JP2021097661A - Ink-jet ink for cell culture, members for cell culture and production methods thereof, and methods for generating cell culture patterns - Google Patents

Abstract

To provide ink-jet ink for cell culture with excellent cell adhesiveness, members for cell culture capable of reproducing in vivo cell arrangement and production methods thereof, and methods for generating cell patterns.SOLUTION: Disclosed is an ink-jet ink (I) for cell culture containing a cell adhesive material (A) that satisfies the following conditions (1) to (3): (1) a content of the cell adhesive material (A) is 0.01 mass% or more and 10 mass% or less in 100 mass% of the ink-jet ink for cell culture; (2) the viscosity of the ink-jet ink for cell culture is 50 mPa s or less at 25°C; (3) the surface tension of the ink-jet ink for cell culture is 20 mN/m or more and 50 mN/m or less.SELECTED DRAWING: Figure 1

Description

The present invention relates to an inkjet ink for cell culture containing a cell adhesive material, a member for cell culture and a method for producing the same, and a method for forming a cell culture pattern.

In recent years, attention has been focused on the development of cell engineering and regenerative medicine, and active attempts have been made to culture cells in an appropriate form in vitro, apply them to drug discovery research and treatment, and use them as biological reaction models. ing. For this purpose, collagen, polystyrene and polymethylmethacrylate are generally known to be good in the development of a substrate for cell culture, which is one of the tools for drug discovery research and regenerative medicine research. Among them, polystyrene has low cytotoxicity, is economical, and has advantages in processability, and in the current tissue culture, polystyrene treated with hydrophilic treatment is used. However, since it is not possible to control the adhesion and non-adhesion of cells with such a culture member, the cells are arranged or oriented in order to reproduce patterns with individual differences such as ganglia and blood vessels of living tissue. It is difficult to do.

On the other hand, a technique of adhering cultured cells only to a minute portion on a substrate and arranging them has been reported. Such technology makes it possible to apply cultured cells to artificial organs, biosensors, bioreactors, and the like. As a method for arranging cultured cells, a substrate having a surface pattern that differs in ease of adhesion to the cells is used, and the cells are cultured on this surface and processed so that the cells adhere. A method is adopted in which cells are arranged by adhering the cells only to the surface of the cells.

As a method of reproducing the arrangement and orientation of cells in a living body, for example,
(1) A charge-retaining medium in which an electrostatic charge pattern is formed is applied to cell culture for the purpose of proliferating nerve cells in a circuit shape (Patent Document 1).
(2) An attempt to arrange cultured cells on a surface obtained by patterning a cell adhesion inhibitory or cell adhesion photosensitive hydrophilic polymer by a photolithography method (Patent Document 2).
(3) A cell culture substrate in which a substance such as collagen that affects the adhesion rate and morphology of cells is patterned, and a substrate prepared by a photolithography method are disclosed (Patent Document 3).
By culturing cells on such a substrate, more cells are adhered to the surface on which collagen or the like is patterned, and cell patterning is realized.
However, in (1), the charge of the charge holding medium is weakened immediately after the production, the positive charge pattern disappears, and there is a problem in stability.

Further, in (2) and (3), when patterning is performed by a photolithography method or the like using a photosensitive material, a high-definition pattern can be obtained, but the cell adhesion material needs to have photosensitive. For example, it is often difficult to chemically modify a biopolymer or the like in order to impart such photosensitivity, and there is a problem that the range of selectivity of the cell adhesion material is extremely narrowed. Further, in the photolithography method using a photoresist, it is necessary to use a developing solution or the like, which may have an adverse effect on cell culture.
Against the background of these problems, many surface design methods have been studied with the aim of realizing excellent biocompatible polymer materials. For example, (4) reports a technique for forming a pattern by inkjet printing using a cell non-adhesive polymer having a polyethylene oxide structure (Patent Document 4). By using the inkjet technology, it is possible to reproduce a cell pattern having more stability than (1). In addition, there is no need for chemical modification to impart photosensitivity as in (2) and (3), and a pattern can be formed without contacting a plurality of materials with the base material during inkjet printing. It is possible to modify surfaces, wet surfaces, and thin films. However, in (4), it is not sufficient to reproduce the cell orientation in the living body, and a technique for easily producing a cell pattern has been required.

Further, since the silicone material has excellent biocompatibility and oxygen permeability, it is used in a wide range of applications including medical materials. For example, Patent Document 5 discloses a technique of designing a mortar-shaped groove on the surface of a silicone substrate, arranging hepatocytes without layering an extracellular matrix gel, and efficiently constructing a bile canaliculus. There is. However, in Patent Document 5, although the bile canaliculus tissue can be formed by the arrangement of hepatocytes, fine irregularities are produced on a silicon wafer by utilizing anisotropic wet etching, and further, fine irregularities are formed on the silicon wafer. Since the collagen is coated, there is a problem that the molding process is not easy and the processing cost is high. In addition, the operation of exchanging the medium and collecting the tissue was complicated.

Japanese Unexamined Patent Publication No. 2-245181 Japanese Unexamined Patent Publication No. 3-7576 Japanese Unexamined Patent Publication No. 5-176753 JP-A-2017-104027 JP 2014-187971

An object of the present invention is to provide an inkjet ink for cell culture having excellent cell adhesion, a cell culture member capable of reproducing cell orientation in a living body, a method for producing the same, and a method for forming a cell pattern.

As a result of diligent studies to solve the above problems, the present invention not only controls cell orientation but also broadens the range of material selection by using an inkjet ink containing a cell adhesion material (A). , Found that it is possible to form a pattern that does not adversely affect cytotoxicity.
This provides a method for reproducing cell orientation in a living body, and is expected to be applied to a wide range of applications such as medical materials and drug discovery screening. For example, in a cell culture member called a microchip, by observing or measuring the chemical reaction of cells held on the patterning member, it is expected to bring about merits such as shortening of analysis time.

That is, the present invention
The present invention relates to an inkjet ink (I) for cell culture containing the cell adhesion material (A) and satisfying the following conditions (1) to (3).
(1) The content of the cell adhesive material (A) in 100% by weight of the inkjet ink for cell culture is 0.01% by mass or more and 10% by mass or less.
(2) The viscosity at 25 ° C. is 50 mPa · s or less.
(3) The surface tension is 20 mN / m or more and 50 mN / m or less.

The present invention also relates to the cell culture inkjet ink (I), wherein the cell adhesion material (A) contains at least one selected from the group consisting of extracellular matrix and synthetic substrate.

The present invention further relates to the cell culture inkjet ink (I) containing a surface tension adjusting agent (B) and a solvent (C).

The present invention also relates to a cell culture member having a cell adhesion pattern layer formed by the cell culture inkjet ink (I) on a substrate.

The present invention also relates to a cell culture member in which a cell non-adhesive layer and a cell adhesion pattern layer formed by the cell culture inkjet ink (I) are laminated in this order on a substrate.

The present invention also relates to a cell culture member in which a cell adhesion layer and a cell non-adhesion pattern layer formed by the cell culture inkjet ink (I) are laminated in this order on a substrate.

The present invention also relates to a cell culture member having a cell adhesion pattern layer formed by the cell culture inkjet ink (I) on a substrate and a cell non-adhesion pattern layer.

The present invention also relates to the cell culture member in which a cell non-adhesive layer or a cell non-adhesive pattern layer is formed of the cell non-adhesive composition (D).

The present invention also relates to the cell culture member in which the cell non-adhesive composition (D) has a viscosity at 25 ° C. of 50 mPa · s or less and a surface tension of 20 mN / m or more and 50 mN / m or less.

The present invention also relates to the cell culture member containing 0.1% by mass or more and 50% by mass or less of the cell non-adhesive resin (D1) in 100% by weight of the cell non-adhesive composition (D).

The present invention also relates to the cell culture member having an average surface roughness Ra of the culture surface of 1.0 μm or less.

Further, the present invention is the base material is a silicone base material, ISO18369-4 (FATT Method) with oxygen permeability coefficient (Dk) ^{is, 200Barrer (1Barrer = 1 × 10} -11 O 2 (STP) cm / cm 2 The present invention relates to the cell culture member having mmHg) or more.

The present invention also relates to the cell culture member for reproducing cell orientation in a living body.

The present invention also relates to a method for producing a cell culture member, which forms a culture layer by inkjet printing the cell culture inkjet ink (I) on a substrate.

The present invention also relates to a method for producing a cell culture member, which forms a culture layer by inkjet printing the cell culture inkjet ink (I) and the cell non-adhesive composition (D) on a substrate.

The present invention also relates to a method for forming a cell pattern in which cells are oriented by the cell culture member.

The present invention also relates to a cell pattern formed by the above method.

The present invention also relates to a method for forming a biological tissue by inducing differentiation using the cell pattern.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an inkjet ink for cell culture having excellent cell adhesion, a cell culture member capable of reproducing cell orientation in a living body, a method for producing the same, and a method for forming a cell pattern.
Further, it is possible to provide a cell culture member having excellent oxygen permeability, controlling cell arrangement, and not adversely affecting cytotoxicity, a method for producing the same, and a method for forming a cell pattern. In addition, it is possible to provide a cell culture member having excellent cell adhesion, controlling cell arrangement, and forming a living tissue by inducing differentiation, and a method for producing the same.

