Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0062—General methods for three-dimensional culture
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
  • B29C64/10—Processes of additive manufacturing
  • B29C64/106—Processes of additive manufacturing using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material
  • B29C64/112—Processes of additive manufacturing using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material using individual droplets, e.g. from jetting heads
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
  • B29C64/40—Structures for supporting 3D objects during manufacture and intended to be sacrificed after completion thereof
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B33—ADDITIVE MANUFACTURING TECHNOLOGY
  • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
  • B33Y10/00—Processes of additive manufacturing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B33—ADDITIVE MANUFACTURING TECHNOLOGY
  • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
  • B33Y70/00—Materials specially adapted for additive manufacturing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B33—ADDITIVE MANUFACTURING TECHNOLOGY
  • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
  • B33Y80/00—Products made by additive manufacturing
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F126/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F226/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen
  • C08F226/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen by a single or double bond to nitrogen
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L39/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen; Compositions of derivatives of such polymers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29L—INDEXING SCHEME ASSOCIATED WITH SUBCLASS B29C, RELATING TO PARTICULAR ARTICLES
  • B29L2031/00—Other particular articles
  • B29L2031/753—Medical equipment; Accessories therefor
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F2800/00—Copolymer characterised by the proportions of the comonomers expressed
  • C08F2800/20—Copolymer characterised by the proportions of the comonomers expressed as weight or mass percentages
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F2810/00—Chemical modification of a polymer
  • C08F2810/20—Chemical modification of a polymer leading to a crosslinking, either explicitly or inherently
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F2810/00—Chemical modification of a polymer
  • C08F2810/50—Chemical modification of a polymer wherein the polymer is a copolymer and the modification is taking place only on one or more of the monomers present in minority

Definitions

• the present invention relates to a composition for 3D printing supports or 3D cell culture supports.
• 3D printing refers to three-dimensional modeling based on 3D model data
• 3D bioprinting which uses this technology to produce three-dimensional cell patterns, is attracting attention in the field of regenerative medicine.
• a three-dimensional cell pattern needs to be as complex and flexible as an actual living tissue or organ, and in order to create such a pattern, it is necessary to 3D bioprinting is performed using a support (a support exhibiting Bingham plastic behavior) that becomes liquid during drawing (at the time of drawing) and becomes solid when stress-free after drawing (patent document 1).
• Patent Document 2 a 3D cell culture support using anionic microgel particles derived from methacrylic acid or the like is known, and 3D bioprinting using this support has also been proposed (Patent Document 2).
• Patent Document 2 the above-mentioned support described in Patent Document 2 has insufficient salt resistance, and when salt or ions are added, charge shielding or chelation occurs, resulting in a decrease in viscosity and poor shape retention as a support.
• it is difficult to use ink for 3D printing that uses ink that hardens in response to calcium ions, for example.
• the problem to be solved by the present invention is to provide a composition that is useful as a 3D printing support or a 3D cell culture support and has excellent salt tolerance.
• composition for 3D printing support or 3D cell culture support containing the following components (A) and (B) (hereinafter, composition of the present invention, composition for 3D printing support of the present invention or (also referred to as 3D cell culture support composition).
• R 1 and R 2 each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, or R 1 and R 2 combine with each other to form a hydrogen atom having 3 to 10 carbon atoms. may form a ring structure.
• R 3 and R 4 each independently represent a hydrogen atom or a methyl group
• R 5 represents an alkylene group having 2 to 4 carbon atoms
• n is an average value of 1 to 4. Shows 1000.
• the structural unit derived from the crosslinkable monomer is a structural unit derived from a vinyl crosslinkable monomer, a structural unit derived from an allyl crosslinkable monomer, a structural unit derived from a (meth)acrylate crosslinkable monomer, and (meth)acrylamide-based crosslinkable monomer, or one or more structural units derived from a (meth)acrylamide-based crosslinkable monomer.
• ⁇ 5> The composition according to any one of ⁇ 1> to ⁇ 4>, wherein the polymer is a particulate polymer.
• Viscosity change rate (%) ⁇ (Viscosity before addition of NaCl) - (Viscosity after addition of NaCl) ⁇ /(Viscosity before addition of NaCl) x 100 ... ( ⁇ )
• the viscosity before adding NaCl means the viscosity (mPa s) of the composition when measured using a rotational viscometer at a measurement temperature of 23 ° C. and a shear rate of 100 sec -1 .
• composition (II) of the present invention containing the following components (A2) and (B).
• A2 When the viscosity of the dispersion was measured using a rotational viscometer at a measurement temperature of 23°C and a shear rate of 100 sec -1 while being dispersed in pure water, the viscosity of the dispersion was 1000 mPa ⁇ s.
• a nonionic polymer whose pure water content reaching a smaller value is 70% by mass or more
• a method for manufacturing a three-dimensional structure comprising the following steps (i) and (ii). (i) Filling a container with the composition according to any one of ⁇ 1> to ⁇ 8> (ii) Bringing the second composition into contact with the composition filled in the container in step (i)
• step (ii) is a step of injecting the second composition into the composition filled in the container in step (i) while applying shear.
• step (ii) is a step of injecting the second composition into the composition filled in the container in step (i) while applying shear.
• step (ii) is a step of injecting the second composition into the composition filled in the container in step (i) while applying shear.
• the second composition contains cells and an aqueous medium.
• the second composition further contains an extracellular matrix.
