JP4930544B2 - Method for producing carrier for cell culture - Google Patents

Description

  The present invention relates to a method for producing a cell culture carrier having both a cell adhesive region and a cell non-adhesive region.

  In recent years, a technique for arranging cells at a predetermined position on a substrate, that is, a cell micropatterning technique has attracted attention. Until now, cell micropatterning technology has been used for basic research in cell biology, but recently, application to tissue engineering, regenerative medicine, biosensor construction, cell imaging support tools, etc. has begun to be examined. Typical methods for obtaining a cell micropattern include a microcontact printing method (Patent Document 1, Non-Patent Document 1), a photolithography method (Patent Document 2, Non-Patent Document 2), and a laser ablation method (Patent Document 3, Non-Patent Document 1). A cell-adhesive region and a cell non-adhesive region are provided on a cell culture carrier by using literature 3).

Non-Patent Document 4 describes a method for producing a cell culture carrier having a cell adhesive region and a cell non-adhesive region using a microcontact printing method. In Non-Patent Document 4, a functional group is introduced onto the surface of a gold substrate or Si / SiO ₂ substrate, and a methacrylic acid derivative in which oligoethylene glycol is introduced into a side chain carboxyl group from the functional group is used as an atom transfer radical weight. Poly (oligo (ethylene glycol) methacrylate), a comb-shaped polymer polymerized by a legal method, is introduced, and N, N'-disuccinimidyl carbonate (DSC) is added to the hydroxyl group at the end of oligoethylene glycol in each side chain. Is activated by binding. The substrate thus obtained is brought into contact with biotin or poly-L-lysine by a microcontact printing method using a stamp on which a fine pattern is formed, and bonded to the activated hydroxyl group. Finally, the amino group of diglycolamine is bonded to the activated hydroxyl group remaining on the substrate for blocking. In this way, a cell culture carrier having a cell adhesive region and a cell non-adhesive region is obtained.

JP 2002-355031 A JP2007-312736 JP2003-033177

Langmuir, 19, 1493-1499 (2003). Langmuir, 19, 9855-9862 (2003) Biomaterials, 26, 5395-5404 (2005) Biomacromolecules, 8, 3922-3929 (2007).

  However, conventional carriers for cell culture provided with a cell adhesive region and a cell non-adhesive region have problems in terms of cell pattern fineness, maintenance power, production cost, etc., and have not been put into practical use. For example, in Patent Document 1, although a fine cell pattern can be constructed, it is difficult to maintain the cell pattern for a long period of time, or an expensive material is required. In Patent Document 2, although the manufacturing cost is relatively low and the cell pattern can be maintained for a long time, the fineness of the cell pattern and the cell affinity of the cell adhesion region are insufficient. In Patent Document 3, it is possible to maintain the cell pattern for a long period of time, but the cell affinity of the cell adhesion region is insufficient. In the method of Non-Patent Document 4, an atom transfer radical polymerization method is used for polymerization of a methacrylic acid derivative. This polymerization method has extremely severe restrictions on polymerization conditions and raw material purity as compared with conventional radical polymerization methods. The presence of a small amount of oxygen and impurities in the reaction system has a great influence on the polymerization reaction in the atom transfer radical polymerization method, and thus it is difficult to control the reaction. Therefore, the method of Non-Patent Document 4 has a problem that it is difficult to use industrially. Further, when a gold substrate is used as the substrate in this method, there is a problem that the cost is increased.

  The present invention has been made in view of such circumstances, and includes a cell culture carrier that includes a cell adhesive region and a cell non-adhesive region and can stably maintain a cell pattern for a long period of time. An object of the present invention is to provide a simple manufacturing method.

  The present invention employs the following means in order to solve the above problems.

  That is, in the present invention, a hydrophilic compound is bonded to a support into which a functional group has been introduced, the hydrophilic compound is activated in a two-step process, and then a functional group capable of binding to an active carbonyl group by a microcontact printing method. The gist is to immobilize a cytophilic substance having a group regioselectively. The present invention includes the following group of inventions.

(1)
A method for producing a carrier for cell culture comprising a surface comprising a cell adhesive region and a cell non-adhesive region, wherein a functional group is introduced into the surface of a support, and a reaction with the functional group is performed. A hydrophilic compound binding step of binding a hydrophilic compound having a binding group capable of forming a covalent bond and a hydroxyl group to the functional group via a bond between the functional group and the binding group; A carboxyl group is formed by a ring-opening half esterification reaction of a cyclic acid anhydride with a hydroxyl group on the bonded hydrophilic compound, and the carboxyl group formed in the carboxyl group forming step and the carboxyl group forming step is activated. An activation step for converting to a carbonyl group, and an active carbonyl group in a part of the support surface on which the active carbonyl group is formed in the activation step. A cytophilic substance having a functional group capable of forming a covalent bond by contact is brought into contact, and a covalent bond is formed by a reaction between an active carbonyl group in the partial region and a functional group of the cytophilic substance. The cell affinity substance is immobilized on the partial region, and after the cell affinity substance immobilization step and the cell affinity substance immobilization step, the surface of the support reacts with an active carbonyl group. A low molecular compound having a functional group capable of forming a covalent bond is contacted, and a covalent bond is formed by a reaction between the active carbonyl group remaining on the surface and the functional group of the low molecular compound, and the active carbonyl group is And a blocking step of substituting with a group containing the low molecular weight compound, and a method for producing a cell culture carrier.

(2)
The cytophilic substance immobilization step includes a convex portion having an upper surface having a shape corresponding to the partial region, and at least a stamp having the cytophilic substance attached to the upper surface is prepared. The method according to (1), comprising a step of bringing the upper surface into contact with the surface of the support on which an active carbonyl group is formed, and bringing the cytophilic substance into contact with the partial region.

(3)
The method according to (1) or (2), wherein the functional group introduced into the support is at least one selected from the group consisting of an epoxy group, an aldehyde group and an amino group.

