Cell culture substrate, cell culture vessel, cell culture member, method for manufacturing cell culture substrate, and method for culturing cells

Abstract

A cell culture substrate according to the present invention is provided with a diamond-like carbon film on the surface of the substrate. The ratio of oxygen atoms to carbon atoms (O / C) on the surface of the diamond-like carbon film is 0.1 or more, and the contact angle of the diamond-like carbon film and water is 58° or less.　It is possible to provide a cell culture substrate that allows cells to adhere well and has high cell proliferation, regardless of the presence or absence of serum.

Description

Cell culture substrate, cell culture vessel, cell culture component, method for manufacturing cell culture substrate, and method for culturing cells

[0001] The present invention relates to a cell culture substrate, a cell culture vessel, a cell culture member, a method for producing a cell culture substrate, and a method for culturing cells.

[0002] Cell culture, which allows the proliferation of target cells, is an important method in medical and biological research. When cell culture is performed in vitro, many cells must adhere to the surface of a culture substrate, which serves as a scaffold. Therefore, many cell culture substrates for growing cells have a surface necessary for cell adhesion and growth. To create such a surface, sufficient mechanical strength and stability are required, and polymeric materials are often used.

[0003] For cell culture substrates, a method of surface-treating molded polymeric materials to make the surface hydrophilic and enhance cell adhesion is known. Hydrophilic groups known to enhance cell growth include amino groups, amide groups, carbonyl groups, carboxyl groups, ester groups, and hydroxyl groups, and plasma treatments and the like are used to generate these groups. For example, Patent Document 1 proposes a method and apparatus for microwave plasma treatment of polymeric substrates to form a surface favorable for cell growth.

[0004] Furthermore, Patent Document 2 proposes a biopolymer scaffold for the growth of mammalian nerve cells and for the creation of a scaffold, specifically, a sheet-shaped biopolymer scaffold coated with DLC (diamond-like carbon) or a microparticle-shaped biopolymer scaffold coated with DLC.

[0005] Special table 2005-504855 publication Special table 2007-521114 publication

[0006] Demand for serum-free media is increasing for cell culture due to their safety and reproducibility. However, serum-free culture using such media requires more sophisticated adjustments to establish the culture environment than serum-containing culture.

[0007] Therefore, an object of the present invention is to provide a novel cell culture substrate that allows good cell adhesion and high cell proliferation regardless of the presence or absence of serum.

[0008] After extensive research, the inventors focused on the surface composition and physical properties of a diamond-like carbon (hereinafter, sometimes referred to as "DLC") film provided on the surface of a substrate. Culture media contain various proteins necessary for cell culture. In particular, the extracellular matrix plays an important role in cell adhesion, and extracellular matrix proteins (specifically, collagen, laminin, fibronectin, etc.) contained in the extracellular matrix are adsorbed to the substrate prior to cell adhesion, thereby serving as a scaffold for cell adhesion. The inventors succeeded in preferentially enhancing the adsorption of extracellular matrix proteins by adjusting the surface composition and physical properties of the DLC film. They discovered that a cell culture substrate with a specific composition and specific physical properties can solve the above-mentioned problems, leading to the completion of the present invention.

[0009] That is, the present invention has the following aspects. [1] A cell culture substrate having a diamond-like carbon film on the surface of a substrate, wherein the ratio of oxygen atoms to carbon atoms (O / C) on the surface of the diamond-like carbon film is 0.1 or more, and the diamond-like carbon film has a water contact angle of 58° or less. [2] The cell culture substrate according to [1] above, wherein the diamond-like carbon film has a water contact angle of 35 to 58°. [3] The diamond-like carbon film has a surface free energy d component of 20 to 40 mJ / m ２ and the h component is 17 to 31 mJ / m ２[4] The cell culture substrate according to any one of [1] to [3] above, wherein the ratio of nitrogen atoms to carbon atoms (N / C) on the surface of the diamond-like carbon film is 0.1 or more. [5] The cell culture substrate according to any one of [1] to [4] above, wherein the ratio of nitrogen atoms to carbon atoms (N / C) on the surface of the diamond-like carbon film is 0.15 or more. [6] The cell culture substrate according to [4] or [5] above, wherein, in XANES measurement of the surface of the diamond-like carbon film, there is at least one peak between 395 and 403 eV of the N K-edge. [7] The cell culture substrate according to any one of [1] to [6] above, which is used for serum-free culture. [8] The cell culture substrate according to any one of [1] to [7] above, wherein the diamond-like carbon film has been subjected to gamma-ray treatment. [9] The cell culture substrate according to any one of [1] to [8] above, wherein the diamond-like carbon film has a thickness of 0.5 to 100 nm.

[10] The cell culture substrate according to any one of [1] to [9] above, wherein the substrate has a thickness of 0.01 to 10 mm.

[11] The cell culture substrate according to any one of [1] to

[10] above, wherein the cell culture substrate provided with the diamond-like carbon film has the property of preferentially adsorbing fibronectin, and as an indicator of this property, when 2.5 μg / mL or less of fibronectin is added, the ratio of the amount of fibronectin adsorbed to the substrate in a medium containing 2 mg / mL of albumin to the amount of fibronectin adsorbed to the substrate in PBS buffer is 10% or more.

[12] A cell culture vessel formed from the cell culture substrate according to any one of [1] to

[11] above.

[13] The cell culture vessel according to

[12] above, wherein the cell culture vessel is a bag, and the thickness of the substrate is 0.01 to 1 mm.

[14] A cell culture component formed from the cell culture substrate according to any one of [1] to

[11] above.

[15] The cell culture component according to

[14] above, wherein the cell culture component is a microcarrier and the substrate has a diameter of 0.001 to 1 mm.

[16] A method for producing a cell culture substrate according to any one of [1] to

[11] above, characterized in that the diamond-like carbon film comprises the steps of depositing the film using an acetylene-containing gas as an introduced gas and subjecting the surface of the diamond-like carbon film to radiation treatment.

[17] A method for producing a cell culture substrate according to

[16] above, wherein the method for depositing the diamond-like carbon film is plasma CVD.

[18] A method for producing a cell culture substrate according to

[16] or

[17] above, wherein the radiation is gamma rays or electron beams.

[19] A method for producing a cell culture substrate according to any one of

[16] to

[18] above, wherein the introduced gas further contains nitrogen.

[20] A method for culturing cells using the cell culture substrate according to any one of [1] to

[11] above.

[21] A method for culturing cells according to

[20] above, wherein a serum-free medium is used.

[22] A method for culturing cells according to

[20] or

[21] above, wherein the cells are mesenchymal stem cells or iPS cells.

[23] A cell culture method according to any one of

[20] to

[22] above, comprising a step of adsorbing fibronectin to the surface of the cell culture substrate.

[24] A cell culture method using the cell culture vessel according to

[12] or

[13] above.

[25] A cell culture method using the cell culture member according to

[14] or

[15] above.

[0010] According to the present invention, it is possible to provide a cell culture substrate that allows cells to adhere well and promotes high cell proliferation, regardless of the presence or absence of serum.

[0011] 1 is a diagram showing the results of XANES measurement of the DLC film surface of the present invention. 2 is a diagram showing the results of cell counting in Examples 1 and 3, and Comparative Examples 1 and 2. 3 is a diagram showing the amount of hFN adsorption in PBS in Examples 1, 3, and Comparative Examples 1 and 2. 4 is a diagram showing the amount of hFN adsorption in PRIME-XV medium in Examples 1, 3, and Comparative Examples 1 and 2. 5 is a diagram showing the effect of hFN precoat concentration on cell adhesion in Examples 1, 3, and Comparative Examples 1 and 2.

[0012] [Cell Culture Substrate] The cell culture substrate of the present invention has a DLC film on the surface of the substrate, and is particularly suitable for use in serum-free culture, as described below. The substrate and DLC film are described in detail below.

[0013] <Substrate> The substrate constituting the cell culture substrate of the present invention is not particularly limited in material, shape, usage form, etc., as long as it can achieve the effects of the present invention, and examples thereof include resin containers, microparticles used in microcarriers, pouch-shaped bags, etc. Resins, glass, etc. can be suitably used as materials constituting the substrate, as they are less susceptible to changes caused by cell culture-related reagents, culture media, etc. It is more preferable to use resins in terms of lightness during handling, ease of molding processability, strength, etc.

[0014] As for materials for microcarriers, any material that can be processed to a size of several tens to several hundreds of micrometers and can be surface-treated with DLC can be used without any particular problems. As with the containers, resins and glass can be used, as well as metals, gels such as collagen, alginic acid, and carrageenan, cellulose, and dextran.

