WO2012029882A1 - 細胞培養用温度応答性基材及びその製造方法 - Google Patents
細胞培養用温度応答性基材及びその製造方法 Download PDFInfo
- Publication number
- WO2012029882A1 WO2012029882A1 PCT/JP2011/069839 JP2011069839W WO2012029882A1 WO 2012029882 A1 WO2012029882 A1 WO 2012029882A1 JP 2011069839 W JP2011069839 W JP 2011069839W WO 2012029882 A1 WO2012029882 A1 WO 2012029882A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- temperature
- substrate
- responsive
- cell culture
- block copolymer
- Prior art date
Links
Images
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
- C12N5/0068—General culture methods using substrates
-
- 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
- C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
- C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
-
- 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
- C08F2438/00—Living radical polymerisation
- C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]
-
- 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
- C12N2539/00—Supports and/or coatings for cell culture characterised by properties
- C12N2539/10—Coating allowing for selective detachment of cells, e.g. thermoreactive coating
Definitions
- the present invention relates to a cell culture substrate useful in fields such as biology and medicine.
- This application is a priority claim application based on a Japanese patent application filed on August 31, 2010 (Japanese Patent Application No. 2010-208506).
- animal cell culture technology has made significant progress, and research and development for animal cells has been extended to various fields.
- the target animal cells can be used not only to commercialize the original cells, but also to produce useful products by analyzing the cells and their surface proteins.
- the cells are returned to the patient's living body to be treated.
- many researchers have focused on the technology for culturing animal cells.
- Patent Document 1 Japanese Patent Application Laid-Open No. 2-211865 (Patent Document 1) describes cell culture support in which a substrate surface is coated with a polymer having an upper or lower critical solution temperature of 0 to 80 ° C. in water. A new cell that cultivates cells on the body at a temperature lower than the upper critical lysis temperature or higher than the lower critical lysis temperature and then exfoliates the cultured cells without enzyme treatment by setting the temperature to the upper critical lysis temperature or lower or the lower critical lysis temperature or lower. A culture method is described.
- Patent Document 2 discloses that skin cells are treated at a temperature lower than the upper critical lysis temperature using the temperature-responsive cell culture substrate described in Japanese Patent Laid-Open No. 2-211865 (Patent Document 1). Alternatively, it is described that the cultured skin cells are detached with low damage by culturing at a temperature lower than the lower critical solution temperature or higher and then lower than the upper critical solution temperature or lower than the lower critical solution temperature. Further, Japanese Patent Application No. 2007-105311 (Patent Document 3) discloses a method for repairing surface protein of cultured cells using a temperature-responsive cell culture substrate described in JP-A-2-21865 (Patent Document 1). Are listed. By utilizing the temperature-responsive cell culture substrate as described above, various new developments have been made with respect to conventional culture techniques.
- the conventional technology coats the surface of a chemically inert engineer plastic using high energy such as an electron beam, which requires a large-scale device such as an electron beam irradiation device. Therefore, there is a problem that the price of the base material is inevitably high.
- Non-Patent Document 1 Soft Matter, 5, 2937-2946 (2009) (Non-Patent Document 1), Interface, 4, 1151-1157 (2007) (Non-Patent Document 2)
- Non-Patent Document 2 it has a specific molecular structure.
- a method of synthesizing a polymer and coating the surface of a substrate is known.
- both technologies can cultivate cells in the same way as conventional cell culture substrates, and the cells can be detached by simply changing the temperature as in the temperature responsive substrate for cell cultures prepared using the electron beam. Or, when the cultured cells become confluent, they have not reached a technical level that can be detached as a cell sheet.
- JP-A-2-21865 JP 05-192138 A Japanese Patent Application No. 2007-105311
- an object of the present invention is to provide a novel temperature-responsive substrate for cell culture created based on a completely different idea from the prior art and a method for producing the same.
- a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded is 0.8 to 3.0 ⁇ g as a temperature-responsive polymer component on the substrate surface. It has been found that when the coating is performed at a rate of / cm 2 , cells can be cultured efficiently, and the cultured cells or cell sheets can be efficiently peeled simply by changing the temperature of the substrate surface.
- a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded is coated on the substrate surface at a rate of 0.8 to 3.0 ⁇ g / cm 2 as a temperature-responsive polymer component.
- a temperature-responsive substrate for cell culture is provided.
- the responsive base material for cell cultures of this invention is not limited, The following aspect is included as a preferable aspect.
- the surface of the substrate is coated at a rate of 0.09 to 7.0 ⁇ g / cm 2 as a water-insoluble polymer component.
- the substrate surface has a phase separation structure.
- the content of the temperature-responsive polymer in the block copolymer is 30 to 90 wt%.
- the average molecular weight of the temperature-responsive polymer in the block copolymer is 3000 or more. As shown in FIG. 2, since the ratio of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is close to 1, any average molecular weight is adopted in order to obtain the effect of the present invention. May be.
- the temperature-responsive polymer is a poly-N-substituted acrylamide derivative, a poly-N-substituted methacrylamide derivative, a copolymer thereof, polyvinyl methyl ether, or two or more thereof.
- the temperature responsive polymer is poly-N-isopropylacrylamide.
- the base material is a single plate or a combination of two or more.
- a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded is dissolved or dispersed in an organic solvent, and the block copolymer solution is spin-coated on the substrate surface.
- a method for producing a temperature-responsive substrate for cell culture which is uniformly applied and dried.
- the manufacturing method of the responsive base material for cell cultures of this invention is not limited, The following aspect is included as a preferable aspect.
- the block copolymer is obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization.
- the solvent for dissolving the block copolymer is a mixed solution of acetonitrile and N, N-dimethylformamide.
- the mixing ratio of the mixed solvent of acetonitrile and N, N-dimethylformamide is 5: 1 by volume.
- the temperature-responsive substrate for cell culture By using the temperature-responsive substrate for cell culture according to the present invention, cells can be efficiently cultured, and the cultured cells or cell sheets can be efficiently detached simply by changing the temperature of the substrate surface. it can. Moreover, according to this invention, such a functional surface can be produced simply.
- FIG. 1 is a diagram showing a synthesis route of a block copolymer of Example 1.
- FIG. 3 is a view showing the characteristics of the block copolymer obtained in Example 1. It is a figure which shows the characteristic of the temperature-responsive surface obtained in Example 1.
- FIG. The amount of temperature-responsive polymer modification and the stability of the coating film were evaluated by ATR / FT-IR measurement. It is a figure which shows the characteristic of the temperature-responsive surface obtained in Example 1.
- FIG. Surface wettability was evaluated by measuring the fixed amount of PIPAAm and static contact angle (underwater bubble method). It is a figure which shows the relationship between the temperature-responsive surface of each modification amount obtained in Example 1, and cell behavior.