FIG. 1 is a top view and a cross-sectional view showing an embodiment (first form) of the cell culture member of the present invention. FIG. 2 is a top view and a cross-sectional view showing one embodiment (second form) of the cell culture member of the present invention. FIG. 3 is a top view and a cross-sectional view showing one embodiment (third form) of the cell culture member of the present invention. FIG. 4 is a top view and a cross-sectional view showing one embodiment (fourth form) of the cell culture member of the present invention. FIG. 5 is a transmission optical micrograph showing the orientation of cells immediately after seeding or 7 days after culturing in the cell culture member of the present invention. FIG. 6 is a diagram showing cell orientation immediately after seeding or 7 days after culturing in the silicone cell culturing member of the present invention. FIG. 7 is a transmission optical micrograph showing confluence of myoblasts on the cell culture member of the present invention 3 days after sowing or 10 days after culture. FIG. 8 is a fluorescence transmission type optical microscope photograph showing myotube formation on the 10th day after culturing myoblasts in the cell culture member of the present invention.

<Inkjet ink for cell culture (I)>
The cell culture inkjet ink (I) of the present invention has good ejection properties from an inkjet nozzle, and the cell culture inkjet ink (I) is printed on a substrate to form a patterning culture layer having cell adhesion. can do.
In addition, by seeding cells on a cell culture member having this cell adhesion pattern layer, the cells are adhered to the cell adhesion pattern, and by culturing, a cell pattern that reproduces the orientation of the cells in the living body is formed. Can be done.

In addition, a biological tissue can be formed by inducing differentiation using the cell pattern formed by the cell culture member of the present invention.

[cell]
The cells used in the present invention are generally not particularly limited as long as they need to adhere themselves to an appropriate surface for proliferation, but cells exhibiting orientation include myoblasts, fibroblasts, and the like. Examples include osteoblasts and nerve cells. For example, a nerve tissue in which nerve cells are cultured in a pattern can be formed, or a myoblast can be cultured by induction of differentiation to form a continuous myotube tissue. In addition, although mainly human-derived cells are used, cells derived from non-human animals (specifically, mice, rats, rabbits, pigs, dogs, monkeys, cows, horses, sheep, chickens, etc.) may be used. ..
In addition, hepatocytes can be used, and the method for isolating hepatocytes from human origin or animals can be carried out according to a known method. The origin of hepatocytes may be fetal, neonatal, or adult. Also, embryonic stem (ES) cells and induced pluripotent (iPS) stem cells, or hepatocytes induced to differentiate from cord blood, bone marrow, fat, and blood-derived tissue stem cells can be used. The method for inducing hepatocytes from these cells can be carried out according to a known method.

[Cell Adhesive Material (A)]
As the cell adhesion material (A) of the present invention, any material having cell adhesion can be used, but it is preferable to use an extracellular matrix and / or a synthetic substrate.

[Extracellular matrix]
Specific examples of the extracellular matrix include at least one consisting of collagen, laminin, fibronectin, hyaluronic acid, vitronectin, and cadherin.

Preferred are collagen, laminin, hyaluronic acid and fibronectin.
Collagen is a major protein component of connective tissue and basement membrane, and there are many types with different stretch strength and tissue distribution. Structural changes in the hardness, flexibility, and structure of many body tissues are due to changes in collagen composition, as well as cell restriction and segmentation. Commercially available collagen products include Collagen type i to v (acid-extracted collagen manufactured by Nippi), pepsin-solubilized collagen (manufactured by Nippi), AteloCell series (high-purity collagen acidic solution derived from cowhide manufactured by Koken), and Cellmatrix series (pepsin solubilized collagen derived from pig tendon manufactured by Nitta Gelatin Co., Ltd.), cell campus series (collagen derived from scales manufactured by Taki Chemical Co., Ltd.) and the like can be used.

Laminin is a glycoprotein present in the basement membrane of most tissues. It is composed of three strands of α1, β1 and γ1. It binds to collagen and heparan sulfate and is responsible for the composition and function of the basement membrane. In the culture system, it mainly adheres epithelial cells, endothelial cells, hepatocytes, etc., promotes axon extension of nerve cells, and is involved in differentiation and chemotaxis. It is closely related to the metastatic potential of cancer, and it is known that highly metastatic cancer cells have a large amount of laminin receptors on the cell surface. Commercially available products of laminin include laminin solution derived from mouse EHS sarcoma (Fujifilm Wako Pure Chemical Industries, Ltd.), Recombinant Human Laminin (Oriental Yeast), laminin 521 (manufactured by BioLamine), which is usually expressed and secreted by human ES cells, i. Matrix-511 (manufactured by Takara Bio Co., Ltd .: composed of C-terminal regions of laminin α chain, β chain, and γ chain) and the like can be used.
Hyaluronic acid is abundantly contained in the subcutaneous tissue such as the dermis, and retains water between cells and functions as a buffer. It is also abundant in eye tissue such as the vitreous body, and functions as a buffer and maintains the shape of the tissue. It is also abundant in joint tissues such as joint fluid and cartilage, and functions as a lubricant and buffer. Therefore, hyaluronic acid is often used as an ingredient in pharmaceuticals and cosmetics.
Examples of commercially available products of hyaluronic acid include hyalooligo (Kewpie Corp.: low molecular weight hyaluronic acid) biohyaluro 12, acetylated hyaluronic acid (Shiseido Co., Ltd.), and hyaluronic acid (Tokyo Chemical Industry Co., Ltd.).
There are four types of fibronectin: plasma fibronectin, cellular fibronectin, fetal fibronectin, and single-chain fibronectin. Fibronectin is a glycoprotein that forms an extracellular matrix, and a polypeptide forms a dimer.
As commercially available products of fibronectin, a fibronectin solution derived from human plasma (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a fibronectin solution derived from bovine plasma (manufactured by Sigma Aldrich), and the like can be used.

These extracellular matrices may be used alone or in combination of two or more. As a commercially available product in which a plurality of products are mixed and used, Matrigel (manufactured by Corning Inc .: a mixture of laminin, iv collagen, heparin sulfate proteoglycan, entactin / nidgen and a growth factor) can be used.

[Synthetic substrate]
Synthetic substrates include oligopeptide-supporting polymers, polylysine, and heparin-mimicking hyaluronic acid. An oligopeptide-supporting polymer is a substrate in which an oligopeptide having an arginine-glycine-aspartic acid (RGD) sequence is covalently bonded to the polymer. As a commercially available product, Synthemax series (manufactured by Corning Inc.) and the like can be used. Polylysine is a substrate to which the lysine molecule of an essential amino acid is peptide-bonded. A commercially available product such as ε-poly-L-lysine coating solution (manufactured by Cosmo Bio Co., Ltd.) can be used. Heparin-mimicking hyaluronic acid is a substrate obtained by substituting the hydroxyl groups of hyaluronic acid with sulfonic acid. As a commercially available product, sulfated hyaluronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) and the like can be used.

The content of the cell adhesion material (A) in 100% by mass of the inkjet ink (I) for cell culture of the present invention is 0.01% by mass or more and 10% by mass or less, 0.01% by mass or more. It is preferably contained in an amount of 8% by mass or less. By setting the content of the cell adhesion material (A) to 0.01% by mass or more, the effect of promoting cell adhesion due to the structure can be exhibited. By setting the cell adhesive material (A) to 10% by mass or less, the ejection from the inkjet head becomes good, and a high-definition pattern can be formed.

Further, the cell adhesion control inkjet ink (I) of the present invention may contain components other than the cell adhesion material (A).

[Surface tension adjuster (B)]
The inkjet ink (I) for cell culture of the present invention preferably contains a surface tension adjusting agent (B). By containing the surface tension adjusting agent (B), it is possible to control the ejection property from the inkjet head and the wettability to the substrate.
Examples of the surface tension adjusting agent (B) include surfactants, antifoaming agents, wetting agents, and wetting adjusting agents, and those described in "Pharmaceutical Additives Standard 2018" issued by the Ministry of Health, Labor and Welfare from the viewpoint of cytotoxicity. preferable.

Examples of the surfactant include sucrose fatty acid ester, cetanol, cholesterol, alkylallyl polyether alcohol, sodium N-cocoyl-N-methylaminoethylsulfonate, laurylpyrrolindone and the like. Examples of the defoaming agent include ethanol, glycerin fatty acid ester, polyoxyl stearate and the like. Examples of the wetting agent include isobutyl adipate, allantoin, magnesium chloride, sodium dibunato, urea, glycerin, dioctylsodium sulfosuccinate and the like. Examples of the wetting modifier include olive oil, sodium hydroxide, lactic acid, potassium hydroxide and the like.

Among these, it is preferable to contain sucrose fatty acid ester. Examples of the sucrose fatty acid ester include the Ryoto sugar ester series (manufactured by Mitsubishi Chemical Foods Co., Ltd.) and the DK ester series (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.).
The content of the surface tension adjusting agent (B) in 100% by mass of the inkjet ink (I) for cell culture of the present invention is preferably 0.001% by mass or more and 10% by mass or less, preferably 0.01% by mass or more. %, More preferably 1.0 mass or less.
By setting the content of the surface tension adjusting agent (B) to 0.001% by mass or more, it is possible to exert the effect of controlling the ejection property from the inkjet head and the wettability to the substrate. By setting the surface tension adjusting agent (B) to 10% by mass or less, high biocompatibility can be ensured.

