JP7604801B2 - Inkjet ink for cell culture, cell culture member and its manufacturing method, and method for forming cell culture pattern - Google Patents

Description

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

In recent years, the development of cell engineering and regenerative medicine have attracted attention, and there have been active attempts to culture cells in appropriate forms outside the body and use them in drug discovery research, treatment applications, and as biological reaction models. For such purposes, in the development of cell culture substrates, which are one of the tools in drug discovery research and regenerative medicine research, collagen, polystyrene, and polymethyl methacrylate are generally known to be good materials, and polystyrene is particularly advantageous in terms of low cytotoxicity, economy, and workability, and hydrophilic-treated polystyrene is currently used in tissue culture. However, because it is not possible to control the adhesion or non-adhesion of cells with such culture materials, it is considered difficult to arrange or orient cells in order to reproduce patterns that vary from individual to individual, such as ganglia and blood vessels in biological tissues.

Meanwhile, a technique has been reported for adhering and arranging cultured cells only on minute areas of a substrate. This technique makes it possible to apply cultured cells to artificial organs, biosensors, bioreactors, and so on. One method for arranging cultured cells is to use a substrate with a patterned surface that varies in ease of adhesion to cells, culture cells on this surface, and arrange the cells by adhering them only to the surface that has been treated to allow cells to adhere.

Methods for reproducing the arrangement and orientation of cells in a living body include, for example:
(1) The application of a charge-retaining medium on which a static charge pattern is formed to cell culture for the purpose of, for example, growing nerve cells in a circuit-like manner (Patent Document 1).
(2) An attempt was made to arrange cultured cells on a surface patterned by photolithography using a cell adhesion-inhibiting or cell adhesive photosensitive hydrophilic polymer (Patent Document 2).
(3) A cell culture substrate on which a substance such as collagen that affects the adhesion rate and morphology of cells is patterned, and this substrate produced by a photolithography method are disclosed (Patent Document 3).
By culturing cells on such a substrate, more cells can be attached to the collagen or other patterned surface, thereby achieving cell patterning.
However, in the case of (1), the charge of the charge storage medium weakens immediately after production, and the positive charge pattern disappears, and there is a problem with stability.

In addition, in the cases of (2) and (3), when patterning is performed by photolithography using a photosensitive material, a highly precise pattern can be obtained, but the cell adhesive material must have photosensitivity, and it is often difficult to chemically modify a biopolymer or the like to impart such photosensitivity, which results in a very narrow range of selectivity for the cell adhesive material. In addition, in the case of photolithography using a photoresist, it is necessary to use a developer, etc., which may have an adverse effect on cell culture.
In light of these problems, many surface design methods have been studied with the aim of realizing excellent biocompatible polymeric 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 technique, it becomes possible to reproduce a cell pattern with greater stability than (1). In addition, there is no need for chemical modification to impart photosensitivity as in (2) and (3), and since a pattern can be formed without contacting multiple materials with the substrate during inkjet printing, modification of uneven surfaces, wet surfaces, and thin films is possible. However, (4) is not sufficient to reproduce the cell orientation in a living body, and a technique for easily producing a cell pattern has been required.

In addition, silicone materials are used in a wide range of applications, including medical materials, because of their excellent biocompatibility and oxygen permeability. For example, Patent Document 5 discloses a technique for designing a cone-shaped groove on the surface of a silicone substrate, arranging hepatocytes, and constructing bile canaliculi efficiently without layering extracellular matrix gel. However, Patent Document 5 discloses a technique for forming bile canaliculi tissue by arranging hepatocytes, but has a problem in that molding is not easy and processing costs are high because fine irregularities are created on a silicon wafer using anisotropic wet etching, and then collagen is coated on the silicon wafer. In addition, the operation of changing the culture medium and recovering the tissue is also complicated.

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

The objective of the present invention is to provide an inkjet ink for cell culture that has excellent cell adhesiveness, a cell culture component that can reproduce cell orientation in a living body and a method for producing the same, and a method for forming a cell pattern.

As a result of extensive research conducted to solve the above problems, the present inventors have found that the use of an inkjet ink containing a cell adhesive material (A) not only controls the orientation of cells, but also widens the range of material options and enables the formation of patterns that do not have adverse effects on cytotoxicity.
This provides a method for reproducing in vivo cell orientation, and is expected to be applied to a wide range of applications, including medical materials and drug screening. For example, in cell culture components called microchips, observing or measuring the chemical reactions of cells held on a patterning component is expected to bring benefits such as shortening analysis time.

That is, the present invention provides
The present invention relates to an ink-jet ink for cell culture (I) that contains a cell adhesive material (A), and that satisfies the following conditions (1) to (3).
(1) The content of the cell adhesive material (A) is 0.01% by mass or more and 10% by mass or less, based on 100% by weight of the ink-jet ink for cell culture.
(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 inkjet ink for cell culture (I) in which the cell adhesive material (A) contains at least one material selected from the group consisting of extracellular matrices and synthetic substrates.

The present invention also relates to the inkjet ink for cell culture (I) which further contains a surface tension adjuster (B) and a solvent (C).

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

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

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

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

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

The present invention also relates to the cell culture member, in which the non-cell-adhesive composition (D) has a viscosity of 50 mPa·s or less at 25°C 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, which contains 0.1% by mass or more and 50% by mass or less of a non-cell-adhesive resin (D1) in 100% by weight of a non-cell-adhesive composition (D).

The present invention also relates to the cell culture member, in which the average surface roughness Ra of the culture surface is 1.0 μm or less.

The present invention also relates to the cell culture member as described above, wherein the substrate is a silicone substrate and the oxygen permeability coefficient (Dk) according to ISO18369-4 (FATT method) is 200 Barrer (1 Barrer=1×10 ⁻¹¹ O ₂ (STP) cm/cm ² 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 comprises inkjet printing the inkjet ink (I) for cell culture onto a substrate to form a culture layer.

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

The present invention also relates to a method for forming a cell pattern, in which cells are aligned using 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 biological tissue by inducing differentiation using the cell pattern.

INDUSTRIAL APPLICABILITY The present invention provides an inkjet ink for cell culture having excellent cell adhesiveness, a cell culture component capable of reproducing in vivo cell orientation and a method for producing the same, and a method for forming a cell pattern.
In addition, a cell culture member having excellent oxygen permeability, capable of controlling cell arrangement and not adversely affecting cytotoxicity, a manufacturing method thereof, and a method for forming a cell pattern can be provided. In addition, a cell culture member having excellent cell adhesiveness, capable of controlling cell arrangement, and capable of forming a biological tissue by inducing differentiation, and a manufacturing method thereof can be provided.

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

<Inkjet ink for cell culture (I)>
The inkjet ink for cell culture (I) of the present invention has good ejection properties from an inkjet nozzle, and by printing the inkjet ink for cell culture (I) on a substrate, a patterned culture layer having cell adhesive properties can be formed.
Furthermore, by seeding cells onto a cell culture component having this cell adhesion pattern layer, the cells adhere to the cell adhesion pattern and are cultured, thereby forming a cell pattern that reproduces the orientation of cells in a living body.

In addition, the cell patterns formed by the cell culture component of the present invention can be used to form biological tissues by inducing differentiation.

