JP6024004B2 - Method for producing corneal cell and method for producing corneal cell sheet - Google Patents

Description

  The present invention relates to a method for culturing corneal cells on a cell culture substrate containing a copolymer (A) of a polyethylene glycol acrylic monomer (a) and methoxyethyl acrylate (b) and an inorganic material (B). , And cultured corneal cells obtained thereby.

  Conventionally, plastic (for example, polystyrene) containers have been mainly used as cell culture substrates for animal tissues and the like. These containers are subjected to surface treatment such as plasma treatment or coating of silicon, cell adhesion factor, or the like in order to effectively perform cell culture. When these cell culture vessels are used as a culture substrate, cultured (growth) cells adhere to the vessel surface, and in order to isolate and recover the cells, trypsin or other protein hydrolase or chemical It was necessary to peel from the container surface using chemicals. The operation of peeling cells with such enzymes and chemicals not only cuts the binding part of the cell and the substrate, but also cuts the bond between cells, so that the cell is growing (for example, a sheet) It could not be removed as it was, or could cause unexpected changes in cell properties.

  In recent years, using a base material in which a polymer having a lower critical solution temperature (LCST) such as poly N-isopropylacrylamide is coated on the surface of a cell culture vessel, the polymer shows a hydrophobic state at the cell culture temperature. Adheres to the polymer, and after the cultivation, the polymer is treated at a low temperature to make it hydrophilic, thereby reducing the adhesion between the cell and the polymer, and the cell is removed from the substrate without using hydrolase or chemicals. Has been reported (see, for example, Patent Documents 1 and 2 and Non-Patent Document 1).

However, there are many cases where such a culture substrate cannot be peeled off from the surface of the substrate after culturing depending on the cell type. For example, corneal cells are one of them, and cultured corneal cells could not be completely detached even after low-temperature treatment after culturing.
In addition, when the culture substrate is sterilized by radiation (for example, γ-rays), the temperature responsiveness of the polymer having LCST is greatly reduced, and the original cells are not easily detached.

On the other hand, an organic-inorganic composite particle (X) in which a polymer (P) of a monomer containing a (meth) acrylic acid ester monomer (a) and a water-swellable clay mineral (B) form a three-dimensional network. A cell culture substrate having a dry film of an organic-inorganic composite (X) formed by drying a dispersion of the above on its surface is disclosed (for example, see Patent Document 3).
However, in the above-mentioned conventional documents, specific means relating to corneal cell culture and detachment methods are not disclosed.

Japanese Patent Publication No. 6-104061 JP-A-5-192138 Japanese Patent No. 4430124

Masayuki Yamato, Mitsuo Okano, “Frontiers of Nanobiotechnology”, Chapter 6, P.A. 340-P. 347, CMC Publishing (published in 2003)

  Problems to be solved by the present invention include a method for culturing corneal cells, which can control the adhesive force between the surface of a culture substrate and corneal cells and can be easily detached without breaking the cell membrane by low-temperature treatment after culturing, and the cultured cornea To provide cells.

  As a result of earnest research to solve the above-mentioned problems, the present inventors contain a copolymer (A) of a polyethylene glycol acrylic monomer (a) and methoxyethyl acrylate (b), and an inorganic material (B). The corneal cells are cultured on the cell culture substrate to maintain low adhesion between the substrate surface and the cells, and the corneal cell membrane (or cell sheet) is not broken by the low-temperature treatment after the culture. The inventors have found a method for culturing corneal cells that can be easily detached from the present invention, and have completed the present invention.

That is, the present invention provides a cell containing a copolymer (A) of a monomer (a) represented by the following formula (1) and methoxyethyl acrylate (b) and colloidal silica as an inorganic material (B). There is provided a method for producing a corneal cell, comprising a step of culturing a corneal cell on a culture substrate to obtain a cultured cell, and a step of peeling the cultured cell from the cell culture substrate.

（式中、Ｒ_１は水素原子またはメチル基、Ｒ_２は炭素原子数１〜３のアルキル基、ｎは４〜３０の整数を表す。）

(In the formula, R ₁ represents a hydrogen atom or a methyl group, R ₂ represents an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 4 to 30.)

