JP2016214121A - Cell culture vessel and method for producing cell sheet - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture vessel which makes it possible to obtain a cell sheet by cell culture, and also easily peel the obtained cell sheet.SOLUTION: A cell culture vessel comprises a support comprising plastic having a hydrophilic treated face, a silicon oxide layer disposed on the hydrophilic treated face of the support, and a hydrophobic polysiloxane layer disposed on the silicon oxide layer.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell culture container and a method for producing a cell sheet using the cell culture container.

  One technique for regenerative medicine is to transplant cells. Examples of the cell form for transplantation include a cell sheet obtained by culturing cells in a sheet form. The cell sheet is a sheet-like cell aggregate in which cells are connected by at least one layer by intercellular bonding. Cell sheets can be obtained by culturing cells on a support such as a petri dish. In general, cell sheets formed on a support are firmly bonded to the support surface via adhesion molecules and the like. Therefore, it is not easy to peel the cell sheet from the support without breaking the bond between cells. Therefore, research is being conducted on a technique that can easily recover a cell sheet with little damage to cells.

  Non-Patent Document 1 discloses a culture vessel in which a temperature-responsive polymer is applied to the surface. Non-Patent Document 1 describes a method of recovering a cell sheet by performing low-temperature treatment after culturing cells using the culture vessel. According to this method, since an enzyme is not used for peeling, it is described that a cell sheet can be obtained without losing an extracellular matrix effective for treatment while maintaining an intercellular bond.

  However, in the method described in Non-Patent Document 1, there is a case where cells are damaged particularly when the low temperature treatment for peeling the cell sheet takes a long time. Moreover, in the case of cells with strong adhesive strength, it may be necessary to intentionally weaken the cell adhesive strength by performing subculture in order to obtain a cell sheet by peeling off at low temperature treatment.

  Therefore, research has been conducted on a culture container having a surface on which a cell sheet can be easily peeled off without special treatment after the cell sheet is formed.

Patent Document 1 discloses a method of obtaining a cell sheet that can be easily detached without requiring special treatment by seeding cells on a surface having a cell adhesive region and a cell non-adhesive region. Yes.
Patent Document 2 discloses a method for forming a cell sheet by high-density seeding.

  Patent Document 3 has a polymer hydrous gel layer as a support and a cell adhesive gel layer on the cell culture surface side outermost surface layer, and the polymer hydrous gel layer and the cell adhesive gel layer are interposed between them. Discloses a cell culture carrier having a gel layer containing an anionic synthetic polymer and a polyvalent metal ion. Also disclosed is a method of culturing cells on the cell culture carrier to form a cell sheet.

  Patent Document 4 discloses a cell culture carrier having a gel layer containing an anionic polysaccharide, a polyvalent metal ion, and a water-soluble polymer on a gel containing at least chitosan. Also disclosed is a method of recovering a cell sheet obtained by culturing cells on the cell culture carrier by dissolving a gel layer.

  Patent Document 5 discloses a method of obtaining a cell sheet by culturing cells while applying a centrifugal force in the bottom direction to cells added to the culture solution.

JP 2014-138557 A International Publication No. 2011/142364 JP 2006-217878 A JP 2007-259735 A JP 2010-161952 A

Japan Linyi, Yamato et al., Vol. 64, No. 2, pp. 335-341, 2006

  However, in the method of Patent Document 1, since a fine pattern of a cell adhesive region and a cell non-adhesive region is formed, the cost tends to increase, and quality control of the culture vessel may be difficult.

  In the method of Patent Document 2, the type of cells is limited to epithelial or endothelial cells, and there is a problem that versatility is poor.

  In the method of Patent Document 3, when the gel layer is peeled from the cell sheet, a solubilization process or a gel layer removal process using tweezers is performed, which makes the operation complicated. In addition, the cell sheet may be chemically or physically damaged.

  Even in the method of Patent Document 4, similarly to the method of Patent Document 3, there is a possibility that the cell sheet is damaged when the gel layer is dissolved.

  In the method of Patent Document 5, since it is necessary to treat the cultured cells with an enzyme such as trypsin before the cells are accumulated on the bottom of the container by centrifugal force, there is a problem that the extracellular matrix is once lost, Operation becomes complicated.

  Accordingly, an object of the present invention is to provide a cell culture container that can obtain a cell sheet by culturing cells and can easily peel the obtained cell sheet.

(1) a support comprising a plastic having a hydrophilic treated surface;
A silicon oxide layer disposed on the hydrophilic treated surface of the support;
A hydrophobic polysiloxane layer disposed on the silicon oxide layer;
A cell culture vessel.
(2) The cell culture container according to (1), wherein the hydrophobic polysiloxane layer is formed using a silicon compound represented by the following formula (1);
(R ¹ ) _p (R ² ) _4-pq Si (Y) _q (1)
(In formula (1), Y is independently a group capable of generating a silanol group by hydrolysis, p is 1 or 2, q is 2 or 3, and p + q is 3 or 4. R ¹ is independently an organic group containing a carbon atom directly attached to a silicon atom and containing a hydrophobic group, and R ² is an organic group containing a carbon atom directly attached to a silicon atom).
(3) The hydrophobic group is a methyl group, ethyl group, propyl group, butyl group, vinyl group, styryl group, acryloyl group, methacryloyl group, acryloyloxy group, methacryloyloxy group, phenyl group, glycidoxy group, epoxy group, or The cell culture container according to (2), which is an epoxycyclohexyl group.
(4) The cell culture container according to any one of (1) to (3), wherein the silicon oxide layer has a thickness of 1 μm to 350 μm.
(5) The cell culture container according to any one of (1) to (3), wherein the silicon oxide layer has a thickness of 10 μm to 120 μm.
(6) The cell culture container according to any one of (1) to (5), wherein the silicon oxide layer is formed by a chemical vapor deposition method.
(7) A method for producing a cell sheet using the cell culture container according to any one of (1) to (6).