• ⁇ 13> The manufacturing method according to any one of ⁇ 9> to ⁇ 12>, wherein the three-dimensional structure is an organoid or a spheroid.
• ⁇ 14> A three-dimensional structure obtained by the manufacturing method according to any one of ⁇ 9> to ⁇ 13>.
• composition of the present invention is useful as a 3D printing support or a 3D cell culture support, and has excellent salt tolerance.
• FIG. 3 is a diagram showing a test pattern when using the support composition of Example 1.
• 3 is a diagram showing a test pattern when using the support composition of Comparative Example 1.
• FIG. 3 is a diagram showing a test pattern when using the support composition of Comparative Example 1.
• composition The composition for 3D printing supports or 3D cell culture supports of the present invention contains the following components (A) and (B).
• R 1 and R 2 each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, or R 1 and R 2 combine with each other to form a hydrogen atom having 3 to 10 carbon atoms. may form a ring structure.
• composition of the present invention contains (A) a polymer having a structural unit represented by the above formula (1).
• component (A) By containing such component (A), excellent salt tolerance can be obtained while satisfying 3D printing performance and ease of three-dimensional cell culture.
• the number of carbon atoms in the alkyl groups represented by R 1 and R 2 is preferably 1 to 8, more preferably 1, in order to obtain the desired viscosity when stress is applied while satisfying salt resistance. -4, particularly preferably 1 or 2.
• the alkyl group may be linear or branched. Examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group, heptyl group, octyl group, and nonyl group. , decyl group, etc.
• R 1 is preferably a hydrogen atom or an alkyl group having 1 to 10 carbon atoms in order to obtain the desired viscosity when stress is applied while satisfying salt resistance. More preferred is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and particularly preferred is a hydrogen atom or an alkyl group having 1 or 2 carbon atoms.
• R 2 a hydrogen atom is preferable in order to obtain a desired viscosity upon application of stress.
• the ring structure formed by bonding R 1 and R 2 to each other preferably has 4 to 8 carbon atoms, more preferably 4 to 6 carbon atoms.
• the structural unit (1) is the structural unit represented by the following formula (1-1), the following formula ( At least one kind selected from the structural units represented by the following formula (1-2) and the following formula (1-3) is preferred, and the structural unit represented by the following formula (1-1) is particularly preferred.
• Examples of monomers inducing the structural unit (1) include N-vinylformamide, N-vinylacetamide, N-vinylpropionamide, N-vinylbutyramide, N-vinylisobutyramide, and N-vinyl-2-methylbutane.
• the content ratio of the structural unit (1) is determined so that the content of the structural unit (1) in the polymer of component (A) is determined in order to obtain the desired viscosity when stress is applied while satisfying the salt resistance, and to improve the printing performance and to increase the ease of culturing. It is preferably 50% by mass or more, more preferably 55% by mass or more, particularly preferably 64% by mass or more based on the structural unit, and also achieves desired viscosity characteristics before and after applying stress and satisfies ease of manufacture. Therefore, it is preferably 95% by mass or less, more preferably 90% by mass or less, particularly preferably 85% by mass or less, based on the total structural units in the polymer of component (A).
• the specific range is preferably 50% by mass or more and 95% by mass or less, more preferably 55% by mass or more and 90% by mass or less, and 64% by mass or more and 85% by mass or less, based on all structural units in the polymer of component (A). Particularly preferably less than % by mass.
• the content ratio of the structural unit (1) can be measured by pyrolysis gas chromatography mass spectrometry (PyGC-MS), CHN elemental analysis, 1 H-NMR, 13 C-NMR, etc.
• the polymer of component (A) is designed to obtain the desired viscosity characteristics before and after stress application by adjusting the interaction between the polymers, and to satisfy ease of manufacture. Those having a structural unit represented by formula (2) are preferred.
• R 3 and R 4 each independently represent a hydrogen atom or a methyl group
• R 5 represents an alkylene group having 2 to 4 carbon atoms
• n is an average value of 1 to 4. Shows 1000.
• R 5 represents an alkylene group having 2 to 4 carbon atoms, and n R 5 may be the same or different.
• the number of carbon atoms in the alkylene group represented by R 5 is preferably 2 or 3, more preferably 2.
• the alkylene group represented by R 5 may be linear or branched. Examples of the alkylene group include ethane-1,2-diyl group, propane-1,2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group, and butane-1,2-diyl group. group, butane-1,3-diyl group, butane-1,4-diyl group, and the like. Among these, ethane-1,2-diyl group is preferred.
• the average value of n is 1 to 1000, but in order to obtain desired viscosity characteristics before and after applying stress to improve printing performance, to increase ease of culturing, and to satisfy ease of manufacture, the average value is set.
• the average value is preferably 2 or more, the average value is more preferably 4 or more, the average value is even more preferably 8 or more, and the average value is particularly preferably 10 or more.
• desired viscosity characteristics can be obtained before and after applying stress to improve printing performance.
• the average value is preferably 500 or less, more preferably the average value is 250 or less, even more preferably the average value is 100 or less, and the average value is 50 or less.
• the average value is particularly preferably 35 or less.
• the average value is preferably 2 to 500 in order to obtain desired viscosity characteristics before and after applying stress to improve printing performance, to increase ease of culturing, and to satisfy ease of manufacture. , more preferably an average value of 4 to 250, still more preferably an average value of 8 to 100, even more preferably an average value of 10 to 50, particularly preferably an average value of 10 to 35.