Moreover, this invention provides the manufacturing method of a corneal cell whose mass ratio ((silica) / (A)) of the said polymer (A) and the said silica exists in the range of 0.01-0.5. .

Moreover, this invention provides the manufacturing method of a corneal cell sheet which peels the said cultured cell from the said cell culture base material in a sheet form .

  The greatest feature of the cell culture substrate of the present invention is that the constituent part of the inorganic material (B) is responsible for the growth of corneal cells, and the copolymer (A) of monomer (a) and methoxyethyl acrylate (b) is It is responsible for maintaining low adhesion to corneal cells and for detaching corneal cells due to temperature changes. These two parts can be individually adjusted according to the state of proliferation and detachment of corneal cells. For example, at the time of culture (37 ° C.), corneal cells proliferate with high proliferation ability while maintaining weak adhesion with the culture surface, and after completion of the culture, the temperature is lowered to 30 ° C. or less (for example, room temperature) The portion of the copolymer (A) exhibits higher hydrophilicity, the adhesive force between the culture surface and the cells is further weakened, and the cells can be peeled off in the form of a sheet (or film) without being broken. A corneal cell as used herein is a cell that is present on the basement membrane of the corneal limbus where corneal epithelial stem cells located near the cornea and the corneal cortex are present. It refers to corneal limbal epithelial cells and corneal endothelial cells.

The copolymer (A) interacts and bonds with the inorganic material (B) mainly by ionic bonds or hydrogen bonds. This bonding force is strong, and the polymer and the inorganic material (B) cannot be easily separated.

The method for culturing corneal cells of the present invention has a high proliferative ability while maintaining a weak adhesive force between the cells and the culture surface, and the cultured cells can be used without using a drug (such as trypsin). It has the feature that it can be easily peeled off and recovered from the surface of the culture substrate without breaking.
In addition, the culture substrate used in the present culture method has a feature that radiation sterilization such as γ rays and electron beams is possible.

  The monomer (a) used in the present invention is suitably used as long as the copolymer (A) with methoxyethyl acrylate (b) can interact with the inorganic material (B) to form an organic-inorganic composite. Among them, (meth) acrylic acid polypropylene glycol and polyethylene glycol ester monomers are preferably used, and the monomer (a) of the following formula (1) is particularly preferably used.

（式中、Ｒ_１は水素原子またはメチル基、Ｒ_２は炭素原子数１〜３のアルキル基、ｎは４〜３０の整数を表す。）

(In the formula, R ₁ represents a hydrogen atom or a methyl group, R ₂ represents an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 4 to 30.)

  By using these monomers (a), the initial adhesion of corneal cells can be kept low, and a cell culture substrate having good cell growth and detachability can be obtained. Moreover, when this cell culture substrate is laminated on the surface of a support such as a plastic substrate such as polystyrene by using methoxyethyl acrylate (b), the adhesiveness between the two is strong and the production can be simplified. .

  One or more of the above monomers (a) may be mixed and used depending on the required surface properties. In addition, as necessary, other copolymerization monomers, such as acrylic monomers having an anion group such as a sulfone group or a carboxyl group, quaternary ammonium groups, etc., to the extent that does not affect the culture properties and physical properties. An acrylic monomer having a cationic group, an acrylic monomer having a zwitterionic group having a quaternary ammonium group and a phosphate group, an acrylic monomer having an amino acid residue having a carboxyl group and an amino group, and an acrylic having a sugar residue -Based monomers, acrylic monomers having hydroxyl groups, amphiphilic acrylic monomers having a hydrophilic chain such as polyethylene glycol and a hydrophobic group such as nonylphenyl group, N-substituted (meth) acrylamide derivatives, N , N-disubstituted (meth) acrylamide derivatives, N, N′-methylenebi It can be used in combination, such as acrylamide.