  According to the present invention, it is possible to provide a cell culture container capable of obtaining a cell sheet by culturing cells and easily peeling the obtained cell sheet. In addition, according to the present invention, a method for producing a cell sheet using the cell culture container can be provided.

It is a schematic sectional drawing for demonstrating the structure of the cell culture container which concerns on this embodiment. It is a figure which shows the result of having measured the infrared absorption spectrum of the silicon oxide layer using the FT-IR apparatus in Example 1. FIG. 4 is a graph showing the results of surface analysis performed by XPS measurement in Example 1. It is a schematic sectional drawing which shows the layer structure of the cell culture dish (E1) and (C1)-(C4) produced in Example 1 and Comparative Examples 1-4. It is an image which shows the cell sheet obtained by the cell culture using the cell culture dish (E1) of Example 1 in Evaluation 1. It is a photograph which shows the cell state in the cell sheet obtained by the cell culture using the cell culture dishes (E1) and (C1) to (C2) in Evaluation 1. It is an image which shows the result of having dye | stained the cell sheet obtained by the cell culture using the cell culture dish (E1) in the evaluation 3. FIG. It is a graph which shows the result of the XPS measurement in evaluation 6.

[Cell culture vessel]
The cell culture container according to the present invention includes a support including a plastic having a hydrophilic treatment surface, a silicon oxide layer disposed on the hydrophilic treatment surface of the support, and a silicon oxide layer disposed on the silicon oxide layer. And a hydrophobic polysiloxane layer.

  Since the cell culture container according to the present invention has cell adhesiveness, a cell sheet can be obtained by culturing cells. Moreover, since the cell culture container of this invention has moderate cell adhesiveness, the cell sheet obtained by culture | cultivation can be peeled easily. Specifically, the cell sheet can be easily peeled off from the adhesive surface by applying a water flow to the cell sheet obtained by culturing the cells by intentional rocking or pipetting. Moreover, it can also peel easily by picking the edge part of a cell sheet using instruments, such as tweezers.

  The cell culture container of the present embodiment is preferably capable of culturing cells until it becomes confluent. In the cell culture container of the present embodiment, more preferably, the cells can be cultured until they reach a confluent state, and when the cells are cultured until they reach a confluent state, the cell sheet does not perform any operation or It peels from the adhesive surface of the container by shaking the container. This is because when the cells proliferate and reach a confluent state, the contraction force acting between the cells becomes stronger than the adhesion force between the cell sheet and the adhesion surface of the container, and the cell sheet peels off from the adhesion surface. It is done. Therefore, according to the preferred cell culture container of this embodiment, cell adhesion is performed uniformly, a uniform confluent state is achieved, and a cell sheet with very little damage is obtained.

The structure of the cell culture container of this embodiment is demonstrated with reference to FIG. FIG. 1A is a schematic cross-sectional view showing the layer configuration of the cell culture container of the present embodiment. The cell culture container has a support 3 containing plastic having a hydrophilic treatment surface, and a silicon oxide layer 2 is disposed on the hydrophilic treatment surface of the support 3. A hydrophobic polysiloxane layer 1 is disposed on the silicon oxide layer 2. The support 3 can be a member constituting at least a part of the outer wall of the cell culture container. Moreover, the support body 3 can also be made into a member different from the member which comprises an outer wall. FIG. 1 (B) shows a schematic cross-sectional view of the cell culture container of the present embodiment having a dish shape. In FIG. 1B, a dish-type plastic container functions as the support 3. In FIG. 1 (B), the support body 3 constitutes the side wall and the bottom wall of the outer wall, and the silicon oxide layer 2 is disposed on the entire top surface of the bottom wall of the support body 3. A hydrophobic polysiloxane layer 1 is disposed. The upper surface of the hydrophobic polysiloxane layer 1 is in contact with a space (culture part) in which the culture solution inside the container is arranged. Further, the upper surface of the bottom wall of the support 3 is subjected to a hydrophilic treatment. Although not shown in FIG. 1, the cell culture container can have a lid of an appropriate size. The cell culture container is preferably liquid-tight when closed with the lid. Note that the present invention is not limited to this embodiment.
Hereinafter, each component of the present invention will be described.

(Support)
The support 3 preferably has a structure capable of holding cells and a culture solution. Typically, as shown in FIG. 1 (B), a dish having a circular bottom plate and a side wall erected upward from the periphery of the bottom plate so as to hold cells and culture medium The shape may be a shape, or may be another shape capable of holding a cell and a culture solution. More specifically, the support can be in the shape of a petri dish, a flask, a beaker, a well plate, a tube, or the like. In addition, it is not essential that the support has a structure capable of holding cells and culture medium by the support alone.

  The support is configured to include a plastic having a hydrophilic treatment surface. Examples of the plastic include polystyrene, polypropylene, polyvinyl chloride, polyethylene, cyclic polyolefin, acrylic resin, and polyethylene terephthalate. Of these, polystyrene is preferred.

  The support has on its surface a hydrophilic treated surface formed by performing a hydrophilic treatment. The hydrophilic treatment is not particularly limited as long as the treatment can modify the surface of the plastic to be hydrophilic. Examples of such surface treatment include corona discharge treatment, plasma treatment, UV ozone treatment, electron beam irradiation treatment, and laser treatment.

A commercially available support having a hydrophilic treatment surface may be used, for example, a Nunc ^™ cell culture dish (trade name, manufactured by Thermo Fisher Scientific), a Falcon cell culture dish (manufactured by Corning), a cell, and the like. It is commercially available as a petri dish for culture (manufactured by Sumitomo Bakelite) or a dish for tissue culture (manufactured by Asahi Glass).

Moreover, the support body which has a hydrophilic treatment surface can also be prepared by performing a hydrophilic treatment to the support body made from an untreated plastic which has not been subjected to the hydrophilic treatment, for example. As an untreated support, for example, Nunc ^™ Petri dish (trade name, manufactured by Thermo Fisher Scientific), Falcon Petri dish (Corning), suspension culture dish (Sumitomo Bakelite), or untreated dish ( Asahi Glass Co., Ltd.) can be used.