[cell]
The cells used in the present invention are not particularly limited as long as they generally need to adhere to an appropriate surface for proliferation, but examples of cells that exhibit orientation include myoblasts, fibroblasts, osteoblasts, and nerve cells. For example, nerve tissue can be formed by culturing nerve cells in a pattern, and continuous myotube tissue can be formed by culturing myoblasts through differentiation induction. In addition, cells derived from humans are mainly used, but cells derived from animals other than humans (specifically, mice, rats, rabbits, pigs, dogs, monkeys, cows, horses, sheep, chickens, etc.) may also be used.
Hepatocytes can also be used, and methods for isolating hepatocytes from humans or animals can be carried out according to known methods. Hepatocytes may be derived from fetuses, newborns, or adults. Hepatocytes induced to differentiate from embryonic stem (ES) cells and induced pluripotent (iPS) stem cells, or from umbilical cord blood, bone marrow, adipose, or blood-derived tissue stem cells can also be used. Hepatocytes can be induced from these cells according to known methods.

[Cell adhesive material (A)]
Any cell adhesive material (A) can be used in the present invention so long as it has cell adhesiveness, 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 of collagen, laminin, fibronectin, hyaluronic acid, vitronectin, and cadherin.

Preferred are collagen, laminin, hyaluronic acid and fibronectin.
Collagen is the main protein component of connective tissues and basement membranes and exists in many types with different tensile strengths and tissue distributions. The stiffness, flexibility, and structural changes in many body tissues are due to changes in collagen composition, as well as cellular restriction and compartmentalization. Commercially available collagen products include Collagentypes i-v (acid-extracted collagen from Nippi Corporation), pepsin-solubilized collagen (from Nippi Corporation), AteloCell series (high-purity collagen acid solution derived from bovine hide from Koken Co., Ltd.), Cellmatrix series (pepsin-solubilized collagen derived from porcine tendons from Nitta Gelatin Co., Ltd.), and Cellcampus series (collagen derived from scales from Taki Chemical Co., Ltd.).

Laminin is a glycoprotein present in the basement membrane of most tissues. It is composed of three chains: α1, β1, and γ1. It binds to collagen and heparan sulfate to form and function as a basement membrane. In culture systems, it mainly adheres epithelial cells, endothelial cells, hepatic cells, etc., and also promotes axon extension in nerve cells and is involved in differentiation, chemotaxis, etc. It is closely related to the metastatic ability of cancer, and it is known that highly metastatic cancer cells have more laminin receptors on the cell surface. Commercially available laminin products include a laminin solution derived from mouse EHS sarcoma (FUJIFILM Wako Pure Chemical Industries, Ltd.), Recombinant Human Laminin (Oriental Yeast Co., Ltd.), Laminin 521 (BioLamina, Inc.), which is normally expressed and secreted by human ES cells, and i Matrix-511 (Takara Bio Inc.: composed of the C-terminal regions of the α-chain, β-chain, and γ-chain of laminin).
Hyaluronic acid is found in large amounts in subcutaneous tissues such as the dermis, where it retains moisture between cells and functions as a buffer. It is also found in large amounts in eye tissues such as the vitreous body, where it functions as a buffer and helps maintain the shape of the tissue. It is also found in large amounts in joint tissues such as synovial fluid and cartilage, where it functions as a lubricant and buffer. For this reason, hyaluronic acid is often used as an ingredient in pharmaceuticals and cosmetics.
Commercially available products of hyaluronic acid include Hyalo-Oligo (Kewpie Corporation: low molecular weight hyaluronic acid), Bio-Hyalo 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 the extracellular matrix, and its polypeptides form dimers.
Commercially available fibronectin products include a human plasma-derived fibronectin solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and a bovine plasma-derived fibronectin solution (Sigma-Aldrich Co., Ltd.).

These extracellular matrices may be used alone or in combination. A commercially available product that can be used in combination is Matrigel (manufactured by Corning: a mixture of laminin, IV collagen, heparin sulfate proteoglycan, entactin/nidogen, and growth factors).

[Synthetic substrate]
Examples of synthetic substrates include oligopeptide-supported polymers, polylysine, and heparin-mimicking hyaluronic acid. The oligopeptide-supported polymer is a substrate in which an oligopeptide having an arginine-glycine-aspartic acid (RGD) sequence is covalently bonded to a polymer. Commercially available products include the Synthemax series (manufactured by Corning). Polylysine is a substrate in which an essential amino acid lysine molecule is bound by a peptide bond. Commercially available products include ε-poly-L-lysine coating solution (manufactured by Cosmo Bio). Heparin-mimicking hyaluronic acid is a substrate in which the hydroxyl groups of hyaluronic acid are replaced with sulfonic acid. Commercially available products include sulfated hyaluronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.).

The content of the cell adhesive 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, and preferably 0.01% by mass or more and 8% by mass or less. By making the content of the cell adhesive material (A) 0.01% by mass or more, the effect of promoting cell adhesion due to the structure can be exerted. By making the content of the cell adhesive material (A) 10% by mass or less, ejection from the inkjet head becomes good and a highly precise pattern can be formed.

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

[Surface tension adjuster (B)]
The ink-jet ink for cell culture (I) of the present invention preferably contains a surface tension adjuster (B), which makes it possible to control the ejection properties from an ink-jet head and the wettability to a substrate.
Examples of the surface tension adjuster (B) include surfactants, antifoaming agents, wetting agents, and wetting adjusters. From the viewpoint of cytotoxicity, those listed in the "Pharmaceutical Additives Standards 2018" issued by the Ministry of Health, Labor and Welfare are preferred.

Surfactants include sucrose fatty acid esters, cetanol, cholesterol, alkylaryl polyether alcohols, sodium N-cocoyl-N-methylaminoethyl sulfonate, lauryl pyrrolindone, etc. Antifoaming agents include ethanol, glycerin fatty acid esters, polyoxyl stearate, etc. Wetting agents include isobutyl adipate, allantoin, magnesium chloride, sodium dibutyryl acetate, urea, glycerin, dioctyl sodium sulfosuccinate, etc. Wetting regulators include olive oil, sodium hydroxide, lactic acid, potassium hydroxide, etc.

Among these, it is preferable to contain sucrose fatty acid esters. Examples of sucrose fatty acid esters include the Ryoto Sugar Ester series (manufactured by Mitsubishi Chemical Foods Corporation) and the DK Ester series (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.).
The content of the surface tension adjuster (B) in 100% by mass of the ink-jet ink for cell culture (I) of the present invention is preferably 0.001% by mass or more and 10% by mass or less, and more preferably 0.01% by mass or more and 1.0% by mass or less.
By making the content of the surface tension modifier (B) 0.001% by mass or more, it is possible to exert the effect of controlling the ejection property from an inkjet head and the wettability to a substrate, and by making the content of the surface tension modifier (B) 10% by mass or less, it is possible to ensure high biocompatibility.

[Solvent (C)]
The ink-jet ink for cell culture (I) of the present invention preferably contains a solvent (C). By containing the solvent (C), the viscosity of the ink-jet head and the wettability to the substrate can be suitably controlled.
As the solvent (C), it is preferable to use a water-soluble solvent so as not to dissolve the substrate, and glycol ethers and diols are good, among which (poly)alkylene glycol monoalkyl ethers, (poly)alkylene glycol dialkyl ethers, and alkane diols having 3 to 6 carbon atoms are effective. These can be dried quickly during inkjet printing, and can realize accurate printing. In addition, because of their high boiling points, they also function as moisturizers. 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, 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 monomethyl ether, tripropylene glycol monomethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, and dipropylene glycol dimethyl ether. Specific examples of diols include 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,2-pentanediol, 1,5-pentanediol, 1,2-hexanediol, 1,6-hexanediol, and 2-methyl-2,4-pentanediol. Among these, 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 are highly effective. These solvents may be used alone, or multiple 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 ink-jet ink for cell culture (I) 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 an effect of controlling the ejection property from an inkjet head and the wettability to a substrate.