  The inorganic material (B) used in the present invention is one or more inorganic materials selected from water-swellable clay minerals and silica. Examples of water-swellable clay minerals include water-swellable clay minerals that can be peeled off in layers, preferably clay minerals that swell and uniformly disperse in water or a mixed solution of water and an organic solvent, particularly preferably water. An inorganic clay mineral that can be uniformly dispersed at a molecular level (single layer) or at a level close thereto is used. Specific examples include water-swellable hectorite containing sodium as an interlayer ion, water-swellable montmorillonite, water-swellable saponite, and water-swellable synthetic mica. You may mix and use these clay minerals.

Examples of the silica (SiO ₂ ) used in the present invention include colloidal silica, preferably colloidal silica that can be uniformly dispersed in an aqueous solution and has a particle size of 10 nm to 500 nm, and particularly preferably a colloidal particle having a particle size of 10 to 50 nm. Silica is used.

In the cell culture substrate of the present invention, the mass ratio of monomer (a) to methoxyethyl acrylate (b) ((a) / (b)) is preferably 0.1 to 1.0, 0.1 -0.5 is more preferable, and 0.1-0.3 is particularly preferable. When the mass ratio ((a) / (b)) is within this range, low adhesion between the corneal cells and the culture surface and good proliferation can be maintained, and the cells after culture can be easily detached, which is preferable.
In the cell culture substrate of the present invention, the mass ratio ((B) / (A)) of the polymer (A) and the inorganic material (B) is preferably 0.01 to 0.5, 0.01 to 0.3 is more preferable, and 0.04 to 0.2 is particularly preferable. When the mass ratio ((B) / (A)) is within this range, it is preferable because it can have both good culturing properties and high peelability for corneal cells.

Furthermore, with the corneal cells cultured on the cell culture substrate of the present invention, the medium temperature is lowered to 30 ° C. or less, the medium is removed, and a small amount of medium is left, and a hydrophilic porous support is placed on the cells. After being placed, it can be peeled off by lifting the support, but since the adhesive force between the cell culture substrate of the present invention and corneal cells is relatively weak, the medium is not subjected to low-temperature treatment, for example, The cells can be easily detached by the physical peeling force listed in the above.
(1) A method in which the cell sheet is directly sandwiched and lifted by using tweezers after culturing,
(2) A method in which a glass rod, pipette tip, rubber spatula or the like is inserted between the cell sheet and the cell culture substrate, and the cell sheet is lifted off,
(3) A method of peeling the cell sheet while sucking it with a straw-shaped device,
(4) There is a method of peeling by a pipetting operation in which a medium is sucked or taken out by a pipette.

  Since the corneal cell sheet produced by the culture method of the present invention does not use a proteolytic enzyme such as trypsin, the basal protein of the cell is not damaged, is in a state closer to the cell morphology in the living body, and has high cell activity. It is considered that the post-transplant fixation and curability are high.

The method for producing a culture substrate of the present invention is such that a copolymer (A) of a monomer (a) and methoxyethyl acrylate (b) interacts with an inorganic material (B) to form an organic-inorganic composite. If there is, it will not be specifically limited. For example, an aqueous medium (C) in which the monomer (a), methoxyethyl acrylate (b), the inorganic material (B) and the polymerization initiator (D) are mixed is applied to a support, and the monomer (a) And methoxyethyl acrylate (b) are copolymerized to form a thin layer of the composite (X) of the polymer (A) and the inorganic material (B).
The aqueous medium (C) used in the production method may contain monomers (a), (b), an inorganic material (B), etc., and an organic-inorganic composite having good physical properties may be obtained by polymerization. It is not limited. For example, it may be water or an aqueous solution containing a solvent miscible with water and / or other compounds, and further includes antiseptics and antibacterial agents, coloring agents, fragrances, enzymes, proteins, collagen, sugars, Amino acids, DNAs, salts, water-soluble organic solvents, surfactants, polymer compounds, leveling agents and the like can be included.
As the polymerization initiator (D) used in the present invention, a known radical polymerization initiator can be appropriately selected and used. Preferably, those having water solubility or water dispersibility and uniformly contained in the entire system are preferably used. Specifically, as a polymerization initiator, a water-soluble peroxide such as potassium peroxodisulfate or ammonium peroxodisulfate, a water-soluble azo compound such as VA-044, V-50, V-501 (all of which are Wako Pure Chemical Industries, Ltd.) In addition to Yaku Kogyo Co., Ltd., a mixture of Fe ²⁺ and hydrogen peroxide is exemplified.