[Solvent (C)]
The inkjet ink (I) for cell culture of the present invention preferably contains a solvent (C). By containing the solvent (C), the viscosity of the inkjet head and the wettability with respect to the substrate can be suitably controlled.
As the solvent (C), it is preferable to use a water-soluble solvent from the viewpoint of not dissolving the base material, and glycol ethers and diols are preferable, and among them, (poly) alkylene glycol monoalkyl ether and (poly) alkylene glycol dialkyl. Ether and alcandiol having 3 to 6 carbon atoms are effective. These can be quickly dried during inkjet printing and can realize accurate printing. In addition, since it has a high boiling point, it also has a function as a moisturizer. Specific examples of glycol ethers include ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monopropyl ether, diethylene glycol monobutyl ether, diethylene glycol monopentyl ether, diethylene glycol monohexyl ether, and triethylene. Glycol monomethyl ether, triethylene glycol monoethyl ether, triethylene glycol monopropyl ether, triethylene glycol monobutyl ether, tetraethylene glycol monomethyl ether, tetraethylene glycol monobutyl ether, propylene glycol monopropyl ether, propylene glycol monobutyl ether, dipropylene glycol Examples thereof include monomethyl ether, tripropylene glycol monomethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, and dipropylene glycol dimethyl ether. Specific examples of the diols include 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,2-pentanediol, and 1,5-pentanediol. Examples thereof include 1,2-hexanediol, 1,6-hexanediol, 2-methyl-2,4-pentanediol and the like. Among these, the most effective are diethylene glycol monobutyl ether, diethylene glycol monohexyl ether, triethylene glycol monomethyl ether, diethylene glycol diethyl ether, 1,2-propanediol, 1,2-butanediol, 1,3-butanediol, 1, 2-Hexanediol and 2-methyl-2,4-pentanediol. These solvents may be used alone, or a plurality of aqueous solutions such as water, an aqueous acetic acid solution, and an aqueous lactic acid solution may be mixed and used.
The content of the solvent (C) in 100% by mass of the inkjet ink (I) for cell culture of the present invention is more preferably 90% by mass or more.
By setting the content of the solvent (C) to 90% by mass or more, it is possible to exert the effect of controlling the ejection property from the inkjet head and the wettability to the substrate.

[Viscosity of (I), surface tension]
The inkjet ink for cell culture of the present invention has a viscosity at 25 ° C. of 50 mPa · s or less from the viewpoint of a balance between ejection property from the inkjet head and reliability of dot formation after landing. Further, it is preferably 2 mPa · s or more and 20 mPa · s or less, and more preferably 5 mPa · s or more and 15 mPa · s or less.
From the same viewpoint, the surface tension at 25 ° C. is 20 mN / m or more and 50 mN / m. Further, it is preferably 25 mN / m or more and 40 mN / m or less. The viscosity and surface tension may be appropriately adjusted according to the blending amount of the resin (A), and further may be appropriately adjusted by using a surface tension adjusting agent, a solvent or the like.
The surface tension can be measured by checking the surface tension when the platinum plate is wetted with ink in an environment of 25 ° C. using an automatic surface tension meter CBVP-Z manufactured by Kyowa Interface Science Co., Ltd. The viscosity can be measured by reading the viscosity at 50 rpm in an environment of 25 ° C. using a TVE25L type viscometer manufactured by Toki Sangyo Co., Ltd.

[Manufacturing method of (I)]
The inkjet ink (I) for cell culture of the present invention can be produced by a known method. Specifically, after adding the surface tension adjusting agent (B) and the solvent (C) to the cell adhesive material (A) so as to exhibit the desired characteristics of the inkjet ink for cell culture (I), It can be obtained by removing coarse particles with a filter or the like.

<Cell culture member>
By having the cell adhesion pattern on the culture surface, the cell culture member in the present invention can control the cell adhesion and further reproduce the cell orientation in the living body.
The cell culture member in the present invention can be divided into the following four forms depending on the method of producing the pattern.
The method for producing the cell culture member of the present invention is not particularly limited, but from the viewpoint of pattern printing accuracy, a method of inkjet printing the cell culture inkjet ink (I) to form a culture layer, a cell culture inkjet. A method of forming a culture layer by inkjet printing the ink (I) and the cell non-adhesive composition (D) is preferable.

[First form]
First, the cell culture member in the first embodiment has a cell adhesion pattern layer formed by the cell culture inkjet ink (I) on the substrate.
As a method for producing a cell culture member in the first embodiment, for example, an inkjet carriage for cell culture (I) is filled in an inkjet carriage, and the inkjet ink (I) for cell culture is discharged from an inkjet head on a substrate. A method of forming a culture layer by inkjet printing an inkjet ink (I) for cell culture can be mentioned.

[Second form]
Further, the cell culture member according to the second embodiment of the present invention is formed by laminating a cell non-adhesive layer and a cell adhesion pattern layer formed by the cell culture inkjet ink (I) on a substrate in this order. Is.
The cell non-adhesive layer is preferably coated with the cell non-adhesive composition (D). The cell non-adhesive composition (D) is not particularly limited as long as it has cell non-adhesiveness, but is preferably in the form of an ink. Further, it is preferable to contain a cell non-adhesive resin (D1). The cell non-adhesive resin (D1) is not particularly limited as long as it is a resin having cell non-adhesiveness, but it is preferable to use a resin having a polyethylene oxide structure or a resin having a betaine structure. Specifically, the resin having a polyethylene oxide structure has an acrylic polymer portion formed from a monomer having an average value of water / 1-octanol partitioning coefficient (cLogPow) of 0 or more and 2 or less, and a polyethylene oxide portion. Examples of block polymers and commercially available products include the Pluronic series (manufactured by BASF). Examples of the polymer having a betaine structure include a Lipidure series (manufactured by NOF Corporation) and a RAM resin series (manufactured by Osaka Organic Chemical Co., Ltd.).
The content of the polyethylene oxide structure is preferably 0.1% by mass to 20% by mass, more preferably 1.0% by mass to 10% by mass in the cell non-adhesive resin (D1). Within this range, when culturing myoblasts, it becomes difficult for the cells to exfoliate from the surface of the cell culture member, and it is easy to confluent. The cell non-adhesive composition (D) preferably contains a solvent in addition to containing the cell non-adhesive resin (D1). The content of the cell non-adhesive resin (D1) is preferably 0.1 to 50% by mass, more preferably 0.5 to 3% by mass, based on 100% by mass of the cell non-adhesive composition (D). Within this range, it is easy to adjust the average surface roughness Ra of the surface of the cell culture member to a suitable range.
In addition, the cell non-adhesive composition (D) can contain a cross-linking agent. As a result, when culturing myoblasts, the cells are less likely to detach from the surface of the cell culture member, and it is easy to confluent the cells. When a cross-linking agent is contained, the cell non-adhesive resin (D1) preferably has a functional group serving as a cross-linking point such as a carboxyl group, a hydroxyl group, an epoxy group, an amino group and an isocyanate group.

The method for coating the cell non-adhesive composition (D) on the substrate is not particularly limited. As a method of coating the base material with the cell non-adhesive composition (D), a spin coating method is preferable from the viewpoint of ease of coating and adjustment of the average surface roughness Ra, but brush, dipping, roller, and spraying are suitable. The solution can also be applied to the substrate by various application methods such as injection and coating machine. After coating, the film thickness of the coating film after removing the solvent is not limited. The solvent is not particularly limited as long as it can form a surface layer by coating and does not erode the substrate, but water, methanol, ethanol, acetone, methyl ethyl ketone (hereinafter referred to as MEK), acetonitrile, dimethylformamide, etc. The solvent and the like can be mentioned. Further, those described in the solvent (C) can also be used.

In the method for producing a cell culture member in the second embodiment, for example, the cell non-adhesive composition (D) is coated on a substrate to form a cell non-adhesive layer, and then the cell culture is performed on the cell non-adhesive layer. Examples thereof include a method of forming a culture layer by inkjet printing the inkjet ink (I) for use.

[Third form]
Further, the cell culture member according to the third embodiment of the present invention is formed by laminating a cell adhesion layer and a cell non-adhesion pattern layer formed by the cell culture inkjet ink (I) on a substrate in this order. Is.
The cell non-adhesive pattern layer is preferably one in which the cell non-adhesive composition (D) is formed on the cell adhesion layer by inkjet printing. The cell non-adhesive composition (D) preferably has a viscosity of 50 mPa · s or less (25 ° C.) and a surface tension of 20 to 50 mN / m. The content of the cell non-adhesive resin (D1) in 100% by weight of the cell non-adhesive composition (D) is preferably 0.1 to 50% by mass. Further, the cell non-adhesive composition (D) preferably contains a surface tension adjusting agent (B) and a solvent (C), similarly to the cell culture inkjet ink (I). By containing the surface tension adjusting agent (B) and the solvent (C), it is possible to control the ejection property from the inkjet head and the wettability to the substrate.

As a method for producing a cell culture member in the third embodiment, a cell culture inkjet ink (I) is inkjet-printed on a substrate to form a cell adhesion layer, and then a cell non-adhesive composition is formed on the cell adhesion layer. Examples thereof include a method of forming a culture layer by inkjet printing the substance (D).