• n in formula (2) is set to an average value of 35 or less, printing performance and ease of culturing are particularly good.
• each "average value" in this specification can be measured by NMR.
• R 4 in formula (2) is a methyl group
• 1 H-NMR is measured for the structure of formula (2) above, and an alkylene group having 2 to 4 carbon atoms represented by R 5 and The average value of n can be calculated by comparing the integral values of each proton peak with the methyl group represented by R 4 .
• the content ratio of the structural unit (1) described above is 64% by mass or more, and n in formula (2) is set to 35 or less on average, the content ratio of the structural unit (1) is 64% by mass or more.
• the content is 85% by mass or less and n in formula (2) is set to an average value of 10 to 35, printing performance and ease of culturing become particularly good.
• Examples of monomers inducing the structural unit (2) include ethylene glycol mono(meth)acrylate, diethylene glycol mono(meth)acrylate, triethylene glycol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, and (meth)acrylate.
• Examples include 2-methoxyethyl acid, methoxydiethylene glycol (meth)acrylate, methoxytriethylene glycol (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, polypropylene glycol mono(meth)acrylate, methoxypolypropylene glycol (meth)acrylate, and the like. These monomers can be used alone or in combination of two or more.
• the content ratio of the structural unit (2) is preferably 5 mass based on all the structural units in the polymer of component (A) in order to obtain desired viscosity characteristics before and after stress application and to satisfy ease of manufacture. % or more, more preferably 7% by mass or more, particularly preferably 10% by mass or more, and also for improving printing performance, increasing ease of culturing, and maintaining the shape of a three-dimensional structure by obtaining sufficient viscosity.
• the content is preferably 50% by mass or less, more preferably 40% by mass or less, particularly preferably 30% by mass or less, based on the total structural units in the polymer of component (A).
• the specific range is preferably 5% by mass or more and 50% by mass or less, more preferably 7% by mass or more and 40% by mass or less, and 10% by mass or more and 30% by mass or less, based on all structural units in the polymer of component (A). Particularly preferably less than % by mass.
• the content ratio of the structural unit (2) may be measured in the same manner as the content ratio of the structural unit (1).
• the content of the structural unit (2) is 30% by mass or less, and n in the above formula (2) is set to 35 or less on average, the content of the structural unit (2) is 10% by mass or less.
• the content is 30% by mass or less and n in the above-mentioned formula (2) is set to 10 to 35 on average, printing performance and ease of culturing become particularly good.
• the content ratio [(1):(2)] of structural units (1) and structural units (2) contained in the polymer of component (A) is determined to obtain desired viscosity characteristics before and after stress application and to achieve printing performance.
• the mass ratio is preferably 50:50 to 95:5, more preferably 58:42 to 92:8, and 68:32. ⁇ 89:11 is particularly preferred.
• the content ratio [(1):(2)] is 68:32 or more, printing performance and ease of culturing are particularly good.
• the content ratio [(1):(2)] is set to 68:32 or more, and n in the above formula (2) is set to 35 or less on average
• the content ratio [(1):(2)] ] is set to 68:32 to 89:11
• n in the above-mentioned formula (2) is set to an average value of 10 to 35, printing performance and ease of culturing become particularly good.
• the polymer of component (A) preferably has a structural unit derived from a crosslinkable monomer in addition to the structural unit (1) in order to reduce spinnability and obtain the desired viscosity before and after stress application.
• the polymer of component (A) is preferably one having, in addition to the structural unit (1), the above-mentioned structural unit (2) and a structural unit derived from a crosslinkable monomer.
• Structural units derived from crosslinkable monomers include structural units derived from vinyl crosslinkable monomers, structural units derived from allyl crosslinkable monomers, structural units derived from (meth)acrylate crosslinkable monomers, and structural units derived from (meth)acrylate crosslinkable monomers. ) One or more types selected from structural units derived from acrylamide crosslinkable monomers can be mentioned. Further, as the crosslinking monomer, a di- to penta-functional cross-linking monomer is preferable, and a di- to tetra-functional cross-linking monomer is more preferable.
• crosslinking monomers in order to obtain high visibility, allyl crosslinking monomers and (meth)acrylate crosslinking monomers are preferred, and allyl crosslinking monomers are more preferred. Furthermore, as the crosslinking monomer, nonionic crosslinking monomers are preferred in order to improve salt resistance.
• a crosslinkable monomer having a degradable partial structure may be preferable.
• the degradable partial structure include a disulfide bond, an ester bond, a thioester bond, an acetal bond, a benzyl ester structure, and a nitrobenzyl structure.
• a crosslinkable monomer having a disulfide bond is used and a reducing agent such as dithiothreitol or glutathione is added, a three-dimensional structure can be easily obtained from a container.
• an allylic crosslinking monomer having a degradable partial structure is preferable because of its high availability.
• vinyl crosslinking monomers include aromatic vinyl crosslinking monomers such as divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylene, and divinylethylbenzene; N,N'-methylenebis(N-vinylacetamide), N, N'-ethylenebis(N-vinylacetamide), N,N'-propylenebis(N-vinylacetamide), N,N'-butylenebis(N-vinylacetamide), N,N'-hexylenebis(N-vinylacetamide) ), N,N'-alkylene bis(N-vinylacetamide); N,N'-alkylene bis(N-vinylformamide) such as N,N'-butylene bis(N-vinylformamide), divinyl ether, Examples include N,N'-(diacetyl)-N,N'-(divinyl)-1,3-bis(aminomethyl)cyclohexan
• allyl-based crosslinking monomer examples include pentaerythritol diallyl ether, pentaerythritol triallyl ether, pentaerythritol tetraallyl ether, tetraallyloxyethane, triallyl phosphate, trimethylolpropane diallyl ether, trimethylolpropane triallyl ether, and allyl chloride.