(4)
The method according to (3), wherein the functional group introduction step includes a step of applying the silane coupling agent having the functional group to the surface of the support by a sol-gel method.

(5)
(1) to (4), wherein the hydrophilic compound is at least one selected from the group consisting of ethylene glycol, a polymer of ethylene glycol, and a copolymer of ethylene glycol and propylene glycol. The method in any one of.

(6)
A carrier for cell culture, comprising a surface comprising a cell adhesive region and a cell non-adhesive region produced by the method according to any one of (1) to (5).

(7)
A cell culture carrier comprising a surface comprising a cell adhesion region and a cell non-adhesion region, a support having a functional group on the surface, and a binding group capable of reacting with the functional group to form a covalent bond And a hydrophilic compound having a hydroxyl group, a carboxylic acid compound having two carboxylic acid groups, a cytophilic substance having a functional group capable of forming a covalent bond with the carboxylic acid group, and a covalent bond with the carboxylic acid group. In (a) the cell adhesion region formed by a low molecular compound having a functional group that can be formed, a bond is formed between the functional group on the support and the binding group of the hydrophilic compound. An ester bond is formed between the hydroxyl group of the hydrophilic compound component and one carboxylic acid group of the carboxylic acid compound, and the other carboxylic acid group of the carboxylic acid compound and the cell parent are formed. A covalent bond is formed between the functional group of the active substance and (b) in the cell non-adhesive region, a bond is formed between the functional group on the support and the binding group of the hydrophilic compound. An ester bond is formed between the hydroxyl group of the hydrophilic compound component and one carboxylic acid group of the carboxylic acid compound, and the other carboxylic acid group of the carboxylic acid compound and the low molecule A carrier for cell culture, wherein a covalent bond is formed with a functional group of the compound.

  In the cell culture substrate produced by the method of the present invention, the cell non-adhesive region is covered with a hydrophilic polymer at a high density, and the cell adhesion inhibitory property of the cell non-adhesive region becomes extremely high. At the same time, since the cell affinity substance is covalently immobilized in the cell adhesion region, the cell affinity of the cell adhesion region is extremely high. This makes it possible to maintain the cell pattern stably for a long time.

  The method of the present invention is advantageous in that the operation of each step is simple compared with the prior art.

FIG. 1 is a diagram schematically illustrating each step in the method for producing a cell culture carrier of the present invention. FIG. 2 is a diagram schematically illustrating a process of selectively immobilizing a cell affinity substance using a microcontact printing method and blocking the remaining region with a low molecular weight compound. FIG. 3 shows an embodiment of the present invention including the step of introducing epoxy groups on the glass surface. FIG. 4 shows an embodiment of the present invention that includes the step of introducing aldehyde groups onto the glass surface. FIG. 5 is a microscope image of the line-shaped cell pattern obtained in Example 1. FIG. 6 is a microscope image of the dot-like cell pattern obtained in Example 1.

  Each process in the manufacturing method of the support | carrier for cell cultures of this invention is typically shown in FIG. Hereinafter, each process is explained in full detail.

(Functional group introduction process)
In the present invention, the “functional group introduction step” is a step of introducing a functional group onto the surface of the support.

  The support of the present invention is not particularly limited as long as it can provide a stable surface against water. Specific materials constituting the support include inorganic materials such as metal, metal oxide, glass, quartz, silicon, and ceramic, elastomer, plastic, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin And synthetic polymers such as fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin and vinyl chloride resin, and natural polymers such as chitin, chitosan and cellulose. The shape of the support is not particularly limited, and may be, for example, a flat shape such as a flat plate, a flat membrane, a film, or a porous membrane, or a three-dimensional shape such as a cylinder, stamp, well, microchannel, or fine particle. In particular, considering the industrial applicability of the present invention, a support made of glass, quartz or silicon is desirable.

  The functional group introduced into the surface of the support is not particularly limited as long as it can be covalently bonded to a binding group on the hydrophilic compound described later. However, when the support is glass, quartz, or silicon, the functional group is preferably an epoxy group, an aldehyde group, or an amino group that can be easily introduced with a general-purpose silane coupling agent. The group is particularly preferred. In addition, an N-hydroxysuccinimide group, a hydroxyl group, an isocyanate group, a maleimide group, a thiol group, a carboxyl group, a carbodiimide group, and the like are conceivable, but not limited thereto.

  General-purpose silane coupling agents that can be used in the present invention include silane coupling agents having an epoxy group, an aldehyde group, or an amino group.

（式中、
Ｒ_１、Ｒ_２、Ｒ_３は互いに独立にメチル、メトキシまたはエトキシを示し、ただしＲ_１、Ｒ_２およびＲ_３のうち少なくとも１つはメトキシまたはエトキシであり、Ｅはエポキシ基を含有する基、例えば３−グリシドキシプロピル基または２−（３，４−エポキシシクロヘキシル）エチル基である）で表されるものが使用できる。具体的には３−グリシドキシプロピルトリメトキシシラン、３−グリシドキシプロピルトリエトキシシラン、３−グリシドキシプロピルメチルジメトキシシラン、３−グリシドキシプロピルメチルジエトキシシラン、３−グリシドキシプロピルジメチルエトキシシラン、２−（３，４−エポキシシクロヘキシル）エチルトリメトキシシラン、および２−（３，４−エポキシシクロヘキシル）エチルトリエトキシシランが挙げられる。 Examples of the silane coupling agent having an epoxy group include the following general formula (I):

(Where
R ₁ , R ₂ , R ₃ independently of one another represent methyl, methoxy or ethoxy, provided that at least one of R ₁ , R ₂ and R ₃ is methoxy or ethoxy, E is a group containing an epoxy group, For example, those represented by 3-glycidoxypropyl group or 2- (3,4-epoxycyclohexyl) ethyl group) can be used. Specifically, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropyltriethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxy Examples include propyldimethylethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, and 2- (3,4-epoxycyclohexyl) ethyltriethoxysilane.

