophilic by an addition reaction. A polymer layer is formed (S3). If necessary, free polymer molecules and the like are washed and removed by the above-mentioned means by a roll-to-roll method (S4). Thus, a long hydrophilized film is obtained (end).

  As shown in FIG. 8, the following method (1) is used as a method for producing a hydrophilic film 810 in which a pressure-sensitive adhesive layer 803 is further laminated and a release film 804 for protecting it is bonded. There is ~ (3).

(1) A step of preparing a long hydrophilized film intermediate product 1 in which a primer layer 602 and a hydrophilic polymer layer 603 are laminated on a film base material layer 601 by the above-described method, and a long length wound in a roll shape The intermediate hydrophilized film product 1 is fed out and conveyed, and the pressure-sensitive adhesive layer 803 is formed on the side of the film base layer 601 of the fed hydrophilized film intermediate product 1 by the formation method described above with respect to the pressure-sensitive adhesive layer. A method comprising the steps of: bonding a long release film 804 to the pressure-sensitive adhesive layer 803 and winding the resulting hydrophilized film 810 into a roll following the formation of the pressure-sensitive adhesive layer.

(2) A step of preparing the long hydrophilized film intermediate product 1 in which the primer layer 602 and the hydrophilic polymer layer 603 are laminated on the film base layer 601 by the above-described method, and the long release film 804 A step of producing a long hydrophilized film intermediate product 2 laminated with a pressure-sensitive adhesive layer 803 by a roll-to-roll method, or preparing a preliminarily produced long hydrophilized film intermediate product 2; The long hydrophilized film intermediate product 1 and the hydrophilized film intermediate product 2 are brought into contact with the film base layer 601 of the hydrophilized film intermediate product 1 and the adhesive layer 803 of the hydrophilized film intermediate product 2. And a step of manufacturing a long hydrophilized film 810 provided with a release film 804 and winding the manufactured long hydrophilized film 810 into a roll.

(3) The long hydrophilic intermediate film 3 in which the film base 601, the pressure-sensitive adhesive layer 803, and the release film 804 are laminated is manufactured by a roll-to-roll method, or is manufactured in advance. The step of preparing the long hydrophilized film intermediate product 3 and the primer layer 602 and the hydrophilic polymer layer 603 on the side of the film base layer 601 of the long hydrophilized film intermediate product 3 by the above-described method. And a step of winding the elongated hydrophilized film 810 provided with the release film 804 into a roll, which are sequentially formed and manufactured.

  As shown in FIG. 9, a long hydrophilic film 910 in which a heat-sealable resin layer 905 is further laminated similarly includes a film base layer 601, a heat-sealable resin layer 905, a primer layer 602, and a hydrophilic polymer. The layer 603 can be formed and manufactured by a roll-to-roll method in an arbitrary order. A hydrophilized film intermediate product 4 in which a heat-sealable resin layer 905 is laminated on a long film base material layer 601 is prepared in advance, and a primer layer 602 and a hydrophilic polymer are prepared on the hydrophilized film intermediate product 4. The layer 603 may be formed sequentially.

<Cut process>
The cutting step is a step of obtaining a sheet-like hydrophilized film by cutting the long hydrophilized film. As shown in FIG. 11, the cutting step is typically a step of cutting a long hydrophilized film 1100 and obtaining a plurality of single-wafer hydrophilized films 1101 separated from each other. . However, it is not limited to this, and the cutting step may be a step of cutting the hydrophilized film into a sheet by half cutting. Specifically, as shown in FIG. 12, the release film 1202 can be peeled from the long hydrophilic film 1201 on the surface of the hydrophilic film on which the primer layer and the hydrophilic polymer layer are not formed. By bonding, a long half-cut laminate 1200 is prepared. In the cutting step, a cut line (half-cut line) formed from the surface of the hydrophilized film 1201 to the thickness direction position that cuts the hydrophilized film but does not cut the release film 1202 in the thickness direction of the film. ) 1210 is formed, and a plurality of regions 1230 surrounded by the cut lines 1210 and separable as sheet pieces are formed in the hydrophilic film 1201. In this manner, a long, half-cut hydrophilized film with a release film 1220 is obtained. In this embodiment, the plurality of regions 1230 of the hydrophilized film 1201 surrounded by the score line 1210 can be independently separated from other parts of the hydrophilized film and the release film as a sheet-like hydrophilized film. It can be used in the method for producing a hydrophilic substrate of the present invention. The laminated body for half-cut 1200 is typically a hydrophilized film 810 laminated with a release film 804 shown in FIG. 8, but is not limited to this, and a release film is formed on a hydrophilic film having another structure. It may be bonded in a peelable manner.