  As the catalyst, tertiary amine compounds such as N, N, N ′, N′-tetramethylethylenediamine are preferably used. However, the catalyst is not necessarily used. The polymerization temperature is, for example, 0 ° C. to 100 ° C. according to the type of polymerization catalyst or initiator. The polymerization time can also be carried out for several tens of seconds to several tens of hours.

  On the other hand, the photopolymerization initiator is less susceptible to oxygen inhibition and has a high polymerization rate, and therefore is suitably used as the polymerization initiator (D). Specifically, acetophenones such as p-tert-butyltrichloroacetophenone, benzophenones such as 4,4′-bisdimethylaminobenzophenone, ketones such as 2-methylthioxanthone, benzoin ethers such as benzoin methyl ether, hydroxy Examples include α-hydroxy ketones such as cyclohexyl phenyl ketone, phenyl glyoxylates such as methyl benzoyl formate, and metallocenes.

As the light used in this step, electron beam, γ-ray, X-ray, ultraviolet ray, visible light, etc. can be used. Among them, the apparatus and handling are easy and the viewpoint of not causing crosslinking simultaneously with polymerization of the monomer (b). It is preferable to use ultraviolet rays. The intensity of the irradiated ultraviolet light is preferably 10 to 500 mW / cm ² and the irradiation time is generally about 0.1 to 200 seconds. In radical polymerization by normal heating, oxygen works as an inhibitor of polymerization. However, in the present invention, it is not always necessary to prepare a solution in an atmosphere in which oxygen is blocked and to perform polymerization by ultraviolet irradiation, and these are performed in an air atmosphere. It is possible. However, it may be desirable that the polymerization rate can be further increased by performing ultraviolet irradiation in an inert gas atmosphere.

Moreover, as a second production example of the culture substrate of the present invention, the concentration of the inorganic material (B) in the aqueous medium (C) is in a range represented by the following formula (2) or formula (3). As described above, after the monomers (a) and (b), the inorganic material (B), and the polymerization initiator (D) are mixed in an aqueous medium (C), the monomers (a) and (b) are combined. A first step of producing a dispersion (L) of a composite (X) of the polymer (A) and the inorganic material (B) by polymerization;
A method for producing a cell culture substrate comprising sequentially performing the second step of forming the thin layer of the complex (X) by applying the dispersion (L) to a substrate and then drying the dispersion. It is done.
Formula (2) When Ra <0.19
Concentration (mass%) of inorganic material (B) <12.4Ra + 0.05
Formula (3) When Ra ≧ 0.19
Concentration (mass%) of inorganic material (B) <0.87Ra + 2.17
(In the formula, the concentration (mass%) of the inorganic material (B) is a value obtained by dividing the mass of the inorganic material (B) by the total mass of the aqueous medium (C) and the inorganic material (B) and multiplying by 100, Ra Is a mass ratio ((B) / (A)) between the inorganic material (B) and the polymer (A).)

When the concentration (% by mass) of the inorganic material (B) in the aqueous medium is within the range represented by the formula (2) or the formula (3), a good dispersion (L) of the composite (X) can be obtained. The coating on the support is easy, and a smooth and uniform thin coating is obtained, which is preferable.

The dispersion (L) produced by the production method of the present invention may be used as it is, or may be used after undergoing a purification step such as washing with water. Further, a leveling agent, a surfactant, a peptide, a protein, collagen, an amino acid, a polymer compound or the like may be added to the dispersion (L).

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not limited only to these Examples.