[Fourth form]
Further, the cell culture member according to the fourth embodiment of the present invention has a cell adhesion pattern layer formed by the cell culture inkjet ink (I) and a cell non-adhesion pattern layer on the substrate. ..

The cell non-adhesive pattern layer is the same as that shown in the third form, and the composition, viscosity, surface tension, content of the cell non-adhesive resin (D1), etc. of the cell non-adhesive composition (D), etc. The characteristics are the same. Thereby, the ejection property of the cell non-adhesive composition (D) from the inkjet head and the wettability to the substrate can be controlled.
In the method for producing the cell culture member in the fourth embodiment, for example, the cell culture inkjet ink (I) and the cell non-adhesive composition (D) are filled in different inkjet carriages, and the cell culture inkjet ink (I) is used. ), The cell non-adhesive composition (D) is ejected from the inkjet head, and inkjet printing is performed on the substrate to form a culture layer.

The average surface roughness Ra of the surface of the cell culture member is adjusted to a suitable range by simultaneously inkjet printing the cell culture inkjet ink (I) and the cell non-adhesive composition (D) to form a pattern. In the present invention, the preferred form of the cell culture member is such that the cell culture region can be separated from the cell adhesion region and the non-adhesion region more accurately, and the cell orientation in the living body can be reproduced. Is.

[Inkjet printing]
The inkjet printing in the present invention is a single-pass method in which a cell culture inkjet ink (I) and / or a cell non-adhesive composition (D) is ejected and printed only once on a substrate, and the maximum recording medium. Either of a serial type method in which recording is performed while reciprocally scanning a short shuttle head in a direction orthogonal to the transport direction of the recording medium between the recording widths may be adopted.

[Inkjet printing device]
Further, the inkjet printing apparatus needs to include an inkjet head that ejects the inkjet ink (I) for cell culture or the cell non-adhesive composition (D), and a drying step that dries the ejected ink from the inkjet head. .. When the ink is ejected from the inkjet head, the ejected inkjet ink for cell culture (I) or the cell non-adhesive composition (D) lands on the base material to form a pattern, and the base material conveys the pattern. As the ink is processed, it is transported into the drying apparatus and the drying process is performed.

[Inkjet method]
The inkjet method is not particularly limited, and is known, for example, a charge control method for ejecting ink using an electrostatic attraction, a drop-on-demand method (pressure pulse method) for using the vibration pressure of a piezo element, and an electric signal. The acoustic inkjet method that converts the ink into an acoustic beam and irradiates the ink to eject the ink using the radiation pressure, and the thermal inkjet that heats the ink to form bubbles and uses the generated pressure (Bubble Jet (registered trademark)). Any method may be used. The inkjet head used in the inkjet method may be an on-demand method or a continuous method. Further, as the discharge method, an electric-mechanical conversion method (example: single cavity type, double cavity type, bender type, piston type, shared mode type, shared wall type, etc.), an electric-heat conversion method (example: thermal inkjet). Specific examples include molds, bubble jets (registered trademarks), electrostatic suction methods (eg, electrolytic control types, slit jets, etc.), and discharge methods (eg, spark jets, etc.). However, any discharge method may be used. The ink nozzle or the like used when recording by the inkjet method is not particularly limited, and can be appropriately selected according to the purpose.
The shape of the pattern formed by the inkjet method is not particularly limited and can be appropriately selected depending on the intended purpose. Specific examples include stripes, dots, and triangles, but any pattern may be used. In this report, in order to evaluate the orientation during cell culture, specifically, stripes are preferable. The line width of the stripe having the bonded portion and the non-bonded portion is not particularly limited and can be appropriately selected depending on the purpose, but the bonded portion is preferably 30 μm or more and 120 μm or less, and 50 μm or more and 100 μm or less. Is more preferable. The non-adhesive portion is preferably 10 μm or more and 70 μm or less, and more preferably 30 μm or more and 50 μm or less.

[Base material]
The substrate of the present invention is not particularly limited as long as it can be used for cell culture. Specifically, (meth) acrylic resin, styrene resin, polycarbonate resin, polyolefin resin, polyvinyl chloride, polyester, polyvinyl acetate, vinyl-acetate copolymer, styrene-methylmethacrylate copolymer, acrylic. Consists of nitrile-styrene copolymer, acrylic nitrile-butadiene-styrene copolymer, nylon, polymethylpentene, silicone resin, amino resin, polysulphon, polyethersulphon, polyetherimide, fluororesin, and polyimide resin material. In addition to one or more kinds of resins selected from the group, dust-free paper, glass base material, porous ceramic base material, cellulose base material and the like can be mentioned. The form of the base material may be a sheet shape, a plate shape, or a dish shape as well as a three-dimensional molded body. Examples of the sheet-like material include film, paper and the like. Examples of the plate-shaped plate include flat-bottomed plates having 6 holes, 12 holes, 24 holes, 96 holes and the like. Examples of the dish shape include dishes having diameters of 35 mm, 60 mm, 90 mm, 100 mm and the like.

As the substrate, a silicone substrate, a porous ceramic substrate, a cellulose substrate and the like are preferable from the viewpoint of oxygen permeability. The silicone base material, the porous ceramic base material, the cellulose base material and the like are not particularly limited as long as they can be used for cell culture. It is preferably a silicone base material, specifically, a base material containing a silicone-based resin, and the silicone-based resin has a main chain of polyorganosiloxane such as polydimethylsiloxane, polyphenylmethylsiloxane, and polydiphenylsiloxane. And so on. Preferably, it is polydimethylsiloxane. Specific examples of such a silicone base material include a silicone-containing medical device, a silicone-containing culture container, a silicone base material having a microchannel, and preferably a silicone-containing microchannel device.

[Oxygen permeability coefficient]
The cell culture member of the present invention has an oxygen permeability coefficient (Dk) according to ISO18369-4 (FATT method) of 200 Barrer (1 Barrer = 1 × 10 ^-11 O ₂ (STP) cm / cm ² mmHg) or more and 300 Barrer or more. Is preferable.

[Average Surface Roughness Ra]
The average surface roughness Ra of the culture surface in the cell culture member of the present invention is preferably 1.0 μm or less, more preferably 0.5 μm or less. By setting the average surface roughness of the culture surface of the present invention to 1.0 μm, cell adhesion and extensibility are improved, and orientation is exhibited. The culture surface means the surface of the cell culture region including the culture layer.

The present invention will be described in more detail with reference to the following examples, but the following examples do not limit the scope of rights of the present invention in any way.

<Preparation of cell adhesion control inkjet ink>
[Manufacturing Example 1]
-Preparation of cell adhesion material (A-1) Cell campus AQ-03A (collagen solution manufactured by Taki Chemical Co., Ltd., active ingredient: 0.3%) solvent is completely volatilized with a vacuum pump to completely volatilize the cell adhesion material. (A-1) was obtained.
[Manufacturing Example 2]
-Preparation of cell adhesion material (A-2) The solvent of the laminin solution derived from mouse EHS sarcoma (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., active ingredient: 0.05%) is completely volatilized with a vacuum pump to completely volatilize the cell adhesion material. (A-2) was obtained.
[Manufacturing Example 3]
-Preparation of cell adhesion material (A-3) The solvent of human plasma-derived fibronectin solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., active ingredient: 0.05%) is completely volatilized with a vacuum pump to completely volatilize the cell adhesion material (cell adhesion material (A-3). A-3) was obtained.
[Manufacturing Example 4]
-Preparation of cell adhesion material (A-4) The solvent of Matrigel (Matrigel solution manufactured by Corning Co., Ltd., active ingredient: 0.10%) was completely volatilized with a vacuum pump to prepare the cell adhesion material (A-4). Obtained.
[Manufacturing Example 5]
-Preparation of cell adhesion material (A-5) The solvent of Synthemax (synthmax solution manufactured by Corning Co., Ltd., active ingredient: 0.10%) is completely volatilized by a vacuum pump, and the cell adhesion material (A-5) is prepared. Got

[Manufacturing Example 6]
-Preparation of surface tension adjuster (B1) Surface tension adjustment by completely volatilizing the solvent of sucrose myristic acid ester aqueous solution "Ryoto Sugar Ester M1695" (manufactured by Mitsubishi Chemical Foods Co., Ltd., active ingredient: 99%) with a vacuum pump. Agent (B1) was obtained.
[Manufacturing Example 7]
-Preparation of surface tension adjuster (B2) Surface tension adjustment by completely volatilizing the solvent of sucrose lauryl ester aqueous solution "Ryoto Sugar Ester L1695" (manufactured by Mitsubishi Chemical Foods Co., Ltd., active ingredient: 99%) with a vacuum pump. Agent (B2) was obtained.
[Manufacturing Example 8]
-Preparation of surface tension adjuster (B3) Surface tension adjustment by completely volatilizing the solvent of sucrose stearic acid ester aqueous solution "Ryoto Sugar Ester S1670" (manufactured by Mitsubishi Chemical Foods Co., Ltd., active ingredient: 99%) with a vacuum pump. Agent (B3) was obtained.