• Examples of (meth)acrylate crosslinking monomers include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, and polyethylene glycol di(meth)acrylate.
• meth)acrylate propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, tetrapropylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, 1,4 -Butanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, glycerin di(meth)acrylate, trimethylolethane di(meth)acrylate, trimethylolpropane di(meth)acrylate, trimethylolpropane di(meth)acrylate (meth)acrylate, butanetriol di(meth)acrylate, pentaerythritol di(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, glucose di(meth)acrylate, glucose
• Examples of (meth)acrylamide crosslinking monomers include N,N'-methylenebis(meth)acrylamide, N,N'-ethylenebis(meth)acrylamide, N,N'-propylenebis(meth)acrylamide, N, Examples include N'-butylenebis(meth)acrylamide, N,N'-hexylenebis(meth)acrylamide, N,N'-bis((meth)acryloyl)cystamine, and the like. These can be used alone or in combination of two or more.
• the content ratio of the structural unit derived from the crosslinkable monomer (hereinafter also referred to as "structural unit (3)”) is determined in order to reduce the spinnability and obtain the desired viscosity before and after applying stress.
• 5% by mass or more, and in order to maintain a high degree of swelling and obtain high viscosity preferably 10% by mass or less, more preferably 7% by mass, based on all structural units in the polymer of component (A).
• the content is particularly preferably 5% by mass or less.
• the specific range is preferably 0.1% by mass or more and 10% by mass or less, more preferably 0.5% by mass or more and 7% by mass or less, based on the total structural units in the polymer of component (A).
• the content is more preferably 2% by mass or more and 7% by mass or less, even more preferably 2% by mass or more and 7% by mass or less, and particularly preferably 2.5% by mass or more and 5% by mass or less.
• the content ratio of the structural unit (3) may be measured in the same manner as the content ratio of the structural unit (1).
• the content ratio [(1):(3)] of structural units (1) and structural units (3) contained in the polymer of component (A) is based on transparency (visibility during printing and cell culture).
• the mass ratio is preferably 83:17 to 99.99:0.01, more preferably 89:11 to 99.9:0.1, and 93: More preferably 7 to 99:1, particularly preferably 94:6 to 99:1.
• Transparency is particularly good when the content ratio [(1):(3)] is 94:6 or more.
• the polymer of component (A) may have structural units other than the structural unit (1), the structural unit (2), and the structural unit derived from the crosslinkable monomer.
• structural units derived from non-crosslinkable monomers other than structural unit (1) and structural unit (2) may be mentioned, and preferably structural units derived from nonionic non-crosslinkable monomers.
• non-crosslinking monomers include methyl (meth)acrylate, ethyl (meth)acrylate, hydroxymethyl (meth)acrylate, 2,3-dihydroxypropyl (meth)acrylate, and (meth)acrylamide.
• polymer of component (A) examples include particulate polymers, monolithic polymers, plate-like polymers, film-like polymers, fibrous polymers, and chip-like polymers. To obtain viscosity, particulate polymers are preferred, and gel particulate polymers are more preferred. As the polymer of component (A), nonionic polymers are preferred in order to improve salt resistance.
• the volume average particle diameter is preferably 0.05 to 100 ⁇ m, more preferably 0.1 to 50 ⁇ m. Further, the coefficient of variation of the volume average particle diameter is preferably 30% or less, more preferably 25% or less. Note that the volume average particle diameter and coefficient of variation can be measured by observation using a liquid atomic force microscope, observation using a phase contrast microscope, laser diffraction/scattering particle size distribution measurement, and the like. Alternatively, after fluorescently staining the particles, the measurement can be performed by confocal laser microscopy or the like.
• the polymer of component (A) was dispersed in pure water using a rotational viscometer (for example, ViscoQC 300R manufactured by Anton Paar) at a measurement temperature of 23°C and a shear rate of 100sec -1 .
• the pure water content at which the viscosity of the dispersion reaches a value smaller than 1000 mPa s when the viscosity of the dispersion is measured under the following conditions is preferably 70% by mass or more, more preferably 80% by mass or more, and 90% by mass. % or more is more preferable, and 95% or more by mass is particularly preferable. Further, it is usually 99.9% by mass or less.
• the pure water content may be measured according to the method described in the Examples described later.
• the polymer of component (A) can be produced by appropriately combining known methods described in JP-A-10-226715, JP-A-2002-239380, and the like.
• the content ratio of the polymer of component (A) is preferably 0.1% by mass or more, more preferably 0.2% by mass or more, particularly Preferably it is 0.5% by mass or more, and in order to increase the ease of three-dimensional culture and improve visibility, it is preferably 30% by mass or less, more preferably 20% by mass in the composition of the present invention. % or less, particularly preferably 10% by mass or less.
• the specific range is preferably 0.1% by mass or more and 30% by mass or less, more preferably 0.2% by mass or more and 20% by mass or less, and 0.5% by mass or more and 10% by mass in the composition of the present invention. The following are particularly preferred.
• composition of the present invention contains (B) an aqueous medium.
