WO2020262609A1 - 細胞足場材、細胞培養支持体、及び細胞の培養方法 - Google Patents
細胞足場材、細胞培養支持体、及び細胞の培養方法 Download PDFInfo
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Images
Classifications
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- 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
-
- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/64—Polyesters containing both carboxylic ester groups and carbonate groups
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- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/20—Material Coatings
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- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
-
- 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
- C12N5/0068—General culture methods using substrates
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
- C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
Definitions
- One embodiment of the present disclosure relates to a cell scaffold material, a cell culture support, and a method for culturing cells.
- Cell culture is performed by seeding cells on a substrate and adding a medium.
- a method of coating a base material with a cell scaffold material in order to improve the adhesiveness between the base material and cells is known.
- stem cells represented by iPS cells and the like in vitro
- proliferate them and apply them to the body of animals or humans.
- cell culture cells are proliferated while subcultured, but it is preferable that cell differentiation is suppressed and cells proliferate. When some cells differentiate, the progress of cell proliferation is hindered, and cell proliferation may be suppressed especially in a large-scale culture. Further, when cells having different differentiation stages are present in the culture system, it is necessary to isolate cells at a specific differentiation stage, but it is technically difficult to isolate cells with high purity.
- a protein component is used as a medium, a scaffold material, or the like.
- This protein can become an antigen when contaminated with the final cultured cell product.
- the protein is derived from animals and humans, there is a large variation between lots, which may affect the stable culture of cells. Therefore, there is an increasing demand for culturing cells in a serum-free medium.
- a cell scaffold material suitable for culturing cells particularly stem cells in a serum-free medium, is required. So far, in culturing stem cells, laminin, fibronectin and the like have been studied as cell scaffolding materials, but sufficient performance has not been obtained.
- Japanese Unexamined Patent Publication No. 2018-068192 discloses that a compound having a structure consisting of an aromatic ring and a hydrogen bond unit between blocks of an amphipathic block copolymer acts on cell proliferation and morphological control.
- Patent Document 1 describes a block of a block copolymer of polyethylene glycol (PEG) and polyL-lactic acid (PLLA), poly ⁇ -caprolactone (PCL), or trimethylene carbonate (PTMC) as a specific compound.
- PEG polyethylene glycol
- PLLA polyL-lactic acid
- PCL poly ⁇ -caprolactone
- PTMC trimethylene carbonate
- Compounds having a structure of N- (4- (ureidomethyl) benzyl) benzamide are listed between them.
- Japanese Unexamined Patent Publication No. 2018-068192 proposes that this compound is used as a cell growth / growth regulator by adding it to a medium.
- Japanese Unexamined Patent Publication No. 2018-068192 only discloses the use of the cell growth / growth regulator added to the medium, and has not fully studied the cell scaffold material.
- the cell scaffold material is required to have adhesiveness between the base material and the cells and not adversely affect the proliferation of the cells. Furthermore, in cell culture using a cell scaffold material, it may be desired to proliferate cells while suppressing differentiation.
- One object of the present disclosure is to provide a cell scaffold material, a cell culture support, and a method for culturing cells, which suppress cell differentiation and promote cell proliferation.
- a cell scaffold material according to [1] wherein the polycarbonate structural unit (B) contains at least one unit having an alkoxyalkyloxycarbonyl group as a substituent.
- the polycarbonate structural unit (B) contains at least one unit having a methoxyethyloxycarbonyl group as a substituent.
- a cell culture support comprising the cell scaffold material according to any one of [1] to [9] and a substrate.
- the cell scaffold material according to any one of [1] to [9] is prepared, the cell scaffold material is placed in a culture system, and cells are cultured in the presence of the cell scaffold material.
- a method of culturing cells including the above.
- a cell scaffolding material that suppresses cell differentiation and promotes cell proliferation, a cell culture support, and a method for culturing cells.
- FIG. 1 is a graph showing the fluorescence intensity of fluorescently labeled BSA when a support coated with each polymer is used.
- FIG. 2 is a fluorescence difference micrograph of human normal fibroblasts cultured using a support coated with each polymer, and is (a) 1 hour, (b) 1 day, and (c) 2 from the start of cell culture. One day later, (d) three days later, and (e) seven days later.
- FIG. 3 is a graph showing the number of cells with respect to the cell culture time in culturing human normal fibroblasts using a support coated with each polymer.
- FIG. 4 is a graph showing the FGF-2 concentration with respect to the cell culture time in culturing human normal fibroblasts using a support coated with each polymer.
- the cell scaffolding material includes a copolymer having a polylactic acid structural unit (A) and a polycarbonate structural unit (B). According to one embodiment, cell differentiation can be suppressed and cell proliferation can be promoted.
- both the polylactic acid structural unit and the polycarbonate structural unit constituting the cell scaffold material according to the present disclosure have adhesiveness to cells and high biocompatibility.
- the polylactic acid structural unit has a hydrophobic property
- the polycarbonate structural unit is water-insoluble but has a hydrophilic property as compared with the polylactic acid structural unit. Based on these properties, due to the properties of each structural unit, the copolymer tends to self-assemble while retaining its ability to adhere to cells. It is presumed that this structural property has an effect of suppressing cell differentiation and promoting proliferation when used as a cell scaffold material. However, it is not bound by this theory.
- the cell scaffold material preferably contains a copolymer having a polylactic acid structural unit (A) and a polycarbonate structural unit (B). It is preferable that this copolymer constitutes a block copolymer by the structural unit (A) and the structural unit (B).
- the polylactic acid structural unit (A) means a structural unit composed of a polylactic acid structure, and may be a structural unit containing a plurality of lactic acid units.
- the polylactic acid structural unit (A) include poly D-lactic acid structural unit, poly L-lactic acid structural unit, poly DL-lactic acid structural unit, and the like.
- the polylactic acid structural unit (A) is a poly D-lactic acid structural unit or a poly L-lactic acid structural unit, and more preferably a poly D-lactic acid structural unit.
- the copolymer contains two or more polylactic acid structural units (A)
- the polylactic acid structural units (A) contained in one molecule of the copolymer are all or partly the same. May be different, but it is preferable that they are all the same.
- the polycarbonate structural unit (B) means a structural unit composed of a polycarbonate structure, and may be a structural unit containing a plurality of carbonate units.
- the polycarbonate structural unit may be a structural unit of a polymer having a carbonate bond in the main chain and may be either an aliphatic polycarbonate structural unit or an aromatic polycarbonate structural unit, but it must be an aliphatic polycarbonate structural unit. Is preferable.
- Examples of the aliphatic polycarbonate structural unit include a polyethylene carbonate structural unit, a polypropylene carbonate structural unit, a polytrimethylene carbonate structural unit, a structural unit of a copolymer thereof, a structural unit of a derivative having a side chain introduced therein, and the like. Can be mentioned.
- Examples of the aromatic polycarbonate structural unit include polyaryl carbonate structural units and the like, and derivatives thereof and the like.
- Examples of the polyaryl carbonate structural unit include a polyphenyl carbonate structural unit and the like.
- the carbonate unit constituting the polycarbonate structural unit (B) may have a substituent.
- the polycarbonate structural unit (B) preferably has at least one carbonate unit having an alkoxyalkyloxycarbonyl group as a substituent.
- the carbonate unit having this substituent may be contained in one or more in the polycarbonate structural unit (B), and is contained in an amount of 80 mol% or more with respect to all the carbonate units constituting the polycarbonate structural unit (B). All of the carbonate units may have this substituent.
- the alkoxyalkyloxycarbonyl group is preferably introduced by directly bonding to the carbon atom of the carbonate bond of the main chain.
- the alkoxy group may be linear or branched, preferably an alkoxy group having 5 or less carbon atoms, more preferably an alkoxy group having 3 or less carbon atoms, and further preferably ethoxy. It is a group or a methoxy group, and particularly preferably a methoxy group.
- the alkylene group interposed between the alkoxy group and the carbonyl group may be linear or branched, preferably having 5 or less carbon atoms, and more preferably 3 or less carbon atoms. , More preferably a propylene group or an ethylene group, and particularly preferably an ethylene group.
- examples of the alkoxyalkyloxycarbonyl group include a methoxyethyloxycarbonyl group, a methoxypropyloxycarbonyl group, an ethoxyethyloxycarbonyl group, an ethoxypropyloxycarbonyl group and the like, and a methoxyethyloxycarbonyl group is particularly preferable.
- an alkoxyalkyloxycarbonyl group for example, a methoxyethyloxycarbonyl group as a substituent of the polycarbonate structural unit (B) in the copolymer
- the adhesiveness to the cell can be further enhanced, and the cell grows. Differentiation can be further suppressed.
- this substituent it becomes easy to form a layer structure containing water molecules between the molecular chains of the water-insoluble structural unit, the cells are stably retained in the polycarbonate structural unit (B) portion, and the cell proliferation is promoted. It is believed that it is more promoted and cell differentiation is more suppressed, but it is not bound by this theory.
- polycarbonate structural unit (B) it is preferable to contain at least one carbonate unit represented by the following general formula (II).
- the polycarbonate structural unit (B) may be a unit obtained by polymerizing a plurality of carbonate units represented by the general formula (II).
- the degree of polymerization of the unit represented by the general formula (II) may be, for example, 2 to 2000.