- FIG. 4 is a photograph showing the relationship between the temperature-responsive surface and cell behavior of each modification amount obtained in Example 1.
- FIG. Temperature response of B79-IP120 coated on TCPS (A) and TCPS at 0.1 w / v% (B), 0.3 w / v% (C), and 0.5 w / v% (D), respectively.
- a micrograph of adherent cells at 72 hours after seeding cells at 37 ° C. on the sexual surface is shown. Thereafter, the temperature is lowered to 20 ° C., and the cell morphology after further culturing for 2 hours is shown in (E) to (H), respectively.
- Cell seeding 5.0 ⁇ 10 3 cells / cm 2 .
- the present invention relates to a temperature response for cell culture in which a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded is coated on a substrate surface at a rate of 0.8 to 3.0 ⁇ g / cm 2. It is a conductive substrate.
- the water-insoluble polymer portion of the block copolymer coated on the surface of the substrate has an effect that it is not released from the surface of the substrate during cell culture and also when the cultured cells or cell sheets are detached based on temperature changes.
- block copolymer means a polymer having at least two compositionally distinct segments, and as understood by those skilled in the art, for example, diblock copolymers, triblock copolymers, random block copolymers, Examples include star-branched block copolymers or hyperbranched block copolymers.
- the block copolymer used in the present invention typically has a water-insoluble polymer segment (A) and a temperature-responsive polymer segment (B), but has the following structure: an AB structure comprising two block segments; Any of ABA or BAB including three block segments; and — (AB) n — (where n is an integer greater than or equal to 2) including a plurality of blocks. Also good.
- a block copolymer that can be used for the temperature-responsive substrate for cell culture of the present invention preferably has an AB structure.
- a block copolymer having an AB structure includes, for example, a poly-n-butyl methacrylate (PBMA) segment as a water-insoluble polymer segment (A) and a poly-N as a temperature-responsive polymer segment (B).
- PBMA poly-n-butyl methacrylate
- A water-insoluble polymer segment
- B temperature-responsive polymer segment
- PIPAAm isopropylacrylamide
- the block copolymer is expressed as “B79-IP120” etc. using the number of monomer units of each polymer (for example, 79 BMA, 120 IPAAm) (See Example 1).
- water-insoluble polymer used in the present specification is not particularly limited as long as it is insoluble in water, and examples thereof include polyalkyl acrylates such as poly-n-butyl acrylate and poly-t-butyl acrylate. , Poly-n-butyl methacrylate, poly-t-butyl methacrylate, polyalkyl methacrylates such as polymethyl methacrylate, polystyrene and the like.
- temperature responsive polymer means a polymer having a lower critical solution temperature (LCST) and / or a polymer having an upper critical solution temperature (UCST), but homopolymers, copolymers thereof, Or any of a mixture may be sufficient.
- LCST lower critical solution temperature
- UST upper critical solution temperature
- examples of such a polymer include polymers described in Japanese Patent Publication No. 06-104061. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers.
- the monomer that can be used include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, vinyl ether derivatives, and polyvinyl alcohol partially acetylated products.
- any two or more of these monomers can be used.
- copolymers with monomers other than the above monomers, grafts or copolymers of polymers, or a mixture of polymers and / or copolymers may be used.
- the substance to be separated is a biological substance
- the temperature-responsive polymer that can be used in the present invention is a poly-N-substituted acrylamide derivative, a poly-N-substituted methacrylamide derivative, a copolymer thereof, a polyvinyl methyl ether, or a combination of two or more. There may be.
- poly-Nn-propyl acrylamide lower critical dissolution temperature of homopolymer 21 ° C.
- poly-Nn-propyl methacrylamide 27 ° C.
- poly -N-isopropylacrylamide 32 ° C
- poly-N-isopropylmethacrylamide 43 ° C
- poly-N-cyclopropylacrylamide 45 ° C
- poly-N-ethoxyethylacrylamide about 35 ° C
- Poly-N-ethoxyethyl methacrylamide about 45 ° C.
- poly-N-tetrahydrofurfuryl acrylamide about 28 ° C.
- poly-N-tetrahydrofurfuryl methacrylamide about 35 ° C.
- poly-N N-ethylmethylacrylamide 56 ° C
- poly-N, N-diethylacrylamide 32 ° C
- the temperature-responsive polymer used in the present invention has a content in the block copolymer of 30 to 90 wt%, preferably 40 to 85 wt%, more preferably 45 to 60 wt%. .
- the amount is less than 30 wt%, the cultured cells on the polymer are hardly detached even if the temperature is changed, and the working efficiency is remarkably deteriorated.
- it is more than 90 wt%, it is difficult for cells to adhere to the region, making it difficult to sufficiently attach the cells, which is not preferable as the cell culture substrate of the present invention. It is not preferable because it decreases and is easily released from the substrate surface.
- the temperature-responsive polymer used in the present invention has an average molecular weight of 3000 or more, preferably 5000 or more, more preferably 10,000 or more, and most preferably 12000 or more.
- the molecular weight of the temperature-responsive polymer used by this invention is the said lower limit, it will not be restrict
- the upper limit of the average molecular weight of the temperature-responsive polymer may be 35000, preferably 30000, more preferably 25000.
- the temperature-responsive base material for cell culture according to the present invention is coated with a temperature-responsive polymer content in the block copolymer in the range of 0.8 to 3.0 ⁇ g / cm 2 , preferably 0.9 to a 2.0 [mu] g / cm 2, more preferably from 1.3 ⁇ 1.8 ⁇ g / cm 2.
- the coating amount is less than 0.8 ⁇ g / cm 2 , the cultured cells on the block copolymer are hardly detached even if the temperature is changed, and the working efficiency is remarkably deteriorated.
- it is more than 3.0 ⁇ g / cm 2 it is difficult for cells to adhere to the region, and it becomes difficult to sufficiently attach the cells, which is not preferable as the cell culture substrate of the present invention.
- the coating amount may be measured according to a conventional method, for example, FT-IR-ATR method, elemental analysis method, ESCA or the like may be used, and any method may be used.
- the temperature-responsive substrate for cell culture is characterized in that a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded at a predetermined ratio is coated on the surface of the substrate.
- the ratio of the “water-insoluble polymer” is, as described above, when the temperature-responsive polymer exceeds 90 wt%, that is, when the water-insoluble polymer is less than 10 wt%, the block copolymer is released from the substrate surface. Since it becomes easy, the ratio of the water-insoluble polymer in the block copolymer may be 10 wt% or more.