[Manufacturing Example 9]
-Preparation of solvent (C1):
A 5 mmol / L acetic acid aqueous solution and 1,2-propanediol were mixed at a weight ratio of 2: 1 to obtain a solvent (C1).
[Manufacturing Example 10]
-Preparation of solvent (C2):
A 5 mmol / L acetic acid aqueous solution and 1,2-hexanediol were mixed at a weight ratio of 2: 1 to obtain a solvent (C2).
[Manufacturing Example 11]
-Preparation of solvent (C3):
A 5 mmol / L acetic acid aqueous solution and diethylene glycol diethyl ether were mixed at a weight ratio of 2: 1 to obtain a solvent (C3).

[Example 1]
-Preparation of inkjet ink for cell culture (I-1) 0.18 parts by mass of cell adhesion material (A-1), 0.01 parts by mass of surface tension adjuster (B1), 99 parts of solvent (C1). The mixture was stirred with a shaker for 81 parts by mass until it became sufficiently uniform. Then, filtration was performed with a membrane filter having an opening of 1.0 μm to remove coarse particles causing head clogging, and an inkjet ink (I-1) was prepared.

[Examples 2 to 5], [Comparative Examples 1 to 3]
-Preparation of inkjet ink for cell culture (I-2 to 8) Inkjet for cell culture in the same manner as in Example 1 except that the viscosity and surface tension were adjusted by changing the blending ratios in Table 1. Inks (I-2-8) were prepared.

[Manufacturing Example 12]
-Preparation of non-cell adhesive resin (D1-1) The solvent of the resin solution having the polyethylene oxide structure obtained by the method described in Example 2 of JP-A-2017-104027 is completely volatilized by a vacuum pump, and the cell is non-adhesive. A sex resin (D1-1) was obtained.
[Manufacturing Example 13]
-Preparation of cell non-adhesive resin (D1-2) The solvent of BASF's Pluronic F-127 aqueous solution (active ingredient 10%) was completely volatilized with a vacuum pump to completely volatilize the solvent, and the cell non-adhesive resin (D1-2) was prepared. Got
[Manufacturing Example 14]
-Preparation of cell non-adhesive resin (D1-3) The solvent of RAM resin 1000 solution (active ingredient 30%) manufactured by Osaka Organic Chemical Co., Ltd. was completely volatilized by a vacuum pump to completely volatilize the solvent, and the cell non-adhesive resin (D1-). 3) was obtained.
[Manufacturing Example 15]
-Preparation of non-cell adhesive resin (D1-4) The solvent of Lipidure-PMB (active ingredient 5%) manufactured by NOF Corporation was completely volatilized by a vacuum pump to completely volatilize the solution, and the non-cell adhesive resin (D1-4) was prepared. Got

[Manufacturing Example 16]
According to the method described in Production Example 8 of JP-A-2016-112396, VPE0201 (manufactured by Wako Pure Chemical Industries, Ltd .: Macroazo initiator) was changed to 5.6 parts by weight to contain 10% by mass of a polyethylene oxide structure as a polymerization initiator. A resin solution was obtained. The monomer composition of the resin (D1-5) was methoxyethyl acrylate: methacrylic acid = 49.4: 0.6. The solvent of the resin solution was completely volatilized with a vacuum pump to obtain a cell non-adhesive resin (D1-5).

[Manufacturing Example 17]
According to the method of Production Example 16, the blending amount of VPE0201 was changed to obtain a resin solution containing 20% by mass of a polyethylene oxide structure. Then, the solvent of the resin solution was completely volatilized by a vacuum pump to obtain a cell non-adhesive resin (D1-6).

[Manufacturing Example 18]
According to the method of Production Example 16, the blending amount of VPE0201 was changed to obtain a resin solution containing 0.1% by mass of a polyethylene oxide structure. Then, the solvent of the resin solution was completely volatilized by a vacuum pump to obtain a cell non-adhesive resin (D1-7).

[Manufacturing Example 19]
According to the method of Production Example 16, the blending amount of VPE0201 was changed to obtain a resin solution containing 1.0% by mass of a polyethylene oxide structure. Then, the solvent of the resin solution was completely volatilized by a vacuum pump to obtain a cell non-adhesive resin (D1-8).

-Preparation of Cell Non-Adhesive Composition (D) [Production Example 20]
-Preparation of cell non-adhesive composition (D-1) 16.00 parts by mass of cell non-adhesive resin (D1-1), 0.01 part by mass of surface tension modifier (B1), solvent (C1) The mixture was stirred with a shaker at 83.99 parts by mass until it became sufficiently uniform. Then, filtration was performed with a membrane filter having an opening of 1.0 μm to remove coarse particles causing head clogging, and a cell non-adhesive composition (D-1) was prepared.

[Manufacturing Examples 21 to 24]
-Preparation of cell non-adhesive composition (D-2 to 5) Instead of cell non-adhesive resin (D1-1), the cell non-adhesive resin shown in Table 1 was used, and the compounding ratio was changed to Table 2. The cell non-adhesive compositions (D-2 to 5) were prepared in the same manner as in Production Example 20 except that the viscosity and surface tension were adjusted.
[Production Examples 25 to 28]
-Preparation of non-cell adhesive composition (D-6-9) V02 (Nisshinbo Co., Ltd.) was used as the non-adhesive cell resin and cross-linking agent obtained in Production Examples 16 to 19 in place of the non-adhesive resin (D1-1). , Aqueous carbodiimide group-containing compound) was mixed as a solid content at a mass ratio of 100: 13.5, and the viscosity and surface tension were adjusted by changing to the blending ratio shown in Table 2, but the same as in Production Example 20. A cell non-adhesive composition (D-6-9) was prepared by the method.

<Evaluation-1>
[Surface tension of (I) and (D)]
For the surface tension, an automatic surface tension meter CBVP-Z manufactured by Kyowa Interface Science Co., Ltd. was used, and a platinum plate was subjected to an inkjet ink for cell culture (I-1 to 8) in an environment of 25 ° C., and a cell non-adhesive composition (cell non-adhesive composition). It was measured by confirming the surface tension when wet with D-1 to 9). The results are shown in Tables 1 and 2.

[Viscosities of (I) and (D)]
As for the viscosity, 1 mL each of the inkjet ink for cell culture (I-1 to 8) and the cell non-adhesive composition (D-1 to 9) was placed in a TVE25L type viscometer manufactured by Toki Sangyo Co., Ltd. and placed in an environment of 25 ° C. Then, it was measured by reading the viscosity at 50 rpm. The results are shown in Tables 1 and 2.

[Evaluation of dischargeability of (I) and (D)]
Immediately after filling the carriage A of the patterning jet (inkjet evaluation machine manufactured by Tritec) with the prepared inkjet ink (I) for cell culture and the cell non-adhesive composition (D), and using the ejection observation function of the patterning jet. Flying observation was carried out, and it was verified whether it could fly normally after being ejected from the nozzle. The results of evaluation using the following indicators are shown in Tables 1 and 2.
⊚: Keeping a spherical shape, go straight, and discharge regularly. Good dischargeability.
〇: When dropped, the tail was pulled and discharged. Dischargeability is slightly good.
Δ: Immediately after the discharge, the droplet was divided into two or more drops and discharged. It can be used.
X: Discharged irregularly. Poor dischargeability.

[Cytotoxicity evaluation of (I) and (D)]
In line with the cytotoxicity of the guideline issued by the Ministry of Health, Labor and Welfare "Basic concept of biological safety evaluation necessary for application for manufacturing and marketing approval of medical equipment" (Yakushokuki No. 0301 No. 20), the following 1 Samples (Y1 to 17) shown in Tables 1 and 2 were prepared using an inkjet ink for cell culture or a non-adhesive composition in the order of 7 to 7 and evaluated for cytotoxicity. Tables 1 and 2 show the evaluation results classified by the value of the colony formation rate.

1. 1. Samples (Y1-17), preparation of negative / positive controls:
(Y1 to 17): Inkjet ink for cell culture (I-1 to 8) and cell non-adhesive composition (D-) on an untreated polyimide film (Kapton 200EN manufactured by Toray DuPont Co., Ltd.) having an ^{area of 30 cm 2.} 2 mL of 1 to 9) was added, and spin coating was performed at 3000 rpm for 30 seconds. Samples (Y1 to 17) whose inner surface was coated with a coating agent were prepared by drying at room temperature for 24 hours.
-Negative / positive control: A high-density polyethylene sheet (provided by Hatano Research Institute, Food and Drug Safety Center) was used as the negative control material. As the positive control material A, a 0.1% ZDEC-containing polyurethane film was used, and as the positive control material B, a 0.25% ZDBC-containing polyurethane film was used.
-For the blank, a multiple well plate 24-well flat bottom 3526 (manufactured by Falcon) was used.

2. Extract preparation:
Samples (Y1-17), negative and positive control materials A and B were ^{adjusted to have an area of 15 cm 2} on one side and placed in a 15 mL centrifuge tube, respectively. 5 mL of E-MEM medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to which MO5 medium (10% fetal bovine serum (FBS; manufactured by BioWest) was added at an external ratio of 5% by mass) was added, and the temperature was 37 ° C., 5% CO ₂ incubator. Stored in 24 hours.