• aqueous medium include one or more selected from water, alcohol, and a culture medium, and water, a culture medium, a mixture of water and alcohol, and a mixture of water and a culture medium are preferred.
• component (B) is particularly preferably a medium or a mixture of water and a medium.
• the alcohol is preferably a lower alcohol, more preferably a linear or branched monohydric alcohol having 1 to 6 carbon atoms.
• examples include ethanol, isopropanol, n-propanol, and the like. One of these can be used alone or two or more can be used in combination.
• the content of water is preferably 80% by mass or more and less than 100% by mass, more preferably 90% by mass or more and less than 100% by mass, in order to act as a stable medium when used for cell culture. Less than % by mass.
• the medium is not particularly limited as long as cells can survive or grow, but examples include Eagle's medium, Ham's medium, Fisher's medium, Dulbecco's modified MEM (DMEM) medium, MEM medium, F12 medium, and RPMI1640 medium. Examples include medium, MCDB104 medium, 199 medium, MCDB153 medium, L15 medium, SkBM medium, Basal medium, and a medium containing a mixed medium thereof. Further, the medium may be either a serum medium or a serum-free medium. Serum includes fetal bovine serum. Additionally, minerals, carbon sources (glucose, carbon dioxide, etc.), nitrogen sources (glutamine, etc.), antibiotics, vitamin sources, mineral sources, proteins, peptides, etc. may be added to the medium.
• polyvalent ion sources are preferred.
• multivalent ion sources include calcium ion sources such as calcium carbonate, calcium hydrogen phosphate, and calcium chloride. According to the present invention, three-dimensional cell culture can be efficiently performed even when such a multivalent ion source is used as a mineral.
• the medium used in the present invention may be a medium containing the above-mentioned plurality of mediums or additives.
• the content of the aqueous medium as component (B) is preferably 70% by mass or more, more preferably 80% by mass in the composition of the present invention, in order to facilitate three-dimensional culture and improve visibility. Above, it is particularly preferably 90% by mass or more, and in order to improve the shape retention of the three-dimensional structure, preferably 99.9% by mass or less, more preferably 99.8% by mass in the composition of the present invention.
• the content is particularly preferably 99.5% by mass or less.
• the specific range is preferably 70% by mass or more and 99.9% by mass or less, more preferably 80% by mass or more and 99.8% by mass, and 90% by mass or more and 99.5% by mass in the composition of the present invention. The following are particularly preferred.
• the content ratio [(A):(B)] of the component (A) polymer and the component (B) aqueous medium contained in the composition of the present invention is determined by the shape retention of the three-dimensional structure and the three-dimensional culture.
• the mass ratio is preferably 0.1:99.9 to 30:70, more preferably 0.2:99.8 to 20:80, and 0.5:99.5. to 10:90 is particularly preferred.
• composition of the present invention may contain components other than the above (hereinafter also referred to as other components) as necessary.
• Other components include surfactants, tonicity agents (eg, sodium chloride), chelating agents, pH adjusters, buffers, thickeners, stabilizers, and the like. One of these can be used alone or two or more can be used in combination.
• the composition of the present invention is preferably a plastic fluid composition.
• plastic fluid is a type of non-Newtonian fluid, and means a fluid that does not flow until a certain shear stress (yield stress) is applied.
• a slurry composition is preferable.
• a slurry refers to a mixture of a solid substance and a liquid.
• the viscosity when measured using a rotational viscometer (for example, rotational viscometer ViscoQC 300R manufactured by Anton Paar) at a measurement temperature of 23°C and a shear rate of 100 sec -1 is as follows: 1 mPa ⁇ s or more and 1 ⁇ 10 5 mPa ⁇ s or less is preferable, 5 mPa ⁇ s or more and 5 ⁇ 10 4 mPa ⁇ s or less is more preferable, 10 mPa ⁇ s or more and 1 ⁇ 10 4 mPa ⁇ s or less is even more preferable, and 50 mPa ⁇ s It is particularly preferable that the pressure is above 5 ⁇ 10 3 mPa ⁇ s or below.
• a rotational viscometer for example, rotational viscometer ViscoQC 300R manufactured by Anton Paar
• the viscosity when measured at a measurement temperature of 23° C. and a shear rate of 0.1 sec -1 is preferably 1 ⁇ 10 3 mPa ⁇ s or more and 1 ⁇ 10 8 mPa ⁇ s or less, and 5 ⁇ 10 3 mPa ⁇ s. s or more and 5 ⁇ 10 7 mPa ⁇ s or less, more preferably 1 ⁇ 10 4 mPa ⁇ s or more and 1 ⁇ 10 7 mPa ⁇ s or less, and 5 ⁇ 10 4 mPa ⁇ s or more and 5 ⁇ 10 6 mPa ⁇ s or less is particularly preferred.
• the above viscosity can be determined according to JIS Z 8803:2011. Specifically, it may be measured according to the method described in Examples described later.
• the viscosity The ratio (X/Y) of viscosity Y when measured using the same viscometer at a measurement temperature of 23°C and a shear rate of 100 sec -1 is preferably 10 or more, more preferably 50 or more, and 75
• the number is more preferably 100 or more, particularly preferably 100 or more. Moreover, it is usually 10,000 or less. The larger this ratio is, the more desirable the viscosity characteristics are, similar to plastic fluid.
• the viscosity change rate when NaCl is added is preferably -50% or more and 50% or less, more preferably -30% or more and 20% or less, More preferably -20% or more and 10% or less, particularly preferably -10% or more and 5% or less.