- the water-insoluble polymer content in the block copolymer is the above-mentioned desired amount of the culture substrate. It is only necessary to adjust appropriately so that a range amount of temperature-responsive polymer can be coated. More specifically, the temperature-responsive substrate for cell culture according to the present invention may be coated with a water-insoluble polymer content in the block copolymer in the range of 0.09 to 7.0 ⁇ g / cm 2. It is preferably 0.14 to 4.5 ⁇ g / cm 2 , more preferably 0.2 to 3.0 ⁇ g / cm 2 .
- the block copolymer of the present invention is a combination of a water-insoluble polymer segment and a temperature-responsive polymer segment having affinity for water. Therefore, when this block copolymer is coated on the substrate surface and dried, it is expected that a phase separation structure such as a fine lamellar structure, a sea-island structure, a cylinder structure, or a bicontinuous structure is formed on the substrate surface.
- the form, size, etc. of the phase separation structure are not particularly limited. However, when cells adhere to the surface of the substrate, it is advantageous to have a phase separation structure on the surface of the substrate, which can suppress cell degeneration. is there.
- RAFT reversible addition-fragmentation chain transfer
- ATRP atom transfer radical polymerization
- anionic polymerization method or a living radical polymerization method
- the production method of the block copolymer is not limited to the above polymerization method and other polymerization methods, but the RAFT polymerization method is preferably used.
- the dithioester functional group that is a part of the structure of the RAFT agent remains at the end of the produced block copolymer.
- the temperature responsive substrate for cell culture of the present invention it is possible to impart new functionality to the surface of the temperature responsive substrate for cell culture of the present invention.
- a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, an aldehyde group, a sulfonic acid group etc. are mentioned.
- a peptide or protein that promotes cell adhesion may be immobilized on the end of the polymer chain produced as described above.
- the lower critical solution temperature (LCST) of poly-N-isopropylacrylamide changes depending on the hydrophilicity / hydrophobicity of the terminal functional group, the polymer chain end as in the present invention is changed.
- This functional group introduction is expected as a new technique for controlling the temperature responsiveness of the substrate surface from another viewpoint.
- the block copolymer of the present invention can be obtained by the RAFT polymerization method, that is, the method of growing a temperature-responsive polymer by radical polymerization in the presence of the RAFT agent.
- the initiator used in this case is not particularly limited.
- 2,2′-azobis isobutyronitrile
- V-70 2,2′-azobis [(2-carboxyethyl) -2- (methylpropionamidine)]
- polymer chains can be grown from this initiator.
- the RAFT agent used is not particularly limited, but benzyldithiobenzoate, cumyl dithiobenzoate, 2-cyanopropyldithiobenzoate, 1-phenylethylphenyl dithioacetate, cumylphenyl dithioacetate, benzyl 1- Examples include pyrrole carbodithioate and cumyl 1-pyrrole carbodithioate.
- the solvent used in the polymerization in the present invention is not particularly limited, but benzene, 1,4-dioxane, dimethylformaldehyde (DMF) and the like are preferable.
- the solvent is not limited in any way, but can be appropriately selected depending on the type of monomer, RAFT agent and polymerization initiator used in the polymerization reaction.
- the present invention uses a solvent such as 1,4-dioxane in the presence of a RAFT agent in the presence of a RAFT agent to perform hydration power within a temperature range of 0 to 80 ° C. from the monomer by a surface initiated radical polymerization method. It is a method of growing a polymer in which changes. Other initiator concentrations, RAFT agent concentration, reaction temperature, reaction time, and the like during polymerization are not particularly limited, and may be changed according to the purpose. Furthermore, the state of the reaction solution may be left standing or stirred. In the RAFT method used in the present invention, the reaction can proceed without using a metal ion or the like, so that the product after the reaction can be easily purified, and the reaction step itself can be conveniently operated. .
- a solvent such as 1,4-dioxane
- the temperature-responsive substrate for cell culture of the present invention is typically obtained by dissolving or dispersing the block copolymer thus obtained in a solvent and coating the copolymer uniformly on the substrate surface.
- the solvent used is not particularly limited as long as it can dissolve or disperse the block copolymer and appropriately select a solvent that does not dissolve the substrate surface as much as possible.
- the mixing ratio is not particularly limited.
- the volume ratio of acetonitrile is 5 and N, N-dimethylformamide.
- the method is not particularly limited, and examples thereof include a method using a spin coater and a method of allowing a substrate to stand on a horizontal table.
- the temperature responsive substrate for cell culture according to the present invention is obtained by removing the solvent.
- the method for removing the solvent is not particularly limited, but is a method of slowly evaporating in room temperature / atmosphere over time, a method of evaporating slowly in room temperature / solvent saturated environment over time, or evaporation by heating.
- the method of evaporating under reduced pressure, etc. but the first two methods are good for producing a clean surface of the final temperature-responsive substrate for cell culture, particularly in a room temperature / solvent saturated environment.
- the method of slowly evaporating over time is preferred.
- the cell culture substrate on which the coating is applied is not limited to glass, modified glass, polystyrene, polymethyl methacrylate, polyethylene terephthalate, polycarbonate, etc. that are usually used for cell culture, and materials that can generally be given form. However, for example, polymer compounds other than those described above, ceramics, metals, and the like can be used.
- the shape of the cell culture substrate is not limited to a cell culture dish such as a Petri dish, but is a plate (plate), fiber (thread), (porous) particle (granular), tube or film base.
- the base material which combined materials or 2 or more types may be sufficient. Moreover, even if it has the shape (flask etc.) of the container generally used for cell culture etc., it does not interfere.
- the shape of the cell culture substrate used in the present invention is a single plate or a combination of two or more.
- the surface of the cell culture substrate is preferably hydrophobic. Therefore, when using a cell culture substrate having a hydrophilic surface such as a glass substrate, it is preferable to perform a hydrophobic treatment in advance.
- the hydrophobizing treatment is not particularly limited, but may be a treatment using a silane coupling agent (for example, hexyltriethoxysilane).
- the cells that can be used on the surface of the temperature-responsive substrate for cell culture obtained in the present invention are not limited to its origin, and the source and production method are not particularly limited.
- Examples of the cells used in the present invention include cells of animals, insects, plants, etc., and bacteria.
- animal cells are preferably derived from humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, cattle, marmoset, African green monkeys, and the like.
- the medium used in the present invention is not particularly limited as long as it is a medium for animal cells, and examples thereof include a serum-free medium and a serum-containing medium. Such a medium may further contain a differentiation inducer such as retinoic acid or ascorbic acid.
- the seeding density of cells on the surface of the substrate can be appropriately changed, and the seeding density can be determined according to a conventional method.
- the temperature of the culture substrate is simply set to the upper critical solution temperature of the coating polymer on the culture substrate or less than the lower critical solution temperature.
