JP7790068B2 - Culture substrate and method for producing same - Google Patents

Description

This disclosure relates to a culture substrate and a method for producing the same.

Various proposals have been made regarding technical means used in cell culture. For example, Patent Document 1 discloses a culture product having a surface for cell culture, characterized in that the culture surface has a substrate derived from an extracellular matrix and a stabilizer containing a sugar containing at least one of maltose and lactose.

Japanese Patent Application Laid-Open No. 2018-19654

The purpose of this disclosure is to provide a novel culture substrate and a method for producing the same.

One aspect of the present disclosure relates to a culture substrate. The culture substrate includes a substrate and a resin portion that is provided on the surface of the substrate and contains a photo-fusible resin.

The shape of the resin portion of the culture substrate can be easily controlled by light irradiation. Therefore, the shape of the resin portion and the recesses that can be formed by the resin portion of the culture substrate can be freely designed.

The shape of the above culture substrate can be controlled by light irradiation, so the shape of the resin portion and the recesses that can be formed by the resin portion can be changed during culture.

The photo-fusible resin may have a disulfide bond.

Another aspect of the present disclosure relates to a method for producing a culture substrate. The method includes step A of irradiating light onto a portion of a resin portion formed on the substrate and containing a photofusible resin, and step B of removing the irradiated resin portion to form a recess on the substrate.

This method makes it easy to produce a culture substrate with a desired surface shape.

The resin portion irradiated with light is water-soluble, and in step B, the resin portion irradiated with light may be removed with an aqueous solvent.

Another aspect of the present disclosure relates to a method for culturing cells, the method comprising culturing cells using the culture substrate described above.

The culturing step may include irradiating a portion of the resin portion with light and removing the irradiated resin portion to change the shape of the resin portion.

This disclosure provides a novel culture substrate.

FIG. 1 is a schematic cross-sectional view showing a culture substrate according to one embodiment. FIG. 2 is a schematic cross-sectional view showing one embodiment of a method for producing a culture substrate and a method for culturing cells using the culture substrate. FIG. 3 is a schematic cross-sectional view showing one embodiment of a method for producing a culture substrate having a resin portion with a step. FIG. 4 is a schematic cross-sectional view showing a culture substrate according to one embodiment.

Embodiments of the present disclosure will be described below with appropriate reference to the drawings. However, the present disclosure is not limited to the following embodiments.

In this specification, the term "process" refers not only to an independent process, but also to processes that cannot be clearly distinguished from other processes, as long as the intended effect of that process is achieved. Furthermore, numerical ranges indicated using "to" indicate ranges that include the numerical values before and after "to" as the minimum and maximum values, respectively.

Unless otherwise specified, the exemplified materials may be used alone or in combination of two or more.

Furthermore, in the numerical ranges described in stages in this specification, the upper or lower limit of a numerical range in one stage may be replaced with the upper or lower limit of a numerical range in another stage. "A or B" may include either A or B, or may include both.

Figure 1 is a schematic cross-sectional view of a culture substrate according to one embodiment. The culture substrate 10 shown in Figure 1 comprises a substrate 1 and a resin portion 2 provided on the surface of the substrate 1. The resin portion 2 may be provided on at least a portion of the main surface of the substrate 1. In the culture substrate 10, the resin portion 2 forms a recess on the main surface of the substrate 1. That is, the culture substrate 10 comprises a substrate 1 and a resin portion (partition) 2 provided on the surface of the substrate 1 and forming the recess. The resin portion 2 may have a predetermined pattern.

The substrate 1 may be formed from at least one material selected from the group consisting of, for example, polystyrene, polypropylene, polyethylene, acrylic resin, PTFE (polytetrafluoroethylene; Teflon (registered trademark)), glass, stainless steel, and anodized aluminum.

The substrate may have the shape of a container typically used for cell culture. For example, a plastic petri dish typically used for cell culture can be used as the substrate. A culture flask or a multi-well plate, for example, can also be used as the substrate.

The resin portion 2 includes a photo-fusible resin. A photo-fusible resin is a resin that exhibits the property of melting when irradiated with light. The photo-fusible resin may be a polymer having one or more (two or more) functional groups (photodegradable functional groups) that decompose when irradiated with light. The number of photodegradable functional groups may be, for example, 1 to 1,000, or 4 to 50. The photo-fusible resin melts when, for example, the photodegradable functional groups decompose when irradiated with light, resulting in a lower molecular weight.