[0015] Examples of materials used for such substrates include polyethylene terephthalate, polyethylene naphthalate, polyethersulfone, polyimide, nylon, polystyrene (PS), polyvinyl alcohol, ethylene-vinyl alcohol copolymer, fluororesin, polyvinyl chloride, polyethylene, polypropylene, cyclic polyolefin, cellulose, dextran, alginic acid, carrageenan, acetylcellulose, polyvinylidene chloride, aramid, polyphenylene sulfide, polyurethane, thermoplastic polyurethane elastomer (TPU), polycarbonate (PC), polymethyl methacrylate (PMMA), phenolic resin, epoxy resin, polyarylate, polynorbornene, styrene-isobutylene-styrene block copolymer (SIBS), allyl diglycol carbonate, poly-4-methylpentene-1 (TPX), alicyclic acrylic resin, alicyclic polyolefin resin, and the like, but are not limited to these as long as the resin satisfies the above conditions. Among these, it is preferable to contain at least one resin selected from the group consisting of polystyrene (PS), polyvinyl alcohol, polyethylene, cellulose, polymethyl methacrylate (PMMA), polycarbonate (PC), poly-4-methylpentene-1 (TPX), alicyclic acrylic resin, and alicyclic polyolefin resin, and polyethylene or polystyrene is particularly suitable because it is non-cytotoxic, versatile, and transparent.

[0016] Examples of polystyrene include styrene homopolymers and copolymers of styrene with styrene-based monomers such as α-methylstyrene, vinyltoluene, chlorostyrene, ethylstyrene, i-propylstyrene, dimethylstyrene, and bromostyrene. Also usable are copolymers of styrene-based monomers with vinyl monomers polymerizable therewith, and copolymers of styrene-based monomers with rubber components such as butadiene. Polyethylene may be ethylene homopolymers or copolymers with α-olefins such as propylene, 1-butene, 4-methyl-1-pentene, and 1-octene, and these copolymers may be either linear or branched. Furthermore, mixed resins of ethylene homopolymers and the above α-olefins may also be used. The polyethylene can be appropriately selected from a wide range of polyethylenes, regardless of whether they are high-density or low-density.

[0017] The surface of the substrate may be subjected to a surface treatment to promote the formation of a DLC film. Examples of surface treatments include corona treatment, plasma treatment, UV irradiation treatment, etc. However, in the present invention, excessive surface treatment for cell adhesion is not necessary, and any treatment that improves the film-forming properties of the DLC film will suffice.

[0018] The thickness of the substrate is not particularly limited as long as the effects of the present invention are achieved, but from the viewpoint of the strength and handleability of the substrate, it is preferably 0.01 to 10 mm. A thickness of 0.01 mm or more provides excellent strength. On the other hand, a thickness of 10 mm or less provides light weight and easy handling. From the above viewpoints, the thickness of the substrate is more preferably in the range of 0.1 to 6 mm.

[0019] <Diamond-Like Carbon Film (DLC Film)> The cell culture substrate of the present invention has a DLC film formed on the surface of the substrate. The diamond-like carbon (DLC) film is a film made of diamond sp ３ Bonding and sp in graphite ２ It is an amorphous film that contains both bonds as a skeletal structure of carbon atoms.

[0020] (Ratio of oxygen atoms to carbon atoms, O / C (atom %)) The DLC film contains oxygen atoms, and in XPS (X-ray photoelectron spectroscopy) analysis, the ratio of oxygen atoms to carbon atoms (O / C) on the surface of the DLC film is 0.1 or more. If the O / C is less than 0.1, there is the disadvantage that cells are difficult to adhere or proliferate. For cell adhesion and proliferation, the O / C is more preferably 0.10 or more, even more preferably 0.11 or more, and particularly preferably 0.12 or more. The upper limit of the O / C is not particularly limited, but from the viewpoint of the manufacturing process and cost, it is preferably 0.30 or less, more preferably 0.25 or less, and even more preferably 0.20 or less. The ratio of oxygen atoms to carbon atoms (O / C) on the surface of the DLC film is preferably 0.1 or more and 0.30 or less, more preferably 0.11 or more and 0.25 or less, and even more preferably 0.12 or more and 0.20 or less. To incorporate oxygen atoms into the DLC film, for example, a DLC film may be formed using acetylene gas alone, followed by plasma treatment in the presence of oxygen gas or an oxygen-containing gas.

[0021] (Ratio of Nitrogen Atoms to Carbon Atoms, N / C (atom %)) Nitrogen atoms are preferably contained in the DLC film, and in XPS (X-ray photoelectron spectroscopy) analysis, the ratio of nitrogen atoms to carbon atoms (N / C) on the surface of the DLC film is preferably 0.1 or more. An N / C ratio of 0.1 or more is advantageous in terms of cell adhesion and proliferation. From the above perspectives, N / C is more preferably 0.15 or more, and even more preferably 0.16 or more. There is no particular upper limit for N / C, but from the perspectives of the manufacturing process and cost, it is preferably 0.50 or less, more preferably 0.45 or less, and even more preferably 0.40 or less. N / C is preferably 0.1 or more and 0.50 or less, more preferably 0.15 or more and 0.45 or less, and even more preferably 0.16 or more and 0.40 or less. Nitrogen atoms can be incorporated into the DLC film by incorporating nitrogen into the introduced gas. For example, if it is desired to increase the nitrogen content in the DLC film, this can be adjusted by increasing the nitrogen content in the introduced gas.

[0022] (Thickness of DLC film) The thickness of the DLC film is preferably in the range of 0.5 to 100 nm, more preferably in the range of 1 to 70 nm, and even more preferably in the range of 5 to 50 nm. The thinner the DLC film, the shorter the DLC film formation process time, which is advantageous. Furthermore, the thinner the DLC film, the less likely it is to become discolored, improving visibility when observing cell culture. On the other hand, the thicker the DLC film, the more stable the film and the better the cells can be attached. The thickness of the DLC film can be adjusted by the film formation process time, the concentration of the introduced gas, etc.

[0023] (Contact angle of water on DLC film) In the cell culture substrate having the DLC film of the present invention, the contact angle of the DLC film when using pure water at a measurement temperature of 25°C is 58° or less. This is because the wettability with the culture medium is improved, making it easier for cells to approach the DLC film surface, thereby improving cell adhesion and proliferation. From the above perspectives, the contact angle is preferably 56° or less, more preferably 54° or less, even more preferably 52° or less, and particularly preferably 50° or less. The contact angle is preferably 35° or more and 56° or less, more preferably 37° or more and 54° or less, even more preferably 39° or more and 52° or less, and particularly preferably 40° or more and 50° or less. On the other hand, the lower limit of the water contact angle is preferably 35° or more. A water contact angle of 35° or more is preferable because excessive hydrophilicity prevents the interaction between water molecules and the DLC film from becoming too strong, thereby preventing inhibition of cell adhesion and proliferation. Furthermore, due to the influence of the hydrophilic functional group, it is possible to eliminate the possibility that trace components such as proteins necessary for the growth or proliferation of cells in the culture medium will be adsorbed and adversely affect cell proliferation. From the above viewpoints, the lower limit of the water contact angle is preferably 37° or more, more preferably 39° or more, and even more preferably 40° or more.

[0024] The contact angle of a DLC film can be adjusted by controlling the type and duration of gas introduction during DLC ​​film formation and the applied power during discharge (hereinafter sometimes referred to as "discharge power"). To reduce the contact angle, methods such as adding functional groups containing oxygen or nitrogen atoms to the surface or adjusting film formation conditions to increase the atomic ratio of oxygen or nitrogen are preferably selected. Furthermore, the water contact angle can also be adjusted by irradiating the substrate with gamma rays during sterilization, as described below. The desired contact angle can be obtained by appropriately controlling the intensity of gamma ray irradiation. Because the contact angle of a DLC film depends on the surface properties of the substrate, even if the substrate is made of fine particles or other materials and is not suitable for directly measuring the contact angle of a DLC film, the contact angle of the DLC film surface for each material can be determined by preparing a substrate such as a plate of the same material and measuring the contact angle.