- at least one carbonate unit represented by the general formula (II) may be contained, or two or more carbonate units may be contained, and 80 for all the carbonate units contained in the polycarbonate structural unit. It may be contained in a molar% or more.
- all of the carbonate units contained in the polycarbonate structural unit may be units represented by the general formula (II).
- the units represented by the plurality of general formulas (II) may all be the same or different from each other. Or part may be different.
- M is a hydrogen atom, a linear or branched alkyl group having 5 or less carbon atoms, preferably 3 or less, and more preferably 2 or less, or a group represented by —LZ.
- the group represented by ⁇ LZ is as described later in Y.
- M a methyl group or an ethyl group is preferable, and a methyl group is more preferable.
- the main chain of the polycarbonate structural unit becomes the skeleton of polytrimethylene carbonate.
- Z may be a chain ether group, a cyclic ether group, a group having an acetal structure, an alkoxy group, an alkoxyalkyl group, or a monovalent group having a combination thereof, and among them, a chain ether group or an alkoxy. Alkoxy groups are preferred.
- the chain ether group preferably has a structure of alkylene glycol such as ethylene glycol or propylene glycol or a polymer thereof.
- R is an ethylene group or a propylene group
- R' is a hydrogen atom or a linear or branched alkyl group having 5 or less carbon atoms
- n is 1 to 1. It is an integer of 30.
- R is preferably an ethylene group.
- R' is preferably a methyl group or an ethyl group, and more preferably a methyl group.
- n is preferably 1 to 5, more preferably 1 or 2.
- Z is an alkoxyalkyl group, and examples thereof include a methoxyethyl group, a methoxypropyl group, an ethoxyethyl group, an ethoxypropyl group, a propyloxyethyl group, a propyloxypropyl group, and the like, and among them, a methoxyethyl group. Is preferable.
- -L-Z examples include -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 CH 3 , -CH 2 OCH 3 , -CH 2 OCH 2 CH 3 , and-.
- polycarbonate structural unit (B) it is preferable to contain at least one carbonate unit represented by the following general formula (I).
- X is a hydrogen atom or an alkyl group having 5 or less carbon atoms.
- the alkyl group having 5 or less carbon atoms may be linear or branched.
- X is preferably a methyl group or an ethyl group, and more preferably a methyl group.
- the polycarbonate structural unit (B) may be a unit obtained by polymerizing a plurality of carbonate units represented by the general formula (I).
- the degree of polymerization of the unit represented by the general formula (I) may be, for example, 2 to 2000.
- at least one carbonate unit represented by the general formula (I) may be contained, or two or more carbonate units may be contained, which is 80 with respect to all the carbonate units contained in the polycarbonate structural unit. It is preferably contained in an amount of mol% or more.
- all of the carbonate units contained in the polycarbonate structural unit may be units represented by the general formula (I).
- the units represented by the plurality of general formulas (I) may all be the same or different from each other. Or part may be different.
- the copolymer according to one embodiment may be a block copolymer containing a polylactic acid structural unit (A) and a polycarbonate structural unit (B).
- the block structure of the block copolymer preferably has at least one end of the polylactic acid structural unit (A). That is, the block structure of the block copolymer preferably has one end or both ends of the polylactic acid structural unit (A).
- AB type, ABA type, ABAB type and the like can be mentioned, and ABA type is preferable.
- the polylactic acid structural unit (A) has adhesiveness to the substrate, so that the cell scaffold is attached to the substrate.
- the material can be adhered more stably. Further, as in the ABA type, since both ends are polylactic acid structural units (A), the polylactic acid structural units (A) are adhered to the base material at both ends of the block copolymer, and the polycarbonate structure in the intermediate portion is formed. It is considered that the block copolymer is attached to the base material so that the unit (B) is lifted from the base material. In this configuration, the adherence of the cells to the substrate can be further enhanced while capturing the cells in the portion of the raised polycarbonate structural unit (B).
- An example of a block copolymer is a copolymer represented by the following general formula (IV). More specifically, a copolymer represented by the following general formula (IV) is preferable.
- X is the same as the general formula (I) described above, and thus the description thereof will be omitted.
- a, b, c and d are the same as the general formula (IV) described above, and thus the description thereof will be omitted.
- the number average molecular weight (Mn) of one molecule of the copolymer according to one embodiment is preferably 5,000 to 100,000, more preferably 10,000 to 80,000, and even more preferably 12,500 to 70,000. , 15,000 to 50,000 are particularly preferable.
- the molecular weight distribution (Mw / Mn) of one molecule of the copolymer according to one embodiment is not particularly limited, but is, for example, 2.0 or less, preferably 1.5 or less, and more preferably 1.2 or less. Is.
- the number average molecular weight (Mn) of the polylactic acid structural unit (A) is preferably 1,000 to 50,000, more preferably 3,000 to 40,000, further preferably 4,000 to 35,000, and 5, More preferably 000 to 30,000.
- the molecular weight distribution (Mw / Mn) of the polylactic acid structural unit (A) is not particularly limited, but is preferably 1.0 to 10, more preferably 1.0 to 8, and even more preferably 1.05 to 5.
- the number average molecular weight (Mn) of the polycarbonate structural unit (B) is preferably 2,000 to 50,000, more preferably 5,000 to 40,000, further preferably 6,000 to 35,000, and 7,000. ⁇ 20,000 is particularly preferable.
- the molecular weight distribution (Mw / Mn) of the polycarbonate structural unit (B) is not particularly limited, but is preferably 1.0 to 10, more preferably 1.0 to 8, and even more preferably 1.05 to 5.
- the number average molecular weight of the polylactic acid structural unit (A) is the number average molecular weight of one block, and when the block copolymer contains two or more blocks of the polylactic acid structural unit (A), It is preferable that each satisfies this range.
- the polycarbonate structural unit (B) the number average molecular weight (Mn) and the weight average molecular weight (Mw) can be measured by using gel permeation chromatography (GPC) calibrated with standard polystyrene. Further, the molecular weight distribution can be obtained from the ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn).
- the copolymer according to one embodiment it is preferable that all the units constituting the polylactic acid structural unit (A) are lactic acid units. Further, in the copolymer according to one embodiment, it is preferable that all the units constituting the polycarbonate structural unit (B) are carbonate units.
- the total unit of the monomer unit constituting the polylactic acid structural unit (A) is preferably 10 mol% or more, preferably 30 mol, with respect to the total unit of the monomer unit constituting the entire copolymer. % Or more is more preferable, and 40 mol% or more is further preferable.
- the total unit of the monomer unit constituting the polylactic acid structural unit (A) is 90 mol% or less with respect to the total unit of the monomer unit constituting the entire copolymer.
- 70 mol% or less is more preferable, and 60 mol% or less is further preferable.
- the total unit of the monomer unit constituting the polylactic acid structural unit (A) is preferably 10 to 90 mol%, more preferably 30 to 70 mol%, and 40 to 40 mol% of the total unit of the monomer unit constituting the entire copolymer. -60 mol% is more preferred.
- the total unit of the monomer unit constituting the polycarbonate structural unit (B) is preferably 10 mol% or more, preferably 30 mol%, with respect to the total unit of the monomer unit constituting the entire copolymer. The above is more preferable, and 40 mol% or more is further preferable. Further, in the copolymer according to another embodiment, the total unit of the monomer unit constituting the polycarbonate structural unit (B) is preferably 90 mol% or less with respect to the total unit of the monomer unit constituting the entire copolymer. , 70 mol% or less is more preferable, and 60 mol% or less is further preferable.
- the total unit of the monomer unit constituting the polycarbonate structural unit (B) is preferably 10 to 90 mol%, more preferably 30 to 70 mol%, and 40 to 40 to all of the monomer units constituting the entire copolymer. 60 mol% is more preferred.
- the total number of monomer units constituting the polylactic acid structural unit (A) and the total number of monomer units constituting the polycarbonate structural unit (B) are 10 in molar ratio. : 90 to 90:10 is preferable, 30:70 to 70:30 is more preferable, and 40:50 to 50:40 is even more preferable.
- all the units of the monomer units constituting the entire copolymer all the units of the monomer units constituting the polylactic acid structural unit (A) and the monomers constituting the polycarbonate structural unit (B).
- the total unit of all the units is preferably 80 mol% or more, preferably 90 mol% or more.
- the copolymer according to one embodiment may be composed of a polylactic acid structural unit (A) and a polycarbonate structural unit (B).
- the copolymer according to one embodiment is a block copolymer and the block copolymer contains two or more blocks of the polylactic acid structural unit (A), two or more polylactic acid structural units It is preferable that the total amount of all the monomer units constituting the block (A) satisfies the above range.
- the polycarbonate structural unit (B) is preferable.
- the copolymer according to one embodiment is not limited to that obtained by the following synthesis method.
- the copolymer can be synthesized by preparing polylactic acid and polycarbonate and binding the polylactic acid and polycarbonate. It is also possible to prepare polycarbonate and synthesize lactic acid, lactic acid lactide, or a combination thereof as a monomer on one end or both ends of the polycarbonate by polymerizing and bonding them. It is also possible to prepare polylactic acid and polymerize and bond carbonate, which is a monomer, to one end or both ends of polylactic acid for synthesis. Further, random polymerization may be carried out using lactic acid and carbonate, or block copolymerization may be carried out using a polymerization reagent.