[式中、Ｒ_１１およびＲ_１２の一方は支持体とエポキシ環とを連結する連結基、例えば：

（式中*はエポキシ基への結合を示し、Ｒ_１、Ｒ_２、Ｒ_３は上記で定義したとおりである）であり、他方は水素または他の置換基、好ましくは水素であり、或いは、Ｒ_１１とＲ_１２とが一緒になって、連結基を介して支持体と連結された環、例えば

（式中*はそれぞれエポキシ基への結合を示し、Ｒ_１、Ｒ_２、Ｒ_３は上記で定義したとおりである）を形成している］ That is, the epoxy group introduced in this functional group introduction step has the following structure.

[Wherein one of R ₁₁ and R ₁₂ is a linking group for linking the support and the epoxy ring, for example:

(Wherein * represents a bond to an epoxy group, R ₁ , R ₂ , R ₃ are as defined above), the other is hydrogen or another substituent, preferably hydrogen, or R ₁₁ and R _{12 taken} together to form a ring connected to the support through a linking group, for example

(Wherein * represents a bond to an epoxy group, and R ₁ , R ₂ and R ₃ are as defined above)]

（式中、Ｒ_１、Ｒ_２、Ｒ_３は上記で定義したとおりであり、Ａはアミノ基を含有する基、例えば３−アミノプロピル基またはＮ−（２−アミノエチル）−３−アミノプロピル基である）で表されるものが使用できる。具体的にはＮ−（２−アミノエチル）−３−アミノプロピルトリメトキシシラン、Ｎ−（２−アミノエチル）−３−アミノプロピルメチルジメトキシシラン、Ｎ−（２−アミノエチル）−３−アミノプロピルトリエトキシシラン、３−アミノプロピルトリメトキシシラン、３−アミノプロピルトリエトキシシラン、３−アミノプロピルメチルジメトキシシラン、３−アミノプロピルメチルジエトキシシラン、および３−アミノプロピルジメチルエトキシシランが挙げられる。 Examples of the silane coupling agent having an amino group include the following general formula (II):

Wherein R ₁ , R ₂ and R ₃ are as defined above, and A is a group containing an amino group, such as a 3-aminopropyl group or N- (2-aminoethyl) -3-aminopropyl Can be used. Specifically, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropylmethyldimethoxysilane, N- (2-aminoethyl) -3-amino Examples include propyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropylmethyldimethoxysilane, 3-aminopropylmethyldiethoxysilane, and 3-aminopropyldimethylethoxysilane.

[Ｒ_１３は支持体とアミノ基とを連結する連結基、例えば

（式中*はアミノ基への結合を示し、Ｒ_１、Ｒ_２、Ｒ_３は上記で定義したとおりである）である] That is, the amino group introduced in this functional group introduction step has the following structure.

[R ₁₃ is a linking group for linking the support and the amino group, for example,

(Wherein * represents a bond to an amino group and R ₁ , R ₂ and R ₃ are as defined above)]

  As a method for introducing an aldehyde group to the support surface, in addition to a method of directly introducing it using a silane coupling agent having an aldehyde group, an aldehyde group is obtained after introducing an epoxy group or an amino group to the support surface. A method is mentioned.

  After introducing an epoxy group onto the support surface using a general-purpose silane coupling agent or the like, it can be hydrolyzed to give a diol, and then converted to an aldehyde group by oxidative cleavage. When the introduced epoxy group is present in the acyclic structure as in the case where the group E in the general formula (I) is a 3-glycidoxypropyl group, one aldehyde group from one epoxy group Will occur.

[Ｒ_１１’は、Ｒ_１１およびＲ_１２について上記で定義した、支持体とエポキシ環とを連結する連結基であり、Ｒ_１２’は、Ｒ_１１またはＲ_１２について上記で定義した、水素または他の置換基である。] [Scheme 1]

[R _{11 ′} is a linking group connecting the support and the epoxy ring as defined above for R ₁₁ and R ₁₂ , and R _{12 ′} is hydrogen or other as defined above for R ₁₁ or R ₁₂ Is a substituent. ]

  When the introduced epoxy group is formed in a ring structure, such as when the group E in the general formula (I) is a 2- (3,4-epoxycyclohexyl) ethyl group, one epoxy Two aldehyde groups are generated from the group.

［Ｒ_１１’’とＲ_１２’’とは一緒になって、Ｒ_１１およびＲ_１２について上記で定義した、連結基を介して支持体と連結された環を形成している。］ [Scheme 2]

[R _{11 ″} and R _{12 ″} together form a ring connected to the support via the linking group as defined above for R ₁₁ and R ₁₂ . ]

In addition, after introducing an amino group to the support surface using a general-purpose silane coupling agent, etc., the aldehyde group is also introduced by reacting the introduced amino group with a compound having two aldehyde groups (for example, glutaraldehyde). can do. Furthermore, if necessary, the carbon nitrogen double bond (C = N) can be reduced with a reducing agent such as sodium borohydride (NaBH ₄ ).

[Ｒ_１３は上記で定義したとおりである] [Scheme 3]

[R ₁₃ is as defined above]

  Examples of the application method of the general-purpose silane coupling agent to the support surface include, but are not limited to, an immersion method, a sol-gel method, a vapor deposition method, a spray method, and an integral blend method. The most desirable method in the present invention is the sol-gel method. Application of the silane coupling agent by the sol-gel method brings about a suitable result for subsequent processes such as an increase in the amount of hydrophilic compound bound and the amount of target substance immobilized. Here, the sol-gel method is a method in which a sol obtained by hydrolyzing a metal alkoxide is dried to obtain a gel that loses fluidity, and the gel is fired to obtain a metal oxide. As a method for forming a thin film of a sol solution on the substrate surface in the sol-gel method, there are a spin coating method and a dip coating method. The spin coating method is a method of applying a sol by dropping a sol solution onto a substrate and rotating the substrate at a high speed. The dip coating method is a method of applying a sol by immersing a substrate in a sol solution and pulling it up at an appropriate speed. A typical procedure for the application of a silane coupling agent by the sol-gel method is described below. First, a silane coupling agent and weakly acidic water (pH 2-3) are mixed at an appropriate ratio, for example, 1: 3 (molar ratio), and stirred for 15 minutes to 24 hours. This is diluted with isopropyl alcohol (IPA) to prepare a 0.1% to 1.0% sol solution. This is applied to the substrate surface using a spin coater. The number of rotations may be set to 500 to 1500 rpm, and the rotation time may be set to 1 to 10 seconds. After drying, it is heated in an oven at 80 to 120 ° C. for 15 to 60 minutes. Finally, ultrasonic cleaning is performed in pure water.