Examples of photodegradable functional groups include disulfide bonds (-S-S-), trithiocarbonates, nitrobenzyl, alkylphenones, acylphosphine oxides, oxime esters, hexaarylbisimidazoles, allyl sulfides, and styrylpyrene dimers.

The photo-fusible resin may be a resin having disulfide bonds, or a resin having disulfide bonds in the main chain of the polymer. In the case of a photo-fusible resin having a cross-linked structure, the disulfide bonds may be present in the main chain and/or at the cross-linking sites.

The main skeleton of the photo-fusible resin may be a hydrophilic skeleton. The hydrophilic skeleton may be derived from at least one skeleton selected from the group consisting of polyalkylene glycol, polyethyleneimine, (meth)acrylic polymers having hydroxyl groups in their side chains, polysaccharides, and proteins. Photo-fusible resins containing a hydrophilic skeleton tend to melt upon irradiation with light, resulting in resin components that exhibit hydrophilic properties, making it easier to remove the resin components with an aqueous solvent.

The main skeleton of the photo-fusible resin may be a hydrophobic skeleton. The hydrophobic skeleton may be derived from at least one skeleton selected from the group consisting of rubbers and silicones. The rubber may be polybutadiene, polyisoprene, or the like. Photo-fusible resins containing a hydrophobic skeleton tend to melt upon irradiation with light, resulting in resin components that exhibit hydrophobicity, making it easier to remove the resin components with an organic solvent.

The content of the photo-fusible resin may be, for example, 70% by mass or more, 80% by mass or more, or 90% by mass or more, based on the mass of the resin portion 2, and may be 99% by mass or less, or 95% by mass or less.

The resin part 2 may contain a photoradical generator in addition to the photofusible resin. By including a photoradical generator, the photofusibility of the resin part 2 becomes even more efficient. Examples of photoradical generators include hydrogen abstraction-type photoradical polymerization initiators, which generate radicals by abstracting hydrogen from other molecules upon light irradiation, and intramolecular cleavage-type photoradical polymerization initiators, which photocleave themselves upon light irradiation to generate two radicals. The photoradical generator is preferably an intramolecular cleavage-type photoradical generator.

Examples of hydrogen abstraction-type photoradical generators include hexaarylbisimidazole (HABI) compounds, benzophenone compounds, thioxanthone compounds, fluorenone compounds, and α-diketone compounds.

Examples of HABI compounds include 2,2'-bis(o-chlorophenyl)-4,4',5,5'-tetraphenylbiimidazole (e.g., 2,2'-bis(2-chlorophenyl)-4,4',5,5'-tetraphenyl-1,2'-biimidazole), 2,2'-bis(o-bromophenyl)-4,4',5,5'-tetraphenylbiimidazole, and 2,2'-bis(o,p-dichlorophenyl)-4,4',5,5'-tetraphenylbiimidazole. Examples include 2,2'-bis(o-chlorophenyl)-4,4',5,5'-tetra(m-methoxyphenyl)biimidazole, 2,2'-bis(o,o'-dichlorophenyl)-4,4',5,5'-tetraphenylbiimidazole, 2,2'-bis(o-nitrophenyl)-4,4',5,5'-tetraphenylbiimidazole, and 2,2'-bis(o-methylphenyl)-4,4',5,5'-tetraphenylbiimidazole.

Examples of benzophenone compounds include 4,4'-bis(dimethylamino)benzophenone and 4,4'-bis(diethylamino)benzophenone.

Thioxanthone compounds include thioxanthone, 2-isopropylthioxanthone, 2-dodecylthioxanthone, 2-cyclohexylthioxanthone, 2,4-diethylthioxanthone, 2,4-dimethylthioxanthone, 1-phenoxythioxanthone, 1-methoxycarbonylthioxanthone, 2-ethoxycarbonylthioxanthone, 3-(2-methoxyethoxycarbonyl)-thioxanthone, 4-butoxycarbonylthioxanthone, 3-butoxycarbonyl-7-methylthioxanthone, 3,4-di-[2-(2-methoxyethoxy)-ethoxycarbonyl]-thioxanthone, 2-chlorothioxanthone, and 1-ethoxycarbonyl 1-3-ethoxythioxanthone, 1-ethoxycarbonyl-3-chlorothioxanthone, 1-chloro-4-n-propoxythioxanthone, 2-methyl-6-dimethoxymethyl-thioxanthone, 2-methyl-6-(1,1-dimethoxybenzyl)-thioxanthone, 6-ethoxycarbonyl-2-methoxy-thioxanthone, 6-ethoxycarbonyl-2-methylthioxanthone, 1-ethoxycarbonyl-3-(1-methyl-1-morpholinoethyl)-thioxanthone, 2-morpholinomethylthioxanthone, 2-methyl-6-morpholinomethylthioxanthone, thioxanthone-2-carboxylic acid polyethylene glycol ester, etc.