- polylactic acid poly D-lactic acid, poly L-lactic acid, poly DL-lactic acid and the like can be used alone or in combination thereof, but poly D-lactic acid or poly L-lactic acid is used as a single component. It is preferable, and it is more preferable to use poly D-lactic acid as a single component. It is preferable that the number average molecular weight of polylactic acid is appropriately set according to the target molecular weight of the structural unit (A) to be introduced into the block copolymer.
- Examples of the monomer component constituting polylactic acid include lactic acid, lactic acid lactide, and a combination of lactic acid and lactic acid lactide.
- lactic acid which is a monomer component constituting polylactic acid D-lactic acid, L-lactic acid, DL-lactic acid and the like can be used alone or in combination thereof, but D-lactic acid or L-lactic acid is used alone. It is preferably used as a component, and more preferably D-lactic acid is used as a single component.
- lactic acid lactide which is a monomer component
- D-lactic acid lactide, L-lactic acid lactide, DL-lactic acid lactide and the like can be used alone or in combination thereof, but D-lactic acid lactide or L-lactic acid lactide is used alone. It is preferable to use it as one component, and it is more preferable to use D-lactic acid lactide as a single component. Lactic acid and lactic acid lactide may be used in combination as the monomer component, or each may be used alone.
- an aliphatic polycarbonate and an aromatic polycarbonate can be used alone or in combination thereof.
- a polycarbonate derivative containing at least one carbonate unit having an alkoxyalkyloxycarbonyl group as a substituent can be preferably used, and further, the substituent is preferably a methoxyethyloxycarbonyl group.
- the polycarbonate it is preferable to use a polycarbonate derivative containing at least one carbonate unit represented by the general formula (II). Furthermore, it is preferable to use a polycarbonate derivative containing at least one carbonate unit represented by the general formula (I).
- the carbonate unit represented by the general formula (II) a carbonate unit having an alkoxyalkyloxycarbonyl group as a substituent is preferable. Further, as the carbonate unit represented by the general formula (I), a carbonate unit having a methoxyethyloxycarbonyl group as a substituent is preferable. It is preferable that the number average molecular weight of the polycarbonate is appropriately set according to the target molecular weight of the polycarbonate structural unit (B) to be introduced into the block copolymer.
- an aliphatic carbonate is preferable, and examples thereof include ethylene carbonate, propylene carbonate, trimethylene carbonate and the like, or derivatives thereof. These may be used alone or in combination of two or more.
- a carbonate having a structure represented by the general formula (II) or a cyclic carbonate which is a cyclic body thereof is more preferable.
- a trimethylene carbonate derivative described later can be used.
- Copolymerization of polylactic acid or the monomer component of polylactic acid with polycarbonate or the monomer component of polycarbonate is preferably carried out by solution polymerization, and if necessary, a polymerization initiator, a polymerization catalyst or the like is added to the synthetic system. It can be carried out.
- the polymerization solvent include dichloromethane, chloroform, diethyl ether, tetrahydrofuran (THF), toluene and the like.
- polymerization initiator examples include alcohol-based polymerization initiators such as 1-pyrenebutanol, lauryl alcohol, decanol, and stearyl alcohol; 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU), dimethyl.
- Cyclic amine polymerization initiators such as aminopyridine (DMAP), triethylenediamine (DABCO), (+)-spartane, and (-)-spartane can be mentioned.
- polymerization catalyst examples include bifunctionalized thiourea such as 1- (3,5-bis (trifluoromethyl) phenyl) -3-cyclohexyl-2-thiourea (TU).
- the polymerization is preferably carried out in the temperature range from room temperature to a temperature that does not affect the synthetic system, more specifically in the range from room temperature to 50 ° C., in an atmospheric atmosphere or an inactive atmosphere, preferably in an inert atmosphere such as a nitrogen atmosphere. It is preferably carried out for 1 minute to 12 hours, more preferably for 30 minutes to 6 hours.
- the completion of the polymerization reaction can be determined by whether or not the monomer component or the like is present in the reaction system, and can be confirmed by a method such as 1 H-NMR or TLC.
- the polymerization reaction can be terminated by adding a reaction terminator.
- the reaction terminator include acetic acid, hydrochloric acid, sulfuric acid, benzoic acid and the like.
- This polycarbonate derivative can be obtained by ring-opening polymerization of a monomer in which a functional group introduced as a side chain into a polymer is introduced into the skeleton of cyclic carbonate.
- a method for synthesizing a polycarbonate derivative containing a carbonate unit represented by the general formula (II) will be described.
- the carbon atom of trimethylene carbonate is represented by a group represented by M and Y.
- a trimethylene carbonate derivative having the above-mentioned group introduced therein can be used as a monomer.
- M and Y are as described in the above general formula (II).
- trimethylene carbonate examples include 5-methyl-5- (2-methoxyethyloxycarbonyl) -1,3-dioxane-2-one and 5-methyl-5- (2-ethoxyethyloxycarbonyl).
- the ring-opening polymerization of the monomer in which the functional group introduced as a side chain into the polymer is introduced into the skeleton of the cyclic carbonate is not particularly limited. It can be done by various methods. For example, in ring-opening polymerization, a cationic polymerization reaction, an anionic polymerization reaction or the like can be used using various polymerization initiators, and a polymerization catalyst or the like may be used if necessary.
- the polymerization solvent, the polymerization initiator, and the polymerization catalyst are not particularly limited, and those described in the above-mentioned synthesis of the copolymer can be used. More specifically, it can be synthesized according to the method described in JP-A-2014-161675.
- the cell scaffold material according to one embodiment may contain the copolymer according to one embodiment described above as a single component. Moreover, the cell scaffold material according to one embodiment may be provided as a cell scaffold material composition in a state of being added to a solvent.
- the solvent include water, an organic solvent, or a mixed solvent thereof.
- the water in addition to the water used as the solvent, the intermediate water of the copolymer according to one embodiment may be used.
- the organic solvent include dichloromethane, methanol and the like.
- the cell scaffolding material composition can further contain a medium additive, other additive components, etc., which will be described later, if necessary.
- the cell culture support containing the cell scaffold material and the base material according to the above-described embodiment. Since the cell culture support according to one embodiment can exert an action of suppressing cell differentiation and promoting proliferation, it can be suitably used for proliferating cells at a specific differentiation stage by culturing.
- the shape of the base material contained in the support is not particularly limited, but may be one or more selected from the group consisting of a flat flat plate, a curved flat plate, a sphere, a block body, and the like.
- the base material those used as a cell culture base material can be used without particular limitation, and for example, well plates such as dishes, flat-bottomed well plates, and round-bottomed well plates; petri dishes and the like.
- Cell containers; microbeads, microcarriers, three-dimensional blocks; cell sheets and the like can be used.
- the material of the base material is not particularly limited, but a material that does not show toxicity to cells is preferable.
- ester resins such as polyethylene terephthalate, (meth) acrylic resins, epoxy resins, urethane resins, styrene resins, thiol resins, silicone resins and other resins; pure nickel, titanium, platinum, gold, tungsten , Metals such as renium, palladium, rhodium, ruthenium; alloys such as stainless steel, titanium / nickel, nitinol, cobalt chromium, platinum iridium alloys; glass, ceramics and the like.
- substrates suitable for regenerative medicine include silicone, polyether blockamide (PEBAX), polyurethane, silicone-polyurethane copolymer, ceramics, collagen, hydroxyapatite, nylon, polyethylene terephthalate, Goretex (registered trademark) and the like.
- Biocompatible materials such as ultra-high molecular weight polyethylene, polyvinyl chloride, and other bio-derived materials; polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and their copolymers, PHB-PHV-based poly. Examples thereof include natural polymers such as (alkanoic acid), polyesters, starch, cellulose and chitosan, and biodegradable materials such as derivatives thereof.
- the cell culture support can be obtained by applying a cell scaffold material to the substrate.
- the cell scaffold material can be dissolved in an organic solvent and then applied to the substrate in the form of a liquid composition.
- a liquid composition As the solvent, dichloromethane, methanol and the like can be used.
- the liquid composition may further contain a medium additive, other additive components, and the like, which will be described later.
- the cell scaffold material is preferably 0.05 to 10% by mass, more preferably 0.1 to 1% by mass, based on the total amount of the liquid composition.
- the method of applying the liquid composition to the substrate is not particularly limited, and examples thereof include a method using a spin coater.
- the amount of the cell scaffolding material applied to the base material can be appropriately adjusted depending on the type of the base material, the type of the cell scaffolding material, the type of cells to be cultured, the method of application, and the like.
- the amount of the cell scaffolding material applied to the base material is the amount of solid content per unit area, and can be, for example, 0.05 ⁇ g / mm 2 to 500 ⁇ g / mm 2 .
- the solid content per unit area can be, for example, 0.01 ⁇ g / mm 2 to 10 ⁇ g / mm 2, and 0.05 ⁇ g / mm 2 to 5 ⁇ g / mm. be a 2, or 0.1 [mu] g / mm 2 can be a ⁇ 3.0 [mu] g / mm 2.
- the cell scaffold material Before applying the cell scaffold material to the substrate, it may be treated with a polymer other than the copolymer according to one embodiment.
- a polymer other than the copolymer examples include (meth) acrylic resins and the like. It is preferable that the other polymer is imparted to the substrate in a liquid state by adding a solvent as appropriate. By interposing another polymer between the base material and the cell scaffold material, the adhesion of the scaffold material to the base material can be further enhanced.
- the cell culture support according to one embodiment can be placed in the culture system of the cells to be grown.