  The sheet-like hydrophilized film obtained in the cutting step can have an arbitrary shape according to the shape of the region to which the hydrophilized film is fixed of the substrate to which the hydrophilized film is fixed. For example, the shape may be a polygon such as a triangle, a rectangle (rectangle, square, parallelogram, rhombus, etc.), a pentagon, a hexagon, a heptagon, an octagon, a circle, an ellipse, or the like. The sheet-like hydrophilized film is particularly preferably polygonal. This is because if it is a polygon, a sheet piece can be efficiently obtained from a long hydrophilic film. Of the polygons, a rectangle or a square is particularly preferable.

<Hydrophilic film fixing process>
The hydrophilic film fixing step is a step of fixing a sheet-like hydrophilic film on the surface of the substrate. When the substrate is composed of a plurality of members, it is a part having an inner surface to which the hydrophilic film is fixed, and a member (first member) having a shape in which the side on which the inner surface exists is opened is a single wafer Fix the hydrophilic film.

  The first member is a part of a substrate having an inner surface to which a hydrophilized film is fixed, as in the first members 401 and 501 in FIGS. 4 and 5, and the side where the inner surface exists is open. It is not particularly limited as long as it is a member. Since the first member has an open inner surface to which the hydrophilic film is fixed, it is easy to fix the hydrophilic film.

  Various methods can be employed as a method of fixing the hydrophilic film to the first member. Typical examples are given below. When the substrate is not composed of a plurality of members, the following fixing method for the first member can be applied as it is as a method for fixing the hydrophilic film to various types of substrates.

  As an example of the embodiment, a hydrophilized film fixing step of attaching a sheet-like hydrophilized film to the first member via an adhesive can be mentioned. In this embodiment, the hydrophilic film 610 is fixed to the first member 720 via an adhesive 703 as shown in FIG. For this purpose, an adhesive may be applied to one or both of the hydrophilic film 610 and the first member 720, and both may be bonded together, but preferably a release film 804 is applied as shown in FIG. A hydrophilic film 810 in a state is prepared, the release film 804 is removed, and the film is fixed to the first member via the adhesive layer 803. 13A to 13C show a process of bonding the hydrophilized film 810 to the first member 401 shown in FIG. 4 via the adhesive layer 803 (corresponding to the I-I ′ cross section of FIG. 1). Affixing of the hydrophilic film 810 can also be performed using a labeling labeler.

  If necessary, the first member after application of the hydrophilic film can be autoclaved to remove bubbles on the application surface of the hydrophilic film.

  As another example of the embodiment, a hydrophilic film fixing step of fixing a sheet-like hydrophilic film to the first member by heat sealing can be mentioned. In this embodiment, as shown in FIG. 9, the hydrophilic film 910 on which the heat-sealable resin layer 905 is formed is disposed on the surface of the first member so that the heat-sealable resin layer 905 is in contact with the first member. In this method, both are fixed by heating. 14A to 14C show a process of bonding the hydrophilic film 910 to the first member 401 shown in FIG. 4 via the adhesive layer 905 (corresponding to the I-I ′ cross section of FIG. 1).

  Note that the heat-sealable resin layer 905 is not necessarily required for fixing by heat sealing. For example, in the hydrophilized film 610 as shown in FIG. 6, when the film base layer 601 is a thermoplastic resin film and the first member is also a thermoplastic resin member, the two layers are overlapped to obtain an appropriate temperature. Heat sealing is possible by heating and melting both to cool.