  It is considered that the hydrophilic functional group (for example, a hydroxy group or a carbonyl group) present on the plastic surface is increased by the hydrophilic treatment. In the present embodiment, the surface of the plastic support is subjected to a hydrophilic treatment, whereby the adhesiveness of the cells is improved. In this embodiment, since the silicon oxide layer and the hydrophobic polysiloxane layer are provided on the support, the hydrophilic treatment surface of the support is not in direct contact with the culture solution. However, the surface of the support is hydrophilically treated, so that nutrients that are the materials of the intercellular matrix such as proteins contained in the culture solution are easily collected on the adhesion surface. As a result, the untreated support It is considered that the adhesiveness of the cells is improved as compared with the case of using. Further, it is considered that the silicon oxide layer is easily added on the surface of the support because the surface of the support is subjected to hydrophilic treatment.

  The static water contact angle of the hydrophilic treated surface is preferably less than 70 °. For example, the static water contact angle of hydrophilically treated polystyrene is 64 °. In addition, the water contact angle can use the value measured using the contact angle measuring device within 1 minute after dripping water from a micro syringe under the temperature of 25 degreeC, 30% of humidity, and atmospheric pressure. Moreover, as a contact angle measuring device, Kyowa Interface Science Co., Ltd. CA-Z type can be used, for example.

(Silicon oxide layer)
The silicon oxide layer is disposed on the hydrophilic treated surface of the support.
The silicon oxide layer is not particularly limited, but can be formed by, for example, chemical vapor deposition (CVD) or sputtering, and is preferably formed by chemical vapor deposition. Among chemical vapor deposition methods, it is preferable to use a plasma CVD method capable of forming a silicon oxide layer at a relatively low temperature. That is, it is preferable to form a silicon oxide layer by a low temperature vacuum plasma CVD method using an organosilicon compound and an inert gas as raw materials. The low temperature vacuum plasma CVD method does not need to heat the support and can form a film at a low temperature of, for example, 10 ° C. or more and 70 ° C. or less. In the case of forming a film by a low temperature vacuum plasma CVD method, it is preferable to keep the inside of a film formation chamber (vacuum chamber) before film formation at a vacuum degree of 1 × 10 ⁻⁵ Pa or more and 1 × 10 ⁻³ Pa or less for a certain time.

  Examples of the vacuum plasma CVD method include a capacitively coupled plasma CVD method, an inductively coupled plasma CVD method, and an ECR (electron cyclotron resonance) plasma CVD method. Among these, it is preferable to use an inductively coupled plasma CVD method. The inductively coupled plasma CVD method can generate a uniform and high-density plasma, can produce a high-quality silicon oxide layer, and can form a high-quality silicon oxide layer having excellent adhesion to a support.

  Although it does not restrict | limit especially as an organosilicon compound as source gas, For example, hexamethyldisiloxane, tetramethyldisiloxane, triethyltrimethyldisiloxane etc. are mentioned. From the viewpoint of being sufficiently decomposed in vacuum plasma and being able to form a film easily, it is preferable to use a relatively low molecular weight disiloxane compound. As the inert gas, for example, argon gas, helium gas, neon gas, krypton gas, or xenon gas can be used.

  By supplying oxygen gas together with the organosilicon compound to the reaction chamber to cause plasma discharge, silicon oxide can be deposited on the support.

  Moreover, as a sputtering method, DC (direct current) sputtering system, AC (alternating current) sputtering system, RF (high frequency) sputtering system, etc. can be used, for example. These sputtering methods may be either a bipolar sputtering method or a magnetron sputtering method. Further, among these, the RF magnetron sputtering method or the AC sputtering method is advantageous in terms of film thickness control because it uses a DC power source or an AC power source with a stable process and a simple apparatus structure. . As the target, one having silicon or silicon oxide as a main component is used.

  The thickness of the silicon oxide layer is preferably 1 μm or more and 350 μm or less, more preferably 10 μm or more and 120 μm or less, and further preferably 15 μm or more and 70 μm or less. When the film thickness of the silicon oxide layer is less than 1 μm, the adhesive force between the cell sheet formed by culture and the adhesive surface becomes strong, and the cell sheet tends to be difficult to peel off. Moreover, when the film thickness of a silicon oxide layer is larger than 350 micrometers, there exists a tendency for cell adhesiveness to fall and to become difficult to form a cell sheet.

  As a method for measuring the thickness of the silicon oxide layer, for example, a method using the stylus type step meter used in the present embodiment can be mentioned. Other examples include a method using an optical interference film thickness meter, a method using reflectance spectroscopy, a method using various microscopes such as an optical and electron microscope, and a method using ellipsometry. As for the thickness of a silicon oxide layer, it is desirable to show as an average value of the thickness in 3-10 points, for example.

  Examples of the method for confirming that the silicon oxide layer is formed on the support include a method using FT-IR and a method based on surface element analysis using an X-ray photoelectron analyzer.

(Hydrophobic polysiloxane layer)
The hydrophobic polysiloxane layer is disposed on the silicon oxide layer. In the present embodiment, it is preferable that the surface of the hydrophobic polysiloxane layer is a region (adhesion surface) that comes into contact with cells during culture. In this embodiment, since the adhesive surface becomes hydrophobic, the adhesive force between the cell sheet formed by culture and the hydrophobic polysiloxane layer can be in an appropriate range, and the cell sheet is easily peeled off. be able to. Further, since the hydrophobic polysiloxane layer has no toxicity to cells, the cells can be grown in a state of being adhered until they reach a confluent state.

  Hydrophobic polysiloxane is a polymer composed of repeating units of siloxane bonds (Si—O—Si), and can be obtained by condensation polymerization of silanol compounds having a hydrophobic group.

  In addition to a silanol group (Si—OH), the silanol compound in this embodiment has an organic group containing a carbon atom directly connected to a silicon atom and containing a hydrophobic group. This organic group becomes the side chain of the polysiloxane. The silanol compound preferably has a structure represented by the following formula (1).