- the cell sheet can be peeled without enzyme treatment.
- the above peeling can be performed in a culture solution or in another isotonic solution, and can be performed according to the purpose.
- the method of tapping or shaking the substrate, or the method of stirring the medium using a pipette, alone or in combination It may be used.
- the temperature-responsive substrate for cell culture of the present invention By using the temperature-responsive substrate for cell culture of the present invention, cells obtained from each tissue can be efficiently cultured. If this culturing method is used, the cultured cells or cell sheets can be efficiently peeled off without being damaged only by changing the temperature. Conventionally, such work has required labor and operator's skill, but according to the present invention, this need is eliminated and a large amount of cells can be processed. According to the present invention, it is shown that such a culture substrate surface can be produced by using a living radical polymerization method. In particular, the surface of the culture substrate can be easily and precisely designed by the RAFT polymerization method (one of the living radical polymerization methods), and a functional group can be easily introduced at the end of the molecular chain. Temperature responsive substrates and methods for producing them are highly advantageous cell culture techniques.
- Example 1 Preparation and Properties of Temperature Responsive Surface
- PIPAAm poly-N-isopropylacrylamide
- PBMA poly-n-butyl methacrylate
- a temperature-responsive block copolymer consisting of the following segments was prepared and spin-coated on the substrate surface.
- physical properties such as the amount of polymer introduced and surface wettability were evaluated, and the difference in cell adhesion to the substrate surface due to temperature change was also investigated.
- PBMA-b-PIPAAm block copolymer was prepared using PBMA, which is a water-insoluble polymer synthesized by RAFT polymerization, as a macro RAFT agent (FIG. 1).
- PBMA water-insoluble polymer synthesized by RAFT polymerization
- FIG. 1 analytical values of PBMA and PBMA-b-PIPAAm synthesized with RAFT polymerization and controlled in molecular weight are shown in FIG.
- a PBMA-b-PIPAAm block copolymer having a ratio of monomer units of BMA and IPAAm of 70:24 is represented as “B79-IP24”.
- TCPS polystyrene
- the surface polymer amount of this substrate was determined by the total reflection Fourier transform infrared spectroscopy (ATR / FT-IR) method, and the difference in the polymer amount before and after the treatment was compared (see FIG. 3).
- the wettability at 20 ° C. and 37 ° C. was evaluated by the static contact angle measurement method using the underwater bubble method.
- the phase-separation structure is formed according to the ratio (refer FIG. 2) of the PIPAAm chain to the block copolymer which consists of a PBMA chain and a PIPAAm chain.
- Example 2 Examination of graft amount and cell behavior According to the description in Example 1, a substrate in which TCPS was coated with various concentrations of B79-IP120 was prepared, and bovine carotid artery-derived vascular endothelial cells ( BAEC) was seeded at a concentration of 1.0 ⁇ 10 4 cells / cm 2 , and the adhesion of the cells at 37 ° C. onto the surface of the polymer-coated substrate and the desorption behavior of the cells by low-temperature treatment at 20 ° C. were evaluated.
- BAEC bovine carotid artery-derived vascular endothelial cells
- FIG. 5 indicates the TCPS base material not coated with the polymer, and the black rhombus and white rhombus indicate the cell behavior on the base material obtained by coating TCPS with B79-IP120 at 0.3 and 0.5 w / v%, respectively.
- FIG. 5 shows changes in cell adhesion rate (%) when BAEC cells are seeded on each substrate and cultured for 72 hours, and then the culture temperature is changed from 37 ° C. to 20 ° C. More specifically, the cell adhesion rate is increased by culturing at 37 ° C., but the adhesion rate is high in TCPS for cell culture, and then the substrate coated with B79-IP120 at 0.3 w / v% is high. It was.
- the cell adhesion rate was low in the base material coated with B79-IP120 at 0.5 w / v%.
- concentration of 0.3 w / v% is 1.44 ⁇ g / cm 2
- the cell adhesion rate tends to decrease as the amount of PIPAAm covered increases.
- the change in the cell adhesion rate when the temperature was changed from 37 ° C. to 20 ° C. was noticeable when a substrate having a PIPAAm coating amount of 1.44 ⁇ g / cm 2 was used.
- Example 3 Detailed analysis of changes in cell behavior by block copolymer (1) Relationship between PIPAAm molecular chain length and cell behavior The number of monomer units (number of monomers) of BMA in the block copolymer was kept constant, and the monomer unit of IPAAm was changed. The prepared block copolymer was prepared according to the production method of Example 1. As in Example 1, TCPS was coated with a block copolymer, and changes in the adhesion rate of BAEC cells with changes in temperature were examined (FIG. 7). As in Example 2 above, the cell adhesion rate increased as the amount of PIPAAm covered decreased.
- the base material having a low coating amount of PIPAAm (1.18 ⁇ g / cm 2 ) has a higher coating amount (1.44, 1.60, 1.63 ⁇ g / It was found that a base material (1.44, 1.60, 1.63 ⁇ g / cm 2, etc.) that desorbs more slowly than (cm 2 ) and has a high coating amount desorbs quickly after a temperature change.
- a base material (1.44, 1.60, 1.63 ⁇ g / cm 2, etc.) that desorbs more slowly than (cm 2 ) and has a high coating amount desorbs quickly after a temperature change.
- Example 2 In the same manner as in Example 2, a substrate coated with these block copolymers on TCPS was prepared, and then the adhesion of BAEC cells seeded on each substrate, followed by the change in the cell desorption behavior due to the temperature change, was determined by the cell adhesion rate. Evaluation was made (FIG. 8).
- the three block copolymers used differed significantly in their chain length, but there was little difference in the amount of PIPAAm coated on TCPS.
- the adhesion and detachment of the cells seeded on each substrate there was no significant difference due to the chain length of the block copolymer of each substrate.
- FIG. 9 shows the cell sheet formed on a substrate coated with PBMA-b-PIPAAm block copolymer (0.3 w / v% concentration B79-IP120), and the temperature The photograph after peeling of the cell sheet by a change is shown.
- PBMA-b-PIPAAm block copolymer 0.3 w / v% concentration of B79-IP120
- the cell sheet was formed while maintaining the intercellular connection between adjacent cells.
- various block copolymers having different chain lengths, monomer units, and graft amounts were prepared, and the time required for cells to become confluent on the substrate coated with each block copolymer (Period for confluent), and 20 The time (Cell sheet harvesting at 20 ° C.) required until the cell sheet was peeled after changing to ° C. was examined. As a result, it was found that when the amount of PIPAAm was high (for example, 1.45 ⁇ g / cm 2 or more), it took 7 days or more for the cells to become confluent.