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

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

<Cell culture materials>
The cell culture member of the present invention has a cell adhesion pattern on the culture surface, which makes it possible to control cell adhesiveness and further reproduce the cell orientation in vivo.
The cell culture member of the present invention can be divided into the following four types depending on the method for producing the pattern.
The method for producing the cell culture component of the present invention is not particularly limited, but from the viewpoint of printing accuracy of the pattern, a method of inkjet printing the inkjet ink for cell culture (I) to form a culture layer, or a method of inkjet printing the inkjet ink for cell culture (I) and the non-cell-adhesive composition (D) to form a culture layer is preferred.

[First form]
First, the cell culture member in the first embodiment has a cell adhesion pattern layer formed on a substrate by the ink-jet ink for cell culture (I).
An example of a method for producing a cell culture member in the first embodiment is a method in which the inkjet ink for cell culture (I) is filled into an inkjet carriage, the inkjet ink for cell culture (I) is ejected from an inkjet head, and the inkjet ink for cell culture (I) is inkjet printed onto a substrate to form a culture layer.

[Second Form]
In addition, the cell culture member in the second embodiment of the present invention is formed by laminating, in this order, a non-cell adhesive layer and a cell adhesive pattern layer formed by the ink-jet ink for cell culture (I) on a substrate.
The cell non-adhesive layer is preferably a layer coated with a cell non-adhesive composition (D). The cell non-adhesive composition (D) is not particularly limited as long as it has cell non-adhesive properties, but is preferably in the form of an ink. It also preferably contains a cell non-adhesive resin (D1). The cell non-adhesive resin (D1) is not particularly limited as long as it has cell non-adhesive properties, 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 is a block polymer having an acrylic polymer portion and a polyethylene oxide portion formed from a monomer having an average water/1-octanol partition coefficient (cLogPow) of 0 or more and 2 or less, and commercially available products such as the Pluronic series (manufactured by BASF Corporation). Examples of polymers having a betaine structure include the Lipidure series (manufactured by NOF Corporation) and the RAM resin series (manufactured by Osaka Organic Chemical Industry Co., Ltd.).
The content of the polyethylene oxide structure in the non-cell-adhesive resin (D1) is preferably 0.1% by mass to 20% by mass, more preferably 1.0% by mass to 10% by mass. In this range, when culturing myoblasts, the cells are less likely to detach from the surface of the cell culture member, and it is easy to make the cells confluent. In addition to containing the non-cell-adhesive resin (D1), the non-cell-adhesive composition (D) preferably contains a solvent. Furthermore, the content of the non-cell-adhesive resin (D1) is preferably 0.1 to 50% by mass, more preferably 0.5 to 3% by mass, in 100% by mass of the non-cell-adhesive composition (D). In this range, it is easy to adjust the average surface roughness Ra of the surface of the cell culture member to a suitable range.
The non-cell-adhesive composition (D) may contain a crosslinking agent. This makes it easier to culture myoblasts and makes the cells less likely to detach from the surface of the cell culture substrate, making it easier to achieve confluence. When the crosslinking agent is contained, the non-cell-adhesive resin (D1) preferably has a functional group that serves as a crosslinking point, such as a carboxyl group, a hydroxyl group, an epoxy group, an amino group, or an isocyanate group.

The method of coating the cell non-adhesive composition (D) on the substrate is not particularly limited. The method of coating the cell non-adhesive composition (D) on the substrate is preferably a spin coating method from the viewpoint of ease of coating and adjustment of the average surface roughness Ra, but the solution can also be applied to the substrate by various coating methods such as brushing, immersion, roller, spraying, injection, and coating machine. The thickness of the coating film after the solvent is removed after coating is not limited. The solvent is not particularly limited as long as it is capable of forming a surface layer by coating and does not corrode the substrate, and examples of the solvent include water, methanol, ethanol, acetone, methyl ethyl ketone (hereinafter referred to as MEK), acetonitrile, dimethylformamide, and the like. In addition, the solvents described in the solvent (C) can also be used.

In the second embodiment, the method for producing the cell culture member includes, for example, coating a substrate with a non-cell-adhesive composition (D) to form a non-cell-adhesive layer, and then inkjet printing an inkjet ink for cell culture (I) onto the non-cell-adhesive layer to form a culture layer.

[Third Form]
Furthermore, the cell culture member in the third embodiment of the present invention is formed by laminating, in this order, a cell adhesive layer formed from the ink-jet ink for cell culture (I) and a cell non-adhesive pattern layer on a substrate.
The cell non-adhesive pattern layer is preferably formed by inkjet printing the cell non-adhesive composition (D) on the cell adhesive layer. 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. In addition, the cell non-adhesive composition (D) preferably contains a surface tension adjuster (B) and a solvent (C) like the inkjet ink for cell culture (I). By containing the surface tension adjuster (B) and the solvent (C), the ejection property from the inkjet head and the wettability to the substrate can be controlled.

In the third embodiment, a method for producing a cell culture member includes inkjet printing a cell culture inkjet ink (I) onto a substrate to form a cell adhesive layer, and then inkjet printing a non-cell adhesive composition (D) onto the cell adhesive layer to form a culture layer.

[Fourth Form]
A cell culture member according to a fourth embodiment of the present invention has a cell adhesive pattern layer and a cell non-adhesive pattern layer formed on a substrate using the ink-jet ink for cell culture (I).

The cell non-adhesive pattern layer is the same as that shown in the third embodiment, and the properties of the cell non-adhesive composition (D) such as composition, viscosity, surface tension, content of the cell non-adhesive resin (D1), etc. are also the same, which allows the ejection property of the cell non-adhesive composition (D) from an inkjet head and the wettability of the cell non-adhesive composition (D) to a substrate to be controlled.
In the fourth embodiment, a method for producing a cell culture member includes, for example, filling different inkjet carriages with the inkjet ink for cell culture (I) and the non-cell-adhesive composition (D), ejecting the inkjet ink for cell culture (I) and the non-cell-adhesive composition (D) from inkjet heads, and inkjet printing the ink onto a substrate to form a culture layer.

By simultaneously inkjet printing the cell culture inkjet ink (I) and the cell non-adhesive composition (D) to form a pattern, it becomes easy to adjust the average surface roughness Ra of the surface of the cell culture component to a suitable range, and it becomes possible to more precisely separate the cell adhesive and non-adhesive regions, thereby reproducing the cell orientation in a living body. This is therefore a preferred form of cell culture component in the present invention.

[Inkjet printing]
The inkjet printing in the present invention may be a single-pass method in which the inkjet ink for cell culture (I) and/or the non-cell-adhesive composition (D) is ejected onto a substrate only once for printing, or a serial type method in which a short shuttle head is reciprocally scanned in a direction perpendicular to the transport direction of the recording medium to record within the maximum recording width of the recording medium.

[Inkjet printing device]
The inkjet printing device must be equipped with an inkjet head for ejecting the inkjet ink for cell culture (I) or the non-cell-adhesive composition (D), and a drying process for drying what is ejected from the inkjet head. When the ink is ejected from the inkjet head, the ejected inkjet ink for cell culture (I) or the non-cell-adhesive composition (D) lands on the substrate to form a pattern, and the pattern is transported into a drying device as the substrate is transported, where it is dried.