(R ¹ ) _p (R ² ) _4-pq Si (OH) _q (1)
(P is 1 or 2, q is 2 or 3, p + q is 3 or 4, and R ¹ is independently an organic group containing a carbon atom directly connected to a silicon atom and containing a hydrophobic group. And 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 an organic group composed of a hydrophobic group containing a carbon atom directly bonded to a silicon atom, or a linker structure (for example, an alkylene group or an aryl group) containing a carbon atom directly bonded to a silicon atom and the linker An organic group having a hydrophobic group bonded to a silicon atom through a structure. The number of carbon atoms of R ¹ is, for example, 1 to 20, preferably from 1 to 15, more preferably 1 to 10, particularly preferably 1-8. Carbon number of a linker structure is 1-6, for example, Preferably it is 1-4. Examples of the linker structure include a methylene group, an ethylene group, a propylene group, a butylene group, and a phenylene group. Examples of the hydrophobic group include a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 2 to 6 carbon atoms (for example, a vinyl group), a substituted or unsubstituted group having 2 to 6 carbon atoms. Examples include substituted alkynyl groups, acryloyl groups, methacryloyl groups, acryloyloxy groups, methacryloyloxy groups, substituted or unsubstituted aryl groups having 6 to 12 carbon atoms, glycidoxy (glycidyloxy) groups, epoxy groups, and epoxycyclohexyl groups. . The alkyl group, alkenyl group, and alkynyl group may be linear, branched, or cyclic. Examples of the substituent for the alkyl group, alkenyl group, alkynyl group or aryl group include an alkyl group having 1 to 4 carbon atoms, an alkenyl group having 2 to 4 carbon atoms (for example, a vinyl group), a phenyl group, and the like. Specific examples of hydrophobic groups include methyl, ethyl, propyl, butyl, vinyl, styryl, acryloyl, methacryloyl, acryloyloxy, methacryloyloxy, phenyl, glycidoxy (glycidyloxy) groups , Epoxy group, epoxycyclohexyl group and the like.

Particularly preferred embodiments of R ¹ include a 3-methacryloxypropyl group, a 3-acryloxypropyl group, a 3-glycidoxypropyl group, or a 2- (3,4-epoxycyclohexyl) ethyl group.

R ² is an organic group containing a carbon atom directly connected to a silicon atom, preferably a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, or a substituted or unsubstituted aryl group having 6 to 12 carbon atoms. is there. The alkyl group may be linear, branched, or cyclic. R ² is more preferably a linear or branched alkyl group having 1 to 6 carbon atoms, and still more preferably a methyl group or an ethyl group.

  The silanol compound can be produced by hydrolyzing a silicon compound having a group capable of producing a silanol group (Si-OH) by hydrolysis (hereinafter also referred to as a silane coupling agent). Such a silicon compound has a structure represented by the following formula (2).

(R ¹ ) _p (R ² ) _4-pq Si (Y) _q (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 above for the silanol compound).

  Examples of Y include 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, or an alkylcarbonyloxy group. Can be mentioned. Y is an alkoxy group having 1 to 6 carbon atoms (for example, 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) , An alkylcarbonyloxy group having 1 to 6 carbon atoms (for example, an acetoxy group) or a chlorine atom is preferable.

  Examples of the silicon compound of the formula (2) include vinyltrimethoxysilane, vinyltriethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycol. Sidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltri Examples include methoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane. Of these, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, 3-methacryloxypropyl Methyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, or 3-acryloxypropyltrimethoxysilane are preferred.

  Other silicon compounds include, for example, methyltrimethoxysilane, methyltriethoxysilane, ethyltriethoxysilane, n-propyltrimethoxysilane, n-propyltriethoxysilane, isopropyltrimethoxysilane, isopropyltriethoxy. Silane, n-butyltrimethoxysilane, n-butyltriethoxysilane, n-pentyltrimethoxysilane, n-hexyltrimethoxysilane, vinyltriethoxysilane, cyclohexyltrimethoxysilane, cyclohexyltriethoxysilane, phenyltrimethoxysilane, Phenyltriethoxysilane, dimethyldimethoxysilane, dimethyldiethoxysilane, diethyldimethoxysilane, diethyldiethoxysilane, di-n-propyldimethoxysilane, -N-propyldiethoxysilane, diisopropyldimethoxysilane, diisopropyldiethoxysilane, di-n-butyldimethoxysilane, di-n-butyldiethoxysilane, di-n-pentyldimethoxysilane, di-n-pentyldiethoxysilane , Di-n-hexyldimethoxysilane, di-n-hexyldiethoxysilane, di-n-cyclohexyldimethoxysilane, di-n-cyclohexyldiethoxysilane, diphenyldimethoxysilane, diphenyldiethoxysilane, and the like.

  As the silicon compound of the formula (2), a compound commercially available as a silane coupling agent can be suitably used. Moreover, the silicon compound of Formula (2) may be used individually by 1 type, and may be used in combination of 2 or more type.

  When forming the hydrophobic polysiloxane layer, the silicon compound of formula (2) can be hydrolyzed as follows to produce a silanol compound of formula (1). Hydrolysis conditions are not particularly limited, but for example, the following method can be employed. First, dilute hydrochloric acid is added to the silicon compound of formula (2) to hydrolyze the group Y. The pH of the diluted hydrochloric acid is desirably adjusted to 2.0 to 3.0. The molar ratio of water molecules to silicon compound is, for example, 2-4. By this operation, the group Y is converted into a silanol group to produce a silanol compound of the formula (1). Next, a silanol compound is applied to the surface of the silicon oxide layer, and a polysiloxane layer is formed by condensation polymerization. The silanol compound of formula (1) is dissolved in alcohol together with a base. The concentration of the silanol compound in the silanol solution is preferably adjusted to be in the range of 0.1 to 10% (v / v). As the base, for example, triethylamine, N, N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, or the like can be used. The concentration of the base in the silanol solution is preferably adjusted to be in the range of 0.1 to 10% (v / v). As the alcohol, for example, ethanol, 2-propanol, tert-butyl alcohol, or the like can be used. The silanol solution containing the silanol compound is brought into contact with the surface of the silicon oxide layer and left for 10 minutes to 24 hours. The reaction temperature can be set, for example, in the range of 4 to 80 ° C., but is preferably room temperature (20 to 25 ° C.). By the above method, a hydrophobic polysiloxane layer can be formed on the silicon oxide layer. The coating density of the hydrophobic polysiloxane layer can be controlled by, for example, the concentration of the silanol compound or base, or the time for which the silanol solution is brought into contact with the silicon oxide layer surface.