- ⁇ / RTI> By using the temperature-responsive cell culture substrate of the present invention, cells obtained from each tissue can be efficiently cultured. If this culturing method is used, the cultured cells or the cell sheet can be efficiently peeled off without being damaged only by changing the temperature.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Graft Or Block Polymers (AREA)
Abstract
Description
本発明の一態様では、基材表面が相分離構造を有する。
本発明の一態様では、ブロックコポリマー内の温度応答性ポリマーの含量が30~90wt%である。
本発明の一態様では、ブロックコポリマー内の温度応答性ポリマーの平均分子量が3000以上である。なお、図2で示したように、重量平均分子量(Mw)と数平均分子量(Mn)との比が1に近いことから、本発明の効果を得るためには、いずれの平均分子量を採用してもよい。
本発明の一態様では、温度応答性ポリマーが、ポリ-N-置換アクリルアミド誘導体、ポリ-N-置換メタクリルアミド誘導体、これらの共重合体、ポリビニルメチルエーテルのいずれか一つ、もしくは二つ以上からなる。
本発明の一態様では、温度応答性ポリマーがポリ-N-イソプロピルアクリルアミドである。
本発明の一態様では、基材が板状の単独、もしくは2種以上を組み合わせたものであることを特徴とする。
本発明の一態様では、ブロックコポリマーを溶解する溶媒が、アセトニトリルとN,N-ジメチルホルムアミドの混合液である。
本発明の一態様では、アセトニトリルとN,N-ジメチルホルムアミドの混合溶媒の混合比率が、体積比で5:1である。
本実施例においては、温度応答性ポリマーであるポリ-N-イソプロピルアクリルアミド(PIPAAm)のセグメントと水不溶性ポリマーであるポリ-n-ブチルメタクリレート(PBMA)のセグメントからなる温度応答性ブロックコポリマーを調製し、基材表面にスピンコーティングした。作製した温度応答性ブロックコポリマー被覆表面について、ポリマーの表面導入量や表面ぬれ性などの物性評価、並びに、温度変化による基材表面への細胞接着性の違いについても検討した。
具体的には、RAFT重合により合成した水不溶性ポリマーであるPBMAをマクロRAFT剤として、PBMA-b-PIPAAmブロックコポリマーを調製した(図1)。また、RAFT重合により分子量を制御して合成されたPBMAおよびPBMA-b-PIPAAmの分析値を図2に示す。なお、本明細書において、例えば、BMAとIPAAmのモノマー単位の比が70:24であるPBMA-b-PIPAAmブロックコポリマーを「B79-IP24」として表した。
得られたポリマーを核磁気共鳴分光法(1H-NMR)やゲル浸透クロマトグラフィー法(GPC)により評価した。PBMA-b-PIPAAmを0.1w/v%、0.3w/v%、または0.5w/v%の各濃度でアセトニトリル/N,N-ジメチルホルムアミド混合溶媒(5/1 in v/v)に溶解した。またコントロールとして、PBMA溶液(0.5w/v%)およびPIPPAm(0.5w/v%)を同様に調製した。次に、ポリマー溶液を細胞培養用ポリスチレン(TCPS)基材表面にスピンコートし(3000rpm,30sec)、一晩室温で乾燥した後、水で基材表面を洗浄し、6時間室温で減圧乾燥させることによって、温度応答性表面を調製した。また、ポリマー溶液で被覆していないTCPS、およびUpCell(登録商標)(セルシード社製、日本)もコントロールとして用いた。
実施例1の記載に従って、TCPSに各種濃度のB79-IP120を被覆した基材を調製し、該作製した基材上にウシ頸動脈由来血管内皮細胞(BAEC)を1.0×104cell/cm2濃度で播種し、ポリマー被覆基材表面上への37℃における細胞の接着性および20℃での低温処理による細胞の脱着挙動について評価した。図5中の黒丸はポリマーを被覆していないTCPS基材を示し、黒菱形および白菱形はTCPSにB79-IP120をそれぞれ0.3および0.5w/v%で被覆した基材上における細胞挙動の変化を表す。図5は、BAEC細胞を各基材に播種し、72時間培養後に、培養温度を37℃から20℃に変化させた場合の細胞接着率(%)の変化を示す。より具体的には、37℃での培養により細胞接着率が上昇するが、細胞培養用のTCPSでは接着率が高く、次いで、0.3w/v%でB79-IP120を被覆した基材が高かった。一方、0.5w/v%でB79-IP120を被覆した基材では細胞接着率が低かった。実際にTCPSに被覆されたPIPAAmの量を測定すると、濃度が0.3w/v%の場合では1.44μg/cm2であり、0.5w/v%の場合では1.81μg/cm2であり、PIPAAmの被覆量が多くなると細胞接着率が減少する傾向にあると言える。次に、37℃から20℃に温度変化させた場合の細胞接着率の変化は、PIPAAmの被覆量が1.44μg/cm2である基材を用いた場合に顕著に現れた。TCPSでは、温度変化に伴う細胞接着率にほとんど変化が見られなかった。一方、PIPAAmの被覆量が1.81μg/cm2である基材では、37℃から20℃へ温度変化させた場合、速やかに細胞が剥離する現象が見られたが、37℃における細胞接着率が低く、細胞を培養するには好ましくない条件であると考えられる。
さらに、上記各種基材上で培養したBAEC細胞の温度変化による脱着を観察した結果を写真に示す。0.3w/v%濃度のB79-IP120で被覆した基材上において37℃、3日間培養したところ、細胞の接着および伸展を確認した(図6)。次に、細胞接着した基材を20℃で2時間低温処理した結果、細胞は基材表面から自発的に脱着することが明らかとなった。一方、PBMA被覆基材において同様の操作を行ったが、細胞の接着および伸展は観察されたが、低温処理による細胞の脱着は観察されなかった(データ示さず)。以上の結果から、基材表面に導入されたPIPAAm鎖がLCST以下の低温処理により水和することで、基材表面の特性が変化し、接着した細胞が自発的に剥離したと考えられる。
(1)PIPAAm分子鎖長と細胞挙動の関係
ブロックコポリマー中のBMAのモノマー単位数(量体数)を一定にし、IPAAmのモノマー単位を変化させたブロックコポリマーを実施例1の製造法に従って調製した。実施例1と同様に、TCPSにブロックコポリマーを被覆させ、温度変化に伴うBAEC細胞の接着率の変化を調べた(図7)。上記実施例2と同様に、PIPAAmの被覆量の減少に伴って、細胞接着率が増加した。また、温度変化により細胞の脱着については、PIPAAmの被覆量が低い基材(1.18μg/cm2)においては、より被覆量の高い基材(1.44,1.60,1.63μg/cm2)に比べて緩やかに脱着し、被覆量が高い基材(1.44,1.60,1.63μg/cm2など)では、温度変化後、速やかに脱着することが分かった。
(2)ブロックコポリマー鎖長と細胞挙動の関係
鎖長が異なる3種のブロックコポリマー(B36-IP52、B79-IP120、およびB142-IP231)を調製した。実施例2と同様に、これらのブロックコポリマーをTCPSに被覆した基材を調製し、その後、各基材に播種したBAEC細胞の接着、続く温度変化による細胞の脱着挙動の変化を細胞接着率によって評価した(図8)。使用した3種のブロックコポリマーは、それらの鎖長は著しく異なるが、TCPSに被覆されたPIPAAmの量にほとんど差がなかった。また、各基材に播種された細胞の接着および脱着についても、各基材のブロックコポリマーの鎖長による顕著な違いは見られなかった。このことから、温度変化による細胞の接着および脱着の制御には、基材表面へのPIPAAmの修飾量が重要な要因の1つであると言える。
(3)ブロックコポリマーによる細胞シートの形成