- the cell culture support can be placed in a medium in advance and the target cells can be seeded in this medium, or the cell culture support can be supplied to the culture system in which the target cells are present. Can be done.
- the target cells come into contact with the cell culture support, and the target cells adhere to the cell culture support and grow.
- the method for culturing cells according to one embodiment can include preparing a cell scaffold material, arranging the cell scaffold material in a culture system, and culturing cells in the presence of the cell scaffold material.
- the cell scaffold material the cell scaffold material according to the above-described embodiment can be used. This makes it possible to suppress cell differentiation, promote cell proliferation, and produce a large amount of undifferentiated cells.
- the cell scaffold material In the step of preparing the cell scaffold material, the cell scaffold material according to the above-described embodiment is prepared. At this time, the cell scaffold material may be provided in the form of the above-mentioned composition containing the cell scaffold material as one component, or may be provided in the form of a cell culture support containing the cell scaffold material and the base material. Good.
- the cell scaffold material is arranged in the culture system by appropriately selecting an arrangement method according to the form in which the cell scaffold material is provided.
- the cell scaffold material can be arranged in the culture system in advance before cell seeding.
- the target cells can be seeded in advance in a culture system, and the cell scaffold material can be placed in the subsequent culture system as a cell scaffold material composition or a microbead-shaped cell culture support.
- the target cells are cultured in the presence of the cell scaffold material.
- the cells that can be cultured for example, primary cultured cells, cultured cell lines, recombinant cultured cell lines and the like can be used.
- the origin of the cells is not particularly limited, and examples thereof include mammals such as humans, chimpanzees, monkeys, cows, horses, pigs, dogs, cats, rabbits, rats, mice, and hamsters; and birds such as chickens.
- cells obtained by hybridizing two or more cells of different species may be used.
- the organs and tissues from which the cells are derived are not particularly limited, and for example, blood cells / lymphatic system, vascular system, brain / nervous system, bone marrow, muscle tissue, thymus, salivary gland, oral cavity, esophagus, stomach, liver, bile sac, and pancreas.
- stem cells and cancer cells include stem cells and cancer cells.
- stem cells can be preferably used as cells that can be cultured.
- ES cells embryonic stem cells
- iPS cells artificial pluripotent stem cells
- EC cells embryonic tumor cells
- EG cells embryonic germ stem cells
- nuclear transplant ES cells somatic cell-derived ES cells, etc.
- Differentiating pluripotent stem cells hematopoietic stem cells, bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, other interstitial-derived stem cells, mouse cells, neural stem cells and other tissue stem cells; , Precursor cells in various tissues such as pancreas, adipose tissue, bone tissue, cartilage tissue, and nerve tissue, and various stem cells such as fibroblasts.
- the conditions usually used for culturing the target cell can be used as they are depending on the cell type.
- the medium used for cell culture is not particularly limited as long as the cells are viable and proliferative, and can be appropriately selected depending on the type of cells to be cultured.
- the medium may be either a serum medium or a serum-free medium, but the cell scaffold material according to the embodiment can be preferably used for culturing in the serum-free medium.
- serum-free medium include Eagle's medium, Dalbeco's modified Eagle's medium (low glucose or high glucose), Eagle's MEM medium, ⁇ MEM medium, IMDM medium, ham F10 medium, ham F12 medium, RPMI1640 medium, and the like, or a blend thereof. The medium is mentioned.
- the serum medium can be prepared by adding serum to a serum-free medium.
- serum-free medium for example, fetal bovine serum (FBS), horse serum, human serum or the like can be used.
- FBS fetal bovine serum
- horse serum horse serum
- human serum human serum
- concentration of serum is preferably 30% or less.
- Additives may be added to the medium as needed.
- additives include vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C and vitamin D; coenzymes such as folic acid; glycine, alanine, arginine, asparagine, glutamine, isoleucine and leucine.
- Equivalent amino acids sugars or organic acids as carbon sources such as lactic acid; growth factors such as EGF, FGF, PFGF, TGF- ⁇ ; interleukins such as IL-1, IL-6; TNF- ⁇ , TNF- ⁇ , leptin
- growth factors such as EGF, FGF, PFGF, TGF- ⁇
- interleukins such as IL-1, IL-6
- TNF- ⁇ , TNF- ⁇ leptin
- cytokines such as transferase; metal ions such as iron ions, selenium ions and zinc ions; SH reagents such as ⁇ -mercaptoethanol and glutathione; proteins such as albumin and the like.
- the cell culturing method is not particularly limited, and a method suitable for each cell may be used. Generally, cell cultures are carried out in the range of 30-40 ° C, preferably at a temperature of 37 ° C, in the range of 70-100%, preferably in the range of 95-100%, and 2% -7% CO. 2. It can be carried out in a 5% CO 2 environment, preferably.
- the time and method of cell passage are also not particularly limited, and a method suitable for each cell may be used.
- the form of the culture may be a two-dimensional culture in which cells are easily attached, or a three-dimensional culture in which cells are suspended in a culture system.
- the culture form can be appropriately selected depending on the cell type, medium composition, and the like.
- the cell scaffolding material, the base material, and the medium according to the above-mentioned one embodiment may be stored in separate containers.
- the cell culture support and the medium may be stored in separate containers by using the cell scaffold support provided with the base material and the cell scaffold material according to the embodiment.
- the kit may also include cells to be cultured.
- the kit may be provided with an instrument or the like used for culturing.
- the cell scaffold material and the cell scaffold material support according to the embodiment are as described above.
- a copolymer having a polylactic acid structural unit (A) and a polycarbonate structural unit (B) for a cell scaffold material it is possible to provide the use of a copolymer having a polylactic acid structural unit (A) and a polycarbonate structural unit (B) for producing a cell scaffold material.
- a copolymer having a polylactic acid structural unit (A) and a polycarbonate structural unit (B) for producing a cell scaffold material for the copolymer having the polylactic acid structural unit (A) and the polycarbonate structural unit (B), the matters described regarding the copolymer in the cell scaffold material according to the above-described embodiment are incorporated as they are.
- the polycarbonate structural unit (B) may contain at least one unit having an alkoxyalkyloxycarbonyl group as a substituent, and the polycarbonate structural unit (B) is described above. It may contain at least one unit represented by the general formula (I).
- the polylactic acid structural unit (A) may be a poly D-lactic acid structural unit or a poly L-lactic acid structural unit.
- the present copolymer may be an ABA type block copolymer. Any combination of these may be used.
- the polycarbonate structure (B) contains at least one unit having an alkoxyalkyloxycarbonyl group as a substituent, or a unit represented by the above-mentioned general formula (I).
- the polylactic acid structural unit (A), which comprises at least one, is a poly D-lactic acid structural unit or a poly L-lactic acid structural unit.
- ABA may be a block copolymer.
- Dehydrated grade THF and DCM were supplied from the solvent supply system manufactured by Kanto Chemical Co., Inc. (water content ⁇ 10 ppm).
- Azobisisobutyric acid (35.0-37.0% by mass) and magnesium sulfate (95.0%) are available from Fujifilm Wako Pure Chemical Industries, Ltd.
- Triphosgene (98%), benzyl alcohol, 2-methoxyethyl acrylate (> 98.0). %), 1,4-dioxane (> 99.0%), 2,2-azobis (isobutyronitrile) (AIBN> 98.0%) were purchased from Tokyo Kasei Kogyo Co., Ltd. and used as they were.
- Synthesis Example 2 Synthesis of Polylactic Acid (PDLA)" TU (5.8 mg, 1.49 mmol) and D-lactide (77.1 mg, 0.535 mmol) were dissolved in DCM (240 mg) and stirred at room temperature. After the reaction for 2 hours, the reaction solution was reprecipitated in 2-propanol (30 mL) and dried under vacuum to give a white solid PDLA.
- PDLA Polylactic Acid
- Synthesis Example 3 Synthesis of PTMC (Polytrimethylene Carbonate)" Benzyl alcohol (4.32 mg, 0.04 mmol), trimethylene carbonate (2.01 g, 20 mmol), DBU (182.4 mg, 1.4 mmol) and TU (447.1 mg, 1.4 mmol) in DCM (20 mL) It was dissolved and stirred. After 24 hours, the reaction was stopped by adding benzoic acid, the reaction solution was reprecipitated in 2-propanol, and the supernatant was centrifuged. The obtained viscous material was dissolved in DCM and recovered, concentrated with an evaporator, and dried to obtain PTMC.
- PTMC Polytrimethylene Carbonate
- the polymerization solvent was added dropwise to 1000 ml of n-hexane to recover the polymer produced by the polymerization as a precipitate.
- the obtained by-product was purified by repeating the precipitation operation three times with a THF / n-hexane system.
- the purified polymer was dissolved in THF and recovered, concentrated on an evaporator, and vacuum dried at 60 ° C. for 30 hours to obtain PMEA.
- a polymer-coated PET film (hereinafter referred to as a support) is irradiated in a clean bench with a spot UV irradiation device (“SP-11” manufactured by Ushio, Inc.) at an output of 4 W / cm 2 for 10 minutes. And sterilized. Further, as a control, an untreated support (PET support) to which no polymer was applied after the degreasing treatment was prepared.
- SP-11 spot UV irradiation device manufactured by Ushio, Inc.