• Viscosity change rate (%) ⁇ (Viscosity before addition of NaCl) - (Viscosity after addition of NaCl) ⁇ /(Viscosity before addition of NaCl) x 100 ...
• the viscosity before addition of NaCl was measured using a rotational viscometer (for example, ViscoQC 300R, a rotational viscometer manufactured by Anton Paar) at a measurement temperature of 23°C and a shear rate of 100 sec -1
• the viscosity after adding NaCl refers to the viscosity (mPa ⁇ s) of the composition when NaCl is added to the composition so that the NaCl concentration is 0.15 mol/L, and after 60 minutes, the composition is rotated. It means the viscosity (mPa ⁇ s) measured using a type viscometer at a measurement temperature of 23°C.
• the above-mentioned viscosity change rate may be measured according to the method described in Examples described later.
• the viscosity of the composition of the present invention after addition of NaCl when measured at a measurement temperature of 23° C. and a shear rate of 100 sec -1 is preferably 1 mPa ⁇ s or more and 1 ⁇ 10 5 mPa ⁇ s or less, and 5 mPa ⁇ s or less. ⁇ S or more and 5 ⁇ 10 4 mPa ⁇ s or less are more preferable, 10 mPa ⁇ s or more and 1 ⁇ 10 4 mPa ⁇ s or less are still more preferable, and 50 mPa ⁇ s or more and 5 ⁇ 10 3 mPa ⁇ s or less are particularly preferable.
• composition (II) The present invention also provides a composition for a 3D printing support or a 3D cell culture support, which contains the following component (A2) and component (B).
• A2 While dispersing in pure water, measure the viscosity of the dispersion using a rotational viscometer (for example, Anton Paar's rotational viscometer ViscoQC 300R) at a temperature of 23°C and a shear rate of 100 sec -1 .
• a nonionic polymer having a pure water content of 70% by mass or more, at which the viscosity of the dispersion reaches a value smaller than 1000 mPa ⁇ s when measured.
• B Aqueous medium
• the polymer of component (A2) was dispersed in pure water using a rotational viscometer (for example, ViscoQC 300R manufactured by Anton Paar) at a measurement temperature of 23°C and a shear rate of 100 sec -1 .
• the pure water content at which the viscosity of the dispersion reaches a value smaller than 1000 mPa s when the viscosity of the dispersion is measured under the following conditions is preferably 70% by mass or more, more preferably 80% by mass or more, and 90% by mass. % or more is more preferable, and 95% or more by mass is particularly preferable. Further, it is usually 99.9% by mass or less.
• the pure water content may be measured according to the method described in the Examples described later.
• the polymer of component (A2) is preferably one having the structural unit (1).
• the structural unit (1) is the same as that of the polymer of component (A).
• the same polymer as the above-mentioned component (A) is preferable.
• the composition (II) of the present invention is preferably the same as the composition of the present invention.
• the aqueous medium (B) contained in the composition (II) of the present invention is the same as the aqueous medium (B) contained in the composition of the present invention.
• composition of the present invention and the composition (II) of the present invention are useful as 3D printing supports or 3D cell culture supports.
• it has excellent 3D printing performance and ease of 3D cell culture, and is less susceptible to contaminants when used in 3D printing or 3D cell culture. Also suitable for manufacturing original structures.
• due to its high transparency, visibility during printing and cell culture is high.
• the composition of the present invention and the composition (II) of the present invention have excellent salt tolerance. Therefore, even when salts or ions are added, the viscosity does not decrease easily, and when used as a 3D cell culture support and a multivalent ion source (such as a calcium ion source) is added, the ions can easily reach the cells and the cells can be grown. Cheap.
• 3D printing support is used for 3D printing that can create a desired structure in three-dimensional space, and is configured so that it can be placed to embed structure material. This means a material that supports a structure material in three-dimensional space.
• 3D cell culture support is used for 3D cell culture in which desired cells can be cultured in a three-dimensional space, and when culturing the desired cells in a three-dimensional space, the cells are refers to a material that is arranged to embed a composition containing cells and supports the composition containing cells in three-dimensional space. Note that "for 3D printing supports or for 3D cell culture supports” is a concept that includes applications for both 3D printing supports and 3D cell culture supports.
• the three-dimensional structure manufacturing method of the present invention includes the following steps (i) and (ii).
• the method for producing a three-dimensional structure of the present invention is similar to the known 3D printing method and 3D bioprinting method, except that the composition of the present invention or the composition (II) of the present invention is used.
• it can be manufactured by an inkjet method, a material extrusion method, a stereolithography method, etc. Specifically, this may be done with reference to the descriptions in WO2018/187595 pamphlet, WO2018/165584 pamphlet, WO2015/129881 pamphlet, etc.
• Step of filling a container with the composition of the present invention or composition (II) of the present invention ii) Step of bringing the second composition into contact with the composition filled in the container in step (i)
• Step (i) Examples of containers used in step (i) include glass containers, plastic containers, metal containers, etc., but plastic containers are preferred because they allow the status of 3D printing to be confirmed and can be directly used for cell culture. is preferred. Also, a translucent or transparent container is preferred.
• the second composition may be anything that acts as an ink or bioink in 3D printing or three-dimensional cell culture, and includes, for example, a curable composition as well as one containing cells and an aqueous medium.
• a curable composition as well as one containing cells and an aqueous medium.