(Example 1) (Reference Example)
[Preparation of Reaction Solution Containing Monomer (a), (b), Inorganic Material (B), and Aqueous Medium (C))
Polyethylene glycol acrylate (trade name “AM-90G” manufactured by Shin-Nakamura Chemical Co., Ltd.) as the monomer (a), R ₁ in the formula (1) is a hydrogen atom, R ₂ is an alkyl group having 1 carbon atom, and n is 9. Compound which is an integer) 1.2 g, methoxyethyl acrylate (b) 2.9 g, water-swellable clay mineral Laponite XLG (manufactured by Rockwood Additives Ltd.) 0.2 g as inorganic material (B), water as water medium (C) 100 g was uniformly mixed to prepare a reaction solution (1).

[Preparation of solution in which polymerization initiator (D) is dissolved in solvent (E)]
A solution (2) is prepared by uniformly mixing 9.8 g of methanol as a solvent (E) and 0.2 g of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (manufactured by Ciba Geigy) as a polymerization initiator (D). did.

[Preparation of dispersion (L) of complex (X) (first step)]
250 μl of the solution (2) is added to the total amount of the reaction solution (1) and dispersed uniformly, and then irradiated with ultraviolet rays having an ultraviolet intensity of 40 mW / cm ² at 365 nm for 180 seconds, and a milky white complex (X) dispersion liquid (L1) was produced.
Ra = 0.05 in this reaction system, concentration (mass%) of inorganic material (B) = 0.20 (%) <12.4Ra + 0.05 = 0.67

[Preparation of culture substrate (thin layer of complex (X)) (second step)]
A dispersion (L1) of the above complex (X) is placed in a polystyrene petri dish (IWAKI tissue culture dish 3000-035) having a diameter of 35 mm, and the dispersion is thinly applied to the surface of the petri dish at 3000 revolutions using a spin coater. After coating, the cell dish is dried for 10 minutes in a hot air dryer at 80 ° C., and then the petri dish is washed with sterilized water, and then the petri dish is dried at 40 ° C. for 5 hours in a sterile bag to obtain the cell culture substrate 1. It was.

[Culture of normal human corneal epithelial cells]
The cell culture substrate 1 obtained above was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), and then an appropriate amount of medium DMEM / F12 (B27 supplement, 20 ng / mL EGF) was added to normal human corneal limbal epithelium. Cells (collected from imported human cornea for research provided by Rocky Mountain Lions' Eye Bank) are seeded (seeding concentration is 2 × 10 ⁴ cells / cm ² ) and cultured at 37 ° C. in 5% carbon dioxide. It was. After confirming that the cells grew sufficiently, the culture substrate 1 was allowed to stand on ice water for 5 minutes, and then the medium was sucked off, leaving a small amount of medium, and a ring-shaped polyvinylidene fluoride having a width of about 5 mm. A (PVDF) porous membrane was placed on the cells, the cells were sucked onto the PVDF membrane, and then the PVDF membrane was lifted. As a result, all cultured cells were completely detached from the substrate in a single sheet form.

(Example 2)
[Preparation of Reaction Solution Containing Monomer (a), (b), Inorganic Material (B), and Aqueous Medium (C))
Polyethylene glycol acrylate (trade name “AM-90G” manufactured by Shin-Nakamura Chemical Co., Ltd.) as the monomer (a), R ₁ in the formula (1) is a hydrogen atom, R ₂ is an alkyl group having 1 carbon atom, and n is 9. Compound which is an integer.) 0.8 g, methoxyethyl acrylate (b) 3.0 g, SNOWTEX 20 (20% by weight colloidal silica aqueous solution, manufactured by Nissan Chemical Industries, Ltd.) 1.0 g (solid) Min. 0.2 g) and 100 g of water as an aqueous medium (C) were uniformly mixed to prepare a reaction solution (2).

[Preparation of dispersion (L) of complex (X) (first step)]
250 μl of the solution (2) of Example 1 was added to the total amount of the reaction solution (2) and dispersed uniformly, and then irradiated with ultraviolet rays having an ultraviolet intensity of 40 mW / cm ² at 365 nm for 180 seconds to give a milky white composite ( A dispersion (L2) of X) was prepared.
Ra = 0.05 in this reaction system, concentration (mass%) of inorganic material (B) = 0.20 (%) <12.4Ra + 0.05 = 0.67

[Preparation of culture substrate (thin layer of complex (X)) (second step)]
The dispersion (L2) of the complex (X) is placed in a polystyrene petri dish (IWAKI tissue culture dish 3000-035) having a diameter of 35 mm, and the dispersion is thinly applied to the surface of the petri dish at 3000 revolutions using a spin coater. After the coating, it is dried for 10 minutes in a hot air dryer at 80 ° C., and then the petri dish is washed with sterilized water, and then the petri dish is dried at 40 ° C. for 5 hours in a sterile bag to obtain the cell culture substrate 2. It was.