図9には、PBMA-b-PIPAAmブロックコポリマー(0.3w/v%濃度のB79-IP120)で被覆した基材上で形成させた細胞シートと、温度変化による細胞シートの剥離後の写真を示す。PBMA-b-PIPAAmブロックコポリマー(0.3w/v%濃度のB79-IP120)を被覆した基材上でBAEC細胞を37℃にて培養し、コンフルエントになるまで培養した後、20℃で保温すると、隣接した細胞同士の細胞間結合は維持したまま、細胞シートを形成した。
図10では、鎖長、モノマー単位、およびグラフト量の異なる各種ブロックコポリマーを調製し、各ブロックコポリマーで被覆した基材上での細胞がコンフルエントになるまでに要する時間(Period for confluent)、および20℃へ変化させた後に細胞シートとして剥離するまでに要する時間(Cell sheet harvest at 20℃)を検討した。その結果、PIPAAmの被覆量が高いもの(例えば、1.45μg/cm2以上)になると、細胞がコンフルエントになるまでに7日以上の時間を要するようになることが分かった。一方で、PIPAAmの被覆量が0.8μg/cm2より低くなると、細胞がうまく剥離できず、細胞シートが形成されないことが分かった。以上の結果から、PBMA-b-PIPAAmを被覆した基材表面の使用により、細胞培養、並びに温度変化に応じた培養細胞または細胞シートの剥離を効率良く実現できることを確信した。
実施例2で用いたB79-IP120を0.1w/v%の濃度で水中に分散させることを試みた。その結果、本ポリマーは水中で分散、乳化させられず均一な溶液を得ることはできなかった。得られた溶液をTCPS基材表面へ被覆したが、目視できる程度に器材表面が不均一となり、本発明として好ましいものではなかった。
Claims (12)
- 水不溶性ポリマーセグメントと温度応答性ポリマーセグメントが結合した構造をとるブロックコポリマーが、基材表面に温度応答性ポリマー分として0.8~3.0μg/cm2の割合で被覆されている、細胞培養用温度応答性基材。
- 基材表面に水不溶性ポリマー分として0.09~7.0μg/cm2の割合で被覆されている、請求項1記載の細胞培養用温度応答性基材。
- 基材表面が相分離構造を有する、請求項1または2記載の細胞培養用温度応答性基材。
- ブロックコポリマー内の温度応答性ポリマーの含量が30~90wt%である、請求項1~3のいずれか1項記載の細胞培養用温度応答性基材。
- ブロックコポリマー内の温度応答性ポリマーの平均分子量が3000以上である、請求項1~4のいずれか1項記載の細胞培養用温度応答性基材。
- 温度応答性ポリマーが、ポリ-N-置換アクリルアミド誘導体、ポリ-N-置換メタクリルアミド誘導体、これらの共重合体、ポリビニルメチルエーテルのいずれか一つ、もしくは二つ以上からなる、請求項1~5のいずれか1項記載の細胞培養用温度応答性基材。
- 温度応答性ポリマーがポリ-N-イソプロピルアクリルアミドである、請求項1~6のいずれか1項記載の細胞培養用温度応答性基材。
- 基材が板状の単独、もしくは2種以上を組み合わせたものであることを特徴とする請求項1~7いずれか1項記載の温度応答性基材。
- 水不溶性ポリマーセグメントと温度応答性ポリマーセグメントが結合した構造をとるブロックコポリマーを有機溶媒に溶解、もしくは分散させ、当該ブロックコポリマー溶液を基材表面へスピンコートすることで均一に塗布し、乾燥させることを特徴とする細胞培養用温度応答性基材の製造方法。
- ブロックコポリマーが可逆的付加開裂連鎖移動(RAFT)重合によって得られるものである、請求項9記載の細胞培養用温度応答性基材の製造方法。
- ブロックコポリマーを溶解する溶媒が、アセトニトリルとN,N-ジメチルホルムアミドの混合液である、請求項9または10記載の細胞培養用温度応答性基材の製造方法。
- アセトニトリルとN,N-ジメチルホルムアミドの混合溶媒の混合比率が、体積比で5:1である、請求項11記載の細胞培養用温度応答性基材の製造方法。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201180042162.6A CN103080295B (zh) | 2010-08-31 | 2011-08-31 | 细胞培养用温度应答性基材及其制造方法 |
EP11821897.3A EP2612902B1 (en) | 2010-08-31 | 2011-08-31 | Temperature-responsive substrate for cell culture and method for producing same |
JP2012531949A JP5846584B2 (ja) | 2010-08-31 | 2011-08-31 | 細胞培養用温度応答性基材及びその製造方法 |
US13/819,685 US9279102B2 (en) | 2010-08-31 | 2011-08-31 | Temperature-responsive substrate for cell culture and production method thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010208506 | 2010-08-31 | ||
JP2010-208506 | 2010-08-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012029882A1 true WO2012029882A1 (ja) | 2012-03-08 |
Family
ID=45772959
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2011/069839 WO2012029882A1 (ja) | 2010-08-31 | 2011-08-31 | 細胞培養用温度応答性基材及びその製造方法 |
Country Status (5)
Country | Link |
---|---|
US (1) | US9279102B2 (ja) |
EP (1) | EP2612902B1 (ja) |
JP (1) | JP5846584B2 (ja) |
CN (1) | CN103080295B (ja) |
WO (1) | WO2012029882A1 (ja) |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013181713A1 (en) * | 2012-06-07 | 2013-12-12 | The University Of Queensland | Release media |
WO2014133168A1 (ja) * | 2013-02-28 | 2014-09-04 | Sakai Hideaki | 新規グラフトポリマー、それを用いた細胞培養用温度応答性基材及びその製造方法、並びに当該ポリマーが固定化された液体クロマトグラフィー担体及びそれを用いた液体クロマトグラフィー法 |
JP2015221851A (ja) * | 2014-05-22 | 2015-12-10 | 積水化学工業株式会社 | 粘着剤組成物、細胞支持用基材、細胞培養用基材、並びに、細胞の培養方法 |
WO2016159153A1 (ja) * | 2015-03-31 | 2016-10-06 | 東ソー株式会社 | 細胞培養基材、その製造方法、およびそれを用いた細胞培養方法 |
JP2016194054A (ja) * | 2015-03-31 | 2016-11-17 | 東ソー株式会社 | ブロック共重合体 |
JP2017012018A (ja) * | 2015-06-26 | 2017-01-19 | 国立研究開発法人国立循環器病研究センター | 細胞培養器の製造方法 |
WO2018116902A1 (ja) | 2016-12-22 | 2018-06-28 | Dic株式会社 | 細胞培養基材 |
JP2018154752A (ja) * | 2017-03-17 | 2018-10-04 | 東ソー株式会社 | 共重合体およびその製造方法 |
WO2018198495A1 (ja) * | 2017-04-25 | 2018-11-01 | ダイキン工業株式会社 | 温度応答性細胞培養基材及びその製造方法 |