(Hydrophilic compound binding step)
In the present invention, the “hydrophilic compound binding step” refers to a hydrophilic compound having a binding group and a hydroxyl group capable of forming a covalent bond by reacting with a functional group introduced onto the support surface by the above procedure. It is a step of bonding to the functional group via a bond between the functional group and the binding group.

  In the present invention, since the hydrophilic compound is directly bonded to the functional group on the support, it is possible to introduce a hydrophilic compound having a large molecular weight as compared with the atom transfer radical polymerization method described in Non-Patent Document 4. . For this reason, in the present invention, the hydrophilic compound functions well as a hydrophilic spacer interposed between the support and the cytophilic substance in the area where the cytophilic substance is immobilized (cell adhesive area). In the region where the cell affinity substance is not immobilized (cell non-adhesive region), it functions well as an inhibitor that inhibits non-specific adsorption of proteins and cell adhesion. Here, the hydrophilic spacer means a water-soluble organic chemical structure having at least a function of tethering the cell affinity substance to the support by covalent bond. A hydrophilic compound having a chain structure, in particular, a hydrophilic compound having a chain structure with an average molecular weight of 1000 or more, as described later, has a high function as both a hydrophilic spacer and a substance that inhibits cell adhesion. This is particularly preferable. It is particularly preferable that the chain-like hydrophilic compound has a binding group at one end of the chain structure and a hydroxyl group at the other end. The “binding group” capable of binding to the functional group introduced on the support surface may be a hydroxyl group, or a different type of functional group (for example, an amino group, an epoxy group, an aldehyde group, A carboxyl group, an N-hydroxysuccinimide group, an isocyanate group, a maleimide group, a thiol group, a carbodiimide group, etc.), but more preferably a hydroxyl group. When the “bonding group” is a hydroxyl group, a ring-opening half-esterification reaction between a hydroxyl group for functioning as a bonding group and a cyclic acid anhydride described later on one molecule of a hydrophilic compound Each have a hydroxyl group to be used in (i.e., there are two hydroxyl groups).

  Considering the production cost, the hydrophilic compound is preferably a chain-like hydrophilic compound having one hydroxyl group at each end, specifically, ethylene glycol, a polymer of ethylene glycol, and ethylene glycol At least one selected from the group consisting of copolymers with propylene glycol is preferred, and polymers of ethylene glycol are most preferred. The polymer of ethylene glycol is not particularly limited as long as it is a polymer in which two or more molecules of ethylene glycol are polymerized, for example, one having a number average molecular weight of 1,000 or more, but one having a number average molecular weight of 10,000 or less is preferred, and one having 4000 or less Is particularly preferred. The copolymer of ethylene glycol and propylene glycol is preferably a block copolymer, and in particular, at least one unit of ethylene glycol at each end of a (poly) propylene glycol block composed of one or more units of propylene glycol unit. A block copolymer obtained by copolymerizing a (poly) ethylene glycol block composed of units is preferred. The number average molecular weight of the copolymer of ethylene glycol and propylene glycol is not particularly limited as long as it is 1000 or more, but the number average molecular weight is preferably 12000 or less, and particularly preferably 4000 or less. In the present invention, “propylene glycol” refers to 1,2-propanediol.

Example 1 of hydrophilic compound binding reaction
When a hydrophilic compound having two hydroxyl groups is bonded to the epoxy group introduced on the support surface, one hydrophilic compound is bonded to one epoxy group.

[Ｒ_１１およびＲ_１２は上記で定義したとおりである。ただし、Ｒ_１１およびＲ_１２の一方が、支持体とエポキシ環とを連結する連結基であり、他方が水素である場合には、本スキームにおいては、Ｒ_１１が支持体とエポキシ環とを連結する連結基であり、Ｒ_１２が水素となる割合が多い。] [Scheme 4]

[R ₁₁ and R ₁₂ are as defined above. However, when one of R ₁₁ and R ₁₂ is a linking group that connects the support and the epoxy ring, and the other is hydrogen, in this scheme, R ₁₁ connects the support and the epoxy ring. In many cases, R ₁₂ is hydrogen. ]

HO—R ₁₄ —OH represents a hydrophilic compound having two hydroxyl groups, for example, the above-mentioned ethylene glycol, a polymer of ethylene glycol, or a copolymer of ethylene glycol and propylene glycol. R ₁₄ denotes for example, the above ethylene glycol, polymers of ethylene glycol, or a structure obtained by removing hydroxyl groups at both ends of a copolymer of ethylene glycol and propylene glycol. ]

Example 2 of hydrophilic compound binding reaction
When a hydrophilic compound having two hydroxyl groups is bonded to the aldehyde group introduced on the support surface, two hydrophilic compounds are bonded to one aldehyde group.

[ＨＯ−Ｒ_１４−ＯＨおよびＲ_１４は上記で定義したとおりである。Ｒ_１５は上記で定義したＲ_１１’、Ｒ_１１’’とＲ_１２’’とが一緒になって形成する基、或いは、

（Ｒ_１３は上記で定義した通りであり、*はアルデヒド基への結合を示す）を示す。］ [Scheme 5]

_[HO-R 14 -OH, and _{R 14} are as defined above. R ₁₅ is a group formed by R _{11 ′} , R _{11 ″} and R _{12 ″ as} defined above, or

(R ₁₃ is as defined above, * indicates a bond to an aldehyde group). ]

(Carboxyl group forming step)
In the present invention, the “carboxyl group forming step” means a hydroxyl group on the hydrophilic compound bonded by the hydrophilic compound bonding step (in the case where the bonding group is a hydroxyl group, the reaction in the hydrophilic compound bonding step). Is a step of forming a carboxyl group derived from the cyclic acid anhydride by subjecting the cyclic acid anhydride to a ring-opening half esterification reaction.