• three-dimensional cells can be produced as a three-dimensional structure. Examples of three-dimensional cells include organoids, spheroids, embryoid bodies, tumors, cysts, and fine tissues. Organoids and spheroids are preferred, and organoids are particularly preferred.
• the three-dimensional structure manufacturing method of the present invention is suitable for manufacturing complex and flexible three-dimensional structures such as biological tissues and organs, and is particularly suitable for manufacturing organoids.
• Examples of the cells include anchorage-dependent cells and floating cells (eg, blood cells such as white blood cells, red blood cells, and platelets).
• anchorage-dependent cells include cancer cells such as HeLa cells and F9 cells; fibroblasts such as 3T3 cells; stem cells such as ES cells, iPS cells, and mesenchymal stem cells; renal cells such as HEK293 cells; NT2 cells.
• Neuronal cells such as UV ⁇ 2 cells, HMEC-1 cells, and HUVEC; endothelial cells such as H9c2 cells; and epithelial cells such as Caco-2 cells.
• the aqueous medium include one or more selected from water, alcohol, and a culture medium.
• the composition may further contain a surfactant, a tonicity agent (for example, sodium chloride), a chelating agent, a pH adjuster, a buffer, a thickener, a stabilizer, and the like.
• the second composition when using a second composition containing cells and an aqueous medium, preferably further contains at least one selected from an extracellular matrix and a hydrogel; It is more preferable to include.
• extracellular matrix components include components contained in basement membranes and glycoproteins present in intercellular spaces.
• Components contained in the basement membrane include, for example, type IV collagen, laminin, heparan sulfate proteoglycan, and entactin.
• Glycoproteins present in intercellular spaces include collagen, laminin, entactin, fibronectin, fibrinogen, heparin sulfate, and the like. One of these can be used alone or two or more can be used in combination.
• the hydrogel include a combination of a polysaccharide and, if necessary, a coagulant corresponding to the polysaccharide.
• polysaccharides include hyaluronic acid, hyaluronate, alginic acid, alginate, carrageenan, glucomannan, agarose, cellulose, pectin, gellan gum, chitin, chitosan, chondroitin sulfate, etc.
• acid or base Those subjected to hydrolysis treatment or chemical modification treatment such as acetylation can also be used. One of these can be used alone or two or more can be used in combination.
• the coagulant include divalent metal salts.
• divalent metal salts include barium salts, calcium salts, magnesium salts, and the like. According to the present invention, three-dimensional cell culture can be efficiently performed even when such a divalent metal salt is used as a coagulant.
• the curable composition when used as the second composition, a 3D printed object can be manufactured as a three-dimensional structure.
• shaped objects include models for design images, industrial parts, and medical equipment.
• the curable composition includes thermoplastic resins such as ABS resin, polyethylene, polypropylene, vinyl chloride resin, polyethylene terephthalate, polycarbonate, polyacetal, and polyimide; phenolic resin, epoxy resin, Examples include compositions containing thermosetting resins such as melamine resins and silicone resins; photocurable resins such as acrylic resins; and inorganic substances such as silica and hydroxyapatite.
• thermoplastic resins such as ABS resin, polyethylene, polypropylene, vinyl chloride resin, polyethylene terephthalate, polycarbonate, polyacetal, and polyimide
• phenolic resin epoxy resin
• examples include compositions containing thermosetting resins such as melamine resins and silicone resins; photocurable resins such as acrylic resins; and inorganic substances such as silica and
• step (ii) it is preferable to inject the second composition into the composition filled in the container in step (i) while applying shear.
• the force applied by shearing is less than or equal to the yield value of the composition of the present invention or the composition (II) of the present invention
• the composition of the present invention or the composition (II) of the present invention filled in the container is solid or A composition of the invention or a composition of the invention filled into a container, which is semi-solid but when the force applied by shearing exceeds the yield value of the composition of the invention or composition (II) of the invention
• Product (II) becomes a liquid.
• drawing (stress application) is performed by injecting the second composition into the composition of the present invention or the composition (II) of the present invention.
• the composition of the present invention or the composition (II) of the present invention becomes solid or semi-solid again.
• the shear may be applied by any energy such as mechanical, electrical, radiation, or light.
• the second composition is injected, for example, via an injector, dispenser, microchannel, or the like.
• injection may be performed using a syringe, pipette, or autocell injector.
• a computer-controlled cell injector while applying shear to the composition of the present invention or the composition (II) of the present invention filled in a container, multiple At the location, a second composition comprising cells and an aqueous medium is injected.
• the desired result can be obtained. It is possible to manufacture three-dimensional structures of different shapes. In particular, when a second composition containing cells and an aqueous medium is used, the cells can be placed at desired positions. Note that by directly including cells, etc. in the composition of the present invention without using the second composition, and filling the container with the present composition, the desired shape and position as described above will not be obtained; It is possible to place
• a second composition containing cells and an aqueous medium it is preferable to culture the injected cells after injecting the second composition. This allows the cells to adhere, spread and proliferate to form three-dimensional cells.
• the obtained three-dimensional structure can be recovered according to a conventional method.
• the three-dimensional cells can be collected by suctioning with a syringe or pipette, for example.
• a three-dimensional structure can be manufactured easily and efficiently.
• the method for manufacturing a three-dimensional structure of the present invention is not easily affected by contaminants during 3D printing (drawing) or three-dimensional cell culture, and is suitable for manufacturing complex and flexible three-dimensional structures such as biological tissues and organs. Also suitable for manufacturing.