3. 3. Cell seeding:
The cryopreserved V-79 cells (Chinese hamster lung-derived fibroblasts) are thawed at 37 ° C. and then transferred to MEM10 medium (E-MEM medium in which FBS is added at an external ratio of 10% by mass) to 100 cells / mL. Soaked in a 50 mL centrifuge tube. Multiple well plates were seeded in 0.5 mL each on a 24-well flat bottom 3526 (manufactured by Falcon) and _{stored in a 5% CO 2} incubator at 37 ° C. for 24 hours.

4. Cell culture:
Prepare 5 centrifuge tubes for each extract and extract with different concentrations diluted with MO5 medium so that the extract is 50%, 25%, 12.5%, 6.25%, 3.13%. The liquid was prepared. Subsequently, the MEM10 medium in the multiple well plate was sucked up with an ejector, and 0.5 mL of extracts having different concentrations was added. In the blank, the MEM10 medium was sucked up with an ejector, 0.5 mL of MO5 medium was added, and the medium was _{stored in a 5% CO 2} incubator at 37 ° C. for 7 days.

5. Immobilization:
The MO5 medium in the multiple well plate cultured for 7 days was sucked up with an ejector. Mildform 10 nM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well in an amount of 0.5 mL, and after 1 minute, the mixture was sucked up with an ejector.

6. staining:
A staining reagent was prepared by mixing the mass ratio of Giemsa stain (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and phosphate buffered saline (PBS) at a ratio of 1: 9. 0.5 mL of the staining reagent was added to each well, and the mixture was allowed to stand for about 30 minutes and sucked with an ejector. Washed with 0.5 mL of PBS and air dried.

7. Colony count:
The colonies formed were counted. The number of blank colonies was used as the control group, and the average value of the three times was set to 100%. Samples (Y1~17), the number of colonies obtained by extract negative / positive controls averaged for each concentration was determined the ratio IC ₅₀ for the number of colonies in the control group.
-Test establishment conditions: Based on the following A to C, it was judged that this experiment was established.
A: The colony formation rate in the control group was good.
B: The plating efficiency of the negative control material was similar to that of the control group.
C: positive control material _{IC 50} of A and B, were each less than 7% and 80%.
The IC ₅₀ is an extract concentration at which the colony formation rate is 50%.
・ Criteria for grading based on the value of colony formation rate:
◯: IC ₅₀ > 30% Cytotoxicity is negative.
×: 30% ≧ _{IC 50} cytotoxicity is positive.
-The evaluation results are shown in Tables 1 and 2.

<Result>
The cell culture inkjet inks (I-1 to 5) shown in Table 1 had good ejection properties due to the relationship between viscosity and surface tension. Moreover, since the cytotoxicity is negative, it is shown that the cells are not adversely affected. Furthermore, the cell non-adhesive compositions (D-1 to 9) shown in Table 2 also have good ejection properties due to the relationship between viscosity and surface tension, and have negative cytotoxicity, so that they do not adversely affect cells. Indicated.
On the other hand, in the cell culture inkjet inks (I-6, 7) of [Comparative Examples 1 and 2], the surface tension is too high or too low, so that the inkjet ink ejects the ink irregularly from the nozzle. It could not be used as ink. Further, since the viscosity of the cell culture inkjet ink (I-8) of [Comparative Example 3] was too high, the inkjet ink was divided into two or more drops immediately after ejection and ejected.

<Preparation of cell culture members (polystyrene base material)>
[Example 6]
-Preparation of cell culture member (S-1) The carriage of the LaboJet-500 inkjet evaluation machine manufactured by Microjet Co., Ltd. was filled with the inkjet ink (I-1) of Example 1 and treated with an untreated dish having a diameter of 35 mm (polystyrene manufactured by Corning). ) Was printed at design intervals of a line width of 50 μm to obtain a cell culture member (S-1).

[Example 7], [Comparative example 5]
-Preparation of cell culture members (S-2) and (S-19) Cell culture members (S-2) and (S) in the same manner as in (S-1) except that the materials shown in Table 3 were used. -19) was prepared.

[Example 8]
-Preparation of cell culture member (S-3) 1 mL of cell non-adhesive composition (D-1) was added to the inner surface of an untreated dish (polystyrene manufactured by Corning) having a diameter of 35 mm, and spin-coated at 3000 rpm for 30 seconds. After drying at room temperature for 24 hours, the carriage of the LaboJet-500 inkjet evaluation machine manufactured by Microjet Co., Ltd. was filled with (I-3), printed on the cell non-adhesive layer at a design interval of a line width of 50 μm, and the cell culture member. (S-3) was obtained.

[Examples 9 to 12]
-Preparation of cell culture members (S-4 to 7) Cell culture members (S-4 to 7) were prepared in the same manner as in (S-3) except that the materials shown in Table 3 were used.

[Example 13]
-Preparation of cell culture member (S-8) The carriage of the LaboJet-500 inkjet evaluation machine manufactured by Microjet Co., Ltd. is filled with the cell culture inkjet ink (I-1), and the diameter is 0 μm at the design interval. After printing on the inner surface of a 35 mm untreated dish (polystyrene manufactured by Corning), the cells were dried at room temperature for 24 hours. Then, the carriage of the LaboJet-500 inkjet evaluation machine manufactured by Microjet Co., Ltd. was filled with the cell non-adhesive composition, printed on the cell adhesion layer at a design interval of a line width of 50 μm, and a cell culture member (S- 8) was obtained.

[Examples 14 to 17]
-Preparation of cell culture members (S-9 to 12) Cell culture members (S-9 to 12) were prepared in the same manner as in (S-8) except that the materials shown in Table 3 were used. ..

[Example 18]
-Preparation of cell culture member (S-13) Cell culture inkjet ink (I-2) on carriage A of Microjet's BioSpot inkjet evaluation machine, and cell non-adhesive composition (D-1) on carriage B. At the same time, the cells were printed on the inner surface of an untreated dish (polystyrene manufactured by Corning) having a diameter of 35 mm at a design interval of a line width of 50 μm to obtain a cell culture member (S-13).

[Examples 19 to 22]
-Preparation of cell culture members (S-14 to 17) Cell culture members (S-14 to 17) were prepared in the same manner as in (S-13) except that the materials shown in Table 3 were used. ..

[Comparative Example 4]
-Cell culture member (S-18)
An untreated dish (polystyrene made by Corning) having a diameter of 35 mm was prepared.

[Comparative Example 6]
-Preparation of cell culture member (S-20) Cell culture inkjet ink (I-8) was spin-coated on an untreated dish (Coning polystyrene) with a diameter of 35 mm at 3000 rpm for 30 seconds and dried at room temperature for 24 hours. After that, a cell culture member (S-20) was prepared.

<Evaluation-2>
[Average Surface Roughness Ra]
The average surface roughness Ra (arithmetic mean surface roughness Ra) of the culture surface of the cell culture member was measured by JIS B 0601. The average surface roughness Ra represents the average value of the absolute deviation from the average line. The evaluation results are shown in Table 3.
[Evaluation criteria]
〇: Average surface roughness ≤ 0.5 μm Smooth surface Δ: 0.5 μm <Average surface roughness ≤ 1.0 μm Smoothness is inferior, but usable ×: 1.0 μm <Average surface roughness Due to unevenness of cells Movement is restricted and cannot be used

[Pattern of cell culture member: interval between width (L) and width (S)]
-Pattern of cell culture member (S-1, 2, 19):
The width of the cell adhesion pattern formed by (I); L (μm) and the width of the base material; S (μm) are photographed with a transmission optical microscope at 10 times and evaluated by measuring the widths of L and S. did.
-Pattern of cell culture member (S-3 to 17):
The width of the cell adhesion pattern formed by (I); L (μm) and the width of the cell non-adhesion pattern formed by (D); S (μm) were photographed with a transmission optical microscope at 10 times, and L and S were photographed. It was evaluated by measuring the width of.
The closer the width L (μm) and the width S (μm) are to the design width of 50 μm, the higher the printing accuracy. The evaluation results are shown in Table 3.

[Formation of cell pattern (morphology of cell orientation)]
A cell line for mouse fibroblasts (NIH / 3T3 cells) was added to 10% FBS-DMEM medium to prepare a cell suspension having a NIH / 3T3 concentration of 80,000 cells / ml. 2 mL of the obtained cell suspension was seeded on the above cell culture members (S1 to 20). Thereafter, and cultured until day 7 with 5% _CO 2/37 ℃ incubator.
In order to confirm whether the cells are oriented, the cell culture state immediately after seeding and 7 days after seeding is photographed with a transmission optical microscope at 4x, and compared with the cell morphology immediately after seeding, according to the following criteria. Judgment was made and the results are shown in Table 3.

FIG. 5 is a transmission optical micrograph showing the cell orientation morphology observed immediately after seeding and after cell culture on the 7th day with the cell culture member prepared in the example of the present invention.
In FIG. 5, each numerical panel has the following features.
1: Immediately after seeding the cells, it is observed that the cells are randomly dispersed on the cell culture member.
After cell culture on the 2: 7th day, it is observed that the cells adhere, extend, and orient along the adhesive line. Excellent cell orientation.
After cell culture on the 3: 7th day, the cells adhere and extend on the adhesive line, but it is observed that they are oriented irregularly. Poor cell orientation.
After cell culture on day 4: 7, it is observed that the cells adhere and stretch non-uniformly and do not orient. Poor cell orientation.