  In terms of production cost, the cyclic acid anhydride is preferably succinic anhydride or glutaric anhydride, but is not limited thereto. A scheme of the ring-opening half esterification reaction when succinic anhydride or glutaric anhydride is used as the cyclic acid anhydride is shown below.

（ｎは１または２を示す。Ｒ_１４は上記で定義したとおりである。） [Scheme 6]

(N represents 1 or 2. R ₁₄ is as defined above.)

In addition, when this reaction is applied to the R ₁₄ —OH group bonded along Scheme 4, the hydroxyl group on the bonded carbon of R ₁₁ generated in Scheme 4 is also a ring-opening half ester. Undergo chemical reaction.

  The catalyst used in the ring-opening half esterification reaction is not particularly limited as long as it promotes this reaction, and specific examples include triethylamine, isobutylethylamine, pyridine, 4-dimethylaminopyridine, and the like. -Dimethylaminopyridine is preferable, and 4-dimethylaminopyridine is most preferable in view of reaction rate and yield.

  The ring-opening half esterification reaction is preferably performed in an inert organic solvent such as toluene to which the above catalyst is added.

  When succinic anhydride was used as the cyclic acid anhydride and 4-dimethylaminopyridine was used as the catalyst, the concentrations of succinic anhydride and 4-dimethylaminopyridine were 1 to 50 mM, the reaction temperature was 4 to 100 ° C., and the reaction time was 2 It is desirable to set in the range of minutes to 16 hours. Here, by adjusting various reaction conditions (reagent concentration, reaction temperature, reaction time), the rate of introduction of carboxyl groups into the hydrophilic compound can be controlled. That is, if the reagent concentration or reaction temperature is set low or the reaction time is set short, the surface density of the active carbonyl group formed in the activation step decreases, and conversely if set high or long, the surface density of the active carbonyl group increases. Becomes higher. It is desirable that the surface density of the active carbonyl group is appropriately adjusted according to the type of cytophilic substance to be immobilized. For example, in the case of low molecular weight compounds such as peptides and hydrocarbons, the surface density of the active carbonyl group is preferably as high as possible, and conversely, in the case of polymers such as proteins, it is better not to be too high.

(Activation process)
In the present invention, the “activation step” is a step of converting the carboxyl group formed in the carboxyl group formation step into an active carbonyl group.

  The active carbonyl group is a functional group for binding the hydrophilic compound and the cell affinity substance by a covalent bond. Here, the active carbonyl group means a chemical structure of R—C (═O) —X. X represents a leaving group such as halogen, N-hydroxysuccinimide group or derivatives thereof, 1-hydroxybenzotriazole group or derivatives thereof, pentafluorophenyl group, paranitrophenyl group, but is not limited thereto. The active carbonyl group is preferably an N-hydroxysuccinimide ester group in terms of reactivity, safety, and production cost. Conversion of the carboxyl group to an N-hydroxysuccinimide ester group is achieved by reacting the carboxyl group with N-hydroxysuccinimide and carbodiimide simultaneously. Here, carbodiimide means an organic compound having a chemical structure of -N = C = N-, for example, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, etc. Although considered, it is not limited to these. The concentration of N-hydroxysuccinimide and carbodiimide is preferably set in the range of 1 to 100 mM, the reaction temperature is 4 to 100 ° C., and the reaction time is in the range of 2 minutes to 16 hours. As the reaction solvent, N, N'-dimethylformamide (DMF), toluene or the like can be used.

(Cell affinity substance immobilization process)
In the present invention, the “cytophilic substance immobilization step” means that a covalent bond can be formed by reacting with an active carbonyl group in a part of the support surface on which an active carbonyl group is formed in the activation step. A cytophilic substance having a different functional group is contacted, and a covalent bond is formed by a reaction between the active carbonyl group in the partial area and the functional group of the cytophilic substance, and the partial area has the This is a step of immobilizing a cell affinity substance. In this step, the region where the cell affinity substance is selectively immobilized constitutes the cell adhesion region in the cell culture carrier.

The cell affinity substance is not particularly limited as long as it has a functional group capable of reacting with an active carbonyl group to form a covalent bond, and has a direct or indirect affinity for cells. Here, “indirectly” means that a substance that reacts with an active carbonyl group to form a covalent bond does not directly interact with the cell, but the substance that directly interacts with the cell is physicochemical or This refers to the case where the substance acquires cell affinity by biological binding. Specific examples include gelatin, collagen, laminin, Matrigel ^™ , fibronectin, poly-L-lysine, polyethyleneimine, antibody, antigen, growth factor, hormone, cytokine, peptide, hydrocarbon and the like. It is desirable that these cytophilic substances are appropriately selected depending on the cell type. For example, gelatin, collagen, Matrigel ^™ , fibronectin are used for culturing endovascular cells, laminin, Matrigel ^™ are used for culturing epithelial cells, and poly-L-lysine, polyethyleneimine, laminin, Matrigel are used for culturing neurons. ^For fibroblast culture, ^TM is suitable for fibronectin, poly-L-lysine, polyethyleneimine and the like. Even if the cell affinity substance to be immobilized has no functional group capable of reacting with an active carbonyl group to form a covalent bond, such as an amino group, an amino group or the like is added to these substances. It can be used for immobilization by artificial introduction.

  Examples of the “functional group capable of reacting with an active carbonyl group to form a covalent bond” on the cell affinity substance include an amino group, a thiol group, a hydroxyl group, and a carboxyl group.