Examples of fluorenone compounds include 9-fluorenone, 3,4-benzo-9-fluorenone, 2-dimethylamino-9-fluorenone, 2-methoxy-9-fluorenone, 2-chloro-9-fluorenone, 2,7-dichloro-9-fluorenone, 2-bromo-9-fluorenone, 2,7-dibromo-9-fluorenone, 2-nitro-9-fluorenone, and 2-acetoxy-9-fluorenone.

Examples of α-diketone compounds include benzil (a compound also known as diphenylethanedione or dibenzoyl).

Examples of intramolecular cleavage-type photoradical generators include benzyl ketal-based photoradical generators, α-aminoalkylphenone-based photoradical generators, α-hydroxyalkylphenone-based photoradical generators, α-hydroxyacetophenone-based photoradical generators, and acylphosphine oxide-based photoradical generators.

Examples of benzyl ketal photoradical generators include 2,2-dimethoxy-1,2-diphenylethan-1-one (Omnirad 651).

Examples of α-aminoalkylphenone photoradical generators include 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)-butanone-1 (Omnirad 369), 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one (Omnirad 907), and 2-dimethylamino-2-(4-methyl-benzyl)-1-(4-morpholin-4-yl-phenyl)-butan-1-one (Omnirad 379EG).

Examples of α-hydroxyalkylphenone-based photoradical generators include 1-hydroxycyclohexylphenyl ketone (Omnirad 184).

Examples of α-hydroxyacetophenone-based photoradical generators include 2-hydroxy-1-{4-[4-(2-hydroxy-2-methyl-propionyl)-benzyl]-phenyl}-2-methyl-propan-1-one (Omnirad 127) and 2-hydroxy-2-methyl-1-phenyl-propan-1-one (Omnirad 1173).

Examples of acylphosphine oxide photoradical generators include 2,4,6-trimethylbenzoyl-diphenyl-phosphine oxide (OmniradTPO H) and bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (Omnirad819).

The content of the photoradical generator may be, for example, 1% by mass or more, 5% by mass or more, or 10% by mass or more, and 30% by mass or less, 20% by mass or less, or 15% by mass or less, based on the mass of the resin part 2.

The resin portion 2 may further contain components (other components) other than the photo-fusible resin and the photo-radical generator. Examples of other components include culture medium components such as water, phosphate-buffered saline (PBS), amino acids, vitamins, inorganic salts, glucose, and serum.

The resin portion 2 preferably does not have cell adhesive properties. "Not having cell adhesive properties" means that cells do not adhere to the resin portion 2 under culture conditions. A resin portion 2 that does not have cell adhesive properties can be formed by using the above-mentioned photofusible resin. A culture substrate 10 in which the resin portion 2 does not have cell adhesive properties can be suitably used as a culture substrate for suspension culture, in which cells are cultured in a suspended state in a medium.

One example of the composition of the resin portion 2 is a mixed cured product of a polymer having a highly hydrophilic main skeleton and disulfide bonds in the main chain or crosslinking sites, and an intramolecular cleavage-type photoradical generator. This mixed cured product melts upon exposure to light, producing resin components that are easily removed with an aqueous solvent, and it is possible to form a resin portion 2 that does not have cell adhesive properties. The highly hydrophilic main skeleton in this mixed cured product may be derived from one selected from the group consisting of polyalkylene glycol, polyethyleneimine, (meth)acrylic polymers with hydroxyl groups in their side chains, polysaccharides, and proteins.

Another example of the composition of the resin portion 2 is a mixed cured product of a polymer having a highly hydrophobic main skeleton and disulfide bonds in the main chain or crosslinking sites, and an intramolecular cleavage-type photoradical generator. This mixed cured product melts upon exposure to light, producing resin components that are easily removed with an organic solvent, and it is possible to form a resin portion 2 to which cells do not adhere. The highly hydrophobic main skeleton in this mixed cured product may be composed of at least one skeleton selected from the group consisting of rubbers such as polybutadiene and polyisoprene, and silicones.