[0025] (XANES of DLC Film) In measuring the X-ray absorption near-edge structure (XANES) spectrum of the surface of the DLC film, it is preferable that at least one peak be present between 395 and 403 eV of the N K-edge. Satisfying this condition has the advantage of improving cell adhesion and proliferation. Figure 1 shows an example of the results of XANES measurement of the DLC film surface of the cell culture substrate of the present invention. In the example shown in Figure 1, two peaks are observed between 395 and 403 eV, confirming the preferred embodiment of the present invention in which at least one peak is present. Both of these peaks are attributed to the π* peak of N. Furthermore, XANES measurement was performed according to the method described in the Examples. In order to form a DLC film having at least one peak between 395 and 403 eV of the N K-edge, for example, acetylene and nitrogen may be mixed as the introduced gas during the DLC film formation, and parameters such as discharge power may be controlled.

[0026] (Surface Free Energy of DLC Film) Surface free energy is generally composed of a d component (dispersion component), a p component (polar component), an i component (induction component), and an h component (hydrogen bond component). In the surface free energy of the DLC film according to the present invention, the d component (dispersion component) calculated from the contact angles with water and diiodomethane using the Owens-Wendt equation is 20 to 40 mJ / m ２ and preferably in the range of 22 mJ / m ２ 38mJ / m or more ２ More preferably, 24 mJ / m or less ２ 36mJ / m or more ２ More preferably, 26 mJ / m or less ２ 34mJ / m or more ２ Even more preferably, 30 mJ / m or less ２ 33mJ / m or more ２ The following is particularly preferred: Furthermore, the h component (hydrogen bonding component) is 17 to 31 mJ / m ２ It is preferable that the ２ 29mJ / m or more ２ More preferably, 21 mJ / m or less ２ 28mJ / m or more ２ More preferably, 22 mJ / m or less ２ 27mJ / m or more ２ Even more preferably, 25 mJ / m ２ 27mJ / m or more ２ The lower limit of the component d is 22 mJ / m ２ More preferably, 24 mJ / m or more ２ More preferably, 26 mJ / m or more ２ More preferably, 30 mJ / m or more ２ On the other hand, the upper limit of the component d is 38 mJ / m ２ More preferably, 36 mJ / m or less ２ More preferably, 34 mJ / m or less ２ Even more preferably, 33 mJ / m ２ The following is particularly preferred: The lower limit of the component h is 19 mJ / m ２ More preferably, 21 mJ / m or more ２ More preferably, 22 mJ / m or more ２More preferably, 25 mJ / m or more ２ On the other hand, the upper limit of the h component is 29 mJ / m ２ More preferably, 28 mJ / m or less ２ More preferably, 27 mJ / m or less ２ The following is even more preferable. Although the reason is unclear, DLC films having a surface free energy within this range are likely to promote adhesion of adherent cells, such as mesenchymal stem cells (MSCs), thereby promoting cell proliferation and suppressing the adsorption of proteins necessary for cell proliferation, such as BSA, which is added in small amounts to the culture medium. To adjust the surface free energy of a DLC film, methods include controlling the type, time, and discharge power of the gas introduced during DLC ​​film formation, as well as controlling the intensity of gamma-ray irradiation during sterilization. Generally, a smaller water contact angle tends to result in a smaller d component (dispersion component) and a larger h component. However, the surface free energy of a DLC film is complex and is not determined solely by the water contact angle. Therefore, trial and error is required to control the process conditions during DLC ​​film formation and gamma-ray irradiation, etc.

[0027] <Adsorption of Extracellular Matrix Proteins to Substrates> Extracellular matrix proteins such as collagen, laminin, and fibronectin adsorb onto a substrate, providing a scaffold for adherent cells to adhere to the substrate. To efficiently adsorb extracellular matrix proteins to a substrate, they are precoated or added to the culture medium. The culture method of the present invention enables successful cell culture with reduced amounts of extracellular matrix proteins such as collagen, laminin, and fibronectin precoated or added, or even without precoating. Precoating is cumbersome and contributes to inconsistent cell culture results, so eliminating the need for precoating is a significant advantage. Culture media contain large amounts of other proteins, such as albumin, which competitively inhibit the adsorption of extracellular matrix proteins to the substrate. A desirable property of a substrate for culturing adherent cells is the ability to preferentially adsorb extracellular matrix proteins.

[0028] The preferential adsorption of extracellular matrix proteins to a substrate can be determined by the ratio of the amount of fibronectin adsorbed to the substrate in a medium containing a certain amount of albumin to which fibronectin has been added to a PBS buffer (phosphate buffered saline) to the amount of fibronectin adsorbed to the substrate in which fibronectin has been added. This ratio varies depending on the amount of albumin contained in the medium and the amount of fibronectin added. In the cell culture substrate on which the DLC film of the present invention has been formed, the amount of fibronectin at which the above ratio is 10% or more when a medium containing 2 mg / mL of albumin is used is preferably 2.5 μg / mL or less, more preferably 1.25 μg / mL or less, and even more preferably 0.625 μg / mL or less.

[0029] <Cell Culturing Method> The scope of the present invention also includes a cell culturing method using the cell culture substrate of the present invention. As described above, the cell culture substrate of the present invention is preferable in that it allows adhesive cells to be cultured, and adhesive cells can be efficiently proliferated by the cell culturing method of the present invention.

[0030] (Cells) The cell culture method of the present invention is effective for culturing adherent cells, as described above, and the cells are cultured by adhering to a DLC film disposed on a substrate. Adherent cells can be grown until the surface of the DLC film is covered with cells or until the nutrients in the culture medium become insufficient. Cells can be passaged before reaching the above-mentioned stage. While there is no limitation on the cells that can be used in the cell culture method of the present invention, adherent cells are preferred. Examples include established cell lines such as Sf9 cells, HEK293 cells, CHO cells, and Vero cells, as well as neurons, epithelial cells, mesenchymal stem cells, and fibroblasts, and also include induced pluripotent stem cells (iPS cells). Of these, the present invention is preferably applied to mesenchymal stem cells or iPS cells, which can be cultured efficiently.

[0031] (Culture Medium) The culture medium provides a nutrient source for cells, and sugars, nitrogen-containing compounds, etc. are used. As sugars, typically, sugars such as maltose, sucrose, glucose, fructose, and mixtures thereof are used. The sugar concentration in the culture solution is not particularly limited, but is preferably set at 0.1 to 10 w / v%. Furthermore, as nitrogen-containing compounds, in addition to various proteins, ammonium chloride, ammonium sulfate, corn steep liquor, yeast extract, meat extract, peptone, etc. are used, and the concentration is preferably set at 0.1 to 10 w / v%. Furthermore, in addition to sugars and nitrogen-containing compounds, vitamins, inorganic salts, etc. can be added to the culture solution as needed.

[0032] (Serum-Free Medium) Serum derived from living organisms, such as fetal bovine serum, has traditionally been used to support cell survival and proliferation. Serum provides supplementary nutrients and / or growth factors, promoting cell survival and proliferation as well as adhesion to cell culture vessels. While serum is beneficial for cell growth, it can also have adverse effects due to the introduction of infectious agents or the abnormal induction of unnecessary gene expression upon exposure to serum. Furthermore, serum is composed of various components, and lot-to-lot variation during production has become a problem. In recent years, attempts have been made to culture cells in so-called serum-free media. Under these circumstances, the cell culture method of the present invention is advantageous in that it allows the use of serum-free media. That is, even in serum-free media, cells can be uniformly attached to the DLC film of the present invention, providing conditions for cell survival and proliferation while eliminating the adverse effects of using serum. Bovine serum albumin (BSA), a component of serum, is a useful component for cell culture, as it binds and supplies essential proteins and other substances to cells in the media, and may therefore be added to serum-free media. Furthermore, to avoid the risks of using animal-derived components, recombinant human albumin (HSA) produced using a human cell expression system may be used instead of BSA. In the present invention, serum-free culture refers to culture without serum. Specifically, it refers to culture using, for example, a serum-free medium.

[0033] <Method for Manufacturing Cell Culture Substrate> (Step of Forming Diamond-Like Carbon Film) The method for manufacturing a cell culture substrate of the present invention involves forming a diamond-like carbon film (DLC film) on a substrate. The coating method for the DLC film is not particularly limited as long as it is a method that can form a DLC film. Known methods such as sputtering, DC magnetron sputtering, RF magnetron sputtering, chemical vapor deposition (CVD), plasma CVD, plasma ion implantation, superimposed RF plasma ion implantation, ion plating, arc ion plating, ion beam deposition, and laser ablation can be used. Among these, plasma CVD is preferred from the viewpoint of the film's ability to conform to various shapes. In the plasma CVD method, the substrate is first placed on one side of an electrode in a plasma CVD film-forming device, the device is evacuated to 1 Pa or less, and then an introduction gas is introduced. In the present invention, an acetylene-containing gas is preferably used as the introduction gas, and an introduction gas further containing oxygen and / or nitrogen is used as needed. After the pressure has stabilized, a high frequency voltage is applied to generate plasma, which is then used to form the film.