- each polymer was dissolved in DCM or methanol to adjust the concentration to 0.2% by mass. 100 ⁇ L of this solution was applied twice to the surface of the treated PET film with a spin coater, allowed to stand at room temperature for one day, and then immersed in pure water overnight for washing. The obtained support was submerged in a 24-well plate for cell culture (“Coster 24 wells” manufactured by Corning), fluorescently labeled BSA (FITC labeled) was dissolved in PBS at 0.1 w / v%, and 500 ⁇ L / well was added. Then, it shook at 37 ° C. and 60 min (tilt angle: 6 °, 30 r / min).
- FIG. 1 is a graph showing the fluorescence intensity of the fluorescently labeled BSA when the support coated with each polymer is used with reference to the fluorescence intensity of the PET support.
- FGF-2 fibroblast growth factor 2
- FIG. 2 is a photograph observed by a fluorescence difference microscope, which is (a) 1 hour, (b) 1 day, (c) 2 days, (d) 3 days, and (e) 7 days after the start of cells.
- FIG. 3 shows a graph of the number of cells with respect to the cell culture time.
- (Ii) Evaluation method for suppressing cell differentiation The culture supernatant collected at each cell culture time was measured with a human FGF-2 measurement ELISA kit (manufactured by R & D system).
- FIG. 4 shows a graph of the concentration of FGF-2 with respect to the cell culture time.
- the cell proliferation number of the plate using PDMED (hereinafter referred to as PDMED well) is smaller than that of the plate using PET as a blank (hereinafter referred to as PET well), but generally good proliferation is achieved. It turns out to keep.
- PMB the plate using PMB (hereinafter referred to as PMB well) does not adhere to the cells and hardly contributes to the growth.
- PMB well the plate using PMB (hereinafter referred to as PMB well) does not adhere to the cells and hardly contributes to the growth.
- the PET wells proliferated while expanding greatly, and when the cell density increased, the proliferation in the mass characteristic of fibroblasts was observed.
- the PDMED well it was observed that the cells did not grow well, and even if the cell density increased, the cells adhered to the support and proliferated without forming a mass.
- (3-2) iPS cells In the same manner as in the preparation of the cell scaffold material described in (1) above, the diameter of the support was set to 50 mm, and the PDMED support, PMB support, untreated PET support as a control, and the control Laminin (“iMatrix511” manufactured by Nippi Co., Ltd.) support was prepared. Each support was set on the bottom surface of a 60 mm dish (“3010-060” manufactured by IWAKI). Feeder-free iPS cells derived from Kyoto University were seeded on each dish (referred to as "PDMED dish”, “PMB dish”, “PET dish”, and "iMatrix dish”, respectively) in an amount of 2.5 x 10 4 cells / dish. ..
- a medium (“StemFitAK02” manufactured by Ajinomoto Healthy Supply Co., Ltd.) was added to each dish, and the cells were cultured at 37 ° C. in a 5% CO 2 atmosphere. After culturing the cells for 7 days, the state of the cells was observed using an inverted microscope (“CKX31SF” manufactured by Olympus Corporation), and undifferentiated cells were separated and quantified by FACS (flow cytometry). Further, in the same manner as in (3-1) above, the culture supernatant was obtained from the first day to the 7th day of the culture and used for quantitative evaluation of IL-6 (interleukin-6) and TNF ⁇ (tumor necrosis factor ⁇ ).
- IL-6 interleukin-6
- TNF ⁇ tumor necrosis factor ⁇
- Table 1 shows the undifferentiated cell ratio on the 7th day of culture and the concentrations of IL-6 and TNF ⁇ on the 5th day of culture. The closer the undifferentiated cell ratio (%) is to 100 (%), the more the cell differentiation is suppressed. Further, the closer the concentration of IL-6 is to 0, the more the cell differentiation is suppressed. Furthermore, the closer the concentration of TNF ⁇ is to 0, the more the cell differentiation is suppressed.
- IL-6 is known as a cytokine involved in the induction of differentiation. From the results of this experiment, in the control iMatrix dish, the undifferentiated cell rate of viable cells was lower than the others, and the release of IL-6 and TNF ⁇ on the 5th day of culture was also extremely high compared to the others. From this, it was found that the iMatrix dish did not sufficiently suppress cell differentiation. In the PMB dish and PET dish, the undifferentiated cell rate on the 7th day of culture was lower than that on the PDMED dish, and IL-6 was also released on the 5th day of culture. Regarding the PMB dish, the release of about 2 pg / mL TNF ⁇ was confirmed on the second day of culture (data not shown).