[Inkjet method]
The inkjet method is not particularly limited, and may be any of known methods, such as a charge control method that uses electrostatic attraction to eject ink, a drop-on-demand method (pressure pulse method) that uses the vibration pressure of a piezoelectric element, an acoustic inkjet method that converts an electric signal into an acoustic beam and irradiates the ink to eject ink using radiation pressure, and a thermal inkjet (Bubble Jet (registered trademark)) method that heats the ink to form bubbles and uses the resulting pressure. In addition, the inkjet head used in the inkjet method may be an on-demand type or a continuous type. Furthermore, specific examples of the ejection method include an electromechanical conversion method (e.g., single cavity type, double cavity type, bender type, piston type, share mode type, shared wall type, etc.), an electrothermal conversion method (e.g., thermal inkjet type, bubble jet (registered trademark) type, etc.), an electrostatic suction method (e.g., electrolytic control type, slit jet type, etc.), and a discharge method (e.g., spark jet type, etc.), but any ejection method may be used. The ink nozzles and the like used when recording by the ink-jet method are not particularly limited, and can be appropriately selected depending on the purpose.
The shape of the pattern formed by the inkjet method is not particularly limited and can be appropriately selected depending on the purpose. Specifically, stripes, dots, triangles, etc. can be given as specific examples, but any pattern may be used. In this report, stripes are specifically preferred in order to evaluate the orientation during cell culture. The line width of the stripe having an adhesive portion and a non-adhesive portion is not particularly limited and can be appropriately selected depending on the purpose, but the adhesive portion is preferably 30 μm or more and 120 μm or less, and more preferably 50 μm or more and 100 μm or less. 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.

[Substrate]
The substrate of the present invention is not particularly limited as long as it can be used for cell culture. Specifically, the substrate may be one or more resins selected from the group consisting of (meth)acrylic resins, styrene resins, polycarbonate resins, polyolefin resins, polyvinyl chloride, polyester, polyvinyl acetate, vinyl-acetate copolymers, styrene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, acrylonitrile-butadiene-styrene copolymers, nylon, polymethylpentene, silicone resins, amino resins, polysulfones, polyethersulfones, polyetherimides, fluororesins, and polyimide resin materials, as well as dust-free paper, glass substrates, porous ceramic substrates, cellulose substrates, and the like. The substrate may be in the form of a three-dimensional molded body, or a sheet, plate, or dish shape. Examples of sheet-shaped substrates include films and papers. Examples of plate-shaped substrates include flat-bottom plates with 6 holes, 12 holes, 24 holes, 96 holes, and the like. Examples of dish-shaped substrates include dishes with diameters of 35 mm, 60 mm, 90 mm, 100 mm, and the like.

The substrate is preferably a silicone substrate, a porous ceramic substrate, a cellulose substrate, etc., from the viewpoint of oxygen permeability. The silicone substrate, the porous ceramic substrate, the cellulose substrate, etc. are not particularly limited as long as they can be used for cell culture. A silicone substrate is preferable, specifically a substrate containing a silicone-based resin, and examples of the silicone-based resin include those having a polyorganosiloxane main chain such as polydimethylsiloxane, polyphenylmethylsiloxane, and polydiphenylsiloxane. Polydimethylsiloxane is preferable. Specific examples of such silicone substrates include silicone-containing medical devices, silicone-containing culture vessels, and silicone substrates having microchannels, preferably silicone-containing microchannel devices.

[Oxygen permeability coefficient]
The cell culture component of the present invention has an oxygen permeability (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 preferably 300 Barrer or more.

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

The present invention will be explained in more detail with reference to the following examples, but the following examples are not intended to limit the scope of the invention in any way.

<Preparation of cell adhesion control inkjet ink>
[Production Example 1]
Preparation of cell adhesive material (A-1) The solvent of Cell Campus AQ-03A (collagen solution manufactured by Taki Chemical Industry, active ingredient: 0.3%) was completely evaporated using a vacuum pump to obtain cell adhesive material (A-1).
[Production Example 2]
Preparation of cell adhesive material (A-2) The solvent of a mouse EHS sarcoma-derived laminin solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., active ingredient: 0.05%) was completely evaporated using a vacuum pump to obtain a cell adhesive material (A-2).
[Production Example 3]
Preparation of cell adhesive material (A-3) The solvent of a human plasma-derived fibronectin solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., active ingredient: 0.05%) was completely evaporated using a vacuum pump to obtain a cell adhesive material (A-3).
[Production Example 4]
Preparation of Cell Adhesive Material (A-4) The solvent of Matrigel (Matrigel solution manufactured by Corning, active ingredient: 0.10%) was completely evaporated using a vacuum pump to obtain cell adhesive material (A-4).
[Production Example 5]
Preparation of Cell Adhesive Material (A-5) The solvent of Synthemax (Synthemax solution manufactured by Corning Incorporated, active ingredient: 0.10%) was completely evaporated using a vacuum pump to obtain cell adhesive material (A-5).

[Production Example 6]
Preparation of Surface Tension Adjuster (B1) The solvent of an aqueous solution of sucrose myristate ester "Ryoto Sugar Ester M1695" (manufactured by Mitsubishi Chemical Foods Corporation, active ingredient: 99%) was completely evaporated using a vacuum pump to obtain surface tension adjuster (B1).
[Production Example 7]
Preparation of Surface Tension Adjuster (B2) The solvent of an aqueous solution of sucrose laurate "Ryoto Sugar Ester L1695" (manufactured by Mitsubishi Chemical Foods Corporation, active ingredient: 99%) was completely evaporated using a vacuum pump to obtain surface tension adjuster (B2).
[Production Example 8]
Preparation of Surface Tension Adjuster (B3) The solvent of an aqueous solution of sucrose stearate "Ryoto Sugar Ester S1670" (manufactured by Mitsubishi Chemical Foods Corporation, active ingredient: 99%) was completely evaporated using a vacuum pump to obtain surface tension adjuster (B3).

[Production Example 9]
Preparation of solvent (C1):
A 5 mmol/L aqueous solution of acetic acid and 1,2-propanediol were mixed in a weight ratio of 2:1 to obtain a solvent (C1).
[Production Example 10]
Preparation of solvent (C2):
A 5 mmol/L aqueous solution of acetic acid and 1,2-hexanediol were mixed in a weight ratio of 2:1 to obtain a solvent (C2).
[Production Example 11]
Preparation of solvent (C3):
A 5 mmol/L aqueous solution of acetic acid and diethylene glycol diethyl ether were mixed in 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 adhesive material (A-1), 0.01 parts by mass of surface tension adjuster (B1), and 99.81 parts by mass of solvent (C1) were stirred with a shaker until sufficiently homogenous. After that, the mixture was filtered with a membrane filter having an opening of 1.0 μm to remove coarse particles that may cause head clogging, and inkjet ink (I-1) was prepared.

[Examples 2 to 5], [Comparative Examples 1 to 3]
Preparation of Inkjet Inks for Cell Culture (I-2 to 8) Inkjet inks for cell culture (I-2 to 8) were prepared in the same manner as in Example 1, except that the viscosity and surface tension were adjusted by changing the compounding ratio to be as shown in Table 1.