JP2019094421A (ja) * | 2017-11-22 | 2019-06-20 | 東ソー株式会社 | ブロック共重合体コートビーズおよびその製造方法 |
JP2020014453A (ja) * | 2018-07-13 | 2020-01-30 | 東ソー株式会社 | 幹細胞の培養基材及び幹細胞の製造方法 |
JP2020015892A (ja) * | 2018-07-13 | 2020-01-30 | 東ソー株式会社 | ブロック共重合体及び培養基材、幹細胞の製造方法 |
JP2020041107A (ja) * | 2018-09-13 | 2020-03-19 | 東ソー株式会社 | 温度応答性共重合体の精製法 |
JP2020171891A (ja) * | 2019-04-11 | 2020-10-22 | 東ソー株式会社 | 温度応答性ブロック共重合体膜の製造方法 |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105566567A (zh) * | 2016-01-27 | 2016-05-11 | 清华大学 | 硬度可调节的温敏聚合物材料及其制备方法和应用 |
EP3561042A4 (en) * | 2016-12-22 | 2020-10-07 | DIC Corporation | SCAFFOLDING MATERIAL FOR CELL CULTURE |
US11441120B2 (en) | 2016-12-22 | 2022-09-13 | Fujifilm Corporation | Cell culture substrate |
TWI693283B (zh) * | 2019-03-14 | 2020-05-11 | 三顧股份有限公司 | 一種微細胞層片的製造方法 |
WO2020230884A1 (ja) * | 2019-05-15 | 2020-11-19 | 積水化学工業株式会社 | 細胞培養用足場材料及び細胞培養用容器 |
CN112831005B (zh) | 2019-11-25 | 2022-08-12 | 青岛金典生化器材有限公司 | 细胞培养用的温敏性智能型基材及其制备方法 |
JPWO2022202911A1 (ja) | 2021-03-26 | 2022-09-29 | ||
CN113121860A (zh) * | 2021-04-23 | 2021-07-16 | 广州洁特生物过滤股份有限公司 | 一种温敏型细胞培养基材及其制备方法 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH02211865A (ja) | 1989-02-10 | 1990-08-23 | Kao Corp | 細胞培養支持体材料 |
JPH05192138A (ja) | 1992-01-22 | 1993-08-03 | Kao Corp | 皮膚細胞培養法及び培養皮膚 |
JPH06104061A (ja) | 1992-09-21 | 1994-04-15 | Shinohara Tekkosho:Kk | 印刷機における回転軸への伝達装置 |
JPH09169850A (ja) * | 1995-12-20 | 1997-06-30 | Kagaku Gijutsu Shinko Jigyodan | ポリアクリルアミド誘導体を有するブロック共重合体および温度応答性高分子ミセル |
JP2007105311A (ja) | 2005-10-14 | 2007-04-26 | Nakamura Shinkyu Sekkotsuin:Kk | 半月板矯正用ベルト |
WO2010010837A1 (ja) * | 2008-07-25 | 2010-01-28 | コニカミノルタホールディングス株式会社 | 細胞培養支持体および細胞培養方法 |
WO2010027081A1 (ja) * | 2008-09-08 | 2010-03-11 | 学校法人東京理科大学 | スフェロイド複合体およびスフェロイド含有ハイドロゲルならびにその製造方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5641669B2 (ja) | 2007-03-14 | 2014-12-17 | 株式会社セルシード | 細胞表層蛋白修復方法 |
CN101293943B (zh) * | 2008-06-23 | 2011-05-11 | 天津工业大学 | 半乳糖基温度敏感型高分子水凝胶及其制备方法 |
-
2011
- 2011-08-31 US US13/819,685 patent/US9279102B2/en active Active
- 2011-08-31 CN CN201180042162.6A patent/CN103080295B/zh active Active
- 2011-08-31 WO PCT/JP2011/069839 patent/WO2012029882A1/ja active Application Filing
- 2011-08-31 EP EP11821897.3A patent/EP2612902B1/en active Active
- 2011-08-31 JP JP2012531949A patent/JP5846584B2/ja active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH02211865A (ja) | 1989-02-10 | 1990-08-23 | Kao Corp | 細胞培養支持体材料 |
JPH05192138A (ja) | 1992-01-22 | 1993-08-03 | Kao Corp | 皮膚細胞培養法及び培養皮膚 |
JPH06104061A (ja) | 1992-09-21 | 1994-04-15 | Shinohara Tekkosho:Kk | 印刷機における回転軸への伝達装置 |
JPH09169850A (ja) * | 1995-12-20 | 1997-06-30 | Kagaku Gijutsu Shinko Jigyodan | ポリアクリルアミド誘導体を有するブロック共重合体および温度応答性高分子ミセル |
JP2007105311A (ja) | 2005-10-14 | 2007-04-26 | Nakamura Shinkyu Sekkotsuin:Kk | 半月板矯正用ベルト |
WO2010010837A1 (ja) * | 2008-07-25 | 2010-01-28 | コニカミノルタホールディングス株式会社 | 細胞培養支持体および細胞培養方法 |
WO2010027081A1 (ja) * | 2008-09-08 | 2010-03-11 | 学校法人東京理科大学 | スフェロイド複合体およびスフェロイド含有ハイドロゲルならびにその製造方法 |
Non-Patent Citations (3)
Title |
---|
INTERFACE, vol. 4, 2007, pages 1151 - 1157 |
See also references of EP2612902A4 * |
SOFT MATTER, vol. 5, 2009, pages 2937 - 2946 |
Cited By (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9587104B2 (en) | 2012-06-07 | 2017-03-07 | The University Of Queensland | Release media |
WO2013181713A1 (en) * | 2012-06-07 | 2013-12-12 | The University Of Queensland | Release media |
CN104640972A (zh) * | 2012-06-07 | 2015-05-20 | 昆士兰大学 | 释放介质 |
JP2015519902A (ja) * | 2012-06-07 | 2015-07-16 | ザ ユニバーシティー オブ クイーンズランド | 放出媒体 |