  As another example of the embodiment, a hydrophilized film fixing step in which the first member and the sheet-like hydrophilized film are welded and fixed by ultrasonic welding can be exemplified. Ultrasonic welding is generally used as a method of joining resin members together. Typically, as shown in FIG. 15, an energy director (width is about 100 to 1000 μm, height is necessary for ultrasonic welding at the periphery of the region where the hydrophilic film 610 is fixed on the first member 401. A first member 401 ′ in which a thin resin protrusion (50 to 1000 μm) 1501 is integrally formed with other parts is prepared. And the hydrophilization film 610 provided with the film base material layer 601, the primer layer 602, and the hydrophilic polymer layer 603 is made to contact the inner bottom face area | region of 1st member 401 '. More specifically, in FIG. 16 (viewed from the VV ′ cross section shown in FIG. 15), the hydrophilized film 610 is placed on the first member 401 ′ and ultrasonic waves are applied to the portion where the energy director 1501 exists. The hydrophilized film 610 is fixed to the bottom surface of the first member 401 ′ by applying ultrasonic vibration while being pressed by the welding ultrasonic horn (FIG. 16C).

  As another example of the embodiment, a hydrophilized film fixing step of fixing the first member and a sheet-like hydrophilized film by laser welding may be mentioned. Laser welding is a method in which a light-transmitting resin member and a light-absorbing resin member are overlapped, and a laser beam is irradiated while pressing the two members together so that the interface between the members is melted and bonded. As shown in FIG. 17, when the hydrophilic member 610 having the film base layer 601, the primer layer 602, and the hydrophilic polymer layer 603 and the first member 401 are joined, the film base layer 601 and the first member 401 are joined. Laser welding is possible by using one of the fixed surfaces as a light-transmitting resin member and the other as a light-absorbing resin member.

  As another example of the embodiment, the hydrophilized film fixing that obtains the film-fixing first member by filling the resin into an injection mold in which the sheet-like hydrophilizing film is arranged in advance is manufactured. A process can be mentioned. This method is a kind of in-mold molding. A specific example of manufacturing the first member 401 in which the film base layer 601 is fixed to the bottom surface by this method will be described with reference to FIG. In an injection mold that combines a convex mold (core mold) 1801 and a concave mold (cavity mold) 1802 that form a mold space 1804 corresponding to the first member 401 when the first member 401 is clamped, a film substrate layer 601 and a primer The sheet-like hydrophilized film 610 provided with the layer 602 and the hydrophilic polymer layer 603 is disposed so that the hydrophilized film 610 is positioned in the first member 401 after the resin is filled and solidified. To do. Next, through the gate 1803, the molten resin is filled in the mold by applying injection pressure (FIG. 18B). After filling, the mold is opened after the resin is solidified, and the first member 401 to which the hydrophilic film 610 is fixed is obtained (FIG. 18C).

  As a modification of this embodiment, instead of the hydrophilized film 610, a hydrophilic material further comprising a heat-sealable resin layer 905 on the surface of the film base layer 601 opposite to the primer layer 602 and the hydrophilic polymer layer 603. An embodiment using the fluorinated film 910 can be given. As shown in FIG. 19, in an injection mold in which a convex mold 1801 and a concave mold 1802 are combined, at least a part (all in FIG. 19) of the surface of the heat-sealable resin layer 905 is exposed to the mold space, and the hydrophilic polymer layer The hydrophilized film 910 is placed in the mold so that the surface of 603 is in contact with the inner wall surface of the mold, and the mold is clamped. Next, molten resin is filled into the mold through the gate 1803 by applying injection pressure, and after the resin is solidified, the mold is opened to obtain the first member 401 to which the hydrophilic film 910 is fixed (FIG. 21C). . In this embodiment, since the heat-sealable resin layer 905 is melted by contact with a high-temperature molten resin and solidifies together with the solidification of the resin, the film base layer 601 and the first member 401 are firmly bonded via the heat-sealable resin layer 905. To do.