The cell culture members (S-1 to 17) of the examples shown in Table 3 had an average roughness Ra of 1.0 μm or less, and after cell culture on the 7th day, adhered so that the cells were aligned along the fine lines in the visual field. In contrast to [Comparative Example 4], the cells did not adhere to each other and became a non-uniform mass, and the orientation could not be confirmed. Further, in [Comparative Example 5], since the ink jet ink for cell culture (I-8) has a high viscosity, the printing accuracy of the design pattern is low, and as a result, some of the design patterns deviate from the pattern, are arranged irregularly, and have no orientation. There was a place. In [Comparative Example 6], it was observed that the cells adhered non-uniformly to the visual field and did not orient.

<Preparation of culture members (silicone base material)>
[Example 23]
-Preparation of cell culture member (S-21) The carriage of the LaboJet-500 inkjet evaluation machine manufactured by Microjet Co., Ltd. was filled with the inkjet ink (I-1) of Example 1 and treated with an untreated silicone 6-well plate (Bethel). A cell culture member (S-21) was obtained by printing on the inner surface of one well of VECELL (registered trademark) manufactured by the same company at a design interval of a line width of 50 μm.

[Example 24]
-Preparation of cell culture member (S-22) A cell culture member (S-22) was prepared in the same manner as in (S-21) except that the materials shown in Table 4 were used.

[Example 25]
-Preparation of cell culture member (S-23) A cell non-adhesive composition (D-1) was placed on the inner surface of one well of an untreated silicone 6-well plate (VECELL (registered trademark) manufactured by Bethel). 1 mL was added and spin-coated at 3000 rpm for 30 seconds. After drying at room temperature for 24 hours, the carriage of the LaboJet-500 inkjet evaluation machine manufactured by Microjet Co., Ltd. is filled with (I-3), printed on a non-adhesive layer of cells at a design interval of a line width of 50 μm, and used for cell culture. A member (S-23) was obtained.

[Examples 26 and 27]
-Preparation of cell culture members (S-24, 25) Cell culture members (S-24, 25) were prepared in the same manner as in (S-23) except that the materials shown in Table 4 were used.

[Example 28]
-Preparation of cell culture member (S-26) The carriage of the BioSpot inkjet evaluation machine manufactured by Microjet Co., Ltd. is filled with the cell culture inkjet ink (I-1), and an untreated silicone 6-well plate (manufactured by Bethel Co., Ltd.) is filled. After printing on the inner surface of one well of VECELL (registered trademark) at a design interval of 0 μm in line width, the cells were dried at room temperature for 24 hours. Then, the carriage of the LaboJet-500 inkjet evaluation machine manufactured by Microjet Co., Ltd. was filled with the composition (D-1) having cell non-adhesion, and printed on the cell adhesion layer at a design interval of a line width of 50 μm, and the cells were printed. A culture member (S-26) was obtained.

[Examples 29 and 30]
-Preparation of cell culture member (S-27, 28) A cell culture member (S-27, 28) was prepared in the same manner as in (S-26) except that the materials shown in Table 4 were used.

[Example 31]
-Preparation of cell culture member (S-29) Cell culture inkjet ink (I-4) on carriage A of Microjet's BioSpot inkjet evaluation machine, and cell non-adhesive composition (D-2) on carriage B. On the inner surface of one well of an untreated silicone 6-well plate (VECELL (registered trademark) manufactured by Bethel), the cells were simultaneously printed at design intervals of a line width of 50 μm, and a cell culture member (cell culture member). S-29) was obtained.

[Examples 32 and 33]
-Preparation of cell culture members (S-30, 31) Cell culture members (S-30 to 31) were prepared in the same manner as in (S-29) except that the materials shown in Table 4 were used.

[Comparative Example 7]
-Cell culture member (S-32)
An untreated dish (polystyrene made by Corning) having a diameter of 35 mm was used.

[Comparative Example 8]
Preparation of Cell Culture Member (S-33) The inner surface of one well of an untreated silicone 6-well plate (VECELL®) manufactured by Bethel was used.

[Comparative Example 9]
-Preparation of cell culture member (S-34) Cell culture inkjet ink (I-1) was spin-coated on an untreated dish (Coning polystyrene) having a diameter of 35 mm at 3000 rpm for 30 seconds and dried at room temperature for 24 hours. A cell culture member (S-34) was prepared.

<Evaluation-3>
[Average Surface Roughness Ra]
Evaluation-2 was evaluated by the method described.
[Pattern of cell culture member: interval between width (L) and width (S)]
Evaluation-2 was evaluated by the method described.

[Oxygen permeability coefficient (Dk)]
Using a planar electrode cell of an oxygen permeability measuring device (201T, Rehder), the oxygen permeability coefficient (S-21-34) of the cell culture member (S-21 to 34) is determined by the method specified in ISO18369-4 (FATT method). dk) were measured _{^{(1Barrer = 1 × 10 -11 O}} 2 (STP) cm / cm 2 mmHg). The evaluation results are shown in Table 4.
[Evaluation criteria]
⊚: Dk ≧ 300 Barrer (extremely good)
◯: 300> Dk ≧ 200 (good)
Δ: 200> Dk ≧ 20 (slightly defective)
X: 20> Dk (defective)

[Cell activity]
After sterilizing the cell culture member (S-21 to 34) with ethylene oxide gas, 10% of fetal bovine serum (FBS) was added to Dulbecco's modified Eagle's medium (DMEM) (hereinafter, "10% FBS-DMEM medium"). and also called.) and medium, human liver cell line (Huh7 cells) were seeded 1.5 × 10 ⁵ per well, were cultured to 5 days in 5% CO _2/37 ℃ incubator.
Cell activity was assessed by performing an ATP assay after 1 and 5 days. Specifically, 1 ml of ATP reagent (ATP measurement reagent for "cells": manufactured by Toyo Venet Co., Ltd.) was added to the cell culture member after culturing, pipetting was performed 5 times, and the mixture was allowed to stand at room temperature for 5 minutes. 100 ml
The reagent and cytolysis solution of No. 1 were separated into a separate plate and stirred for 1 minute. The amount of light emitted was measured using MisrasLB940 (manufactured by Berthold), and the evaluation results are shown in Table 4.
Cytotoxicity = (luminance after 5 days of culture) / (luminance after 1 day of culture) x 100
〇: 100% ≤ cell activity Δ: 20% ≤ cell activity <100%
×: Cell activity <20%

[Formation of cell pattern (morphology of cell orientation)]
Hepatocytes prepared from rat liver (Slc / Wister provided by Sankyo Lab Service; hereinafter referred to as hepatocytes) are added to 10% FBS-DMEM medium, and the concentration of hepatocytes is 8 × 10 ⁴ cells / A cell suspension to be ml was prepared. 2 mL of the obtained cell suspension was seeded on the above-mentioned cell culture member (S21 to 34). Thereafter, and cultured until day 1 5% _CO 2/37 ℃ incubator.
In order to confirm whether the cells are arranged, the cell culture state immediately after seeding and one day later is photographed with a transmission optical microscope at 4x, and compared with the cell morphology immediately after seeding, according to the following criteria. Judged. The evaluation results are shown in Table 4.
In addition, FIG. 6 is a schematic view showing the morphology of the cell arrangement observed immediately after seeding and after the cell culture on the first day with the cell culture member prepared in the example of the present invention. In FIG. 6, the schematic diagram of each number has the following features.
1: Immediately after seeding the cells, it is observed that the cells are randomly dispersed on the cell culture member in the visual field.
2: After 1 day of cell culture, it is observed that the cells adhere and are arranged along the adhesive line. Excellent cell arrangement.
3: After 1 day of cell culture, cells adhere to the adhesive lines, but it is observed that they are arranged irregularly. The cell arrangement is good.
4: After 1 day of cell culture, it is observed that the cells adhere unevenly and do not arrange. The cell arrangement is poor.

The cell culture members (S-21 to 31) used in the examples shown in Table 4 have an average roughness Ra of 1.0 μm or less, and the width L (μm) and width S (μm) after printing are about. It was 50 μm, indicating that the printing accuracy was high. Moreover, since the oxygen permeability was good, the cell activity by the ATP assay was good. Furthermore, one day after culturing, it was observed that the cells adhered and arranged along the thin line in the visual field.
On the other hand, the cell culture member (S-32) used in Comparative Example 7 had low oxygen permeation and did not supply sufficient oxygen from the bottom surface of the member to the cell layer. It adversely affected the morphology of. Further, since the cell culture member (S-33) used in Comparative Example 8 does not have a pattern layer formed by the cell culture inkjet ink on the silicone substrate, after the cell culture on the first day. , It was observed that the cells adhered unevenly and did not arrange, and the cell activity was low. Further, since the cell culture member (S-34) used in Comparative Example 9 does not have a pattern layer formed by the inkjet ink for cell culture on the silicone substrate, the cells adhere unevenly. It was observed that they were not aligned.

<Preparation of members for culturing myoblasts>
[Examples 34 and 35]
-Preparation of members for myoblast culture (S-1, S-2) In the same manner as in Examples 6 and 7, cell culture members (S-1, S-2) were obtained.