[Production Example 12]
Preparation of non-cell-adhesive resin (D1-1) The solvent of the resin solution having a polyethylene oxide structure obtained by the method described in Example 2 of JP 2017-104027 was completely evaporated using a vacuum pump to obtain a non-cell-adhesive resin (D1-1).
[Production Example 13]
Preparation of Non-Cell-Adhesive Resin (D1-2) The solvent in an aqueous solution of Pluronic F-127 (active ingredient 10%) manufactured by BASF was completely evaporated using a vacuum pump to obtain a non-cell-adhesive resin (D1-2).
[Production Example 14]
Preparation of non-cell-adhesive resin (D1-3) The solvent in RAM Resin-1000 solution (active ingredient 30%) manufactured by Osaka Organic Chemical Industry Co., Ltd. was completely evaporated using a vacuum pump to obtain non-cell-adhesive resin (D1-3).
[Production Example 15]
Preparation of non-cell-adhesive resin (D1-4) The solvent of Lipidure-PMB (active ingredient 5%) manufactured by NOF Corporation was completely evaporated using a vacuum pump to obtain non-cell-adhesive resin (D1-4).

[Production Example 16]
According to the method described in Example 8 of JP 2016-112396 A, the polymerization initiator was changed to 5.6 parts by weight of VPE0201 (macroazo initiator manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a resin solution containing 10% by mass of polyethylene oxide structure. The monomer composition of 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 non-cell-adhesive resin (D1-5).

[Production Example 17]
A resin solution containing 20% by mass of a polyethylene oxide structure was obtained by changing the blending amount of VPE0201 according to the method of Production Example 16. Next, the solvent of the resin solution was completely evaporated using a vacuum pump to obtain a non-cell-adhesive resin (D1-6).

[Production Example 18]
A resin solution containing 0.1% by mass of a polyethylene oxide structure was obtained by changing the amount of VPE0201 blended according to the method of Production Example 16. The solvent in the resin solution was then completely evaporated using a vacuum pump to obtain a non-cell-adhesive resin (D1-7).

[Production Example 19]
A resin solution containing 1.0 mass% of a polyethylene oxide structure was obtained by changing the blending amount of VPE0201 according to the method of Production Example 16. Next, the solvent of the resin solution was completely evaporated using a vacuum pump to obtain a non-cell-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 parts by mass of surface tension adjuster (B1), and 83.99 parts by mass of solvent (C1) were stirred with a shaker until sufficiently homogenous. Then, the mixture was filtered with a membrane filter having an opening of 1.0 μm to remove coarse particles that may cause head clogging, and cell non-adhesive composition (D-1) was prepared.

[Production Examples 21 to 24]
Preparation of non-cell-adhesive compositions (D-2 to 5) Non-cell-adhesive compositions (D-2 to 5) were prepared in the same manner as in Production Example 20, except that the non-cell-adhesive resins shown in Table 1 were used instead of the non-cell-adhesive resin (D1-1) and the viscosity and surface tension were adjusted by changing the compounding ratio to that shown in Table 2.
[Production Examples 25 to 28]
Preparation of non-cell-adhesive compositions (D-6 to 9) The non-cell-adhesive resins obtained in Production Examples 16 to 19 were replaced with the non-cell-adhesive resin (D1-1), and V02 (Nisshinbo, aqueous carbodiimide group-containing compound) was used as a crosslinking agent. The resins were mixed in a mass ratio of 100:13.5 (solid content), and the blending ratio was changed to that shown in Table 2 to adjust the viscosity and surface tension. The non-cell-adhesive compositions (D-6 to 9) were prepared in the same manner as in Production Example 20.

<Rating - 1>
[Surface tension of (I) and (D)]
The surface tension was measured by checking the surface tension when a platinum plate was wetted with the ink-jet inks for cell culture (I-1 to 8) and the non-cell-adhesive compositions (D-1 to 9) in an environment of 25° C. using an automatic surface tensiometer CBVP-Z manufactured by Kyowa Interface Science Co., Ltd. The results are shown in Tables 1 and 2.

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

[Evaluation of ejection properties of (I) and (D)]
The prepared inkjet ink for cell culture (I) and the cell non-adhesive composition (D) were filled into carriage A of a patterning jet (an inkjet evaluation machine manufactured by Tritec), and the flight observation immediately after filling was performed using the ejection observation function of the patterning jet to verify whether the ink could fly normally after being ejected from the nozzle. The results of the evaluation using the following indexes are shown in Tables 1 and 2.
: The particles are discharged regularly while maintaining their spherical shape.
◯: The product was discharged with a trail when dropped. The dischargeability was somewhat good.
Δ: Immediately after ejection, the ink was ejected in two or more divided droplets. It is now possible to use the ink.
×: Irregular ejection. The ejection properties are poor.

[Evaluation of cytotoxicity of (I) and (D)]
In accordance with the cytotoxicity criteria set forth in the guideline issued by the Ministry of Health, Labor and Welfare, "Basic principles of biological safety assessment required for applications for manufacturing and sales approval of medical devices" (Yakushokuki-Hatsu 0301 No. 20), samples (Y1 to 17) listed in Tables 1 and 2 were prepared using inkjet inks for cell culture or non-cell-adhesive compositions in the order of 1 to 7 below, and cytotoxicity was evaluated. The evaluation results, graded according to the value of the colony formation rate, are shown in Tables 1 and 2.

1. Preparation of samples (Y1-17) and negative/positive controls:
(Y1 to 17): ² mL of the inkjet ink for cell culture (I-1 to 8) and the non-cell adhesive composition (D-1 to 9) were added to an untreated polyimide film (Kapton 200EN, manufactured by DuPont-Toray Co., Ltd.) with an area of 30 cm2, and spin-coated at 3000 rpm for 30 seconds. The film was dried at room temperature for 24 hours to prepare samples (Y1 to 17) whose inner surface was coated with the coating agent.
Negative/positive controls: A high-density polyethylene sheet (provided by the Food and Drug Safety Center, Hadano Research Institute) was used as the negative control material. A polyurethane film containing 0.1% ZDEC was used as the positive control material A, and a polyurethane film containing 0.25% ZDBC was used as the positive control material B.
For the blank, a 24-well flat-bottom multiple well plate 3526 (manufactured by Falcon) was used.

2. Preparation of extract:
Samples (Y1-17), negative and positive control materials A and B were adjusted to have a surface area of 15 ^cm2 on one side and placed in a 15 mL centrifuge tube. 5 mL of MO5 medium (E-MEM medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS; manufactured by BioWest) at an external ratio of 5% by mass) was added, and the tubes were stored in a 37°C, 5% _CO2 incubator for 24 hours.

3. Cell seeding:
Cryopreserved V-79 cells (fibroblasts derived from Chinese hamster lung) were thawed at 37°C, transferred to MEM10 medium (E-MEM medium containing 10% by mass of FBS) and diluted in a 50 mL centrifuge tube to 100 cells/mL. 0.5 mL of each was seeded in a 24-well flat-bottom 3526 multiple well plate (Falcon) and stored in a 37°C, 5% _CO2 incubator for 24 hours.

4. Cell culture:
Five centrifuge tubes were prepared for each extract, and extracts of different concentrations were prepared by diluting them with MO5 medium to 50%, 25%, 12.5%, 6.25%, and 3.13%. Next, the MEM10 medium in the multiple well plate was aspirated with an aspirator, and 0.5 mL of the extracts of different concentrations was added. For the blank, the MEM10 medium was aspirated with an aspirator, and 0.5 mL of MO5 medium was added, and the blank was stored in a 37°C, 5% _CO2 incubator for 7 days.

5. Fixation:
After 7 days of incubation, the MO5 medium in the multiple well plate was aspirated using an aspirator. 0.5 mL of 10 nM mild formaldehyde (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well, and after 1 minute, the medium was aspirated using an aspirator.