• the obtained three-dimensional cells can be used, for example, to evaluate the toxicity and drug efficacy of substances, elucidate the biochemical functions of cells, and search for biomarkers.
• Example 1 (1) 270 g of ethyl acetate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 21.6 g of N-vinylacetamide (manufactured by Showa Denko Co., Ltd.), M-230G (manufactured by Shin Nakamura Chemical Industries, Ltd.) as methoxypolyethylene glycol (23) monomethacrylate ), 0.9 g of pentaerythritol allyl ether (manufactured by Sigma-Aldrich (a mixture whose main components are pentaerythritol diallyl ether, pentaerythritol triallyl ether, and pentaerythritol tetraallyl ether)) were put into a separable flask.
• ethyl acetate manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
• N-vinylacetamide manufactured by Showa Denko Co., Ltd.
• Example 2 Polymer A2 and support composition B2 were obtained in the same manner as in Example 1, except that the amount of N-vinylacetamide used was changed to 17.8 g and the amount of M-230G was changed to 11.3 g. .
• Example 3 Polymer A3 and support composition B3 were obtained in the same manner as in Example 1, except that M-230G was changed to M-450G (manufactured by Shin-Nakamura Chemical Industries, Ltd.) as methoxypolyethylene glycol (45) monomethacrylate. .
• Example 4 Polymer A4 and support composition B4 were obtained in the same manner as in Example 1, except that the amount of N-vinylacetamide used was changed to 21.0 g and the amount of pentaerythritol allyl ether was changed to 1.5 g. Ta.
• Test example 1 Measurement of viscosity
• JIS Z 8803:2011 the viscosity ( mPa ⁇ s) was measured.
• Test Example 2 Measurement of water content when viscosity is less than 1000 mPa ⁇ s.
• Each support composition was diluted with pure water so that the water content increased by 0.1% by mass, and the viscosity at each dilution was measured until the viscosity value became less than 1000 mPa ⁇ s. .
• the water content (% by mass) when the viscosity became less than 1000 mPa ⁇ s was recorded. The results are shown in Table 1. If the water content is 90% by mass or more when the viscosity is less than 1000 mPa ⁇ s, the polymer easily absorbs water and when the support composition is used for 3D printing or 3D cell culture, contaminants can be removed. It can be said that it is not easily affected.
• Test example 3 Evaluation of shear rate dependence of viscosity
• the shear rate dependence of the viscosity of the support composition diluted in Test Example 2 to just below the viscosity of 1000 mPa ⁇ s was evaluated. That is, using a rotational viscometer ViscoQC 300R and an adapter jig CC18 (manufactured by Anton Paar), the viscosity of the above support composition was measured at 23° C. and a shear rate of 0.1 (1/s). The obtained viscosity value was designated as X. Furthermore, the adapter jig was changed to CC12 manufactured by Anton Paar, and the viscosity was measured under the conditions of 23° C.
• Test example 4 Evaluation of transparency
• the absorbance of the support composition diluted to just below the viscosity of 1000 mPa ⁇ s in Test Example 2 at a wavelength of 600 nm and an optical path length of 10 mm was measured using a spectrophotometer BioSpectrometer (manufactured by Eppendorf).
• the results are shown in Table 1.
• the support compositions of Examples 1 to 4 had high transparency.
• the support compositions of Examples 1 to 3 had particularly high transparency and high visibility during printing and cell culture.
• Test example 5 Evaluation of salt tolerance
• Sodium chloride manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
• the viscosity was measured according to the conditions of Test Example 1 60 minutes after the addition of sodium chloride.
• the viscosity change rate (%) was calculated according to the following formula ( ⁇ ), with the viscosity measured in Test Example 2 when it became less than 1000 mPa ⁇ s ( ⁇ 1000 mPa ⁇ s) as the “viscosity before addition of NaCl”. The results are shown in Table 1.
• Viscosity change rate (%) ⁇ (Viscosity before addition of NaCl) - (Viscosity after addition of NaCl) ⁇ /(Viscosity before addition of NaCl) x 100 ... ( ⁇ )
• Test example 6 Evaluation of 3D printing performance
• Sodium chloride manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
• the support composition diluted in Test Example 2 until the viscosity was just below 1000 mPa ⁇ s, so that the final concentration was 0.15 mol/L.
• Calcium chloride manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.
• test pattern was modeled in a calcium ion-containing 3D printing support.
• CELLINK XPLORE manufactured by Cell Ink Co., Ltd.
• Test example 7 (cell culture evaluation) In Test Example 2, 100 g of the support composition diluted to just below the viscosity of 1000 mPa ⁇ s was sterilized in an autoclave, and 1.34 g of Dulbecco's modified Eagle medium powder (manufactured by Sigma-Aldrich) and fetal bovine serum (Sigma-Aldrich) were added. 11 mL of (manufactured by) was added and mixed uniformly. HEK293 cells were mixed here at a concentration of 1.0 ⁇ 10 5 /mL, and 2 mL was added to a 12-well plate.
• Dulbecco's modified Eagle medium powder manufactured by Sigma-Aldrich
• fetal bovine serum Sigma-Aldrich
• the proliferation rate of living cells was evaluated according to the following criteria. The results are shown in Table 1. (Proliferation rate of living cells) AA: The number of viable cells is 5 times or more than the time of seeding. A: The number of viable cells is 2 times or more and less than 5 times the time of seeding. B: The number of viable cells is less than 2 times the time of seeding.

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