[0034] (Radiation Treatment Step) It is preferable to subject a cell culture substrate having a DLC film formed on its surface to radiation treatment before cell culture. Examples of radiation include electron beams and gamma rays, with gamma rays being more preferred. One aspect of the method for producing a cell culture substrate of the present invention is a method in which a DLC film is formed on a substrate and then subjected to gamma ray irradiation. Gamma ray irradiation of the DLC film changes the surface structure of the DLC film, making it easier to obtain a cell culture substrate with a suitable contact angle. Sterilization using gamma rays must be performed under conditions that do not deteriorate the cell culture substrate itself. The dose is preferably 5 to 40 kGy, more preferably 10 to 30 kGy. A dose that is too low can result in insufficient sterilization, potentially adversely affecting cell culture, while a dose that is too high can cause deterioration of the substrate, resulting in problems such as reduced strength and discoloration of the substrate. Furthermore, gamma ray irradiation can also control the water contact angle (wettability) and surface free energy, and it is important to control the gamma ray irradiation intensity and the environment during irradiation (oxygen concentration, moisture, etc.), and the method of encapsulating the substrate, which affects these, is also important. It is preferable to sterilize a cell culture substrate having a DLC film formed on its surface before cell culture. Sterilization methods include irradiation with radiation such as electron beams or gamma rays (gamma rays), and gas sterilization such as ethylene oxide gas (EOG), and any method may be used. The radiation treatment may also serve as a sterilization treatment.

[0035] [Cell Culture Vessel and Cell Culture Component] The cell culture vessel and cell culture component of the present invention are formed from the cell culture substrate described above. The shape of the cell culture vessel is not limited, and examples include a petri dish, dish, plate, flask, bottle, bag, tube, etc. Furthermore, examples of the cell culture component include microcarriers, and as long as they are minute carriers used as microcarriers, there are no limitations on their shape, and examples include particles, beads, powder, and nonwoven fabrics. When the cell culture vessel is a petri dish, dish, plate, flask, or bottle, the thickness of the cell culture vessel is preferably in the range of 0.01 to 10 mm, similar to the cell culture substrate, and more preferably in the range of 0.1 to 6 mm. A thicker thickness provides superior strength, but is heavier and more difficult to handle. Conversely, a thinner thickness provides lighter weight and easier handling, but is disadvantageous in terms of strength. When the cell culture vessel is a bag, from the viewpoint of maintaining its shape, the thickness and size of the substrate constituting the cell culture substrate forming it are preferably in the range of 10 to 1000 μm (0.01 to 1 mm), more preferably 20 to 500 μm. When the cell culture vessel is a tube, the thickness and size of the substrate constituting the cell culture substrate forming it are preferably in the range of 50 to 5000 μm, more preferably 100 to 2000 μm. Furthermore, when the cell culture component is a microcarrier, the thickness and size of the substrate constituting the cell culture substrate forming it are preferably in the range of 1 to 1000 μm (0.001 to 1 mm) in diameter, more preferably 5 to 500 μm in diameter.

[0036] <Method for manufacturing cell culture vessel and cell culture member> A preferred method for manufacturing the cell culture vessel and cell culture member of the present invention is to first prepare a vessel or member and then form a DLC film thereon. For example, various molding methods can be used to manufacture a resin vessel depending on the shape, such as injection molding, extrusion molding, and injection blow molding.

[0037] The cell culture vessel of the present invention is obtained by forming a DLC film on the bottom and / or inner wall of a container molded as described above. Similarly to the cell culture vessel, a DLC film is formed on the outer surface of the molded cell culture member after molding to form the cell culture member of the present invention. The DLC film is formed on the culture surface that comes into contact with cells. The DLC film coating method is not particularly limited as long as it is a method that forms a DLC film, and is the same as the method used for the cell culture substrate described above. For example, known methods such as sputtering, DC magnetron sputtering, RF magnetron sputtering, chemical vapor deposition (CVD), plasma CVD, plasma ion implantation, superimposed RF plasma ion implantation, ion plating, arc ion plating, ion beam deposition, and laser ablation can be used.

[0038] For example, injection molding is preferred for resin petri dishes, dishes, etc. A specific example involves melting polystyrene resin (hereinafter sometimes referred to as "PS resin") in a heated cylinder, injecting and filling the molten resin into a mold, and then cooling and solidifying it in the mold to form a shape. A DLC film is then formed on the bottom or other portion of a PS resin petri dish, dish, etc., to produce the petri dish or dish-shaped cell culture vessel of the present invention. Cell culture is performed in the DLC film-covered portion of the cell culture vessel, such as the bottom. In the case of resin plates, similar to the cell culture substrate manufacturing method described above, extrusion molding is preferred, with a DLC film formed on an extruded PS resin plate. For flasks, bottles, etc., injection blow molding is preferred, with a DLC film preferably formed on the inner wall, including the bottom, of the molded PS resin flask, bottle, etc. In the case of a resin bag or tube, a PS resin film is prepared by extrusion molding, a DLC film is formed on the PS resin film, and the resulting DLC-coated PS resin film is formed into a bag shape so that the DLC film is on the inside, and then the bag or tube can be welded to produce the bag or tube.

[0039] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0040] <Evaluation Method> (1) Surface Composition The bottom of the well was cut out from each test plate of each Example and Comparative Example to serve as a test piece. The surface on which the DLC film was formed was analyzed for carbon C using an XPS (X-ray photoelectron spectroscopy) device K-Alpha (X-ray source: Al Kα monochromatic rays) manufactured by Thermo Fisher Scientific. １ｓ , oxygen O １ｓ , nitrogen N １ｓ Next, using the analysis software attached to the instrument, sensitivity coefficient correction and background removal were performed for each spectrum, and the atom % of each element, carbon, oxygen, and nitrogen, was analyzed from each peak area ratio so that the total of the three atoms was 100%.

[0041] (2) Contact angle with water The bottom of the well was cut out from each test plate of each Example and Comparative Example to serve as a test piece, and the contact angle of the surface on which the DLC film was formed with a droplet of distilled water was measured using a contact angle meter ("DropMaster 500" manufactured by Kyowa Interface Science Co., Ltd.).

[0042] (3) Surface Free Energy: Test specimens were prepared by cutting out the bottom surface of each well from each test plate in each Example and Comparative Example. Using distilled water and diiodomethane as test liquids, the contact angle of each test liquid was measured on the surface on which a DLC film had been formed using a contact angle meter. The dispersion component (d component) and hydrogen bonding force component (h component) of the surface free energy were then calculated using the Owens-Wendt theory using the analysis software provided with the measurement device. The contact angle was measured using a "PCA-11" measurement device manufactured by Kyowa Interface Science Co., Ltd., and the accompanying analysis software, "FAMS" software version 5.0.10 manufactured by Kyowa Interface Science Co., Ltd., was used. The contact angle was calculated as the average of five measurements for one DLC film surface. The characteristic values ​​of the two test liquids and the contact angles measured using each test liquid were input into the analysis software. The characteristic values ​​of each test liquid are shown in Table 1.

[0043]

[0044] (4) XANES Measurement The X-ray absorption near-edge structure (XANES) spectrum was measured for the DLC films produced in each example and comparative example. A case where there is at least one peak between 395 and 403 eV of the N K-edge is described as "peak present." The measurement conditions are as follows: Experimental facility: Aichi Synchrotron Light Center Experimental station: BL7U Absorption edge: Nitrogen K (409.9 eV) absorption edge Detection method: Total fluorescence yield method with MCP plate detection Incident angle: 35.3° Number of accumulations: 20 Incident X-ray energy sweep steps: As shown in the table below

[0045]

[0046] Energy correction: The energy of the incident X-ray was corrected using the kinetic energy of Au 4f photoelectrons from the Au plate excited by X-rays with a measurement start energy (385 eV). The binding energy of Au 4f was 84.04 eV, and the device work function was 4.74 eV.