  In this embodiment, it is considered that a chemical bond such as a covalent bond is formed between the hydrophobic polysiloxane layer and the silicon oxide layer. The hydrophobic polysiloxane layer may be bonded to the surface of the silicon oxide layer by physical adsorption. Physical adsorption is thought to occur by van der Waals forces or interactions between functional groups.

  The state of polysiloxane in the hydrophobic polysiloxane layer is not always clear, but in general, when polysiloxane is added to the surface of the inorganic layer, organic groups are present on the surface (opposite side of the inorganic layer). It is thought to be oriented.

  The surface of the hydrophobic polysiloxane layer has excellent releasability with respect to the cell sheet. For example, in a culture vessel in which the temperature-responsive polymer described in Non-Patent Document 1 is applied to the surface, the surface is obtained by low-temperature treatment. It can also be understood from the phenomenon that cells become easily detached when they become hydrophobic.

  The static water contact angle on the surface of the hydrophobic polysiloxane layer is preferably 70 ° or more, more preferably 80 ° or more.

  Judging from the product information regarding the silane coupling agent, the thickness of the hydrophobic polysiloxane layer formed on the inorganic silicon oxide layer is considered to be on the order of nm. Therefore, the thickness of the hydrophobic polysiloxane layer is small compared to the thickness of the silicon oxide layer formed on the order of μm, and the surface structure (silicon oxide layer and hydrophobic polysiloxane layer) is almost oxidized. It may be considered that it originates from the silicon layer.

[Method for producing cell sheet]
By using the cell culture container of the present embodiment, a cell sheet can be easily obtained.
The cells used in this embodiment are not particularly limited as long as they are adherent cells. Examples of such cells include liver cells that are liver parenchymal cells, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, Epidermal cells such as epidermal keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, mammary cells, pericytes, muscle cells such as smooth muscle cells and cardiomyocytes, kidneys Examples include cells, pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells. These cells may be primary cells collected directly from tissues or organs, or may be passaged from one generation to another. Furthermore, these cells are undifferentiated embryonic stem cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells that have unipotency, and differentiation has ended. Any of cells may be sufficient. In addition, the cells may be cultured as a single species, or two or more cells may be co-cultured.

  Any culture medium can be used without particular limitation as long as it is a cell culture medium generally used in the art. For example, depending on the type of cells used, published by Asakura Shoten, such as MEM medium, BME medium, DME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, and RPMI1640 medium A basal medium as described in "Tissue Culture Technology 3rd Edition" edited by the Japanese Society for Tissue Culture, page 581 can be used. Furthermore, serum (such as fetal bovine serum), various growth factors, antibiotics, amino acids, etc. may be added to the basal medium. A commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used. Considering the clinical application of the finally obtained cell tissue, it is preferable to use a medium that does not contain animal-derived components.

  From the viewpoint of forming a cell sheet, it is preferable to culture the cells to a confluent state in a culture vessel. When cells are cultured to form a cell sheet, a cell sheet in a confluent state may be obtained by previously seeding a sufficient number of cells to reach a confluent state without proliferating.

  Since the cell sheet obtained by culturing can be easily peeled off, a cell sheet having no damage or almost no damage can be obtained. The obtained cell sheet is suitable for use in regenerative medicine. In addition, cell sheets can be used for application to detection devices such as biosensors.

EXAMPLES The present invention will be specifically described below based on examples, but the present invention is not limited to the examples.

Example 1
A culture dish (trade name: Nunc ^™ cell culture dish, catalog number: 153066, manufactured by Thermo Fisher Scientific, diameter: 3.5 cm) with a lid removed is a CVD deposition apparatus (manufactured by ANELVA, model number: PED-401) ). The culture dish is made of plastic (polystyrene), and the bottom surface of the dish is subjected to corona discharge treatment to form a hydrophilic treated surface. The static water contact angle of this hydrophilic treated surface was 64 °. The static water contact angle was determined by measuring within 1 minute after dropping a water droplet from a microsyringe using a contact angle measuring device (Kyowa Interface Science Co., Ltd., CA-Z type).

  Subsequently, the vaporized mixed gas containing hexamethyldisiloxane (HMDSO) and oxygen is converted into a CVD deposition apparatus (product name: PED) so that the flow rate of HMDSO is 3 sccm (Standard cubic centimeter per minute) and the flow rate of oxygen is 50 sccm. -401, manufactured by ANELVA) for chemical cultivation in which a chemical vapor deposition reaction was performed for 10 minutes under the conditions of pressure 15 Pa, power 100 W and 18 ° C. (chiller display temperature), and a silicon oxide layer was formed on the bottom of the dish A dish (hereinafter also referred to as a silicon oxide deposition dish (1)) was obtained.

It was confirmed using a FT-IR apparatus (manufactured by DIGILAB, model number: FTS-7000) that a silicon oxide layer was formed on the bottom surface of the dish. When the infrared absorption spectrum of the silicon oxide layer is measured by an FT-IR apparatus, peaks (1075 cm ⁻¹ and 1150 cm ⁻¹ ) derived from asymmetric stretching of silicon oxide are detected. FIG. 2A shows the result of measuring the bottom surface of the dish before forming the silicon oxide layer, and FIG. 2B shows the result of measuring the bottom surface of the dish after forming the silicon oxide layer. As shown in FIG. 2B, a target peak indicating the presence of the silicon oxide layer is detected, and it can be seen that the silicon oxide layer is formed on the bottom of the dish.