[Example 36]
-Preparation of myoblast cultivation member (S-21) Add 1 mL of cell non-adhesive composition (D-6) to the inner surface of an untreated dish (polystyrene made by Corning) with a diameter of 35 mm, and add 1 mL of the cell non-adhesive composition (D-6) at 3000 rpm for 30 seconds. Spin coated. After drying at room temperature for 24 hours, the carriage of the LaboJet-500 inkjet evaluation machine manufactured by Microjet Co., Ltd. is filled with (I-1) and printed on the cell non-adhesive layer at a design interval of 50 μm in line width and 30 μm in line interval. , A cell culture member (S-21) was obtained.

[Examples 37 to 41]
-Preparation of cell culture member (S-22-27) A cell culture member (S-22-27) was prepared in the same manner as in (S-21) except that the L / S shown in Table 5 was formed. did.

[Examples 42 to 46]
-Preparation of members for myoblast culture (S-13 to 17) Cell culture members (S-13 to 17) were obtained in the same manner as in Examples 18 to 22.

[Comparative Examples 10 to 12]
-Preparation of members for myoblast culture (S-18 to 20) Cell culture members (S-18 to 20) were obtained in the same manner as in Comparative Examples 4 to 6.

<Evaluation-4>
[Induction of myoblast differentiation]
By observing a mouse-derived myoblast cell line (C2C12 cells) by fluorescent staining of myosin heavy chains and nuclei, the effect on differentiation into myotube cells was confirmed. Myosin heavy chain is a muscle contraction-related protein expressed in the process of myotube formation, and the expression of myosin heavy chain and multinucleation by cell fusion are the characteristics of differentiation into myotube cells. By observing the myosin heavy chain by fluorescent immunostaining and nuclear staining, the presence or absence of differentiation into myotube cells and the orientation in a specific direction can be evaluated more clearly, and the formation of myosin tissue can be observed.

A mouse-derived myoblast cell line (C2C12 cells) was added to 10% FBS-DMEM medium to prepare a cell suspension having a C2C12 concentration of 50,000 cells / ml. 2 mL of the obtained cell suspension was seeded so as to serve as the above-mentioned cell culture member.
After being confluent, it was replaced with a differentiation-inducing medium (2% Horse Serum (Gibco), 1% antibiotic-antifungal agent mixed solution (anti-anti, Gibco) -added High-glucose DMEM (D5790, sigma)).
In order to confirm whether the myoblasts are arranged, the cell culture state 3 days and 10 days after seeding was photographed with a transmission optical microscope 4 times, and compared with the cell morphology immediately after seeding, as follows. Judgment was made according to the criteria of.
FIG. 7 is a schematic view showing the morphology of the cell arrangement observed immediately after seeding and after the cell culture on the first day with the myoblast culture member prepared in the examples of the present invention. In FIG. 7, each numerical panel has the following features.
1: During myoblast culture, it is confirmed that the cells adhere and are arranged along the adhesive line, and it is observed that the cells are confluent on the substrate. (Good)
2: During myoblast culture, it is confirmed that the cells adhere and are arranged along the adhesive line, and it is observed that the cells are not confluent on the substrate. (Yes)
3: When culturing myoblasts, the cells adhere to the adhesive lines, but it is confirmed that they are arranged irregularly, and it is observed that the cells are confluent on the substrate. (Defective)
4: When culturing myoblasts, it is observed that the cells adhere to the line having adhesiveness, but the adhesiveness is weak and the cells are detached from the substrate. (Defective)

[Cell fluorescence staining]
On the 10th day of culture, confluentized C2C12 cells were fluorescently stained according to a conventional method. The nucleus was stained with Hoechst 33342 (H3570, Thermo Fisher Scientific, USA) and the myosin heavy chain was stained with Anti-MYH (SC-20641, Cosmo Bio, Tokyo) and GoatAnti-LabbitIgGH & L Alexafluoro488 (ab150047, Abcam). .. The stained cells were observed with a transmission optical microscope.
FIG. 8 is a schematic view showing the morphology of the cell arrangement observed immediately after seeding and after the cell culture on the first day with the myoblast culture member prepared in the examples of the present invention. In FIG. 8, each numerical panel has the following features.
〇: It is observed that the myosin heavy chain is oriented in the horizontal axis direction and mimics the myotube tissue.
X: Myosin heavy chain was confirmed in a random direction, and the myosin tissue could not be imitated.
-: The cells are exfoliated and the tissue is not constructed, which is defective.

In the myoblast culture substrate used in Examples 34 and 35 shown in Table 5, the cell adhesion site did not peel off from the substrate, and after 10 days of culturing, the myoblast mimicking tissue was confluent while maintaining the orientation. Was formed. In Examples 36 to 41, the curing agent in the non-adhesive resin prevented peeling from the substrate and formed a myocardial mimicry tissue. In Examples 36 to 38, myoblasts were able to efficiently differentiate into myotubes and form myocardial mimicry tissue depending on the blending ratio of polyethylene oxide in the non-adhesive portion.
In Comparative Example 10, the cells did not adhere to each other and formed a non-uniform mass, which was exfoliated from the substrate over time. In Comparative Example 11, since the ink jet ink for cell culture (I-8) had a high viscosity, cells flowed out from the adhesive coating surface and showed orientation, but some of them deviated from the pattern, arranged irregularly, and were oriented. There was a part where there was no ink, and it peeled off from the substrate. In Comparative Example 12, it was observed that the cells adhered unevenly to the visual field and did not orient.
FIG. 5 is an immunostaining diagram of cells cultured in the differentiation medium shown in FIG. Blue indicates the nucleus and green indicates the myosin heavy chain. By using the differentiation medium from the 3rd day of the culture to the 10th day of the culture, the cells were oriented in the horizontal axis direction, and the fusion of the cells and the formation of a long myotube could be observed.

<Industrial applicability>
The cell orientation control technology makes it possible to produce organ chips, which is expected to be developed as a drug discovery screening method. In addition, since it can be applied to biodegradable materials, it can also be applied to cell sheets and artificial organs.

1 Cell culture member 2 Width of cell adhesion pattern formed by cell culture inkjet ink (I); L (μm)
3 Width of substrate pattern; S (μm)
4 Base material 5 Culture layer 6 Width of cell non-adhesive pattern formed by cell non-adhesive composition (D); S (μm)

Claims (18)

An inkjet ink (I) for cell culture containing the cell adhesion material (A), which satisfies the following conditions (1) to (3).
(1) The content of the cell adhesive material (A) in 100% by mass of the inkjet ink for cell culture is 0.01% by mass or more and 10% by mass or less.
(2) The viscosity at 25 ° C. is 50 mPa · s or less.
(3) The surface tension is 20 mN / m or more and 50 mN / m or less. The inkjet ink (I) for cell culture according to claim 1, wherein the cell adhesion material (A) contains at least one selected from the group consisting of an extracellular matrix and a synthetic substrate. The inkjet ink (I) for cell culture according to claim 1 or 2, further comprising a surface tension adjusting agent (B) and a solvent (C). A cell culture member having a cell adhesion pattern layer formed by the cell culture inkjet ink (I) according to any one of claims 1 to 3 on a substrate. A cell culture member obtained by laminating a cell non-adhesive layer and a cell adhesion pattern layer formed by the cell culture inkjet ink (I) according to any one of claims 1 to 3 on a substrate in this order. A cell culture member formed by laminating a cell adhesion layer and a cell non-adhesion pattern layer formed by the cell culture inkjet ink (I) according to any one of claims 1 to 3 on a substrate in this order. A cell culture member having a cell adhesion pattern layer formed by the cell culture inkjet ink (I) according to any one of claims 1 to 3 and a cell non-adhesion pattern layer on a substrate. The cell culture member according to any one of claims 5 to 7, wherein the cell non-adhesive layer or the cell non-adhesive pattern layer is formed of the cell non-adhesive composition (D). The cell culture member according to claim 8, wherein the cell non-adhesive composition (D) has a viscosity at 25 ° C. of 50 mPa · s or less and a surface tension of 20 mN / m or more and 50 mN / m or less. The cell culture member according to claim 8 or 9, wherein the cell non-adhesive composition (D) contains 0.1% by mass or more and 50% by mass or less of the cell non-adhesive resin (D1) in 100% by weight. The cell culture member according to any one of claims 4 to 10, wherein the average surface roughness Ra of the culture surface is 1.0 μm or less. Billing substrate is the silicone substrate, ISO18369-4 (FATT Method) with oxygen permeability coefficient (Dk) is at ^{200Barrer (1Barrer = 1 × 10 -11} O 2 (STP) cm / cm 2 mmHg) or higher Item 4. The cell culture member according to any one of Items 4 to 11. The cell culture member according to any one of claims 4 to 12, for reproducing cell orientation in a living body. A method for producing a cell culture member, wherein the cell culture inkjet ink (I) according to any one of claims 1 to 3 is inkjet printed on a substrate to form a culture layer. A cell culture member for forming a culture layer by inkjet printing the cell culture inkjet ink (I) and the cell non-adhesive composition (D) according to any one of claims 1 to 3 on a substrate. Manufacturing method. A method for forming a cell pattern, wherein cells are oriented by the cell culture member according to any one of claims 4 to 13. A cell pattern formed by the method according to claim 16. A method for forming a living tissue by inducing differentiation using the cell pattern according to claim 17.

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