6. Dyeing:
A staining reagent was prepared by mixing Giemsa staining solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and phosphate buffered saline (PBS) at a mass ratio of 1:9. 0.5 mL of the staining reagent was added to each well, left to stand for about 30 minutes, and then aspirated with an aspirator. The wells were washed with 0.5 mL of PBS and air-dried.

7. Colony count:
The colonies formed were counted. The blank colony count was used as the control group, and the average of three times was set as 100%. The colony counts obtained with the samples (Y1-17) and the negative and positive control extracts were averaged for each concentration, and the percentage _IC50 relative to the colony count in the control group was calculated.
-Conditions for test validity: This experiment was determined to be valid based on the following A to C.
A: The colony formation rate in the control group was good.
B: The colony formation rate in the negative control material was equivalent to that of the control group.
C: The IC ₅₀ of positive control materials A and B was less than 7% and less than 80%, respectively.
_IC50 is the extract concentration at which the colony formation rate is 50%.
-Grading criteria based on colony formation rate:
○: IC ₅₀ >30% Cytotoxicity is negative.
×: 30% or more _IC50 cytotoxicity is positive.
The evaluation results are shown in Tables 1 and 2.

<Results>
The inkjet inks for cell culture (I-1 to I-5) of Examples 1 to 5 shown in Table 1 had good dischargeability due to the relationship between viscosity and surface tension. In addition, the negative cytotoxicity indicated that they had no adverse effect on cells. Furthermore, the non-cell-adhesive compositions (D-1 to D-9) shown in Table 2 also had good dischargeability due to the relationship between viscosity and surface tension, and the negative cytotoxicity indicated that they had no adverse effect on cells.
On the other hand, the inkjet inks for cell culture (I-6, 7) of [Comparative Examples 1 and 2] had too high or too low surface tension, so the inkjet ink was irregularly discharged from the nozzle, making it impossible to use as an ink. Also, the inkjet ink for cell culture (I-8) of [Comparative Example 3] had too high a viscosity, so the inkjet ink was discharged in two or more droplets immediately after discharge.

<Preparation of cell culture materials (polystyrene substrate)>
[Example 6]
Preparation of cell culture component (S-1) The inkjet ink (I-1) of Example 1 was filled into the carriage of a LaboJet-500 inkjet evaluation machine manufactured by Microjet Corporation, and printed on the inner surface of an untreated dish (polystyrene manufactured by Corning) with a diameter of 35 mm at a designed interval with a line width of 50 μm to obtain a cell culture component (S-1).

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

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

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

[Example 13]
Preparation of cell culture member (S-8) The carriage of a Microjet LaboJet-500 inkjet evaluation machine was filled with the cell culture inkjet ink (I-1), printed on the inner surface of a 35 mm diameter untreated dish (polystyrene manufactured by Corning) at a designed interval of a line width of 0 μm, and then dried at room temperature for 24 hours. Thereafter, the carriage of a Microjet LaboJet-500 inkjet evaluation machine was filled with a cell non-adhesive composition, printed on the cell adhesive layer at a designed 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 components (S-9 to 12) Cell culture components (S-9 to 12) were prepared in the same manner as (S-8) except that the materials shown in Table 3 were used.

[Example 18]
Preparation of cell culture component (S-13) The cell culture inkjet ink (I-2) was filled into carriage A of a BioSpot inkjet evaluation machine manufactured by Microjet Corporation, and the cell non-adhesive composition (D-1) was filled into carriage B. At the same time, they were printed on the inner surface of a 35 mm diameter untreated dish (polystyrene manufactured by Corning) at a designed interval with a line width of 50 μm, to obtain a cell culture component (S-13).

[Examples 19 to 22]
Preparation of cell culture components (S-14 to 17) Cell culture components (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 materials (S-18)
An untreated dish (polystyrene, Corning) with a diameter of 35 mm was prepared.

[Comparative Example 6]
Preparation of cell culture member (S-20) The inkjet ink for cell culture (I-8) was spin-coated at 3,000 rpm for 30 seconds onto an untreated dish (polystyrene manufactured by Corning) with a diameter of 35 mm, and then dried at room temperature for 24 hours to prepare a cell culture member (S-20).

<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 according to JIS B 0601. The average surface roughness Ra represents the average value of absolute deviations from the mean line. The evaluation results are shown in Table 3.
[Criteria]
◯: Average surface roughness ≦ 0.5 μm Smooth surface △: 0.5 μm < average surface roughness ≦ 1.0 μm Poor smoothness, but usable ×: 1.0 μm < average surface roughness Unusable because cell movement is restricted by unevenness

[Pattern of cell culture member: Spacing between width (L) and width (S)]
Patterns of cell culture components (S-1, 2, 19):
The width L (μm) of the cell adhesion pattern formed by (I) and the width S (μm) of the substrate were photographed using a 10x transmission optical microscope and evaluated by measuring the widths L and S.
Patterns of cell culture components (S-3 to 17):
The width L (μm) of the cell adhesion pattern formed by (I) and the width S (μm) of the cell non-adhesion pattern formed by (D) were photographed using a 10x transmission optical microscope and evaluated by measuring the widths L and S.
The closer the width L (μm) and the width S (μm) are to the designed width of 50 μm, the higher the printing accuracy. The evaluation results are shown in Table 3.

[Cell Pattern Formation (Cell Orientation Morphology)]
A mouse fibroblast cell line (NIH/3T3 cells) was added to 10% FBS-DMEM medium to prepare a cell suspension with a concentration of 80,000 cells/ml of NIH/3T3. 2 mL of the obtained cell suspension was seeded on the above cell culture components (S1 to 20). After that, the cells were cultured in a 5% CO ₂ /37°C incubator until the 7th day.
To confirm whether the cells were oriented, the cell culture conditions immediately after seeding and 7 days later were photographed using a 4x transmission optical microscope, and the photographs were compared with the cell morphology immediately after seeding to evaluate the orientation according to the following criteria. The results are shown in Table 3.

FIG. 5 is a set of transmission optical microscope photographs showing the cell orientation morphology observed immediately after seeding and after 7 days of cell culture in the cell culture components prepared in the examples of the present invention.
In FIG. 5, each number panel has the following characteristics:
1: Immediately after the cells are seeded, the cells are observed to be randomly dispersed on the cell culture substrate.
2: After 7 days of cell culture, it is observed that the cells adhere, spread, and are oriented along the adhesive lines. Cell orientation is excellent.
3: After 7 days of cell culture, cells are observed to adhere and spread on the adhesive lines, but are irregularly oriented. Poor cell orientation.
4: After 7 days of cell culture, it is observed that the cells adhere and spread unevenly and are not oriented. The cell orientation is poor.

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 the seventh day of cell culture, it was observed that the cells adhered, spread, and were oriented along thin lines in the field of view, whereas in [Comparative Example 4] the cells did not adhere and formed uneven clumps, and no orientation was observed. In [Comparative Example 5], the viscosity of the inkjet ink for cell culture (I-8) was high, so the printing accuracy of the design pattern was low, and as a result, some parts were outside the pattern, arranged irregularly, and had no orientation. In [Comparative Example 6], it was observed that the cells adhered unevenly in the field of view and were not oriented.

<Preparation of culture member (silicone substrate)>
[Example 23]
Preparation of cell culture member (S-21) The inkjet ink (I-1) of Example 1 was filled into the carriage of a LaboJet-500 inkjet evaluation machine manufactured by Microjet Corporation, and printed on the inner surface of one well of an untreated silicone 6-well plate (VECELL (registered trademark) manufactured by Vecell Corporation) with a line width of 50 μm at a designed interval to obtain a cell culture member (S-21).