  In addition, a silicon wafer temporarily fixed with a tape is placed in the vapor deposition apparatus at the same time, and after the vapor deposition reaction, the level difference on the vapor deposition surface that appears after peeling off the tape is measured using a stylus type step gauge DekTakII (product name, manufactured by ULVAC). The film thickness of the silicon oxide layer was measured by measuring at three points and calculating the average value. The film thickness of the silicon oxide layer was 60 μm.

  Next, a hydrophobic polysiloxane layer was formed on the bottom surface of the silicon oxide vapor-deposited dish (1) using 3-methacryloxypropyltrimethoxysilane (3-MPTS) by the following procedure. First, 1.0 ml of hydrochloric acid (pH 2.4) was added to 2.0 ml of a silane coupling agent (product names: KBM-503, 3-methacryloxypropyltrimethoxysilane, manufactured by Shin-Etsu Chemical Co., Ltd.) The mixture was stirred for a minute to conduct a hydrolysis reaction. The obtained solution was added to 100 ml of 2-propanol (manufactured by Junsei Chemical Co., Ltd.), and further 500 μl of triethylamine (Kanto Chemical Co., Inc.) was added thereto and stirred to prepare a silanol solution (1). Next, 3 ml of silanol solution (1) was applied to the bottom surface of the silicon oxide vapor-deposited dish (1) and allowed to stand at room temperature for 135 minutes to conduct a condensation reaction. After forming the polysiloxane layer, the surface was washed with pure water, water droplets were removed by nitrogen air blow, and then a drying treatment was performed in an oven at 80 ° C. for 15 minutes. This produced the cell culture dish (E1) in which the hydrophobic polysiloxane layer was formed on the silicon oxide layer.

  In order to confirm whether or not a polysiloxane layer was formed, surface analysis by XPS was performed on the obtained silicon oxide vapor-deposited dish (1) and cell culture dish (E1) according to the following procedure. First, a sample was prepared by cutting the dish bottom to an appropriate size with an ultrasonic cutter. Subsequently, about this sample, each spectrum of C1s, O1s, and Si2p was acquired using the X-ray photoelectron analyzer (The product name: ESCA-3400 by Shimadzu Corporation Corp.). And after calculating the area of each spectrum, the area ratio was calculated and compared. In FIG. 3, the calculation result of the element ratio by XPS measurement is shown. As shown in FIG. 3, the values of C1s, O1s and Si2p are different between the silicon oxide deposition dish (1) and the cell culture dish (E1), and the polysiloxane layer is formed on the surface of the silicon oxide deposition dish (1). It was confirmed that was formed. In FIG. 3, error bars indicate standard deviation.

(Comparative Example 1)
A culture dish (trade name: Nunc ^™ cell culture dish, manufactured by Thermo Fisher Scientific, diameter: 3.5 cm), which was a product before forming the silicon oxide layer, was used as the cell culture dish (C1).

(Comparative Example 2)
The silicon oxide deposition dish (1) in Example 1 was used as the cell culture dish (C2).

(Comparative Example 3)
Hydrophobic polysiloxane layer by applying the above silanol solution (1) to a culture dish (trade name: Nunc ^™ cell culture dish, manufactured by Thermo Fisher Scientific, diameter: 3.5 cm) without forming a silicon oxide layer A cell culture dish (C3) was prepared in the same manner as in Example 1 except that the above was formed. In other words, in Comparative Example 3, 3 ml of the silanol solution (1) was applied to the bottom surface of the culture dish, allowed to stand at room temperature for 135 minutes, washed and dried to produce a cell culture dish (C3).

(Comparative Example 4)
Except for using an untreated culture dish (trade name: Nunc ^™ Petri dish, untreated polystyrene, manufactured by Thermo Fisher Scientific, static water contact angle: 90 °) whose surface was not subjected to hydrophilic treatment. A cell culture dish (C4) was prepared in the same manner as in Example 1.

  A schematic diagram of the layer structure of the cell culture dishes (E1) and (C1) to (C4) prepared in Example 1 and Comparative Examples 1 to 4 is shown in FIG. 4A is a layer configuration of the cell culture dish (E1) of Example 1, FIG. 4B is a layer configuration of the cell culture dish (C1) of Comparative Example 1, and FIG. 4C is a comparative example. 4 is a layer configuration of the cell culture dish (C3) of Comparative Example 3, and FIG. 4E is a layer of the cell culture dish (C4) of Comparative Example 4. The configuration is shown. Reference numeral 1 denotes a polysiloxane layer, reference numeral 2 denotes a silicon oxide layer, reference numeral 3 denotes a support (made of polystyrene) whose surface is subjected to hydrophilic treatment, and reference numeral 4 denotes a support (made of polystyrene) whose surface is not processed.

<Evaluation 1>
When the cells were cultured using the obtained cell culture dishes (E1) and (C1) to (C4), the ability to form a cell sheet (cell proliferation ability) and the peelability of the cell sheet were evaluated according to the following procedure.

Each cell culture dish was seeded with 3 to 5 × 10 ⁵ mouse fibroblast HH cells (adherent cells), and 10% by volume in DMEM medium containing fetal bovine serum (FBS) at 37 ° C. Cultured for 7-10 days. Then, the cell sheet forming ability and the sheet peelability were evaluated.

The ability to form cell sheets was evaluated according to the following criteria.
○: The cells proliferate until they reach 90% confluence to form a cell sheet.
×: Cannot proliferate until cells reach 90% confluence.
The 90% confluent state refers to a state in which 90% or more of the adhesion surface of the container is covered with grown cells, and can be determined by observing the adhesion surface with a microscope. It is also possible to quantitatively determine by acquiring an image with a microscope and performing image analysis using software. In addition, if a cell can proliferate to a 90% confluent state, the cell sheet will inevitably be formed.