In the culture substrate 10, the resin portion 2 functions as a partition wall that forms the recess. A culture target can be cultured in the recess formed by the resin portion 2. The culture target is not particularly limited and may be various cells or tissues. The culture target may be, for example, a spheroid. A spheroid is a cell aggregate formed by the aggregation of cells. Spheroids are formed by aggregating cells and organizing them through three-dimensional culture (3D culture).

The bottom shape of the recess may be circular, elliptical, or polygonal (e.g., rectangular). The recess formed by the resin portion 2 on the surface of the substrate 1 can also function as a flow path.

The shape of the resin part 2 and the recess formed by the resin part 2 can be set appropriately depending on the type of culture subject and the use of the recess. For example, a resin part 2 having a step may be formed on the substrate 1. The shape of the resin part 2 and the recess formed by the resin part 2 can be controlled to the desired shape by changing the photomask and exposure conditions (exposure direction, etc.) during light irradiation.

Because the resin portion 2 contains a photo-fusible resin and its shape can be controlled by light irradiation, the culture substrate 10 allows for flexible design of the shape of the resin portion 2 and the recesses formed by the resin portion 2. In other words, the shape of the culture substrate surface of the culture substrate 10 can be easily changed depending on the culture subject.

The shape of the resin portion 2 and the recesses formed by the resin portion 2 can be changed by light irradiation even during culture. For example, with the culture substrate 10, it is easy to enlarge the recesses, which are the areas where cells are cultured, after a predetermined period of time has passed since the start of culture.

The culture substrate can be manufactured by a method including step A of irradiating light onto a portion of a resin portion formed on the substrate and containing a photofusible resin, and step B of removing the irradiated resin portion to form a recess on the substrate.

The light-irradiated resin portion is preferably water-soluble. In this case, in step B, the light-irradiated resin portion may be removed with an aqueous solvent. If the light-irradiated resin portion can be removed with an aqueous solvent, the culture substrate and its manufacturing method will be more desirable from an environmental perspective, as well as from the perspectives of safety, substrate damage, workability, etc.

Figure 2 is a schematic cross-sectional view illustrating an example of a method for producing a culture substrate and an example of a method for culturing cells using the culture substrate.

In the method for manufacturing the culture substrate shown in Figures 2(a) to (c), first, a substrate 1 is prepared, on whose surface a resin film 3 is provided. The resin film 3 can be formed on the main surface of the substrate by a method that includes applying or filling a photofusible resin or its precursor onto the substrate, and curing the photofusible resin or its precursor.

Curing conditions can be set appropriately depending on the type of photofusible resin, etc. For example, the heating temperature for curing may be, for example, 40 to 120°C, or 60 to 110°C, or 80 to 100°C. The heating time for curing may be, for example, 0.1 to 24 hours, and may be 16 hours or less, 12 hours or less, 8 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less.

Water 4 may be present on part or the entire surface of the resin film 3 opposite the surface that contacts the substrate. By having water present on the surface of the resin film 3, the photofusion reaction of the photofusible resin is less likely to be inhibited by oxygen.

As shown in Figure 2(a), light (actinic rays) L is irradiated in a pattern onto predetermined portions of the resin film 3 through a photomask 5. After light irradiation, the irradiated portions on the substrate melt, forming molten portions 6 (irradiated resin portions), as shown in Figure 2(b). The portions not irradiated with light (resin portions 2) form partition walls that form recesses.

The wavelength of the light L can be appropriately set depending on the type of photofusible resin, etc. The light L may be, for example, light containing light with a wavelength of 365 nm. The exposure dose of light irradiation may be, for example, 1000 mJ/cm ² or more. In this specification, the exposure dose means the product of the illuminance (mW/cm ² ) and the irradiation time (seconds). Light irradiation may be performed directly on the object to be irradiated or may be performed through glass or the like. The light source used for light irradiation is not particularly limited, and examples include an LED lamp, a mercury lamp (low pressure, high pressure, ultra-high pressure, etc.), a metal halide lamp, an excimer lamp, a xenon lamp, etc. Among these, the light source used for light irradiation may be an LED lamp, a mercury lamp, or a metal halide lamp.

As shown in Figure 2(c), the melted portion 6 on the substrate is removed by immersing it in water 4. The melted portion 6 can be removed, for example, by immersing it in a container 7 containing water 4 for 10 minutes or more. The melted portion 6 can also be removed using a solvent other than water. The melted portion 6 can also be removed by blotting.