  As a method of bringing the active carbonyl group and the cytophilic substance into position selective contact with only a partial region of the surface where the active carbonyl group is formed, a convex having an upper surface having a shape corresponding to the region to be contacted can be used. Prepared at least on the upper surface (typically, the entire surface of the stamp, or the entire surface on the side where the convex portion is formed), and the upper surface of the convex portion, A method in which a cytophilic substance is brought into contact with the region and transferred is brought into contact with the support surface on which an active carbonyl group is formed. This method is known as a microcontact printing (μCP) method, and has many applications especially to cell micropatterning. The upper surface of the convex portion of the stamp is in contact with the surface of the carrier, but the concave portion is not in contact, so that the cytophilic substance can be immobilized position-selectively. A stamp having a desired uneven pattern used in the μCP method is usually formed of polydimethylsiloxane (PDMS). Examples of contact methods other than the μCP method include an inkjet printing method.

  By using the μCP method, it is possible to form a very fine cell-adhesive region pattern with a width of about 1 μm. In particular, a pattern having a width of about 5 μm or more is created with good reproducibility. be able to.

  A typical application method of the μCP method in the present invention will be described with reference to FIG. First, a PDMS stamp having a desired concavo-convex pattern manufactured by a known method is manufactured (FIG. 2A). This stamp is immersed in a solution of a cytophilic substance, for example, a PBS solution of gelatin, and left for 5 minutes or longer (FIG. 2 (b)). The gelatin concentration is preferably set to 1 to 5 mg / ml. As a result, gelatin molecules are adsorbed on the PDMS stamp. Rinse the stamp lightly with distilled water and dry with nitrogen blow. The surface of the stamp on which the gelatin molecules are adsorbed is brought into contact with the surface of the activated carbonyl side of the activated carrier surface (referred to as “substance solidifying carrier” in FIGS. 1 and 2) (FIG. 2 ( c) and (d)). After leaving in that state for 10 minutes or more, the stamp is carefully pulled away from the surface of the carrier (FIG. 2 (e)). By the above operation, gelatin molecules are transferred from the stamp surface to the carrier surface. At this time, a covalent bond is formed between the gelatin and the active carbonyl group on the surface of the carrier.

(Blocking process)
In the present invention, the “blocking step” refers to contacting the low molecular weight compound having a functional group capable of reacting with an active carbonyl group to form a covalent bond after the cell affinity substance immobilization step. A covalent bond is formed by a reaction between the active carbonyl group remaining on the surface and the functional group of the low molecular compound, and the remaining active carbonyl group is converted into a group containing the low molecular compound (usually the low molecular compound A carbonyl group to which a compound is bonded). In the process of the present invention, the active carbonyl group remaining in the region where the cell affinity substance is not immobilized on the surface of the carrier is replaced with a group containing a low molecular compound, thereby forming a cell non-adhesive region.

  Examples of the “functional group capable of reacting with an active carbonyl group to form a covalent bond” on the low molecular compound include an amino group, a thiol group, a hydroxyl group, and a carboxyl group.

  The blocking agent (low molecular weight compound) is not particularly limited as long as it is a low molecular weight compound having a functional group capable of forming a covalent bond by reacting with an active carbonyl group, but “reacting with an active carbonyl group to form a covalent bond. It is preferable to use a low molecular compound further having a hydrophilic group (in particular, a hydrophilic group that is electrically neutral in water) in addition to “possible functional group”. This is because, when such a blocking agent is used, the hydrophilicity of the cell non-adhesive region is further increased, and thus the action of inhibiting nonspecific adsorption of the biological substance is further enhanced. Examples of the hydrophilic group that is electrically neutral in water include a hydroxyl group, a thiol group, a cyano group, and an aldehyde group.

  As the “low molecular compound” used in the present invention, a part of hydrogen of the hydrocarbon compound having 6 or less carbon atoms, more preferably 4 or less is the functional group (and the hydrophilic group if necessary). The compound substituted by is preferable, and specific examples include ethanolamine, trishydroxymethylaminomethane, and diglycolamine (IUPAC name: 2- (2-aminoethoxy) ethanol). Glycolamine is most preferred.

  These low molecular weight compounds are dissolved in a buffer solution such as PBS so as to have a concentration of 10 to 50 mM, and a carrier on which a desired cytophilic substance is immobilized is immersed therein (FIG. 2 (f)). The reaction temperature may be set in the range of 4 to 37 ° C. and the reaction time in the range of 1 minute to 1 hour. As a result, cell adhesion to a region where the cell affinity substance is not immobilized (cell non-adhesive region) can be almost completely prevented (FIGS. 2 (g) and (h)).

  Hereinafter, the present invention will be described using specific examples.

Example 1
This example is an example of the embodiment shown in FIG. 3, which is a step of introducing an epoxy group on the glass surface, a step of bonding polyethylene glycol (PEG) to the epoxy group, and a hydroxyl existing at the free end of the PEG. A step of reacting succinic anhydride (SuA) with a group to form a carboxyl group, a step of converting the carboxyl group into an active ester group, a step of regioselectively fixing gelatin to the active ester group by μCP method, And blocking an unreacted active ester group with diglycolamine (DGA). A specific procedure will be described below.

  2205 μl of 3-glycidoxypropyltrimethoxysilane (GPTS, GE Toshiba Silicone) and 480 μl of diluted hydrochloric acid (pH 2.4) were mixed and stirred for 1 hour. This was diluted with isopropyl alcohol (IPA) to prepare a 0.5% sol solution. This was applied to a glass substrate (100 mm × 100 mm) that had been UV-cleaned by spin coating. After drying, the substrate was heated in an oven at 100 ° C. for 15 minutes. On this substrate, PEG4000 (Kanto Chemical) containing a catalytic amount of concentrated sulfuric acid was dropped and heated at 80 ° C. for 1 hour. After the reaction, the substrate was thoroughly washed with water and dried by nitrogen blowing. Next, 50 mg of succinic anhydride (SuA, Kanto Chemical) and 60 mg of 4-dimethylaminopyridine (DMAP, Wako Pure Chemical Industries, Ltd.) are heated and dissolved in 43.5 g of toluene (Kanto Chemical), and the substrate is immersed therein. did. After heating at 80 ° C. for 1 hour, the substrate was washed with ethanol and pure water and dried by nitrogen blowing. Next, 58 mg of N-hydroxysuccinimide (NHS, Wako Pure Chemical Industries) and 78 μl of N, N′-diisopropylcarbodiimide (DIC, Wako Pure Chemical Industries) were heated and dissolved in 43.5 g of toluene, and the substrate was immersed therein. . After heating at 80 ° C. for 1 hour, the substrate was washed with ethanol and pure water and dried by nitrogen blowing. After the substrate was cut into a size of 25 mm × 10 mm, gelatin (Sigma-Aldrich) was regioselectively immobilized on the substrate surface by the μCP method.