[Culture of normal human corneal epithelial cells]
The cell culture substrate 2 obtained above was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), and then an appropriate amount of medium DMEM / F12 (B27supplement, 20 ng / mL EGF) was added to normal human corneal epithelial cells ( (Collected from imported human cornea for research provided by Rocky Mountain Lions' Eye Bank) was seeded (seeding concentration 2 × 10 ⁴ cells / cm ² ) and cultured at 37 ° C. in 5% carbon dioxide. After confirming that the cells had grown sufficiently, the culture substrate 2 was allowed to stand at room temperature for 5 minutes, and then the cells at the edge of the petri dish were picked and lifted with two tweezers. The sheet was completely peeled from the substrate.

(Comparative Example 1)
Normal human corneal epithelial cells were cultured in the same manner as in Example 1 using two temperature-responsive cell culture equipment “UpCell 3.5cm Petri Dishes” (manufactured by Cellseed Co., Ltd.) purchased from Wako Pure Chemical Industries, Ltd. It was. After confirming that the cells have grown sufficiently, let the first culture substrate stand on ice water for 5 minutes, and then suck out the medium and leave a small amount of medium in a ring-shaped polyhedron with a width of about 5 mm. When a porous membrane made of vinylidene fluoride (PVDF) is placed on the cells, the cells are sucked into the PVDF membrane, and then the PVDF membrane is lifted, the cultured cells are broken and completely peeled off from the substrate in a single sheet I couldn't.
In addition, after leaving the second culture substrate at room temperature for 5 minutes, the cells at the edge of the petri dish were picked and lifted with two tweezers. It was not possible to peel completely.

(Example 3) (Reference Example)
The dispersion liquid (L1) of the composite (X) of Example 1 was placed on a polystyrene dish (IWAKI tissue culture 3000-035) having a diameter of 35 mm and a slide glass “S9224” (manufactured by Matsunami Glass Industrial Co., Ltd.). After coating in the same manner as in Example 1, it was dried in a hot air dryer at 80 ° C. for 10 minutes. Next, using a cutter knife on these coatings, cut 1 × 1 mm square grids, and then strongly press the cellophane tape on the grids and place the edges of the tape at an angle of 45 °. When it peeled off rapidly and the state of the grid was observed with the optical microscope, it was confirmed that the coating film was not peeled off at all and the adhesiveness with the substrate was good.

From the said Example, the cell culture substratum of this invention has favorable adhesiveness between the support bodies of another material, and has the outstanding cell culture and peeling function with respect to a corneal cell.
It was also clear that this cell culture substrate can be easily produced in a very short time without removing oxygen.

  The corneal cell culture method of the present invention can be used in the field of regenerative medicine to prepare sheet-like corneal cells for transplantation.

Claims (3)

A cornea on a cell culture substrate containing a copolymer (A) of a monomer (a) represented by the following formula (1) and methoxyethyl acrylate (b) and colloidal silica as an inorganic material (B) Culturing cells to obtain cultured cells;
Peeling the cultured cells from the cell culture substrate;
A method for producing a corneal cell comprising:

(In the formula, R ₁ represents a hydrogen atom or a methyl group, R ₂ represents an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 4 to 30.) The method for producing a corneal cell according to claim 1, wherein a mass ratio ((silica) / (A)) between the polymer (A) and the colloidal silica is in a range of 0.01 to 0.5.   It is a manufacturing method of Claim 1 or 2, Comprising: The manufacturing method of the corneal cell sheet which peels the said cultured cell from the said cell culture base material in a sheet form.