  As another example of the embodiment, a hydrophilized film fixing step of locking a sheet-like hydrophilized film to the first member via a physical locking means can be mentioned. The physical locking means is not particularly limited as long as the hydrophilic film can be fixed at a predetermined position on the first member. For example, a hook provided on the first member and a sheet-like hydrophilic film are provided. A combination with a through-hole that functions as a hook receiving portion that engages with the hook is provided. Or the combination of the recessed part provided in the 1st member, and the convex part which functions as a recessed part receiving part engaged with the said recessed part provided in the sheet-like hydrophilization film etc. can be considered.

<Member joining process>
In the member joining step, when the base is composed of a plurality of members, the film-fixed first member in which the hydrophilic film is fixed at a predetermined position of the first member and one or more other members are joined. Is a step of forming a hydrophilic substrate.

  In the film fixing first member, as illustrated in FIGS. 4 and 5, the hydrophilic film is fixed to the opened inner surface. The other members of the substrate are configured such that when combined with the first member, the substrate in which the inner chamber in which the hydrophilic film on the first member is disposed on the inner surface is formed is completed. In the obtained substrate, the facing direction of the hydrophilic film is not opened and is closed by an inner wall surface (may be an inner wall surface having a through hole). The other members can be a combination of one or more, preferably 3 or less, more preferably 2 or 1.

  The first member and the one or more other members constituting the base are made of various materials such as the resin material described for the base. The first member and one or more other members can be joined in a manner according to the purpose of cell culture or the like, and if necessary, joined together in a liquid-tight manner so that the culture solution does not leak. Joining between members can be performed by a method such as ultrasonic welding, laser welding, heat sealing, etc., as long as the members are made of resin materials. Even if the members are made of other materials, an adhesive is used. It can be performed by an arbitrary method such as adhesion using.

  In any of the examples of FIGS. 13, 14, 16, 17, and 18, the base member 100 is completed by bonding the second member 402 to the first member 401 or 401 'after fixing the hydrophilic film. A hydrophilic substrate such as the cell culture container of the present invention can be obtained by combining the hydrophilic substrate produced by bonding with another member such as a lid, if necessary.

  In the above description, the case where a hydrophilic film having a film base layer, a primer layer, and a hydrophilic polymer layer is separately arranged in one region of the base surface has been described. Also in the case where the primer layer and the hydrophilic polymer are directly provided on the surface, the hydrophilic substrate of the present invention can be obtained according to the above description. That is, the primer layer and the hydrophilic polymer layer are formed on the surface of the substrate by a method similar to the method in which the primer layer and the hydrophilic polymer layer are sequentially provided on the film substrate layer, and are reacted. The hydrophilic substrate of the invention can be obtained.

<Sterilization process>
The method of the present invention preferably further comprises a step of sterilizing the hydrophilic substrate of the present invention. As the sterilization method, methods such as γ-ray irradiation sterilization, ethylene oxide gas sterilization, electron beam sterilization, ultraviolet irradiation sterilization, hydrogen peroxide sterilization, ethanol sterilization and the like can be used. In particular, γ-irradiation sterilization is preferable in that all biological species can be killed.

  The hydrophilic substrate as described above is preferably used for cell culture, for example, as a petri dish, flask, multiwell plate, cell factory type culture container, roller bottle type culture container, etc. used for suspension culture. For example, the surface of the hydrophilic polymer layer can be exposed in the culture solution, and cells can be seeded in the culture solution for culturing. It can also be used as a solid phase carrier in ELISA. In addition to the above-described cell culture uses and the like, any application that needs to suppress cell adhesion and protein adsorption can be applied. Examples thereof include protein storage and homogeneous assay.

  Next, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.