The peelability of the cell sheet was evaluated for the cell culture dish in which the cells grew to 90% confluence and formed the cell sheet. The peelability of the cell sheet was evaluated according to the following criteria.
A: The cell sheet peels from the adhesive surface by shaking the dish.
B: The cell sheet can be detached by a water flow by pipetting.
C: The cell sheet cannot be peeled off by a water flow by pipetting, but can be peeled off by grasping with a tweezers.
D: Even if the cell sheet is gripped with tweezers, it cannot be peeled off.

  The results are shown in Table 1.

  In the cell culture dish (E1) of Example 1, the cells could be grown to a confluent state, and the formed cell sheet peeled off the adhesive surface only by shaking the dish. A photograph of the obtained cell sheet is shown in FIG.

  In the cell culture dish (C1) of Comparative Example 1, cells adhered to the bottom surface and proliferated and reached a 90% confluent state, but the cell sheet could not be peeled from the adhesive surface.

In FIG. 6, the photograph which shows the cell state at the time of culture | cultivating using cell culture dish (E1) and (C1)-(C2) is shown. As shown in the photograph of FIG. 6, in the cell culture dish (E1) of Example 1 and the cell culture dish (C1) of Comparative Example 1, the cells reached a confluent state of 90% or more. However, in the cell culture dish (C2) in which the silicon oxide layer of Comparative Example 2 was present on the outermost surface, the cells died during the culture and did not reach 90% confluence. FIG. 6C also shows that in the cell culture dish (C2), the number of cells adhered to the bottom surface is smaller than that in the cell culture dish (E1) and the cell culture dish (C1). Therefore, it was suggested that a cell culture dish having a silicon oxide layer on the outermost surface is not suitable for cell culture. In addition, as a document suggesting the toxicity of silicon oxide to cells (Yang et al., Particle and Fiber Toxicology, Vol. 7, 1 2010), the document mentions the toxicity of SiO ₂ nanoparticles.

  In the cell culture dish (C3) of Comparative Example 3, the cells grew while adhering to the bottom surface, and reached a confluent state of 90% or more, but the cell sheet could not be peeled off using tweezers. Therefore, it can be seen that it is not possible to obtain good cell sheet detachability simply by forming a hydrophobic polysiloxane layer on a plastic support having a hydrophilic treated surface. That is, it can be seen that a silicon oxide layer is required to obtain good cell sheet peelability.

  The reason for this is as follows. When a hydrophobic polysiloxane layer is formed directly on a support having a hydrophilic treatment surface, a relatively large amount of proteins contained in the culture solution gathers on the adhesion surface, and a cell sheet having a relatively strong adhesion to the adhesion surface is formed. Will be. On the other hand, by disposing a silicon oxide layer between the hydrophilic treatment surface of the support and the hydrophobic polysiloxane layer, the distance between the hydrophilic treatment surface and the adhesion surface can be appropriately increased, and gather on the adhesion surface. Protein can be relatively low. Therefore, it is considered that a cell sheet having a relatively weak adhesion to the adhesion surface can be formed. In addition, the silicon oxide layer also functions as a primer layer, and the hydrophobic polysiloxane layer can be efficiently formed at a high density, which is considered to improve the hydrophobicity of the adhesive surface. For the above reasons, it is considered that the cell culture container of the present embodiment can achieve both the cell sheet forming ability and the excellent peelability of the cell sheet. In addition, the above reason is estimation and does not restrict | limit this invention.

  In the cell culture dish (C4) of Comparative Example 4, cells did not adhere. Thereby, in order for a cell to adhere | attach and to proliferate, it turns out that the hydrophilic treatment is required for the surface of the support body. If the surface of the support is subjected to a hydrophilic treatment, nutrients such as proteins contained in the culture solution can be easily attracted to the adhesion surface, and the cell adhesion of the formed cell sheet can be improved.

<Evaluation 2>
Whether the cell sheet can be recovered without increasing the number of cells seeded in the cell culture dish was confirmed by the following procedure.

Under the same culture conditions as in Evaluation 1 above, HH cells were cultured in a culture dish (manufactured by Thermo Fisher Scientific, trade name: Nunc ^™ cell culture dish, diameter 10 cm). Next, the HH cells were collected after trypsin treatment, and 2.5 × 10 ⁶ HH cells, which were in excess of the dish size, were collected from the cell culture dishes (E1) and (C1) to (C4). ). Subsequently, the cell culture dishes (E1) and (C1) to (C4) seeded with HH cells were incubated at 37 ° C., and further cultured for 4 days. Thereafter, the cell sheet forming ability and the cell sheet peelability were evaluated according to the above criteria. The results are shown in Table 2.

  Further, when the cell culture dish (C2) of Comparative Example 2 was used, the cells did not adhere and a cell sheet was not formed. That is, when the cell culture dish (C2) having the silicon oxide layer on the outermost surface was used, the number of cells to be adhered tended to decrease. When the silicon oxide layer is present on the outermost surface, the cell adhesiveness is considered to decrease.

<Evaluation 3>
The cell sheet obtained in Evaluation 1 using the cell culture dish (E1) was analyzed by the following cell staining in order to determine the viability of the cells constituting the cell sheet.

  First, the cell sheet was washed with a phosphate buffer (PBS). Thereafter, live cells diluted with PBS are stained with Calcein-AM (trade name, manufactured by Dojindo Chemical Co., Ltd.) at a concentration of 1 μg / ml, and dead cells are stained with PI (Propidium Iodide; manufactured by Dojindo Chemical Co., Ltd.) at a concentration of 1 μg / ml. The cell nuclei were stained with Hoechst 33342 (trade name, manufactured by Molecular Probes) diluted to a 1/1000 concentration. Staining was performed by allowing to stand in a 37 ° C. incubator for 15 minutes. After staining, the plate was washed with PBS, and then observed with a confocal microscope (Carl Zeiss) to obtain an image.