The melted portion 6 may be removed using an aqueous solvent. Examples of aqueous solvents include water and a mixed solvent of water and a hydrophilic organic solvent. In the mixed solvent of water and a hydrophilic organic solvent, the water content may be, for example, 80% by mass or more. The aqueous solvent may contain, for example, an organic acid. The aqueous solvent may also be phosphate-buffered saline (PBS), a culture medium, or the like.

Examples of water include tap water, natural water, purified water, distilled water, ion-exchanged water, pure water, and ultrapure water (such as Milli-Q water). Milli-Q water refers to ultrapure water obtained using a Milli-Q water production system manufactured by Merck Millipore (Merck). Since water has reduced impurities, it may be purified water, distilled water, ion-exchanged water, pure water, or ultrapure water.

Hydrophilic organic solvents include alcohols such as methanol, ethanol, 2-propanol, and 1,2-propanediol; and glycol ethers such as ethylene glycol monomethyl ether, propylene glycol monomethyl ether, ethyl cellosolve, propylene glycol monopropyl ether, propylene glycol monoisopropyl ether, butyl cellosolve, ethylene glycol monoisobutyl ether, propylene glycol monobutyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, and dipropylene glycol monomethyl ether.

The culture substrate 10 may be washed, sterilized, or otherwise treated as needed. Washing can be performed using, for example, water or phosphate-buffered saline (PBS). Washing may be performed once or multiple times.

Figures 2(d) and (e) show a method for culturing cells using the above-mentioned culture substrate. In this method, as shown in Figure 2(d), before starting cell culture, culture medium 8 is added to culture substrate 10. The culture medium 8 can be selected appropriately depending on the type of cells to be used, etc.

As shown in Figure 2(e), spheroids 9, which are the subject of culture, are cultured in recesses formed by the resin portion 2 on the surface of the substrate 1. Any ordinary culture method can be used, with no particular restrictions. For example, suspension culture can be used as the culture method.

In the culture substrate 10, the shape of the resin portion 2, which serves as a partition, can be changed by light irradiation. Therefore, during cell culture, the shape of the recess, which is the area where cells are cultured, can be changed by light irradiation. In other words, the process of culturing cells using the culture substrate 10 may include irradiating light onto a portion of the resin portion 2 and removing the irradiated resin portion, thereby changing the shape of the resin portion 2.

Figure 3 is a schematic cross-sectional view showing an example of a method for manufacturing a culture substrate having a resin portion 2 with a step.

As shown in FIG. 3(a), light L1 is irradiated in a pattern onto predetermined portions of the resin film 3 from the resin film 3 side through a photomask 5a. The irradiation with light L1 may be performed while water 4 is present on the resin film 3.

Next, as shown in Figure 3(b), light L2 is irradiated in a pattern onto predetermined portions of the resin film 3 from the substrate 1 side through a photomask 5b. The shape of the resin portion 2 formed can be controlled by adjusting the shape of the photomasks 5a and 5b and the light irradiation conditions, etc.

For example, as shown in Figure 3(c), by adjusting the shape of the photomasks 5a and 5b and the light irradiation conditions, a culture substrate comprising a resin portion 2 with a step and a substrate 1 can be obtained.

The culture substrate can also have the configuration shown in Figure 4. The culture substrate 10' shown in Figure 4 comprises a substrate 1 and a resin portion 2 that is provided on the surface of the substrate 1 and forms a recess. The resin portion 2 is provided over the entire main surface of the culture substrate 10'. The resin portion 2 forms the partition wall and bottom surface of the recess.

1...substrate, 2...resin part, 3...resin film, 4...water, 5, 5a, 5b...photomask, 6...melted part, 7...container, 8...culture medium, 9...spheroid, 10, 10'...culture substrate, L, L1, L2...light.

Claims (5)

a substrate; and a resin portion provided on a surface of the substrate and containing a photo-fusible resin ;
A culture substrate , wherein the photo-fusible resin has a disulfide bond . a step A of irradiating a part of a resin portion formed on a substrate and containing a photo-fusible resin with light;
and a step B of removing the resin portion irradiated with light to form a recess on the base material,
A method for producing a culture substrate, wherein the photo-fusible resin has a disulfide bond . the resin portion irradiated with light exhibits water solubility,
The method according to claim 2 , wherein in step B, the resin portion irradiated with light is removed with a water-based solvent. A method for culturing cells, comprising the step of culturing cells using the culture substrate according to claim 1 . The method according to claim 4 , wherein the culturing step includes irradiating a part of the resin portion with light and removing the irradiated resin portion to change the shape of the resin portion.