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

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

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

[Example 28]
Preparation of cell culture component (S-26) The carriage of a BioSpot inkjet evaluation machine manufactured by Microjet was filled with the inkjet ink for cell culture (I-1), and printed on the inner surface of one well of an untreated silicone 6-well plate (VECELL (registered trademark) manufactured by Vecell) with a line width of 0 μm at a designed interval, and then dried at room temperature for 24 hours. Thereafter, the carriage of a LaboJet-500 inkjet evaluation machine manufactured by Microjet was filled with the composition (D-1) having non-adhesive properties for cells, and printed on the cell adhesion layer with a line width of 50 μm at a designed interval, to obtain a cell culture component (S-26).

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

[Example 31]
Preparation of cell culture member (S-29) The cell culture inkjet ink (I-4) was filled into carriage A of a BioSpot inkjet evaluation machine manufactured by Microjet Corporation, and the cell non-adhesive composition (D-2) was filled into carriage B. Both were printed simultaneously on the inner surface of one well of an untreated silicone 6-well plate (VECELL (registered trademark) manufactured by Vecell Corporation) with a line width of 50 μm at a designed interval to obtain a cell culture member (S-29).

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

[Comparative Example 7]
・Cell culture materials (S-32)
Untreated dishes (polystyrene, Corning) with a diameter of 35 mm were 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 (registered trademark) manufactured by Vecell Corporation) was used.

[Comparative Example 9]
Preparation of cell culture member (S-34) The inkjet ink for cell culture (I-1) was spin-coated on an untreated dish (polystyrene manufactured by Corning) with a diameter of 35 mm at 3000 rpm for 30 seconds, and dried at room temperature for 24 hours to prepare a cell culture member (S-34).

<Rating - 3>
[Average surface roughness Ra]
Evaluation was performed according to the method described in 2.
[Pattern of cell culture member: Spacing between width (L) and width (S)]
Evaluation was performed according to the method described in 2.

[Oxygen permeability coefficient (Dk)]
The oxygen permeability coefficient (Dk) of the cell culture components (S-21 to S-34) was measured by the method specified in ISO18369-4 (FATT method) using a flat electrode cell of an oxygen permeability measuring device (201T, Rehder) (1 Barrer = 1 x ^10-11 _O2 (STP) cm/ ^cm2 mmHg). The evaluation results are shown in Table 4.
[Criteria]
◎: Dk≧300 Barrer (very good)
○: 300>Dk≧200 (good)
△: 200>Dk≧20 (slightly poor)
×: 20>Dk (defective)

[Cell Activity]
After the cell culture components (S-21 to 34) were sterilized with ethylene oxide gas, Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (hereinafter referred to as "10% FBS-DMEM medium") was used as the culture medium, and 1.5 x ¹⁰⁵ human liver cell lines (Huh7 cells) were seeded per well and cultured in a 5% _CO2 /37°C incubator until the fifth day.
The cell activity was evaluated by performing an ATP assay after 1 and 5 days. Specifically, 1 ml of ATP reagent (ATP measurement reagent for cells: manufactured by Toyo B-Net Co., Ltd.) was added to the cell culture device after the culture, pipetted five times, and allowed to stand at room temperature for 5 minutes. Then, 100 ml of ATP was added to the cell culture device after the culture.
The reagent and cell lysate were dispensed onto a separate plate and stirred for 1 minute. The luminescence intensity was measured using Mithras LB940 (manufactured by Berthold). The evaluation results are shown in Table 4.
Cytotoxicity = (luminescence amount after 5 days of culture) / (luminescence amount after 1 day of culture) x 100
〇 : 100% ≦ Cell activity △ : 20% ≦ Cell activity < 100%
×: Cell activity <20%

[Cell Pattern Formation (Cell Orientation Morphology)]
Hepatocytes prepared from rat liver (Slc/Wister provided by Sankyo Labo Services; hereafter referred to as hepatocytes) were added to 10% FBS-DMEM medium to prepare a cell suspension with a hepatocyte concentration of 8 x 10 ⁴ cells/ml. 2 mL of the obtained cell suspension was seeded on the above cell culture members (S21-34). After that, the cells were cultured in a 5% CO ₂ /37°C incubator until day 1.
To confirm whether the cells were aligned, the cell culture conditions immediately after seeding and one day later were photographed with a 4x transmission optical microscope and compared with the cell morphology immediately after seeding to evaluate the alignment according to the following criteria. The evaluation results are shown in Table 4.
6 is a schematic diagram showing the morphology of cell arrangement observed immediately after seeding and after one day of cell culture in the cell culture device prepared in the Example of the present invention. In FIG. 6, the schematic diagrams indicated by the numbers have the following characteristics.
1: Immediately after the cells are seeded, the cells are observed to be randomly dispersed on the cell culture substrate in the visual field.
2: One day after cell culture, cells were observed to adhere and align along the adhesive lines. Cell alignment was excellent.
3: After one day of cell culture, cells are observed to adhere in lines with adhesiveness, but are irregularly arranged. Cells are well arranged.
4: After one day of cell culture, it is observed that the cells are unevenly attached and not aligned. The alignment of the cells is poor.

The cell culture components (S-21 to 31) used in the examples shown in Table 4 had an average roughness Ra of 1.0 μm or less, and the width L (μm) and width S (μm) after printing were approximately 50 μm, indicating high printing accuracy. In addition, the oxygen permeability was good, and therefore the cell activity measured by the ATP assay was good. Furthermore, after one day of culture, it was observed that the cells were adhered and aligned along the fine lines in the field of view.
On the other hand, the cell culture member (S-32) used in Comparative Example 7 had low oxygen permeability and was not able to supply sufficient oxygen from the bottom surface of the member to the cell layer, which adversely affected the cell activity and cell arrangement morphology determined by the ATP assay. In addition, the cell culture member (S-33) used in Comparative Example 8 did not have a pattern layer formed by an inkjet ink for cell culture on a silicone substrate, and therefore, after the first day of cell culture, it was observed that the cells adhered non-uniformly and did not arrange, and the cell activity was low. Furthermore, the cell culture member (S-34) used in Comparative Example 9 also did not have a pattern layer formed by an inkjet ink for cell culture on a silicone substrate, and therefore, it was observed that the cells adhered non-uniformly and did not arrange.

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

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

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

[Examples 42 to 46]
Preparation of myoblast culture members (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 myoblast culture members (S-18 to 20) Cell culture members (S-18 to 20) were obtained in the same manner as in Comparative Examples 4 to 6.

<Rating - 4>
[Induction of myoblast differentiation]
The effect on differentiation into myotube cells was confirmed by observing mouse-derived myoblast cell line (C2C12 cells) by fluorescent staining of myosin heavy chain and nuclei. Note that myosin heavy chain is a muscle contraction-related protein expressed during the myotube formation process, and the expression of myosin heavy chain and multinucleation due to cell fusion are characteristics of differentiation into myotube cells. By observing by fluorescent immunostaining of myosin heavy chain and nuclear staining, it is possible to more clearly evaluate the presence or absence of differentiation into myotube cells and their orientation in a specific direction, and to observe the formation of myotube tissue.