Here, a template for a polydimethylsiloxane (PDMS) stamp was produced as follows. First, a negative photoresist, SU-8 2002 (Kayaku Microchem) was spin-coated on a glass substrate (10 cm square), and then the substrate was heated at 95 ° C. for 1 minute. The resist-coated surface was brought into contact with a 5-inch photomask having a stripe pattern in which opening lines with a width of 60 μm were formed at a pitch of 200 μm, and a mercury lamp was irradiated for 3 seconds from the photomask side. The illuminance of the mercury lamp was 20 mW / cm ² at a wavelength of 365 nm. After exposure, the substrate was heated at 95 ° C. for 1 minute. Finally, the exposure pattern was developed with propylene glycol monomethyl ether acetate (Pure Chemical). The mold thus obtained had a stripe pattern in which recesses having a width of 140 μm and a depth of about 3 μm were formed at a pitch of 200 μm. The PDMS stamp was obtained by applying SYLPOT184 (Toray Dow Corning) to the above mold, heating it in an oven at 150 ° C. for 15 minutes, and then carefully peeling the polymerized PDMS from the mold. This PDMS stamp had a stripe pattern in which convex portions having a width of 140 μm and a height of about 3 μm were formed at a pitch of 200 μm. The stamp was immersed in a PBS solution containing 1 mg / ml gelatin, allowed to stand for 5 minutes, rinsed lightly with distilled water, and dried with nitrogen blow. The surface on the side of the concavo-convex pattern of the stamp on which the gelatin molecules were adsorbed was brought into contact with the surface on the activated carbonyl side of the activated carrier surface. After standing in that state for 10 minutes, the stamp was carefully pulled away from the carrier surface. The thus obtained substrate on which gelatin was immobilized was immediately immersed in a DGA in PBS and subjected to a blocking treatment. The concentration of DGA in the blocking treatment was 50 mM, the reaction temperature was room temperature, and the reaction time was 5 minutes.

The cell culture carrier produced as described above was sterilized with 70% ethanol, and then seeded with bovine vascular endothelial cells. As the medium, DMEM (Dulbecco's modified Eagle medium, Sigma-Aldrich) containing 10% fetal bovine serum was used. As a result, a linear cell pattern as shown in FIG. 5 was obtained. Similar cell patterns were obtained when type I collagen (Nitta gelatin) or Matrigel ^TM (Becton Dickinson) was used instead of gelatin (FIG. 5).

A dot-like cell pattern was obtained in the same procedure. In this case as well, the procedure is the same as that described above except that the design of the photomask used for producing the mold is different. The photomask used for producing the mold has a lattice pattern composed of opening lines with a width of 30 μm, and the interval between the opening lines is 30 μm, 40 μm or 50 μm. The obtained template had a dot-like pattern in which square concave portions each having a side of 30 μm, 40 μm or 50 μm and a depth of about 3 μm were formed in an array. In addition, the PDMS stamp obtained by this mold had a dot pattern in which square convex portions each having a side of 30 μm, 40 μm or 50 μm and a height of about 3 μm were formed in an array. FIG. 6 shows a dot-like cell pattern obtained on a cell culture carrier having a dot-like cell adhesive region pattern formed by the μCP method using this stamp. In FIG. 6, the area of each cell adhesion region is 900 μm ² (30 μm × 30 μm), 1600 μm ² (40 μm × 40 μm), and 2500 μm ² (50 μm × 50 μm) from the left.

  The cell pattern shown in FIGS. 5 and 6 was maintained for at least one week.

Example 2
This example is an example of the embodiment shown in FIG. 4, in which an aldehyde group is introduced onto the glass surface, PEG is covalently bonded to the aldehyde group, and hydroxyl groups present at the free ends of the PEG are anhydrous. A step of reacting succinic acid to form a carboxyl group, a step of converting the carboxyl group to an active ester group, a step of regioselectively fixing gelatin to the active ester group by μCP method, an unreacted active ester group A step of blocking with DGA. A specific procedure will be described below.

  2205 μl of GPTS and 480 μl of diluted hydrochloric acid (pH 2.4) were mixed and stirred for 1 hour. This was diluted with IPA to prepare a 0.5% sol solution. This was applied to a glass substrate (100 mm × 100 mm) that had been UV-cleaned by spin coating. After drying, the substrate was heated in an oven at 100 ° C. for 15 minutes. Next, the substrate was immersed in 10 mM dilute sulfuric acid and heated at 80 ° C. for 2 hours. Thereafter, the substrate was immersed in a 20 mM sodium periodate aqueous solution (Kanto Chemical) and left at room temperature for 15 minutes. On this substrate, PEG 4000 containing a catalytic amount of concentrated sulfuric acid was dropped and heated at 80 ° C. for 1 hour. After the reaction, the substrate was thoroughly washed with water and dried by nitrogen blowing. Next, 50 mg of SuA and 60 mg of DMAP were dissolved by heating in 43.5 g of toluene, and the substrate was immersed therein. After heating at 80 ° C. for 1 hour, the substrate was washed with ethanol and pure water and dried by nitrogen blowing. Next, 58 mg of NHS and 78 μl of DIC were dissolved by heating in 43.5 g of toluene, and the substrate was immersed therein. After heating at 80 ° C. for 1 hour, the substrate was washed with ethanol and pure water and dried by nitrogen blowing. After the substrate was cut into a size of 25 mm × 10 mm, gelatin was regioselectively immobilized on the substrate surface by the μCP method. As the stamp used in the μCP method, a line pattern and three types of dot patterns are used as in Example 1, and gelatin is applied to the substrate surface in the same procedure as described in Example 1. Immobilization was performed. The substrate on which the gelatin was immobilized was immediately immersed in the DGA solution by the same procedure as in Example 1 to perform a blocking treatment. The cell culture carrier produced as described above was sterilized with 70% ethanol, and then seeded with bovine vascular endothelial cells. As the medium, DMEM containing 10% fetal bovine serum was used. As a result, the same cell pattern as in Example 1 was obtained. This cell pattern was maintained for at least one week.