[0047] <Cell proliferation test using MSCs> A cell proliferation test was performed using 6-well plates prepared under the conditions shown in the following Examples and Comparative Examples. The cell types, serum-free medium, and main reagents used are as follows: Mesenchymal stem cells (MSCs): JCRB1110 (human umbilical cord blood-derived immortalized cell line) Serum-free medium: Prime-XV-MSC Expansion XSFM-B (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as "XV") Cell detachment reagent: TrypeLE ＴＭ -Select Enzyme (1X), no phenol red (Thermo Fisher Scientific) (hereinafter referred to as "Triple Select") -Reagent for measuring viable cell quantity: Premix WST-1 Cell Proliferation Assay System (Takara Bio Inc.) (hereinafter referred to as "WST-1"). The procedure for the cell proliferation test is as follows: 1. MSCs maintained in a T225 flask (Thermo Fisher Scientific) in serum-containing MSC maintenance medium were detached using Triple Select and cultured at 2 x 10 cells / ml in XV medium.４ A cell suspension was prepared at 2 mL of medium per well and seeded onto a 6-well plate. After seeding, the cells were incubated at 37°C and 5% CO for 96 hours. ２ 2. After 96 hours of culture, 200 μl of WST-1 was added to each well (2 mL of cell-containing solution), and the cells were incubated at 37°C and 5% CO ２ After 96 hours, 200 μl of WST-1 was added to 2 ml of blank control medium, and the mixture was incubated at 37°C and 5% CO ２ After 2 hours, 100 μL of each sample was collected in triplicate into a 96-well plate, and the difference in absorbance at a dominant wavelength of 450 nm and a secondary wavelength of 650 nm was measured using a plate reader. The difference in absorbance at the dominant and secondary wavelengths obtained with the blank control was subtracted from the sample value to determine the final absorbance value for each sample. Each sample was measured in triplicate, and the final absorbance values ​​were averaged. The ratio of the average absorbance value to the absorbance measured with CellBIND (Corning, 6-well plate) used for comparison was used as an index of cell proliferation.

[0048] Example 1: A 6-well plate (Corning, #351146) (thickness: 1.52 mm) was used as a polystyrene test plate. A DLC film was formed on the well surface to obtain an evaluation plate for cell culture. Film formation was performed using a plasma CVD film formation device (U-TECH Co., Ltd.). The plate was placed on one side of a flat electrode, and the chamber was evacuated to 1 Pa or less, followed by gas introduction. After the pressure stabilized, a high-frequency voltage was applied to generate plasma and form a film. The film formation time was adjusted to achieve a film thickness of approximately 20 nm. Only acetylene was introduced as the gas, and the discharge power was 300 W. The DLC film thickness was 20 nm. The DLC film thickness was measured using the following method. During film formation, a partially masked Si wafer was attached to one of the bottom surfaces of the six wells. After the film formation, the Si wafer was collected, and the step between the DLC filmed portion and the non-DLC filmed portion was measured using a high-precision micro-profile measuring instrument (Kosaka Laboratory Co., Ltd., Surfcorder ET4000A). The same applies to the other examples.

[0049] Example 2 A DLC film was formed on a polystyrene test plate in the same manner as in Example 1, except that the introduced gas was a mixed gas of acetylene and hydrogen (gas flow ratio (sccm) acetylene:hydrogen = 2:5), to obtain an evaluation plate for cell culture. The thickness of the DLC film was 19 nm.

[0050] Example 3 A DLC film was formed on a polystyrene test plate in the same manner as in Example 1, except that the introduced gas was a mixed gas of acetylene and nitrogen (gas flow ratio (sccm) acetylene:nitrogen = 4:5), to obtain an evaluation plate for cell culture. The thickness of the DLC film was 20 nm.

[0051] Example 4: After forming a DLC film by the method described in Example 1, plasma treatment was performed using only oxygen, with oxygen introduced at 200 sccm, a discharge power of 50 W, and a discharge time of 5 seconds, to obtain an evaluation plate for cell culture. The DLC film had a thickness of 20 nm.

[0052] Example 5 A DLC film was formed on a polystyrene test plate in the same manner as in Example 1, except that the introduced gas was a mixed gas of acetylene and nitrogen (gas flow ratio (sccm) acetylene:nitrogen = 2:5), to obtain an evaluation plate for cell culture. The thickness of the DLC film was 20 nm.

[0053] Example 6 A DLC film was formed on a polystyrene test plate in the same manner as in Example 1, except that the introduced gas was a mixed gas of acetylene and nitrogen (gas flow ratio (sccm) acetylene:nitrogen = 1:5), to obtain an evaluation plate for cell culture. The thickness of the DLC film was 20 nm.

[0054] In each of Examples 1 to 6, the plates after the DLC film formation and the plasma treatment were vacuum-sealed in a resin bag, heat-sealed, and sterilized by gamma-ray irradiation at a dose of approximately 25 kGy. The plates were removed from the sealed bag and used for each evaluation test.

[0055] Comparative Example 1 The above evaluation was carried out using the polystyrene test plate used in Example 1 without forming a DLC film on it. The plate used had been sterilized in advance by electron beam irradiation.

[0056] Comparative Example 2: Evaluation was carried out in the same manner as in Example 1 using a commercially available CellBIND (Corning, 6-well plate). Note that the plate used had been sterilized in advance. The WST-1 absorbance in Comparative Example 2 was used as the reference value.

[0057] Comparative Example 3 Evaluation was carried out in the same manner as in Example 1, except that sterilization was carried out using ethylene oxide gas (referred to as EOG in Table 3) instead of gamma ray sterilization. The results are shown in Table 3.

[0058] Comparative Example 4 Evaluation was carried out in the same manner as in Example 3, except that sterilization was carried out using ethylene oxide gas (referred to as EOG in Table 3) instead of gamma ray sterilization. The results are shown in Table 3.

[0059]

[0060] The results shown in Table 3 indicate that in Examples 1 to 6, which have DLC films with an O / C ratio of 0.1 or greater and a water contact angle of 58° or less, cell proliferation is significantly improved based on the WST-1 absorbance. Furthermore, compared to CellBIND (Comparative Example 2), which is known to have high cell proliferation, the WST-1 absorbance evaluation results confirm that the DLC films exhibit equal or superior performance, demonstrating the extremely high effectiveness of the present invention. In particular, significantly higher cell proliferation was observed in Examples 3, 5, and 6, which contain nitrogen atoms with an N / C ratio of 0.1 or greater in the DLC film. In these cases, XANES measurements revealed at least one peak between 395 and 403 eV of the N K-edge, indicating that the presence of the π* peak of nitrogen atoms creates a favorable environment for cell proliferation. Figure 1 shows the results of XANES measurements on the DLC film surface of Example 3. Furthermore, it can be seen that gamma-ray treatment of the DLC film significantly improved cell proliferation.

[0061] <Cell proliferation test using iPS cells> To examine the effect of the amount of extracellular matrix protein added on iPS cell proliferation, we used iMatrix-511 silk, a commercially available E8 fragment of human laminin 511. The cell types, serum-free medium, and main reagents used are as follows: - Induced pluripotent stem cells (human iPS cells): 253G1 strain - Medium and reagents: serum-free medium StemFit ＴＭ AK02N (product number AK02N, Ajinomoto Co., Inc.) iMatrix-511 silk (product number 387-10131, Nippi Corporation) CultureSure Y-27632 (product number 034-24024, Fujifilm Wako Pure Chemical Industries, Ltd.) 0.5 mol / L-EDTA solution (pH 8.0) (product number 06894-85, Nacalai Tesque, Inc.) PBS(-) (phosphate-buffered saline, product number 166-23555, Fujifilm Wako Pure Chemical Industries, Ltd.) Trypan blue (product number 145-0022, Bio-Rad Laboratories, Inc.)

[0062] (A) Preparation of medium and reagents StemFit ＴＭ AK02N: Bring to room temperature before use. The medium used when thawing iPS cells was supplemented with Y-27632 to a concentration of 3 μM. Detachment solution: 0.5 mol / L-EDTA solution (pH 8.0) adjusted to 0.5 mM with PBS (-). Warmed to 37°C before use. Y-27632: Adjusted to 10 mM with PBS (-). Device: CellDrop FL cell counter

[0063] (B) Thawing of iPS cells 1) 10 mL of medium was prepared in a tube and heated in a 37°C water bath. 2) The frozen iPS cell tube was semi-thawed in a 37°C water bath and transferred to a tube containing 10 mL of medium that had been heated in a clean bench. 3) After centrifugation at 100 x g for 3 minutes at room temperature, the supernatant was removed. 4) 10 mL of new medium was added, and the concentration was adjusted to 0.13 μg / cm. ２ iMatrix-511 silk was added to the cell suspension so that the cells were mixed well and seeded onto a 10 cm dish.２ The cells were cultured in an incubator.