A mouse myoblast cell line (C2C12 cells) was added to 10% FBS-DMEM medium to prepare a cell suspension with a C2C12 concentration of 50,000 cells/ml. 2 mL of the obtained cell suspension was seeded onto the cell culture member.
After the cells became confluent, the medium was replaced with a differentiation induction medium (High-glucose DMEM (D5790, Sigma) supplemented with 2% Horse Serum (Gibco) and 1% antibiotic-antimycotic mixed solution (anti-anti, Gibco)).
To confirm whether the myoblasts were aligned, the cell culture conditions 3 and 10 days after seeding were photographed using a 4x transmission optical microscope and compared with the cell morphology immediately after seeding, and judged according to the following criteria.
Fig. 7 is a schematic diagram showing the morphology of cell arrangement observed immediately after seeding and after one day of cell culture in the myoblast culture component prepared in the Example of the present invention. In Fig. 7, the panels with numbers have the following characteristics.
1: During myoblast culture, it was confirmed that the cells adhered and aligned along the adhesive lines, and it was observed that the cells were confluent on the substrate. (Good)
2: During myoblast culture, it was confirmed that the cells adhered and aligned along the adhesive lines, and it was observed that the cells were not confluent on the substrate. (Acceptable)
3: During myoblast culture, the cells adhere to the adhesive lines, but are irregularly arranged, and the cells are observed to be confluent on the substrate (poor).
4: During myoblast culture, the cells adhere to the adhesive lines, but the adhesion is weak and the cells are observed to peel off from the substrate. (Poor)

[Cytofluorescence staining]
On day 10 of culture, confluent C2C12 cells were stained with fluorescent dyes according to standard methods. Nuclei were stained with Hoechst 33342 (H3570, Thermo Fisher Scientific, USA), and myosin heavy chain was stained with anti-MYH (SC-20641, Cosmo Bio, Tokyo) and Goat Anti-Rabbit IgG H&L Alexa Fluor 488 (ab150077, Abcam). Stained cells were observed under a transmitted light microscope.
Fig. 8 is a schematic diagram showing the morphology of cell arrangement observed immediately after seeding and after one day of cell culture in the myoblast culture component prepared in the Example of the present invention. In Fig. 8, the panels with numbers have the following characteristics.
○: Myosin heavy chains are observed to be oriented along the transverse axis, mimicking myotube tissue.
×: Myosin heavy chains were observed in random directions, failing to mimic myotube tissue.
-: Cells peel off and do not form tissue, resulting in poor results.

In the myoblast culture substrate used in Examples 34 and 35 shown in Table 5, the cell adhesion sites did not detach from the substrate, and after 10 days of culture, the cells became confluent while maintaining their orientation, forming a myocardial mimetic tissue. In Examples 36 to 41, the hardener in the non-adhesive resin prevented the cells from detaching from the substrate, forming a myocardial mimetic tissue. In Examples 36 to 38, the compounding ratio of polyethylene oxide in the non-adhesive portion allowed the myoblasts to efficiently differentiate into myotubes, forming a myocardial mimetic tissue.
In Comparative Example 10, the cells did not adhere but formed non-uniform clumps, and peeled off from the substrate over time. In Comparative Example 11, because the viscosity of the inkjet ink for cell culture (I-8) was high, the cells flowed out from the adhesive coating surface and showed orientation, but in some places the cells were out of the pattern, arranged irregularly, and had no orientation, and peeled off from the substrate. In Comparative Example 12, it was observed that the cells adhered non-uniformly within the field of view and were not oriented.
Immunostaining of cells cultured in the differentiation medium shown in Figure 8. Blue indicates nuclei, and green indicates myosin heavy chain. By using the differentiation medium from day 3 to day 10 of culture, the cells were oriented in the horizontal axis direction, and cell fusion and the formation of long myotubes could be observed.

<Industrial Applicability>
The cell orientation control technology enables the creation of organ chips, which is expected to be used as a drug discovery screening method. In addition, since the technology 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 inkjet ink for cell culture (I); L (μm)
3 Width of the pattern on the substrate: S (μm)
4 Substrate 5 Culture layer 6 Width of the cell non-adhesive pattern formed by the cell non-adhesive composition (D); S (μm)

Claims (15)

An inkjet ink (I) for cell culture containing a cell adhesive material (A), a surface tension adjuster (B) and a solvent (C), the inkjet ink (I) for cell culture satisfying the following conditions (1) to ( 5 ), wherein the solvent (C) contains water and a water-soluble solvent.
(1) The content of the cell adhesive material (A) is 0.01% by mass or more and 0.18 % by mass or less based on 100% by mass of the ink-jet ink for cell culture.
(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.
(4) The content of the surface tension adjuster (B) is 0.001% by mass or more and 10% by mass or less, based on 100% by mass of the ink-jet ink for cell culture.
(5) The content of the solvent (C) is 90% by mass or more based on 100% by mass of the ink-jet ink for cell culture.
With the proviso that said inkjet ink for cell culture (I) is a composition comprising a solution comprising, in order, an amino acid chain gelatin polymer derived from a natural source of a cold adapted marine species at a concentration of 1% (w/v) to 20% (w/v), and further comprising a polymerization initiator such as a photoinitiator, wherein said amino acid chains are chemically functionalized with a chemical selected from the group consisting of methacryloyl groups or acryloyl groups to render them reactive to polymerization or crosslinking in the presence of free radicals;
And,
This excludes the case where the ink-jet ink for cell culture (I) further contains a hydrogel precursor (excluding the cell adhesive material (A)) and the cell adhesive material (A) is in the form of particles. The inkjet ink (I) for cell culture according to claim 1, wherein the cell adhesive material (A) comprises at least one material selected from the group consisting of extracellular matrices and synthetic substrates. A cell culture member having a cell adhesion pattern layer formed on a substrate using the ink-jet ink for cell culture (I) according to claim 1 or 2 . 3. A cell culture member comprising a substrate, a non-cell adhesive layer, and a cell adhesive pattern layer formed by the ink-jet ink (I) for cell culture according to claim 1 , laminated in this order on the substrate. A cell culture member comprising a substrate, a cell adhesion layer formed from the ink-jet ink (I) for cell culture according to claim 1 or 2 , and a non-cell adhesion pattern layer laminated in this order on the substrate. A cell culture member having a cell adhesive pattern layer and a cell non-adhesive pattern layer formed on a substrate using the ink-jet ink for cell culture (I) according to claim 1 or 2 . The cell culture member according to any one of claims 4 to 6 , wherein the non-cell-adhesive layer or the non-cell-adhesive patterned layer is formed from a non-cell-adhesive composition (D). 8. The cell culture member according to claim 7 , wherein the non-cell-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. 9. The cell culture member according to claim 7 or 8 , comprising 0.1 mass % or more and 50 mass % or less of the non-cell-adhesive resin (D1) relative to 100 mass % of the non-cell-adhesive composition (D). The cell culture member according to any one of claims 3 to 9 , wherein the average surface roughness Ra of the culture surface is 1.0 µm or less. 11. The cell culture member according to claim 3 , wherein the substrate is a silicone substrate and has an oxygen permeability (Dk) according to ISO18369-4 (FATT method) of 200 Barrer (1 Barrer = 1 x ^10-11 _O2 (STP)cm/ ^cm2 mmHg) or more. The cell culture member according to any one of claims 3 to 11, for reproducing cell orientation in a living body. A method for producing a cell culture member, comprising ink-jet printing the ink-jet ink for cell culture (I) according to claim 1 or 2 onto a substrate to form a culture layer. A method for producing a cell culture member, comprising inkjet printing the inkjet ink for cell culture (I) and the non-cell-adhesive composition (D) according to claim 1 or 2 onto a substrate to form a culture layer. A method for forming a cell pattern, comprising orienting cells using the cell culture member according to any one of claims 3 to 12 .