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Abstract
Description
近年拡大している再生医療では、iPS細胞等に代表される幹細胞を生体外で細胞培養して増殖し、動物又はヒト等の体内へ適用することが研究されている。細胞培養では、細胞を継代しながら細胞を増殖させていくが、細胞の分化が抑制されて細胞が増殖していくことが好ましい。一部の細胞が分化すると細胞増殖の進行が妨げられ、特に大量培養においては細胞増殖が抑制される場合がある。また、培養系に分化段階が異なる細胞が存在する場合、特定の分化段階の細胞を単離する必要が生じるが、純度高く細胞を単離することは技術的に困難を伴う。
これまで、幹細胞の培養において、ラミニン、フィブロネクチン等が細胞足場材として検討されているが、十分な性能が得られていない。
特開2018-068192号公報には、この化合物を細胞伸展・増殖調節剤として、培地に添加して用いることが提案されている。
細胞足場材には、基材と細胞との接着性を備えながら、細胞の増殖に悪影響を与えないことが求められる。さらに、細胞足場材を用いた細胞培養においては、分化を抑制しつつ細胞を増殖させることが望まれる場合がある。
[1]ポリ乳酸構造単位(A)と、ポリカーボネート構造単位(B)と、を有する共重合体を含む、細胞足場材。
[2]前記ポリカーボネート構造単位(B)は、置換基として、アルコキシアルキルオキシカルボニル基を有する単位を少なくとも1つ含む、[1]記載の細胞足場材。
[3]前記ポリカーボネート構造単位(B)は、置換基として、メトキシエチルオキシカルボニル基を有する単位を少なくとも1つ含む、[1]又は[2]に記載の細胞足場材。
(一般式(I)中、Xは水素原子又は炭素数5以下のアルキル基である。)
[5]前記一般式(I)中、Xはメチル基である、[4]に記載の細胞足場材。
[6]前記共重合体は、一方端又は両端部がポリ乳酸構造単位(A)であるブロック共重合体である、[1]から[5]のいずれかに記載の細胞足場材。
[7]前記共重合体は、AB型、ABA型、又はABAB型のブロック共重合体である、[6]に記載の細胞足場材。
[8]前記共重合体は、ABA型ブロック共重合体である、[7]に記載の細胞足場材。
[9]前記ポリ乳酸構造単位(A)は、ポリD-乳酸構造単位、又はポリL-乳酸構造単位である、[1]から[8]のいずれか記載の細胞足場材。
[11][1]から[9]のいずれかに記載の細胞足場材を用意すること、前記細胞足場材を培養系に配置すること、及び前記細胞足場材の存在下で、細胞を培養することを含む、細胞の培養方法。
[12]前記培養系が、無血清培地で構成される、[11]に記載の細胞の培養方法。
一実施形態によれば、細胞の分化を抑制して、細胞の増殖を促進することができる。
ポリ乳酸構造単位(A)としては、例えば、ポリD-乳酸構造単位、ポリL-乳酸構造単位、ポリDL-乳酸構造単位等が挙げられる。好ましくは、ポリ乳酸構造単位(A)は、ポリD-乳酸構造単位、又はポリL-乳酸構造単位であり、より好ましくはポリD-乳酸構造単位である。共重合体に2個以上のポリ乳酸構造単位(A)が含まれる場合は、共重合体の1分子中に含まれるポリ乳酸構造単位(A)は、全て同一であっても全て又は一部が異なってもよいが、全て同一であることが好ましい。
ポリカーボネート構造単位としては、主鎖にカーボネート結合を有する重合体の構造単位であって、脂肪族ポリカーボネート構造単位又は芳香族ポリカーボネート構造単位のいずれであってもよいが、脂肪族ポリカーボネート構造単位であることが好ましい。
脂肪族ポリカーボネート構造単位としては、例えば、ポリエチレンカーボネート構造単位、ポリプロピレンカーボネート構造単位、ポリトリメチレンカーボネート構造単位等、これらの共重合体の構造単位、又はこれらに側鎖を導入した誘導体の構造単位等が挙げられる。芳香族ポリカーボネート構造単位としては、例えば、ポリアリールカーボネート構造単位等、又はこれらの誘導体等が挙げられる。ポリアリールカーボネート構造単位としては、例えば、ポリフェニルカーボネート構造単位等が挙げられる。
共重合体に2個以上のポリカーボネート構造単位(B)が含まれる場合は、共重合体の1分子中に含まれる構造単位(B)は、全て同一であっても全て又は一部が異なってもよいが、全て同一であることが好ましい。
アルコキシアルキルオキシカルボニル基は、主鎖のカーボネート結合の炭素原子に直接結合して導入されることが好ましい。
また、アルコキシアルキルオキシカルボニル基において、アルコキシ基は、直鎖又は分岐であってもよく、炭素数5以下のアルコキシ基が好ましく、より好ましくは炭素数3以下のアルコキシ基であり、さらに好ましくはエトキシ基又はメトキシ基であり、特に好ましくはメトキシ基である。
具体的には、アルコキシアルキルオキシカルボニル基は、メトキシエチルオキシカルボニル基、メトキシプロピルオキシカルボニル基、エトキシエチルオキシカルボニル基、エトキシプロピルオキシカルボニル基等が挙げられ、特にメトキシエチルオキシカルボニル基が好ましい。
ポリカーボネート構造単位において、一般式(II)で表されるカーボネート単位は少なくとも1個含まれてもよく、2個以上含まれてもよく、ポリカーボネート構造単位に含まれるカーボネート単位の全単位に対して80モル%以上含まれてよい。さらに、ポリカーボネート構造単位に含まれるカーボネート単位の全てが一般式(II)で表される単位であってよい。
また、ポリカーボネート構造単位に複数の一般式(II)で表される単位が含まれる場合では、複数の一般式(II)で表される単位は全てが互いに同一であっても異なってもよく、又は一部が異なってもよい。
Lは、主鎖とZとのリンカーであり、アルキレン基、エーテル結合、エステル結合、単結合、-C(=O)-、又はこれらの組み合わせを有する2価の基であってよく、なかでも、エーテル結合、エステル結合、単結合、-C(=O)-、又はこれらの組み合わせを有する2価の基が好ましく、エステル結合、又は-C(=O)-を有する2価の基がより好ましい。
鎖状エーテル基としては、例えば、エチレングリコール、プロピレングリコール等のアルキレングリコール又はその重合体の構造を有することが好ましい。
Zの具体例としては、下記一般式(III)において、Rがエチレン基又はプロピレン基であり、R’が水素原子又は炭素数5以下の直鎖又は分岐のアルキル基であり、nは1~30の整数である。
-(R-O)n-R’ (III)
一般式(III)において、Rは好ましくはエチレン基である。また、R’は好ましくはメチル基又はエチル基であり、より好ましくはメチル基である。また、nは好ましくは1~5であり、より好ましくは1又は2である。
より好ましくは、Zは、アルコキシアルキル基であり、例えば、メトキシエチル基、メトキシプロピル基、エトキシエチル基、エトキシプロピル基、プロピルオキシエチル基、プロピルオキシプロピル基等が挙げられ、なかでもメトキシエチル基が好ましい。
Xにおいて、炭素数5以下のアルキル基は直鎖又は分岐であってよい。また、Xは好ましくはメチル基又はエチル基であり、より好ましくはメチル基である。
ポリカーボネート構造単位において、一般式(I)で表されるカーボネート単位は少なくとも1個含まれてもよく、2個以上含まれてもよく、ポリカーボネート構造単位に含まれるカーボネート単位の全単位に対して80モル%以上含まれていることが好ましい。さらに、ポリカーボネート構造単位に含まれるカーボネート単位の全てが一般式(I)で表される単位であってよい。
また、ポリカーボネート構造単位に複数の一般式(I)で表される単位が含まれる場合では、複数の一般式(I)で表される単位は全てが互いに同一であっても異なってもよく、又は一部が異なってもよい。
ブロック共重合体のブロック構造において、一方端又は両端部がポリ乳酸構造単位(A)であることで、ポリ乳酸構造単位(A)は基材への接着性を備えるため、基材に細胞足場材をより安定して付着させることができる。
さらに、ABA型のように、両端部がポリ乳酸構造単位(A)であることで、ブロック共重合体の両端部においてポリ乳酸構造単位(A)が基材に接着し、中間部分のポリカーボネート構造単位(B)が基材から浮き上がるようにして、ブロック共重合体が基材に付着されると考えられる。この構成では、浮き上がったポリカーボネート構造単位(B)の部分で細胞を捕獲しながら、基材への細胞の接着性をより高めることができる。
一般式(V)において、a、b、c及びdは、上記した一般式(IV)と同じであるため、説明を省略する。
一実施形態による共重合体の1分子の分子量分布(Mw/Mn)は、特に限定されるものではないが、例えば、2.0以下、好ましくは1.5以下、より好ましくは1.2以下である。
また、ポリ乳酸構造単位(A)の分子量分布(Mw/Mn)は、特に限定されないが、1.0~10が好ましく、1.0~8がより好ましく、1.05~5がさらに好ましい。
また、ポリカーボネート構造単位(B)の分子量分布(Mw/Mn)は、特に限定されないが、1.0~10が好ましく、1.0~8がより好ましく、1.05~5がさらに好ましい。
また、数平均分子量(Mn)及び重量平均分子量(Mw)は、標準ポリスチレンで校正したゲル浸透クロマトグラフィー(GPC)を用いて測定することができる。また、分子量分布は、数平均分子量(Mn)に対する重量平均分子量(Mw)の比(Mw/Mn)から求めることができる。
一実施形態による共重合体において、共重合体全体を構成するモノマー単位の全単位に対し、ポリ乳酸構造単位(A)を構成するモノマー単位の全単位は、10モル%以上が好ましく、30モル%以上がより好ましく、40モル%以上がさらに好ましい。
また、別の一実施形態による共重合体において、共重合体全体を構成するモノマー単位の全単位に対し、ポリ乳酸構造単位(A)を構成するモノマー単位の全単位は、90モル%以下が好ましく、70モル%以下がより好ましく、60モル%以下がさらに好ましい。
共重合体全体を構成するモノマー単位の全単位に対し、ポリ乳酸構造単位(A)を構成するモノマー単位の全単位は、10~90モル%が好ましく、30~70モル%がより好ましく、40~60モル%がさらに好ましい。
また、別の一実施形態による共重合体において、共重合体全体を構成するモノマー単位の全単位に対し、ポリカーボネート構造単位(B)を構成するモノマー単位の全単位は、90モル%以下が好ましく、70モル%以下がより好ましく、60モル%以下がさらに好ましい。
共重合体全体を構成するモノマー単位の全単位に対し、ポリカーボネート構造単位(B)を構成するモノマー単位の全単位は、10~90モル%が好ましく、30~70モル%がより好ましく、40~60モル%がさらに好ましい。