Comparative Example A cell culture carrier was produced according to the method described in Patent Document 2, and the resulting cell pattern was compared with the present invention. The specific procedure will be described below.

39 g of toluene and 13.5 g of GPTS were mixed, and 450 μl of triethylamine (Wako Pure Chemical Industries, Ltd.) was added with stirring. A glass substrate (100 mm × 100 mm) that had been cleaned with UV was immersed therein and left at room temperature for 16 hours. Thereafter, the glass substrate was washed with ethanol and pure water and dried by nitrogen blowing. This substrate was immersed in tetraethylene glycol containing a catalytic amount of concentrated sulfuric acid and heated in an oven at 80 ° C. for 15 minutes. Next, the hydrophilic thin film on the substrate surface was oxidatively decomposed using a photomask with a photocatalyst. The photomask used here is designed so that the same cell pattern as in FIG. 6 can be obtained if successful. That is, this photomask has a lattice pattern composed of light shielding lines with a width of 30 μm, and the interval between the light shielding lines is 30 μm, 40 μm or 50 μm. The mercury lamp had an illuminance of 20 mW / cm ² and an exposure time of 100 seconds. This substrate was cut into a size of 25 mm × 15 mm. The cell culture carrier produced as described above was sterilized with 70% ethanol, and then seeded with bovine vascular endothelial cells. As the medium, DMEM containing 10% fetal bovine serum was used. However, with this carrier, a dot-like cell pattern as shown in FIG. 6 could not be obtained. The cause is considered that the cell affinity of the cell adhesion region is not sufficient to obtain a fine cell pattern.

Claims (6)

A method for producing a carrier for cell culture comprising a surface comprising a cell adhesive region and a cell non-adhesive region,
A functional group introduction step of introducing a functional group onto the surface of the support;
A hydrophilic compound having a chain structure having a binding group capable of reacting with the functional group to form a covalent bond at one end and a hydroxyl group at the other end, the functional group and the binding property A hydrophilic compound binding step for binding via a bond with a group;
Forming a carboxyl group by subjecting a hydroxyl group on the bound hydrophilic compound to a ring-opening half-esterification reaction with a cyclic acid anhydride;
An activation step of converting the carboxyl group formed in the carboxyl group formation step into an active carbonyl group;
A cell affinity substance having a functional group capable of reacting with the active carbonyl group to form a covalent bond is brought into contact with a part of the surface of the support on which the active carbonyl group has been formed in the activation step; Cytophilic substance immobilization in which a covalent bond is formed by a reaction between an active carbonyl group in a part of the region and a functional group of the cytophilic substance to immobilize the cytophilic substance in the part of the region. Conversion process,
After the cell affinity substance immobilization step, the surface of the support is contacted with a low molecular compound having 6 or less carbon atoms having a functional group capable of reacting with an active carbonyl group to form a covalent bond, A blocking step of forming a covalent bond by a reaction between the active carbonyl group remaining on the functional group of the low molecular weight compound and replacing the active carbonyl group with a group containing the low molecular weight compound. A method for producing a carrier for cell culture, which is characterized.   The cytophilic substance immobilization step includes a convex portion having an upper surface having a shape corresponding to the partial region, and at least a stamp having the cytophilic substance attached to the upper surface is prepared. The method according to claim 1, comprising a step of bringing the upper surface into contact with the surface of the support on which an active carbonyl group is formed, thereby bringing the cytophilic substance into contact with the partial region.   The method according to claim 1 or 2, wherein the functional group introduced into the support is at least one selected from the group consisting of an epoxy group, an aldehyde group and an amino group.   The method according to claim 3, wherein the functional group introduction step includes a step of applying the silane coupling agent having the functional group to the surface of the support by a sol-gel method.   The hydrophilic compound is at least one selected from the group consisting of ethylene glycol, a polymer of ethylene glycol, and a copolymer of ethylene glycol and propylene glycol. The method according to claim 1. A cell culture carrier comprising a surface including a cell adhesion region and a cell non-adhesion region,
A support having a functional group on the surface;
A chain-structured hydrophilic compound having a binding group capable of reacting with the functional group to form a covalent bond at one end and a hydroxyl group at the other end ;
A carboxylic acid compound having two carboxylic acid groups;
A cytophilic substance having a functional group capable of forming a covalent bond with a carboxylic acid group;
Formed with a carboxylic acid group and a low molecular weight compound having a functional group capable of forming a covalent bond and having 6 or less carbon atoms ,
(A) In the cell adhesive region, a bond is formed between the functional group on the support and the binding group of the hydrophilic compound, and the hydroxyl group of the hydrophilic compound component and the carboxylic acid compound An ester bond is formed with one of the carboxylic acid groups, and a covalent bond is formed between the other carboxylic acid group of the carboxylic acid compound and the functional group of the cytophilic substance,
(B) In the cell non-adhesive region, a bond is formed between the functional group on the support and the binding group of the hydrophilic compound, and the hydroxyl group of the hydrophilic compound component and the carboxylic acid An ester bond is formed with one carboxylic acid group of the compound, and a covalent bond is formed between the other carboxylic acid group of the carboxylic acid compound and the functional group of the low molecular weight compound. A cell culture carrier.

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