  FIG. 7 shows the acquired image. Fig. 7 (A) is an image of a living cell stained with Calcein-AM, Fig. 7 (B) is an image of a dead cell stained with PI, and Fig. 7 (C) is an image of a cell nucleus stained with Hoechst. is there. FIG. 7D is an image obtained by superimposing these three images. From FIG. 7D, it can be seen that the living cells in FIG. 7A and the cell nuclei in FIG. 7C overlap. From this result, it was proved that the obtained cell sheet was composed of many living cells and was not a floating structure due to cell death.

(Example 2)
A silane coupling agent (product name: A-187, 3-glycidyloxypropyltrimethoxysilane) instead of a silane coupling agent (product name: KBM-503, 3-methacryloxypropyltrimethoxysilane, manufactured by Shin-Etsu Chemical Co., Ltd.) A cell culture dish (E2) was produced in the same manner as in Example 1 except that Momentive Performance Material was used.

Example 3
Instead of a silane coupling agent (product name: KBM-503, 3-methacryloxypropyltrimethoxysilane, manufactured by Shin-Etsu Chemical Co., Ltd.), a silane coupling agent (product name: KBM-5103, 3-acryloxypropyltrimethoxysilane, A cell culture dish (E3) was prepared in the same manner as in Example 1 except that Shin-Etsu Chemical Co., Ltd. was used.

(Comparative Example 5)
A silane coupling agent (product name: KBM-903, 3-aminopropyltrimethoxy) which is an aminosilane instead of a silane coupling agent (product name: KBM-503, 3-methacryloxypropyltrimethoxysilane, manufactured by Shin-Etsu Chemical Co., Ltd.) A cell culture dish (C5) was prepared using Silane (manufactured by Shin-Etsu Chemical Co., Ltd.). In Comparative Example 5, 1 ml of the above aminosilane was added dropwise to 50 ml of 2-propanol with stirring, and 250 μl of triethylamine was further added to prepare a silanol solution. Next, in the same manner as in Experimental Example 1, 3 ml of a silanol solution was applied to the bottom surface of the silicon oxide layer deposition dish (1) and allowed to stand at room temperature for 135 minutes to perform a condensation reaction. After the reaction, washing and drying steps were performed to prepare a cell culture dish (C5).

In addition, the surface of the polysiloxane layer formed of the aminosilane is hydrophilic.
Further, as in Example 1, it was confirmed by XPS measurement that a polysiloxane layer was formed on the surface of the silicon oxide layer in the cell culture dishes (E2) to (E3) and (C5).

<Evaluation 4>
According to the method described in Evaluation 1 above, the cell sheet forming ability and the cell sheet peelability were evaluated. The results are shown in Table 3.

  From these Examples 2-3 and Comparative Example 5, it can be seen that the release property of the cell sheet can be improved by the hydrophobic polysiloxane layer.

<Examples 4 to 10>
Cell culture dishes (E4) to (E10) were produced in the same manner as in Example 1 except that the silicon oxide layer having a predetermined film thickness was formed by adjusting the CVD reaction time. Example 8 corresponds to Example 1.
<Evaluation 5>
In addition, the formed cell culture dishes (E4) to (E10) were evaluated for the ability to form a cell sheet and the peelability of the cell sheet according to Evaluation 1 described above. The results are shown in Table 4.

  From the above results, the thickness of the silicon oxide layer is preferably 10 μm or more and 120 μm or less, and more preferably 15 μm or more and 70 μm or less.

<Evaluation 6>
Separately, dishes that were made up to the steps of forming the silicon oxide layers of Examples 4, 5 and 9 were prepared, and X-ray photoelectron analyzers (manufactured by Shimadzu Corporation, product name: ESCA-3400) were obtained for the obtained samples. Each spectrum of C1s, O1s, and Si2p was acquired using. The results are shown in FIG. 8, and no significant difference in film composition was observed on each sample surface.

<Example 11>
The cell culture dish (1) is seeded with 3 × 10 ⁵ bovine vascular endothelial B23 cells (adherent cells) and cultured at 37 ° C. in DMEM medium containing 10% by volume of fetal bovine serum (FBS). did. As a result, it reached 90% confluence after 7 days of culture. The formed cell sheet could be easily detached.

DESCRIPTION OF SYMBOLS 1 Hydrophobic polysiloxane layer 2 Silicon oxide layer 3 Support body (surface-treated)
4 Support (untreated surface)

Claims (7)

A support comprising a plastic having a hydrophilic treated surface;
A silicon oxide layer disposed on the hydrophilic treated surface of the support;
A hydrophobic polysiloxane layer disposed on the silicon oxide layer;
A cell culture vessel. The cell culture container according to claim 1, wherein the hydrophobic polysiloxane layer is formed using a silicon compound represented by the following formula (1).
(R ¹ ) _p (R ² ) _4-pq Si (Y) _q (1)
(In formula (1), Y is independently a group capable of generating a silanol group by hydrolysis, p is 1 or 2, q is 2 or 3, and p + q is 3 or 4. R ¹ is independently an organic group containing a carbon atom directly attached to a silicon atom and containing a hydrophobic group, and R ² is an organic group containing a carbon atom directly attached to a silicon atom).   Hydrophobic group is methyl, ethyl, propyl, butyl, vinyl, styryl, acryloyl, methacryloyl, acryloyloxy, methacryloyloxy, phenyl, glycidoxy, epoxy, or epoxycyclohexyl The cell culture container according to claim 2, wherein   The cell culture container according to any one of claims 1 to 3, wherein the silicon oxide layer has a thickness of 1 µm to 350 µm.   The cell culture container according to any one of claims 1 to 3, wherein a thickness of the silicon oxide layer is 10 µm or more and 120 µm or less.   The cell culture container according to claim 1, wherein the silicon oxide layer is formed by a chemical vapor deposition method. The method to manufacture a cell sheet using the cell culture container in any one of Claims 1 thru | or 6.

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