JPWO2014133168A1 (ja) * | 2013-02-28 | 2017-02-09 | 秀昭 坂井 | 新規グラフトポリマー、それを用いた細胞培養用温度応答性基材及びその製造方法、並びに当該ポリマーが固定化された液体クロマトグラフィー担体及びそれを用いた液体クロマトグラフィー法 |
JP2021059742A (ja) * | 2013-02-28 | 2021-04-15 | 秀昭 坂井 | 細胞培養用温度応答性基材 |
WO2014133168A1 (ja) * | 2013-02-28 | 2014-09-04 | Sakai Hideaki | 新規グラフトポリマー、それを用いた細胞培養用温度応答性基材及びその製造方法、並びに当該ポリマーが固定化された液体クロマトグラフィー担体及びそれを用いた液体クロマトグラフィー法 |
US11371015B2 (en) | 2013-02-28 | 2022-06-28 | Hideaki Sakai | Graft polymer, temperature-responsive substrate for cell culture using the same and production method therefor, as well as liquid chromatographic carrier having the novel graft polymer immomibilized thereon and liquid chromatographic method using the same |
JP7049487B2 (ja) | 2013-02-28 | 2022-04-06 | 秀昭 坂井 | 細胞培養用温度応答性基材 |
JP2015221851A (ja) * | 2014-05-22 | 2015-12-10 | 積水化学工業株式会社 | 粘着剤組成物、細胞支持用基材、細胞培養用基材、並びに、細胞の培養方法 |
WO2016159153A1 (ja) * | 2015-03-31 | 2016-10-06 | 東ソー株式会社 | 細胞培養基材、その製造方法、およびそれを用いた細胞培養方法 |
JP2016194054A (ja) * | 2015-03-31 | 2016-11-17 | 東ソー株式会社 | ブロック共重合体 |
JP2017012018A (ja) * | 2015-06-26 | 2017-01-19 | 国立研究開発法人国立循環器病研究センター | 細胞培養器の製造方法 |
WO2018116902A1 (ja) | 2016-12-22 | 2018-06-28 | Dic株式会社 | 細胞培養基材 |
US11427803B2 (en) | 2016-12-22 | 2022-08-30 | Fujifilm Corporation | Cell culture substrate |
KR20190094149A (ko) | 2016-12-22 | 2019-08-12 | 디아이씨 가부시끼가이샤 | 세포 배양 기재 |
JP2018154752A (ja) * | 2017-03-17 | 2018-10-04 | 東ソー株式会社 | 共重合体およびその製造方法 |
WO2018198495A1 (ja) * | 2017-04-25 | 2018-11-01 | ダイキン工業株式会社 | 温度応答性細胞培養基材及びその製造方法 |
JP2019094421A (ja) * | 2017-11-22 | 2019-06-20 | 東ソー株式会社 | ブロック共重合体コートビーズおよびその製造方法 |
JP2020015892A (ja) * | 2018-07-13 | 2020-01-30 | 東ソー株式会社 | ブロック共重合体及び培養基材、幹細胞の製造方法 |
JP2020014453A (ja) * | 2018-07-13 | 2020-01-30 | 東ソー株式会社 | 幹細胞の培養基材及び幹細胞の製造方法 |
JP7293683B2 (ja) | 2018-07-13 | 2023-06-20 | 東ソー株式会社 | ブロック共重合体及び培養基材、幹細胞の製造方法 |
JP2020041107A (ja) * | 2018-09-13 | 2020-03-19 | 東ソー株式会社 | 温度応答性共重合体の精製法 |
JP7135631B2 (ja) | 2018-09-13 | 2022-09-13 | 東ソー株式会社 | 温度応答性共重合体の精製法 |
JP2020171891A (ja) * | 2019-04-11 | 2020-10-22 | 東ソー株式会社 | 温度応答性ブロック共重合体膜の製造方法 |
Also Published As
Publication number | Publication date |
---|---|
EP2612902A4 (en) | 2015-04-01 |
US9279102B2 (en) | 2016-03-08 |
EP2612902B1 (en) | 2016-10-26 |
EP2612902A1 (en) | 2013-07-10 |
JPWO2012029882A1 (ja) | 2013-10-31 |
CN103080295B (zh) | 2014-08-27 |
CN103080295A (zh) | 2013-05-01 |
JP5846584B2 (ja) | 2016-01-20 |
US20140212973A1 (en) | 2014-07-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5846584B2 (ja) | 細胞培養用温度応答性基材及びその製造方法 | |
JP6206930B2 (ja) | 直鎖型温度応答性高分子が固定化された温度応答性細胞培養基材、及びその製造方法 | |
JP7049487B2 (ja) | 細胞培養用温度応答性基材 | |
JP5907661B2 (ja) | 直鎖型温度応答性高分子が固定化された温度応答性細胞培養基材、及びその製造方法 | |
JP5349728B2 (ja) | 細胞培養基材及び細胞培養方法 | |
JP2013059312A (ja) | 温度応答性細胞培養用ビーズ及びその製造方法 | |
WO2018116902A1 (ja) | 細胞培養基材 | |
JP6313822B2 (ja) | 温度応答性細胞培養用ビーズ及びその製造方法 | |
WO2022114074A1 (ja) | 細胞塊の製造方法 | |
JP2019083761A (ja) | 温度応答性細胞培養基材及びその製造方法 | |
JP2021180653A (ja) | 細胞培養用温度応答性基材及びその製造方法 | |
CN112831005B (zh) | 细胞培养用的温敏性智能型基材及其制备方法 | |
JP2023073152A (ja) | 細胞培養用温度応答性基材及びその製造方法 | |
JP3428133B2 (ja) | 細胞培養材料、製造および培養方法 | |
JP2006280206A (ja) | 細胞培養基材及び細胞培養方法 | |
JP5911678B2 (ja) | 伸縮可能な温度応答性基材、製造方法及びその利用方法 | |
JP6123247B2 (ja) | 温度応答性を有する細胞培養基材の製造方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 201180042162.6 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11821897 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
ENP | Entry into the national phase |
Ref document number: 2012531949 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13819685 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2011821897 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011821897 Country of ref document: EP |