一実施形態による共重合体において、共重合体全体を構成するモノマー単位の全単位に対し、ポリ乳酸構造単位(A)を構成するモノマー単位の全単位及びポリカーボネート構造単位(B)を構成するモノマー単位の全単位の合計単位は、80モル%以上が好ましく、90モル%以上が好ましい。さらには、一実施形態による共重合体は、ポリ乳酸構造単位(A)及びポリカーボネート構造単位(B)から構成されてもよい。
また、ポリカーボネートを用意し、ポリカーボネートの一方端又は両端にモノマーとして乳酸、乳酸ラクチド、又はこれらの組み合わせを重合させて結合させて合成することも可能である。
また、ポリ乳酸を用意し、ポリ乳酸の一方端又は両端にモノマーであるカーボネートを重合させて結合させて合成することも可能である。
また、乳酸とカーボネートとを用いて、ランダム重合してもよく、又は重合試薬を用いてブロック共重合することも可能である。
ポリ乳酸の数平均分子量は、ブロック共重合体に導入する構造単位(A)の目的とする分子量に応じて適宜設定することが好ましい。
ポリ乳酸を構成するモノマー成分である乳酸としては、D-乳酸、L-乳酸、DL-乳酸等を単独で、又はこれらを組み合わせて用いることができるが、D-乳酸又はL-乳酸を単一成分として用いることが好ましく、D-乳酸を単一成分として用いることがより好ましい。
モノマー成分である乳酸ラクチドとしては、D-乳酸ラクチド、L-乳酸ラクチド、DL-乳酸ラクチド等を単独で、又はこれらを組み合わせて用いることができるが、D-乳酸ラクチド又はL-乳酸ラクチドを単一成分として用いることが好ましく、D-乳酸ラクチドを単一成分として用いることがより好ましい。
モノマー成分として乳酸と乳酸ラクチドとを組み合わせて用いてもよいが、それぞれ単独で用いてもよい。
ポリカーボネートとしては、置換基としてアルコキシアルキルオキシカルボニル基を有するカーボネート単位を少なくとも1個含むポリカーボネート誘導体を好ましく用いることができ、さらには、この置換基はメトキシエチルオキシカルボニル基であることが好ましい。
また、ポリカーボネートとしては、一般式(II)で表されるカーボネート単位を少なくとも1個含むポリカーボネート誘導体を用いることが好ましい。さらには、一般式(I)で表されるカーボネート単位を少なくとも1個含むポリカーボネート誘導体を用いることが好ましい。一般式(II)で表されるカーボネート単位としては、置換基としてアルコキシアルキルオキシカルボニル基を有するカーボネート単位が好ましい。また、一般式(I)で表されるカーボネート単位としては、置換基としてメトキシエチルオキシカルボニル基を有するカーボネート単位が好ましい。
ポリカーボネートの数平均分子量は、ブロック共重合体に導入するポリカーボネート構造単位(B)の目的とする分子量に応じて適宜設定することが好ましい。
重合溶媒としては、例えば、ジクロロメタン、クロロホルム、ジエチルエーテル、テトラヒドロフラン(THF)、トルエン等が挙げられる。
重合触媒としては、例えば、1-(3,5-ビス(トリフルオロメチル)フェニル)-3-シクロヘキシル-2-チオウレア(TU)等の二官能基化チオウレア等が挙げられる。
重合反応の終了は、モノマー成分等が反応系に存在しているか否かで判断することができ、1H-NMR、TLC等の方法により確認することができる。重合反応が十分に進行したら、反応停止剤を加えることによって重合反応を終了させることができる。反応停止剤としては、例えば、酢酸、塩酸、硫酸、安息香酸等が挙げられる。
より具体的な例として、一般式(II)で表されるカーボネート単位を含むポリカーボネート誘導体の合成方法について説明する。
例えば、一般式(II)においてm=m’=1である単位を有するポリトリメチレンカーボネートの誘導体を重合するためには、トリメチレンカーボネートの炭素原子に、Mで表される基及びYで表される基を導入したトリメチレンカーボネート誘導体をモノマーとして用いることができる。M及びYは、上記一般式(II)において説明した通りである。
より詳細には、特開2014-161675号公報に記載の方法にしたがって合成することができる。
また、一実施形態による細胞足場材は、溶媒に添加された状態で細胞足場材組成物として提供されてもよい。溶媒としては、水、有機溶媒、又はこれらの混合溶媒を挙げることができる。水としては、溶媒として使用する水に加えて、一実施形態による共重合体の中間水であってもよい。有機溶媒としては、ジクロロメタン、メタノール等が挙げられる。細胞足場材組成物は、必要に応じて、後述する培地用添加剤、その他の添加成分等をさらに含むことができる。
一実施形態による細胞培養支持体は、細胞の分化を抑制し且つ増殖を促す作用を奏することができるため、特定の分化段階の細胞を培養により増殖させることに好適に用いることができる。
具体的には、基材として、細胞培養基材として使用されているものを特に制限することなく用いることができ、例えば、ディッシュ、平底ウェルプレート、丸底ウェルプレート等のウェルプレート;シャーレ等の細胞容器;マイクロビーズ、マイクロキャリア、三次元ブロック体;細胞シート等を用いることができる。
例えば、細胞足場材を有機溶媒に溶解した後、液体組成物の状態で基材に付与することができる。
溶媒としては、ジクロロメタン、メタノール等を用いることができる。また、液体組成物は、細胞足場材に加えて、後述する培地用添加剤、その他の添加成分等をさらに含むことができる。液体組成物全量に対して細胞足場材は0.05~10質量%が好ましく、0.1~1質量%がより好ましい。
基材への細胞足場材の付与量としては、基材の種類、細胞足場材の種類、培養対象となる細胞の種類、付与の方法等によって適宜調整が可能である。基材への細胞足場材の付与量は、単位面積当たりの固形分量で、例えば0.05μg/mm2~500μg/mm2とすることができる。スピンコーターを用いた付与方法を用いる場合には、単位面積当たりの固形分量で、例えば、0.01μg/mm2~10μg/mm2とすることができ、0.05μg/mm2~5μg/mm2とすることができ、又は0.1μg/mm2~3.0μg/mm2とすることができる。
一実施形態による細胞の培養方法は、細胞足場材を用意すること、細胞足場材を培養系に配置すること、及び細胞足場材の存在下で、細胞を培養することを含むことができる。
ここで、細胞足場材には、上記した一実施形態による細胞足場材を用いることができる。これによって、細胞の分化を抑制し、細胞の増殖を促進し、未分化の細胞を大量に生産することができる。
培養対象となり得る細胞として、例えば、初代培養細胞、培養細胞株、組換培養細胞株等を用いることができる。細胞の由来については、特に限定されず、例えば、ヒト、チンパンジー、サル、ウシ、ウマ、ブタ、イヌ、ネコ、ウサギ、ラット、マウス、ハムスター等の哺乳類;ニワトリ等の鳥類等が挙げられる。また、種の異なる2つ以上の細胞をハイブリッドさせた細胞を用いてもよい。
細胞が由来する器官、組織としては、特に限定されず、例えば、血球・リンパ系、血管系、脳・神経系、骨髄、筋組織、胸腺、唾液腺、口腔、食道、胃、肝臓、胆嚢、脾臓、小腸、大腸、直腸、皮膚、角膜、肺、甲状腺、哺乳器、子宮、子宮頸部、卵巣、精巣、膵臓、腎臓、副腎皮質、膀胱、胎盤、臍帯、胎仔、胎子、尾、間葉系幹細胞、癌細胞等が挙げられる。
細胞培養に用いる培地としては、細胞が生存し、増殖可能であるものであれば特に限定されず、培養する細胞の種類に応じて適宜選択することができる。
培地としては、血清培地又は無血清培地のいずれであってもよいが、一実施形態による細胞足場材は無血清培地での培養に好ましく用いることができる。
無血清培地としては、例えば、イーグル培地、ダルベッコ変法イーグル培地(低グルコース又は高グルコース)、イーグルMEM培地、αMEM培地、IMDM培地、ハムF10培地、ハムF12培地、RPMI1640培地等、又はこれらのブレンド培地が挙げられる。
血清培地は、無血清培地に血清を添加して調製することができる。無血清培地に血清を添加する場合は、例えば、ウシ胎仔血清(FBS)、ウマ血清、ヒト血清等の血清等を用いることができる。血清を添加する場合は、血清の濃度は30%以下が好ましい。
また、一実施形態によれば、細胞足場材の製造のための、ポリ乳酸構造単位(A)と、ポリカーボネート構造単位(B)とを有する共重合体の使用を提供することができる。
ここで、ポリ乳酸構造単位(A)と、ポリカーボネート構造単位(B)とを有する共重合体については、上述した一実施形態による細胞足場材における共重合体に関して記述した事項をそのまま援用する。例えば、本共重合体中、ポリカーボネート構造単位(B)は、置換基として、アルコキシアルキルオキシカルボニル基を有する単位を少なくとも1つ含んでいてもよく、また、ポリカーボネート構造単位(B)は、上述した一般式(I)で表される単位を少なくとも1つ含んでいてもよい。また、本共重合体中、ポリ乳酸構造単位(A)は、ポリD-乳酸構造単位、又はポリL-乳酸構造単位であってもよい。さらに、本共重合体は、ABA型ブロック共重合体であってもよい。これらの任意の組み合わせであってもよい。一例では、本共重合体中、ポリカーボネート構造体(B)は、置換基として、アルコキシアルキルオキシカルボニル基を有する単位を少なくとも1つ含むか、又は上述した一般式(I)で表される単位を少なくとも1つを含み、ポリ乳酸構造単位(A)は、ポリD-乳酸構造単位、又はポリL-乳酸構造単位である。この一例の共重合体は、ABAがブロック共重合体であってもよい。
実施例において得られた生成物の分子量、構造の確認、重合の進行度は、以下の手順によって評価した。
(1)分子量
数平均分子量(Mn)及び重量平均分子量(Mw)は、Chromaster GPC分析システム(株式会社日立ハイテクサイエンス製)にGPCカラム(株式会社日立化成テクノサービス製「Gelpack GL-R420,R430,R440」)を接続して、THFを溶出液として1.75mL/minで溶出し測定した。
分子量分布は、上記(1)の方法で求めた重量平均分子量(Mw)と数平均分子量(Mn)の値を用い、その比(Mw/Mn)として求めた。
モノマー及びポリマーの構造解析については、NMR測定装置(日本電子株式会社製、JEOL 500MHz JNM-ECX)を用い、1H-NMR測定を行った。なお、ケミカルシフトはCDCl3(1H:7.26ppm)を基準とした。