[0064] (C) Culturing of iPS cells in well plates Cell proliferation tests were carried out using the well plates prepared in Examples 1, 3, Comparative Examples 1 and 2. The procedure for the cell proliferation test was as follows. ＴＭ 1.13 × 10 iPS cells suspended in AK02N medium (containing 3 μM Y-27632) ４ Cells were seeded at 1000 cells / well and cultured for 72 hours. The final concentration of iMatrix-511 silk was 0.1 μg / mL, and each condition was cultured in triplicate. 72 hours after seeding, iPS cells were detached in a detachment solution (0.5 mM EDTA) and recovered, followed by cell counting. The results are shown in Figure 2. In contrast to Comparative Example 1, in which no proliferation from the initial seeding number was observed, good cell proliferation comparable to that observed in Comparative Example 2 was observed in Examples 1 and 3, which included a DLC film. While no significant difference was observed between Example 1 and Example 3, Example 3 tended to have a slightly higher cell number.

[0065] Next, it was investigated whether the DLC film formation process was also effective in bag-shaped culture vessels. Example 7 <Preparation of bag-shaped culture vessel> A culture bag (model number: 30515) manufactured by Fukoku Co., Ltd. was used as the bag-shaped culture vessel. The thickness of the film used in the bag was 95 μm. The bag was opened with scissors, and a DLC film was formed on only one side. The film formation conditions were the same as in Example 1. After the DLC film formation, the films were thermocompressed together using a heat sealer to return to the original bag shape. The prepared bag-shaped culture vessel was placed in a resin bag for sealing, and then sterilized by gamma ray irradiation in the same manner as in Examples 1 to 6. The length of the inside of the bag seal was measured to determine the base area to be 185 cm ２ It was calculated as follows.

[0066] Example 8 A bag-shaped culture vessel was prepared in the same manner as in Example 7, except that the film formation conditions were the same as in Example 3. The length of the inside of the sealed bag was measured to determine the bottom area of ​​185 cm. ２ It was calculated as follows.

[0067] Comparative Example 5: For comparison, a culture bag manufactured by Fukoku Co., Ltd. that had not been subjected to a coating treatment was used. The bottom area was 215 cm², referring to the length information in the catalog. ２ The culture bag used was sterilized in advance by gamma ray treatment.

[0068] Comparative Example 6: A culture bag manufactured by Fukoku (sterilized by gamma rays) used in Example 7 was aseptically coated with fibronectin instead of the DLC film. Sigma-Aldrich fibronectin (product number: F0895-1MG) was used as the fibronectin, and the concentration was 1 μg / cm ２ Fibronectin coating was started the day before cell seeding, and the fibronectin solution was removed 1 hour before cell seeding.

[0069] <Culturing of MSCs using a bag-shaped culture vessel> In this test, the bag-shaped culture vessels of Comparative Example 5, Comparative Example 6, and Example 8 were used. MSCs (product number: JCRB1110, lot number: 10142011) derived from human umbilical cord blood that had been maintained in Ex-MSC·XF Medium (manufactured by Myoridge Co., Ltd.: ME-09H00152) were cultured at 2 × 10 ３ Cells / cm ２ The cell density was adjusted to 0.5 mL / cm ２ The cell suspension was seeded into each test bag so that the liquid volume was 100 ml. The bag was pressed to remove as much air as possible, and then the bag was stored at 37°C, 5% CO ２ After 5 days, the culture supernatant was discarded, and the cells were washed with 10 mL of PBS. ＴＭ Add 10 mL of Select Enzyme (1x) (Invitrogen) and incubate at 37°C, 5% CO ２The cells were detached by placing them in an incubator and allowed to stand for 20 minutes, then collected in a 50 mL tube. The cells were washed with 40 mL of DMEM (product number D5796-1L, Sigma-Aldrich) and collected in the same tube to a total volume of 50 mL. The 50 mL tube was inverted to mix well and the cell suspension was then counted using the AO / PI method. The AO / PI method was used to count cells after AO / PI solution treatment using an automated cell counter, Cell Drop FL (DeNovix). The results are shown in Table 4 (viable cell counts during MSC culture using a bag-shaped culture substrate with a DLC film). As can be seen from Table 4, MSCs did not proliferate in untreated culture bags. However, by performing the DLC film formation treatment under the same conditions as in Example 3, cell proliferation was possible even in the bag-shaped culture equipment.

[0070]

[0071] <Culturing iPS cells using a bag-shaped culture vessel> In this test, the bag-shaped culture vessels of Comparative Example 5, Example 7, and Example 8 were used. iMatrix pre-coating was performed the day before cell seeding. As a 1 / 1000 dilution condition for iMatrix-511 silk (product number 387-10131, Nippi Corporation), 430 μL was diluted with 100 mL of PBS, and 50 mL was added to each bag of Comparative Example 5, Example 7, and Example 8. The coating time before seeding was approximately 24 hours. iPS cells that had been passaged were detached and collected, and the resulting cell suspension was subjected to a cell count. The required amount was divided equally into two 50 mL tubes and centrifuged (100 x g, 3 minutes, room temperature). The supernatant was removed with an aspirator, and the cells were then resuspended in serum-free medium StemFit. ＴＭ The cells were resuspended in 100 mL of AK02N (product number AK02N, Ajinomoto Co., Inc.) (+3 μM Y-27632 (product number HY-10583, MedChem Express)). The iMatrix solution in the bag was discarded, and 100 mL of cell suspension was added. The bag was compressed to remove as much air as possible. The number of cells at the time of seeding was 1.28 × 10 ４ Cells / cm ２ 37°C, 5% CO ２Static culture was performed in an incubator, and after two days, 50 mL of the same medium was added to each well. After static culture for three days after seeding, photographs were taken and the cells were recovered. The entire supernatant was discarded, and the cells were washed with 10 mL of PBS(-) (product number 166-23555, Fujifilm Wako Pure Chemical Industries, Ltd.), which was also discarded. Then, 10 L of 0.5 mM EDTA solution (product number 13567-84, Nacalai Tesque Inc.) was added, and the cells were incubated at 37°C and 5% CO ２ The cells were placed in an incubator and allowed to stand for 10 minutes. The entire EDTA solution was collected in a 50 mL tube, washed with 40 mL of DMEM (product number D5796-1L, Sigma-Aldrich), and collected in the same tube to bring the total volume to 50 mL. The 50 mL tube was inverted to mix well the cell suspension, and after treatment with the AO / PI solution, the cell number was measured using an automated cell counter, Cell drop FL (DeNovix).

[0072] Similarly, using the bag-shaped culture vessels of Comparative Example 5, Example 7, and Example 8, the amount of iMatrix-511 silk to be precoated was halved (1 / 2000 dilution), and the number of cells at the time of seeding was 1.13 × 10 ４ Cells / cm ２ The culture was performed using a bag-shaped culture vessel with a DLC film. The results of two experiments in which the iMatrix concentration was varied (cell counts during iPS cell culture using a bag-shaped culture vessel with a DLC film) are summarized in Table 5. As can be seen from Table 5, no iPS cell proliferation was observed in the bag without film formation treatment in Comparative Example 5, but iPS cell proliferation was observed in Examples 7 and 8, in which a DLC film was formed. This demonstrates that DLC film formation treatments under the same conditions as Examples 1 and 3 are effective for iPS cell proliferation even in bag-shaped culture substrates. Furthermore, the cell count was higher in Example 8 when a more diluted iMatrix was used than in Example 7, supporting the conclusion that a nitrogen-containing DLC ​​film formation treatment is more preferable.

[0073]

[0074] Next, we investigated the effectiveness of DLC film formation on microcarriers for cell proliferation. Example 9 (C-DLC Microcarrier) <Preparation of Polystyrene Particles> An aqueous phase was prepared by mixing 5.9 parts of TCP-10·U (Taihei Chemical Industry Co., Ltd., 10 wt% aqueous dispersion of tricalcium phosphate), 0.033 parts of a 10 wt% aqueous solution of sodium dodecylbenzenesulfonate, and 189 parts of ion-exchanged water. An oil phase was prepared by mixing 58.5 parts of styrene (Fujifilm Wako Pure Chemical Corporation), 6.5 parts of divinylbenzene (Fujifilm Wako Pure Chemical Corporation, 55% purity), and 0.65 parts of 2,2'-azobis(2,4-dimethylvaleronitrile) (Fujifilm Wako Pure Chemical Corporation). The aqueous and oil phases were placed in a reaction vessel equipped with a stirrer, heating / cooling device, and nitrogen introduction device, and droplets of the oil phase were formed in the aqueous phase by stirring. After droplet formation, the internal temperature was raised to 60°C, and stirring at 60°C was continued for 5 hours. The internal temperature was then raised to 90°C, and stirring at 90°C was continued for 4 hours. After cooling to room temperature, hydrochloric acid was added to decompose the tribasic calcium phosphate, and the polystyrene particles were collected by filtration. The polystyrene particles were then dried by heating at 85°C for 7 hours, passed through a mesh with 250 μm openings (manufactured by Tokyo Screen Co., Ltd.), and the polystyrene particles remaining on a mesh with 150 μm openings (manufactured by Tokyo Screen Co., Ltd.) were collected to obtain classified polystyrene particles.