Example 1
Silanol was prepared by adding 350 μl of dilute hydrochloric acid (pH 2.4) to 1.8 ml of 3-glycidoxypropyltrimethoxysilane (manufactured by Momentive Performance Materials). This was added to 100 ml of 2-propanol (manufactured by Junsei Chemical Co., Ltd.). To this, 500 μl of triethylamine (Wako Pure Chemical Industries, Ltd.) was further added. The obtained silanol solution was dispensed into a polystyrene flask (bottom area 75 cm ² ; trade name EASYFLASKS NUNCLON SURFACE; manufactured by Thermo Fisher Scientific Co., Ltd.) 20 ml at a time, and the primer layer and the hydrophilic polymer layer were coated. The surface to be applied was left for 135 minutes at room temperature. Subsequently, the inside of the flask was washed with pure water and then dried at 80 ° C. for 10 minutes. Next, after dispensing PEG400 (number average molecular weight 380-420; manufactured by Kanto Chemical Co., Inc.) diluted to 5% by volume with tert-butyl alcohol (manufactured by Kanto Chemical Co.) containing a catalytic amount of concentrated sulfuric acid, Incubated at 80 ° C. for 45 minutes. It was washed with pure water and dried at 80 ° C. for 10 minutes. By this operation, a hydrophilic substrate (hereinafter sometimes referred to as “low adsorption treatment flask”) in which a primer layer containing polysiloxane and a hydrophilic polymer layer containing PEG were provided on the substrate was produced.

(Comparative Example 1)
As Comparative Example 1, the same polystyrene flask as in Example 1 having no primer layer and hydrophilic polymer layer was used. About the flask of this comparative example 1 and the low adsorption process flask manufactured in the said Example 1, the protein adsorption amount and cell adhesiveness were investigated as follows.

(Measurement of protein adsorption)
5 ml each of phosphate buffer (concentration 1 μg / ml) in which horseradish peroxidase (HRP) -labeled anti-rabbit IgG antibody (manufactured by Nordic Immunological Laboratories) was dissolved in the low adsorption treatment flask of Example 1 and the flask of Comparative Example 1 Then, in Example 1, the surface on which the primer layer and the hydrophilic polymer layer were provided was set as the bottom surface and allowed to stand at room temperature overnight. In Comparative Example 1 as well, the same surface was used as the bottom surface and allowed to stand overnight. After standing, it was washed with a phosphate buffer buffer containing 0.05% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) three times. After drying, HRP of the antibody adsorbed by Immunostar LD (manufactured by Wako Pure Chemical Industries, Ltd.) as a luminescent substrate was emitted. Thereafter, an image image was obtained with a CCD imager LAS4000mini (manufactured by GE Healthcare), and the amount of luminescence was quantified to determine the amount of protein adsorbed. The result is shown in FIG. In FIG. 21, the average value of the protein adsorption amount of Comparative Example 1 is set to 100, and the protein adsorption amount of Example 1 is represented by a relative value. As shown in FIG. 21, in Example 1, the protein adsorption amount was suppressed to 5.4%. From the above results, it was revealed that the hydrophilic substrate of the present invention suppresses protein adsorption.

(Measurement of cell adhesion)
In order to examine cell adhesion, cell culture was performed using the low adsorption treatment flask of Example 1 and the flask of Comparative Example 1. DMEM medium (manufactured by Sigma) containing 5% fetal bovine serum and 5% bovine serum was placed in the flasks of Example 1 and Comparative Example 1, and 6 × 10 ⁶ NIH3T3 cells were seeded in each medium. Incubated for 24 hours at ° C. After 24 hours, cell adhesion was observed with a microscope (Olympus) (FIG. 22). As shown in FIG. 22, cell adhesion was observed in Comparative Example 1, whereas in Example 1, it was observed that cells were floating. From the above results, it was revealed that the hydrophilic substrate of the present invention suppresses cell adhesion.

(Example 2)
A polystyrene 6-hole multi-dish (manufactured by Thermo Fisher Scientific Hook) was used as the base, and a primer layer and a PEG layer were formed on the bottom of the multi-dish in the same manner as in Example 1 above to form a hydrophilic base (hereinafter, “ Sometimes called “low-adsorption treated multi-dish”). In this example, PEG was used in the reaction as it was without being diluted with tert-butyl alcohol.

  As Comparative Example 2, the same 6-hole multidish made of polystyrene as in Example 2 having no primer layer and hydrophilic polymer layer was used. About the multi-dish of this comparative example 1 and the low adsorption process multi-dish manufactured in the said Example 2, the protein adsorption amount and cell adhesiveness were investigated as follows.