2,2-ビス(メチロール)プロピオン酸(bis-MPA:98%)、2-メトキシエタノール(99.8%)、Amberlyst-15(登録商標)(dry,moisture≦1.5%)はシグマアルドリッチジャパン株式会社より購入し、そのまま使用した。酢酸エチル(99.5%)、ジクロロメタン(DCM:99.5%)、ピリジン(99.5%)、塩化アンモニウム(98.5%)、炭酸水素ナトリウム(99.5~100.3%)、1,8-ジアザビシクロ[5.4.0]ウンデカ-7-エン(DBU:99.0%)、安息香酸(99.5%)、ジエチルエーテル(99.0%)、ヘキサン(95.0%)、トリメチレンカーボネート(TMC:98.0%)、メタノール(99.8%)、テトラヒドロフラン(THF:99.5%)、(+)-sparteine(Sp)、2-プロパノール(99.7%)は関東化学株式会社より購入した。脱水グレードのTHF、DCMは関東化学株式会社製ソルベントサプライシステムから供給した(水分量<10ppm)。塩酸(35.0~37.0質量%)、硫酸マグネシウム(95.0%)は富士フイルム和光純薬株式会社より、トリホスゲン(98%)、ベンジルアルコール、2-メトキシエチルアクリレート(>98.0%)、1,4-ジオキサン(>99.0%)、2,2-アゾビス(イソブチロニトリル)(AIBN>98.0%)は東京化成工業株式会社より購入し、そのまま使用した。1-(3,5-ビス(トリフルオロメチル)フェニル)-3-シクロヘキシル-2-チオウレア(TU)は既報の手法を参考に合成した。モノマー類、TUはTHFに溶解させCaH2(水素化カルシウム)にて乾燥させた。DBUはCaH2を用いて減圧蒸留したものを使用した。
D-乳酸ラクチド(D-lactide)は、Purac社より購入した。
Lipidure(登録商標)-CM5206(以下、PMBとも記す)は、日油株式会社より購入した。
TU(5.8mg,1.49mmol)及びD-lactide(77.1mg,0.535mmol)をDCM(240mg)中に溶解させ、室温で撹拌した。2時間の反応後、反応溶液を2-プロパノール(30mL)中に再沈殿し、真空下で乾燥させて白色の固体PDLAを得た。
ベンジルアルコール(4.32mg,0.04mmol)、トリメチレンカーボネート(2.01g,20mmol)、DBU(182.4mg,1.4mmol)及びTU(447.1mg,1.4mmol)をDCM(20mL)に溶解させ、撹拌した。24時間後、安息香酸を加えて反応を停止し、反応溶液を2-プロパノール中に再沈殿後、上澄み液を遠心分離した。得られた粘性体をDCMに溶解させて回収し、エバポレーターで濃縮後、乾燥させ、PTMCを得た。
15gの2-メトキシエチルアクリレート(130.14g/mol,115mmmol)、60gの1,4-ジオキサンに溶解し、30分間窒素バブリングを行った。その後、開始剤である15mgのAIBN(0.091mmol)を少量の1,4-ジオキサンに溶解して加え、窒素バブリングしながら75℃で6時間重合を行った。その後、重合溶媒をn-ヘキサン1000mlに滴下することで重合で生成したポリマーを沈殿物として回収した。得られた副生成物を、THF/n-ヘキサン系で沈殿操作を3回繰り返し、精製した。精製後のポリマーはTHFに溶解させて回収し、エバポレーターで濃縮後、60℃で30時間、真空乾燥させ、PMEAを得た。
ポリマーとして、合成例1で得られたPDMED、合成例1の中間体であるPMEMTC、合成例2で得られたPDLA、合成例3で得られたPTMC、合成例4で得られたPMEA、PMB(日油株式会社製「LIPIDURE(登録商標)-CM5206」)を用いて、以下の評価を行った。
直径15mmで円形に打ち抜いた厚み125μmのPETフィルム(三菱ケミカル株式会社製「Diafoil、T100E125 E07」)を、メタノールに一晩浸漬し脱脂処理を行った。その後、各ポリマーをDCMもしくはメタノールに溶解し、0.2質量%の濃度に調整した。この溶液を上記処理済みのPETフィルムに100μLを2度スピンコーターで塗布して、室温にて一日静置した後に一晩純水に浸漬して洗浄した。これを乾燥した後、ポリマー塗布PETフィルム(以下、支持体)をクリーンベンチ内で、スポットUV照射装置(ウシオ電機株式会社製「SP-11」)にて4W/cm2の出力で10分間照射して滅菌した。また、対照として、脱脂処理をした後に、ポリマーを塗布しない未処理の支持体(PET支持体)を用意した。
直径15mmで円形に打ち抜いた厚み125μmのPETフィルム(三菱ケミカル株式会社製「Diafoil、T100E125 E07」)を、メタノールに一晩浸漬し脱脂処理を行った。基板の裏面に0.1質量%のBSA(ウシ血清アルブミン)を1質量%TritonX-100含有のリン酸バッファー液(PBS pH7.4)に溶解し、100μLを2度スピンコーターで塗布して、室温にて一日静置した後に一晩純水に浸漬して洗浄、これを乾燥することでPETへの非特異吸着を防止した。その後、各ポリマーをDCMもしくはメタノールに溶解し、0.2質量%の濃度に調整した。この溶液を上記処理済みのPETフィルムの表面に100μLを2度スピンコーターで塗布して、室温にて一日静置した後に一晩純水に浸漬して洗浄した。
得られた支持体を細胞培養向け24穴プレート(コーニング社製「Coster 24 wells」)に沈め、蛍光標識BSA(FITC標識)を0.1w/v%でPBSに溶解し、500μL/wellを添加して、37℃、60minで搖動した(傾斜角:6°,30r/min)。対照として、ポリマーを塗布しない未処理の支持体(PET支持体)を用いて、同様に処理した。
搖動後、PBSで洗浄し、蛍光顕微鏡(株式会社キーエンス製「BZ-X」)にて、吸着の状態を観察し、吸着量を蛍光強度から評価した。その結果を図1に示す。図1は、PET支持体の蛍光強度を基準として、各ポリマーを塗布した支持体を用いた場合の蛍光標識BSAの蛍光強度を相対的に示すグラフである。
(3-1)ヒト正常線維芽細胞
上記(1)細胞足場材の調製で調製したPDMED支持体、PMB支持体、PET支持体を、24穴組織培養用プレートの各ウェルの底面にセットした。また、支持体をセットしないウェルを用意した。各ウェルに、5×103cells/cm2の播種密度でヒト皮膚正常線維芽細胞(NHDF)を播種した。各ウェルにEagle MEM培地に10%ウシ胎仔血清(FBS)を添加して調製した培地を添加した。
このプレートを37℃、5%CO2雰囲気下で培養した。細胞培養開始から1時間後、1日後、2日後、3日後、7日後にそれぞれ細胞の状況を観察するとともに、培養上清を1000μL取り出し、7000rpmで1分間遠心分離し、その上澄みを500μL取り出し、FGF-2(線維芽細胞成長因子2)の定量評価に供試した。
蛍光差顕微鏡(オリンパス株式会社製「MVX10」)を用いて、細胞増殖の状態を観察するとともに、細胞数を計測した。図2は、蛍光差顕微鏡によって観察した写真であり、細胞開始から(a)1時間後、(b)1日後、(c)2日後、(d)3日後、(e)7日後である。図3に、細胞培養時間に対する細胞数のグラフを示す。
(ii)細胞分化抑制の評価方法
各細胞培養時間に採取した培養上清をヒトFGF-2測定ELISAキット(R&D system社製)にて測定した。図4に、細胞培養時間に対するFGF-2の濃度のグラフを示す。
細胞の増殖の観察からも、PDMEDは、ヒト線維芽細胞としての機能である塊状増殖をすることなく、増殖のみが優先して起こっていることが示唆された。これはFGF-2の安定した放出からも明らかである。
上記(1)細胞足場材の調製と同様の方法で、支持体の直径を50mmとし、PDMED支持体、PMB支持体、対照としての未処理のPET支持体、対照としてのラミニン(株式会社ニッピ製「iMatrix511」)支持体を用意した。各支持体を60mmディッシュ(IWAKI社製「3010-060」)の底面にセットした。京都大学由来フィーダーフリーiPS細胞を各ディッシュ(それぞれ、「PDMEDディッシュ」、「PMBディッシュ」、「PETディッシュ」、「iMatrixディッシュ」という)に、2.5×104cells/ディッシュの量で播種した。
表1に、培養7日目の未分化細胞率と、培養5日目のIL-6及びTNFαの濃度を示す。
未分化細胞率(%)が100(%)に近いほど、細胞の分化が抑制されていることを意味する。また、IL-6の濃度が0に近いほど、細胞の分化が抑制されていることを意味する。さらに、TNFαの濃度が0に近いほど、細胞の分化が抑制されていることを意味する。
また、PMBディッシュ及びPETディッシュでは、培養7日目での未分化細胞率がPDMEDディッシュよりも低く、また、培養5日目でのIL-6の放出も認められた。なお、PMBディッシュについては、培養2日目で約2pg/mLのTNFαの放出が確認された(データ示さず)。このことから、PMBディッシュ及びPETディッシュでも、細胞の分化が十分に抑制されていないことが示された。
これらに対して、PDMEDディッシュでは、培養7日目での未分化細胞率が高く、また培養初日から5日目までの間で各サイトカインの放出が認められなかった。このことからも、PDMEDを細胞足場材として使用した場合、細胞の分化を抑制しながら、増殖に寄与できることがわかった。
Claims (12)
- ポリ乳酸構造単位(A)と、ポリカーボネート構造単位(B)と、を有する共重合体を含む、細胞足場材。
- 前記ポリカーボネート構造単位(B)は、置換基として、アルコキシアルキルオキシカルボニル基を有する単位を少なくとも1つ含む、請求項1記載の細胞足場材。
- 前記ポリカーボネート構造単位(B)は、置換基として、メトキシエチルオキシカルボニル基を有する単位を少なくとも1つ含む、請求項1又は2に記載の細胞足場材。
- 前記一般式(I)中、Xはメチル基である、請求項4に記載の細胞足場材。
- 前記共重合体は、一方端又は両端部がポリ乳酸構造単位(A)であるブロック共重合体である、請求項1から5のいずれか1項に記載の細胞足場材。
- 前記共重合体は、AB型、ABA型、又はABAB型のブロック共重合体である、請求項6に記載の細胞足場材。
- 前記共重合体は、ABA型ブロック共重合体である、請求項7に記載の細胞足場材。
- 前記ポリ乳酸構造単位(A)は、ポリD-乳酸構造単位、又はポリL-乳酸構造単位である、請求項1から8のいずれか1項に記載の細胞足場材。
- 請求項1から9のいずれか1項に記載の細胞足場材と、基材とを含む、細胞培養支持体。
- 請求項1から9のいずれか1項に記載の細胞足場材を用意すること、前記細胞足場材を培養系に配置すること、及び前記細胞足場材の存在下で、細胞を培養することを含む、細胞の培養方法。
- 前記培養系が、無血清培地で構成される、請求項11に記載の細胞の培養方法。
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EP3992276A1 (en) | 2022-05-04 |
JP7414067B2 (ja) | 2024-01-16 |
CN114051530A (zh) | 2022-02-15 |
US20220228096A1 (en) | 2022-07-21 |
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JPWO2020262609A1 (ja) | 2020-12-30 |
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