[0075] <DLC Film Formation on Particulate Substrate> A DLC film was formed on the surface of polystyrene particles to obtain evaluation particles for cell culture. Film formation was performed using a plasma CVD film formation device (manufactured by U-TECH Co., Ltd.). Polystyrene particles were placed in the recessed portion of one side of a flat electrode, and the chamber was evacuated to 1 Pa or less, followed by gas introduction. After the pressure stabilized, a high-frequency voltage was applied to generate plasma and form a film. To ensure uniform film formation over the entire polystyrene particle, the high-frequency voltage application was stopped midway through film formation. The polystyrene particle placement area was then agitated, and the film formation process was repeated twice. The film formation time was adjusted to achieve a film thickness of approximately 20 nm or greater. Only acetylene was introduced as the gas, and the discharge power was 300 W. Following the DLC film formation process, sterilization was performed by gamma-ray irradiation, as in Examples 1 to 6.

[0076] Example 10 The DLC film formation process was carried out on a particulate culture substrate prepared in the same manner as in Example 9 under the same conditions as in Example 3, in which a mixed gas of acetylene and nitrogen was introduced, and then sterilization was carried out by gamma ray irradiation.

[0077] Comparative Example 7 Polystyrene particles were prepared by the method described in Example 9, and sterilized by gamma ray irradiation without carrying out a DLC film formation treatment to prepare a particulate culture substrate.

[0078] Comparative Example 8: Commercially available CellBIND microcarriers (Corning, #3779) were used. The particle size was 125 to 212 μm. The cell proliferation rate of the particulate substrate was shown as the relative cell number to that of Comparative Example 8.

[0079] <MSC proliferation and culture using particulate culture substrate> (A) Cell thawing Frozen cells (human adipose-derived mesenchymal stem cells, LONZA, PT-5006) were thawed in a water bath set at 37°C. After thawing, the cells were suspended in 5 mL of medium in a sterilized tube. The cells were centrifuged at 25°C and 600 x g for 5 minutes. The supernatant was removed, and the cells were suspended in 1 mL of serum-free medium (R:STEM Medium for hMSC High Growth (Rohto Pharmaceutical Co., Ltd., EMI-500)) at 37°C. The cell suspension and trypan blue staining solution were mixed at a 1:1 ratio, poured into a cell counting board, and the cell number was counted using a cell counter (Waken B Tech, FACSCOPE B). 5000 cells / cm were plated in a 6-well plate. ２ and placed in an incubator (37°C, 5% CO ２ ) and statically cultured for 3 days.

[0080] (B) Detachment The medium and detachment agent were warmed in a water bath set at 37°C. The cell status and confluence were observed using a phase-contrast microscope. The medium in the culture vessel was removed using an aspirator. The cells were washed once with DPBS(-), a Dulbecco's phosphate-buffered saline (hereinafter referred to as "DPBS"). After adding the detachment agent, the culture vessel was returned to a 37°C incubator and allowed to stand for approximately 2-5 minutes. Cell detachment was confirmed by observation using a phase-contrast microscope. Trypsin inhibitor was added to inactivate the detachment agent, and the entire volume was transferred to a centrifuge tube. The mixture was centrifuged at 25°C and 600 x g for 5 minutes, the supernatant was removed, and the cell pellet was resuspended in fresh medium. Approximately 50 μL of the cell suspension was taken and the cell count was counted.

[0081] <Preparation of Microcarriers> The required amount of microcarriers was taken, and DPBS was added dropwise in Examples 9, 10, and Comparative Example 7, and sterilized water in Comparative Example 8, and the mixture was gently mixed to completely wet the microcarriers. The required amount of microcarrier suspension was placed in a centrifuge tube and allowed to stand for several minutes. After the microcarriers had settled, the supernatant was removed with an electric pipette, and the microcarriers were resuspended in an appropriate amount of medium and then placed in a spinner flask (Corning, #3125). The amount of microcarriers used was determined so that the surface area was uniform, with each being 150 cm. ２ It was made to be like this.

[0082] <Culture of MSCs using microcarriers> 7.5 x 10 cells per spinner flask ５ The cells were seeded to a volume of 15 mL, and the medium was added to a total volume of 15 mL. The culture vessel was gently shaken to evenly distribute the cells and microcarriers, and the cells were then incubated in an incubator (37°C, 5% CO ２ The cells were allowed to stand in a refrigerator for 20 hours. After 20 hours, 30 mL of fresh medium was added dropwise to bring the final volume to 45 mL. The cells were placed on a magnetic stirrer (Wakken B Tech, WKN-1104-1L) in an incubator, and stirring was started at 30 rpm. The culture was continued for 4 days.

[0083] <MSC cell recovery and cell count measurement> The medium and detachment agent (Trypsin-EDTA (0.5%), Gibco, 15400054) were warmed in a water bath set at 37°C. The microcarriers in the spinner flask were uniformly dispersed using an electric pipette, and a 10 mL sample was taken. The sample was transferred to a centrifuge tube and left to stand for 10 minutes to allow the microcarriers to settle, and the culture supernatant was removed using an electric pipette. The cells and microcarriers were washed twice with DPBS, and 5 mL of a 0.05% detachment agent solution was added and mixed with the microcarriers using an electric pipette. After leaving the mixture to stand for 5-10 minutes in an incubator, the entire solution and microcarriers were gently mixed, passed through a 70 μm cell strainer, and transferred to a new centrifuge tube. 0.5 mg / mL Trypsin Inhibitor (Fujifilm Wako Pure Chemical Corporation, 202-09226) was added to the centrifuge tube beforehand. After washing the cell strainer with DPBS, the cell suspension was centrifuged at 600 x g for 10 minutes, the supernatant was removed, and the cell pellet was resuspended in a small amount of DPBS. The cell suspension was dispersed by pipetting, and the cell count and viability were measured. The cell count and viability were measured by staining the cells with trypan blue staining solution using a cell counter FACSCOPE B (Wakken B Tech).

[0084] The viable cell counts and viability rates in the culture results of Comparative Example 7, Comparative Example 8, and Example 9 are summarized in Table 6 (MSC culture results using DLC-treated microcarriers). The viable cell counts are shown as relative numbers, with Comparative Example 8 set as 1. Next, the viable cell counts and viability rates in the culture results of Comparative Example 8, Example 9, and Example 10 are summarized in Table 7 (MSC culture results using nitrogen-containing DLC-treated microcarriers). The viable cell counts are shown as the number per mL of medium.

[0085]

[0086]

[0087] Since no MSC proliferation was observed in Comparative Example 7 but MSC proliferation was observed in Example 9, it can be seen that the DLC film formation treatment under the same conditions as Example 1 aids adhesion and proliferation of MSCs to the substrate. Furthermore, the number of viable cells was higher in Example 10 than in Example 9, which indicates that the nitrogen-containing DLC ​​film formation treatment further enhances adhesion and proliferation of cells to the substrate.

[0088] Next, the influence of DLC film formation treatment on the adsorption of fibronectin and cell adhesion was investigated using a plate-shaped culture substrate.

[0089] <Fibronectin adsorption treatment for measuring fibronectin adsorption amount> Human fibronectin (hereinafter referred to as "hFN") (Corning, 354008) was added to PBS (phosphate-buffered saline) or PRIME-XV MSC Expansion SFM (Fujifilm Wako Pure Chemical Industries, Ltd.) to the following concentrations (0, 0.625, 1.25, 2.5, 5, 10 μg / mL). Next, a solution containing hFN was added to each culture substrate (Comparative Example 1, Comparative Example 2, Example 1, Example 3) at 100 μL / well, and the substrates were incubated at 37°C. After incubation, each well was washed five times with 200 μL of PBS.