(Measurement of protein adsorption)
For the low-adsorption treated multi-dish of Example 2 and the multi-dish of Comparative Example 2, the protein adsorption amount was measured by the same method as in Example 1 and Comparative Example 1. The amount of the phosphate buffer containing the antibody was 2 ml per well, and the amount of immunostar LD used for the luminescence reaction was 1 ml per well. The measurement results are shown in FIG. In FIG. 23, the average value of the protein adsorption amount of Comparative Example 2 is set to 100, and the protein adsorption amount of Example 2 is expressed as a relative value. As shown in FIG. 23, in Example 2, the protein adsorption amount was suppressed to 0.28%. From the above results, it was revealed that the hydrophilized substrate of the present invention suppresses protein adsorption.

(Measurement of cell adhesion)
In order to examine the cell adhesion, the low-adsorption treatment multi-dish of Example 2 and the multi-dish of Comparative Example 2 were subjected to cell culture in the same manner as in Example 1 and Comparative Example 1, and the state of cell adhesion was observed. (FIG. 24). The cell seeding number was 1 × 10 ⁶ per well. As shown in FIG. 24, cell adhesion was observed in Comparative Example 2, whereas in Example 2, it was observed that cells were floating. From the above results, it was revealed that the hydrophilic substrate of the present invention suppresses cell adhesion.

DESCRIPTION OF SYMBOLS 100 Base body 101 Bottom part 102 Side wall part 103 Top surface part 104 Through-hole 105 Neck part 106 Locking part 110 Lid 120 Cell culture container 130 Inner chamber 140 Hydrophilization film

Claims (8)

Comprising a substrate,
A hydrophilic polymer containing a film base layer, a primer layer containing polysiloxane provided on the film base layer, and a polyalkylene glycol provided on the primer layer in at least one region of the surface of the substrate A method for producing a hydrophilic substrate on which a hydrophilic film having a layer is fixed,
The following steps:
A cutting process for obtaining a sheet-like hydrophilized film by cutting a long hydrophilized film;
The said method including the hydrophilization film fixing process which fixes a sheet-like hydrophilization film on the surface of a base | substrate. Further comprising a hydrophilized film production process for producing a long hydrophilized film,
The hydrophilized film manufacturing process unwinds and conveys a long film base layer wound in a roll shape, and sequentially forms a primer layer and a hydrophilic polymer layer on the unrolled film base layer. The manufacturing method of the hydrophilization base | substrate of Claim 1 including the process of winding up the subsequent hydrophilization film in roll shape.   The method for producing a hydrophilic substrate according to claim 1 or 2, wherein the hydrophilic film fixing step includes a step of sticking a sheet-like hydrophilic film to the surface of the substrate via an adhesive.   The hydrophilized film fixing step includes a step of obtaining a hydrophilized substrate by filling a resin in an injection mold in which a sheet-like hydrophilized film is previously disposed and molding the substrate. A method for producing a hydrophilized substrate. A container-shaped substrate;
A hydrophilized substrate in which a primer layer containing polysiloxane and a hydrophilic polymer layer containing polyalkylene glycol are provided on at least one region of the inner surface of the substrate. A container-shaped substrate;
At least one region of the inner surface of the substrate has a film base layer, a primer layer containing polysiloxane provided on the film base layer, and a hydrophilic containing polyalkylene glycol provided on the primer layer A hydrophilic substrate on which a hydrophilic film having a polymer layer is disposed.   The hydrophilized substrate according to claim 5 or 6, which is used for cell culture. An elongated hydrophilization having a film base layer, a primer layer containing polysiloxane provided on the film base layer, and a hydrophilic polymer layer containing polyalkylene glycol provided on the primer layer With film,
An elongated release film disposed on the side of the hydrophilic film on which the primer layer and the hydrophilic polymer layer are not provided,
The hydrophilic film is formed with a plurality of regions surrounded by a cut line that can be separated as sheet pieces, and the cut line extends from the surface of the hydrophilic film in the thickness direction of the film. The half-cut line is formed up to a position in the thickness direction that cuts the hydrophilic film but does not completely cut the release film.
Half-cut hydrophilized film with long release film.

Images