[0090] <ELISA antibody reaction> 150 μL / well of Blocking One (Nacalai Tesque, Inc.) was added to the culture substrate coated with hFN and incubated at 37°C for 30 minutes. After incubation, the plate was washed five times with 200 μL of PBS. Next, an anti-hFN antibody (Clone:HFN7.1, Novus Biologicals) was diluted 4000-fold with Blocking One, added at 100 μL / well, and incubated at 37°C for 2 hours. After incubation, the plate was washed five times with 200 μL of PBS. Then, an HRP-conjugated anti-mouse antibody (Promega Corporation) was diluted 4000-fold with Blocking One, added at 100 μL / well, and incubated at 37°C for 2 hours. After incubation, the plate was washed five times with 200 μL of PBS.

[0091] <Color Reaction> OPD tablets (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in 0.1 M citrate buffer (pH 5) at a ratio of 1 tablet per 5 mL, and 2 μL of approximately 30% hydrogen peroxide solution was added to 5 mL of the solution. The adjusted OPD solution was added at 100 μL / well and incubated at room temperature for 10 minutes. After incubation, 3 M hydrochloric acid was added at 50 μL / well to stop the color reaction. To prevent differences in the color of each culture substrate from affecting the measured values, the solution after the color reaction was stirred with a pipette and transferred to a new 24-well plate at 100 μL / well. The optical density (OD490) of the transferred reaction solution at a wavelength of 490 nm was then measured using a microplate reader. The absorbance of the OPD solution in unreacted wells was measured as the background, and the absorbance was calculated by subtracting the background OD490 from the OD490 of the tested wells.

[0092] <Measurement of the amount of albumin contained in the medium> The amount of albumin, which is the main protein contained in the medium, was measured. The amount of albumin contained in the PRIME-XV medium was measured using a Human Albumin ELISA Kit (KE00076, manufactured by Proteintech) according to the protocol attached to the kit. As a result, it was found that the medium contained 2 mg / mL of albumin.

[0093] The amount of hFN adsorption (absorbance at 490 nm) for each hFN concentration added is summarized in Figure 3 (PBS) and Figure 4 (PRIME-XV medium). Figure 3 shows that there was no significant difference in the amount of hFN adsorption among all plates. On the other hand, Figure 4 shows that the amount of hFN adsorption was greater in Examples 1 and 3 than in Comparative Examples 1 and 2, with the greatest amount of hFN adsorption observed in Example 3, which was treated with a nitrogen-containing DLC ​​film. Table 8 summarizes the ratio of the amount of fibronectin adsorbed to the substrate in PRIME-XV medium to the amount of fibronectin adsorbed to the substrate in PBS buffer. In Examples 1 and 3, the adsorption ratio was 10% or more when fibronectin was added at 0.625 μg / mL or more, demonstrating the preferential adsorption of fibronectin.

[0094]

[0095] <Fibronectin Adsorption Treatment for Cell Adhesion Test> hFN was added to PRIME-XV MSC Expansion SFM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at the following concentrations (0, 0.625, 1.25, 2.5, 5, 10 μg / mL). The prepared solution was added to each culture substrate (Comparative Example 1, Comparative Example 2, Example 1, Example 3) at 1 mL / well and incubated at 37°C for 1 hour. After incubation, the substrate was washed with PBS (phosphate-buffered saline), and PBS containing 2% bovine serum albumin was added at 1 mL / well, followed by incubation at 37°C for 30 minutes. After incubation, the substrate was washed once with PBS and subjected to subsequent experiments.

[0096] <Cell adhesion experiment> Human mesenchymal stem cells (MSCs) at the third passage were passaged twice to obtain cells for testing. The obtained MSCs at the fifth passage were suspended in PRIME-XV MSC Expansion SFM medium and 1 × 10 ４ pieces / cm ２ The cells were seeded at a density of 1000 × 1000. After seeding, the cells were cultured for 2 hours in a 5% carbon dioxide atmosphere. After the culture, the cells were washed once with PBS to remove non-adherent cells, and then PBS containing 1% glutaraldehyde was added and incubated at room temperature overnight for chemical fixation.

[0097] <Measurement of Adherent Cell Number> After chemical fixation, the cells were washed once with PBS and reacted with PBS containing 10 μg / mL Hoechst 33342 to label the cell nuclei. Cell counts were performed using images of Hoechst 33342-labeled cell nuclei observed with a live cell imaging system (Carl Zeiss "Celldiscover7"). Using a 5x objective lens, 3 x 3 images were captured using the tiling function of the device. The captured images were binarized using the device's functions to separate the fluorescence from the cell nuclei and the background, and the number of fluorescently labeled cell nuclei was automatically counted. Five fields of view were imaged for each well, and the counted number of cell nuclei was used as the number of adherent cells. The number of adherent cells was converted to the number of adherent cells per unit area.

[0098] The number of cells attached for each hFN concentration added is summarized in Figure 5. The number of cells attached was converted to a relative number, with the number of attached cells in the plate of Comparative Example 2 under adsorption conditions of 0 μg / mL hFN being set to 1. As with fibronectin adsorption under culture medium conditions (Figure 4), the number of attached cells was higher in Examples 1 and 3 than in Comparative Examples 1 and 2, with the greatest number of attached cells observed in Example 3, which was treated with a nitrogen-containing DLC ​​film. hFN serves as a scaffold for cell adhesion, and it is expected that greater adsorption to the culture substrate will promote cell adhesion. Figures 4 and 5 support this conclusion. This clearly demonstrates that a DLC film with an O / C of 0.1 or greater and a contact angle of 58° or less, particularly a nitrogen-containing DLC ​​film treatment, is advantageous for cell adhesion.

[0099] According to the present invention, a cell culture substrate that allows good cell adhesion and high cell proliferation regardless of the presence or absence of serum can be provided. In other words, since the substrate allows target cells to be effectively proliferated in vitro, it is an extremely important technology in medical and biological research.

Claims

1. A cell culture substrate having a diamond-like carbon film on the surface of the substrate, wherein the ratio of oxygen atoms to carbon atoms (O / C) on the surface of the diamond-like carbon film is 0.1 or more, and the water contact angle of the diamond-like carbon film is 58° or less.

2. The d component of the surface free energy of the diamond-like carbon film is 20 to 40 mJ / m ２ and the h component is 17 to 31 mJ / m ２ The cell culture substrate according to claim 1 , wherein 3. The cell culture substrate according to claim 1, wherein the ratio of nitrogen atoms to carbon atoms (N / C) on the surface of said diamond-like carbon film is 0.1 or more.

4. The cell culture substrate according to claim 3, wherein the surface of the diamond-like carbon film has at least one peak between 395 and 403 eV of the N K-edge in XANES measurement.

5. The cell culture substrate of claim 1, which is used for serum-free culture.

6. The cell culture substrate of claim 1, wherein the diamond-like carbon film has been treated with gamma rays.

7. The cell culture substrate according to claim 1, wherein the diamond-like carbon film has a thickness of 0.5 to 100 nm.

8. The cell culture substrate according to claim 1, wherein the thickness of the substrate is 0.01 to 10 mm.

9. The cell culture substrate according to claim 1, wherein the cell culture substrate provided with the diamond-like carbon membrane has the property of preferentially adsorbing fibronectin, and as an indicator of this property, when 2.5 μg / mL or less of fibronectin is added, the ratio of the amount of fibronectin adsorbed to the substrate in a medium containing 2 mg / mL of albumin to the amount of fibronectin adsorbed to the substrate in PBS buffer is 10% or more.

10. A cell culture vessel formed from the cell culture substrate according to any one of claims 1 to 9.

11. The cell culture vessel according to claim 10, wherein the cell culture vessel is a bag and the thickness of the substrate is 0.01 to 1 mm.

12. A cell culture member formed from the cell culture substrate according to any one of claims 1 to 9.

13. The cell culture device according to claim 12, wherein the cell culture device is a microcarrier and the size of the substrate is 0.001 to 1 mm in diameter.

14. A method for producing a cell culture substrate according to any one of claims 1 to 9, characterized in that the diamond-like carbon film is formed using an acetylene-containing gas as an introduced gas, and the surface of the diamond-like carbon film is subjected to radiation treatment.

15. The method for producing a cell culture substrate according to claim 14, wherein the method for forming the diamond-like carbon film is plasma CVD.

16. The method for producing a cell culture substrate according to claim 14, wherein the radiation is gamma rays or electron beams.

17. The method for producing a cell culture substrate according to claim 14, wherein the introduced gas further contains nitrogen.

18. A method for culturing cells, which uses the cell culture substrate according to any one of claims 1 to 9.

19. The method for culturing cells according to claim 18, wherein a serum-free medium is used.

20. The method for culturing cells according to claim 18, wherein the cells are mesenchymal stem cells or iPS cells.

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